Translational and regulatory challenges for exon skipping therapies.

PubMed

Aartsma-Rus, Annemieke; Ferlini, Alessandra; Goemans, Nathalie; Pasmooij, Anna M G; Wells, Dominic J; Bushby, Katerine; Vroom, Elizabeth; Balabanov, Pavel

2014-10-01

Several translational challenges are currently impeding the therapeutic development of antisense-mediated exon skipping approaches for rare diseases. Some of these are inherent to developing therapies for rare diseases, such as small patient numbers and limited information on natural history and interpretation of appropriate clinical outcome measures. Others are inherent to the antisense oligonucleotide (AON)-mediated exon skipping approach, which employs small modified DNA or RNA molecules to manipulate the splicing process. This is a new approach and only limited information is available on long-term safety and toxicity for most AON chemistries. Furthermore, AONs often act in a mutation-specific manner, in which case multiple AONs have to be developed for a single disease. A workshop focusing on preclinical development, trial design, outcome measures, and different forms of marketing authorization was organized by the regulatory models and biochemical outcome measures working groups of Cooperation of Science and Technology Action: "Networking towards clinical application of antisense-mediated exon skipping for rare diseases." The workshop included participants from patient organizations, academia, and members of staff from the European Medicine Agency and Medicine Evaluation Board (the Netherlands). This statement article contains the key outcomes of this meeting.

Intra-Amygdala Injections of CREB Antisense Impair Inhibitory Avoidance Memory: Role of Norepinephrine and Acetylcholine

ERIC Educational Resources Information Center

Canal, Clinton E.; Chang, Qing; Gold, Paul E.

2008-01-01

Infusions of CREB antisense into the amygdala prior to training impair memory for aversive tasks, suggesting that the antisense may interfere with CRE-mediated gene transcription and protein synthesis important for the formation of new memories within the amygdala. However, the amygdala also appears to modulate memory formation in distributed…

Stabilization of the μ-opioid receptor by truncated single transmembrane splice variants through a chaperone-like action.

PubMed

Xu, Jin; Xu, Ming; Brown, Taylor; Rossi, Grace C; Hurd, Yasmin L; Inturrisi, Charles E; Pasternak, Gavril W; Pan, Ying-Xian

2013-07-19

The μ-opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, as illustrated by the identification of an array of splice variants generated by both 5' and 3' alternative splicing. The current study reports the identification of another set of splice variants conserved across species that are generated through exon skipping or insertion that encodes proteins containing only a single transmembrane (TM) domain. Using a Tet-Off system, we demonstrated that the truncated single TM variants can dimerize with the full-length 7-TM μ-opioid receptor (MOR-1) in the endoplasmic reticulum, leading to increased expression of MOR-1 at the protein level by a chaperone-like function that minimizes endoplasmic reticulum-associated degradation. In vivo antisense studies suggested that the single TM variants play an important role in morphine analgesia, presumably through modulation of receptor expression levels. Our studies suggest the functional roles of truncated receptors in other G protein-coupled receptor families.

Antisense oligonucleotides effectively inhibit the co-transcriptional splicing of a Candida group I intron in vitro and in vivo: Implications for antifungal therapeutics.

PubMed

Zhang, Libin; Leibowitz, Michael J; Zhang, Yi

2009-02-18

Self-splicing of group I intron from the 26S rRNA of Candida albicans is essential for maturation of the host RNA. Here, we demonstrated that the co-transcriptional splicing of the intron in vitro was blocked by antisense oligonucleotides (AONs) targeting the P3-P7 core of the intron. The core-targeted AON effectively and specifically inhibited the intron splicing from its host RNA in living C. albicans. Furthermore, flow cytometry experiments showed that the growth inhibition was caused by a fungicidal effect. For the first time, we showed that an AON targeting the ribozyme core folding specifically inhibits the endogenous ribozyme splicing in living cells and specifically kills the intron-containing fungal strains, which sheds light on the development of antifungal drugs in the future.

FAS-antisense 1 lncRNA and production of soluble versus membrane Fas in B-cell lymphoma

PubMed Central

Sehgal, Lalit; Mathur, Rohit; Braun, Frank K.; Wise, Jillian F.; Berkova, Zuzana; Neelapu, Sattva; Kwak, Larry W.; Samaniego, Felipe

2018-01-01

Impaired Fas-mediated apoptosis is associated with poor clinical outcomes and cancer chemoresistance. Soluble Fas receptor (sFas), produced by skipping of exon 6, inhibits apoptosis by sequestering Fas ligand. Serum sFas is associated with poor prognosis of non-Hodgkin's lymphomas. We found that the alternative splicing of Fas in lymphomas is tightly regulated by a lncRNA corresponding to an antisense transcript of Fas (FAS-AS1). Levels of FAS-AS1 correlate inversely with production of sFas and FAS-AS1 binding to the RBM5 inhibits RBM5-mediated exon 6 skipping. EZH2, often mutated or overexpressed in lymphomas, hyper-methylates the FAS-AS1 promoter and represses the FAS-AS1 expression. EZH2-mediated repression of FAS-AS1 promoter can be released by DZNeP or overcome by ectopic expression of FAS-AS1, both of which increase levels of FAS-AS1 and correspondingly decrease expression of sFas. Treatment with Bruton’s tyrosine kinase (BTK) inhibitor or EZH2 knockdown decreases the levels of EZH2, RBM5 and sFas thereby enhances Fas-mediated apoptosis. This is the first report showing functional regulation of Fas repression by its antisense RNA. Our results reveal new therapeutic targets in lymphomas and provide a rationale for the use of EZH2 inhibitors or ibrutinib in combination with chemotherapeutic agents that recruit Fas for effective cell killing. PMID:24811343

MIR846 and MIR842 comprise a cistronic MIRNA pair that is regulated by abscisic acid by alternative splicing in roots of Arabidopsis

PubMed Central

Jia, Fan; Rock, Christopher D.

2013-01-01

MicroRNAs (miRNAs) are ~21-nucleotide long endogenous small RNAs that regulate gene expression through post-transcriptional or transcriptional gene silencing (PTGS/TGS) and/or translational inhibition. miRNAs can arise from the “exon” of a MIRNA gene, from an intron (e.g. mirtrons in animals), or from the antisense strand of a protein coding gene (natural antisense microRNAs, nat-miRNAs). Here we demonstrate that two functionally related miRNAs, miR842 and miR846, arise from the same transcription unit but from alternate splicing isoforms. miR846 is expressed only from Isoform1 while in Isoforms2 and -3, a part of pre-miR846 containing the miRNA* sequence is included in the intron. The splicing of the intron truncates the pre-MIRNA and disrupts the expression of the mature miR846.. We name this novel phenomenon splicing-regulated miRNA. Abscisic acid (ABA) is shown to mediate the alternative splicing event by reducing the functional Isoform1 and increasing the non-functional Isoform3, thus repressing the expression of miR846 concomitant with accumulation of an ABA-inducible target jacalin At5g28520 mRNA, whose cleavage was shown by modified 5′-RACE. This regulation shows the functional importance of splicing-regulated miRNA and suggests possible mechanisms for altered ABA response phenotypes of miRNA biogenesis mutants. A. lyrata-MIR842 and Aly-MIR846 have conserved genomic arrangements with A. thaliana and candidate target jacalins, similar primary transcript structures and intron processing, and better miRNA-miRNA* pairings, suggesting that the interactions between ABA, MIR842, MIR846 and jacalins are similar in A. lyrata. Together, splicing-regulated miRNAs, nat-miRNAs/inc-miRNAs and mirtrons illustrate the complexity of MIRNA genes, the importance of introns in the biogenesis and regulation of miRNAs, and raise questions about the processes and molecular mechanisms that drive MIRNA evolution. PMID:23341152

Antisense Oligonucleotides for the Treatment of Spinal Muscular Atrophy

PubMed Central

Porensky, Paul N.

2013-01-01

Abstract Spinal muscular atrophy (SMA) is an autosomal recessive disease affecting ∼1 in 10,000 live births. The most striking component is the loss of α-motor neurons in the ventral horn of the spinal cord, resulting in progressive paralysis and eventually premature death. There is no current treatment paradigm other than supportive care, though the past 15 years has seen a striking advancement in understanding of both SMA genetics and molecular mechanisms. A variety of disease-modifying interventions are rapidly bridging the translational gap from the laboratory to clinical trials, including the application of antisense oligonucleotide (ASO) therapy for the correction of aberrant RNA splicing characteristic of SMA. Survival motor neuron (SMN) is a ubiquitously expressed 38-kD protein. Humans have two genes that produce SMN, SMN1 and SMN2, the former of which is deleted or nonfunctional in the majority of patients with SMA. These two genes are nearly identical with one exception, a C to T transition (C6T) within exon 7 of SMN2. C6T disrupts a modulator of splicing, leading to the exclusion of exon 7 from ∼90% of the mRNA transcript. The resultant truncated Δ7SMN protein does not oligomerize efficiently and is rapidly degraded. SMA can therefore be considered a disease of too little SMN protein. A number of cis-acting splice modifiers have been identified in the region of exon 7, the steric block of which enhances the retention of the exon and a resultant full-length mRNA sequence. ASOs targeted to these splice motifs have shown impressive phenotype rescue in multiple SMA mouse models. PMID:23544870

Overcoming imatinib resistance conferred by the BIM deletion polymorphism in chronic myeloid leukemia with splice-switching antisense oligonucleotides

PubMed Central

Liu, Jun; Bhadra, Malini; Sinnakannu, Joanna Rajeswary; Yue, Wan Lin; Tan, Cheryl Weiqi; Rigo, Frank; Ong, S.Tiong; Roca, Xavier

2017-01-01

Many tyrosine kinase-driven cancers, including chronic myeloid leukemia (CML), are characterized by high response rates to specific tyrosine kinase inhibitors (TKIs) like imatinib. In East Asians, primary imatinib resistance is caused by a deletion polymorphism in Intron 2 of the BIM gene, whose product is required for TKI-induced apoptosis. The deletion biases BIM splicing from exon 4 to exon 3, generating splice isoforms lacking the exon 4-encoded pro-apoptotic BH3 domain, which impairs the ability of TKIs to induce apoptosis. We sought to identify splice-switching antisense oligonucleotides (ASOs) that block exon 3 but enhance exon 4 splicing, and thereby resensitize BIM deletion-containing cancers to imatinib. First, we mapped multiple cis-acting splicing elements around BIM exon 3 by minigene mutations, and found an exonic splicing enhancer acting via SRSF1. Second, by a systematic ASO walk, we isolated ASOs that corrected the aberrant BIM splicing. Eight of 67 ASOs increased exon 4 levels in BIM deletion-containing cells, and restored imatinib-induced apoptosis and TKI sensitivity. This proof-of-principle study proves that resistant CML cells by BIM deletion polymorphism can be resensitized to imatinib via splice-switching BIM ASOs. Future optimizations might yield a therapeutic ASO as precision-medicine adjuvant treatment for BIM-polymorphism-associated TKI-resistant CML and other cancers. PMID:29100409

Overcoming imatinib resistance conferred by the BIM deletion polymorphism in chronic myeloid leukemia with splice-switching antisense oligonucleotides.

PubMed

Liu, Jun; Bhadra, Malini; Sinnakannu, Joanna Rajeswary; Yue, Wan Lin; Tan, Cheryl Weiqi; Rigo, Frank; Ong, S Tiong; Roca, Xavier

2017-09-29

Many tyrosine kinase-driven cancers, including chronic myeloid leukemia (CML), are characterized by high response rates to specific tyrosine kinase inhibitors (TKIs) like imatinib. In East Asians, primary imatinib resistance is caused by a deletion polymorphism in Intron 2 of the BIM gene, whose product is required for TKI-induced apoptosis. The deletion biases BIM splicing from exon 4 to exon 3, generating splice isoforms lacking the exon 4-encoded pro-apoptotic BH3 domain, which impairs the ability of TKIs to induce apoptosis. We sought to identify splice-switching antisense oligonucleotides (ASOs) that block exon 3 but enhance exon 4 splicing, and thereby resensitize BIM deletion-containing cancers to imatinib. First, we mapped multiple cis -acting splicing elements around BIM exon 3 by minigene mutations, and found an exonic splicing enhancer acting via SRSF1. Second, by a systematic ASO walk, we isolated ASOs that corrected the aberrant BIM splicing. Eight of 67 ASOs increased exon 4 levels in BIM deletion-containing cells, and restored imatinib-induced apoptosis and TKI sensitivity. This proof-of-principle study proves that resistant CML cells by BIM deletion polymorphism can be resensitized to imatinib via splice-switching BIM ASOs. Future optimizations might yield a therapeutic ASO as precision-medicine adjuvant treatment for BIM -polymorphism-associated TKI-resistant CML and other cancers.

[The role of RNA splicing in the pathogenesis of spinal muscular atrophy and development of its therapeutics].

PubMed

Sahashi, Kentaro; Sobue, Gen

2014-12-01

Loss-of-function mutations in SMN1 cause spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. Degeneration of alpha-motor neurons that results in progressive paralysis is a pathological hallmark of SMA. Recently, peripheral-tissue involvement has also been reported in SMA. Patients have low levels of functional SMN which is attributed to alternative splicing in SMN2, a gene closely-related to SMN1. This decrease in the expression of SMN, a ubiquitously expressed protein involved in promoting snRNP assembly required for splicing, is responsible for SMA. However, the mechanism through which decrease in SMN levels causes SMA remains unclear. Currently, no curative treatment is available for SMA, but SMN restoration is thought to be necessary and sufficient for cure. Antisense oligonucleotides (ASOs) can be designed to specifically alter splicing patterns of target pre-mRNAs. We identified an ASO that redirects SMN2 splicing and is currently in clinical trials for use as RNA-targeting therapeutics. Further, we have also reported a novel application of splicing-modulating ASOs--creation of animal phenocopy models of diseases by inducing mis-splicing. Exploring the relationship between the spatial and temporal effects of therapeutic and pathogenic ASOs yields relevant insights into the roles of SMN in SMA pathogenesis and into its normal physiological functions. This knowledge, in turn, contributes to the ongoing development of targeted therapeutics.

Biology Today: Respect for RNA.

ERIC Educational Resources Information Center

Flannery, Maura C., Ed.

1991-01-01

The high points of the story of RNA are presented. The functions of RNA within the cell, how these functions are carried out, and how they evolved are described. The topics of splicing, self-splicing, RNA editing, transcription and translation, and antisense RNA are discussed. (KR)

Therapeutic correction of ApoER2 splicing in Alzheimer's disease mice using antisense oligonucleotides.

PubMed

Hinrich, Anthony J; Jodelka, Francine M; Chang, Jennifer L; Brutman, Daniella; Bruno, Angela M; Briggs, Clark A; James, Bryan D; Stutzmann, Grace E; Bennett, David A; Miller, Steven A; Rigo, Frank; Marr, Robert A; Hastings, Michelle L

2016-04-01

Apolipoprotein E receptor 2 (ApoER2) is an apolipoprotein E receptor involved in long-term potentiation, learning, and memory. Given its role in cognition and its association with the Alzheimer's disease (AD) risk gene, apoE, ApoER2 has been proposed to be involved in AD, though a role for the receptor in the disease is not clear. ApoER2 signaling requires amino acids encoded by alternatively spliced exon 19. Here, we report that the balance of ApoER2 exon 19 splicing is deregulated in postmortem brain tissue from AD patients and in a transgenic mouse model of AD To test the role of deregulated ApoER2 splicing in AD, we designed an antisense oligonucleotide (ASO) that increases exon 19 splicing. Treatment of AD mice with a single dose of ASO corrected ApoER2 splicing for up to 6 months and improved synaptic function and learning and memory. These results reveal an association between ApoER2 isoform expression and AD, and provide preclinical evidence for the utility of ASOs as a therapeutic approach to mitigate Alzheimer's disease symptoms by improving ApoER2 exon 19 splicing. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Building a bridge between neurobiology and mental illness.

PubMed

Costa, E

1992-10-01

GABA (gamma amino butyric acid) is the most abundant and important inhibitory transmitter in mammalian CNS. It counterbalances the glutamate mediated neuronal excitation. Abnormalities of the interaction of these two transmitters might change the mechanisms of neuronal group selection that according to Edelman [Neural Darwinism. Basic Books, New York] play a role in mediating several brain functions including cognition processes. Indeed imbalances in GABAergic functions were shown to elicit psychoses. They can be obtained by administration of drugs that affect synthesis, metabolism and uptake of GABA and thereby cause a persistent stimulation of GABAA receptors or perhaps by genetic abnormalities in DNA transcription, pre-mRNA splicing, mRNA translation and posttranslation modifications of GABAA receptor subunits. The complexities in the regulation of GABAA receptor subunit structure, synthesis, assembly and the brain location of specific mRNA encoding for these subunits are investigated with in situ mRNA hybridization specific for subunits of GABAA receptors. The role of the variability resulting from the complexities in the regulation of GABAA receptor allosteric modulation by drugs and putative endogenous allosteric modulators of GABA action at GABAA receptors is discussed. This discussion gives relevance to the possibility that genetic abnormalities in the expression of proteins participating in GABAergic function are to be considered as a possible target of the genetic defects operative in psychoses. In line with this thinking, it is suggested that partial allosteric modulators (partial agonists) of GABAA receptors and the phosphothioate or methylphosphonate analogs antisense to specific mRNA oligonucleotides that mediate the expression of genetic information concerning GABAA and glutamate receptor subunits may become valuable tools in psychiatric research. Perhaps in the future these studies might generate new ideas useful in the therapy of genetically determined psychiatric illness.

Systemic peptide-mediated oligonucleotide therapy improves long-term survival in spinal muscular atrophy

PubMed Central

Hazell, Gareth; Shabanpoor, Fazel; Saleh, Amer F.; Bowerman, Melissa; Meijboom, Katharina E.; Zhou, Haiyan; Muntoni, Francesco; Talbot, Kevin; Gait, Michael J.; Wood, Matthew J. A.

2016-01-01

The development of antisense oligonucleotide therapy is an important advance in the identification of corrective therapy for neuromuscular diseases, such as spinal muscular atrophy (SMA). Because of difficulties of delivering single-stranded oligonucleotides to the CNS, current approaches have been restricted to using invasive intrathecal single-stranded oligonucleotide delivery. Here, we report an advanced peptide-oligonucleotide, Pip6a-morpholino phosphorodiamidate oligomer (PMO), which demonstrates potent efficacy in both the CNS and peripheral tissues in severe SMA mice following systemic administration. SMA results from reduced levels of the ubiquitously expressed survival motor neuron (SMN) protein because of loss-of-function mutations in the SMN1 gene. Therapeutic splice-switching oligonucleotides (SSOs) modulate exon 7 splicing of the nearly identical SMN2 gene to generate functional SMN protein. Pip6a-PMO yields SMN expression at high efficiency in peripheral and CNS tissues, resulting in profound phenotypic correction at doses an order-of-magnitude lower than required by standard naked SSOs. Survival is dramatically extended from 12 d to a mean of 456 d, with improvement in neuromuscular junction morphology, down-regulation of transcripts related to programmed cell death in the spinal cord, and normalization of circulating insulin-like growth factor 1. The potent systemic efficacy of Pip6a-PMO, targeting both peripheral as well as CNS tissues, demonstrates the high clinical potential of peptide-PMO therapy for SMA. PMID:27621445

How the discovery of ISS-N1 led to the first medical therapy for spinal muscular atrophy

PubMed Central

Singh, Natalia N.; Howell, Matthew D.; Androphy, Elliot J.; Singh, Ravindra N.

2017-01-01

Spinal muscular atrophy (SMA), a prominent genetic disease of infant mortality, is caused by low levels of survival motor neuron (SMN) protein owing to deletions or mutations of the SMN1 gene. SMN2, a nearly identical copy of SMN1 present in humans, cannot compensate for the loss of SMN1 due to predominant skipping of exon 7 during pre-mRNA splicing. With the recent FDA approval of nusinersen (Spinraza™), the potential for correction of SMN2 exon 7 splicing as a SMA therapy has been affirmed. Nusinersen is an antisense oligonucleotide that targets intronic splicing silencer N1 (ISS-N1) discovered in 2004 at the University of Massachusetts Medical School. ISS-N1 has emerged as the model target for testing the therapeutic efficacy of antisense oligonucleotides using different chemistries as well as different mouse models of SMA. Here we provide a historical account of events that led to the discovery of ISS-N1 and describe the impact of independent validations that raised the profile of ISS-N1 as one of the most potent antisense targets for the treatment of a genetic disease. Recent approval of nusinersen provides a much-needed boost for antisense technology that is just beginning to realize its potential. Beyond treating SMA, the ISS-N1 target offers myriad potentials for perfecting various aspects of the nucleic-acid-based technology for the amelioration of the countless number of pathological conditions. PMID:28485722

How the discovery of ISS-N1 led to the first medical therapy for spinal muscular atrophy.

PubMed

Singh, N N; Howell, M D; Androphy, E J; Singh, R N

2017-09-01

Spinal muscular atrophy (SMA), a prominent genetic disease of infant mortality, is caused by low levels of survival motor neuron (SMN) protein owing to deletions or mutations of the SMN1 gene. SMN2, a nearly identical copy of SMN1 present in humans, cannot compensate for the loss of SMN1 because of predominant skipping of exon 7 during pre-mRNA splicing. With the recent US Food and Drug Administration approval of nusinersen (Spinraza), the potential for correction of SMN2 exon 7 splicing as an SMA therapy has been affirmed. Nusinersen is an antisense oligonucleotide that targets intronic splicing silencer N1 (ISS-N1) discovered in 2004 at the University of Massachusetts Medical School. ISS-N1 has emerged as the model target for testing the therapeutic efficacy of antisense oligonucleotides using different chemistries as well as different mouse models of SMA. Here, we provide a historical account of events that led to the discovery of ISS-N1 and describe the impact of independent validations that raised the profile of ISS-N1 as one of the most potent antisense targets for the treatment of a genetic disease. Recent approval of nusinersen provides a much-needed boost for antisense technology that is just beginning to realize its potential. Beyond treating SMA, the ISS-N1 target offers myriad potentials for perfecting various aspects of the nucleic-acid-based technology for the amelioration of the countless number of pathological conditions.

TSUNAMI: an antisense method to phenocopy splicing-associated diseases in animals

PubMed Central

Sahashi, Kentaro; Hua, Yimin; Ling, Karen K.Y.; Hung, Gene; Rigo, Frank; Horev, Guy; Katsuno, Masahisa; Sobue, Gen; Ko, Chien-Ping; Bennett, C. Frank; Krainer, Adrian R.

2012-01-01

Antisense oligonucleotides (ASOs) are versatile molecules that can be designed to specifically alter splicing patterns of target pre-mRNAs. Here we exploit this feature to phenocopy a genetic disease. Spinal muscular atrophy (SMA) is a motor neuron disease caused by loss-of-function mutations in the SMN1 gene. The related SMN2 gene expresses suboptimal levels of functional SMN protein due to alternative splicing that skips exon 7; correcting this defect—e.g., with ASOs—is a promising therapeutic approach. We describe the use of ASOs that exacerbate SMN2 missplicing and phenocopy SMA in a dose-dependent manner when administered to transgenic Smn−/− mice. Intracerebroventricular ASO injection in neonatal mice recapitulates SMA-like progressive motor dysfunction, growth impairment, and shortened life span, with α-motor neuron loss and abnormal neuromuscular junctions. These SMA-like phenotypes are prevented by a therapeutic ASO that restores correct SMN2 splicing. We uncovered starvation-induced splicing changes, particularly in SMN2, which likely accelerate disease progression. These results constitute proof of principle that ASOs designed to cause sustained splicing defects can be used to induce pathogenesis and rapidly and accurately model splicing-associated diseases in animals. This approach allows the dissection of pathogenesis mechanisms, including spatial and temporal features of disease onset and progression, as well as testing of candidate therapeutics. PMID:22895255

Correction of a Cystic Fibrosis Splicing Mutation by Antisense Oligonucleotides.

PubMed

Igreja, Susana; Clarke, Luka A; Botelho, Hugo M; Marques, Luís; Amaral, Margarida D

2016-02-01

Cystic fibrosis (CF), the most common life-threatening genetic disease in Caucasians, is caused by ∼2,000 different mutations in the CF transmembrane conductance regulator (CFTR) gene. A significant fraction of these (∼13%) affect pre-mRNA splicing for which novel therapies have been somewhat neglected. We have previously described the effect of the CFTR splicing mutation c.2657+5G>A in IVS16, showing that it originates transcripts lacking exon 16 as well as wild-type transcripts. Here, we tested an RNA-based antisense oligonucleotide (AON) strategy to correct the aberrant splicing caused by this mutation. Two AONs (AON1/2) complementary to the pre-mRNA IVS16 mutant region were designed and their effect on splicing was assessed at the RNA and protein levels, on intracellular protein localization and function. To this end, we used the 2657+5G>A mutant CFTR minigene stably expressed in HEK293 Flp-In cells that express a single copy of the transgene. RNA data from AON1-treated mutant cells show that exon 16 inclusion was almost completely restored (to 95%), also resulting in increased levels of correctly localized CFTR protein at the plasma membrane (PM) and with increased function. A novel two-color CFTR splicing reporter minigene developed here allowed the quantitative monitoring of splicing by automated microscopy localization of CFTR at the PM. The AON strategy is thus a promising therapeutic approach for the specific correction of alternative splicing. © 2015 WILEY PERIODICALS, INC.

A Novel Intra-U1 snRNP Cross-Regulation Mechanism: Alternative Splicing Switch Links U1C and U1-70K Expression

PubMed Central

Rösel-Hillgärtner, Tanja Dorothe; Hung, Lee-Hsueh; Khrameeva, Ekaterina; Le Querrec, Patrick; Gelfand, Mikhail S.; Bindereif, Albrecht

2013-01-01

The U1 small nuclear ribonucleoprotein (snRNP)-specific U1C protein participates in 5′ splice site recognition and regulation of pre-mRNA splicing. Based on an RNA-Seq analysis in HeLa cells after U1C knockdown, we found a conserved, intra-U1 snRNP cross-regulation that links U1C and U1-70K expression through alternative splicing and U1 snRNP assembly. To investigate the underlying regulatory mechanism, we combined mutational minigene analysis, in vivo splice-site blocking by antisense morpholinos, and in vitro binding experiments. Alternative splicing of U1-70K pre-mRNA creates the normal (exons 7–8) and a non-productive mRNA isoform, whose balance is determined by U1C protein levels. The non-productive isoform is generated through a U1C-dependent alternative 3′ splice site, which requires an adjacent cluster of regulatory 5′ splice sites and binding of intact U1 snRNPs. As a result of nonsense-mediated decay (NMD) of the non-productive isoform, U1-70K mRNA and protein levels are down-regulated, and U1C incorporation into the U1 snRNP is impaired. U1-70K/U1C-deficient particles are assembled, shifting the alternative splicing balance back towards productive U1-70K splicing, and restoring assembly of intact U1 snRNPs. Taken together, we established a novel feedback regulation that controls U1-70K/U1C homeostasis and ensures correct U1 snRNP assembly and function. PMID:24146627

Oligonucleotides targeting TCF4 triplet repeat expansion inhibit RNA foci and mis-splicing in Fuchs' dystrophy.

PubMed

Hu, Jiaxin; Rong, Ziye; Gong, Xin; Zhou, Zhengyang; Sharma, Vivek K; Xing, Chao; Watts, Jonathan K; Corey, David R; Mootha, V Vinod

2018-03-15

Fuchs' endothelial corneal dystrophy (FECD) is the most common repeat expansion disorder. FECD impacts 4% of U.S. population and is the leading indication for corneal transplantation. Most cases are caused by an expanded intronic CUG tract in the TCF4 gene that forms nuclear foci, sequesters splicing factors and impairs splicing. We investigated the sense and antisense RNA landscape at the FECD gene and find that the sense-expanded repeat transcript is the predominant species in patient corneas. In patient tissue, sense foci number were negatively correlated with age and showed no correlation with sex. Each endothelial cell has ∼2 sense foci and each foci is single RNA molecule. We designed antisense oligonucleotides (ASOs) to target the mutant-repetitive RNA and demonstrated potent inhibition of foci in patient-derived cells. Ex vivo treatment of FECD human corneas effectively inhibits foci and reverses pathological changes in splicing. FECD has the potential to be a model for treating many trinucleotide repeat diseases and targeting the TCF4 expansion with ASOs represents a promising therapeutic strategy to prevent and treat FECD.

Steric antisense inhibition of AMPA receptor Q/R editing reveals tight coupling to intronic editing sites and splicing

PubMed Central

Penn, Andrew C.; Balik, Ales; Greger, Ingo H.

2013-01-01

Adenosine-to-Inosine (A-to-I) RNA editing is a post-transcriptional mechanism, evolved to diversify the transcriptome in metazoa. In addition to wide-spread editing in non-coding regions protein recoding by RNA editing allows for fine tuning of protein function. Functional consequences are only known for some editing sites and the combinatorial effect between multiple sites (functional epistasis) is currently unclear. Similarly, the interplay between RNA editing and splicing, which impacts on post-transcriptional gene regulation, has not been resolved. Here, we describe a versatile antisense approach, which will aid resolving these open questions. We have developed and characterized morpholino oligos targeting the most efficiently edited site—the AMPA receptor GluA2 Q/R site. We show that inhibition of editing closely correlates with intronic editing efficiency, which is linked to splicing efficiency. In addition to providing a versatile tool our data underscore the unique efficiency of a physiologically pivotal editing site. PMID:23172291

Genetic therapies for RNA mis-splicing diseases.

PubMed

Hammond, Suzan M; Wood, Matthew J A

2011-05-01

RNA mis-splicing diseases account for up to 15% of all inherited diseases, ranging from neurological to myogenic and metabolic disorders. With greatly increased genomic sequencing being performed for individual patients, the number of known mutations affecting splicing has risen to 50-60% of all disease-causing mutations. During the past 10years, genetic therapy directed toward correction of RNA mis-splicing in disease has progressed from theoretical work in cultured cells to promising clinical trials. In this review, we discuss the use of antisense oligonucleotides to modify splicing as well as the principles and latest work in bifunctional RNA, trans-splicing and modification of U1 and U7 snRNA to target splice sites. The success of clinical trials for modifying splicing to treat Duchenne muscular dystrophy opens the door for the use of splicing modification for most of the mis-splicing diseases. Copyright © 2011 Elsevier Ltd. All rights reserved.

The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis.

PubMed

Bielli, Pamela; Busà, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio

2014-04-01

Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5' splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival.

The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis

PubMed Central

Bielli, Pamela; Busà, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio

2014-01-01

Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5′ splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival. PMID:24514149

Combination Antisense Treatment for Destructive Exon Skipping of Myostatin and Open Reading Frame Rescue of Dystrophin in Neonatal mdx Mice.

PubMed

Lu-Nguyen, Ngoc B; Jarmin, Susan A; Saleh, Amer F; Popplewell, Linda; Gait, Michael J; Dickson, George

2015-08-01

The fatal X-linked Duchenne muscular dystrophy (DMD), characterized by progressive muscle wasting and muscle weakness, is caused by mutations within the DMD gene. The use of antisense oligonucleotides (AOs) modulating pre-mRNA splicing to restore the disrupted dystrophin reading frame, subsequently generating a shortened but functional protein has emerged as a potential strategy in DMD treatment. AO therapy has recently been applied to induce out-of-frame exon skipping of myostatin pre-mRNA, knocking-down expression of myostatin protein, and such an approach is suggested to enhance muscle hypertrophy/hyperplasia and to reduce muscle necrosis. Within this study, we investigated dual exon skipping of dystrophin and myostatin pre-mRNAs using phosphorodiamidate morpholino oligomers conjugated with an arginine-rich peptide (B-PMOs). Intraperitoneal administration of B-PMOs was performed in neonatal mdx males on the day of birth, and at weeks 3 and 6. At week 9, we observed in treated mice (as compared to age-matched, saline-injected controls) normalization of muscle mass, a recovery in dystrophin expression, and a decrease in muscle necrosis, particularly in the diaphragm. Our data provide a proof of concept for antisense therapy combining dystrophin restoration and myostatin inhibition for the treatment of DMD.

Antisense oligonucleotide-induced alternative splicing of the APOB mRNA generates a novel isoform of APOB.

PubMed

Khoo, Bernard; Roca, Xavier; Chew, Shern L; Krainer, Adrian R

2007-01-17

Apolipoprotein B (APOB) is an integral part of the LDL, VLDL, IDL, Lp(a) and chylomicron lipoprotein particles. The APOB pre-mRNA consists of 29 constitutively-spliced exons. APOB exists as two natural isoforms: the full-length APOB100 isoform, assembled into LDL, VLDL, IDL and Lp(a) and secreted by the liver in humans; and the C-terminally truncated APOB48, assembled into chylomicrons and secreted by the intestine in humans. Down-regulation of APOB100 is a potential therapy to lower circulating LDL and cholesterol levels. We investigated the ability of 2'O-methyl RNA antisense oligonucleotides (ASOs) to induce the skipping of exon 27 in endogenous APOB mRNA in HepG2 cells. These ASOs are directed towards the 5' and 3' splice-sites of exon 27, the branch-point sequence (BPS) of intron 26-27 and several predicted exonic splicing enhancers within exon 27. ASOs targeting either the 5' or 3' splice-site, in combination with the BPS, are the most effective. The splicing of other alternatively spliced genes are not influenced by these ASOs, suggesting that the effects seen are not due to non-specific changes in alternative splicing. The skip 27 mRNA is translated into a truncated isoform, APOB87SKIP27. The induction of APOB87SKIP27 expression in vivo should lead to decreased LDL and cholesterol levels, by analogy to patients with hypobetalipoproteinemia. As intestinal APOB mRNA editing and APOB48 expression rely on sequences within exon 26, exon 27 skipping should not affect APOB48 expression unlike other methods of down-regulating APOB100 expression which also down-regulate APOB48.

Antisense RNAs transcribed from the upstream region of the precore/core promoter of hepatitis B virus.

PubMed

Moriyama, Kosei; Hayashida, Kazuhiro; Shimada, Mitsuo; Nakano, Shuji; Nakashima, Yoshiyuki; Fukumaki, Yasuyuki

2003-07-01

The bidirectional activity of the precore/core promoter of hepatitis B virus (HBV) has been demonstrated in cultured cell lines. However, HBV antisense transcripts (asRNAs) have not been demonstrated in vivo. In the present study using liver tissue from patients with chronic hepatitis, an anchored 5'RACE mapping the 5' ends at position 1680/1681, 1655 or 1609/1602 was carried out. In limited cases, RLM-3'RACE detected asRNAs to terminate at four or five consecutive dT residues in the 0.7 kb downstream region. PCR of oligo(dT)-primed cDNA did not amplify a typical polyadenylated asRNA. RT-PCR using various primers did not detect any spliced forms. Competitive RT-PCR estimated the copy numbers of the asRNAs to be 0.05-0.4 % of total sense RNAs. All sequenced asRNAs had ORF6 but, in one patient, the asRNA initiating at position 1680/1681 had additional initiation and termination codons in front of ORF6. Therefore, asRNAs are transcribed by RNA polymerase III at a low level, encompass a dispensable ORF6 gene and might be retained in the nucleus. The endogenous asRNAs complementary to the common ends of all sense RNAs suggest antisense-mediated self-regulation of hepadnavirus.

Inhibition of Human Immunodeficiency Virus Replication by Antisense Oligodeoxynucleotides

NASA Astrophysics Data System (ADS)

Goodchild, John; Agrawal, Sudhir; Civeira, Maria P.; Sarin, Prem S.; Sun, Daisy; Zamecnik, Paul C.

1988-08-01

Twenty different target sites within human immunodeficiency virus (HIV) RNA were selected for studies of inhibition of HIV replication by antisense oligonucleotides. Target sites were selected based on their potential capacity to block recognition functions during viral replication. Antisense oligomers complementary to sites within or near the sequence repeated at the ends of retrovirus RNA (R region) and to certain splice sites were most effective. The effect of antisense oligomer length on inhibiting virus replication was also investigated, and preliminary toxicity studies in mice show that these compounds are toxic only at high levels. The results indicate potential usefulness for these oligomers in the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex either alone or in combination with other drugs.

Successful COG8 and PDF overlap is mediated by alterations in splicing and polyadenylation signals.

PubMed

Pereira-Castro, Isabel; Quental, Rita; da Costa, Luís T; Amorim, António; Azevedo, Luisa

2012-02-01

Although gene-free areas compose the great majority of eukaryotic genomes, a significant fraction of genes overlaps, i.e., unique nucleotide sequences are part of more than one transcription unit. In this work, the evolutionary history and origin of a same-strand gene overlap is dissected through the analysis of COG8 (component of oligomeric Golgi complex 8) and PDF (peptide deformylase). Comparative genomic surveys reveal that the relative locations of these two genes have been changing over the last 445 million years from distinct chromosomal locations in fish to overlapping in rodents and primates, indicating that the overlap between these genes precedes their divergence. The overlap between the two genes was initiated by the gain of a novel splice donor site between the COG8 stop codon and PDF initiation codon. Splicing is accomplished by the use of the PDF acceptor, leading COG8 to share the 3'end with PDF. In primates, loss of the ancestral polyadenylation signal for COG8 makes the overlap between COG8 and PDF mandatory, while in mouse and rat concurrent overlapping and non-overlapping Cog8 transcripts exist. Altogether, we demonstrate that the origin, evolution and preservation of the COG8/PDF same-strand overlap follow similar mechanistic steps as those documented for antisense overlaps where gain and/or loss of splice sites and polyadenylation signals seems to drive the process.

Splice-mediated Variants of Proteins (SpliVaP) - data and characterization of changes in signatures among protein isoforms due to alternative splicing.

PubMed

Floris, Matteo; Orsini, Massimiliano; Thanaraj, Thangavel Alphonse

2008-10-02

It is often the case that mammalian genes are alternatively spliced; the resulting alternate transcripts often encode protein isoforms that differ in amino acid sequences. Changes among the protein isoforms can alter the cellular properties of proteins. The effect can range from a subtle modulation to a complete loss of function. (i) We examined human splice-mediated protein isoforms (as extracted from a manually curated data set, and from a computationally predicted data set) for differences in the annotation for protein signatures (Pfam domains and PRINTS fingerprints) and we characterized the differences & their effects on protein functionalities. An important question addressed relates to the extent of protein isoforms that may lack any known function in the cell. (ii) We present a database that reports differences in protein signatures among human splice-mediated protein isoform sequences. (i) Characterization: The work points to distinct sets of alternatively spliced genes with varying degrees of annotation for the splice-mediated protein isoforms. Protein molecular functions seen to be often affected are those that relate to: binding, catalytic, transcription regulation, structural molecule, transporter, motor, and antioxidant; and the processes that are often affected are nucleic acid binding, signal transduction, and protein-protein interactions. Signatures are often included/excluded and truncated in length among protein isoforms; truncation is seen as the predominant type of change. Analysis points to the following novel aspects: (a) Analysis using data from the manually curated Vega indicates that one in 8.9 genes can lead to a protein isoform of no "known" function; and one in 18 expressed protein isoforms can be such an "orphan" isoform; the corresponding numbers as seen with computationally predicted ASD data set are: one in 4.9 genes and one in 9.8 isoforms. (b) When swapping of signatures occurs, it is often between those of same functional classifications. (c) Pfam domains can occur in varying lengths, and PRINTS fingerprints can occur with varying number of constituent motifs among isoforms - since such a variation is seen in large number of genes, it could be a general mechanism to modulate protein function. (ii) The reported resource (at http://www.bioinformatica.crs4.org/tools/dbs/splivap/) provides the community ability to access data on splice-mediated protein isoforms (with value-added annotation such as association with diseases) through changes in protein signatures.

SON and its alternatively spliced isoforms control MLL complex-mediated H3K4me3 and transcription of leukemia-associated genes

PubMed Central

Kim, Jung-Hyun; Baddoo, Melody C.; Park, Eun Young; Stone, Joshua K.; Park, Hyeonsoo; Butler, Thomas W.; Huang, Gang; Yan, Xiaomei; Pauli-Behn, Florencia; Myers, Richard M.; Tan, Ming; Flemington, Erik K.; Lim, Ssang-Taek; Erin Ahn, Eun-Young

2016-01-01

SUMMARY Dysregulation of MLL complex-mediated histone methylation plays a pivotal role in gene expression associated with diseases, but little is known about cellular factors modulating MLL complex activity. Here, we report that SON, previously known as an RNA splicing factor, controls MLL complex-mediated transcriptional initiation. SON binds to DNA near transcription start sites, interacts with menin, and inhibits MLL complex assembly, resulting in decreased H3K4me3 and transcriptional repression. Importantly, alternatively spliced short isoforms of SON are markedly upregulated in acute myeloid leukemia. The short isoforms compete with full-length SON for chromatin occupancy, but lack the menin-binding ability, thereby antagonizing full-length SON function in transcriptional repression while not impairing full-length SON-mediated RNA splicing. Furthermore, overexpression of a short isoform of SON enhances replating potential of hematopoietic progenitors. Our findings define SON as a fine-tuner of the MLL-menin interaction and reveal short SON overexpression as a marker indicating aberrant transcriptional initiation in leukemia. PMID:26990989

A role for exon sequences in alternative splicing of the human fibronectin gene.

PubMed Central

Mardon, H J; Sebastio, G; Baralle, F E

1987-01-01

Exon EDIIIA of the fibronectin (Fn) gene is alternatively spliced via pathways which either skip or include the whole exon in the messenger RNA (mRNA). We have investigated the role of EDIIIA exon sequences in the human Fn gene in determining alternative splicing of this exon during transient expression of alpha globin/Fn minigene hybrids in HeLa cells. We demonstrate that a DNA sequence of 81bp within the central region of exon EDIIIA is required for alternative splicing during processing of the primary transcript to generate both EDIIIA+ and EDIIIA- mRNA's. Furthermore, alternative splicing of EDIIIA only occurs when this sequence is present in the correct orientation since when it is in antisense orientation splicing always occurs via exon-skipping generating EDIIIA- mRNA. Images PMID:3671064

A Heroin Addiction Severity-Associated Intronic Single Nucleotide Polymorphism Modulates Alternative Pre-mRNA Splicing of the μ Opioid Receptor Gene OPRM1 via hnRNPH Interactions

PubMed Central

Xu, Jin; Lu, Zhigang; Xu, Mingming; Pan, Ling; Deng, Yi; Xie, Xiaohu; Liu, Huifen; Ding, Shixiong; Hurd, Yasmin L.; Pasternak, Gavril W.; Klein, Robert J.; Cartegni, Luca

2014-01-01

Single nucleotide polymorphisms (SNPs) in the OPRM1 gene have been associated with vulnerability to opioid dependence. The current study identifies an association of an intronic SNP (rs9479757) with the severity of heroin addiction among Han-Chinese male heroin addicts. Individual SNP analysis and haplotype-based analysis with additional SNPs in the OPRM1 locus showed that mild heroin addiction was associated with the AG genotype, whereas severe heroin addiction was associated with the GG genotype. In vitro studies such as electrophoretic mobility shift assay, minigene, siRNA, and antisense morpholino oligonucleotide studies have identified heterogeneous nuclear ribonucleoprotein H (hnRNPH) as the major binding partner for the G-containing SNP site. The G-to-A transition weakens hnRNPH binding and facilitates exon 2 skipping, leading to altered expressions of OPRM1 splice-variant mRNAs and hMOR-1 proteins. Similar changes in splicing and hMOR-1 proteins were observed in human postmortem prefrontal cortex with the AG genotype of this SNP when compared with the GG genotype. Interestingly, the altered splicing led to an increase in hMOR-1 protein levels despite decreased hMOR-1 mRNA levels, which is likely contributed by a concurrent increase in single transmembrane domain variants that have a chaperone-like function on MOR-1 protein stability. Our studies delineate the role of this SNP as a modifier of OPRM1 alternative splicing via hnRNPH interactions, and suggest a functional link between an SNP-containing splicing modifier and the severity of heroin addiction. PMID:25122903

Quantitative Antisense Screening and Optimization for Exon 51 Skipping in Duchenne Muscular Dystrophy.

PubMed

Echigoya, Yusuke; Lim, Kenji Rowel Q; Trieu, Nhu; Bao, Bo; Miskew Nichols, Bailey; Vila, Maria Candida; Novak, James S; Hara, Yuko; Lee, Joshua; Touznik, Aleksander; Mamchaoui, Kamel; Aoki, Yoshitsugu; Takeda, Shin'ichi; Nagaraju, Kanneboyina; Mouly, Vincent; Maruyama, Rika; Duddy, William; Yokota, Toshifumi

2017-11-01

Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder, is caused by mutations in the dystrophin (DMD) gene. Exon skipping is a therapeutic approach that uses antisense oligonucleotides (AOs) to modulate splicing and restore the reading frame, leading to truncated, yet functional protein expression. In 2016, the US Food and Drug Administration (FDA) conditionally approved the first phosphorodiamidate morpholino oligomer (morpholino)-based AO drug, eteplirsen, developed for DMD exon 51 skipping. Eteplirsen remains controversial with insufficient evidence of its therapeutic effect in patients. We recently developed an in silico tool to design antisense morpholino sequences for exon skipping. Here, we designed morpholino AOs targeting DMD exon 51 using the in silico tool and quantitatively evaluated the effects in immortalized DMD muscle cells in vitro. To our surprise, most of the newly designed morpholinos induced exon 51 skipping more efficiently compared with the eteplirsen sequence. The efficacy of exon 51 skipping and rescue of dystrophin protein expression were increased by up to more than 12-fold and 7-fold, respectively, compared with the eteplirsen sequence. Significant in vivo efficacy of the most effective morpholino, determined in vitro, was confirmed in mice carrying the human DMD gene. These findings underscore the importance of AO sequence optimization for exon skipping. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Flowering time control: another window to the connection between antisense RNA and chromatin.

PubMed

Ietswaart, Robert; Wu, Zhe; Dean, Caroline

2012-09-01

A high proportion of all eukaryotic genes express antisense RNA (asRNA), which accumulates to varying degrees at different loci. Whether there is a general function for asRNA is unknown, but its widespread occurrence and frequent regulation by stress suggest an important role. The best-characterized plant gene exhibiting a complex antisense transcript pattern is the Arabidopsis floral regulator FLOWERING LOCUS C (FLC). Changes occur in the accumulation, splicing, and polyadenylation of this antisense transcript, termed COOLAIR, in different environments and genotypes. These changes are associated with altered chromatin regulation and differential FLC expression, provoking mechanistic comparisons with many well-studied loci in yeast and mammals. Detailed analysis of these specific examples may shed light on the complex interplay between asRNA and chromatin modifications in different genomes. Copyright © 2012 Elsevier Ltd. All rights reserved.

Antisense oligonucleotide therapeutics for iron-sulphur cluster deficiency myopathy.

PubMed

Kollberg, Gittan; Holme, Elisabeth

2009-12-01

Iron-sulphur cluster deficiency myopathy is caused by a deep intronic mutation in ISCU resulting in inclusion of a cryptic exon in the mature mRNA. ISCU encodes the iron-sulphur cluster assembly protein IscU. Iron-sulphur clusters are essential for most basic redox transformations including the respiratory-chain function. Most patients are homozygous for the mutation with a phenotype characterized by a non-progressive myopathy with childhood onset of early fatigue, dyspnoea and palpitation on trivial exercise. A more severe phenotype with early onset of a slowly progressive severe muscle weakness, severe exercise intolerance and cardiomyopathy is caused by a missense mutation in compound with the intronic mutation. Treatment of cultured fibroblasts derived from three homozygous patients with an antisense phosphorodiamidate morpholino oligonucleotide for 48 h resulted in 100% restoration of the normal splicing pattern. The restoration was stable and after 21 days the correctly spliced mRNA still was the dominating RNA species.

Exploiting differential RNA splicing patterns: a potential new group of therapeutic targets in cancer.

PubMed

Jyotsana, Nidhi; Heuser, Michael

2018-02-01

Mutations in genes associated with splicing have been found in hematologic malignancies, but also in solid cancers. Aberrant cancer specific RNA splicing either results from mutations or misexpression of the spliceosome genes directly, or from mutations in splice sites of oncogenes or tumor suppressors. Areas covered: In this review, we present molecular targets of aberrant splicing in various malignancies, information on existing and emerging therapeutics against such targets, and strategies for future drug development. Expert opinion: Alternative splicing is an important mechanism that controls gene expression, and hence pharmacologic and genetic control of aberrant alternative RNA splicing has been proposed as a potential therapy in cancer. To identify and validate aberrant RNA splicing patterns as therapeutic targets we need to (1) characterize the most common genetic aberrations of the spliceosome and of splice sites, (2) understand the dysregulated downstream pathways and (3) exploit in-vivo disease models of aberrant splicing. Antisense oligonucleotides show promising activity, but will benefit from improved delivery tools. Inhibitors of mutated splicing factors require improved specificity, as alternative and aberrant splicing are often intertwined like two sides of the same coin. In summary, targeting aberrant splicing is an early but emerging field in cancer treatment.

Antisense Oligonucleotide (AON)-based Therapy for Leber Congenital Amaurosis Caused by a Frequent Mutation in CEP290

PubMed Central

Collin, Rob WJ; den Hollander, Anneke I; van der Velde-Visser, Saskia D; Bennicelli, Jeannette; Bennett, Jean; Cremers, Frans PM

2012-01-01

Leber congenital amaurosis (LCA) is the most severe form of inherited retinal degeneration, with an onset in the first year of life. The most frequent mutation that causes LCA, present in at least 10% of individuals with LCA from North-American and Northern-European descent, is an intronic mutation in CEP290 that results in the inclusion of an aberrant exon in the CEP290 mRNA. Here, we describe a genetic therapy approach that is based on antisense oligonucleotides (AONs), small RNA molecules that are able to redirect normal splicing of aberrantly processed pre-mRNA. Immortalized lymphoblastoid cells of individuals with LCA homozygously carrying the intronic CEP290 mutation were transfected with several AONs that target the aberrant exon that is incorporated in the mutant CEP290 mRNA. Subsequent RNA isolation and reverse transcription-PCR analysis revealed that a number of AONs were capable of almost fully redirecting normal CEP290 splicing, in a dose-dependent manner. Other AONs however, displayed no effect on CEP290 splicing at all, indicating that the rescue of aberrant CEP290 splicing shows a high degree of sequence specificity. Together, our data show that AON-based therapy is a promising therapeutic approach for CEP290-associated LCA that warrants future research in animal models to develop a cure for this blinding disease. PMID:23343883

Subgroup Specific Alternative Splicing in Medulloblastoma

PubMed Central

Kloosterhof, Nanne K; Northcott, Paul A; Yu, Emily PY; Shih, David; Peacock, John; Grajkowska, Wieslawa; van Meter, Timothy; Eberhart, Charles G; Pfister, Stefan; Marra, Marco A; Weiss, William A; Scherer, Stephen W; Rutka, James T; French, Pim J; Taylor, Michael D

2014-01-01

Medulloblastoma is comprised of four distinct molecular variants: WNT, SHH, Group 3, and Group 4. We analyzed alternative splicing usage in 14 normal cerebellar samples and 103 medulloblastomas of known subgroup. Medulloblastoma samples have a statistically significant increase in alternative splicing as compared to normal fetal cerebella (2.3-times; P<6.47E-8). Splicing patterns are distinct and specific between molecular subgroups. Unsupervised hierarchical clustering of alternative splicing events accurately assigns medulloblastomas to their correct subgroup. Subgroup-specific splicing and alternative promoter usage was most prevalent in Group 3 (19.4%) and SHH (16.2%) medulloblastomas, while observed less frequently in WNT (3.2%), and Group 4 (9.3%) tumors. Functional annotation of alternatively spliced genes reveals over-representation of genes important for neuronal development. Alternative splicing events in medulloblastoma may be regulated in part by the correlative expression of antisense transcripts, suggesting a possible mechanism affecting subgroup specific alternative splicing. Our results identify additional candidate markers for medulloblastoma subgroup affiliation, further support the existence of distinct subgroups of the disease, and demonstrate an additional level of transcriptional heterogeneity between medulloblastoma subgroups. PMID:22358458

Innate Immune Response and Off-Target Mis-splicing Are Common Morpholino-Induced Side Effects in Xenopus.

PubMed

Gentsch, George E; Spruce, Thomas; Monteiro, Rita S; Owens, Nick D L; Martin, Stephen R; Smith, James C

2018-03-12

Antisense morpholino oligomers (MOs) have been indispensable tools for developmental biologists to transiently knock down (KD) genes rather than to knock them out (KO). Here we report on the implications of genetic KO versus MO-mediated KD of the mesoderm-specifying Brachyury paralogs in the frog Xenopus tropicalis. While both KO and KD embryos fail to activate the same core gene regulatory network, resulting in virtually identical morphological defects, embryos injected with control or target MOs also show a systemic GC content-dependent immune response and many off-target splicing defects. Optimization of MO dosage and increasing incubation temperatures can mitigate, but not eliminate, these MO side effects, which are consistent with the high affinity measured between MO and off-target sequence in vitro. We conclude that while MOs can be useful to profile loss-of-function phenotypes at a molecular level, careful attention must be paid to their immunogenic and off-target side effects. Copyright © 2018 The Francis Crick Institute. Published by Elsevier Inc. All rights reserved.

4β-Hydroxywithanolide E Modulates Alternative Splicing of Apoptotic Genes in Human Hepatocellular Carcinoma Huh-7 Cells.

PubMed

Lee, Chien-Chin; Chang, Wen-Hsin; Chang, Ya-Sian; Liu, Ting-Yuan; Chen, Yu-Chia; Wu, Yang-Chang; Chang, Jan-Gowth

2017-08-04

Alternative splicing is a mechanism for increasing protein diversity from a limited number of genes. Studies have demonstrated that aberrant regulation in the alternative splicing of apoptotic gene transcripts may contribute to the development of cancer. In this study, we isolated 4β-Hydroxywithanolide E (4bHWE) from the traditional herb Physalis peruviana and investigated its biological effect in cancer cells. The results demonstrated that 4bHWE modulates the alternative splicing of various apoptotic genes, including HIPK3, SMAC/DIABLO, and SURVIVIN. We also discovered that the levels of SRSF1 phospho-isoform were decreased and the levels of H3K36me3 were increased in 4bHWE treatment. Knockdown experiments revealed that the splicing site selection of SMAC/DIABLO could be mediated by changes in the level of H3K36me3 in 4bHWE-treated cells. Furthermore, we extended our study to apoptosis-associated molecules, and detected increased levels of poly ADP-ribose polymerase cleavage and the active form of CASPASE-3 in 4bHWE-induced apoptosis. In vivo experiments indicated that the treatment of tumor-bearing mice with 4bHWE resulted in a marked decrease in tumor size. This study is the first to demonstrate that 4bHWE affects alternative splicing by modulating splicing factors and histone modifications, and provides a novel view of the antitumor mechanism of 4bHWE.

Crosstalk between Wnt Signaling and RNA Processing in Colorectal Cancer.

PubMed

Bordonaro, Michael

2013-01-01

RNA processing involves a variety of processes affecting gene expression, including the removal of introns through RNA splicing, as well as 3' end processing (cleavage and polyadenylation). Alternative RNA processing is fundamentally important for gene regulation, and aberrant processing is associated with the initiation and progression of cancer. Deregulated Wnt signaling, which is the initiating event in the development of most cases of human colorectal cancer (CRC), has been linked to modified RNA processing, which may contribute to Wnt-mediated colonic carcinogenesis. Crosstalk between Wnt signaling and alternative RNA splicing with relevance to CRC includes effects on the expression of Rac1b, an alternatively spliced gene associated with tumorigenesis, which exhibits alternative RNA splicing that is influenced by Wnt activity. In addition, Tcf4, a crucial component of Wnt signaling, also exhibits alternative splicing, which is likely involved in colonic tumorigenesis. Modulation of 3' end formation, including of the Wnt target gene COX-2, also can influence the neoplastic process, with implications for CRC. While many human genes are dependent on introns and splicing for normal levels of gene expression, naturally intronless genes exist with a unique metabolism that allows for intron-independent gene expression. Effects of Wnt activity on the RNA metabolism of the intronless Wnt-target gene c-jun is a likely contributor to cancer development. Further, butyrate, a breakdown product of dietary fiber and a histone deacetylase inhibitor, upregulates Wnt activity in CRC cells, and also modulates RNA processing; therefore, the interplay between Wnt activity, the modulation of this activity by butyrate, and differential RNA metabolism in colonic cells can significantly influence tumorigenesis. Determining the role played by altered RNA processing in Wnt-mediated neoplasia may lead to novel interventions aimed at restoring normal RNA metabolism for therapeutic benefit. Therefore, this minireview presents a brief overview of several aspects of RNA processing of relevance to cancer, which potentially influence, or are influenced by, Wnt signaling activity.

Epigenetic Basis of Neuronal and Synaptic Plasticity.

PubMed

Karpova, Nina N; Sales, Amanda J; Joca, Samia R

2017-01-01

Neuronal network and plasticity change as a function of experience. Altered neural connectivity leads to distinct transcriptional programs of neuronal plasticity-related genes. The environmental challenges throughout life may promote long-lasting reprogramming of gene expression and the development of brain disorders. The modifications in neuronal epigenome mediate gene-environmental interactions and are required for activity-dependent regulation of neuronal differentiation, maturation and plasticity. Here, we highlight the latest advances in understanding the role of the main players of epigenetic machinery (DNA methylation and demethylation, histone modifications, chromatin-remodeling enzymes, transposons, and non-coding RNAs) in activity-dependent and long- term neural and synaptic plasticity. The review focuses on both the transcriptional and post-transcriptional regulation of gene expression levels, including the processes of promoter activation, alternative splicing, regulation of stability of gene transcripts by natural antisense RNAs, and alternative polyadenylation. Further, we discuss the epigenetic aspects of impaired neuronal plasticity and the pathogenesis of neurodevelopmental (Rett syndrome, Fragile X Syndrome, genomic imprinting disorders, schizophrenia, and others), stressrelated (mood disorders) and neurodegenerative Alzheimer's, Parkinson's and Huntington's disorders. The review also highlights the pharmacological compounds that modulate epigenetic programming of gene expression, the potential treatment strategies of discussed brain disorders, and the questions that should be addressed during the development of effective and safe approaches for the treatment of brain disorders.

Glycogen Reduction in Myotubes of Late-Onset Pompe Disease Patients Using Antisense Technology.

PubMed

Goina, Elisa; Peruzzo, Paolo; Bembi, Bruno; Dardis, Andrea; Buratti, Emanuele

2017-09-06

Glycogen storage disease type II (GSDII) is a lysosomal disorder caused by the deficient activity of acid alpha-glucosidase (GAA) enzyme, leading to the accumulation of glycogen within the lysosomes. The disease has been classified in infantile and late-onset forms. Most late-onset patients share a splicing mutation c.-32-13T > G in intron 1 of the GAA gene that prevents efficient recognition of exon 2 by the spliceosome. In this study, we have mapped the splicing silencers of GAA exon 2 and developed antisense morpholino oligonucleotides (AMOs) to inhibit those regions and rescue normal splicing in the presence of the c.-32-13T > G mutation. Using a minigene approach and patient fibroblasts, we successfully increased inclusion of exon 2 in the mRNA and GAA enzyme production by targeting a specific silencer with a combination of AMOs. Most importantly, the use of these AMOs in patient myotubes results in a decreased accumulation of glycogen. To our knowledge, this is the only therapeutic approach resulting in a decrease of glycogen accumulation in patient tissues beside enzyme replacement therapy (ERT) and TFEB overexpression. As a result, it may represent a highly novel and promising therapeutic line for GSDII. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Antisense phosphorothioate oligonucleotides: selective killing of the intracellular parasite Leishmania amazonensis.

PubMed

Ramazeilles, C; Mishra, R K; Moreau, S; Pascolo, E; Toulmé, J J

1994-08-16

We targeted the mini-exon sequence, present at the 5' end of every mRNA of the protozoan parasite Leishmania amazonensis, by phosphorothioate oligonucleotides. A complementary 16-mer (16PS) was able to kill amastigotes--the intracellular stage of the parasite--in murine macrophages in culture. After 24 hr of incubation with 10 microM 16PS, about 30% infected macrophages were cured. The oligomer 16PS acted through antisense hybridization in a sequence-dependent way; no effect on parasites was observed with noncomplementary phosphorothioate oligonucleotides. The antisense oligonucleotide 16PS was a selective killer of the protozoans without any detrimental effect to the host macrophage. Using 16PS linked to a palmitate chain, which enabled it to complex with low density lipoproteins, improved the leishmanicidal efficiency on intracellular amastigotes, probably due to increased endocytosis. Phosphorothioate oligonucleotides complementary to the intron part of the mini-exon pre-RNA were also effective, suggesting that antisense oligomers could prevent trans-splicing in these parasites.

Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis

PubMed Central

2014-01-01

Background The circadian clock enables living organisms to anticipate recurring daily and seasonal fluctuations in their growth habitats and synchronize their biology to the environmental cycle. The plant circadian clock consists of multiple transcription-translation feedback loops that are entrained by environmental signals, such as light and temperature. In recent years, alternative splicing emerges as an important molecular mechanism that modulates the clock function in plants. Several clock genes are known to undergo alternative splicing in response to changes in environmental conditions, suggesting that the clock function is intimately associated with environmental responses via the alternative splicing of the clock genes. However, the alternative splicing events of the clock genes have not been studied at the molecular level. Results We systematically examined whether major clock genes undergo alternative splicing under various environmental conditions in Arabidopsis. We also investigated the fates of the RNA splice variants of the clock genes. It was found that the clock genes, including EARLY FLOWERING 3 (ELF3) and ZEITLUPE (ZTL) that have not been studied in terms of alternative splicing, undergo extensive alternative splicing through diverse modes of splicing events, such as intron retention, exon skipping, and selection of alternative 5′ splice site. Their alternative splicing patterns were differentially influenced by changes in photoperiod, temperature extremes, and salt stress. Notably, the RNA splice variants of TIMING OF CAB EXPRESSION 1 (TOC1) and ELF3 were degraded through the nonsense-mediated decay (NMD) pathway, whereas those of other clock genes were insensitive to NMD. Conclusion Taken together, our observations demonstrate that the major clock genes examined undergo extensive alternative splicing under various environmental conditions, suggesting that alternative splicing is a molecular scheme that underlies the linkage between the clock and environmental stress adaptation in plants. It is also envisioned that alternative splicing of the clock genes plays more complex roles than previously expected. PMID:24885185

Novel interactions between the HTLV antisense proteins HBZ and APH-2 and the NFAR protein family: Implications for the HTLV lifecycles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murphy, Jane; Hall, William W.; Ratner, Lee

The human T-cell leukaemia virus type 1 and type 2 (HTLV-1/HTLV-2) antisense proteins HBZ and APH-2 play key roles in the HTLV lifecycles and persistence in the host. Nuclear Factors Associated with double-stranded RNA (NFAR) proteins NF90/110 function in the lifecycles of several viruses and participate in host innate immunity against infection and oncogenesis. Using GST pulldown and co-immunoprecipitation assays we demonstrate specific novel interactions between HBZ/APH-2 and NF90/110 and characterised the protein domains involved. Moreover we show that NF90/110 significantly enhance Tax mediated LTR activation, an effect that was abolished by HBZ but enhanced by APH-2. Additionally we foundmore » that HBZ and APH-2 modulate the promoter activity of survivin and are capable of antagonising NF110-mediated survivin activation. Thus interactions between HTLV antisense proteins and the NFAR protein family have an overall positive impact on HTLV infection. Hence NFARs may represent potential therapeutic targets in HTLV infected cells. - Highlights: • This study demonstrates for the first time interactions between NF90/110 and the HTLV antisense proteins HBZ and APH-2. • We show that NF90/110 significantly enhance LTR activation by the HTLV Tax protein, an effect that is abolished by HBZ but enhanced by APH-2. • The study shows that even though the HTLV antisense proteins activate survivin expression they antagonize the ability of NF90/110 to do so. • Overall we found that NF90/110 positively regulate HTLV infection and as such might represent a therapeutic target in infected cells.« less

Pharmacology of Antisense Drugs.

PubMed

Bennett, C Frank; Baker, Brenda F; Pham, Nguyen; Swayze, Eric; Geary, Richard S

2017-01-06

Recent studies have led to a greater appreciation of the diverse roles RNAs play in maintaining normal cellular function and how they contribute to disease pathology, broadening the number of potential therapeutic targets. Antisense oligonucleotides are the most direct means to target RNA in a selective manner and have become an established platform technology for drug discovery. There are multiple molecular mechanisms by which antisense oligonucleotides can be used to modulate RNAs in cells, including promoting the degradation of the targeted RNA or modulating RNA function without degradation. Antisense drugs utilizing various antisense mechanisms are demonstrating therapeutic potential for the treatment of a broad variety of diseases. This review focuses on some of the advances that have taken place in translating antisense technology from the bench to the clinic.

A Duchenne Muscular Dystrophy Gene Hot Spot Mutation in Dystrophin-Deficient Cavalier King Charles Spaniels Is Amenable to Exon 51 Skipping

PubMed Central

Walmsley, Gemma L.; Arechavala-Gomeza, Virginia; Fernandez-Fuente, Marta; Burke, Margaret M.; Nagel, Nicole; Holder, Angela; Stanley, Rachael; Chandler, Kate; Marks, Stanley L.; Muntoni, Francesco; Shelton, G. Diane; Piercy, Richard J.

2010-01-01

Background Duchenne muscular dystrophy (DMD), which afflicts 1 in 3500 boys, is one of the most common genetic disorders of children. This fatal degenerative condition is caused by an absence or deficiency of dystrophin in striated muscle. Most affected patients have inherited or spontaneous deletions in the dystrophin gene that disrupt the reading frame resulting in unstable truncated products. For these patients, restoration of the reading frame via antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach. The major DMD deletion “hot spot” is found between exons 45 and 53, and skipping exon 51 in particular is predicted to ameliorate the dystrophic phenotype in the greatest number of patients. Currently the mdx mouse is the most widely used animal model of DMD, although its mild phenotype limits its suitability in clinical trials. The Golden Retriever muscular dystrophy (GRMD) model has a severe phenotype, but due to its large size, is expensive to use. Both these models have mutations in regions of the dystrophin gene distant from the commonly mutated DMD “hot spot”. Methodology/Principal Findings Here we describe the severe phenotype, histopathological findings, and molecular analysis of Cavalier King Charles Spaniels with dystrophin-deficient muscular dystrophy (CKCS-MD). The dogs harbour a missense mutation in the 5′ donor splice site of exon 50 that results in deletion of exon 50 in mRNA transcripts and a predicted premature truncation of the translated protein. Antisense oligonucleotide-mediated skipping of exon 51 in cultured myoblasts from an affected dog restored the reading frame and protein expression. Conclusions/Significance Given the small size of the breed, the amiable temperament and the nature of the mutation, we propose that CKCS-MD is a valuable new model for clinical trials of antisense oligonucleotide-induced exon skipping and other therapeutic approaches for DMD. PMID:20072625

Serine/Arginine-rich Splicing Factor 2 Modulates Herpes Simplex Virus Type 1 Replication via Regulating Viral Gene Transcriptional Activity and Pre-mRNA Splicing.

PubMed

Wang, Ziqiang; Liu, Qing; Lu, Jinhua; Fan, Ping; Xie, Weidong; Qiu, Wei; Wang, Fan; Hu, Guangnan; Zhang, Yaou

2016-12-16

Once it enters the host cell, herpes simplex virus type 1 (HSV-1) recruits a series of host cell factors to facilitate its life cycle. Here, we demonstrate that serine/arginine-rich splicing factor 2 (SRSF2), which is an important component of the splicing speckle, mediates HSV-1 replication by regulating viral gene expression at the transcriptional and posttranscriptional levels. Our results indicate that SRSF2 functions as a transcriptional activator by directly binding to infected cell polypeptide 0 (ICP0), infected cell polypeptide 27 (ICP27), and thymidine kinase promoters. Moreover, SRSF2 participates in ICP0 pre-mRNA splicing by recognizing binding sites in ICP0 exon 3. These findings provide insight into the functions of SRSF2 in HSV-1 replication and gene expression. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Cationic PMMA nanoparticles bind and deliver antisense oligoribonucleotides allowing restoration of dystrophin expression in the mdx mouse.

PubMed

Rimessi, Paola; Sabatelli, Patrizia; Fabris, Marina; Braghetta, Paola; Bassi, Elena; Spitali, Pietro; Vattemi, Gaetano; Tomelleri, Giuliano; Mari, Lara; Perrone, Daniela; Medici, Alessandro; Neri, Marcella; Bovolenta, Matteo; Martoni, Elena; Maraldi, Nadir M; Gualandi, Francesca; Merlini, Luciano; Ballestri, Marco; Tondelli, Luisa; Sparnacci, Katia; Bonaldo, Paolo; Caputo, Antonella; Laus, Michele; Ferlini, Alessandra

2009-05-01

For subsets of Duchenne muscular dystrophy (DMD) mutations, antisense oligoribonucleotide (AON)-mediated exon skipping has proven to be efficacious in restoring the expression of dystrophin protein. In the mdx murine model systemic delivery of AON, recognizing the splice donor of dystrophin exon 23, has shown proof of concept. Here, we show that using cationic polymethylmethacrylate (PMMA) (marked as T1) nanoparticles loaded with a low dose of 2'-O-methyl-phosphorothioate (2'OMePS) AON delivered by weekly intraperitoneal (IP) injection (0.9 mg/kg/week), could restore dystrophin expression in body-wide striated muscles. Delivery of an identical dose of naked AON did not result in detectable dystrophin expression. Transcription, western, and immunohistochemical analysis showed increased levels of dystrophin transcript and protein, and correct localization at the sarcolemma. This study shows that T1 nanoparticles have the capacity to bind and convoy AONs in body-wide muscle tissues and to reduce the dose required for dystrophin rescue. By immunofluorescence and electron microscopy studies, we highlighted the diffusion pathways of this compound. This nonviral approach may valuably improve the therapeutic usage of AONs in DMD as well as the delivery of RNA molecules with many implications in both basic research and medicine.

Inhibition of vemurafenib-resistant melanoma by interference with pre-mRNA splicing

PubMed Central

Salton, Maayan; Kasprzak, Wojciech K.; Voss, Ty; Shapiro, Bruce A.; Poulikakos, Poulikos I.; Misteli, Tom

2015-01-01

Mutations in the serine/threonine kinase BRAF are found in more than 60% of melanomas. The most prevalent melanoma mutation is BRAF(V600E), which constitutively activates downstream MAPK signaling. Vemurafenib is a potent RAF kinase inhibitor with remarkable clinical activity in BRAF(V600E)-positive melanoma tumors. However, patients rapidly develop resistance to vemurafenib treatment. One resistance mechanism is the emergence of BRAF alternative splicing isoforms leading to elimination of the RAS-binding domain. Here we identify interference with pre-mRNA splicing as a mechanism to combat vemurafenib resistance. We find that small molecule pre-mRNA splicing modulators reduce BRAF3-9 production and limit in-vitro cell growth of vemurafenib-resistant cells. In xenograft models, interference with pre-mRNA splicing prevents tumor formation and slows growth of vemurafenib-resistant tumors. Our results identify an intronic mutation as a molecular basis for RNA splicing-mediated RAF inhibitor resistance and we identify pre-mRNA splicing interference as a potential therapeutic strategy for drug resistance in BRAF melanoma. PMID:25971842

Inhibition of vemurafenib-resistant melanoma by interference with pre-mRNA splicing.

PubMed

Salton, Maayan; Kasprzak, Wojciech K; Voss, Ty; Shapiro, Bruce A; Poulikakos, Poulikos I; Misteli, Tom

2015-05-14

Mutations in the serine/threonine kinase BRAF are found in more than 60% of melanomas. The most prevalent melanoma mutation is BRAF(V600E), which constitutively activates downstream MAPK signalling. Vemurafenib is a potent RAF kinase inhibitor with remarkable clinical activity in BRAF(V600E)-positive melanoma tumours. However, patients rapidly develop resistance to vemurafenib treatment. One resistance mechanism is the emergence of BRAF alternative splicing isoforms leading to elimination of the RAS-binding domain. Here we identify interference with pre-mRNA splicing as a mechanism to combat vemurafenib resistance. We find that small-molecule pre-mRNA splicing modulators reduce BRAF3-9 production and limit in-vitro cell growth of vemurafenib-resistant cells. In xenograft models, interference with pre-mRNA splicing prevents tumour formation and slows growth of vemurafenib-resistant tumours. Our results identify an intronic mutation as the molecular basis for a RNA splicing-mediated RAF inhibitor resistance mechanism and we identify pre-mRNA splicing interference as a potential therapeutic strategy for drug resistance in BRAF melanoma.

Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds.

PubMed

Arandel, Ludovic; Polay Espinoza, Micaela; Matloka, Magdalena; Bazinet, Audrey; De Dea Diniz, Damily; Naouar, Naïra; Rau, Frédérique; Jollet, Arnaud; Edom-Vovard, Frédérique; Mamchaoui, Kamel; Tarnopolsky, Mark; Puymirat, Jack; Battail, Christophe; Boland, Anne; Deleuze, Jean-Francois; Mouly, Vincent; Klein, Arnaud F; Furling, Denis

2017-04-01

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations. © 2017. Published by The Company of Biologists Ltd.

Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds

PubMed Central

Arandel, Ludovic; Polay Espinoza, Micaela; Matloka, Magdalena; Bazinet, Audrey; De Dea Diniz, Damily; Naouar, Naïra; Rau, Frédérique; Jollet, Arnaud; Edom-Vovard, Frédérique; Mamchaoui, Kamel; Tarnopolsky, Mark; Puymirat, Jack; Battail, Christophe; Boland, Anne; Deleuze, Jean-Francois; Mouly, Vincent; Klein, Arnaud F.

2017-01-01

ABSTRACT Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations. PMID:28188264

Nuclear receptor coactivators function in estrogen receptor- and progestin receptor-dependent aspects of sexual behavior in female rats

PubMed Central

Molenda-Figueira, Heather A.; Williams, Casey A.; Griffin, Andreana L.; Rutledge, Eric M.; Blaustein, Jeffrey D.; Tetel, Marc J.

2008-01-01

The ovarian hormones, estradiol (E) and progesterone (P) facilitate the expression of sexual behavior in female rats. E and P mediate many of these behavioral effects by binding to their respective intracellular receptors in specific brain regions. Nuclear receptor coactivators, including Steroid Receptor Coactivator-1 (SRC-1) and CREB Binding Protein (CBP), dramatically enhance ligand-dependent steroid receptor transcriptional activity in vitro. Previously, our lab has shown that SRC-1 and CBP modulate estrogen receptor (ER)-mediated induction of progestin receptor (PR) gene expression in the ventromedial nucleus of the hypothalamus (VMN) and hormone-dependent sexual receptivity in female rats. Female sexual behaviors can be activated by high doses of E alone in ovariectomized rats, and thus are believed to be ER-dependent. However, the full repertoire of female sexual behavior, in particular, proceptive behaviors such as hopping, darting and ear wiggling, are considered to be PR-dependent. In the present experiments, the function of SRC-1 and CBP in distinct ER- (Exp. 1) and PR- (Exp. 2) dependent aspects of female sexual behavior was investigated. In Exp. 1, infusion of antisense oligodeoxynucleotides to SRC-1 and CBP mRNA into the VMN decreased lordosis intensity in rats treated with E alone, suggesting that these coactivators modulate ER-mediated female sexual behavior. In Exp. 2, antisense to SRC-1 and CBP mRNA around the time of P administration reduced PR-dependent ear wiggling and hopping and darting. Taken together, these data suggest that SRC-1 and CBP modulate ER and PR action in brain and influence distinct aspects of hormone-dependent sexual behaviors. These findings support our previous studies and provide further evidence that SRC-1 and CBP function together to regulate ovarian hormone action in behaviorally-relevant brain regions. PMID:16769066

Identification of cis-Acting Elements and Splicing Factors Involved in the Regulation of BIM Pre-mRNA Splicing

PubMed Central

Juan, Wen Chun; Roca, Xavier; Ong, S. Tiong

2014-01-01

Aberrant changes in the expression of the pro-apoptotic protein, BCL-2-like 11 (BIM), can result in either impaired or excessive apoptosis, which can contribute to tumorigenesis and degenerative disorders, respectively. Altering BIM pre-mRNA splicing is an attractive approach to modulate apoptosis because BIM activity is partly determined by the alternative splicing of exons 3 or 4, whereby exon 3-containing transcripts are not apoptotic. Here we identified several cis-acting elements and splicing factors involved in BIM alternative splicing, as a step to better understand the regulation of BIM expression. We analyzed a recently discovered 2,903-bp deletion polymorphism within BIM intron 2 that biased splicing towards exon 3, and which also impaired BIM-dependent apoptosis. We found that this region harbors multiple redundant cis-acting elements that repress exon 3 inclusion. Furthermore, we have isolated a 23-nt intronic splicing silencer at the 3′ end of the deletion that is important for excluding exon 3. We also show that PTBP1 and hnRNP C repress exon 3 inclusion, and that downregulation of PTBP1 inhibited BIM-mediated apoptosis. Collectively, these findings start building our understanding of the cis-acting elements and splicing factors that regulate BIM alternative splicing, and also suggest potential approaches to alter BIM splicing for therapeutic purposes. PMID:24743263

Identification of cis-acting elements and splicing factors involved in the regulation of BIM Pre-mRNA splicing.

PubMed

Juan, Wen Chun; Roca, Xavier; Ong, S Tiong

2014-01-01

Aberrant changes in the expression of the pro-apoptotic protein, BCL-2-like 11 (BIM), can result in either impaired or excessive apoptosis, which can contribute to tumorigenesis and degenerative disorders, respectively. Altering BIM pre-mRNA splicing is an attractive approach to modulate apoptosis because BIM activity is partly determined by the alternative splicing of exons 3 or 4, whereby exon 3-containing transcripts are not apoptotic. Here we identified several cis-acting elements and splicing factors involved in BIM alternative splicing, as a step to better understand the regulation of BIM expression. We analyzed a recently discovered 2,903-bp deletion polymorphism within BIM intron 2 that biased splicing towards exon 3, and which also impaired BIM-dependent apoptosis. We found that this region harbors multiple redundant cis-acting elements that repress exon 3 inclusion. Furthermore, we have isolated a 23-nt intronic splicing silencer at the 3' end of the deletion that is important for excluding exon 3. We also show that PTBP1 and hnRNP C repress exon 3 inclusion, and that downregulation of PTBP1 inhibited BIM-mediated apoptosis. Collectively, these findings start building our understanding of the cis-acting elements and splicing factors that regulate BIM alternative splicing, and also suggest potential approaches to alter BIM splicing for therapeutic purposes.

Resveratrol, by modulating RNA processing factor levels, can influence the alternative splicing of pre-mRNAs.

PubMed

Markus, M Andrea; Marques, Francine Z; Morris, Brian J

2011-01-01

Alternative pre-mRNA splicing defects can contribute to, or result from, various diseases, including cancer. Aberrant mRNAs, splicing factors and other RNA processing factors have therefore become targets for new therapeutic interventions. Here we report that the natural polyphenol resveratrol can modulate alternative splicing in a target-specific manner. We transfected minigenes of several alternatively spliceable primary mRNAs into HEK293 cells in the presence or absence of 1, 5, 20 and 50 µM resveratrol and measured exon levels by semi-quantitative PCR after separation by agarose gel electrophoresis. We found that 20 µg/ml and 50 µg/ml of resveratrol affected exon inclusion of SRp20 and SMN2 pre-mRNAs, but not CD44v5 or tau pre-mRNAs. By Western blotting and immunofluorescence we showed that this effect may be due to the ability of resveratrol to change the protein level but not the localization of several RNA processing factors. The processing factors that increased significantly were ASF/SF2, hnRNPA1 and HuR, but resveratrol did not change the levels of RBM4, PTBP1 and U2AF35. By means of siRNA-mediated knockdown we depleted cells of SIRT1, regarded as a major target of resveratrol, and showed that the effect on splicing was not dependent on SIRT1. Our results suggest that resveratrol might be an attractive small molecule to treat diseases in which aberrant splicing has been implicated, and justify more extensive research on the effects of resveratrol on the splicing machinery.

Clinical potential of oligonucleotide-based therapeutics in the respiratory system.

PubMed

Moschos, Sterghios A; Usher, Louise; Lindsay, Mark A

2017-01-01

The discovery of an ever-expanding plethora of coding and non-coding RNAs with nodal and causal roles in the regulation of lung physiology and disease is reinvigorating interest in the clinical utility of the oligonucleotide therapeutic class. This is strongly supported through recent advances in nucleic acids chemistry, synthetic oligonucleotide delivery and viral gene therapy that have succeeded in bringing to market at least three nucleic acid-based drugs. As a consequence, multiple new candidates such as RNA interference modulators, antisense, and splice switching compounds are now progressing through clinical evaluation. Here, manipulation of RNA for the treatment of lung disease is explored, with emphasis on robust pharmacological evidence aligned to the five pillars of drug development: exposure to the appropriate tissue, binding to the desired molecular target, evidence of the expected mode of action, activity in the relevant patient population and commercially viable value proposition. Copyright © 2016 Elsevier Inc. All rights reserved.

Small Molecule Modulators of Pre-mRNA Splicing in Cancer Therapy.

PubMed

Salton, Maayan; Misteli, Tom

2016-01-01

Pre-mRNA splicing is a fundamental process in mammalian gene expression and alternative RNA splicing plays a considerable role in generating protein diversity. RNA splicing events are also key to the pathology of numerous diseases, particularly cancers. Some tumors are molecularly addicted to specific RNA splicing isoforms making interference with pre-mRNA processing a viable therapeutic strategy. Several RNA splicing modulators have recently been characterized, some showing promise in preclinical studies. While the targets of most splicing modulators are constitutive RNA processing components, possibly leading to undesirable side effects, selectivity for individual splicing events has been observed. Given the high prevalence of splicing defects in cancer, small molecule modulators of RNA processing represent a potentially promising novel therapeutic strategy in cancer treatment. Here, we review their reported effects, mechanisms, and limitations. Published by Elsevier Ltd.

Design of RNA splicing analysis null models for post hoc filtering of Drosophila head RNA-Seq data with the splicing analysis kit (Spanki)

PubMed Central

2013-01-01

Background The production of multiple transcript isoforms from one gene is a major source of transcriptome complexity. RNA-Seq experiments, in which transcripts are converted to cDNA and sequenced, allow the resolution and quantification of alternative transcript isoforms. However, methods to analyze splicing are underdeveloped and errors resulting in incorrect splicing calls occur in every experiment. Results We used RNA-Seq data to develop sequencing and aligner error models. By applying these error models to known input from simulations, we found that errors result from false alignment to minor splice motifs and antisense stands, shifted junction positions, paralog joining, and repeat induced gaps. By using a series of quantitative and qualitative filters, we eliminated diagnosed errors in the simulation, and applied this to RNA-Seq data from Drosophila melanogaster heads. We used high-confidence junction detections to specifically interrogate local splicing differences between transcripts. This method out-performed commonly used RNA-seq methods to identify known alternative splicing events in the Drosophila sex determination pathway. We describe a flexible software package to perform these tasks called Splicing Analysis Kit (Spanki), available at http://www.cbcb.umd.edu/software/spanki. Conclusions Splice-junction centric analysis of RNA-Seq data provides advantages in specificity for detection of alternative splicing. Our software provides tools to better understand error profiles in RNA-Seq data and improve inference from this new technology. The splice-junction centric approach that this software enables will provide more accurate estimates of differentially regulated splicing than current tools. PMID:24209455

Design of RNA splicing analysis null models for post hoc filtering of Drosophila head RNA-Seq data with the splicing analysis kit (Spanki).

PubMed

Sturgill, David; Malone, John H; Sun, Xia; Smith, Harold E; Rabinow, Leonard; Samson, Marie-Laure; Oliver, Brian

2013-11-09

The production of multiple transcript isoforms from one gene is a major source of transcriptome complexity. RNA-Seq experiments, in which transcripts are converted to cDNA and sequenced, allow the resolution and quantification of alternative transcript isoforms. However, methods to analyze splicing are underdeveloped and errors resulting in incorrect splicing calls occur in every experiment. We used RNA-Seq data to develop sequencing and aligner error models. By applying these error models to known input from simulations, we found that errors result from false alignment to minor splice motifs and antisense stands, shifted junction positions, paralog joining, and repeat induced gaps. By using a series of quantitative and qualitative filters, we eliminated diagnosed errors in the simulation, and applied this to RNA-Seq data from Drosophila melanogaster heads. We used high-confidence junction detections to specifically interrogate local splicing differences between transcripts. This method out-performed commonly used RNA-seq methods to identify known alternative splicing events in the Drosophila sex determination pathway. We describe a flexible software package to perform these tasks called Splicing Analysis Kit (Spanki), available at http://www.cbcb.umd.edu/software/spanki. Splice-junction centric analysis of RNA-Seq data provides advantages in specificity for detection of alternative splicing. Our software provides tools to better understand error profiles in RNA-Seq data and improve inference from this new technology. The splice-junction centric approach that this software enables will provide more accurate estimates of differentially regulated splicing than current tools.

New approaches to the treatment of orphan genetic disorders: Mitigating molecular pathologies using chemicals.

PubMed

Velho, Renata V; Sperb-Ludwig, Fernanda; Schwartz, Ida V D

2015-08-01

With the advance and popularization of molecular techniques, the identification of genetic mutations that cause diseases has increased dramatically. Thus, the number of laboratories available to investigate a given disorder and the number of subsequent diagnosis have increased over time. Although it is necessary to identify mutations and provide diagnosis, it is also critical to develop specific therapeutic approaches based on this information. This review aims to highlight recent advances in mutation-targeted therapies with chemicals that mitigate mutational pathology at the molecular level, for disorders that, for the most part, have no effective treatment. Currently, there are several strategies being used to correct different types of mutations, including the following: the identification and characterization of translational readthrough compounds; antisense oligonucleotide-mediated splicing redirection; mismatch repair; and exon skipping. These therapies and other approaches are reviewed in this paper.

Functional rescue of dystrophin-deficient mdx mice by a chimeric peptide-PMO.

PubMed

Yin, Haifang; Moulton, Hong M; Betts, Corinne; Merritt, Thomas; Seow, Yiqi; Ashraf, Shirin; Wang, Qingsong; Boutilier, Jordan; Wood, Matthew Ja

2010-10-01

Splice modulation using antisense oligonucleotides (AOs) has been shown to yield targeted exon exclusion to restore the open reading frame and generate truncated but partially functional dystrophin protein. This has been successfully demonstrated in dystrophin-deficient mdx mice and in Duchenne muscular dystrophy (DMD) patients. However, DMD is a systemic disease; successful therapeutic exploitation of this approach will therefore depend on effective systemic delivery of AOs to all affected tissues. We have previously shown the potential of a muscle-specific/arginine-rich chimeric peptide-phosphorodiamidate morpholino (PMO) conjugate, but its long-term activity, optimized dosing regimen, capacity for functional correction and safety profile remain to be established. Here, we report the results of this chimeric peptide-PMO conjugate in the mdx mouse using low doses (3 and 6 mg/kg) administered via a 6 biweekly systemic intravenous injection protocol. We show 100% dystrophin-positive fibers and near complete correction of the dystrophin transcript defect in all peripheral muscle groups, with restoration of 50% dystrophin protein over 12 weeks, leading to correction of the DMD pathological phenotype and restoration of muscle function in the absence of detectable toxicity or immune response. Chimeric muscle-specific/cell-penetrating peptides therefore represent highly promising agents for systemic delivery of splice-correcting PMO oligomers for DMD therapy.

Modulation of voltage- and Ca2+-dependent gating of CaV1.3 L-type calcium channels by alternative splicing of a C-terminal regulatory domain.

PubMed

Singh, Anamika; Gebhart, Mathias; Fritsch, Reinhard; Sinnegger-Brauns, Martina J; Poggiani, Chiara; Hoda, Jean-Charles; Engel, Jutta; Romanin, Christoph; Striessnig, Jörg; Koschak, Alexandra

2008-07-25

Low voltage activation of Ca(V)1.3 L-type Ca(2+) channels controls excitability in sensory cells and central neurons as well as sinoatrial node pacemaking. Ca(V)1.3-mediated pacemaking determines neuronal vulnerability of dopaminergic striatal neurons affected in Parkinson disease. We have previously found that in Ca(V)1.4 L-type Ca(2+) channels, activation, voltage, and calcium-dependent inactivation are controlled by an intrinsic distal C-terminal modulator. Because alternative splicing in the Ca(V)1.3 alpha1 subunit C terminus gives rise to a long (Ca(V)1.3(42)) and a short form (Ca(V)1.3(42A)), we investigated if a C-terminal modulatory mechanism also controls Ca(V)1.3 gating. The biophysical properties of both splice variants were compared after heterologous expression together with beta3 and alpha2delta1 subunits in HEK-293 cells. Activation of calcium current through Ca(V)1.3(42A) channels was more pronounced at negative voltages, and inactivation was faster because of enhanced calcium-dependent inactivation. By investigating several Ca(V)1.3 channel truncations, we restricted the modulator activity to the last 116 amino acids of the C terminus. The resulting Ca(V)1.3(DeltaC116) channels showed gating properties similar to Ca(V)1.3(42A) that were reverted by co-expression of the corresponding C-terminal peptide C(116). Fluorescence resonance energy transfer experiments confirmed an intramolecular protein interaction in the C terminus of Ca(V)1.3 channels that also modulates calmodulin binding. These experiments revealed a novel mechanism of channel modulation enabling cells to tightly control Ca(V)1.3 channel activity by alternative splicing. The absence of the C-terminal modulator in short splice forms facilitates Ca(V)1.3 channel activation at lower voltages expected to favor Ca(V)1.3 activity at threshold voltages as required for modulation of neuronal firing behavior and sinoatrial node pacemaking.

Angubindin-1 opens the blood-brain barrier in vivo for delivery of antisense oligonucleotide to the central nervous system.

PubMed

Zeniya, Satoshi; Kuwahara, Hiroya; Daizo, Kaiichi; Watari, Akihiro; Kondoh, Masuo; Yoshida-Tanaka, Kie; Kaburagi, Hidetoshi; Asada, Ken; Nagata, Tetsuya; Nagahama, Masahiro; Yagi, Kiyohito; Yokota, Takanori

2018-05-17

Within the field of RNA therapeutics, antisense oligonucleotide-based therapeutics are a potentially powerful means of treating intractable diseases. However, if these therapeutics are used for the treatment of neurological disorders, safe yet efficient methods of delivering antisense oligonucleotides across the blood-brain barrier to the central nervous system must be developed. Here, we examined the use of angubindin-1, a binder to the tricellular tight junction, to modulate paracellular transport between brain microvascular endothelial cells in the blood-brain barrier for the delivery of antisense oligonucleotides to the central nervous system. This proof-of-concept study demonstrated that intravenously injected angubindin-1 increased the permeability of the blood-brain barrier and enabled transient delivery of subsequently administered antisense oligonucleotides into the mouse brain and spinal cord, leading to silencing of a target RNA without any overt adverse effects. We also found that two bicellular tight junction modulators did not produce such a silencing effect, suggesting that the tricellular tight junction is likely a better target for the delivery of antisense oligonucleotides than the bicellular tight junction. Our delivery strategy of modulating the tricellular tight junction in the blood-brain barrier via angubindin-1 provides a novel avenue of research for the development of antisense oligonucleotide-based therapeutics for the treatment of neurological disorders. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Pharmacology of a Central Nervous System Delivered 2′-O-Methoxyethyl–Modified Survival of Motor Neuron Splicing Oligonucleotide in Mice and Nonhuman Primates

PubMed Central

Chun, Seung J.; Norris, Daniel A.; Hung, Gene; Lee, Sam; Matson, John; Fey, Robert A.; Gaus, Hans; Hua, Yimin; Grundy, John S.; Krainer, Adrian R.; Henry, Scott P.; Bennett, C. Frank

2014-01-01

Spinal muscular atrophy (SMA) is a debilitating neuromuscular disease caused by the loss of survival of motor neuron (SMN) protein. Previously, we demonstrated that ISIS 396443, an antisense oligonucleotide (ASO) targeted to the SMN2 pre-mRNA, is a potent inducer of SMN2 exon 7 inclusion and SMN protein expression, and improves function and survival of mild and severe SMA mouse models. Here, we demonstrate that ISIS 396443 is the most potent ASO in central nervous system (CNS) tissues of adult mice, compared with several other chemically modified ASOs. We evaluated methods of ISIS 396443 delivery to the CNS and characterized its pharmacokinetics and pharmacodynamics in rodents and nonhuman primates (NHPs). Intracerebroventricular bolus injection is a more efficient method of delivering ISIS 396443 to the CNS of rodents, compared with i.c.v. infusion. For both methods of delivery, the duration of ISIS 396443–mediated SMN2 splicing correction is long lasting, with maximal effects still observed 6 months after treatment discontinuation. Administration of ISIS 396443 to the CNS of NHPs by a single intrathecal bolus injection results in widespread distribution throughout the spinal cord. Based upon these preclinical studies, we have advanced ISIS 396443 into clinical development. PMID:24784568

A multifactor regulatory circuit involving H-NS, VirF and an antisense RNA modulates transcription of the virulence gene icsA of Shigella flexneri.

PubMed

Tran, Chi Nhan; Giangrossi, Mara; Prosseda, Gianni; Brandi, Anna; Di Martino, Maria Letizia; Colonna, Bianca; Falconi, Maurizio

2011-10-01

The icsA gene of Shigella encodes a structural protein involved in colonization of the intestinal mucosa by bacteria. This gene is expressed upon invasion of the host and is controlled by a complex regulatory circuit involving the nucleoid protein H-NS, the AraC-like transcriptional activator VirF, and a 450 nt antisense RNA (RnaG) acting as transcriptional attenuator. We investigated on the interplay of these factors at the molecular level. DNase I footprints reveal that both H-NS and VirF bind to a region including the icsA and RnaG promoters. H-NS is shown to repress icsA transcription at 30°C but not at 37°C, suggesting a significant involvement of this protein in the temperature-regulated expression of icsA. We also demonstrate that VirF directly stimulates icsA transcription and is able to alleviate H-NS repression in vitro. According to these results, icsA expression is derepressed in hns- background and overexpressed when VirF is provided in trans. Moreover, we find that RnaG-mediated transcription attenuation depends on 80 nt at its 5'-end, a stretch carrying the antisense region. Bases engaged in the initial contact leading to sense-antisense pairing have been identified using synthetic RNA and DNA oligonucleotides designed to rebuild and mutagenize the two stem-loop motifs of the antisense region.

Repair of pre-mRNA splicing

PubMed Central

Nlend, Rachel Nlend; Meyer, Kathrin

2010-01-01

Recent analyses of complete genomes have revealed that alternative splicing became more prevalent and important during eukaryotic evolution. Alternative splicing augments the protein repertoire—particularly that of the human genome—and plays an important role in the development and function of differentiated cell types. However, splicing is also extremely vulnerable, and defects in the proper recognition of splicing signals can give rise to a variety of diseases. In this review, we discuss splicing correction therapies, by using the inherited disease Spinal Muscular Atrophy (SMA) as an example. This lethal early childhood disorder is caused by deletions or other severe mutations of SMN1, a gene coding for the essential survival of motoneurons protein. A second gene copy present in humans and few non-human primates, SMN2, can only partly compensate for the defect because of a single nucleotide change in exon 7 that causes this exon to be skipped in the majority of mRNAs. Thus SMN2 is a prime therapeutic target for SMA. In recent years, several strategies based on small molecule drugs, antisense oligonucleotides or in vivo expressed RNAs have been developed that allow a correction of SMN2 splicing. For some of these, a therapeutic benefit has been demonstrated in mouse models for SMA. This means that clinical trials of such splicing therapies for SMA may become possible in the near future. PMID:20523126

Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type 1 Mice.

PubMed

Jauvin, Dominic; Chrétien, Jessina; Pandey, Sanjay K; Martineau, Laurie; Revillod, Lucille; Bassez, Guillaume; Lachon, Aline; MacLeod, A Robert; Gourdon, Geneviève; Wheeler, Thurman M; Thornton, Charles A; Bennett, C Frank; Puymirat, Jack

2017-06-16

Myotonic dystrophy type 1 (DM1), a dominant hereditary muscular dystrophy, is caused by an abnormal expansion of a (CTG) n trinucleotide repeat in the 3' UTR of the human dystrophia myotonica protein kinase (DMPK) gene. As a consequence, mutant transcripts containing expanded CUG repeats are retained in nuclear foci and alter the function of splicing regulatory factors members of the MBNL and CELF families, resulting in alternative splicing misregulation of specific transcripts in affected DM1 tissues. In the present study, we treated DMSXL mice systemically with a 2'-4'-constrained, ethyl-modified (ISIS 486178) antisense oligonucleotide (ASO) targeted to the 3' UTR of the DMPK gene, which led to a 70% reduction in CUG exp RNA abundance and foci in different skeletal muscles and a 30% reduction in the heart. Furthermore, treatment with ISIS 486178 ASO improved body weight, muscle strength, and muscle histology, whereas no overt toxicity was detected. This is evidence that the reduction of CUG exp RNA improves muscle strength in DM1, suggesting that muscle weakness in DM1 patients may be improved following elimination of toxic RNAs. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Reduction of methylviologen-mediated oxidative stress tolerance in antisense transgenic tobacco seedlings through restricted expression of StAPX.

PubMed

Sun, Wei-Hong; Wang, Yong; He, Hua-Gang; Li, Xue; Song, Wan; Du, Bin; Meng, Qing-Wei

2013-07-01

Ascorbate peroxidases are directly involved in reactive oxygen species (ROS) scavenging by reducing hydrogen peroxide to water. The tomato thylakoid-bound ascorbate peroxidase gene (StAPX) was introduced into tobacco. RNA gel blot analysis confirmed that StAPX in tomato leaves was induced by methylviologen-mediated oxidative stress. The sense transgenic seedlings exhibited higher tAPX activity than that of the wild type (WT) plants under oxidative stress conditions, while the antisense seedlings exhibited lower tAPX activity. Lower APX activities of antisense transgenic seedlings caused higher malondialdehyde contents and relative electrical conductivity. The sense transgenic seedlings with higher tAPX activity maintained higher chlorophyll content and showed the importance of tAPX in maintaining the optimal chloroplast development under methylviologen stress conditions, whereas the antisense lines maintained lower chlorophyll content than WT seedlings. Results indicated that the over-expression of StAPX enhanced tolerance to methylviologen-mediated oxidative stress in sense transgenic tobacco early seedlings, whereas the suppression of StAPX in antisense transgenic seedlings showed high sensitivity to oxidative stress.

Analysis of Subcellular RNA Fractions Revealed a Transcription-Independent Effect of Tumor Necrosis Factor Alpha on Splicing, Mediated by Spt5.

PubMed

Diamant, Gil; Eisenbaum, Tal; Leshkowitz, Dena; Dikstein, Rivka

2016-05-01

The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) modulates the expression of many genes, primarily through activation of NF-κB. Here, we examined the global effects of the elongation factor Spt5 on nascent and mature mRNAs of TNF-α-induced cells using chromatin and cytosolic subcellular fractions. We identified several classes of TNF-α-induced genes controlled at the level of transcription, splicing, and chromatin retention. Spt5 was found to facilitate splicing and chromatin release in genes displaying high induction rates. Further analysis revealed striking effects of TNF-α on the splicing of 25% of expressed genes; the vast majority were not transcriptionally induced. Splicing enhancement of noninduced genes by TNF-α was transient and independent of NF-κB. Investigating the underlying basis, we found that Spt5 is required for the splicing facilitation of the noninduced genes. In line with this, Spt5 interacts with Sm core protein splicing factors. Furthermore, following TNF-α treatment, levels of RNA polymerase II (Pol II) but not Spt5 are reduced from the splicing-induced genes, suggesting that these genes become enriched with a Pol II-Spt5 form. Our findings revealed the Pol II-Spt5 complex as a highly competent coordinator of cotranscriptional splicing. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Regulation of alternative splicing by the circadian clock and food related cues

PubMed Central

2012-01-01

Background The circadian clock orchestrates daily rhythms in metabolism, physiology and behaviour that allow organisms to anticipate regular changes in their environment, increasing their adaptation. Such circadian phenotypes are underpinned by daily rhythms in gene expression. Little is known, however, about the contribution of post-transcriptional processes, particularly alternative splicing. Results Using Affymetrix mouse exon-arrays, we identified exons with circadian alternative splicing in the liver. Validated circadian exons were regulated in a tissue-dependent manner and were present in genes with circadian transcript abundance. Furthermore, an analysis of circadian mutant Vipr2-/- mice revealed the existence of distinct physiological pathways controlling circadian alternative splicing and RNA binding protein expression, with contrasting dependence on Vipr2-mediated physiological signals. This view was corroborated by the analysis of the effect of fasting on circadian alternative splicing. Feeding is an important circadian stimulus, and we found that fasting both modulates hepatic circadian alternative splicing in an exon-dependent manner and changes the temporal relationship with transcript-level expression. Conclusions The circadian clock regulates alternative splicing in a manner that is both tissue-dependent and concurrent with circadian transcript abundance. This adds a novel temporal dimension to the regulation of mammalian alternative splicing. Moreover, our results demonstrate that circadian alternative splicing is regulated by the interaction between distinct physiological cues, and illustrates the capability of single genes to integrate circadian signals at different levels of regulation. PMID:22721557

microRNA-185 modulates low density lipoprotein receptor expression as a key posttranscriptional regulator.

PubMed

Jiang, Huajun; Zhang, Jin; Du, Yu; Jia, Xiaojian; Yang, Fan; Si, Shuyi; Wang, Li; Hong, Bin

2015-12-01

Low-density lipoprotein receptor (LDLR) mediates endocytosis of LDL particles and is important in maintaining plasma cholesterol levels, thus its expression is under extensive regulation at multiple levels, including transcriptional and posttranscriptional regulation by transcription factors (TFs) and RNA-binding proteins (RBPs). Here, we identified microRNA-185 (miR-185) as a novel direct posttranscriptional regulator of LDLR and an indirect LDLR modulator through KSRP in hepatic cells. Using quantitative real-time PCR (qPCR), we detected the effect of predicted LDLR-targeting miRNAs and found that overexpression of miR-185 repressed LDLR expression and LDL uptake in HepG2 cells by 62.4 ± 6.0% (p = 7.0 × 10(-5)) and 32.5 ± 6.0% (p = 7.7 × 10(-4)) respectively, through directly targeting LDLR 3'UTR. Unexpectedly, the antisense inhibitor of miR-185 had similar repression effect on LDLR although it reduced the association of endogenous miR-185 with LDLR mRNA. Further experiments revealed that KH-type splicing regulatory protein (KSRP), one of the LDLR-destabilizing RBPs, is also a target of miR-185. KSRP silencing reversed the repression effects of miR-185-inhibitor on LDLR. Thus miR-185 regulates LDLR expression not only through directly targeting but also by a RBP-involved indirect pathway. Finally, the in vivo results showed that miR-185-inhibitor upregulated hepatic LDLR expression and correlated with a decrease in plasma cholesterol level and arterial plaque area in ApoE KO mice. These findings reveal that miR-185 controls cholesterol homeostasis as a key posttranscriptional LDLR modulator in hepatic cells, providing novel insight into the regulatory mechanism for LDLR expression and the anti-atherosclerosis effect of miR-185-inhibitor. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Opioid inhibition of N-type Ca2+ channels and spinal analgesia couple to alternative splicing.

PubMed

Andrade, Arturo; Denome, Sylvia; Jiang, Yu-Qiu; Marangoudakis, Spiro; Lipscombe, Diane

2010-10-01

Alternative pre-mRNA splicing occurs extensively in the nervous systems of complex organisms, including humans, considerably expanding the potential size of the proteome. Cell-specific alternative pre-mRNA splicing is thought to optimize protein function for specialized cellular tasks, but direct evidence for this is limited. Transmission of noxious thermal stimuli relies on the activity of N-type Ca(V)2.2 calcium channels in nociceptors. Using an exon-replacement strategy in mice, we show that mutually exclusive splicing patterns in the Ca(V)2.2 gene modulate N-type channel function in nociceptors, leading to a change in morphine analgesia. Exon 37a (e37a) enhances μ-opioid receptor-mediated inhibition of N-type calcium channels by promoting activity-independent inhibition. In the absence of e37a, spinal morphine analgesia is weakened in vivo but the basal response to noxious thermal stimuli is not altered. Our data suggest that highly specialized, discrete cellular responsiveness in vivo can be attributed to alternative splicing events regulated at the level of individual neurons.

Attenuation of the suppressive activity of cellular splicing factor SRSF3 by Kaposi sarcoma–associated herpesvirus ORF57 protein is required for RNA splicing

PubMed Central

Majerciak, Vladimir; Lu, Mathew; Li, Xiaofan

2014-01-01

Kaposi sarcoma–associated herpesvirus (KSHV) ORF57 is a multifunctional post-transcriptional regulator essential for viral gene expression during KSHV lytic infection. ORF57 requires interactions with various cellular proteins for its function. Here, we identified serine/arginine-rich splicing factor 3 (SRSF3, formerly known as SRp20) as a cellular cofactor involved in ORF57-mediated splicing of KSHV K8β RNA. In the absence of ORF57, SRSF3 binds to a suboptimal K8β intron and inhibits K8β splicing. Knockdown of SRSF3 promotes K8β splicing, mimicking the effect of ORF57. The N-terminal half of ORF57 binds to the RNA recognition motif of SRSF3, which prevents SRSF3 from associating with the K8β intron RNA and therefore attenuates the suppressive effect of SRSF3 on K8β splicing. ORF57 also promotes splicing of heterologous non-KSHV transcripts that are negatively regulated by SRSF3, indicating that the effect of ORF57 on SRSF3 activity is independent of RNA target. SPEN proteins, previously identified as ORF57-interacting partners, suppress ORF57 splicing activity by displacing ORF57 from SRSF3–RNA complexes. In summary, we have identified modulation of SRSF3 activity as the molecular mechanism by which ORF57 promotes RNA splicing. PMID:25234929

Attenuation of the suppressive activity of cellular splicing factor SRSF3 by Kaposi sarcoma-associated herpesvirus ORF57 protein is required for RNA splicing.

PubMed

Majerciak, Vladimir; Lu, Mathew; Li, Xiaofan; Zheng, Zhi-Ming

2014-11-01

Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 is a multifunctional post-transcriptional regulator essential for viral gene expression during KSHV lytic infection. ORF57 requires interactions with various cellular proteins for its function. Here, we identified serine/arginine-rich splicing factor 3 (SRSF3, formerly known as SRp20) as a cellular cofactor involved in ORF57-mediated splicing of KSHV K8β RNA. In the absence of ORF57, SRSF3 binds to a suboptimal K8β intron and inhibits K8β splicing. Knockdown of SRSF3 promotes K8β splicing, mimicking the effect of ORF57. The N-terminal half of ORF57 binds to the RNA recognition motif of SRSF3, which prevents SRSF3 from associating with the K8β intron RNA and therefore attenuates the suppressive effect of SRSF3 on K8β splicing. ORF57 also promotes splicing of heterologous non-KSHV transcripts that are negatively regulated by SRSF3, indicating that the effect of ORF57 on SRSF3 activity is independent of RNA target. SPEN proteins, previously identified as ORF57-interacting partners, suppress ORF57 splicing activity by displacing ORF57 from SRSF3-RNA complexes. In summary, we have identified modulation of SRSF3 activity as the molecular mechanism by which ORF57 promotes RNA splicing. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Characterization of fructose-bisphosphate aldolase regulated by gibberellin in roots of rice seedling.

PubMed

Konishi, Hirosato; Yamane, Hisakazu; Maeshima, Masayoshi; Komatsu, Setsuko

2004-12-01

Fructose-bisphosphate aldolase is a glycolytic enzyme whose activity increases in rice roots treated with gibberellin (GA). To investigate the relationship between aldolase and root growth, GA-induced root aldolase was characterized. GA3 promoted an increase in aldolase accumulation when 0.1 microM GA3 was added exogenously to rice roots. Aldolase accumulated abundantly in roots, especially in the apical region. To examine the effect of aldolase function on root growth, transgenic rice plants expressing antisense aldolase were constructed. Root growth of aldolase-antisense transgenic rice was repressed compared with that of the vector control transgenic rice. Although aldolase activity increased by 25% in vector control rice roots treated with 0.1 microM GA3, FBPA activity increased very little by 0.1 microM GA3 treatment in the root of aldolase-antisense transgenic rice. Furthermore, aldolase co-immunoprecipitated with antibodies against vacuolar H+ -ATPase in rice roots. In the root of OsCDPK13-antisense transgenic rice, aldolase did not accumulate even after treatment with GA3. These results suggest that the activation of glycolytic pathway function accelerates root growth and that GA3-induced root aldolase may be modulated through OsCDPK13. Aldolase physically associates with vacuolar H-ATPase in roots and may regulate the vacuolar H-ATPase mediated control of cell elongation that determines root length.

Mechanisms and Regulation of Alternative Pre-mRNA Splicing

PubMed Central

Lee, Yeon

2015-01-01

Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms with limited gene numbers. Split eukaryotic genes contain intervening sequences or introns disrupting protein-coding exons, and intron removal occurs by repeated assembly of a large and highly dynamic ribonucleoprotein complex termed the spliceosome, which is composed of five small nuclear ribonucleoprotein particles, U1, U2, U4/U6, and U5. Biochemical studies over the past 10 years have allowed the isolation as well as compositional, functional, and structural analysis of splicing complexes at distinct stages along the spliceosome cycle. The average human gene contains eight exons and seven introns, producing an average of three or more alternatively spliced mRNA isoforms. Recent high-throughput sequencing studies indicate that 100% of human genes produce at least two alternative mRNA isoforms. Mechanisms of alternative splicing include RNA–protein interactions of splicing factors with regulatory sites termed silencers or enhancers, RNA–RNA base-pairing interactions, or chromatin-based effects that can change or determine splicing patterns. Disease-causing mutations can often occur in splice sites near intron borders or in exonic or intronic RNA regulatory silencer or enhancer elements, as well as in genes that encode splicing factors. Together, these studies provide mechanistic insights into how spliceosome assembly, dynamics, and catalysis occur; how alternative splicing is regulated and evolves; and how splicing can be disrupted by cis- and trans-acting mutations leading to disease states. These findings make the spliceosome an attractive new target for small-molecule, antisense, and genome-editing therapeutic interventions. PMID:25784052

Identification of a Peptide for Systemic Brain Delivery of a Morpholino Oligonucleotide in Mouse Models of Spinal Muscular Atrophy

PubMed Central

Shabanpoor, Fazel; Hammond, Suzan M; Abendroth, Frank; Hazell, Gareth; Wood, Matthew J.A.

2017-01-01

Splice-switching antisense oligonucleotides are emerging treatments for neuromuscular diseases, with several splice-switching oligonucleotides (SSOs) currently undergoing clinical trials such as for Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA). However, the development of systemically delivered antisense therapeutics has been hampered by poor tissue penetration and cellular uptake, including crossing of the blood–brain barrier (BBB) to reach targets in the central nervous system (CNS). For SMA application, we have investigated the ability of various BBB-crossing peptides for CNS delivery of a splice-switching phosphorodiamidate morpholino oligonucleotide (PMO) targeting survival motor neuron 2 (SMN2) exon 7 inclusion. We identified a branched derivative of the well-known ApoE (141–150) peptide, which as a PMO conjugate was capable of exon inclusion in the CNS following systemic administration, leading to an increase in the level of full-length SMN2 transcript. Treatment of newborn SMA mice with this peptide-PMO (P-PMO) conjugate resulted in a significant increase in the average lifespan and gains in weight, muscle strength, and righting reflexes. Systemic treatment of adult SMA mice with this newly identified P-PMO also resulted in small but significant increases in the levels of SMN2 pre-messenger RNA (mRNA) exon inclusion in the CNS and peripheral tissues. This work provides proof of principle for the ability to select new peptide paradigms to enhance CNS delivery and activity of a PMO SSO through use of a peptide-based delivery platform for the treatment of SMA potentially extending to other neuromuscular and neurodegenerative diseases. PMID:28118087

Identification of Small Molecule and Genetic Modulators of AON-Induced Dystrophin Exon Skipping by High-Throughput Screening

PubMed Central

O'Leary, Debra A.; Sharif, Orzala; Anderson, Paul; Tu, Buu; Welch, Genevieve; Zhou, Yingyao; Caldwell, Jeremy S.; Engels, Ingo H.; Brinker, Achim

2009-01-01

One therapeutic approach to Duchenne Muscular Dystrophy (DMD) recently entering clinical trials aims to convert DMD phenotypes to that of a milder disease variant, Becker Muscular Dystrophy (BMD), by employing antisense oligonucleotides (AONs) targeting splice sites, to induce exon skipping and restore partial dystrophin function. In order to search for small molecule and genetic modulators of AON-dependent and independent exon skipping, we screened ∼10,000 known small molecule drugs, >17,000 cDNA clones, and >2,000 kinase- targeted siRNAs against a 5.6 kb luciferase minigene construct, encompassing exon 71 to exon 73 of human dystrophin. As a result, we identified several enhancers of exon skipping, acting on both the reporter construct as well as endogenous dystrophin in mdx cells. Multiple mechanisms of action were identified, including histone deacetylase inhibition, tubulin modulation and pre-mRNA processing. Among others, the nucleolar protein NOL8 and staufen RNA binding protein homolog 2 (Stau2) were found to induce endogenous exon skipping in mdx cells in an AON-dependent fashion. An unexpected but recurrent theme observed in our screening efforts was the apparent link between the inhibition of cell cycle progression and the induction of exon skipping. PMID:20020055

Identification of small molecule and genetic modulators of AON-induced dystrophin exon skipping by high-throughput screening.

PubMed

O'Leary, Debra A; Sharif, Orzala; Anderson, Paul; Tu, Buu; Welch, Genevieve; Zhou, Yingyao; Caldwell, Jeremy S; Engels, Ingo H; Brinker, Achim

2009-12-17

One therapeutic approach to Duchenne Muscular Dystrophy (DMD) recently entering clinical trials aims to convert DMD phenotypes to that of a milder disease variant, Becker Muscular Dystrophy (BMD), by employing antisense oligonucleotides (AONs) targeting splice sites, to induce exon skipping and restore partial dystrophin function. In order to search for small molecule and genetic modulators of AON-dependent and independent exon skipping, we screened approximately 10,000 known small molecule drugs, >17,000 cDNA clones, and >2,000 kinase- targeted siRNAs against a 5.6 kb luciferase minigene construct, encompassing exon 71 to exon 73 of human dystrophin. As a result, we identified several enhancers of exon skipping, acting on both the reporter construct as well as endogenous dystrophin in mdx cells. Multiple mechanisms of action were identified, including histone deacetylase inhibition, tubulin modulation and pre-mRNA processing. Among others, the nucleolar protein NOL8 and staufen RNA binding protein homolog 2 (Stau2) were found to induce endogenous exon skipping in mdx cells in an AON-dependent fashion. An unexpected but recurrent theme observed in our screening efforts was the apparent link between the inhibition of cell cycle progression and the induction of exon skipping.

Short- and long-term memory are modulated by multiple isoforms of the fragile X mental retardation protein.

PubMed

Banerjee, Paromita; Schoenfeld, Brian P; Bell, Aaron J; Choi, Catherine H; Bradley, Michael P; Hinchey, Paul; Kollaros, Maria; Park, Jae H; McBride, Sean M J; Dockendorff, Thomas C

2010-05-12

The diversity of protein isoforms arising from alternative splicing is thought to modulate fine-tuning of synaptic plasticity. Fragile X mental retardation protein (FMRP), a neuronal RNA binding protein, exists in isoforms as a result of alternative splicing, but the contribution of these isoforms to neural plasticity are not well understood. We show that two isoforms of Drosophila melanogaster FMRP (dFMR1) have differential roles in mediating neural development and behavior functions conferred by the dfmr1 gene. These isoforms differ in the presence of a protein interaction module that is related to prion domains and is functionally conserved between FMRPs. Expression of both isoforms is necessary for optimal performance in tests of short- and long-term memory of courtship training. The presence or absence of the protein interaction domain may govern the types of ribonucleoprotein (RNP) complexes dFMR1 assembles into, with different RNPs regulating gene expression in a manner necessary for establishing distinct phases of memory formation.

Alternative splicing regulation in tumor necrosis factor-mediated inflammation.

PubMed

López-Urrutia, Eduardo; Campos-Parra, Alma; Herrera, Luis Alonso; Pérez-Plasencia, Carlos

2017-11-01

It is generally accepted that alternative splicing has an effect on disease when it leads to conspicuous changes in relevant proteins, but that the combinatorial effect of several small modifications can have marked outcomes as well. Inflammation is a complex process involving numerous signaling pathways, among which the tumor necrosis factor (TNF) pathway is one of the most studied. Signaling pathways are commonly represented as intricate cascades of molecular interactions that eventually lead to the activation of one or several genes. Alternative splicing is a common means of controlling protein expression in time and space; therefore, it can modulate the outcome of signaling pathways through small changes in their elements. Notably, the overall process is tightly regulated, which is easily overlooked when analyzing the pathway as a whole. The present review summarizes recent studies of the alternative splicing of key players of the TNF pathway leading to inflammation, and hypothesizes on the cumulative results of those modifications and the impact on cancer development.

Alternative splicing regulation in tumor necrosis factor-mediated inflammation

PubMed Central

López-Urrutia, Eduardo; Campos-Parra, Alma; Herrera, Luis Alonso; Pérez-Plasencia, Carlos

2017-01-01

It is generally accepted that alternative splicing has an effect on disease when it leads to conspicuous changes in relevant proteins, but that the combinatorial effect of several small modifications can have marked outcomes as well. Inflammation is a complex process involving numerous signaling pathways, among which the tumor necrosis factor (TNF) pathway is one of the most studied. Signaling pathways are commonly represented as intricate cascades of molecular interactions that eventually lead to the activation of one or several genes. Alternative splicing is a common means of controlling protein expression in time and space; therefore, it can modulate the outcome of signaling pathways through small changes in their elements. Notably, the overall process is tightly regulated, which is easily overlooked when analyzing the pathway as a whole. The present review summarizes recent studies of the alternative splicing of key players of the TNF pathway leading to inflammation, and hypothesizes on the cumulative results of those modifications and the impact on cancer development. PMID:29113151

Regulation of gene expression in mammalian nervous system through alternative pre-mRNA splicing coupled with RNA quality control mechanisms.

PubMed

Yap, Karen; Makeyev, Eugene V

2013-09-01

Eukaryotic gene expression is orchestrated on a genome-wide scale through several post-transcriptional mechanisms. Of these, alternative pre-mRNA splicing expands the proteome diversity and modulates mRNA stability through downstream RNA quality control (QC) pathways including nonsense-mediated decay (NMD) of mRNAs containing premature termination codons and nuclear retention and elimination (NRE) of intron-containing transcripts. Although originally identified as mechanisms for eliminating aberrant transcripts, a growing body of evidence suggests that NMD and NRE coupled with deliberate changes in pre-mRNA splicing patterns are also used in a number of biological contexts for deterministic control of gene expression. Here we review recent studies elucidating molecular mechanisms and biological significance of these gene regulation strategies with a specific focus on their roles in nervous system development and physiology. This article is part of a Special Issue entitled 'RNA and splicing regulation in neurodegeneration'. Copyright © 2013 Elsevier Inc. All rights reserved.

A multi-model approach to nucleic acid-based drug development.

PubMed

Gautherot, Isabelle; Sodoyer, Regís

2004-01-01

With the advent of functional genomics and the shift of interest towards sequence-based therapeutics, the past decades have witnessed intense research efforts on nucleic acid-mediated gene regulation technologies. Today, RNA interference is emerging as a groundbreaking discovery, holding promise for development of genetic modulators of unprecedented potency. Twenty-five years after the discovery of antisense RNA and ribozymes, gene control therapeutics are still facing developmental difficulties, with only one US FDA-approved antisense drug currently available in the clinic. Limited predictability of target site selection models is recognized as one major stumbling block that is shared by all of the so-called complementary technologies, slowing the progress towards a commercial product. Currently employed in vitro systems for target site selection include RNAse H-based mapping, antisense oligonucleotide microarrays, and functional screening approaches using libraries of catalysts with randomized target-binding arms to identify optimal ribozyme/DNAzyme cleavage sites. Individually, each strategy has its drawbacks from a drug development perspective. Utilization of message-modulating sequences as therapeutic agents requires that their action on a given target transcript meets criteria of potency and selectivity in the natural physiological environment. In addition to sequence-dependent characteristics, other factors will influence annealing reactions and duplex stability, as well as nucleic acid-mediated catalysis. Parallel consideration of physiological selection systems thus appears essential for screening for nucleic acid compounds proposed for therapeutic applications. Cellular message-targeting studies face issues relating to efficient nucleic acid delivery and appropriate analysis of response. For reliability and simplicity, prokaryotic systems can provide a rapid and cost-effective means of studying message targeting under pseudo-cellular conditions, but such approaches also have limitations. To streamline nucleic acid drug discovery, we propose a multi-model strategy integrating high-throughput-adapted bacterial screening, followed by reporter-based and/or natural cellular models and potentially also in vitro assays for characterization of the most promising candidate sequences, before final in vivo testing.

AMPA GluA1-flip targeted oligonucleotide therapy reduces neonatal seizures and hyperexcitability

PubMed Central

Lykens, Nicole M.; Reddi, Jyoti M.

2017-01-01

Glutamate-activated α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-Rs) mediate the majority of excitatory neurotransmission in brain and thus are major drug targets for diseases associated with hyperexcitability or neurotoxicity. Due to the critical nature of AMPA-Rs in normal brain function, typical AMPA-R antagonists have deleterious effects on cognition and motor function, highlighting the need for more precise modulators. A dramatic increase in the flip isoform of alternatively spliced AMPA-R GluA1 subunits occurs post-seizure in humans and animal models. GluA1-flip produces higher gain AMPA channels than GluA1-flop, increasing network excitability and seizure susceptibility. Splice modulating oligonucleotides (SMOs) bind to pre-mRNA to influence alternative splicing, a strategy that can be exploited to develop more selective drugs across therapeutic areas. We developed a novel SMO, GR1, which potently and specifically decreased GluA1-flip expression throughout the brain of neonatal mice lasting at least 60 days after single intracerebroventricular injection. GR1 treatment reduced AMPA-R mediated excitatory postsynaptic currents at hippocampal CA1 synapses, without affecting long-term potentiation or long-term depression, cellular models of memory, or impairing GluA1-dependent cognition or motor function in mice. Importantly, GR1 demonstrated anti-seizure properties and reduced post-seizure hyperexcitability in neonatal mice, highlighting its drug candidate potential for treating epilepsies and other neurological diseases involving network hyperexcitability. PMID:28178321

Small-scale high-throughput sequencing-based identification of new therapeutic tools in cystic fibrosis.

PubMed

Bonini, Jennifer; Varilh, Jessica; Raynal, Caroline; Thèze, Corinne; Beyne, Emmanuelle; Audrezet, Marie-Pierre; Ferec, Claude; Bienvenu, Thierry; Girodon, Emmanuelle; Tuffery-Giraud, Sylvie; Des Georges, Marie; Claustres, Mireille; Taulan-Cadars, Magali

2015-10-01

Although 97-99% of CFTR mutations have been identified, great efforts must be made to detect yet-unidentified mutations. We developed a small-scale next-generation sequencing approach for reliably and quickly scanning the entire gene, including noncoding regions, to identify new mutations. We applied this approach to 18 samples from patients suffering from cystic fibrosis (CF) in whom only one mutation had hitherto been identified. Using an in-house bioinformatics pipeline, we could rapidly identify a second disease-causing CFTR mutation for 16 of 18 samples. Of them, c.1680-883A>G was found in three unrelated CF patients. Analysis of minigenes and patients' transcripts showed that this mutation results in aberrantly spliced transcripts because of the inclusion of a pseudoexon. It is located only three base pairs from the c.1680-886A>G mutation (1811+1.6kbA>G), the fourth most frequent mutation in southwestern Europe. We next tested the effect of antisense oligonucleotides targeting splice sites on these two mutations on pseudoexon skipping. Oligonucleotide transfection resulted in the restoration of the full-length, in-frame CFTR transcript, demonstrating the effect of antisense oligonucleotide-induced pseudoexon skipping in CF. Our data confirm the importance of analyzing noncoding regions to find unidentified mutations, which is essential to designing targeted therapeutic approaches.

An indicator gene to demonstrate intracellular transposition of defective retroviruses.

PubMed Central

Heidmann, T; Heidmann, O; Nicolas, J F

1988-01-01

An indicator gene for detection and quantitation of RNA-mediated transposition was constructed (neoRT). It was inserted into a Moloney murine leukemia provirus (Mo-MLV) deleted for the envelope gene to test for intracellular transposition of defective retroviruses [Mo-MLV(neo)RT]. NeoRT contains the selectable neo gene (which confers resistance to the drug G418), inactivated by a polyadenylylation sequence inserted between the neo promotor and coding sequence. The polyadenylylation sequence is flanked (on the antisense strand of the DNA) by a donor and an acceptor splice site so as to be removed upon passage of the provirus through an RNA intermediate. 3T3 cells transfected with the defective Mo-MLV(neo)RT provirus are sensitive to G418. After trans-complementation with Mo-MLV, viral transcripts confer resistance to G418 upon infection of test cells. In the resistant cells, the polyadenylylation sequence has been removed, as a result in most cases of precise splicing of the intronic domain. Retrotransposition of the defective Mo-MLV(neo)RT provirus was demonstrated by submitting transfected G418-sensitive clones to selection. Between 1 and 10 G418-resistant clones were obtained per 10(7) cells. Several possess additional copies, with evidence for precise removal of the intronic domain. By using target test cells in coculture experiments, extracellular intermediates of retrotransposition could not be detected. Images PMID:2832848

Global regulation of alternative RNA splicing by the SR-rich protein RBM39.

PubMed

Mai, Sanyue; Qu, Xiuhua; Li, Ping; Ma, Qingjun; Cao, Cheng; Liu, Xuan

2016-08-01

RBM39 is a serine/arginine-rich RNA-binding protein that is highly homologous to the splicing factor U2AF65. However, the role of RBM39 in alternative splicing is poorly understood. In this study, RBM39-mediated global alternative splicing was investigated using RNA-Seq and genome-wide RBM39-RNA interactions were mapped via cross-linking and immunoprecipitation coupled with deep sequencing (CLIP-Seq) in wild-type and RBM39-knockdown MCF-7 cells. RBM39 was involved in the up- or down-regulation of the transcript levels of various genes. Hundreds of alternative splicing events regulated by endogenous RBM39 were identified. The majority of these events were cassette exons. Genes containing RBM39-regulated alternative exons were found to be linked to G2/M transition, cellular response to DNA damage, adherens junctions and endocytosis. CLIP-Seq analysis showed that the binding site of RBM39 was mainly in proximity to 5' and 3' splicing sites. Considerable RBM39 binding to mRNAs encoding proteins involved in translation was observed. Of particular importance, ~20% of the alternative splicing events that were significantly regulated by RBM39 were similarly regulated by U2AF65. RBM39 is extensively involved in alternative splicing of RNA and helps regulate transcript levels. RBM39 may modulate alternative splicing similarly to U2AF65 by either directly binding to RNA or recruiting other splicing factors, such as U2AF65. The current study offers a genome-wide view of RBM39's regulatory function in alternative splicing. RBM39 may play important roles in multiple cellular processes by regulating both alternative splicing of RNA molecules and transcript levels. Copyright © 2016 Elsevier B.V. All rights reserved.

Efficient computation of optimal oligo-RNA binding.

PubMed

Hodas, Nathan O; Aalberts, Daniel P

2004-01-01

We present an algorithm that calculates the optimal binding conformation and free energy of two RNA molecules, one or both oligomeric. This algorithm has applications to modeling DNA microarrays, RNA splice-site recognitions and other antisense problems. Although other recent algorithms perform the same calculation in time proportional to the sum of the lengths cubed, O((N1 + N2)3), our oligomer binding algorithm, called bindigo, scales as the product of the sequence lengths, O(N1*N2). The algorithm performs well in practice with the aid of a heuristic for large asymmetric loops. To demonstrate its speed and utility, we use bindigo to investigate the binding proclivities of U1 snRNA to mRNA donor splice sites.

scaRNAs regulate splicing and vertebrate heart development.

PubMed

Patil, Prakash; Kibiryeva, Nataliya; Uechi, Tamayo; Marshall, Jennifer; O'Brien, James E; Artman, Michael; Kenmochi, Naoya; Bittel, Douglas C

2015-08-01

Alternative splicing (AS) plays an important role in regulating mammalian heart development, but a link between misregulated splicing and congenital heart defects (CHDs) has not been shown. We reported that more than 50% of genes associated with heart development were alternatively spliced in the right ventricle (RV) of infants with tetralogy of Fallot (TOF). Moreover, there was a significant decrease in the level of 12 small cajal body-specific RNAs (scaRNAs) that direct the biochemical modification of specific nucleotides in spliceosomal RNAs. We sought to determine if scaRNA levels influence patterns of AS and heart development. We used primary cells derived from the RV of infants with TOF to show a direct link between scaRNA levels and splice isoforms of several genes that regulate heart development (e.g., GATA4, NOTCH2, DAAM1, DICER1, MBNL1 and MBNL2). In addition, we used antisense morpholinos to knock down the expression of two scaRNAs (scarna1 and snord94) in zebrafish and saw a corresponding disruption of heart development with an accompanying alteration in splice isoforms of cardiac regulatory genes. Based on these combined results, we hypothesize that scaRNA modification of spliceosomal RNAs assists in fine tuning the spliceosome for dynamic selection of mRNA splice isoforms. Our results are consistent with disruption of splicing patterns during early embryonic development leading to insufficient communication between the first and second heart fields, resulting in conotruncal misalignment and TOF. Our findings represent a new paradigm for determining the mechanisms underlying congenital cardiac malformations. Copyright © 2015. Published by Elsevier B.V.

The Characterization of GSDMB Splicing and Backsplicing Profiles Identifies Novel Isoforms and a Circular RNA That Are Dysregulated in Multiple Sclerosis.

PubMed

Cardamone, Giulia; Paraboschi, Elvezia Maria; Rimoldi, Valeria; Duga, Stefano; Soldà, Giulia; Asselta, Rosanna

2017-03-07

Abnormalities in alternative splicing (AS) are emerging as recurrent features in autoimmune diseases (AIDs). In particular, a growing body of evidence suggests the existence of a pathogenic association between a generalized defect in splicing regulatory genes and multiple sclerosis (MS). Moreover, several studies have documented an unbalance in alternatively-spliced isoforms in MS patients possibly contributing to the disease etiology. In this work, using a combination of PCR-based techniques (reverse-transcription (RT)-PCR, fluorescent-competitive, real-time, and digital RT-PCR assays), we investigated the alternatively-spliced gene encoding Gasdermin B, GSDMB , which was repeatedly associated with susceptibility to asthma and AIDs. The in-depth characterization of GSDMB AS and backsplicing profiles led us to the identification of an exonic circular RNA (ecircRNA) as well as of novel GSDMB in-frame and out-of-frame isoforms. The non-productive splicing variants were shown to be downregulated by the nonsense-mediated mRNA decay (NMD) in human cell lines, suggesting that GSDMB levels are significantly modulated by NMD. Importantly, both AS isoforms and the identified ecircRNA were significantly dysregulated in peripheral blood mononuclear cells of relapsing-remitting MS patients compared to controls, further supporting the notion that aberrant RNA metabolism is a characteristic feature of the disease.

Targeted nanoparticle delivery overcomes off-target immunostimulatory effects of oligonucleotides and improves therapeutic efficacy in chronic lymphocytic leukemia

PubMed Central

Yu, Bo; Mao, Yicheng; Bai, Li-Yuan; Herman, Sarah E. M.; Wang, Xinmei; Ramanunni, Asha; Jin, Yan; Mo, Xiaokui; Cheney, Carolyn; Chan, Kenneth K.; Jarjoura, David; Marcucci, Guido; Lee, Robert J.; Byrd, John C.

2013-01-01

Several RNA-targeted therapeutics, including antisense oligonucleotides (ONs), small interfering RNAs, and miRNAs, constitute immunostimulatory CpG motifs as an integral part of their design. The limited success with free antisense ONs in hematologic malignancies in recent clinical trials has been attributed to the CpG motif–mediated, TLR-induced prosurvival effects and inefficient target modulation in desired cells. In an attempt to diminish their off-target prosurvival and proinflammatory effects and specific delivery, as a proof of principle, in the present study, we developed an Ab-targeted liposomal delivery strategy using a clinically relevant CD20 Ab (rituximab)–conjugated lipopolyplex nanoparticle (RIT-INP)– and Bcl-2–targeted antisense G3139 as archetypical antisense therapeutics. The adverse immunostimulatory responses were abrogated by selective B cell–targeted delivery and early endosomal compartmentalization of G3139-encapsulated RIT-INPs, resulting in reduced NF-κB activation, robust Bcl-2 down-regulation, and enhanced sensitivity to fludarabine-induced cytotoxicity. Furthermore, significant in vivo therapeutic efficacy was noted after RIT-INP–G3139 administration in a disseminated xenograft leukemia model. The results of the present study demonstrate that CD20-targeted delivery overcomes the immunostimulatory properties of CpG-containing ON therapeutics and improves efficient gene silencing and in vivo therapeutic efficacy for B-cell malignancies. The broader implications of similar approaches in overcoming immunostimulatory properties of RNA-directed therapeutics in hematologic malignancies are also discussed. PMID:23165478

Alternative-splicing-mediated gene expression

NASA Astrophysics Data System (ADS)

Wang, Qianliang; Zhou, Tianshou

2014-01-01

Alternative splicing (AS) is a fundamental process during gene expression and has been found to be ubiquitous in eukaryotes. However, how AS impacts gene expression levels both quantitatively and qualitatively remains to be fully explored. Here, we analyze two common models of gene expression, each incorporating a simple splice mechanism that a pre-mRNA is spliced into two mature mRNA isoforms in a probabilistic manner. In the constitutive expression case, we show that the steady-state molecular numbers of two mature mRNA isoforms follow mutually independent Poisson distributions. In the bursting expression case, we demonstrate that the tail decay of the steady-state distribution for both mature mRNA isoforms that in general are not mutually independent can be characterized by the product of mean burst size and splicing probability. In both cases, we find that AS can efficiently modulate both the variability (measured by variance) and the noise level of the total mature mRNA, and in particular, the latter is always lower than the noise level of the pre-mRNA, implying that AS always reduces the noise. These results altogether reveal that AS is a mechanism of efficiently controlling the gene expression noise.

Upregulation of endothelial receptor for oxidized LDL (LOX-1) by oxidized LDL and implications in apoptosis of human coronary artery endothelial cells: evidence from use of antisense LOX-1 mRNA and chemical inhibitors.

PubMed

Li, D; Mehta, J L

2000-04-01

A specific lectin-like endothelial receptor for oxidized low density lipoprotein (LOX-1), distinct from the scavenger receptor in monocytes/macrophages, has been identified and cloned. In this study, we examined the regulation of LOX-1 by oxidized low density lipoprotein (ox-LDL) and determined the role of LOX-1 in ox-LDL-induced apoptosis of cultured human coronary artery endothelial cells (HCAECs). Incubation of HCAECs with ox-LDL (40 microg/mL), but not native LDL, for 24 hours markedly increased LOX-1 expression (mRNA and protein). After 48 hours of preincubation of HCAECs with a specific antisense to LOX-1 mRNA (antisense LOX-1), ox-LDL-mediated upregulation of LOX-1 was suppressed (P<0.01). In contrast, treatment of HCAECs with sense LOX-1 had no effect. Ox-LDL also induced apoptosis (determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and DNA laddering) of HCAECs in a concentration- and time-dependent fashion. LOX-1 played an important role in ox-LDL-mediated apoptosis of HCAECs because antisense LOX-1 inhibited this effect of ox-LDL. Polyinosinic acid and carrageenan, 2 different chemical inhibitors of LOX-1, also decreased ox-LDL-mediated apoptosis of HCAECs. Nuclear factor (NF)-kappaB was markedly activated in ox-LDL-treated HCAECs. The critical role of NF-kappaB activation became evident in experiments with antisense LOX-1, which abolished ox-LDL-mediated NF-kappaB activation. In this process, an NF-kappaB inhibitor, caffeic acid phenethyl ester, also inhibited ox-LDL-mediated apoptosis of HCAECs. These findings indicate that ox-LDL upregulates its own endothelial receptor. Ox-LDL-induced apoptosis is mediated by the action of LOX-1. In this process, NF-kappaB activation may play an important role as a signal transduction mechanism.

Introduction of a citrus blight-associated gene into Carrizo citrange [Citrus sinensis (L.) Osbc. x Poncirus trifoliata (L.) Raf.] by Agrobacterium-mediated transformation.

PubMed

Kayim, M; Ceccardi, T L; Berretta, M J G; Barthe, G A; Derrick, K S

2004-11-01

The protein p12 accumulates in leaves of trees with citrus blight (CB), a serious decline of unknown cause. The function of p12 is not known, but sequence analysis indicates it may be related to expansins. In studies to determine the function of p12, sense and antisense constructs were used to make transgenic Carrizo citrange using an Agrobacterium-mediated transformation system. Homogeneous beta-glucuronidase+ (GUS+) sense and antisense transgenic shoots were regenerated using kanamycin as a selective agent. Twenty-five sense and 45 antisense transgenic shoots were in vivo grafted onto Carrizo citrange for further analyses. In addition, 20 sense and 18 antisense shoots were rooted. The homogeneous GUS+ plants contained either the p12 sense or antisense gene (without the intron associated with the gene in untransformed citrus) as shown by PCR and Southern blotting. Northern blots showed the expected RNA in the sense and antisense plants. A protein of identical size and immunoreactivity was observed in seven of nine sense plants but not in nine antisense or non-transgenic plants. At the current stage of growth, there are no visual phenotypic differences between the transgenic and non-transgenic plants. Selected plants will be budded with sweet orange for field evaluation for resistance or susceptibility to CB and general rootstock performance.

Noncoding transcripts in sense and antisense orientation regulate the epigenetic state of ribosomal RNA genes.

PubMed

Bierhoff, H; Schmitz, K; Maass, F; Ye, J; Grummt, I

2010-01-01

Alternative transcription of the same gene in sense and antisense orientation regulates expression of protein-coding genes. Here we show that noncoding RNA (ncRNA) in sense and antisense orientation also controls transcription of rRNA genes (rDNA). rDNA exists in two types of chromatin--a euchromatic conformation that is permissive to transcription and a heterochromatic conformation that is transcriptionally silent. Silencing of rDNA is mediated by NoRC, a chromatin-remodeling complex that triggers heterochromatin formation. NoRC function requires RNA that is complementary to the rDNA promoter (pRNA). pRNA forms a DNA:RNA triplex with a regulatory element in the rDNA promoter, and this triplex structure is recognized by DNMT3b. The results imply that triplex-mediated targeting of DNMT3b to specific sequences may be a common pathway in epigenetic regulation. We also show that rDNA is transcribed in antisense orientation. The level of antisense RNA (asRNA) is down-regulated in cancer cells and up-regulated in senescent cells. Ectopic asRNA triggers trimethylation of histone H4 at lysine 20 (H4K20me3), suggesting that antisense transcripts guide the histone methyltransferase Suv4-20 to rDNA. The results reveal that noncoding RNAs in sense and antisense orientation are important determinants of the epigenetic state of rDNA.

SmD1 Modulates the miRNA Pathway Independently of Its Pre-mRNA Splicing Function.

PubMed

Xiong, Xiao-Peng; Vogler, Georg; Kurthkoti, Krishna; Samsonova, Anastasia; Zhou, Rui

2015-08-01

microRNAs (miRNAs) are a class of endogenous regulatory RNAs that play a key role in myriad biological processes. Upon transcription, primary miRNA transcripts are sequentially processed by Drosha and Dicer ribonucleases into ~22-24 nt miRNAs. Subsequently, miRNAs are incorporated into the RNA-induced silencing complexes (RISCs) that contain Argonaute (AGO) family proteins and guide RISC to target RNAs via complementary base pairing, leading to post-transcriptional gene silencing by a combination of translation inhibition and mRNA destabilization. Select pre-mRNA splicing factors have been implicated in small RNA-mediated gene silencing pathways in fission yeast, worms, flies and mammals, but the underlying molecular mechanisms are not well understood. Here, we show that SmD1, a core component of the Drosophila small nuclear ribonucleoprotein particle (snRNP) implicated in splicing, is required for miRNA biogenesis and function. SmD1 interacts with both the microprocessor component Pasha and pri-miRNAs, and is indispensable for optimal miRNA biogenesis. Depletion of SmD1 impairs the assembly and function of the miRISC without significantly affecting the expression of major canonical miRNA pathway components. Moreover, SmD1 physically and functionally associates with components of the miRISC, including AGO1 and GW182. Notably, miRNA defects resulting from SmD1 silencing can be uncoupled from defects in pre-mRNA splicing, and the miRNA and splicing machineries are physically and functionally distinct entities. Finally, photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis identifies numerous SmD1-binding events across the transcriptome and reveals direct SmD1-miRNA interactions. Our study suggests that SmD1 plays a direct role in miRNA-mediated gene silencing independently of its pre-mRNA splicing activity and indicates that the dual roles of splicing factors in post-transcriptional gene regulation may be evolutionarily widespread.

RNA therapeutics: Beyond RNA interference and antisense oligonucleotides

PubMed Central

Kole, Ryszard; Krainer, Adrian R.; Altman, Sidney

2016-01-01

Here we discuss three RNA therapeutic technologies exploiting various oligonucleotides that bind RNA by base-pairing in a sequence-specific manner yet have different mechanisms of action and effects. RNA interference and antisense oligonucleotides downregulate gene expression by enzyme-dependent degradation of targeted mRNA. Steric blocking oligonucleotides block access of cellular machinery to pre-mRNA and mRNA without degrading the RNA. Through this mechanism, blocking oligonucleotides can redirect alternative splicing, repair defective RNA, restore protein production or also downregulate gene expression. Moreover, they can be extensively chemically modified, resulting in more drug-like properties. The ability of RNA blocking oligonucleotides to restore gene function makes them suited for treatment of genetic disorders. Positive results from clinical trials for the treatment of Duchenne muscular dystrophy show that this technology is close to realizing its clinical potential. PMID:22262036

SRSF3 maintains transcriptome integrity in oocytes by regulation of alternative splicing and transposable elements.

PubMed

Do, Dang Vinh; Strauss, Bernhard; Cukuroglu, Engin; Macaulay, Iain; Wee, Keng Boon; Hu, Tim Xiaoming; Igor, Ruiz De Los Mozos; Lee, Caroline; Harrison, Andrew; Butler, Richard; Dietmann, Sabine; Jernej, Ule; Marioni, John; Smith, Christopher W J; Göke, Jonathan; Surani, M Azim

2018-01-01

The RNA-binding protein SRSF3 (also known as SRp20) has critical roles in the regulation of pre-mRNA splicing. Zygotic knockout of Srsf3 results in embryo arrest at the blastocyst stage. However, SRSF3 is also present in oocytes, suggesting that it might be critical as a maternally inherited factor. Here we identify SRSF3 as an essential regulator of alternative splicing and of transposable elements to maintain transcriptome integrity in mouse oocyte. Using 3D time-lapse confocal live imaging, we show that conditional deletion of Srsf3 in fully grown germinal vesicle oocytes substantially compromises the capacity of germinal vesicle breakdown (GVBD), and consequently entry into meiosis. By combining single cell RNA-seq, and oocyte micromanipulation with steric blocking antisense oligonucleotides and RNAse-H inducing gapmers, we found that the GVBD defect in mutant oocytes is due to both aberrant alternative splicing and derepression of B2 SINE transposable elements. Together, our study highlights how control of transcriptional identity of the maternal transcriptome by the RNA-binding protein SRSF3 is essential to the development of fertilized-competent oocytes.

[Deregulation of pre-messenger RNA splicing and rare diseases].

PubMed

de la Grange, Pierre

2016-12-01

Most of protein-coding human genes are subjected to alternative pre-mRNA splicing. This mechanism is highly regulated to precisely modulate detection of specific splice sites. This regulation is under control of the spliceosome and several splicing factors are also required to modulate the alternative usage of splice sites. Splicing factors and spliceosome components recognize splicing signals and regulatory sequences of the pre-mRNAs. These splicing sequences make splicing susceptible to polymorphisms and mutations. Examples of associations between human rare diseases and defects in pre-messenger RNA splicing are accumulating. Although many alterations are caused by mutations in splicing sequence (i.e., cis acting mutations), recent studies described the disruptive impact of mutations within spliceosome components or splicing factors (i.e., trans acting mutations). Following growing of knowledge regarding splicing regulation, several approaches have been developed to compensate for the effect of deleterious mutations and to restore sufficient amounts of functional protein. © 2016 médecine/sciences – Inserm.

Clinical, biochemical, and molecular studies in pyridoxine-dependent epilepsy. Antisense therapy as possible new therapeutic option.

PubMed

Pérez, Belén; Gutiérrez-Solana, Luis González; Verdú, Alfonso; Merinero, Begoña; Yuste-Checa, Patricia; Ruiz-Sala, Pedro; Calvo, Rocio; Jalan, Anil; Marín, Laura López; Campos, Oscar; Ruiz, Maria Ángeles; San Miguel, Marta; Vázquez, Maria; Castro, Margarita; Ferrer, Isaac; Navarrete, Rosa; Desviat, Lourdes Ruiz; Lapunzina, Pablo; Ugarte, Magdalena; Pérez-Cerdá, Celia

2013-02-01

Pyridoxine-dependent epilepsy seizure (PDE; OMIM 266100) is a disorder associated with severe seizures that can be controlled pharmacologically with pyridoxine. In the majority of patients with PDE, the disorder is caused by the deficient activity of the enzyme α-aminoadipic semialdehyde dehydrogenase (antiquitin protein), which is encoded by the ALDH7A1 gene. The aim of this work was the clinical, biochemical, and genetic analysis of 12 unrelated patients, mostly from Spain, in an attempt to provide further valuable data regarding the wide clinical, biochemical, and genetic spectrum of the disease. The disease was confirmed based on the presence of α-aminoadipic semialdehyde (α-AASA) in urine measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) and pipecolic acid (PA) in plasma and/or cerebrospinal fluid (CSF) measured by high performance liquid chromatography (HPLC)/MS/MS and by sequencing analysis of messenger RNA (mRNA) and genomic DNA of ALDH7A1. Most of the patients had seizures in the neonatal period, but they responded to vitamin B6 administration. Three patients developed late-onset seizures, and most patients showed mild-to-moderate postnatal developmental delay. All patients had elevated PA and α-AASA levels, even those who had undergone pyridoxine treatment for several years. The clinical spectrum of our patients is not limited to seizures but many of them show associated neurologic dysfunctions such as muscle tone alterations, irritability, and psychomotor retardation. The mutational spectrum of the present patients included 12 mutations, five already reported (c.500A>G, c.919C>T, c.1429G>C c.1217_1218delAT, and c.1482-1G>T) and seven novel sequence changes (c.75C>T, c.319G>T, c.554_555delAA, c.757C>T, c.787 + 1G>T, c.1474T>C, c.1093-?_1620+?). Only one mutation, p.G477R (c.1429G>C), was recurrent; this was detected in four different alleles. Transcriptional profile analysis of one patient's lymphoblasts and ex vivo splicing analysis showed the silent nucleotide change c.75C>T to be a novel splicing mutation creating a new donor splice site inside exon 1. Antisense therapy of the aberrant mRNA splicing in a lymphoblast cell line harboring mutation c.75C>T was successful. The present results broaden our knowledge of PDE, provide information regarding the genetic background of PDE in Spain, afford data of use when making molecular-based prenatal diagnosis, and provide a cellular proof-of concept for antisense therapy application. Wiley Periodicals, Inc. © 2013 International League Against Epilepsy.

Rescue of cardiomyopathy through U7snRNA-mediated exon skipping in Mybpc3-targeted knock-in mice.

PubMed

Gedicke-Hornung, Christina; Behrens-Gawlik, Verena; Reischmann, Silke; Geertz, Birgit; Stimpel, Doreen; Weinberger, Florian; Schlossarek, Saskia; Précigout, Guillaume; Braren, Ingke; Eschenhagen, Thomas; Mearini, Giulia; Lorain, Stéphanie; Voit, Thomas; Dreyfus, Patrick A; Garcia, Luis; Carrier, Lucie

2013-07-01

Exon skipping mediated by antisense oligoribonucleotides (AON) is a promising therapeutic approach for genetic disorders, but has not yet been evaluated for cardiac diseases. We investigated the feasibility and efficacy of viral-mediated AON transfer in a Mybpc3-targeted knock-in (KI) mouse model of hypertrophic cardiomyopathy (HCM). KI mice carry a homozygous G>A transition in exon 6, which results in three different aberrant mRNAs. We identified an alternative variant (Var-4) deleted of exons 5-6 in wild-type and KI mice. To enhance its expression and suppress aberrant mRNAs we designed AON-5 and AON-6 that mask splicing enhancer motifs in exons 5 and 6. AONs were inserted into modified U7 small nuclear RNA and packaged in adeno-associated virus (AAV-U7-AON-5+6). Transduction of cardiac myocytes or systemic administration of AAV-U7-AON-5+6 increased Var-4 mRNA/protein levels and reduced aberrant mRNAs. Injection of newborn KI mice abolished cardiac dysfunction and prevented left ventricular hypertrophy. Although the therapeutic effect was transient and therefore requires optimization to be maintained over an extended period, this proof-of-concept study paves the way towards a causal therapy of HCM. © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

Temperature characteristics of fusion splicing Hollow Core Photonic Crystal Fiber by sinusoidal modulation CO2 laser

NASA Astrophysics Data System (ADS)

Fu, Guangwei; Li, Kuixing; Fu, Xinghu; Bi, Weihong

2013-07-01

During the fusion splicing Hollow Core Photonic Crystal Fiber (HC-PCF), the air-holes collapse easily due to the improper fusion duration time and optical power. To analyze the temperature characteristics of fusion splicing HC-PCF, a heating method by sinusoidal modulation CO2 laser has been proposed. In the sinusoidal modulation, the variation relationships among laser power, temperature difference and angular frequency are analyzed. The results show that the theoretical simulation is basically in accordance with the experimental data. Therefore, a low-loss fusion splicing can be achieved by modulating the CO2 laser frequency to avoid the air-holes collapse of HC-PCF. Further, the errors are also given.

Natural antisense RNAs as mRNA regulatory elements in bacteria: a review on function and applications.

PubMed

Saberi, Fatemeh; Kamali, Mehdi; Najafi, Ali; Yazdanparast, Alavieh; Moghaddam, Mehrdad Moosazadeh

2016-01-01

Naturally occurring antisense RNAs are small, diffusible, untranslated transcripts that pair to target RNAs at specific regions of complementarity to control their biological function by regulating gene expression at the post-transcriptional level. This review focuses on known cases of antisense RNA control in prokaryotes and provides an overview of some natural RNA-based mechanisms that bacteria use to modulate gene expression, such as mRNA sensors, riboswitches and antisense RNAs. We also highlight recent advances in RNA-based technology. The review shows that studies on both natural and synthetic systems are reciprocally beneficial.

Estrogen anti-inflammatory activity on human monocytes is mediated through cross-talk between estrogen receptor ERα36 and GPR30/GPER1.

PubMed

Pelekanou, Vasiliki; Kampa, Marilena; Kiagiadaki, Foteini; Deli, Alexandra; Theodoropoulos, Panayiotis; Agrogiannis, George; Patsouris, Efstratios; Tsapis, Andreas; Castanas, Elias; Notas, George

2016-02-01

Estrogens are known modulators of monocyte/macrophage functions; however, the underlying mechanism has not been clearly defined. Recently, a number of estrogen receptor molecules and splice variants were identified that exert different and sometimes opposing actions. We assessed the expression of estrogen receptors and explored their role in mediating estrogenic anti-inflammatory effects on human primary monocytes. We report that the only estrogen receptors expressed are estrogen receptor-α 36-kDa splice variant and G-protein coupled receptor 30/G-protein estrogen receptor 1, in a sex-independent manner. 17-β-Estradiol inhibits the LPS-induced IL-6 inflammatory response, resulting in inhibition of NF-κB transcriptional activity. This is achieved via a direct physical interaction of ligand-activated estrogen receptor-α 36-kDa splice variant with the p65 component of NF-κB in the nucleus. G-protein coupled receptor 30/G-protein estrogen receptor 1, which also physically interacts with estrogen receptor-α 36-kDa splice variant, acts a coregulator in this process, because its inhibition blocks the effect of estrogens on IL-6 expression. However, its activation does not mimic the effect of estrogens, on neither IL-6 nor NF-κB activity. Finally, we show that the estrogen receptor profile observed in monocytes is not modified during their differentiation to macrophages or dendritic cells in vitro and is shared in vivo by macrophages present in atherosclerotic plaques. These results position estrogen receptor-α 36-kDa splice variant and G-protein coupled receptor 30 as important players and potential therapeutic targets in monocyte/macrophage-dependent inflammatory processes. © Society for Leukocyte Biology.

DLEU2 encodes an antisense RNA for the putative bicistronic RFP2/LEU5 gene in humans and mouse.

PubMed

Corcoran, Martin M; Hammarsund, Marianne; Zhu, Chaoyong; Lerner, Mikael; Kapanadze, Bagrat; Wilson, Bill; Larsson, Catharina; Forsberg, Lars; Ibbotson, Rachel E; Einhorn, Stefan; Oscier, David G; Grandér, Dan; Sangfelt, Olle

2004-08-01

Our group previously identified two novel genes, RFP2/LEU5 and DLEU2, within a 13q14.3 genomic region of loss seen in various malignancies. However, no specific inactivating mutations were found in these or other genes in the vicinity of the deletion, suggesting that a nonclassical tumor-suppressor mechanism may be involved. Here, we present data showing that the DLEU2 gene encodes a putative noncoding antisense RNA, with one exon directly overlapping the first exon of the RFP2/LEU5 gene in the opposite orientation. In addition, the RFP2/LEU5 transcript can be alternatively spliced to produce either several monocistronic transcripts or a putative bicistronic transcript encoding two separate open-reading frames, adding to the complexity of the locus. The finding that these gene structures are conserved in the mouse, including the putative bicistronic RFP2/LEU5 transcript as well as the antisense relationship with DLEU2, further underlines the significance of this unusual organization and suggests a biological function for DLEU2 in the regulation of RFP2/LEU5. Copyright 2004 Wiley-Liss, Inc.

Intragenic DNA methylation and BORIS-mediated cancer-specific splicing contribute to the Warburg effect

PubMed Central

Singh, Smriti; Narayanan, Sathiya Pandi; Biswas, Kajal; Gupta, Amit; Ahuja, Neha; Yadav, Sandhya; Panday, Rajendra Kumar; Samaiya, Atul; Sharan, Shyam K.

2017-01-01

Aberrant alternative splicing and epigenetic changes are both associated with various cancers, but epigenetic regulation of alternative splicing in cancer is largely unknown. Here we report that the intragenic DNA methylation-mediated binding of Brother of Regulator of Imprinted Sites (BORIS) at the alternative exon of Pyruvate Kinase (PKM) is associated with cancer-specific splicing that promotes the Warburg effect and breast cancer progression. Interestingly, the inhibition of DNA methylation, BORIS depletion, or CRISPR/Cas9-mediated deletion of the BORIS binding site leads to a splicing switch from cancer-specific PKM2 to normal PKM1 isoform. This results in the reversal of the Warburg effect and the inhibition of breast cancer cell growth, which may serve as a useful approach to inhibit the growth of breast cancer cells. Importantly, our results show that in addition to PKM splicing, BORIS also regulates the alternative splicing of several genes in a DNA methylation-dependent manner. Our findings highlight the role of intragenic DNA methylation and DNA binding protein BORIS in cancer-specific splicing and its role in tumorigenesis. PMID:29073069

[Antisense polynucleotides and prospects for their use in fighting viruses].

PubMed

Tikhonenko, T I

1989-01-01

Natural or synthetic anti-sense (as) polynucleotides complementary to distinct functional regions of mRNA (asRNA or asDNA) are able to inhibit the expression of any target gene. If certain viral mRNAs important for virus replication are targeted the inhibition of viral infection by asRNA or asDNA takes place. Inhibitory effects of complementary polynucleotides on gene activity in eukaryotic cells is due to the disturbance of translation of corresponding mRNAs as well as to the impairment of their splicing or transportation from the nuclei to cytoplasm. In prokaryotic cells, obviously, only the first factor is operating. The recombinant genes programming anti-viral asRNA can confer the resistance to the infection by other virus to the transformed cells. The resistance to viral infection observed in transgenic animals, expressing asRNA genes, may be considered as a new unnatural form of informational immunity.

Modulation of lipoprotein metabolism by antisense technology: preclinical drug discovery methodology.

PubMed

Crooke, Rosanne M; Graham, Mark J

2013-01-01

Antisense oligonucleotides (ASOs) are a new class of specific therapeutic agents that alter the intermediary metabolism of mRNA, resulting in the suppression of disease-associated gene products. ASOs exert their pharmacological effects after hybridizing, via Watson-Crick base pairing, to a specific target RNA. If appropriately designed, this event results in the recruitment of RNase H, the degradation of targeted mRNA or pre-mRNA, and subsequent inhibition of the synthesis of a specific protein. A key advantage of the technology is the ability to selectively inhibit targets that cannot be modulated by traditional therapeutics such as structural proteins, transcription factors, and, of topical interest, lipoproteins. In this chapter, we will first provide an overview of antisense technology, then more specifically describe the status of lipoprotein-related genes that have been studied using the antisense platform, and finally, outline the general methodology required to design and evaluate the in vitro and in vivo efficacy of those drugs.

An extracatalytic function of CD45 in B cells is mediated by CD22

PubMed Central

Coughlin, Sarah; Noviski, Mark; Mueller, James L.; Chuwonpad, Ammarina; Raschke, William C.; Weiss, Arthur; Zikherman, Julie

2015-01-01

The receptor-like tyrosine phosphatase CD45 regulates antigen receptor signaling by dephosphorylating the C-terminal inhibitory tyrosine of the src family kinases. However, despite its abundance, the function of the large, alternatively spliced extracellular domain of CD45 has remained elusive. We used normally spliced CD45 transgenes either incorporating a phosphatase-inactivating point mutation or lacking the cytoplasmic domain to uncouple the enzymatic and noncatalytic functions of CD45 in lymphocytes. Although these transgenes did not alter T-cell signaling or development irrespective of endogenous CD45 expression, both partially rescued the phenotype of CD45-deficient B cells. We identify a noncatalytic role for CD45 in regulating tonic, but not antigen-mediated, B-cell antigen receptor (BCR) signaling through modulation of the function of the inhibitory coreceptor CD22. This finding has important implications for understanding how naïve B cells maintain tonic BCR signaling while restraining inappropriate antigen-dependent activation to preserve clonal “ignorance.” PMID:26561584

The C-module-binding factor supports amplification of TRE5-A retrotransposons in the Dictyostelium discoideum genome.

PubMed

Bilzer, Annika; Dölz, Heike; Reinhardt, Alexander; Schmith, Anika; Siol, Oliver; Winckler, Thomas

2011-01-01

Retrotransposable elements are molecular parasites that have invaded the genomes of virtually all organisms. Although retrotransposons encode essential proteins to mediate their amplification, they also require assistance by host cell-encoded machineries that perform functions such as DNA transcription and repair. The retrotransposon TRE5-A of the social amoeba Dictyostelium discoideum generates a notable amount of both sense and antisense RNAs, which are generated from element-internal promoters, located in the A module and the C module, respectively. We observed that TRE5-A retrotransposons depend on the C-module-binding factor (CbfA) to maintain high steady-state levels of TRE5-A transcripts and that CbfA supports the retrotransposition activity of TRE5-A elements. The carboxy-terminal domain of CbfA was found to be required and sufficient to mediate the accumulation of TRE5-A transcripts, but it did not support productive retrotransposition of TRE5-A. This result suggests different roles for CbfA protein domains in the regulation of TRE5-A retrotransposition frequency in D. discoideum cells. Although CbfA binds to the C module in vitro, the factor regulates neither C-module nor A-module promoter activity in vivo. We speculate that CbfA supports the amplification of TRE5-A retrotransposons by suppressing the expression of an as yet unidentified component of the cellular posttranscriptional gene silencing machinery.

miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA

PubMed Central

Hansen, Thomas B; Wiklund, Erik D; Bramsen, Jesper B; Villadsen, Sune B; Statham, Aaron L; Clark, Susan J; Kjems, Jørgen

2011-01-01

MicroRNAs (miRNAs) are ∼22 nt non-coding RNAs that typically bind to the 3′ UTR of target mRNAs in the cytoplasm, resulting in mRNA destabilization and translational repression. Here, we report that miRNAs can also regulate gene expression by targeting non-coding antisense transcripts in human cells. Specifically, we show that miR-671 directs cleavage of a circular antisense transcript of the Cerebellar Degeneration-Related protein 1 (CDR1) locus in an Ago2-slicer-dependent manner. The resulting downregulation of circular antisense has a concomitant decrease in CDR1 mRNA levels, independently of heterochromatin formation. This study provides the first evidence for non-coding antisense transcripts as functional miRNA targets, and a novel regulatory mechanism involving a positive correlation between mRNA and antisense circular RNA levels. PMID:21964070

Characterization of an apparently synonymous F5 mutation causing aberrant splicing and factor V deficiency.

PubMed

Nuzzo, F; Bulato, C; Nielsen, B I; Lee, K; Wielders, S J; Simioni, P; Key, N S; Castoldi, E

2015-03-01

Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. We investigated a patient with severe FV deficiency (FV:C < 3%) and moderate bleeding symptoms. Thrombin generation experiments showed residual FV expression in the patient's plasma, which was quantified as 0.7 ± 0.3% by a sensitive prothrombinase-based assay. F5 gene sequencing identified a novel missense mutation in exon 4 (c.578G>C, p.Cys193Ser), predicting the abolition of a conserved disulphide bridge, and an apparently synonymous variant in exon 8 (c.1281C>G). The observation that half of the patient's F5 mRNA lacked the last 18 nucleotides of exon 8 prompted us to re-evaluate the c.1281C>G variant for its possible effects on splicing. Bioinformatics sequence analysis predicted that this transversion would activate a cryptic donor splice site and abolish an exonic splicing enhancer. Characterization in a F5 minigene model confirmed that the c.1281C>G variant was responsible for the patient's splicing defect, which could be partially corrected by a mutation-specific morpholino antisense oligonucleotide. The aberrantly spliced F5 mRNA, whose stability was similar to that of the normal mRNA, encoded a putative FV mutant lacking amino acids 427-432. Expression in COS-1 cells indicated that the mutant protein is poorly secreted and not functional. In conclusion, the c.1281C>G mutation, which was predicted to be translationally silent and hence neutral, causes FV deficiency by impairing pre-mRNA splicing. This finding underscores the importance of cDNA analysis for the correct assessment of exonic mutations. © 2014 John Wiley & Sons Ltd.

G-quadruplex structure at intron 2 of TFE3 and its role in Xp11.2 translocation and splicing.

PubMed

Verma, Shiv Prakash; Das, Parimal

2018-03-01

Transcription Factor E3 (TFE3) translocation is found in a group of different type of cancers and most of the translocations are located in the 5' region of TFE3 which may be considered as Breakpoint Region (BR). In our In silico study by QGRS mapper and non BdB web servers we found a Potential G-quadruplex forming Sequence (PQS) in the intron 2 of TFE3 gene. In vitro G-quadruplex formation was shown by native PAGE in presence of Pyridostatin(PDS), which with inter molecular secondary structure caused reduced mobility to migrate slower. G-quadruplex formation was mapped at single base resolution by Sanger sequencing and Circular Dichroism showed the formation of parallel G-quadruplex. FRET analysis revealed increased and decreased formation of G-quadruplex in presence of PDS and antisense oligonucleotide respectively. PCR stop assay, transcriptional and translational inhibition by PQS showed stable G-quadruplex formation affecting the biological processes. TFE3 minigene splicing study showed the involvement of this G-quadruplex in TFE3 splicing too. Therefore, G-quadruplex is evident to be the reason behind TFE3 induced oncogenesis executed by translocation and also involved in the mRNA splicing. Copyright © 2017 Elsevier B.V. All rights reserved.

Genetic therapies to lower cholesterol.

PubMed

Khoo, Bernard

2015-01-01

This review surveys the state-of-the-art in genetic therapies for familial hypercholesterolaemia (FH), caused most commonly by mutations in the LDL receptor (LDLR) gene. FH manifests as highly elevated low density lipoprotein (LDL) cholesterol levels and consequently accelerated atherosclerosis. Modern pharmacological therapies for FH are insufficiently efficacious to prevent premature cardiovascular disease, can cause significant adverse effects and can be expensive. Genetic therapies for FH have been mooted since the mid 1990s but gene replacement strategies using viral vectors have so far been unsuccessful. Other strategies involve knocking down the expression of Apolipoprotein B100 (APOB100) and the protease PCSK9 which designates LDLR for degradation. The antisense oligonucleotide mipomersen, which knocks down APOB100, is currently marketed (with restrictions) in the USA, but is not approved in Europe due to its adverse effects. To address this problem, we have devised a novel therapeutic concept, APO-skip, which is based on modulation of APOB splicing, and which has the potential to deliver a cost-effective, efficacious and safe therapy for FH. Copyright © 2014 Elsevier Inc. All rights reserved.

Cyclin D1b splice variant promotes αvβ3-mediated adhesion and invasive migration of breast cancer cells.

PubMed

Wu, Feng-Hua; Luo, Li-Qiong; Liu, Yi; Zhan, Qiu-Xiao; Luo, Chao; Luo, Jing; Zhang, Gui-Mei; Feng, Zuo-Hua

2014-12-01

Cyclin D1b, a splice variant of the cell cycle regulator cyclin D1, holds oncogenic functions in human cancer. However, the mechanisms underlying cyclin D1b function remain poorly understood. Here we introduced wild-type cyclin D1a or cyclin D1b variant into non-metastatic MCF-7 cells. Our results show that ectopic expression of cyclin D1b promotes invasiveness of the cancer cells in a cyclin D1a independent manner. Specifically, cyclin D1b is found to modulate the expression of αvβ3, which characterizes the metastatic phenotype, and enhance tumor cell invasive potential in cooperating with HoxD3. Notably, cyclin D1b promotes αvβ3-mediated adhesion and invasive migration, which are associated with invasive potential of breast cancer cells. Further exploration indicates that cyclin D1b makes breast cancer cells more sensitive to toll-like receptor 4 ligand released from damaged tumor cells. These findings reveal a role of cyclin D1b as a possible mediator of αvβ3 transcription to promote tumor metastasis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Generation and Analysis of the Expressed Sequence Tags from the Mycelium of Ganoderma lucidum

PubMed Central

Huang, Yen-Hua; Wu, Hung-Yi; Wu, Keh-Ming; Liu, Tze-Tze; Liou, Ruey-Fen; Tsai, Shih-Feng; Shiao, Ming-Shi; Ho, Low-Tone; Tzean, Shean-Shong; Yang, Ueng-Cheng

2013-01-01

Ganoderma lucidum (G. lucidum) is a medicinal mushroom renowned in East Asia for its potential biological effects. To enable a systematic exploration of the genes associated with the various phenotypes of the fungus, the genome consortium of G. lucidum has carried out an expressed sequence tag (EST) sequencing project. Using a Sanger sequencing based approach, 47,285 ESTs were obtained from in vitro cultures of G. lucidum mycelium of various durations. These ESTs were further clustered and merged into 7,774 non-redundant expressed loci. The features of these expressed contigs were explored in terms of over-representation, alternative splicing, and natural antisense transcripts. Our results provide an invaluable information resource for exploring the G. lucidum transcriptome and its regulation. Many cases of the genes over-represented in fast-growing dikaryotic mycelium are closely related to growth, such as cell wall and bioactive compound synthesis. In addition, the EST-genome alignments containing putative cassette exons and retained introns were manually curated and then used to make inferences about the predominating splice-site recognition mechanism of G. lucidum. Moreover, a number of putative antisense transcripts have been pinpointed, from which we noticed that two cases are likely to reveal hitherto undiscovered biological pathways. To allow users to access the data and the initial analysis of the results of this project, a dedicated web site has been created at http://csb2.ym.edu.tw/est/. PMID:23658685

Antisense Oligonucleotide-Based Splicing Correction in Individuals with Leber Congenital Amaurosis due to Compound Heterozygosity for the c.2991+1655A>G Mutation in CEP290.

PubMed

Duijkers, Lonneke; van den Born, L Ingeborgh; Neidhardt, John; Bax, Nathalie M; Pierrache, Laurence H M; Klevering, B Jeroen; Collin, Rob W J; Garanto, Alejandro

2018-03-07

Leber congenital amaurosis (LCA) is a rare inherited retinal disorder affecting approximately 1:50,000 people worldwide. So far, mutations in 25 genes have been associated with LCA, with CEP290 (encoding the Centrosomal protein of 290 kDa) being the most frequently mutated gene. The most recurrent LCA-causing CEP290 mutation, c.2991+1655A>G, causes the insertion of a pseudoexon into a variable proportion of CEP290 transcripts. We previously demonstrated that antisense oligonucleotides (AONs) have a high therapeutic potential for patients homozygously harbouring this mutation, although to date, it is unclear whether rescuing one single allele is enough to restore CEP290 function. Here, we assessed the AON efficacy at RNA, protein and cellular levels in samples that are compound heterozygous for this mutation, together with a protein-truncating mutation in CEP290 . We demonstrate that AONs can efficiently restore splicing and increase protein levels. However, due to a high variability in ciliation among the patient-derived cell lines, the efficacy of the AONs was more difficult to assess at the cellular level. This observation points towards the importance of the severity of the second allele and possibly other genetic variants present in each individual. Overall, AONs seem to be a promising tool to treat CEP290 -associated LCA, not only in homozygous but also in compound heterozygous carriers of the c.2991+1655A>G variant.

Spliceosome Protein (SRp) Regulation of Glucocorticoid Receptor Isoforms and Glucocorticoid Response in Human Trabecular Meshwork Cells

PubMed Central

Jain, Ankur; Wordinger, Robert J.; Yorio, Thomas; Clark, Abbot F.

2012-01-01

Purpose. Glaucoma is a leading cause of visual impairment and blindness, with elevated intraocular pressure (IOP) as a major causative risk factor. Glucocorticoid (GC) therapy causes morphologic and biochemical changes in the trabecular meshwork (TM), an ocular tissue involved in regulating IOP, which can lead to the development of glaucoma in susceptible individuals (steroid responders). Steroid responders comprise 40% of the general population and are at higher risk of developing glaucoma. In addition, a majority of glaucoma patients are steroid responders. Differential distribution of various isoforms of GC receptor (GR) may be responsible for this heterogeneity in the steroid response. The alternatively spliced GRβ isoform acts as dominant negative regulator of classical GRα transcriptional activity. mRNA splicing is mediated by spliceosomes, which include serine-arginine rich proteins (SRps). The purpose of this study was to determine whether specific SRps regulate levels of these isoforms and thereby GC response in TM cells. Methods. Quantitative RT-PCR, Western blot analysis, and immunocytochemistry were used to determine the differential expression of different SRps (SRp20, 30c, and 40) in human normal and glaucomatous TM cell strains. Bioinformatics was used to find putative binding sites for SRp20 and SRp40 on exon 9 of the GR gene. A peptide modulator of splicing (bombesin) and SRp expression vectors were used to modulate SRp levels and determine their effects on GRα/GRβ ratios as well as dexamethasone (DEX) responsiveness via GRE- luciferase reporter activity, fibronectin, and myocilin induction in TM cells. Results. SRp20, SRp30c, and SRp40 regulate GR splicing and the GC response in TM cells. Modulation of SRp levels altered the GRβ/α ratio that correlated with DEX responsiveness. Bombesin decreased SRp20; increased SRp30c, SRp40 levels, and GRβ/α ratio, and suppressed DEX response in TM cells. Conclusions. Relative levels of SRp20, SRp30c, and SRp40 in TM cells control differential expression of the two alternatively spliced isoforms of the GR and thereby regulate GC responsiveness. Different levels and/or activities of these SRps may account for differential GC sensitivity among the normal and glaucoma populations. PMID:22205602

CELF1 preferentially binds to exon-intron boundary and regulates alternative splicing in HeLa cells.

PubMed

Xia, Heng; Chen, Dong; Wu, Qijia; Wu, Gang; Zhou, Yanhong; Zhang, Yi; Zhang, Libin

2017-09-01

The current RIP-seq approach has been developed for the identification of genome-wide interaction between RNA binding protein (RBP) and the bound RNA transcripts, but still rarely for identifying its binding sites. In this study, we performed RIP-seq experiments in HeLa cells using a monoclonal antibody against CELF1. Mapping of the RIP-seq reads showed a biased distribution at the 3'UTR and intronic regions. A total of 15,285 and 1384 CELF1-specific sense and antisense peaks were identified using the ABLIRC software tool. Our bioinformatics analyses revealed that 5' and 3' splice site motifs and GU-rich motifs were highly enriched in the CELF1-bound peaks. Furthermore, transcriptome analyses revealed that alternative splicing was globally regulated by CELF1 in HeLa cells. For example, the inclusion of exon 16 of LMO7 gene, a marker gene of breast cancer, is positively regulated by CELF1. Taken together, we have shown that RIP-seq data can be used to decipher RBP binding sites and reveal an unexpected landscape of the genome-wide CELF1-RNA interactions in HeLa cells. In addition, we found that CELF1 globally regulates the alternative splicing by binding the exon-intron boundary in HeLa cells, which will deepen our understanding of the regulatory roles of CELF1 in the pre-mRNA splicing process. Copyright © 2017 Elsevier B.V. All rights reserved.

Ultraconserved elements are associated with homeostatic control of splicing regulators by alternative splicing and nonsense-mediated decay

PubMed Central

Ni, Julie Z.; Grate, Leslie; Donohue, John Paul; Preston, Christine; Nobida, Naomi; O’Brien, Georgeann; Shiue, Lily; Clark, Tyson A.; Blume, John E.; Ares, Manuel

2007-01-01

Many alternative splicing events create RNAs with premature stop codons, suggesting that alternative splicing coupled with nonsense-mediated decay (AS-NMD) may regulate gene expression post-transcriptionally. We tested this idea in mice by blocking NMD and measuring changes in isoform representation using splicing-sensitive microarrays. We found a striking class of highly conserved stop codon-containing exons whose inclusion renders the transcript sensitive to NMD. A genomic search for additional examples identified >50 such exons in genes with a variety of functions. These exons are unusually frequent in genes that encode splicing activators and are unexpectedly enriched in the so-called “ultraconserved” elements in the mammalian lineage. Further analysis show that NMD of mRNAs for splicing activators such as SR proteins is triggered by splicing activation events, whereas NMD of the mRNAs for negatively acting hnRNP proteins is triggered by splicing repression, a polarity consistent with widespread homeostatic control of splicing regulator gene expression. We suggest that the extreme genomic conservation surrounding these regulatory splicing events within splicing factor genes demonstrates the evolutionary importance of maintaining tightly tuned homeostasis of RNA-binding protein levels in the vertebrate cell. PMID:17369403

Splicing Factor 1 Modulates Dietary Restriction and TORC1 Pathway Longevity in C. elegans

PubMed Central

Heintz, Caroline; Escoubas, Caroline; Zhang, Yue; Weir, Heather J.; Dutta, Sneha; Silva-García, Carlos Giovanni; Bruun, Gitte Hoffmann; Morantte, Ianessa; Hoxhaj, Gerta; Manning, Brendan D.; Andresen, Brage S.; Mair, William B.

2016-01-01

Ageing is driven by a loss of transcriptional and protein homeostasis1–3 and is the key risk factor for multiple chronic diseases. Interventions that attenuate or reverse systemic dysfunction seen with age therefore have potential to reduce overall disease risk in the elderly. Pre-mRNA splicing is a fundamental link between gene expression and the proteome, and deregulation of the splicing machinery is linked to multiple age-related chronic diseases4,5. However, the role of splicing homeostasis in healthy ageing remains unclear. Here we demonstrate that pre-mRNA splicing homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans. Using transcriptomics and in-depth splicing analysis in young and old animals fed ad libitum or on dietary restriction (DR), we find defects in global pre-mRNA splicing with age that are reduced by DR via the branch point binding protein (BBP)/splicing factor 1 (SFA-1). We show that SFA-1 is specifically required for lifespan extension both by DR, and modulation of TORC1 pathway components AMPK, RAGA-1 and RSKS-1/S6 Kinase. Lastly, we demonstrate that overexpression of SFA-1 is sufficient to extend lifespan. Together, these data demonstrate a role for RNA splicing homeostasis in DR longevity and suggest modulation of specific spliceosome components can prolong healthy ageing. PMID:27919065

Two Distinct Repressive Mechanisms for Histone 3 Lysine 4 Methylation through Promoting 3′-End Antisense Transcription

PubMed Central

Margaritis, Thanasis; Oreal, Vincent; Brabers, Nathalie; Maestroni, Laetitia; Vitaliano-Prunier, Adeline; Benschop, Joris J.; van Hooff, Sander; van Leenen, Dik

2012-01-01

Histone H3 di- and trimethylation on lysine 4 are major chromatin marks that correlate with active transcription. The influence of these modifications on transcription itself is, however, poorly understood. We have investigated the roles of H3K4 methylation in Saccharomyces cerevisiae by determining genome-wide expression-profiles of mutants in the Set1 complex, COMPASS, that lays down these marks. Loss of H3K4 trimethylation has virtually no effect on steady-state or dynamically-changing mRNA levels. Combined loss of H3K4 tri- and dimethylation results in steady-state mRNA upregulation and delays in the repression kinetics of specific groups of genes. COMPASS-repressed genes have distinct H3K4 methylation patterns, with enrichment of H3K4me3 at the 3′-end, indicating that repression is coupled to 3′-end antisense transcription. Further analyses reveal that repression is mediated by H3K4me3-dependent 3′-end antisense transcription in two ways. For a small group of genes including PHO84, repression is mediated by a previously reported trans-effect that requires the antisense transcript itself. For the majority of COMPASS-repressed genes, however, it is the process of 3′-end antisense transcription itself that is the important factor for repression. Strand-specific qPCR analyses of various mutants indicate that this more prevalent mechanism of COMPASS-mediated repression requires H3K4me3-dependent 3′-end antisense transcription to lay down H3K4me2, which seems to serve as the actual repressive mark. Removal of the 3′-end antisense promoter also results in derepression of sense transcription and renders sense transcription insensitive to the additional loss of SET1. The derepression observed in COMPASS mutants is mimicked by reduction of global histone H3 and H4 levels, suggesting that the H3K4me2 repressive effect is linked to establishment of a repressive chromatin structure. These results indicate that in S. cerevisiae, the non-redundant role of H3K4 methylation by Set1 is repression, achieved through promotion of 3′-end antisense transcription to achieve specific rather than global effects through two distinct mechanisms. PMID:23028359

Permissive Sense and Antisense Transcription from the 5′ and 3′ Long Terminal Repeats of Human T-Cell Leukemia Virus Type 1

PubMed Central

Polakowski, Nicholas; Hoang, Kimson

2016-01-01

ABSTRACT Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus, and, as such, its genome becomes chromosomally integrated following infection. The resulting provirus contains identical 5′ and 3′ peripheral long terminal repeats (LTRs) containing bidirectional promoters. Antisense transcription from the 3′ LTR regulates expression of a single gene, hbz, while sense transcription from the 5′ LTR controls expression of all other viral genes, including tax. Both the HBZ and Tax proteins are implicated in the development of adult T-cell leukemia (ATL), a T-cell malignancy caused by HTLV-1 infection. However, these proteins appear to harbor opposing molecular functions, indicating that they may act independently and at different time points prior to leukemogenesis. Here, we used bidirectional reporter constructs to test whether transcriptional interference serves as a mechanism that inhibits simultaneous expression of Tax and HBZ. We found that sense transcription did not interfere with antisense transcription from the 3′ LTR and vice versa, even with strong transcription emanating from the opposing direction. Therefore, bidirectional transcription across the provirus might not restrict hbz or tax expression. Single-cell analyses revealed that antisense transcription predominates in the absence of Tax, which transactivates viral sense transcription. Interestingly, a population of Tax-expressing cells exhibited antisense but not activated sense transcription. Consistent with the ability of Tax to induce cell cycle arrest, this population was arrested in G0/G1 phase. These results imply that cell cycle arrest inhibits Tax-mediated activation of sense transcription without affecting antisense transcription, which may be important for long-term viral latency. IMPORTANCE The chromosomally integrated form of the retrovirus human T-cell leukemia virus type 1 (HTLV-1) contains identical DNA sequences, known as long terminal repeats (LTRs), at its 5′ and 3′ ends. The LTRs modulate transcription in both forward (sense) and reverse (antisense) directions. We found that sense transcription from the 5′ LTR does not interfere with antisense transcription from the 3′ LTR, allowing viral genes encoded on opposite DNA strands to be simultaneously transcribed. Two such genes are tax and hbz, and while they are thought to function at different times during the course of infection to promote leukemogenesis of infected T cells, our results indicate that they can be simultaneously transcribed. We also found that the ability of Tax to induce cell cycle arrest inhibits its fundamental function of activating viral sense transcription but does not affect antisense transcription. This regulatory mechanism may be important for long-term HTLV-1 infection. PMID:26792732

Construction of a directed hammerhead ribozyme library: towards the identification of optimal target sites for antisense-mediated gene inhibition.

PubMed Central

Pierce, M L; Ruffner, D E

1998-01-01

Antisense-mediated gene inhibition uses short complementary DNA or RNA oligonucleotides to block expression of any mRNA of interest. A key parameter in the success or failure of an antisense therapy is the identification of a suitable target site on the chosen mRNA. Ultimately, the accessibility of the target to the antisense agent determines target suitability. Since accessibility is a function of many complex factors, it is currently beyond our ability to predict. Consequently, identification of the most effective target(s) requires examination of every site. Towards this goal, we describe a method to construct directed ribozyme libraries against any chosen mRNA. The library contains nearly equal amounts of ribozymes targeting every site on the chosen transcript and the library only contains ribozymes capable of binding to that transcript. Expression of the ribozyme library in cultured cells should allow identification of optimal target sites under natural conditions, subject to the complexities of a fully functional cell. Optimal target sites identified in this manner should be the most effective sites for therapeutic intervention. PMID:9801305

A long antisense RNA in plant chloroplasts.

PubMed

Georg, J; Honsel, A; Voss, B; Rennenberg, H; Hess, W R

2010-05-01

Based on computational prediction of RNA secondary structures, a long antisense RNA (asRNA) was found in chloroplasts of Arabidopsis, Nicotiana tabacum and poplar, which occurs in two to three major transcripts. Mapping of primary 5' ends, northern hybridizations and quantitative real-time reverse transcription polymerase chain reaction (qPCR) experiments demonstrated that these transcripts originate from a promoter that is typical for the plastid-encoded RNA polymerase and are over their full length in antisense orientation to the gene ndhB and therefore were designated asRNA_ndhB. The asRNA_ndhB transcripts predominantly accumulate in young leaves and at physiological growth temperatures. Two nucleotide positions in the mRNA that are subject to C-to-U RNA editing and which were previously found to be sensitive to elevated temperatures are covered by asRNA_ndhB. Nevertheless, the correlation between the accumulation of asRNA_ndhB and RNA editing appeared weak in a temperature shift experiment. With asRNA_ndhB, we describe the first asRNA of plant chloroplasts that covers RNA editing sites, as well as a group II intron splice acceptor site, and that is under developmental control, raising the possibility that long asRNAs could be involved in RNA maturation or the control of RNA stability.

MicroRNA-30b-Mediated Regulation of Catalase Expression in Human ARPE-19 Cells

PubMed Central

Haque, Rashidul; Chun, Eugene; Howell, Jennifer C.; Sengupta, Trisha; Chen, Dan; Kim, Hana

2012-01-01

Background Oxidative injury to retinal pigment epithelium (RPE) and retinal photoreceptors has been linked to a number of retinal diseases, including age-related macular degeneration (AMD). Reactive oxygen species (ROS)-mediated gene expression has been extensively studied at transcriptional levels. Also, the post-transcriptional control of gene expression at the level of translational regulation has been recently reported. However, the microRNA (miRNA/miR)-mediated post-transcriptional regulation in human RPE cells has not been thoroughly looked at. Increasing evidence points to a potential role of miRNAs in diverse physiological processes. Methodology/Principal Findings We demonstrated for the first time in a human retinal pigment epithelial cell line (ARPE-19) that the post-transcriptional control of gene expression via miRNA modulation regulates human catalase, an important and potent component of cell's antioxidant defensive network, which detoxifies hydrogen peroxide (H2O2) radicals. Exposure to several stress-inducing agents including H2O2 has been reported to alter miRNA expression profile. Here, we demonstrated that a sublethal dose of H2O2 (200 µM) up-regulated the expression of miR-30b, a member of the miR-30 family, which inhibited the expression of endogenous catalase both at the transcript and protein levels. However, antisense (antagomirs) of miR-30b was not only found to suppress the miR-30b mimics-mediated inhibitions, but also to dramatically increase the expression of catalase even under an oxidant environment. Conclusions/Significance We propose that a microRNA antisense approach could enhance cytoprotective mechanisms against oxidative stress by increasing the antioxidant defense system. PMID:22880027

The C-Module-Binding Factor Supports Amplification of TRE5-A Retrotransposons in the Dictyostelium discoideum Genome ▿

PubMed Central

Bilzer, Annika; Dölz, Heike; Reinhardt, Alexander; Schmith, Anika; Siol, Oliver; Winckler, Thomas

2011-01-01

Retrotransposable elements are molecular parasites that have invaded the genomes of virtually all organisms. Although retrotransposons encode essential proteins to mediate their amplification, they also require assistance by host cell-encoded machineries that perform functions such as DNA transcription and repair. The retrotransposon TRE5-A of the social amoeba Dictyostelium discoideum generates a notable amount of both sense and antisense RNAs, which are generated from element-internal promoters, located in the A module and the C module, respectively. We observed that TRE5-A retrotransposons depend on the C-module-binding factor (CbfA) to maintain high steady-state levels of TRE5-A transcripts and that CbfA supports the retrotransposition activity of TRE5-A elements. The carboxy-terminal domain of CbfA was found to be required and sufficient to mediate the accumulation of TRE5-A transcripts, but it did not support productive retrotransposition of TRE5-A. This result suggests different roles for CbfA protein domains in the regulation of TRE5-A retrotransposition frequency in D. discoideum cells. Although CbfA binds to the C module in vitro, the factor regulates neither C-module nor A-module promoter activity in vivo. We speculate that CbfA supports the amplification of TRE5-A retrotransposons by suppressing the expression of an as yet unidentified component of the cellular posttranscriptional gene silencing machinery. PMID:21076008

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alvarez, Enrique, E-mail: ealvarez@cbm.uam.es; Castello, Alfredo; Carrasco, Luis

Highlights: {yields} Novel role for poliovirus 2A protease as splicing modulator. {yields} Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. {yields} Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A{sup pro} modulating the alternative splicing of pre-mRNAs. Expression of 2A{sup pro} potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A{sup pro} abrogate Fas exon 6 skipping, whereas higher levels of proteasemore » fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A{sup pro}, leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A{sup pro} on splicing is to selectively block the second catalytic step.« less

A Novel Pathway for Sensory-Mediated Arousal Involves Splicing of an Intron in the period Clock Gene

PubMed Central

Cao, Weihuan; Edery, Isaac

2015-01-01

Study Objectives: D. melanogaster is an excellent animal model to study how the circadian (≅ 24-h) timing system and sleep regulate daily wake-sleep cycles. Splicing of a temperature-sensitive 3'-terminal intron (termed dmpi8) from the circadian clock gene period (per) regulates the distribution of daily activity in Drosophila. The role of dmpi8 splicing on daily behavior was further evaluated by analyzing sleep. Design: Transgenic flies of the same genetic background but expressing either a wild-type recombinant per gene or one where the efficiency of dmpi8 splicing was increased were exposed to different temperatures in daily light-dark cycles and sleep parameters measured. In addition, transgenic flies were briefly exposed to a variety of sensory-mediated stimuli to measure arousal responses. Results: Surprisingly, we show that the effect of dmpi8 splicing on daytime activity levels does not involve a circadian role for per but is linked to adjustments in sensory-dependent arousal and sleep behavior. Genetically altered flies with high dmpi8 splicing efficiency remain aroused longer following short treatments with light and non-photic cues such as mechanical stimulation. Conclusions: We propose that the thermal regulation of dmpi8 splicing acts as a temperature-calibrated rheostat in a novel arousal mechanism, so that on warm days the inefficient splicing of the dmpi8 intron triggers an increase in quiescence by decreasing sensory-mediated arousal, thus ensuring flies minimize being active during the hot midday sun despite the presence of light in the environment, which is usually a strong arousal cue for diurnal animals. Citation: Cao W, Edery I. A novel pathway for sensory-mediated arousal involves splicing of an intron in the period clock gene. SLEEP 2015;38(1):41–51. PMID:25325457

Dual roles for coactivator activator and its counterbalancing isoform coactivator modulator in human kidney cell tumorigenesis.

PubMed

Kang, Yun Kyoung; Schiff, Rachel; Ko, Lan; Wang, Tao; Tsai, Sophia Y; Tsai, Ming-Jer; O'Malley, Bert W

2008-10-01

Coactivator activator (CoAA) has been reported to be a coactivator that regulates steroid receptor-mediated transcription and alternative RNA splicing. Herein, we show that CoAA is a dual-function coregulator that inhibits G(1)-S transition in human kidney cells and suppresses anchorage-independent growth and xenograft tumor formation. Suppression occurs in part by down-regulating c-myc and its downstream effectors ccnd1 and skp2 and causing accumulation of p27/Kip1 protein. In this cellular setting, CoAA directly represses the proto-oncogene c-myc by recruiting HDAC3 protein and decreasing both the acetylation of histone H3 and the presence of RNA polymerase II on the c-myc promoter. Interestingly, a splicing isoform of CoAA, coactivator modulator (CoAM), antagonizes CoAA-induced G(1)-S transition and growth inhibition by negatively regulating the mRNA levels of the endogenous CoAA isoform. In addition, we found that expression of CoAA protein is significantly decreased in human renal cell carcinoma compared with normal kidney. Our study presents evidence that CoAA is a potential tumor suppressor in renal carcinoma and that CoAM is a counterbalancing splice isoform. This is, thus far, the only example of a nuclear receptor coregulator involved in suppression of kidney cancer and suggests potentially significant new roles for coregulators in renal cancer biology.

Evolutionary conservation of cold-induced antisense RNAs of FLOWERING LOCUS C in Arabidopsis thaliana perennial relatives.

PubMed

Castaings, Loren; Bergonzi, Sara; Albani, Maria C; Kemi, Ulla; Savolainen, Outi; Coupland, George

2014-07-17

Antisense RNA (asRNA) COOLAIR is expressed at A. thaliana FLOWERING LOCUS C (FLC) in response to winter temperatures. Its contribution to cold-induced silencing of FLC was proposed but its functional and evolutionary significance remain unclear. Here we identify a highly conserved block containing the COOLAIR first exon and core promoter at the 3' end of several FLC orthologues. Furthermore, asRNAs related to COOLAIR are expressed at FLC loci in the perennials A. alpina and A. lyrata, although some splicing variants differ from A. thaliana. Study of the A. alpina orthologue, PERPETUAL FLOWERING 1 (PEP1), demonstrates that AaCOOLAIR is induced each winter of the perennial life cycle. Introduction of PEP1 into A. thaliana reveals that AaCOOLAIR cis-elements confer cold-inducibility in this heterologous species while the difference between PEP1 and FLC mRNA patterns depends on both cis-elements and species-specific trans-acting factors. Thus, expression of COOLAIR is highly conserved, supporting its importance in FLC regulation.

IRAS: High-Throughput Identification of Novel Alternative Splicing Regulators.

PubMed

Zheng, S

2016-01-01

Alternative splicing is a fundamental regulatory process of gene expression. Defects in alternative splicing can lead to various diseases, and modification of disease-causing splicing events presents great therapeutic promise. Splicing outcome is commonly affected by extracellular stimuli and signaling cascades that converge on RNA-binding splicing regulators. These trans-acting factors recognize cis-elements in pre-mRNA transcripts to affect spliceosome assembly and splice site choices. Identification of these splicing regulators and/or upstream modulators has been difficult and traditionally done by piecemeal. High-throughput screening strategies to find multiple regulators of exon splicing have great potential to accelerate the discovery process, but typically confront low sensitivity and low specificity of screening assays. Here we describe a unique screening strategy, IRAS (identifying regulators of alternative splicing), using a pair of dual-output minigene reporters to allow for sensitive detection of exon splicing changes. Each dual-output reporter produces green fluorescent protein (GFP) and red fluorescent protein (RFP) fluorescent signals to assay the two spliced isoforms exclusively. The two complementary minigene reporters alter GFP/RFP output ratios in the opposite direction in response to splicing change. Applying IRAS in cell-based high-throughput screens allows sensitive and specific identification of splicing regulators and modulators for any alternative exons of interest. In comparison to previous high-throughput screening methods, IRAS substantially enhances the specificity of the screening assay. This strategy significantly eliminates false positives without sacrificing sensitive identification of true regulators of splicing. © 2016 Elsevier Inc. All rights reserved.

A serine–arginine-rich (SR) splicing factor modulates alternative splicing of over a thousand genes in Toxoplasma gondii

PubMed Central

Yeoh, Lee M.; Goodman, Christopher D.; Hall, Nathan E.; van Dooren, Giel G.; McFadden, Geoffrey I.; Ralph, Stuart A.

2015-01-01

Single genes are often subject to alternative splicing, which generates alternative mature mRNAs. This phenomenon is widespread in animals, and observed in over 90% of human genes. Recent data suggest it may also be common in Apicomplexa. These parasites have small genomes, and economy of DNA is evolutionarily favoured in this phylum. We investigated the mechanism of alternative splicing in Toxoplasma gondii, and have identified and localized TgSR3, a homologue of ASF/SF2 (alternative-splicing factor/splicing factor 2, a serine-arginine–rich, or SR protein) to a subnuclear compartment. In addition, we conditionally overexpressed this protein, which was deleterious to growth. qRT-PCR was used to confirm perturbation of splicing in a known alternatively-spliced gene. We performed high-throughput RNA-seq to determine the extent of splicing modulated by this protein. Current RNA-seq algorithms are poorly suited to compact parasite genomes, and hence we complemented existing tools by writing a new program, GeneGuillotine, that addresses this deficiency by segregating overlapping reads into distinct genes. In order to identify the extent of alternative splicing, we released another program, JunctionJuror, that detects changes in intron junctions. Using this program, we identified about 2000 genes that were constitutively alternatively spliced in T. gondii. Overexpressing the splice regulator TgSR3 perturbed alternative splicing in over 1000 genes. PMID:25870410

Aberrant Splicing Induced by Dysregulated Rbfox2 Produces Enhanced Function of CaV1.2 Calcium Channel and Vascular Myogenic Tone in Hypertension.

PubMed

Zhou, Yingying; Fan, Jia; Zhu, Huayuan; Ji, Li; Fan, Wenyong; Kapoor, Isha; Wang, Yue; Wang, Yuan; Zhu, Guoqing; Wang, Juejin

2017-12-01

Calcium influx from activated voltage-gated calcium channel Ca V 1.2 in vascular smooth muscle cells is indispensable for maintaining myogenic tone and blood pressure. The function of Ca V 1.2 channel can be optimized by alternative splicing, one of post-transcriptional modification mechanisms. The splicing factor Rbfox2 is known to regulate the Ca V 1.2 pre-mRNA alternative splicing events during neuronal development. However, Rbfox2's roles in modulating the key function of vascular Ca V 1.2 channel and in the pathogenesis of hypertension remain elusive. Here, we report that the proportion of Ca V 1.2 channels with alternative exon 9* is increased by 10.3%, whereas that with alternative exon 33 is decreased by 10.5% in hypertensive arteries. Surprisingly, the expression level of Rbfox2 is increased ≈3-folds, presumably because of the upregulation of a dominant-negative isoform of Rbfox2. In vascular smooth muscle cells, we find that knockdown of Rbfox2 dynamically increases alternative exon 9*, whereas decreases exon 33 inclusion of Ca V 1.2 channels. By patch-clamp studies, we show that diminished Rbfox2-induced alternative splicing shifts the steady-state activation and inactivation curves of vascular Ca V 1.2 calcium channel to hyperpolarization, which makes the window current potential to more negative. Moreover, siRNA-mediated knockdown of Rbfox2 increases the pressure-induced vascular myogenic tone of rat mesenteric artery. Taken together, our data indicate that Rbfox2 modulates the functions of vascular Ca V 1.2 calcium channel by dynamically regulating the expressions of alternative exons 9* and 33, which in turn affects the vascular myogenic tone. Therefore, our work suggests a key role for Rbfox2 in hypertension, which provides a rational basis for designing antihypertensive therapies. © 2017 American Heart Association, Inc.

Kinetic and equilibrium properties of regulatory Ca(2+)-binding domains in sodium-calcium exchangers 2 and 3.

PubMed

Tal, Inbal; Kozlovsky, Tom; Brisker, Dafna; Giladi, Moshe; Khananshvili, Daniel

2016-04-01

In mammals, three sodium-calcium exchanger (NCX) protein isoforms (NCX1, NCX2, and NCX3) mediate Ca(2+) fluxes across the membrane to maintain cellular Ca(2+) homeostasis. NCX isoforms and their splice variants are expressed in a tissue-specific manner to meet physiological demands. NCX1 is ubiquitously expressed, NCX2 is expressed in the brain and spinal cord, and NCX3 is expressed in the brain and skeletal muscle. Eukaryotic NCXs contain two cytosolic regulatory Ca(2+)-binding domains, CBD1 and CBD2, which form a two-domain tandem (CBD12) through a short linker. Ca(2+) binding to the CBDs underlies allosteric regulation of NCX. Previous structural and functional studies in NCX1 have shown that the CBDs synergistically interact, where their interactions are modulated in a splice variant-specific manner by splicing segment at CBD2. Here, we analyze the equilibrium and kinetic properties of Ca(2+) binding to purified preparations of CBD1, CBD2, and CBD12 from NCX2 and from NCX3 splice variants. We show that CBD1 interacts with CBD2 in the context of the CBD12 tandem in all NCX isoforms, where these interactions specifically modulate Ca(2+) sensing at the primary sensor of CBD1 to meet the physiological requirements. For example, the rate-limiting slow dissociation of "occluded" Ca(2+) from the primary allosteric sensor of variants expressed in skeletal muscle is ∼10-fold slower than that of variants expressed in the brain. Notably, these kinetic differences between NCX variants occur while maintaining a similar Ca(2+) affinity of the primary sensor, since the resting [Ca(2+)]i levels are similar among different cell types. Copyright © 2016 Elsevier Ltd. All rights reserved.

TGFβ1-mediated PI3K/Akt and p38 MAP kinase dependent alternative splicing of fibronectin extra domain A in human podocyte culture.

PubMed

Madne, Tarunkumar Hemraj; Dockrell, Mark Edward Carl

2018-04-30

Alternative splicing is an important gene regulation process to distribute proteins in health and diseases. Extra Domain A+ Fibronectin (EDA+Fn) is an alternatively spliced form of fibronectin (Fn) protein, present in the extra cellular matrix (ECM) and a recognised marker of various pathologies. TGFβ1 has been shown to induce alternative splicing of EDA+Fn in many cell types. Podocytes are spectacular cell type and play a key role in filtration and synthesise ECM proteins in renal physiology and pathology. In our previous study we have demonstrated expression and alternative splicing of EDA+Fn in basal condition in human podocytes culture. TGFβ1 further induced the basal expression and alternative splicing of EDA+Fn through Alk5 receptor and SR proteins. In this study, we have investigated TGFβ1 mediated signalling involved in alternative splicing of EDA+Fn in human podocytes. We have performed western blotting to characterise the expression of the EDA+Fn protein and other signalling proteins and RT-PCR to look for signalling pathways involved in regulation of alternative splicing of EDA+Fn in conditionally immortalised human podocytes culture.We have used TGFβ1 as a stimulator and SB431542, SB202190 and LY294002 for inhibitory studies. In this work, we have demonstrated in human podocytes culture TGFβ1 2.5ng/ml induced phosphorylation of Smad1/5/8, Smad2 and Smad3 via the ALK5 receptor. TGFβ1 significantly induced the PI3K/Akt pathway and the PI3K/Akt pathway inhibitor LY294002 significantly downregulated basal as well as TGFβ1 induced alternative splicing of EDA+Fn in human podocytes. In addition to this, TGFβ1 significantly induced the p38 MAP kinase signalling pathway and p38 MAP kinase signalling pathway inhibitor SB202190 downregulated the TGFβ1-mediated alternative splicing of EDA+Fn in human podocytes. The results with PI3K and p38 MAP kinase signalling pathway suggest that inhibiting PI3K signalling pathway downregulated the basal alternative splicing of EDA+Fn in human podocytes and its the inhibition of p38 Map Kinase signalling pathway which had specifically downregulated the TGFβ1 mediated alternative splicing of EDA+Fn in human podocytes culture. Activation of TGFβ1-mediated Smad1/5/8 via Alk5 receptor suggests that TGFβ1 signalling pathway involved Alk5/Alk1 receptor axis signalling in human podocytes.

Role of histone modifications and early termination in pervasive transcription and antisense-mediated gene silencing in yeast.

PubMed

Castelnuovo, Manuele; Zaugg, Judith B; Guffanti, Elisa; Maffioletti, Andrea; Camblong, Jurgi; Xu, Zhenyu; Clauder-Münster, Sandra; Steinmetz, Lars M; Luscombe, Nicholas M; Stutz, Françoise

2014-04-01

Most genomes, including yeast Saccharomyces cerevisiae, are pervasively transcribed producing numerous non-coding RNAs, many of which are unstable and eliminated by nuclear or cytoplasmic surveillance pathways. We previously showed that accumulation of PHO84 antisense RNA (asRNA), in cells lacking the nuclear exosome component Rrp6, is paralleled by repression of sense transcription in a process dependent on the Hda1 histone deacetylase (HDAC) and the H3K4 histone methyl transferase Set1. Here we investigate this process genome-wide and measure the whole transcriptome of various histone modification mutants in a Δrrp6 strain using tiling arrays. We confirm widespread occurrence of potentially antisense-dependent gene regulation and identify three functionally distinct classes of genes that accumulate asRNAs in the absence of Rrp6. These classes differ in whether the genes are silenced by the asRNA and whether the silencing is HDACs and histone methyl transferase-dependent. Among the distinguishing features of asRNAs with regulatory potential, we identify weak early termination by Nrd1/Nab3/Sen1, extension of the asRNA into the open reading frame promoter and dependence of the silencing capacity on Set1 and the HDACs Hda1 and Rpd3 particularly at promoters undergoing extensive chromatin remodelling. Finally, depending on the efficiency of Nrd1/Nab3/Sen1 early termination, asRNA levels are modulated and their capability of silencing is changed.

Role of histone modifications and early termination in pervasive transcription and antisense-mediated gene silencing in yeast

PubMed Central

Castelnuovo, Manuele; Zaugg, Judith B.; Guffanti, Elisa; Maffioletti, Andrea; Camblong, Jurgi; Xu, Zhenyu; Clauder-Münster, Sandra; Steinmetz, Lars M.; Luscombe, Nicholas M.; Stutz, Françoise

2014-01-01

Most genomes, including yeast Saccharomyces cerevisiae, are pervasively transcribed producing numerous non-coding RNAs, many of which are unstable and eliminated by nuclear or cytoplasmic surveillance pathways. We previously showed that accumulation of PHO84 antisense RNA (asRNA), in cells lacking the nuclear exosome component Rrp6, is paralleled by repression of sense transcription in a process dependent on the Hda1 histone deacetylase (HDAC) and the H3K4 histone methyl transferase Set1. Here we investigate this process genome-wide and measure the whole transcriptome of various histone modification mutants in a Δrrp6 strain using tiling arrays. We confirm widespread occurrence of potentially antisense-dependent gene regulation and identify three functionally distinct classes of genes that accumulate asRNAs in the absence of Rrp6. These classes differ in whether the genes are silenced by the asRNA and whether the silencing is HDACs and histone methyl transferase-dependent. Among the distinguishing features of asRNAs with regulatory potential, we identify weak early termination by Nrd1/Nab3/Sen1, extension of the asRNA into the open reading frame promoter and dependence of the silencing capacity on Set1 and the HDACs Hda1 and Rpd3 particularly at promoters undergoing extensive chromatin remodelling. Finally, depending on the efficiency of Nrd1/Nab3/Sen1 early termination, asRNA levels are modulated and their capability of silencing is changed. PMID:24497191

Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

PubMed Central

Redpath, G. M. I.; Woolger, N.; Piper, A. K.; Lemckert, F. A.; Lek, A.; Greer, P. A.; North, K. N.; Cooper, S. T.

2014-01-01

Dysferlin and calpain are important mediators of the emergency response to repair plasma membrane injury. Our previous research revealed that membrane injury induces cleavage of dysferlin to release a synaptotagmin-like C-terminal module we termed mini-dysferlinC72. Here we show that injury-activated cleavage of dysferlin is mediated by the ubiquitous calpains via a cleavage motif encoded by alternately spliced exon 40a. An exon 40a–specific antibody recognizing cleaved mini-dysferlinC72 intensely labels the circumference of injury sites, supporting a key role for dysferlinExon40a isoforms in membrane repair and consistent with our evidence suggesting that the calpain-cleaved C-terminal module is the form specifically recruited to injury sites. Calpain cleavage of dysferlin is a ubiquitous response to membrane injury in multiple cell lineages and occurs independently of the membrane repair protein MG53. Our study links calpain and dysferlin in the calcium-activated vesicle fusion of membrane repair, placing calpains as upstream mediators of a membrane repair cascade that elicits cleaved dysferlin as an effector. Of importance, we reveal that myoferlin and otoferlin are also cleaved enzymatically to release similar C-terminal modules, bearing two C2 domains and a transmembrane domain. Evolutionary preservation of this feature highlights its functional importance and suggests that this highly conserved C-terminal region of ferlins represents a functionally specialized vesicle fusion module. PMID:25143396

ELAV, a Drosophila neuron-specific protein, mediates the generation of an alternatively spliced neural protein isoform.

PubMed

Koushika, S P; Lisbin, M J; White, K

1996-12-01

Tissue-specific alternative pre-mRNA splicing is a widely used mechanism for gene regulation and the generation of different protein isoforms, but relatively little is known about the factors and mechanisms that mediate this process. Tissue-specific RNA-binding proteins could mediate alternative pre-mRNA splicing. In Drosophila melanogaster, the RNA-binding protein encoded by the elav (embryonic lethal abnormal visual system) gene is a candidate for such a role. The ELAV protein is expressed exclusively in neurons, and is important for the formation and maintenance of the nervous system. In this study, photoreceptor neurons genetically depleted of ELAV, and elav-null central nervous system neurons, were analyzed immunocytochemically for the expression of neural proteins. In both situations, the lack of ELAV corresponded with a decrease in the immunohistochemical signal of the neural-specific isoform of Neuroglian, which is generated by alternative splicing. Furthermore, when ELAV was expressed ectopically in cells that normally express only the non-neural isoform of Neuroglian, we observed the generation of the neural isoform of Neuroglian. Drosophila ELAV promotes the generation of the neuron-specific isoform of Neuroglian by the regulation of pre-mRNA splicing. The findings reported in this paper demonstrate that ELAV is necessary, and the ectopic expression of ELAV in imaginal disc cells is sufficient, to mediate neuron-specific alternative splicing.

CELFish ways to modulate mRNA decay

PubMed Central

St. Louis, Irina Vlasova; Dickson, Alexa M.; Bohjanen, Paul R.; Wilusz, Carol J.

2013-01-01

The CELF family of RNA-binding proteins regulates many steps of mRNA metabolism. Although their best characterized function is in pre-mRNA splice site choice, CELF family members are also powerful modulators of mRNA decay. In this review we focus on the different modes of regulation that CELF proteins employ to mediate mRNA decay by binding to GU-rich elements. After starting with an overview of the importance of CELF proteins during development and disease pathogenesis, we then review the mRNA networks and cellular pathways these proteins regulate and the mechanisms by which they influence mRNA decay. Finally, we discuss how CELF protein activity is modulated during development and in response to cellular signals. We conclude by highlighting the priorities for new experiments in this field. PMID:23328451

Identification of sequence motifs significantly associated with antisense activity.

PubMed

McQuisten, Kyle A; Peek, Andrew S

2007-06-07

Predicting the suppression activity of antisense oligonucleotide sequences is the main goal of the rational design of nucleic acids. To create an effective predictive model, it is important to know what properties of an oligonucleotide sequence associate significantly with antisense activity. Also, for the model to be efficient we must know what properties do not associate significantly and can be omitted from the model. This paper will discuss the results of a randomization procedure to find motifs that associate significantly with either high or low antisense suppression activity, analysis of their properties, as well as the results of support vector machine modelling using these significant motifs as features. We discovered 155 motifs that associate significantly with high antisense suppression activity and 202 motifs that associate significantly with low suppression activity. The motifs range in length from 2 to 5 bases, contain several motifs that have been previously discovered as associating highly with antisense activity, and have thermodynamic properties consistent with previous work associating thermodynamic properties of sequences with their antisense activity. Statistical analysis revealed no correlation between a motif's position within an antisense sequence and that sequences antisense activity. Also, many significant motifs existed as subwords of other significant motifs. Support vector regression experiments indicated that the feature set of significant motifs increased correlation compared to all possible motifs as well as several subsets of the significant motifs. The thermodynamic properties of the significantly associated motifs support existing data correlating the thermodynamic properties of the antisense oligonucleotide with antisense efficiency, reinforcing our hypothesis that antisense suppression is strongly associated with probe/target thermodynamics, as there are no enzymatic mediators to speed the process along like the RNA Induced Silencing Complex (RISC) in RNAi. The independence of motif position and antisense activity also allows us to bypass consideration of this feature in the modelling process, promoting model efficiency and reducing the chance of overfitting when predicting antisense activity. The increase in SVR correlation with significant features compared to nearest-neighbour features indicates that thermodynamics alone is likely not the only factor in determining antisense efficiency.

Histone hyperacetylation and exon skipping: a calcium-mediated dynamic regulation in cardiomyocytes

PubMed Central

Sharma, Alok; Nguyen, Hieu; Cai, Lu; Lou, Hua

2015-01-01

In contrast to cell type-specific pre-mRNA alternative splicing, mechanisms controlling activity-dependent alternative splicing is under-studied and not well understood. In a recent study, we conducted a comprehensive analysis of calcium-mediated mechanism that regulates alternative exon skipping in mouse cardiomyocytes. Our results reveal a strong link between histone hyperacetylation and skipping of cassette exons, and provide support to the kinetic coupling model of the epigenetic regulation of alternative splicing at the chromatin level. PMID:26325491

Activations of c-fos/c-jun signaling are involved in the modulation of hypothalamic superoxide dismutase (SOD) and neuropeptide Y (NPY) gene expression in amphetamine-mediated appetite suppression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsieh, Y.-S.; Yang, S.-F.; Chiou, H.-L.

2006-04-15

Amphetamine (AMPH) is known as an anorectic agent. The mechanism underlying the anorectic action of AMPH has been attributed to its inhibitory action on hypothalamic neuropeptide Y (NPY), an appetite stimulant in the brain. This study was aimed to examine the molecular mechanisms behind the anorectic effect of AMPH. Results showed that AMPH treatment decreased food intake, which was correlated with changes of NPY mRNA level, but increased c-fos, c-jun and superoxide dismutase (SOD) mRNA levels in hypothalamus. To determine if c-fos or c-jun was involved in the anorectic response of AMPH, infusions of antisense oligonucleotide into the brain weremore » performed at 1 h before daily AMPH treatment in freely moving rats, and the results showed that c-fos or c-jun knockdown could block this anorectic response and restore NPY mRNA level. Moreover, c-fos or c-jun knockdown could partially block SOD mRNA level that might involve in the modulation of NPY gene expression. It was suggested that c-fos/c-jun signaling might involve in the central regulation of AMPH-mediated feeding suppression via the modulation of NPY gene expression.« less

Size-uniform 200 nm particles: fabrication and application to magnetofection.

PubMed

Mair, Lamar; Ford, Kris; Alam, M d Rowshon; Kole, Ryszard; Fisher, Michael; Superfine, Richard

2009-04-01

We report on the fabrication of arrays of mono- and multimetallic particles via metal evaporation onto lithographically patterned posts, as well as the magnetic force calibration and successful magnetofection of iron particles grown via this method. This work represents the first instance in which metal evaporation onto post structures was used for the formation of released, shape-defined metal particles. Also, our work represents the first use of lithographically defined particles as agents of magnetofection. Using these techniques it is possible to create particles with complex shapes and lateral dimensions as small as 40 nm. Our demonstrated compositionally flexible particles are highly size-uniform due to their photolithographically defined growth substrates, with particle dimensions along two axes fixed at 200 nm; the third axis dimension can be varied from 20 nm to 300 nm during the deposition procedure. Atomic percent of metals incorporated into the particle volume is highly tunable and particles have been synthesized with as many as four different metals. We performed magnetic force calibrations on a single particle size for iron particles using an axially magnetized NeFeB permanent magnet and comparisons are made with commercially available magnetic beads. In order to evalutate their usefulness as magnetofection agents, an antisense oligonucleotide (ODN) designed to correct the aberrant splicing of enhanced green fluorescent protein mRNA, was successfully transfected into a modified HeLa cell line. Magnetically enhanced gene delivery was accomplished in vitro using antisense ODN-laden iron particles followed by application of a field gradient. Magnetically enhanced transfection resulted in a 76% and 139% increase in fluorescence intensity when compared to Lipofectamine and antisense ODN-loaded particles delivered without magnetic treatment, respectively. To our knowledge, these experiments constitute the first use of lithographically defined particles as successful agents for magnetically enhanced transfection of an antisense oligonucleotide.

Morpholino oligomer-mediated exon skipping averts the onset of dystrophic pathology in the mdx mouse.

PubMed

Fletcher, Sue; Honeyman, Kaite; Fall, Abbie M; Harding, Penny L; Johnsen, Russell D; Steinhaus, Joshua P; Moulton, Hong M; Iversen, Patrick L; Wilton, Stephen D

2007-09-01

Duchenne and Becker muscular dystrophies are allelic disorders arising from mutations in the dystrophin gene. Duchenne muscular dystrophy is characterized by an absence of functional protein, whereas Becker muscular dystrophy, commonly caused by in-frame deletions, shows synthesis of partially functional protein. Anti-sense oligonucleotides can induce specific exon removal during processing of the dystrophin primary transcript, while maintaining or restoring the reading frame, and thereby overcome protein-truncating mutations. The mdx mouse has a non-sense mutation in exon 23 of the dystrophin gene that precludes functional dystrophin production, and this model has been used in the development of treatment strategies for dystrophinopathies. A phosphorodiamidate morpholino oligomer (PMO) has previously been shown to exclude exon 23 from the dystrophin gene transcript and induce dystrophin expression in the mdxmouse, in vivo and in vitro. In this report, a cell-penetrating peptide (CPP)-conjugated oligomer targeted to the mouse dystrophin exon 23 donor splice site was administered to mdxmice by intraperitoneal injection. We demonstrate dystrophin expression and near-normal muscle architecture in all muscles examined, except for cardiac muscle. The CPP greatly enhanced uptake of the PMO, resulting in widespread dystrophin expression.

CEF1/CDC5 alleles modulate transitions between catalytic conformations of the spliceosome

PubMed Central

Query, Charles C.; Konarska, Maria M.

2012-01-01

Conformational change within the spliceosome is required between the first and second catalytic steps of pre-mRNA splicing. A prior genetic screen for suppressors of an intron mutant that stalls between the two steps yielded both prp8 and non-prp8 alleles that suppressed second-step splicing defects. We have now identified the strongest non-prp8 suppressors as alleles of the NTC (Prp19 complex) component, CEF1. These cef1 alleles generally suppress second-step defects caused by a variety of intron mutations, mutations in U6 snRNA, or deletion of the second-step protein factor Prp17, and they can activate alternative 3′ splice sites. Genetic and functional interactions between cef1 and prp8 alleles suggest that they modulate the same event(s) in the first-to-second-step transition, most likely by stabilization of the second-step spliceosome; in contrast, alleles of U6 snRNA that also alter this transition modulate a distinct event, most likely by stabilization of the first-step spliceosome. These results implicate a myb-like domain of Cef1/CDC5 in interactions that modulate conformational states of the spliceosome and suggest that alteration of these events affects splice site use, resulting in alternative splicing-like patterns in yeast. PMID:22408182

Peptidic tools applied to redirect alternative splicing events.

PubMed

Nancy, Martínez-Montiel; Nora, Rosas-Murrieta; Rebeca, Martínez-Contreras

2015-05-01

Peptides are versatile and attractive biomolecules that can be applied to modulate genetic mechanisms like alternative splicing. In this process, a single transcript yields different mature RNAs leading to the production of protein isoforms with diverse or even antagonistic functions. During splicing events, errors can be caused either by mutations present in the genome or by defects or imbalances in regulatory protein factors. In any case, defects in alternative splicing have been related to several genetic diseases including muscular dystrophy, Alzheimer's disease and cancer from almost every origin. One of the most effective approaches to redirect alternative splicing events has been to attach cell-penetrating peptides to oligonucleotides that can modulate a single splicing event and restore correct gene expression. Here, we summarize how natural existing and bioengineered peptides have been applied over the last few years to regulate alternative splicing and genetic expression. Under different genetic and cellular backgrounds, peptides have been shown to function as potent vehicles for splice correction, and their therapeutic benefits have reached clinical trials and patenting stages, emphasizing the use of regulatory peptides as an exciting therapeutic tool for the treatment of different genetic diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

Divergently overlapping cis-encoded antisense RNA regulating toxin-antitoxin systems from E. coli: hok/sok, ldr/rdl, symE/symR.

PubMed

Kawano, Mitsuoki

2012-12-01

Toxin-antitoxin (TA) systems are categorized into three classes based on the type of antitoxin. In type I TA systems, the antitoxin is a small antisense RNA that inhibits translation of small toxic proteins by binding to the corresponding mRNAs. Those type I TA systems were originally identified as plasmid stabilization modules rendering a post-segregational killing (PSK) effect on the host cells. The type I TA loci also exist on the Escherichia coli chromosome but their biological functions are less clear. Genetic organization and regulatory elements of hok/sok and ldr/rdl families are very similar and the toxins are predicted to contain a transmembrane domain, but otherwise share no detectable sequence similarity. This review will give an overview of the type I TA modules of E. coli K-12, especially hok/sok, ldr/rdl and SOS-inducible symE/symR systems, which are regulated by divergently overlapping cis-encoded antisense RNAs.

Modulation of KCNQ1 alternative splicing regulates cardiac IKs and action potential repolarization.

PubMed

Lee, Hsiang-Chun; Rudy, Yoram; Po-Yuan, Phd; Sheu, Sheng-Hsiung; Chang, Jan-Gowth; Cui, Jianmin

2013-08-01

Slow delayed-rectifier potassium current (IKs) channels, made of the pore-forming KCNQ1 and auxiliary KCNE1 subunits, play a key role in determining action potential duration (APD) in cardiac myocytes. The consequences of drug-induced KCNQ1 splice alteration remain unknown. To study the modulation of KCNQ1 alternative splicing by amiloride and the consequent changes in IKs and action potentials (APs) in ventricular myocytes. Canine endocardial, midmyocardial, and epicardial ventricular myocytes were isolated. Levels of KCNQ1a and KCNQ1b as well as a series of splicing factors were quantified by using the reverse transcriptase-polymerase chain reaction and Western blot. The effect of amiloride-induced changes in the KCNQ1b/total KCNQ1 ratio on AP was measured by using whole-cell patch clamp with and without isoproterenol. With 50 μmol/L of amiloride for 6 hours, KCNQ1a at transcriptional and translational levels increased in midmyocardial myocytes but decreased in endo- and epicardial myocytes. Likewise, changes in splicing factors in midmyocardial were opposite to that in endo- and epicardial myocytes. In midmyocardial myocytes amiloride shortened APD and decreased isoproterenol-induced early afterdepolarizations significantly. The same amiloride-induced effects were demonstrated by using human ventricular myocyte model for AP simulations under beta-adrenergic stimulation. Moreover, amiloride reduced the transmural dispersion of repolarization in pseudo-electrocardiogram. Amiloride regulates IKs and APs with transmural differences and reduces arrhythmogenicity through the modulation of KCNQ1 splicing. We suggested that the modulation of KCNQ1 splicing may help prevent arrhythmia. Copyright © 2013 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Receptor-Mediated Uptake of Phosphorothioate Antisense Oligonucleotides in Different Cell Types of the Liver.

PubMed

Miller, Colton M; Tanowitz, Michael; Donner, Aaron J; Prakash, Thazha P; Swayze, Eric E; Harris, Edward N; Seth, Punit P

2018-06-01

Oligonucleotide therapeutics have emerged as a third distinct platform for drug discovery within the pharmaceutical industry. Five oligonucleotide-based drugs have been approved by the US FDA and over 100 oligonucleotides drugs are currently at different stages of human trials. Several of these oligonucleotide drugs are modified using the phosphorothioate (PS) backbone modification where one of the nonbridging oxygen atoms of the phosphodiester linkage is replaced with sulfur. In this review, we summarize our knowledge on receptor-mediated uptake of PS antisense oligonucleotides (ASOs) within different cell types of the liver-a privileged organ for the discovery of oligonucleotide-based therapeutics.

NF-κB Hyper-Activation by HTLV-1 Tax Induces Cellular Senescence, but Can Be Alleviated by the Viral Anti-Sense Protein HBZ

PubMed Central

Zhi, Huijun; Yang, Liangpeng; Kuo, Yu-Liang; Ho, Yik-Khuan; Shih, Hsiu-Ming; Giam, Chou-Zen

2011-01-01

Activation of I-κB kinases (IKKs) and NF-κB by the human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, is thought to promote cell proliferation and transformation. Paradoxically, expression of Tax in most cells leads to drastic up-regulation of cyclin-dependent kinase inhibitors, p21CIP1/WAF1 and p27KIP1, which cause p53-/pRb-independent cellular senescence. Here we demonstrate that p21CIP1/WAF1-/p27KIP1-mediated senescence constitutes a checkpoint against IKK/NF-κB hyper-activation. Senescence induced by Tax in HeLa cells is attenuated by mutations in Tax that reduce IKK/NF-κB activation and prevented by blocking NF-κB using a degradation-resistant mutant of I-κBα despite constitutive IKK activation. Small hairpin RNA-mediated knockdown indicates that RelA induces this senescence program by acting upstream of the anaphase promoting complex and RelB to stabilize p27KIP1 protein and p21CIP1/WAF1 mRNA respectively. Finally, we show that down-regulation of NF-κB by the HTLV-1 anti-sense protein, HBZ, delay or prevent the onset of Tax-induced senescence. We propose that the balance between Tax and HBZ expression determines the outcome of HTLV-1 infection. Robust HTLV-1 replication and elevated Tax expression drive IKK/NF-κB hyper-activation and trigger senescence. HBZ, however, modulates Tax-mediated viral replication and NF-κB activation, thus allowing HTLV-1-infected cells to proliferate, persist, and evolve. Finally, inactivation of the senescence checkpoint can facilitate persistent NF-κB activation and leukemogenesis. PMID:21552325

An intronic ncRNA-dependent regulation of SORL1 expression affecting Aβ formation is upregulated in post-mortem Alzheimer's disease brain samples.

PubMed

Ciarlo, Eleonora; Massone, Sara; Penna, Ilaria; Nizzari, Mario; Gigoni, Arianna; Dieci, Giorgio; Russo, Claudio; Florio, Tullio; Cancedda, Ranieri; Pagano, Aldo

2013-03-01

Recent studies indicated that sortilin-related receptor 1 (SORL1) is a risk gene for late-onset Alzheimer's disease (AD), although its role in the aetiology and/or progression of this disorder is not fully understood. Here, we report the finding of a non-coding (nc) RNA (hereafter referred to as 51A) that maps in antisense configuration to intron 1 of the SORL1 gene. 51A expression drives a splicing shift of SORL1 from the synthesis of the canonical long protein variant A to an alternatively spliced protein form. This process, resulting in a decreased synthesis of SORL1 variant A, is associated with impaired processing of amyloid precursor protein (APP), leading to increased Aβ formation. Interestingly, we found that 51A is expressed in human brains, being frequently upregulated in cerebral cortices from individuals with Alzheimer's disease. Altogether, these findings document a novel ncRNA-dependent regulatory pathway that might have relevant implications in neurodegeneration.

The mediator complex in genomic and non-genomic signaling in cancer.

PubMed

Weber, Hannah; Garabedian, Michael J

2018-05-01

Mediator is a conserved, multi-subunit macromolecular machine divided structurally into head, middle, and tail modules, along with a transiently associating kinase module. Mediator functions as an integrator of transcriptional regulatory activity by interacting with DNA-bound transcription factors and with RNA polymerase II (Pol II) to both activate and repress gene expression. Mediator has been shown to affect multiple steps in transcription, including chromatin looping between enhancers and promoters, pre-initiation complex formation, transcriptional elongation, and mRNA splicing. Individual Mediator subunits participate in regulation of gene expression by the estrogen and androgen receptors and are altered in a number of endocrine cancers, including breast and prostate cancer. In addition to its role in genomic signaling, MED12 has been implicated in non-genomic signaling by interacting with and activating TGF-beta receptor 2 in the cytoplasm. Recent structural studies have revealed extensive inter-domain interactions and complex architecture of the Mediator-Pol II complex, suggesting that Mediator is capable of reorganizing its conformation and composition to fit cellular needs. We propose that alterations in Mediator subunit expression that occur in various cancers could impact the organization and function of Mediator, resulting in changes in gene expression that promote malignancy. A better understanding of the role of Mediator in cancer could reveal new approaches to the diagnosis and treatment of Mediator-dependent endocrine cancers, especially in settings of therapy resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification by high-throughput imaging of the histone methyltransferase EHMT2 as an epigenetic regulator of VEGFA alternative splicing

PubMed Central

Salton, Maayan; Voss, Ty C.; Misteli, Tom

2014-01-01

Recent evidence points to a role of chromatin in regulation of alternative pre-mRNA splicing (AS). In order to identify novel chromatin regulators of AS, we screened an RNAi library of chromatin proteins using a cell-based high-throughput in vivo assay. We identified a set of chromatin proteins that regulate AS. Using simultaneous genome-wide expression and AS analysis, we demonstrate distinct and non-overlapping functions of these chromatin modifiers on transcription and AS. Detailed mechanistic characterization of one dual function chromatin modifier, the H3K9 methyltransferase EHMT2 (G9a), identified VEGFA as a major chromatin-mediated AS target. Silencing of EHMT2, or its heterodimer partner EHMT1, affects AS by promoting exclusion of VEGFA exon 6a, but does not alter total VEGFA mRNA levels. The epigenetic regulatory mechanism of AS by EHMT2 involves an adaptor system consisting of the chromatin modulator HP1γ, which binds methylated H3K9 and recruits splicing regulator SRSF1. The epigenetic regulation of VEGFA is physiologically relevant since EHMT2 is transcriptionally induced in response to hypoxia and triggers concomitant changes in AS of VEGFA. These results characterize a novel epigenetic regulatory mechanism of AS and they demonstrate separate roles of epigenetic modifiers in transcription and alternative splicing. PMID:25414343

The binding protein BiP attenuates stress-induced cell death in soybean via modulation of the N-rich protein-mediated signaling pathway.

PubMed

Reis, Pedro A A; Rosado, Gustavo L; Silva, Lucas A C; Oliveira, Luciana C; Oliveira, Lucas B; Costa, Maximiller D L; Alvim, Fátima C; Fontes, Elizabeth P B

2011-12-01

The molecular chaperone binding protein (BiP) participates in the constitutive function of the endoplasmic reticulum (ER) and protects the cell against stresses. In this study, we investigated the underlying mechanism by which BiP protects plant cells from stress-induced cell death. We found that enhanced expression of BiP in soybean (Glycine max) attenuated ER stress- and osmotic stress-mediated cell death. Ectopic expression of BiP in transgenic lines attenuated the leaf necrotic lesions that are caused by the ER stress inducer tunicamycin and also maintained shoot turgidity upon polyethylene glycol-induced dehydration. BiP-mediated attenuation of stress-induced cell death was confirmed by the decreased percentage of dead cell, the reduced induction of the senescence-associated marker gene GmCystP, and reduced DNA fragmentation in BiP-overexpressing lines. These phenotypes were accompanied by a delay in the induction of the cell death marker genes N-RICH PROTEIN-A (NRP-A), NRP-B, and GmNAC6, which are involved in transducing a cell death signal generated by ER stress and osmotic stress through the NRP-mediated signaling pathway. The prosurvival effect of BiP was associated with modulation of the ER stress- and osmotic stress-induced NRP-mediated cell death signaling, as determined in transgenic tobacco (Nicotiana tabacum) lines with enhanced (sense) and suppressed (antisense) BiP levels. Enhanced expression of BiP prevented NRP- and NAC6-mediated chlorosis and the appearance of senescence-associated markers, whereas silencing of endogenous BiP accelerated the onset of leaf senescence mediated by NRPs and GmNAC6. Collectively, these results implicate BiP as a negative regulator of the stress-induced NRP-mediated cell death response.

The Binding Protein BiP Attenuates Stress-Induced Cell Death in Soybean via Modulation of the N-Rich Protein-Mediated Signaling Pathway1[C][W][OA

PubMed Central

Reis, Pedro A.A.; Rosado, Gustavo L.; Silva, Lucas A.C.; Oliveira, Luciana C.; Oliveira, Lucas B.; Costa, Maximiller D.L.; Alvim, Fátima C.; Fontes, Elizabeth P.B.

2011-01-01

The molecular chaperone binding protein (BiP) participates in the constitutive function of the endoplasmic reticulum (ER) and protects the cell against stresses. In this study, we investigated the underlying mechanism by which BiP protects plant cells from stress-induced cell death. We found that enhanced expression of BiP in soybean (Glycine max) attenuated ER stress- and osmotic stress-mediated cell death. Ectopic expression of BiP in transgenic lines attenuated the leaf necrotic lesions that are caused by the ER stress inducer tunicamycin and also maintained shoot turgidity upon polyethylene glycol-induced dehydration. BiP-mediated attenuation of stress-induced cell death was confirmed by the decreased percentage of dead cell, the reduced induction of the senescence-associated marker gene GmCystP, and reduced DNA fragmentation in BiP-overexpressing lines. These phenotypes were accompanied by a delay in the induction of the cell death marker genes N-RICH PROTEIN-A (NRP-A), NRP-B, and GmNAC6, which are involved in transducing a cell death signal generated by ER stress and osmotic stress through the NRP-mediated signaling pathway. The prosurvival effect of BiP was associated with modulation of the ER stress- and osmotic stress-induced NRP-mediated cell death signaling, as determined in transgenic tobacco (Nicotiana tabacum) lines with enhanced (sense) and suppressed (antisense) BiP levels. Enhanced expression of BiP prevented NRP- and NAC6-mediated chlorosis and the appearance of senescence-associated markers, whereas silencing of endogenous BiP accelerated the onset of leaf senescence mediated by NRPs and GmNAC6. Collectively, these results implicate BiP as a negative regulator of the stress-induced NRP-mediated cell death response. PMID:22007022

Protein Kinase C δ (PKCδ) Splice Variants Modulate Apoptosis Pathway in 3T3L1 Cells during Adipogenesis

PubMed Central

Patel, Rekha; Apostolatos, André; Carter, Gay; Ajmo, Joanne; Gali, Meghanath; Cooper, Denise R.; You, Min; Bisht, Kirpal S.; Patel, Niketa A.

2013-01-01

Increased food intake and lack of physical activity results in excess energy stored in adipocytes, and this imbalance contributes to obesity. New adipocytes are required for storage of energy in the white adipose tissue. This process of adipogenesis is widely studied in differentiating 3T3L1 preadipocytes in vitro. We have identified a key signaling kinase, protein kinase C delta (PKCδ), whose alternative splice variant expression is modulated during adipogenesis. We demonstrate that PKCδII splice variant promotes survival in differentiating 3T3L1 cells through the Bcl2 pathway. Here we demonstrate that resveratrol, a naturally occurring polyphenol, increases apoptosis and inhibits adipogenesis along with disruption of PKCδ alternative splicing during 3T3L1 differentiation. Importantly, we have identified a PKCδII splice variant inhibitor. This inhibitor may be a valuable tool with therapeutic implications in obesity. PMID:23902767

The Exon Junction Complex and Srp54 Contribute to Hedgehog Signaling via ci RNA Splicing in Drosophila melanogaster.

PubMed

Garcia-Garcia, Elisa; Little, Jamie C; Kalderon, Daniel

2017-08-01

Hedgehog (Hh) regulates the Cubitus interruptus (Ci) transcription factor in Drosophila melanogaster by activating full-length Ci-155 and blocking processing to the Ci-75 repressor. However, the interplay between the regulation of Ci-155 levels and activity, as well as processing-independent mechanisms that affect Ci-155 levels, have not been explored extensively. Here, we identified Mago Nashi (Mago) and Y14 core Exon Junction Complex (EJC) proteins, as well as the Srp54 splicing factor, as modifiers of Hh pathway activity under sensitized conditions. Mago inhibition reduced Hh pathway activity by altering the splicing pattern of ci to reduce Ci-155 levels. Srp54 inhibition also affected pathway activity by reducing ci RNA levels but additionally altered Ci-155 levels and activity independently of ci splicing. Further tests using ci transgenes and ci mutations confirmed evidence from studying the effects of Mago and Srp54 that relatively small changes in the level of Ci-155 primary translation product alter Hh pathway activity under a variety of sensitized conditions. We additionally used ci transgenes lacking intron sequences or the presumed translation initiation codon for an alternatively spliced ci RNA to provide further evidence that Mago acts principally by modulating the levels of the major ci RNA encoding Ci-155, and to show that ci introns are necessary to support the production of sufficient Ci-155 for robust Hh signaling and may also be important mediators of regulatory inputs. Copyright © 2017 by the Genetics Society of America.

Inhibition of aac(6′)-Ib-mediated amikacin resistance by nuclease-resistant external guide sequences in bacteria

PubMed Central

Soler Bistué, Alfonso J. C.; Martín, Fernando A.; Vozza, Nicolás; Ha, Hongphuc; Joaquín, Jonathan C.; Zorreguieta, Angeles; Tolmasky, Marcelo E.

2009-01-01

Inhibition of bacterial gene expression by RNase P-directed cleavage is a promising strategy for the development of antibiotics and pharmacological agents that prevent expression of antibiotic resistance. The rise in multiresistant bacteria harboring AAC(6′)-Ib has seriously limited the effectiveness of amikacin and other aminoglycosides. We have recently shown that recombinant plasmids coding for external guide sequences (EGS), short antisense oligoribonucleotides (ORN) that elicit RNase P-mediated cleavage of a target mRNA, induce inhibition of expression of aac(6′)-Ib and concomitantly induce a significant decrease in the levels of resistance to amikacin. However, since ORN are rapidly degraded by nucleases, development of a viable RNase P-based antisense technology requires the design of nuclease-resistant RNA analog EGSs. We have assayed a variety of ORN analogs of which selected LNA/DNA co-oligomers elicited RNase P-mediated cleavage of mRNA in vitro. Although we found an ideal configuration of LNA/DNA residues, there seems not to be a correlation between number of LNA substitutions and level of activity. Exogenous administration of as low as 50 nM of an LNA/DNA co-oligomer to the hyperpermeable E. coli AS19 harboring the aac(6′)-Ib inhibited growth in the presence of amikacin. Our experiments strongly suggest an RNase P-mediated mechanism in the observed antisense effect. PMID:19666539

Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sorscher, E.J.; Kirk, K.L.; Weaver, M.L.

The authors have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropryl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment for 24 hr virtually abolished Cl{sup {minus}} transport inmore » sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br{sup {minus}} uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatments, but not after treatments for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl{sup {minus}} transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl{sup {minus}} permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl{sup {minus}} permeability.« less

The Database Management Module of the Splice System.

DTIC Science & Technology

1983-06-01

standardization is the only wise chocs . E. FUNCTIONS OF THE EATABASE MkNAGEMENT MODULE As a result of onqoing research in thmc impl1msntaticn of SPLICE, thns...u an e-v Offset by one or mc--l orders of ma#-inuIs inorcvesnnt --L tue execution time cf user transacdrioas. Purthermore, ’is s-toraqe requlrement

Anti-NGF Local Therapy for Autonomic Dysreflexia in Spinal Cord Injury

DTIC Science & Technology

2013-10-01

growth factor in the urothelium of sham treated rats, which was decreased 5 by antisense treatment (Fig. 4A) and (5) increased nerve growth...sensitization. B Figure 4. A: Antisense OND mediated suppression of acetic acid (AA) induced NGF protein expression in urothelium . AA exposure...upregulation in the bladder urothelium of SCI rats  Detection of hyperexcitability of bladder afferent neurons due to the reduction of A-type K+ channel

Liver as a target for oligonucleotide therapeutics.

PubMed

Sehgal, Alfica; Vaishnaw, Akshay; Fitzgerald, Kevin

2013-12-01

Oligonucleotide-based therapeutics are an emerging class of drugs that hold the promise for silencing "un-druggable" targets,thus creating unique opportunities for innovative medicines. As opposed to gene therapy, oligonucleotides are considered to be more akin to small molecule therapeutics because they are small,completely synthetic in origin, do not integrate into the host genome,and have a defined duration of therapeutic activity after which effects recover to baseline. They offer a high degree of specificity at the genetic level, thereby reducing off-target effects.At the same time, they provide a strategy for targeting any gene in the genome, including transcripts that produce mutated proteins.Oligonucleotide-based therapeutics include short interfering RNA (siRNA), that degrade target mRNA through RISC mediated RNAi; anti-miRs, that target miRNAs; miRNA mimics, that regulate target mRNA; antisense oligonucleotides, that may be working through RNAseH mediated mRNA decay; mRNA upregulation,by targeting long non-coding RNAs; and oligonucleotides induced alternative splicing [1]. All these approaches require some minimal degree of homology at the nucleic acid sequence level for them to be functional. The different mechanisms of action and their relevant activity are outlined in Fig. 1. Besides homology,RNA secondary structure has also been exploited in the case of ribozymes and aptamers, which act by binding to nucleic acids or proteins, respectively. While there have been many reports of gene knockdown and gene modulation in cell lines and mice with all these methods, very few have advanced to clinical stages.The main obstacle to date has been the safe and effective intracellular delivery of these compounds in higher species, including humans. Indeed, their action requires direct interaction with DNA/RNA within the target cell so even when one solves the issues of tissue and cellular access, intracellular/intranuclear location represents yet another barrier to overcome. To date,hepatic delivery of oligonucleotides has been the area with greatest progress, and thus we have focused on liver-targeted therapeutics that have shown promise at the preclinical and/or clinical level.The liver is the largest internal organ in the body, playing a central role in metabolism, detoxification, synthesis, and secretion of major plasma proteins (carrier proteins, coagulation factors,complement components, hormones, and apolipoproteins),and iron homeostasis. It is therefore not surprising that a large number of disease targets reside in the liver where they are susceptible to modulation by oligonucleotide therapies.

Splicing fidelity: DEAD/H-box ATPases as molecular clocks.

PubMed

Koodathingal, Prakash; Staley, Jonathan P

2013-07-01

The spliceosome discriminates against suboptimal substrates, both during assembly and catalysis, thereby enhancing specificity during pre-mRNA splicing. Central to such fidelity mechanisms are a conserved subset of the DEAD- and DEAH-box ATPases, which belong to a superfamily of proteins that mediate RNP rearrangements in almost all RNA-dependent processes in the cell. Through an investigation of the mechanisms contributing to the specificity of 5' splice site cleavage, two related reports, one from our lab and the other from the Cheng lab, have provided insights into fidelity mechanisms utilized by the spliceosome. In our work, we found evidence for a kinetic proofreading mechanism in splicing in which the DEAH-box ATPase Prp16 discriminates against substrates undergoing slow 5' splice site cleavage. Additionally, our study revealed that discriminated substrates are discarded through a general spliceosome disassembly pathway, mediated by another DEAH-box ATPase Prp43. In their work, Tseng et al. described the underlying molecular events through which Prp16 discriminates against a splicing substrate during 5' splice site cleavage. Here, we present a synthesis of these two studies and, additionally, provide the first biochemical evidence for discrimination of a suboptimal splicing substrate just prior to 5' splice site cleavage. Together, these findings support a general mechanism for a ubiquitous superfamily of ATPases in enhancing specificity during RNA-dependent processes in the cell.

Processing of fish lg heavy chain transcripts diverse splicing patterns and unusual nonsense mediated decay

USDA-ARS?s Scientific Manuscript database

Alternate pathways of RNA processing play an important role in the expression of the secreted (S) and membrane (Mb) forms of immunoglobulin (Ig) heavy (H) chain isotypes in all vertebrates. Interestingly, while the differential splicing mechanism and the splice sites that generate the two forms of I...

Integrative Analysis of Many RNA-Seq Datasets to Study Alternative Splicing

PubMed Central

Li, Wenyuan; Dai, Chao; Kang, Shuli; Zhou, Xianghong Jasmine

2014-01-01

Alternative splicing is an important gene regulatory mechanism that dramatically increases the complexity of the proteome. However, how alternative splicing is regulated and how transcription and splicing are coordinated are still poorly understood, and functions of transcript isoforms have been studied only in a few limited cases. Nowadays, RNA-seq technology provides an exceptional opportunity to study alternative splicing on genome-wide scales and in an unbiased manner. With the rapid accumulation of data in public repositories, new challenges arise from the urgent need to effectively integrate many different RNA-seq datasets for study alterative splicing. This paper discusses a set of advanced computational methods that can integrate and analyze many RNA-seq datasets to systematically identify splicing modules, unravel the coupling of transcription and splicing, and predict the functions of splicing isoforms on a genome-wide scale. PMID:24583115

In vitro knockout of human p47phox blocks superoxide anion production and LDL oxidation by activated human monocytes.

PubMed

Bey, E A; Cathcart, M K

2000-03-01

We previously reported that superoxide dismutase (SOD) blocked human monocyte oxidation of LDL and therefore concluded that superoxide anion (O(2)(.-)) was required for oxidation. Others, however, have suggested that SOD may inhibit by mechanisms alternative to the dismutation of O(2)(.-). This study definitively addresses the involvement of O(2)(.-) in monocyte oxidation of LDL. Using an antisense ODN designed to target p47phox mRNA, we found that treatment of monocytes with antisense ODN caused a substantial and selective decrease in expression of p47phox protein, whereas sense ODN was without effect. Corresponding functional assays demonstrated that antisense ODN inhibited production of O(2)(.-). As sense ODN caused no inhibition of O(2)(.-) production, these results suggested that inhibition of p47phox expression caused reduction in O(2)(.-) production. Evaluation of the contribution of O(2)(.-) production to monocyte-mediated oxidation of LDL lipids confirmed that O(2)(.-) production is required for LDL lipid oxidation as antisense ODN treatment significantly inhibited LDL oxidation whereas sense ODN treatment caused no inhibition. This is the first report of the reduction of NADPH oxidase activity in intact human monocytes by directly targeting the mRNA of a significant member of this enzyme complex. Our results provide convincing data that O(2)(.-) is indeed required for monocyte-mediated LDL oxidation.

When SUMO met splicing.

PubMed

Pozzi, Berta; Mammi, Pablo; Bragado, Laureano; Giono, Luciana E; Srebrow, Anabella

2018-05-09

Spliceosomal proteins have been revealed as SUMO conjugation targets. Moreover, we have reported that many of these are in a SUMO-conjugated form when bound to a pre-mRNA substrate during a splicing reaction. We demonstrated that SUMOylation of Prp3 (PRPF3), a component of the U4/U6 di-snRNP, is required for U4/U6•U5 tri-snRNP formation and/or recruitment to active spliceosomes. Expanding upon our previous results, we have shown that the splicing factor SRSF1 stimulates SUMO conjugation to several spliceosomal proteins. Given the relevance of the splicing process, as well as the complex and dynamic nature of its governing machinery, the spliceosome, the molecular mechanisms that modulate its function represent an attractive topic of research. We posit that SUMO conjugation could represent a way of modulating spliceosome assembly and thus, splicing efficiency. How cycles of SUMOylation/de-SUMOylation of spliceosomal proteins become integrated throughout the highly choreographed spliceosomal cycle awaits further investigation.

High temperature attenuates the gravitropism of inflorescence stems by inducing SHOOT GRAVITROPISM 5 alternative splicing in Arabidopsis.

PubMed

Kim, Joo-Young; Ryu, Jae Yong; Baek, Kon; Park, Chung-Mo

2016-01-01

In higher plants, gravitropism proceeds through three sequential steps in the responding organs: perception of gravity signals, signal transduction and asymmetric cell elongation. Light and temperature also influence the gravitropic orientation of plant organs. A series of Arabidopsis shoot gravitropism (sgr) mutants has been shown to exhibit disturbed shoot gravitropism. SGR5 is functionally distinct from other SGR members in that it mediates the early events of gravitropic responses in inflorescence stems. Here, we demonstrated that SGR5 alternative splicing produces two protein variants (SGR5α and SGR5β) in modulating the gravitropic response of inflorescence stems at high temperatures. SGR5β inhibits SGR5α function by forming non-DNA-binding heterodimers. Transgenic plants overexpressing SGR5β (35S:SGR5β) exhibit reduced gravitropic growth of inflorescence stems, as observed in the SGR5-deficient sgr5-5 mutant. Interestingly, SGR5 alternative splicing is accelerated at high temperatures, resulting in the high-level accumulation of SGR5β transcripts. When plants were exposed to high temperatures, whereas gravitropic curvature was reduced in Col-0 inflorescence stems, it was uninfluenced in the inflorescence stems of 35S:SGR5β transgenic plants and sgr5-5 mutant. We propose that the thermoresponsive alternative splicing of SGR5 provides an adaptation strategy by which plants protect the shoots from hot air under high temperature stress in natural habitats. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Fox-2 Splicing Factor Binds to a Conserved Intron Motif to PromoteInclusion of Protein 4.1R Alternative Exon 16

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ponthier, Julie L.; Schluepen, Christina; Chen, Weiguo

Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of hnRNP A/B proteins to silencer elements in the exon and that downregulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This paper demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding tomore » the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2, but not Fox-1. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.« less

Regulation of alternative splicing by local histone modifications: potential roles for RNA-guided mechanisms

PubMed Central

Zhou, Hua-Lin; Luo, Guangbin; Wise, Jo Ann; Lou, Hua

2014-01-01

The molecular mechanisms through which alternative splicing and histone modifications regulate gene expression are now understood in considerable detail. Here, we discuss recent studies that connect these two previously separate avenues of investigation, beginning with the unexpected discoveries that nucleosomes are preferentially positioned over exons and DNA methylation and certain histone modifications also show exonic enrichment. These findings have profound implications linking chromatin structure, histone modification and splicing regulation. Complementary single gene studies provided insight into the mechanisms through which DNA methylation and histones modifications modulate alternative splicing patterns. Here, we review an emerging theme resulting from these studies: RNA-guided mechanisms integrating chromatin modification and splicing. Several groundbreaking papers reported that small noncoding RNAs affect alternative exon usage by targeting histone methyltransferase complexes to form localized facultative heterochromatin. More recent studies provided evidence that pre-messenger RNA itself can serve as a guide to enable precise alternative splicing regulation via local recruitment of histone-modifying enzymes, and emerging evidence points to a similar role for long noncoding RNAs. An exciting challenge for the future is to understand the impact of local modulation of transcription elongation rates on the dynamic interplay between histone modifications, alternative splicing and other processes occurring on chromatin. PMID:24081581

Regulation of alternative mRNA splicing: old players and new perspectives.

PubMed

Dvinge, Heidi

2018-06-01

Nearly all human multi-exon genes are subject to alternative splicing in one or more cell types. The splicing machinery, therefore, has to select between multiple splice sites in a context-dependent manner, relying on sequence features in cis and trans-acting splicing regulators that either promote or repress splice site recognition and spliceosome assembly. However, the functional coupling between multiple gene regulatory layers signifies that splicing can also be modulated by transcriptional or epigenetic characteristics. Other, less obvious, aspects of alternative splicing have come to light in recent years, often involving core components of the spliceosome previously thought to perform a basal rather than a regulatory role in splicing. Together this paints a highly dynamic picture of splicing regulation, where the final splice site choice is governed by the entire transcriptional environment of a gene and its cellular context. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Current status of antisense RNA-mediated gene regulation in Listeria monocytogenes.

PubMed

Schultze, Tilman; Izar, Benjamin; Qing, Xiaoxing; Mannala, Gopala K; Hain, Torsten

2014-01-01

Listeria monocytogenes is a Gram-positive human-pathogen bacterium that served as an experimental model for investigating fundamental processes of adaptive immunity and virulence. Recent novel technologies allowed the identification of several hundred non-coding RNAs (ncRNAs) in the Listeria genome and provided insight into an unexpected complex transcriptional machinery. In this review, we discuss ncRNAs that are encoded on the opposite strand of the target gene and are therefore termed antisense RNAs (asRNAs). We highlight mechanistic and functional concepts of asRNAs in L. monocytogenes and put these in context of asRNAs in other bacteria. Understanding asRNAs will further broaden our knowledge of RNA-mediated gene regulation and may provide targets for diagnostic and antimicrobial development.

Splicing predictions reliably classify different types of alternative splicing

PubMed Central

Busch, Anke; Hertel, Klemens J.

2015-01-01

Alternative splicing is a key player in the creation of complex mammalian transcriptomes and its misregulation is associated with many human diseases. Multiple mRNA isoforms are generated from most human genes, a process mediated by the interplay of various RNA signature elements and trans-acting factors that guide spliceosomal assembly and intron removal. Here, we introduce a splicing predictor that evaluates hundreds of RNA features simultaneously to successfully differentiate between exons that are constitutively spliced, exons that undergo alternative 5′ or 3′ splice-site selection, and alternative cassette-type exons. Surprisingly, the splicing predictor did not feature strong discriminatory contributions from binding sites for known splicing regulators. Rather, the ability of an exon to be involved in one or multiple types of alternative splicing is dictated by its immediate sequence context, mainly driven by the identity of the exon's splice sites, the conservation around them, and its exon/intron architecture. Thus, the splicing behavior of human exons can be reliably predicted based on basic RNA sequence elements. PMID:25805853

New discoveries of old SON: a link between RNA splicing and cancer.

PubMed

Hickey, Christopher J; Kim, Jung-Hyun; Ahn, Eun-Young Erin

2014-02-01

The SON protein is a ubiquitously expressed DNA- and RNA-binding protein primarily localized to nuclear speckles. Although several early studies implicated SON in DNA-binding, tumorigenesis and apoptosis, functional significance of this protein had not been recognized until recent studies discovered SON as a novel RNA splicing co-factor. During constitutive RNA splicing, SON ensures efficient intron removal from the transcripts containing suboptimal splice sites. Importantly, SON-mediated splicing is required for proper processing of selective transcripts related to cell cycle, microtubules, centrosome maintenance, and genome stability. Moreover, SON regulates alternative splicing of RNAs from the genes involved in apoptosis and epigenetic modification. In addition to the role in RNA splicing, SON has an ability to suppress transcriptional activation at certain promoter/enhancer DNA sequences. Considering the multiple SON target genes which are directly involved in cell proliferation, genome stability and chromatin modifications, SON is an emerging player in gene regulation during cancer development and progression. Here, we summarize available information from several early studies on SON, and highlight recent discoveries describing molecular mechanisms of SON-mediated gene regulation. We propose that our future effort on better understanding of diverse SON functions would reveal novel targets for cancer therapy. © 2013 Wiley Periodicals, Inc.

Human Herpesvirus-8-Transformed Endothelial Cells Have Functionally Activated Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor

PubMed Central

Masood, Rizwan; Cesarman, Ethel; Smith, D. Lynne; Gill, Parkash S.; Flore, Ornella

2002-01-01

Kaposi’s sarcoma is a vascular tumor commonly associated with human immunodeficiency virus (HIV)-1 and human herpesvirus (HHV-8) also known as Kaposi’s sarcoma-associated herpesvirus. The principal features of this tumor are abnormal proliferation of vascular structures lined with spindle-shaped endothelial cells. HHV-8 may transform a subpopulation of endothelial cells in vitro via viral and cellular gene expression. We hypothesized that among the cellular genes, vascular endothelial growth factors (VEGFs) and their cognate receptors may be involved in viral-mediated transformation. We have shown that HHV-8-transformed endothelial cells (EC-HHV-8) express higher levels of VEGF, VEGF-C, VEGF-D, and PlGF in addition to VEGF receptors-1, -2, and -3. Furthermore, antibodies to VEGF receptor-2 inhibited cell proliferation and viability. Similarly, inhibition of VEGF gene expression with antisense oligonucleotides inhibited EC-HHV-8 cell proliferation/viability. The growth and viability of primary endothelial cells and a fibroblast cell line however were unaffected by either the VEGF receptor-2 antibody or the VEGF antisense oligodeoxynucleotides. VEGF and VEGF receptors are thus induced in EC-HHV-8 and participate in the transformation. Inhibitors of VEGF may thus modulate the disease process during development and progression. PMID:11786394

Circular non-coding RNA ANRIL modulates ribosomal RNA maturation and atherosclerosis in humans

PubMed Central

Holdt, Lesca M.; Stahringer, Anika; Sass, Kristina; Pichler, Garwin; Kulak, Nils A.; Wilfert, Wolfgang; Kohlmaier, Alexander; Herbst, Andreas; Northoff, Bernd H.; Nicolaou, Alexandros; Gäbel, Gabor; Beutner, Frank; Scholz, Markus; Thiery, Joachim; Musunuru, Kiran; Krohn, Knut; Mann, Matthias; Teupser, Daniel

2016-01-01

Circular RNAs (circRNAs) are broadly expressed in eukaryotic cells, but their molecular mechanism in human disease remains obscure. Here we show that circular antisense non-coding RNA in the INK4 locus (circANRIL), which is transcribed at a locus of atherosclerotic cardiovascular disease on chromosome 9p21, confers atheroprotection by controlling ribosomal RNA (rRNA) maturation and modulating pathways of atherogenesis. CircANRIL binds to pescadillo homologue 1 (PES1), an essential 60S-preribosomal assembly factor, thereby impairing exonuclease-mediated pre-rRNA processing and ribosome biogenesis in vascular smooth muscle cells and macrophages. As a consequence, circANRIL induces nucleolar stress and p53 activation, resulting in the induction of apoptosis and inhibition of proliferation, which are key cell functions in atherosclerosis. Collectively, these findings identify circANRIL as a prototype of a circRNA regulating ribosome biogenesis and conferring atheroprotection, thereby showing that circularization of long non-coding RNAs may alter RNA function and protect from human disease. PMID:27539542

Regulation of alternative VEGF-A mRNA splicing is a therapeutic target for analgesia.

PubMed

Hulse, R P; Beazley-Long, N; Hua, J; Kennedy, H; Prager, J; Bevan, H; Qiu, Y; Fernandes, E S; Gammons, M V; Ballmer-Hofer, K; Gittenberger de Groot, A C; Churchill, A J; Harper, S J; Brain, S D; Bates, D O; Donaldson, L F

2014-11-01

Vascular endothelial growth factor-A (VEGF-A) is best known as a key regulator of the formation of new blood vessels. Neutralization of VEGF-A with anti-VEGF therapy e.g. bevacizumab, can be painful, and this is hypothesized to result from a loss of VEGF-A-mediated neuroprotection. The multiple vegf-a gene products consist of two alternatively spliced families, typified by VEGF-A165a and VEGF-A165b (both contain 165 amino acids), both of which are neuroprotective. Under pathological conditions, such as in inflammation and cancer, the pro-angiogenic VEGF-A165a is upregulated and predominates over the VEGF-A165b isoform. We show here that in rats and mice VEGF-A165a and VEGF-A165b have opposing effects on pain, and that blocking the proximal splicing event - leading to the preferential expression of VEGF-A165b over VEGF165a - prevents pain in vivo. VEGF-A165a sensitizes peripheral nociceptive neurons through actions on VEGFR2 and a TRPV1-dependent mechanism, thus enhancing nociceptive signaling. VEGF-A165b blocks the effect of VEGF-A165a. After nerve injury, the endogenous balance of VEGF-A isoforms switches to greater expression of VEGF-Axxxa compared to VEGF-Axxxb, through an SRPK1-dependent pre-mRNA splicing mechanism. Pharmacological inhibition of SRPK1 after traumatic nerve injury selectively reduced VEGF-Axxxa expression and reversed associated neuropathic pain. Exogenous VEGF-A165b also ameliorated neuropathic pain. We conclude that the relative levels of alternatively spliced VEGF-A isoforms are critical for pain modulation under both normal conditions and in sensory neuropathy. Altering VEGF-Axxxa/VEGF-Axxxb balance by targeting alternative RNA splicing may be a new analgesic strategy. Copyright © 2014. Published by Elsevier Inc.

Gene therapy for spinomuscular atrophy: a biomedical advance, a missed opportunity for more equitable drug pricing.

PubMed

Friedmann, T

2017-09-01

An experimental approach for gene therapy of spinomuscular atrophy has been reported to prevent development of the neuromuscular features of this lethal and previously untreatable disorder. The approach involves treatment of patients suffering from SMN1-associated infantile form of the disease with a splice-switching antisense oligonucleotide (ASO) that corrects aberrant splicing of the nearly identical SMN2 gene to allow the generation of functional SMN protein, thereby mitigating the development of the disease. This technique represents the first apparently effective therapy for spinal muscular atrophy (SMA) and an important documentation for ASO technology for therapy of neurodegenerative disease. These results with one form of SMA are likely to be relevant for similar applications to other SMA types and are likely to inspire application to a number of other intractable neurodegenerative diseases such as Huntington's disease, amyotrophic lateral sclerosis and possibly even the extremely common Parkinson's and Alzheimer's diseases and others. Nevertheless, the scientific and medical importance of this advance is marred by a pricing policy by the corporate sponsors that may complicate accessibility of the drug for some desperate patients.

Assessment of the feasibility of exon 45–55 multiexon skipping for duchenne muscular dystrophy

PubMed Central

van Vliet, Laura; de Winter, Christa L; van Deutekom, Judith CT; van Ommen, Gert-Jan B; Aartsma-Rus, Annemieke

2008-01-01

Background The specific skipping of an exon, induced by antisense oligonucleotides (AON) during splicing, has shown to be a promising therapeutic approach for Duchenne muscular dystrophy (DMD) patients. As different mutations require skipping of different exons, this approach is mutation dependent. The skipping of an entire stretch of exons (e.g. exons 45 to 55) has recently been suggested as an approach applicable to larger groups of patients. However, this multiexon skipping approach is technically challenging. The levels of intended multiexon skips are typically low and highly variable, and may be dependent on the order of intron removal. We hypothesized that the splicing order might favor the induction of multiexon 45–55 skipping. Methods We here tested the feasibility of inducing multiexon 45–55 in control and patient muscle cell cultures using various AON cocktails. Results In all experiments, the exon 45–55 skip frequencies were minimal and comparable to those observed in untreated cells. Conclusion We conclude that current state of the art does not sufficiently support clinical development of multiexon skipping for DMD. PMID:19046429

Targeting Cancer with Antisense Oligomers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hnatowich, DJ

With financial assistance from the Department of Energy, we have shown definitively that radiolabeled antisense DNAs and other oligomers will accumulate in target cancer cells in vitro and in vivo by an antisense mechanism. We have also shown that the number of mRNA targets for our antisense oligomers in the cancer cell types that we have investigated so far is sufficient to provide and antisense image and/or radiotherapy of cancer in mice. These studies have been reported in about 10 publications. However our observation over the past several years has shown that radiolabeled antisense oligomers administered intravenously in their nativemore » and naked form will accumulate and be retained in target xenografts by an antisense mechanism but will also accumulate at high levels in normal organs such as liver, spleen and kidneys. We have investigated unsuccessfully several commercially available vectors. Thus the use of radiolabeled antisense oligomers for the imaging of cancer must await novel approaches to delivery. This laboratory has therefore pursued two new paths, optical imaging of tumor and Auger radiotherapy. We are developing a novel method of optical imaging tumor using antisense oligomers with a fluorophore is administered while hybridized with a shorter complementary oligomer with an inhibitor. In culture and in tumored mice that the duplex remains intact and thus nonfluorescent until it encounters its target mRNA at which time it dissociates and the antisense oligomer binds along with its fluorophore to the target. Simultaneous with the above, we have also observed, as have others, that antisense oligomers migrate rapidly and quantitatively to the nucleus upon crossing cell membranes. The Auger electron radiotherapy path results from this observation since the nuclear migration properties could be used effectively to bring and to retain in the nucleus an Auger emitting radionuclide such as 111In or 125I bound to the antisense oligomer. Since the object becomes radiotherapy rather than imaging, the delivery problem may be obviated by attaching the antisense oligomer to an antitumor antibody to improve delivery following intravenous administration. Since many antibodies are trapped in endosomes following internalization, a cell penetrating peptide such as tat will also be included to ensure transport of the complex without entrapment. Rather than covalent conjugation of the three entities, we are using streptavidin as linker after biotinylated each component. Our recent efforts have concentrated on establishing the influence of the streptavidin linker on the properties of each component within the delivery nanoparticle. Thus, we have shown that the Herceptin antibody, when linked to a labeled oligomer via streptavidin, remains capable of directing the label oligomer to Her2+ tumor cells in vitro and Her2+ tumor xenografts in mice. In addition, we have demonstrated that a labeled antisense oligomer within the nanoparticle remains capable of migrating to the nucleus and binding to its target mRNA in vitro and in vivo. We have shown that the tat peptide also preserves its properties of cell transport when incubated as one component of the nanoparticle. Most recently, we have addressed another of our concerns, namely whether the streptavidin would adversely effect the biodistribution of the antisense oligomer. We were pleased to find that the 99mTc-labeled antisense MORF within the Herceptin three component and two component nanoparticles accumulated and was retained in tumor in a manner suggestive of radiolabeled Herceptin itself. Thus the preserved properties within the streptavidin delivery nanoparticle of the Herceptin antibody, the tat peptide and the 111In labeled antisense MORF oligomer will explain why we have successfully demonstrated an Auger electron-mediated, antisense-mediated radiotherapy in cells in culture. One remaining concern is that the delivery nanoparticle may deliver the Auger electron emitting radionuclide to the nucleus of normal cells as well as tumor cells. We have now performed tumored mice studies of the three component delivery nanoparticle with the antisense MORF labeled with Cy3 so that tissue slices could be examined by immunohistology for evidence of MORF accumulations in the nuclei of both tumor and normal tissues. Microscopic examination shows nuclear staining in approximately 20% of the tumor cells in animals injected with the antisense nanoparticle and 10% of the tumor cells in animals receiving the sense nanoparticle, whereas no nuclear staining is seen in the tumor cells of mice given the PBS injection as another control. No nuclear staining was observed in all sections from all normal organs. Finally, my colleagues and I wish to express our gratitude to the DOE for their generous support of our research at a time when the NIH was unwilling to fund what they believed to be a risky« less

Size-Uniform 200 nm Particles: Fabrication and Application to Magnetofection

PubMed Central

Mair, Lamar; Ford, Kris; Alam, Rowshon; Kole, Ryszard; Fisher, Michael; Superfine, Richard

2009-01-01

We report on the fabrication of arrays of mono- and multimetallic particles via metal evaporation onto lithographically patterned posts, as well as the magnetic force calibration and successful magnetofection of iron particles grown via this method. This work represents the first instance in which metal evaporation onto post structures was used for the formation of released, shape-defined metal particles. Also, our work represents the first use of lithographically defined particles as agents of magnetofection. Using these techniques it is possible to create particles with complex shapes and lateral dimensions as small as 40 nm. Our demonstrated compositionally flexible particles are highly size-uniform due to their photolithographically defined growth substrates, with particle dimensions along two axes fixed at 200 nm; the third axis dimension can be varied from 20 nm to 300 nm during the deposition procedure. Atomic percent of metals incorporated into the particle volume is highly tunable and particles have been synthesized with as many as four different metals. We performed magnetic force calibrations on a single particle size for iron particles using an axially magnetized NeFeB permanent magnet and comparisons are made with commercially available magnetic beads. In order to evalutate their usefulness as magnetofection agents, an antisense oligonucleotide (ODN) designed to correct the aberrant splicing of enhanced green fluorescent protein mRNA, was successfully transfected into a modified HeLa cell line. Magnetically enhanced gene delivery was accomplished in vitro using antisense ODN-laden iron particles followed by application of a field gradient. Magnetically enhanced transfection resulted in a 76% and 139% increase in fluorescence intensity when compared to Lipofectamine and antisense ODN-loaded particles delivered without magnetic treatment, respectively. To our knowledge, these experiments constitute the first use of lithographically defined particles as successful agents for magnetically enhanced transfection of an antisense oligonucleotide. PMID:20055096

Effects of miR-33a-5P on ABCA1/G1-Mediated Cholesterol Efflux under Inflammatory Stress in THP-1 Macrophages

PubMed Central

Mao, Min; Lei, Han; Liu, Qing; Chen, Yaxi; Zhao, Lei; Li, Qing; Luo, Suxin; Zuo, Zhong; He, Quan; Huang, Wei; Zhang, Nan; Zhou, Chao; Ruan, Xiong Z.

2014-01-01

The present study is to investigate whether inflammatory cytokines inhibit ABCA1/ABCG1-mediated cholesterol efflux by regulating miR-33a-5P in THP-1 macrophages. We used interleukin-6 and tumor necrosis factor-alpha in the presence or absence of native low density lipoprotein (LDL) to stimulate THP-1 macrophages. THP-1 macrophages were infected by either control lentivirus vectors or lentivirus encoding miR-33a-5P or antisense miR-33a-5P. The effects of inflammatory cytokines, miR-33a-5P and antisense miR-33a-5P on intracellular lipids accumulation and intracellular cholesterol contents were assessed by oil red O staining and quantitative intracellular cholesterol assay. ApoA-I-mediated cholesterol efflux was examined using the fluorescent sterol (BODIPY-cholesterol). The gene and protein expressions of the molecules involved in cholesterol trafficking were examined using quantitative real-time polymerase chain reaction and Western blotting. Inflammatory cytokines or miR-33a-5P increased intracellular lipid accumulation and decreased apoA-I-mediated cholesterol efflux via decreasing the expression of ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. However, antisense miR-33a-5P reversed the effects of inflammatory cytokines on intracellular lipid accumulation, cholesterol efflux, and the expression of miR-33a-5P, ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. This study indicated that inflammatory cytokines inhibited ABCA1/ABCG1-mediated cholesterol efflux by up-regulating miR-33a-5P in THP-1 macrophages. PMID:25329888

Effects of miR-33a-5P on ABCA1/G1-mediated cholesterol efflux under inflammatory stress in THP-1 macrophages.

PubMed

Mao, Min; Lei, Han; Liu, Qing; Chen, Yaxi; Zhao, Lei; Li, Qing; Luo, Suxin; Zuo, Zhong; He, Quan; Huang, Wei; Zhang, Nan; Zhou, Chao; Ruan, Xiong Z

2014-01-01

The present study is to investigate whether inflammatory cytokines inhibit ABCA1/ABCG1-mediated cholesterol efflux by regulating miR-33a-5P in THP-1 macrophages. We used interleukin-6 and tumor necrosis factor-alpha in the presence or absence of native low density lipoprotein (LDL) to stimulate THP-1 macrophages. THP-1 macrophages were infected by either control lentivirus vectors or lentivirus encoding miR-33a-5P or antisense miR-33a-5P. The effects of inflammatory cytokines, miR-33a-5P and antisense miR-33a-5P on intracellular lipids accumulation and intracellular cholesterol contents were assessed by oil red O staining and quantitative intracellular cholesterol assay. ApoA-I-mediated cholesterol efflux was examined using the fluorescent sterol (BODIPY-cholesterol). The gene and protein expressions of the molecules involved in cholesterol trafficking were examined using quantitative real-time polymerase chain reaction and Western blotting. Inflammatory cytokines or miR-33a-5P increased intracellular lipid accumulation and decreased apoA-I-mediated cholesterol efflux via decreasing the expression of ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. However, antisense miR-33a-5P reversed the effects of inflammatory cytokines on intracellular lipid accumulation, cholesterol efflux, and the expression of miR-33a-5P, ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. This study indicated that inflammatory cytokines inhibited ABCA1/ABCG1-mediated cholesterol efflux by up-regulating miR-33a-5P in THP-1 macrophages.

TGFβ1-mediated expression and alternative splicing of Fibronectin Extra Domain A in human podocyte culture.

PubMed

Madne, Tarunkumar Hemraj; Dockrell, Mark Edward Carl

2018-02-28

Alternative splicing is a fundamental phenomenon to build protein diversity in health and diseases. Extra Domain A+ Fibronectin (EDA+Fn) is an alternatively spliced form of fibronectin protein present in the extra cellular matrix (ECM) in renal fibrosis. Podocytes are spectacular cell type and play a key role in filtration and synthesise ECM proteins in renal physiology and pathology. TGFβ1 is a strong stimulator of ECM proteins in renal injury. In this study, we have investigated alternative splicing of EDA+ Fn in human podocytes in response to TGFβ1. We have performed western blotting and immunofluorescence to characterise the expression of the EDA+Fn protein, real-time PCR for RNA expression and RT-PCR to look for alternative splicing of EDA+Fn in conditionally immortalised human podocytes culture.We used TGFβ1 as a stimulator and SB431542 and SRPIN340 for inhibitory studies. In this work, for the first time we have demonstrated in human podocytes culture EDA+Fn is expressed in the basal condition and TGFβ1 2.5ng/ml induced the Fn mRNA and EDA+Fn protein expression demonstrated by real-time PCR, western blotting and immunofluorescence. TGFβ1 2.5ng/ml induced the alternative splicing of EDA+Fn shown by conventional RT-PCR. Studies with ALK5 inhibitor SB431542 and SRPIN340 show that TGFβ1 induced alternative splicing of EDA+Fn was by the ALK5 receptor and the SR proteins.  In human podocytes culture, alternative splicing of EDA+Fn occurs at basal conditions and TGFβ1 further induced the alternative splicing of EDA+Fn via ALK5 receptor activation and SR proteins. This is the first evidence of basal and TGFβ1 mediated alternative splicing of EDA+Fn in human podocytes culture.

IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells.

PubMed

Fiume, Giuseppe; Scialdone, Annarita; Rizzo, Francesca; De Filippo, Maria Rosaria; Laudanna, Carmelo; Albano, Francesco; Golino, Gaetanina; Vecchio, Eleonora; Pontoriero, Marilena; Mimmi, Selena; Ceglia, Simona; Pisano, Antonio; Iaccino, Enrico; Palmieri, Camillo; Paduano, Sergio; Viglietto, Giuseppe; Weisz, Alessandro; Scala, Giuseppe; Quinto, Ileana

2016-11-07

The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in IBTK -shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in IBTK -shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3'- and 5'-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein.

IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells

PubMed Central

Fiume, Giuseppe; Scialdone, Annarita; Rizzo, Francesca; De Filippo, Maria Rosaria; Laudanna, Carmelo; Albano, Francesco; Golino, Gaetanina; Vecchio, Eleonora; Pontoriero, Marilena; Mimmi, Selena; Ceglia, Simona; Pisano, Antonio; Iaccino, Enrico; Palmieri, Camillo; Paduano, Sergio; Viglietto, Giuseppe; Weisz, Alessandro; Scala, Giuseppe; Quinto, Ileana

2016-01-01

The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in IBTK-shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in IBTK-shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3′- and 5′-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein. PMID:27827994

Comparison of Zebrafish tmem88a mutant and morpholino knockdown phenotypes

PubMed Central

Place, Elsie S.; Smith, James C.

2017-01-01

Tmem88a is a transmembrane protein that is thought to be a negative regulator of the Wnt signalling pathway. Several groups have used antisense morpholino oligonucleotides in an effort to characterise the role of tmem88a in zebrafish cardiovascular development, but they have not obtained consistent results. Here, we generate an 8 bp deletion in the coding region of the tmem88a locus using TALENs, and we have gone on to establish a viable homozygous tmem88aΔ8 mutant line. Although tmem88aΔ8 mutants have reduced expression of some key haematopoietic genes, differentiation of erythrocytes and neutrophils is unaffected, contradicting our previous study using antisense morpholino oligonucleotides. We find that expression of the tmem88a paralogue tmem88b is not significantly changed in tmem88aΔ8 mutants and injection of the tmem88a splice-blocking morpholino oligonucleotide into tmem88aΔ8 mutants recapitulates the reduction of erythrocytes observed in morphants using o-Dianisidine. This suggests that there is a partial, but inessential, requirement for tmem88a during haematopoiesis and that morpholino injection exacerbates this phenotype in tmem88a morpholino knockdown embryos. PMID:28192479

The nuclear receptor ERβ engages AGO2 in regulation of gene transcription, RNA splicing and RISC loading.

PubMed

Tarallo, Roberta; Giurato, Giorgio; Bruno, Giuseppina; Ravo, Maria; Rizzo, Francesca; Salvati, Annamaria; Ricciardi, Luca; Marchese, Giovanna; Cordella, Angela; Rocco, Teresa; Gigantino, Valerio; Pierri, Biancamaria; Cimmino, Giovanni; Milanesi, Luciano; Ambrosino, Concetta; Nyman, Tuula A; Nassa, Giovanni; Weisz, Alessandro

2017-10-06

The RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNA-silencing pathways It exerts a pivotal role in microRNA maturation and activity and can modulate chromatin remodeling, transcriptional gene regulation and RNA splicing. Estrogen receptor beta (ERβ) is endowed with oncosuppressive activities, antagonizing hormone-induced carcinogenesis and inhibiting growth and oncogenic functions in luminal-like breast cancers (BCs), where its expression correlates with a better prognosis of the disease. Applying interaction proteomics coupled to mass spectrometry to characterize nuclear factors cooperating with ERβ in gene regulation, we identify AGO2 as a novel partner of ERβ in human BC cells. ERβ-AGO2 association was confirmed in vitro and in vivo in both the nucleus and cytoplasm and is shown to be RNA-mediated. ChIP-Seq demonstrates AGO2 association with a large number of ERβ binding sites, and total and nascent RNA-Seq in ERβ + vs ERβ - cells, and before and after AGO2 knock-down in ERβ + cells, reveals a widespread involvement of this factor in ERβ-mediated regulation of gene transcription rate and RNA splicing. Moreover, isolation and sequencing by RIP-Seq of ERβ-associated long and small RNAs in the cytoplasm suggests involvement of the nuclear receptor in RISC loading, indicating that it may also be able to directly control mRNA translation efficiency and stability. These results demonstrate that AGO2 can act as a pleiotropic functional partner of ERβ, indicating that both factors are endowed with multiple roles in the control of key cellular functions.

Spliced XBP1 promotes macrophage survival and autophagy by interacting with Beclin-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tian, Ping-Ge; Jiang, Zhi-Xin; Li, Jian-Hua

Macrophage autophagy plays an important role in the development of atherosclerosis, but the precise mechanism mediating this process is unclear. The potential role of the X-box binding protein 1 (XBP1), a crucial transduction factor that is involved in endoplasmic reticulum stress and the unfolded protein response, in bone marrow-derived macrophage autophagy is unknown. This study mainly explores the roles of XBP1 mRNA splicing in bone marrow-derived macrophage autophagy. The present study shows that the transient overexpression of spliced XBP1 via adenovirus-mediated gene transfer induces autophagy and promotes proliferation in bone marrow-derived macrophages via the down-regulation of Beclin-1, but that themore » sustained overexpression of spliced XBP1 leads to apoptosis. When XBP1 is down-regulated in bone marrow-derived macrophages using siRNA, rapamycin-induced autophagosome formation is ablated. Furthermore, we have detected the overexpression of XBP1 in areas of atherosclerotic plaques in the arteries of ApoE−/− mice. These results demonstrate that XBP1 mRNA splicing plays an important role in maintaining the function of bone marrow-derived macrophages and provide new insight into the study and treatment of atherosclerosis. - Highlights: • XBP1 was up-regulated in atherosclerotic plaques of ApoE−/− mice. • Transient spliced XBP1 overexpression induced macrophages autophagy via Beclin-1. • Sustained spliced XBP1 overexpression triggered macrophages apoptosis. • Spliced XBP1 plays a key role in maintaining the macrophages survival.« less

Rescue of peripheral vestibular function in Usher syndrome mice using a splice-switching antisense oligonucleotide.

PubMed

Vijayakumar, Sarath; Depreux, Frederic F; Jodelka, Francine M; Lentz, Jennifer J; Rigo, Frank; Jones, Timothy A; Hastings, Michelle L

2017-09-15

Usher syndrome type 1C (USH1C/harmonin) is associated with profound retinal, auditory and vestibular dysfunction. We have previously reported on an antisense oligonucleotide (ASO-29) that dramatically improves auditory function and balance behavior in mice homozygous for the harmonin mutation Ush1c c.216G > A following a single systemic administration. The findings were suggestive of improved vestibular function; however, no direct vestibular assessment was made. Here, we measured vestibular sensory evoked potentials (VsEPs) to directly assess vestibular function in Usher mice. We report that VsEPs are absent or abnormal in Usher mice, indicating profound loss of vestibular function. Strikingly, Usher mice receiving ASO-29 treatment have normal or elevated vestibular response thresholds when treated during a critical period between postnatal day 1 and 5, respectively. In contrast, treatment of mice with ASO-29 treatment at P15 was minimally effective at rescuing vestibular function. Interestingly, ASO-29 treatment at P1, P5 or P15 resulted in sufficient vestibular recovery to support normal balance behaviors, suggesting a therapeutic benefit to balance with ASO-29 treatment at P15 despite the profound vestibular functional deficits that persist with treatment at this later time. These findings provide the first direct evidence of an effective treatment of peripheral vestibular function in a mouse model of USH1C and reveal the potential for using antisense technology to treat vestibular dysfunction. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

An antisense RNA in a lytic cyanophage links psbA to a gene encoding a homing endonuclease.

PubMed

Millard, Andrew D; Gierga, Gregor; Clokie, Martha R J; Evans, David J; Hess, Wolfgang R; Scanlan, David J

2010-09-01

Cyanophage genomes frequently possess the psbA gene, encoding the D1 polypeptide of photosystem II. This protein is believed to maintain host photosynthetic capacity during infection and enhance phage fitness under high-light conditions. Although the first documented cyanophage-encoded psbA gene contained a group I intron, this feature has not been widely reported since, despite a plethora of new sequences becoming available. In this study, we show that in cyanophage S-PM2, this intron is spliced during the entire infection cycle. Furthermore, we report the widespread occurrence of psbA introns in marine metagenomic libraries, and with psbA often adjacent to a homing endonuclease (HE). Bioinformatic analysis of the intergenic region between psbA and the adjacent HE gene F-CphI in S-PM2 showed the presence of an antisense RNA (asRNA) connecting these two separate genetic elements. The asRNA is co-regulated with psbA and F-CphI, suggesting its involvement with their expression. Analysis of scaffolds from global ocean survey datasets shows this asRNA to be commonly associated with the 3' end of cyanophage psbA genes, implying that this potential mechanism of regulating marine 'viral' photosynthesis is evolutionarily conserved. Although antisense transcription is commonly found in eukaryotic and increasingly also in prokaryotic organisms, there has been no indication for asRNAs in lytic phages so far. We propose that this asRNA also provides a means of preventing the formation of mobile group I introns within cyanophage psbA genes.

Algorithm to Identify Frequent Coupled Modules from Two-Layered Network Series: Application to Study Transcription and Splicing Coupling

PubMed Central

Li, Wenyuan; Dai, Chao; Liu, Chun-Chi

2012-01-01

Abstract Current network analysis methods all focus on one or multiple networks of the same type. However, cells are organized by multi-layer networks (e.g., transcriptional regulatory networks, splicing regulatory networks, protein-protein interaction networks), which interact and influence each other. Elucidating the coupling mechanisms among those different types of networks is essential in understanding the functions and mechanisms of cellular activities. In this article, we developed the first computational method for pattern mining across many two-layered graphs, with the two layers representing different types yet coupled biological networks. We formulated the problem of identifying frequent coupled clusters between the two layers of networks into a tensor-based computation problem, and proposed an efficient solution to solve the problem. We applied the method to 38 two-layered co-transcription and co-splicing networks, derived from 38 RNA-seq datasets. With the identified atlas of coupled transcription-splicing modules, we explored to what extent, for which cellular functions, and by what mechanisms transcription-splicing coupling takes place. PMID:22697243

Splicing stimulates siRNA formation at Drosophila DNA double-strand breaks

PubMed Central

Merk, Karin; Breinig, Marco; Böttcher, Romy; Krebs, Stefan; Blum, Helmut; Boutros, Michael

2017-01-01

DNA double-strand breaks trigger the production of locus-derived siRNAs in fruit flies, human cells and plants. At least in flies, their biogenesis depends on active transcription running towards the break. Since siRNAs derive from a double-stranded RNA precursor, a major question is how broken DNA ends can generate matching sense and antisense transcripts. We performed a genome-wide RNAi-screen in cultured Drosophila cells, which revealed that in addition to DNA repair factors, many spliceosome components are required for efficient siRNA generation. We validated this observation through site-specific DNA cleavage with CRISPR-cas9 followed by deep sequencing of small RNAs. DNA breaks in intron-less genes or upstream of a gene’s first intron did not efficiently trigger siRNA production. When DNA double-strand breaks were induced downstream of an intron, however, this led to robust siRNA generation. Furthermore, a downstream break slowed down splicing of the upstream intron and a detailed analysis of siRNA coverage at the targeted locus revealed that unspliced pre-mRNA contributes the sense strand to the siRNA precursor. Since splicing factors are stimulating the response but unspliced transcripts are entering the siRNA biogenesis, the spliceosome is apparently stalled in a pre-catalytic state and serves as a signaling hub. We conclude that convergent transcription at DNA breaks is stimulated by a splicing dependent control process. The resulting double-stranded RNA is converted into siRNAs that instruct the degradation of cognate mRNAs. In addition to a potential role in DNA repair, the break-induced transcription may thus be a means to cull improper RNAs from the transcriptome of Drosophila melanogaster. Since the splicing factors identified in our screen also stimulated siRNA production from high copy transgenes, it is possible that this surveillance mechanism serves in genome defense beyond DNA double-strand breaks. PMID:28628606

Targeting Promoter-Associated Noncoding RNA In Vivo.

PubMed

Civenni, Gianluca

2017-01-01

There are many classes of noncoding RNAs (ncRNAs), with wide-ranging functionalities (e.g., RNA editing, mediation of mRNA splicing, ribosomal function). MicroRNAs (miRNAs) and long ncRNAs (lncRNAs) are implicated in a wide variety of cellular processes, including the regulation of gene expression. Incorrect expression or mutation of lncRNAs has been reported to be associated with several disease conditions, such a malignant transformation in humans. Importantly, pivotal players in tumorigenesis and cancer progression, such as c-Myc, may be regulated by lncRNA at promoter level. The function of lncRNA can be reduced with antisense oligonucleotides that sequester or degrade mature lncRNAs. In alternative, lncRNA transcription can be blocked by small interference RNA (RNAi), which had acquired, recently, broad interested in clinical applications. In vivo-jetPEI™ is a linear polyethylenimine mediating nucleic acid (DNA, shRNA, siRNA, oligonucelotides) delivery with high efficiency. Different in vivo delivery routes have been validated: intravenous (IV), intraperitoneal (IP), intratumoral, subcutaneous, topical, and intrathecal. High levels of nucleic acid delivery are achieved into a broad range of tissues, such as lung, salivary glands, heart, spleen, liver, and prostate upon systemic administration. In addition, in vivo-jetPEI™ is also an efficient carrier for local gene and siRNA delivery such as intratumoral or topical application on the skin. After systemic injection, siRNA can be detected and the levels can be validated in target tissues by qRT-PCR. Targeting promoter-associated lncRNAs with siRNAs (small interfering RNAs) in vivo is becoming an exciting breakthrough for the treatment of human disease.

Splicing factor gene mutations in the myelodysplastic syndromes: impact on disease phenotype and therapeutic applications.

PubMed

Pellagatti, Andrea; Boultwood, Jacqueline

2017-01-01

Splicing factor gene mutations are the most frequent mutations found in patients with the myeloid malignancy myelodysplastic syndrome (MDS), suggesting that spliceosomal dysfunction plays a major role in disease pathogenesis. The aberrantly spliced target genes and deregulated cellular pathways associated with the commonly mutated splicing factor genes in MDS (SF3B1, SRSF2 and U2AF1) are being identified, illuminating the molecular mechanisms underlying MDS. Emerging data from mouse modeling studies indicate that the presence of splicing factor gene mutations can lead to bone marrow hematopoietic stem/myeloid progenitor cell expansion, impaired hematopoiesis and dysplastic differentiation that are hallmarks of MDS. Importantly, recent evidence suggests that spliceosome inhibitors and splicing modulators may have therapeutic value in the treatment of splicing factor mutant myeloid malignancies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Context-dependent control of alternative splicing by RNA-binding proteins

PubMed Central

Fu, Xiang-Dong; Ares, Manuel

2015-01-01

Sequence-specific RNA-binding proteins (RBPs) bind to pre-mRNA to control alternative splicing, but it is not yet possible to read the ‘splicing code’ that dictates splicing regulation on the basis of genome sequence. Each alternative splicing event is controlled by multiple RBPs, the combined action of which creates a distribution of alternatively spliced products in a given cell type. As each cell type expresses a distinct array of RBPs, the interpretation of regulatory information on a given RNA target is exceedingly dependent on the cell type. RBPs also control each other’s functions at many levels, including by mutual modulation of their binding activities on specific regulatory RNA elements. In this Review, we describe some of the emerging rules that govern the highly context-dependent and combinatorial nature of alternative splicing regulation. PMID:25112293

Antisense oligonucleotides as innovative therapeutic strategy in the treatment of high-grade gliomas.

PubMed

Caruso, Gerardo; Caffo, Mariella; Raudino, Giuseppe; Alafaci, Concetta; Salpietro, Francesco M; Tomasello, Francesco

2010-01-01

Despite the intensive recent research in cancer therapy, the prognosis in patients affected by high-grade gliomas is still very unfavorable. The efficacy of classical anti-cancer strategies is seriously limited by lack of specific therapies against malignant cells. The extracellular matrix plays a pivotal role in processes such as differentiation, apoptosis, and migration in both the normal and the pathologic nervous system. Glial tumors seem to be able to create a favorable environment for the invasion of glioma cells in cerebral parenchyma when they combine with the extracellular matrix via cell surface receptors. Glioma cells synthesize matrix proteins, such as tenascin, laminin, fibronectin that facilitate the tumor cell's motility. New treatments have shown to hit the acting molecules in the tumor growth and to increase the efficacy and minimize the toxicity. Antisense oligonucleotides are synthetic stretches of DNA which hybridize with specific mRNA strands. The specificity of hybridization makes antisense method an interesting strategy to selectively modulate the expression of genes involved in tumorigenesis. In this review we will focus on the mechanisms of action of antisense oligonucleotides and report clinical and experimental studies on the treatment of high-grade gliomas. We will also report the patents of preclinical and/or clinical studies that adopt the antisense oligonucleotide therapy list in cerebral gliomas.

HnRNP C, YB-1 and hnRNP L coordinately enhance skipping of human MUSK exon 10 to generate a Wnt-insensitive MuSK isoform

PubMed Central

Nasrin, Farhana; Rahman, Mohammad Alinoor; Masuda, Akio; Ohe, Kenji; Takeda, Jun-ichi; Ohno, Kinji

2014-01-01

Muscle specific receptor tyrosine kinase (MuSK) is an essential postsynaptic transmembrane molecule that mediates clustering of acetylcholine receptors (AChR). MUSK exon 10 is alternatively skipped in human, but not in mouse. Skipping of this exon disrupts a cysteine-rich region (Fz-CRD), which is essential for Wnt-mediated AChR clustering. To investigate the underlying mechanisms of alternative splicing, we exploited block-scanning mutagenesis with human minigene and identified a 20-nucleotide block that contained exonic splicing silencers. Using RNA-affinity purification, mass spectrometry, and Western blotting, we identified that hnRNP C, YB-1 and hnRNP L are bound to MUSK exon 10. siRNA-mediated knockdown and cDNA overexpression confirmed the additive, as well as the independent, splicing suppressing effects of hnRNP C, YB-1 and hnRNP L. Antibody-mediated in vitro protein depletion and scanning mutagenesis additionally revealed that binding of hnRNP C to RNA subsequently promotes binding of YB-1 and hnRNP L to the immediate downstream sites and enhances exon skipping. Simultaneous tethering of two splicing trans-factors to the target confirmed the cooperative effect of YB-1 and hnRNP L on hnRNP C-mediated exon skipping. Search for a similar motif in the human genome revealed nine alternative exons that were individually or coordinately regulated by hnRNP C and YB-1. PMID:25354590

Identification by high-throughput imaging of the histone methyltransferase EHMT2 as an epigenetic regulator of VEGFA alternative splicing.

PubMed

Salton, Maayan; Voss, Ty C; Misteli, Tom

2014-12-16

Recent evidence points to a role of chromatin in regulation of alternative pre-mRNA splicing (AS). In order to identify novel chromatin regulators of AS, we screened an RNAi library of chromatin proteins using a cell-based high-throughput in vivo assay. We identified a set of chromatin proteins that regulate AS. Using simultaneous genome-wide expression and AS analysis, we demonstrate distinct and non-overlapping functions of these chromatin modifiers on transcription and AS. Detailed mechanistic characterization of one dual function chromatin modifier, the H3K9 methyltransferase EHMT2 (G9a), identified VEGFA as a major chromatin-mediated AS target. Silencing of EHMT2, or its heterodimer partner EHMT1, affects AS by promoting exclusion of VEGFA exon 6a, but does not alter total VEGFA mRNA levels. The epigenetic regulatory mechanism of AS by EHMT2 involves an adaptor system consisting of the chromatin modulator HP1γ, which binds methylated H3K9 and recruits splicing regulator SRSF1. The epigenetic regulation of VEGFA is physiologically relevant since EHMT2 is transcriptionally induced in response to hypoxia and triggers concomitant changes in AS of VEGFA. These results characterize a novel epigenetic regulatory mechanism of AS and they demonstrate separate roles of epigenetic modifiers in transcription and alternative splicing. Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by US Government employees and is in the public domain in the US.

Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1

PubMed Central

Boddu, Ravindra; Yang, Chaozhe; O’Connor, Amber K.; Hendrickson, Robert Curtis; Boone, Braden; Cui, Xiangqin; Garcia-Gonzalez, Miguel; Igarashi, Peter; Onuchic, Luiz F.; Germino, Gregory G.

2014-01-01

Autosomal recessive polycystic kidney disease (ARPKD) results from mutations in the human PKHD1 gene. Both this gene, and its mouse ortholog, Pkhd1, are primarily expressed in renal and biliary ductal structures. The mouse protein product, fibrocystin/polyductin complex (FPC), is a 445-kDa protein encoded by a 67-exon transcript that spans >500 kb of genomic DNA. In the current study, we observed multiple alternatively spliced Pkhd1 transcripts that varied in size and exon composition in embryonic mouse kidney, liver, and placenta samples, as well as among adult mouse pancreas, brain, heart, lung, testes, liver, and kidney. Using reverse transcription PCR and RNASeq, we identified 22 novel Pkhd1 kidney transcripts with unique exon junctions. Various mechanisms of alternative splicing were observed, including exon skipping, use of alternate acceptor/donor splice sites, and inclusion of novel exons. Bioinformatic analyses identified, and exon-trapping minigene experiments validated, consensus binding sites for serine/arginine-rich proteins that modulate alternative splicing. Using site-directed mutagenesis, we examined the functional importance of selected splice enhancers. In addition, we demonstrated that many of the novel transcripts were polysome bound, thus likely translated. Finally, we determined that the human PKHD1 R760H missense variant alters a splice enhancer motif that disrupts exon splicing in vitro and is predicted to truncate the protein. Taken together, these data provide evidence of the complex transcriptional regulation of Pkhd1/PKHD1 and identified motifs that regulate its splicing. Our studies indicate that Pkhd1/PKHD1 transcription is modulated, in part by intragenic factors, suggesting that aberrant PKHD1 splicing represents an unappreciated pathogenic mechanism in ARPKD. PMID:24984783

Engineered U7 snRNA mediates sustained splicing correction in erythroid cells from β-thalassemia/HbE patients.

PubMed

Preedagasamzin, Sarinthip; Nualkaew, Tiwaporn; Pongrujikorn, Tanjitti; Jinawath, Natini; Kole, Ryszard; Fucharoen, Suthat; Jearawiriyapaisarn, Natee; Svasti, Saovaros

2018-04-30

Repair of a splicing defect of β-globin pre-mRNA harboring hemoglobin E (HbE) mutation was successfully accomplished in erythroid cells from patients with β-thalassemia/HbE disorder by a synthetic splice-switching oligonucleotide (SSO). However, its application is limited by short-term effectiveness and requirement of lifelong periodic administration of SSO, especially for chronic diseases like thalassemias. Here, we engineered lentiviral vectors that stably express U7 small nuclear RNA (U7 snRNA) carrying the splice-switching sequence of the SSO that restores correct splicing of β E -globin pre-mRNA and achieves a long-term therapeutic effect. Using a two-step tiling approach, we systematically screened U7 snRNAs carrying splice-switching SSO sequences targeted to the cryptic 5' splice site created by HbE mutation. We tested this approach and identified the most responsive element for mediating splicing correction in engineered U7 snRNAs in HeLa-β E cell model cell line. Remarkably, the U7 snRNA lentiviral vector (U7 βE4+1) targeted to this region effectively restored the correctly-spliced β E -globin mRNA for at least 5 months. Moreover, the effects of the U7 βE4+1 snRNA lentiviral vector were also evident as upregulation of the correctly-spliced β E -globin mRNA in erythroid progenitor cells from β-thalassemia/HbE patients treated with the vector, which led to improvements of pathologies in erythroid progenitor cells from thalassemia patients. These results suggest that the splicing correction of β E -globin pre-mRNA by the engineered U7 snRNA lentiviral vector provides a promising, long-term treatment for β-thalassemia/HbE. Copyright © 2018 Elsevier Inc. All rights reserved.

Antisense antibiotics: a brief review of novel target discovery and delivery.

PubMed

Bai, Hui; Xue, Xiaoyan; Hou, Zheng; Zhou, Ying; Meng, Jingru; Luo, Xiaoxing

2010-06-01

The nightmare of multi-drug resistant bacteria will still haunt if no panacea is ever found. Efforts on seeking desirable natural products with bactericidal property and screening chemically modified derivatives of traditional antibiotics have lagged behind the emergence of new multi-drug resistant bacteria. The concept of using antisense antibiotics, now as revolutionary as is on threshold has experienced ups and downs in the past decade. In the past five years, however, significant technology advances in the fields of microbial genomics, structural modification of oligonucleotides and efficient delivery system have led to fundamental progress in the research and in vivo application of this paradigm. The wealthy information provided in the microbial genomics era has allowed the identification and/or validation of a number of essential genes that may serve as possible targets for antisense inhibition; antisense oligodeoxynucleotides (ODNs) based on the 3rd generation of modified structures, e.g., peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligomers (PMOs) have shown great potency in gene expression inhibition in a sequence-specific and dosedependent manner at low micromolar concentrations; and cell penetrating peptide mediated delivery system has enabled the effective display of intracellular antisense inhibition of targeted genes both in vitro and in vivo. The new methods show promise in the discovery of novel gene-specific antisense antibiotics that will be useful in the future battle against drug-resistant bacterial infections. This review describes this promising paradigm, the targets that have been identified and the recent technologies on which it is delivered.

Modification of antisense phosphodiester oligodeoxynucleotides by a 5' cholesteryl moiety increases cellular association and improves efficacy.

PubMed

Krieg, A M; Tonkinson, J; Matson, S; Zhao, Q; Saxon, M; Zhang, L M; Bhanja, U; Yakubov, L; Stein, C A

1993-02-01

Phosphodiester oligodeoxynucleotides bearing a 5' cholesteryl (chol) modification bind to low density lipoprotein (LDL), apparently by partitioning the chol-modified oligonucleotides into the lipid layer. Both HL60 cells and primary mouse spleen T and B cells incubated with fluorescently labeled chol-modified oligonucleotide showed substantially increased cellular association by flow cytometry and increased internalization by confocal microscopy compared to an identical molecule not bearing the chol group. Cellular internalization of chol-modified oligonucleotide occurred at least partially through the LDL receptor; it was increased in mouse spleen cells by cell culture in lipoprotein-deficient medium and/or lovastatin, and it was decreased by culture in high serum medium. To determine whether chol-modified oligonucleotides are more potent antisense agents, we titered antisense unmodified phosphodiester and chol-modified oligonucleotides targeted against a mouse immunosuppressive protein. Murine spleen cells cultured with 20 microM phosphodiester antisense oligonucleotides had a 2-fold increase in RNA synthesis, indicating the expected lymphocyte activation. Antisense chol-modified oligonucleotides showed an 8-fold increase in relative potency: they caused a 2-fold increase in RNA synthesis at just 2.5 microM. The increased efficacy was blocked by heparin and was further increased by cell culture in 1% (vs. 10%) fetal bovine serum, suggesting that the effect may, at least in part, be mediated via the LDL receptor. Antisense chol-modified oligonucleotides are sequence specific and have increased potency as compared to unmodified oligonucleotides.

Alternative Splicing in Neurogenesis and Brain Development.

PubMed

Su, Chun-Hao; D, Dhananjaya; Tarn, Woan-Yuh

2018-01-01

Alternative splicing of precursor mRNA is an important mechanism that increases transcriptomic and proteomic diversity and also post-transcriptionally regulates mRNA levels. Alternative splicing occurs at high frequency in brain tissues and contributes to every step of nervous system development, including cell-fate decisions, neuronal migration, axon guidance, and synaptogenesis. Genetic manipulation and RNA sequencing have provided insights into the molecular mechanisms underlying the effects of alternative splicing in stem cell self-renewal and neuronal fate specification. Timely expression and perhaps post-translational modification of neuron-specific splicing regulators play important roles in neuronal development. Alternative splicing of many key transcription regulators or epigenetic factors reprograms the transcriptome and hence contributes to stem cell fate determination. During neuronal differentiation, alternative splicing also modulates signaling activity, centriolar dynamics, and metabolic pathways. Moreover, alternative splicing impacts cortical lamination and neuronal development and function. In this review, we focus on recent progress toward understanding the contributions of alternative splicing to neurogenesis and brain development, which has shed light on how splicing defects may cause brain disorders and diseases.

TGF-beta antisense oligonucleotides reduce mRNA expression of matrix metalloproteinases in cultured wound-healing-related cells.

PubMed

Philipp, Katrin; Riedel, Frank; Germann, Günter; Hörmann, Karl; Sauerbier, Michael

2005-02-01

The pathology of chronic dermal ulcers is characterized by excessive proteolytic activity which degrades extracellular matrix. The transforming growth factor-beta (TGF-beta) has been identified as an important component of wound healing. Recent developments in molecular therapy offer exciting prospects for the modulation of wound healing, specifically those targeting TGF-beta. We investigated the effect of TGF-beta antisense oligonucleotides on the mRNA expression of matrix metalloproteinases in cultured human keratinocytes, fibroblasts and endothelial cells using multiplex RT-PCR. The treatment of keratinocytes and fibroblasts with TGF-beta antisense oligonucleotides resulted in a significant decrease of expression of mRNA of MMP-1 and MMP-9 compared to controls. Accordingly, a decreased expression of MMP-1 mRNA in endothelial cells was detectable. Other MMPs were not affected. Affecting all dermal wound-healing-related cell types, TGF-beta antisense oligonucleotide technology may be a potential therapeutic option for the inhibition of proteolytic tissue destruction in chronic wounds. Pharmaceutical intervention in this area ultimately may help clinicians to proactively intervene in an effort to prevent normal wounds from becoming chronic.

When proteome meets genome: the alpha helix and the beta strand of proteins are eschewed by mRNA splice junctions and may define the minimal indivisible modules of protein architecture

PubMed Central

Barik, Sailen

2008-01-01

The significance of the intron-exon structure of genes is a mystery. As eukaryotic proteins are made up of modular functional domains, each exon was suspected to encode some form of module; however, the definition of a module remained vague. Comparison of pre-mRNA splice junctions with the three-dimensional architecture of its protein product from different eukaryotes revealed that the junctions were far less likely to occur inside the α-helices and β-strands of proteins than within the more flexible linker regions (‘turns’ and ‘loops’) connecting them. The splice junctions were equally distributed in the different types of linkers and throughout the linker sequence, although a slight preference for the central region of the linker was observed. The avoidance of the α-helix and the β-strand by splice junctions suggests the existence of a selection pressure against their disruption, perhaps underscoring the investment made by nature in building these intricate secondary structures. A corollary is that the helix and the strand are the smallest integral architectural units of a protein and represent the minimal modules in the evolution of protein structure. These results should find use in comparative genomics, designing of cloning strategies, and in the mutual verification of genome sequences with protein structures. PMID:15381847

When proteome meets genome: the alpha helix and the beta strand of proteins are eschewed by mRNA splice junctions and may define the minimal indivisible modules of protein architecture.

PubMed

Barik, Sailen

2004-09-01

The significance of the intron-exon structure of genes is a mystery. As eukaryotic proteins are made up of modular functional domains, each exon was suspected to encode some form of module; however, the definition of a module remained vague. Comparison of pre-mRNA splice junctions with the three-dimensional architecture of its protein product from different eukaryotes revealed that the junctions were far less likely to occur inside the alpha-helices and beta-strands of proteins than within the more flexible linker regions ('turns' and 'loops') connecting them. The splice junctions were equally distributed in the different types of linkers and throughout the linker sequence, although a slight preference for the central region of the linker was observed. The avoidance of the alpha-helix and the beta-strand by splice junctions suggests the existence of a selection pressure against their disruption, perhaps underscoring the investment made by nature in building these intricate secondary structures. A corollary is that the helix and the strand are the smallest integral architectural units of a protein and represent the minimal modules in the evolution of protein structure. These results should find use in comparative genomics, designing of cloning strategies, and in the mutual verification of genome sequences with protein structures.

Splicing of designer exons informs a biophysical model for exon definition

PubMed Central

Arias, Mauricio A.; Chasin, Lawrence A.

2015-01-01

Pre-mRNA molecules in humans contain mostly short internal exons flanked by longer introns. To explain the removal of such introns, exon recognition instead of intron recognition has been proposed. We studied this exon definition using designer exons (DEs) made up of three prototype modules of our own design: an exonic splicing enhancer (ESE), an exonic splicing silencer (ESS), and a Reference Sequence (R) predicted to be neither. Each DE was examined as the central exon in a three-exon minigene. DEs made of R modules showed a sharp size dependence, with exons shorter than 14 nt and longer than 174 nt splicing poorly. Changing the strengths of the splice sites improved longer exon splicing but worsened shorter exon splicing, effectively displacing the curve to the right. For the ESE we found, unexpectedly, that its enhancement efficiency was independent of its position within the exon. For the ESS we found a step-wise positional increase in its effects; it was most effective at the 3′ end of the exon. To apply these results quantitatively, we developed a biophysical model for exon definition of internal exons undergoing cotranscriptional splicing. This model features commitment to inclusion before the downstream exon is synthesized and competition between skipping and inclusion fates afterward. Collision of both exon ends to form an exon definition complex was incorporated to account for the effect of size; ESE/ESS effects were modeled on the basis of stabilization/destabilization. This model accurately predicted the outcome of independent experiments on more complex DEs that combined ESEs and ESSs. PMID:25492963

Intein-mediated assembly of a functional β-glucuronidase in transgenic plants

PubMed Central

Yang, Jianjun; Fox, George C.; Henry-Smith, Tina V.

2003-01-01

The DnaE intein in Synechocystis sp. strain PCC6803 is the first and only naturally split intein that has been identified so far. It is capable of catalyzing a protein trans-splicing mechanism to assemble a mature protein from two separate precursors. Therefore, it is a powerful tool for protein modification and engineering. Inteins have not been identified, nor have intein-mediated protein splicing reactions been demonstrated, in plant cells. In this paper, we describe the use of the Ssp DnaE split intein in transgenic plants for reconstitution of a protein trans-splicing reaction. We have synthesized artificial genes that encode for N-terminal half (Int-n) and C-terminal half (Int-c) fragments of Ssp DnaE split intein and divided β-glucuronidase (GUS) gene to encode GUS-n and GUS-c parts of the enzyme as reporter. The in-frame fusions of GUSn/Intn and Intc/GUSc were constructed and transfected into Arabidopsis. We have observed in vivo reassembly of functional β-glucuronidase when both GUSn/Intn and Intc/GUSc constructs were introduced into the same Arabidopsis genome either by cotransformation or through genetic crossing, hereby signifying an intein-mediated protein trans-splicing mechanism reconstituted in plant cells. PMID:12629210

Alternative Splicing Control of Abiotic Stress Responses.

PubMed

Laloum, Tom; Martín, Guiomar; Duque, Paula

2018-02-01

Alternative splicing, which generates multiple transcripts from the same gene, is an important modulator of gene expression that can increase proteome diversity and regulate mRNA levels. In plants, this post-transcriptional mechanism is markedly induced in response to environmental stress, and recent studies have identified alternative splicing events that allow rapid adjustment of the abundance and function of key stress-response components. In agreement, plant mutants defective in splicing factors are severely impaired in their response to abiotic stress. Notably, mounting evidence indicates that alternative splicing regulates stress responses largely by targeting the abscisic acid (ABA) pathway. We review here current understanding of post-transcriptional control of plant stress tolerance via alternative splicing and discuss research challenges for the near future. Copyright © 2017 Elsevier Ltd. All rights reserved.

In Silico Screening Based on Predictive Algorithms as a Design Tool for Exon Skipping Oligonucleotides in Duchenne Muscular Dystrophy

PubMed Central

Echigoya, Yusuke; Mouly, Vincent; Garcia, Luis; Yokota, Toshifumi; Duddy, William

2015-01-01

The use of antisense ‘splice-switching’ oligonucleotides to induce exon skipping represents a potential therapeutic approach to various human genetic diseases. It has achieved greatest maturity in exon skipping of the dystrophin transcript in Duchenne muscular dystrophy (DMD), for which several clinical trials are completed or ongoing, and a large body of data exists describing tested oligonucleotides and their efficacy. The rational design of an exon skipping oligonucleotide involves the choice of an antisense sequence, usually between 15 and 32 nucleotides, targeting the exon that is to be skipped. Although parameters describing the target site can be computationally estimated and several have been identified to correlate with efficacy, methods to predict efficacy are limited. Here, an in silico pre-screening approach is proposed, based on predictive statistical modelling. Previous DMD data were compiled together and, for each oligonucleotide, some 60 descriptors were considered. Statistical modelling approaches were applied to derive algorithms that predict exon skipping for a given target site. We confirmed (1) the binding energetics of the oligonucleotide to the RNA, and (2) the distance in bases of the target site from the splice acceptor site, as the two most predictive parameters, and we included these and several other parameters (while discounting many) into an in silico screening process, based on their capacity to predict high or low efficacy in either phosphorodiamidate morpholino oligomers (89% correctly predicted) and/or 2’O Methyl RNA oligonucleotides (76% correctly predicted). Predictions correlated strongly with in vitro testing for sixteen de novo PMO sequences targeting various positions on DMD exons 44 (R2 0.89) and 53 (R2 0.89), one of which represents a potential novel candidate for clinical trials. We provide these algorithms together with a computational tool that facilitates screening to predict exon skipping efficacy at each position of a target exon. PMID:25816009

Osteopontin splice variants expression is involved on docetaxel resistance in PC3 prostate cancer cells.

PubMed

Nakamura, K D M; Tilli, T M; Wanderley, J L; Palumbo, A; Mattos, R M; Ferreira, A C; Klumb, C E; Nasciutti, L E; Gimba, E R

2016-02-01

Osteopontin (OPN) is a phosphoprotein that activates several aspects of tumor progression. Alternative splicing of the OPN primary transcript generates three splicing isoforms, OPNa, OPNb and OPNc. In this report, we investigated some cellular mechanisms by which OPN splice variants could mediate PC3 prostate cancer (PCa) cell survival and growth in response to docetaxel (DXT)-induced cell death. Cell survival before and after DXT treatment was analyzed by phase-contrast microscopy and crystal-violet staining assays. Quantitative real-time PCR and immunocytochemical staining assays were used to evaluate the putative involvement of epithelial-mesenchymal transition (EMT) and OPN isoforms on mediating PC3 cell survival. Upon DXT treatment, PC3 cells overexpressing OPNb or OPNc isoforms showed higher cell densities, compared to cells overexpressing OPNa and controls. Notably, cells overexpressing OPNb or OPNc isoforms showed a downregulated pattern of EMT epithelial cell markers, while mesenchymal markers were mostly upregulated in these experimental conditions. We concluded that OPNc or OPNb overexpression in PC3 cells can mediate resistance and cell survival features in response to DXT-induced cell death. Our data also provide evidence the EMT program could be one of the molecular mechanisms mediating survival in OPNb- or OPNc-overexpressing cells in response to DXT treatment. These data could further contribute to a better understanding of the mechanisms by which PCa cells acquire resistance to DXT treatment.

RNA splicing during terminal erythropoiesis.

PubMed

Conboy, John G

2017-05-01

Erythroid progenitors must accurately and efficiently splice thousands of pre-mRNAs as the cells undergo extensive changes in gene expression and cellular remodeling during terminal erythropoiesis. Alternative splicing choices are governed by interactions between RNA binding proteins and cis-regulatory binding motifs in the RNA. This review will focus on recent studies that define the genome-wide scope of splicing in erythroblasts and discuss what is known about its regulation. RNA-seq analysis of highly purified erythroblast populations has revealed an extensive program of alternative splicing of both exons and introns. During normal erythropoiesis, stage-specific splicing transitions alter the structure and abundance of protein isoforms required for optimized red cell production. Mutation or deficiency of splicing regulators underlies hematopoietic disease in myelopdysplasia syndrome patients via disrupting the splicing program. Erythroid progenitors execute an elaborate alternative splicing program that modulates gene expression posttranscriptionally, ultimately regulating the structure and function of the proteome in a differentiation stage-specific manner during terminal erythropoiesis. This program helps drive differentiation and ensure synthesis of the proper protein isoforms required to produce mechanically stable red cells. Mutation or deficiency of key splicing regulatory proteins disrupts the splicing program to cause disease.

Widespread Use of Non-productive Alternative Splice Sites in Saccharomyces cerevisiae

PubMed Central

Kawashima, Tadashi; Douglass, Stephen; Gabunilas, Jason; Pellegrini, Matteo; Chanfreau, Guillaume F.

2014-01-01

Saccharomyces cerevisiae has been used as a model system to investigate the mechanisms of pre-mRNA splicing but only a few examples of alternative splice site usage have been described in this organism. Using RNA-Seq analysis of nonsense-mediated mRNA decay (NMD) mutant strains, we show that many S. cerevisiae intron-containing genes exhibit usage of alternative splice sites, but many transcripts generated by splicing at these sites are non-functional because they introduce premature termination codons, leading to degradation by NMD. Analysis of splicing mutants combined with NMD inactivation revealed the role of specific splicing factors in governing the use of these alternative splice sites and identified novel functions for Prp17p in enhancing the use of branchpoint-proximal upstream 3′ splice sites and for Prp18p in suppressing the usage of a non-canonical AUG 3′-splice site in GCR1. The use of non-productive alternative splice sites can be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during stress. These results show that alternative splicing is frequent in S. cerevisiae but masked by RNA degradation and that the use of alternative splice sites in this organism is mostly aimed at controlling transcript levels rather than increasing proteome diversity. PMID:24722551

An alternative splicing program promotes adipose tissue thermogenesis

PubMed Central

Vernia, Santiago; Edwards, Yvonne JK; Han, Myoung Sook; Cavanagh-Kyros, Julie; Barrett, Tamera; Kim, Jason K; Davis, Roger J

2016-01-01

Alternative pre-mRNA splicing expands the complexity of the transcriptome and controls isoform-specific gene expression. Whether alternative splicing contributes to metabolic regulation is largely unknown. Here we investigated the contribution of alternative splicing to the development of diet-induced obesity. We found that obesity-induced changes in adipocyte gene expression include alternative pre-mRNA splicing. Bioinformatics analysis associated part of this alternative splicing program with sequence specific NOVA splicing factors. This conclusion was confirmed by studies of mice with NOVA deficiency in adipocytes. Phenotypic analysis of the NOVA-deficient mice demonstrated increased adipose tissue thermogenesis and improved glycemia. We show that NOVA proteins mediate a splicing program that suppresses adipose tissue thermogenesis. Together, these data provide quantitative analysis of gene expression at exon-level resolution in obesity and identify a novel mechanism that contributes to the regulation of adipose tissue function and the maintenance of normal glycemia. DOI: http://dx.doi.org/10.7554/eLife.17672.001 PMID:27635635

Structure of an Arrestin2-clathrin Complex Reveals a Novel Clathrin Binding Domain that Modulates Receptor Trafficking

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, D.; Kern, R; Puthenveedu, M

2009-01-01

Non-visual arrestins play a pivotal role as adaptor proteins in regulating the signaling and trafficking of multiple classes of receptors. Although arrestin interaction with clathrin, AP-2, and phosphoinositides contributes to receptor trafficking, little is known about the configuration and dynamics of these interactions. Here, we identify a novel interface between arrestin2 and clathrin through x-ray diffraction analysis. The intrinsically disordered clathrin binding box of arrestin2 interacts with a groove between blades 1 and 2 in the clathrin {beta}-propeller domain, whereas an 8-amino acid splice loop found solely in the long isoform of arrestin2 (arrestin2L) interacts with a binding pocket formedmore » by blades 4 and 5 in clathrin. The apposition of the two binding sites in arrestin2L suggests that they are exclusive and may function in higher order macromolecular structures. Biochemical analysis demonstrates direct binding of clathrin to the splice loop in arrestin2L, whereas functional analysis reveals that both binding domains contribute to the receptor-dependent redistribution of arrestin2L to clathrin-coated pits. Mutagenesis studies reveal that the clathrin binding motif in the splice loop is (L/I){sub 2}GXL. Taken together, these data provide a framework for understanding the dynamic interactions between arrestin2 and clathrin and reveal an essential role for this interaction in arrestin-mediated endocytosis.« less

Dual roles for CoAA and its counterbalancing isoform CoAM in human kidney cell tumorigenesis

PubMed Central

Kang, Yun Kyoung; Schiff, Rachel; Ko, Lan; Wang, Tao; Tsai, Sophia Y.; Tsai, Ming-Jer; W. O’Malley, Bert

2008-01-01

Co-Activator Activator (CoAA) has been reported to be a coactivator that regulates steroid receptor-mediated transcription and alternative RNA splicing. Herein we show that CoAA is a dual-function coregulator that inhibits G1/S transition in human kidney cells and suppresses anchorage independent growth and xenograft tumor formation. Suppression occurs in part by downregulating c-myc and its downstream effectors ccnd1 and skp2, and causing accumulation of p27/Kip1 protein. In this cellular setting, CoAA directly represses the proto-oncogene, c-myc by recruiting HDAC3 protein and decreasing both the acetylation of histone H3 and the presence of RNA polymerase II on the c-myc promoter. Interestingly, a splicing isoform of CoAA, Coactivator Modulator (CoAM), antagonizes CoAA-induced G1/S transition and growth inhibition by negatively regulating the mRNA levels of the endogenous CoAA isoform. In addition, we found that expression of CoAA protein is significantly decreased in human renal cell carcinoma as compared to normal kidney. Our study presents evidence that CoAA is a potential tumor suppressor in renal carcinoma and that CoAM is a counterbalancing splice-isoform. This is so far the only example of a nuclear receptor coregulator involved in suppression of kidney cancer, and suggests potentially significant new roles for coregulators in renal cancer biology. PMID:18829545

Late-onset spastic paraplegia: Aberrant SPG11 transcripts generated by a novel splice site donor mutation.

PubMed

Kawarai, Toshitaka; Miyamoto, Ryosuke; Mori, Atsuko; Oki, Ryosuke; Tsukamoto-Miyashiro, Ai; Matsui, Naoko; Miyazaki, Yoshimichi; Orlacchio, Antonio; Izumi, Yuishin; Nishida, Yoshihiko; Kaji, Ryuji

2015-12-15

We identified a novel homozygous mutation in the splice site donor (SSD) of intron 30 (c.5866+1G>A) in consanguineous Japanese SPG11 siblings showing late-onset spastic paraplegia using the whole-exome sequencing. Phenotypic variability was observed, including age-at-onset, dysarthria and pes cavus. Coding DNA sequencing revealed that the mutation affected the recognition of the constitutive SSD of intron 30, splicing upstream onto a nearby cryptic SSD in exon 30. The use of constitutive splice sites of intron 29 was confirmed by sequencing. The mutant transcripts are mostly subject to degradation by the nonsense-mediated mRNA decay system. SPG11 transcripts, escaping from the nonsense-mediated mRNA decay pathway, would generate a truncated protein (p.Tyr1900Phefs5X) containing the first 1899 amino acids and followed by 4 aberrant amino acids. This study showed a successful clinical application of whole-exome sequencing in spastic paraplegia and demonstrated a further evidence of allelic heterogeneity in SPG11. The confirmation of aberrant transcript by splice site mutation is a prerequisite for a more precise molecular diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

A mechanism for exon skipping caused by nonsense or missense mutations in BRCA1 and other genes.

PubMed

Liu, H X; Cartegni, L; Zhang, M Q; Krainer, A R

2001-01-01

Point mutations can generate defective and sometimes harmful proteins. The nonsense-mediated mRNA decay (NMD) pathway minimizes the potential damage caused by nonsense mutations. In-frame nonsense codons located at a minimum distance upstream of the last exon-exon junction are recognized as premature termination codons (PTCs), targeting the mRNA for degradation. Some nonsense mutations cause skipping of one or more exons, presumably during pre-mRNA splicing in the nucleus; this phenomenon is termed nonsense-mediated altered splicing (NAS), and its underlying mechanism is unclear. By analyzing NAS in BRCA1, we show here that inappropriate exon skipping can be reproduced in vitro, and results from disruption of a splicing enhancer in the coding sequence. Enhancers can be disrupted by single nonsense, missense and translationally silent point mutations, without recognition of an open reading frame as such. These results argue against a nuclear reading-frame scanning mechanism for NAS. Coding-region single-nucleotide polymorphisms (cSNPs) within exonic splicing enhancers or silencers may affect the patterns or efficiency of mRNA splicing, which may in turn cause phenotypic variability and variable penetrance of mutations elsewhere in a gene.

Combined hairpin-antisense compositions and methods for modulating expression

DOEpatents

Shanklin, John; Nguyen, Tam

2014-08-05

A nucleotide construct comprising a nucleotide sequence that forms a stem and a loop, wherein the loop comprises a nucleotide sequence that modulates expression of a target, wherein the stem comprises a nucleotide sequence that modulates expression of a target, and wherein the target modulated by the nucleotide sequence in the loop and the target modulated by the nucleotide sequence in the stem may be the same or different. Vectors, methods of regulating target expression, methods of providing a cell, and methods of treating conditions comprising the nucleotide sequence are also disclosed.

Combined hairpin-antisense compositions and methods for modulating expression

DOEpatents

Shanklin, John; Nguyen, Tam Huu

2015-11-24

A nucleotide construct comprising a nucleotide sequence that forms a stem and a loop, wherein the loop comprises a nucleotide sequence that modulates expression of a target, wherein the stem comprises a nucleotide sequence that modulates expression of a target, and wherein the target modulated by the nucleotide sequence in the loop and the target modulated by the nucleotide sequence in the stem may be the same or different. Vectors, methods of regulating target expression, methods of providing a cell, and methods of treating conditions comprising the nucleotide sequence are also disclosed.

iCLIP Predicts the Dual Splicing Effects of TIA-RNA Interactions

PubMed Central

Briese, Michael; Zarnack, Kathi; Luscombe, Nicholas M.; Rot, Gregor; Zupan, Blaž; Curk, Tomaž; Ule, Jernej

2010-01-01

The regulation of alternative splicing involves interactions between RNA-binding proteins and pre-mRNA positions close to the splice sites. T-cell intracellular antigen 1 (TIA1) and TIA1-like 1 (TIAL1) locally enhance exon inclusion by recruiting U1 snRNP to 5′ splice sites. However, effects of TIA proteins on splicing of distal exons have not yet been explored. We used UV-crosslinking and immunoprecipitation (iCLIP) to find that TIA1 and TIAL1 bind at the same positions on human RNAs. Binding downstream of 5′ splice sites was used to predict the effects of TIA proteins in enhancing inclusion of proximal exons and silencing inclusion of distal exons. The predictions were validated in an unbiased manner using splice-junction microarrays, RT-PCR, and minigene constructs, which showed that TIA proteins maintain splicing fidelity and regulate alternative splicing by binding exclusively downstream of 5′ splice sites. Surprisingly, TIA binding at 5′ splice sites silenced distal cassette and variable-length exons without binding in proximity to the regulated alternative 3′ splice sites. Using transcriptome-wide high-resolution mapping of TIA-RNA interactions we evaluated the distal splicing effects of TIA proteins. These data are consistent with a model where TIA proteins shorten the time available for definition of an alternative exon by enhancing recognition of the preceding 5′ splice site. Thus, our findings indicate that changes in splicing kinetics could mediate the distal regulation of alternative splicing. PMID:21048981

C-fos down-regulation inhibits testosterone-dependent male sexual behavior and the associated learning

PubMed Central

Niessen, Neville-Andrew; Balthazart, Jacques; Ball, Gregory F.; Charlier, Thierry D.

2013-01-01

Environmental stimulation results in an increased expression of transcription factors called immediate early genes (IEG) in specific neuronal populations. In male Japanese quail, copulation with a female increases the expression of the IEGs zenk and c-fos in the medial preoptic nucleus (POM), a key nucleus controlling male sexual behavior. The functional significance of this increased IEG expression that follows performance of copulatory behavior is unknown. We addressed this question by repeatedly quantifying the performance of appetitive (learned social proximity response) and consummatory (actual copulation) sexual behavior in castrated, testosterone-treated males that received daily intracerebroventricular injection of an antisense oligodeoxynucleotide targeting c-fos or control vehicle. Daily antisense injections significantly inhibited expression of copulatory behavior as well as acquisition of the learned social proximity response. A strong reduction of the proximity response was still observed in antisense-treated birds that copulated with a female, ruling out the indirect effect of the absence of interactions with females on the learning process. After a two-day interruption of behavioral testing but not of antisense injections, birds were submitted to a final copulatory test that confirmed the behavioral inhibition in antisense-injected birds. Brains were collected 90 min after the behavioral testing for quantification of c-fos immunoreactive cells. A significant reduction of the number of c-fos-positive cells in POM but not in other brain regions was observed following antisense injection. Together, data suggest that c-fos expression in POM modulates copulatory behavior and sexual learning in male quail. PMID:23895306

Alternative splicing modulates Kv channel clustering through a molecular ball and chain mechanism

NASA Astrophysics Data System (ADS)

Zandany, Nitzan; Marciano, Shir; Magidovich, Elhanan; Frimerman, Teddy; Yehezkel, Rinat; Shem-Ad, Tzilhav; Lewin, Limor; Abdu, Uri; Orr, Irit; Yifrach, Ofer

2015-03-01

Ion channel clustering at the post-synaptic density serves a fundamental role in action potential generation and transmission. Here, we show that interaction between the Shaker Kv channel and the PSD-95 scaffold protein underlying channel clustering is modulated by the length of the intrinsically disordered C terminal channel tail. We further show that this tail functions as an entropic clock that times PSD-95 binding. We thus propose a ‘ball and chain’ mechanism to explain Kv channel binding to scaffold proteins, analogous to the mechanism describing channel fast inactivation. The physiological relevance of this mechanism is demonstrated in that alternative splicing of the Shaker channel gene to produce variants of distinct tail lengths resulted in differential channel cell surface expression levels and clustering metrics that correlate with differences in affinity of the variants for PSD-95. We suggest that modulating channel clustering by specific spatial-temporal spliced variant targeting serves a fundamental role in nervous system development and tuning.

Recognizing the enemy within: licensing RNA-guided genome defense

PubMed Central

Dumesic, Phillip A.; Madhani, Hiten D.

2014-01-01

How do cells distinguish normal genes from transposons? Although much has been learned about RNAi-related RNA silencing pathways responsible for genome defense, this fundamental question remains. The literature points to several classes of mechanisms. In some cases, double-stranded RNA structures produced by transposon inverted repeats or antisense integration trigger endo-siRNA biogenesis. In other instances, DNA features associated with transposons—such as their unusual copy number, chromosomal arrangement, and/or chromatin environment—license RNA silencing. Finally, recent studies have identified improper transcript processing events, such as stalled pre-mRNA splicing, as signals for siRNA production. Thus, the suboptimal gene expression properties of selfish elements can enable their identification by RNA silencing pathways. PMID:24280023

Down-Regulating α-Galactosidase Enhances Freezing Tolerance in Transgenic Petunia1

PubMed Central

Pennycooke, Joyce C.; Jones, Michelle L.; Stushnoff, Cecil

2003-01-01

α-Galactosidase (α-Gal; EC 3.2.1.22) is involved in many aspects of plant metabolism, including hydrolysis of the α-1,6 linkage of raffinose oligosaccharides during deacclimation. To examine the relationship between endogenous sugars and freezing stress, the expression of α-Gal was modified in transgenic petunia (Petunia × hybrida cv Mitchell). The tomato (Lycopersicon esculentum) Lea-Gal gene under the control of the Figwort Mosaic Virus promoter was introduced into petunia in the sense and antisense orientations using Agrobacterium tumefaciens-mediated transformation. RNA gel blots confirmed that α-Gal transcripts were reduced in antisense lines compared with wild type, whereas sense plants had increased accumulation of α-Gal mRNAs. α-Gal activity followed a similar trend, with reduced activity in antisense lines and increased activity in all sense lines evaluated. Raffinose content of nonacclimated antisense plants increased 12- to 22-fold compared with wild type, and 22- to 53-fold after cold acclimation. Based upon electrolyte leakage tests, freezing tolerance of the antisense lines increased from –4°C for cold-acclimated wild-type plants to –8°C for the most tolerant antisense line. Down-regulating α-Gal in petunia results in an increase in freezing tolerance at the whole-plant level in nonacclimated and cold-acclimated plants, whereas overexpression of the α-Gal gene caused a decrease in endogenous raffinose and impaired freezing tolerance. These results suggest that engineering raffinose metabolism by transformation with α-Gal provides an additional method for improving the freezing tolerance of plants. PMID:14500789

The Status of Exon Skipping as a Therapeutic Approach to Duchenne Muscular Dystrophy

PubMed Central

Lu, Qi-Long; Yokota, Toshifumi; Takeda, Shin'ichi; Garcia, Luis; Muntoni, Francesco; Partridge, Terence

2011-01-01

Duchenne muscular dystrophy (DMD) is associated with mutations in the dystrophin gene that disrupt the open reading frame whereas the milder Becker's form is associated with mutations which leave an in-frame mRNA transcript that can be translated into a protein that includes the N- and C- terminal functional domains. It has been shown that by excluding specific exons at, or adjacent to, frame-shifting mutations, open reading frame can be restored to an out-of-frame mRNA, leading to the production of a partially functional Becker-like dystrophin protein. Such targeted exclusion can be achieved by administration of oligonucleotides that are complementary to sequences that are crucial to normal splicing of the exon into the transcript. This principle has been validated in mouse and canine models of DMD with a number of variants of oligonucleotide analogue chemistries and by transduction with adeno-associated virus (AAV)-small nuclear RNA (snRNA) reagents encoding the antisense sequence. Two different oligonucleotide agents are now being investigated in human trials for splicing out of exon 51 with some early indications of success at the biochemical level. PMID:20978473

[Inhibition of monocytes adhesion to the intima of arterial wall by local expression of antisense monocyte chemotactic protein-1].

PubMed

Wu, Q; Qiao, H; Wang, Z; Zhang, H; Liu, P; Xu, M; Ren, G; Zhao, S; She, M

2000-04-01

To study the mechanism of monocyte recruitment in atherogenesis and to clarify the effect of monocyte chemotactic protein-1 (MCP-1) in this process. Femoral arteries isolated from the rabbits which had been fed with a high cholesterol diet and locally perfused with MM-LDL within the artery beforehand, were used as the models. Antisense MCP-1cDNA was transferred into the arterial wall by injecting recombinant LNCX-anti-MCP-1/liposomal complex in the femoral sheath and the periarterial tissue. Expression of antisense MCP-1 mediated by recombinant LNCX plasmid/lipsomal complex gene transfer enabled to inhibit MCP-1 gene expression and adhesion of monocyte to the intima. MCP-1 plays an important role on the recruitment of monocytes in the arterial wall, which provides a potential clue in developing a gene therapy project for the prevention and treatment of atherogenesis.

Antisense oligonucleotide inhibition of cholesteryl ester transfer protein enhances RCT in hyperlipidemic, CETP transgenic, LDLr-/- mice.

PubMed

Bell, Thomas A; Graham, Mark J; Lee, Richard G; Mullick, Adam E; Fu, Wuxia; Norris, Dan; Crooke, Rosanne M

2013-10-01

Due to their ability to promote positive effects across all of the lipoprotein classes, cholesteryl ester transfer protein (CETP) inhibitors are currently being developed as therapeutic agents for cardiovascular disease. In these studies, we compared an antisense oligonucleotide (ASO) inhibitor of CETP to the CETP small molecule inhibitor anacetrapib. In hyperlipidemic CETP transgenic (tg) mice, both drugs provided comparable reductions in total plasma cholesterol, decreases in CETP activity, and increases in HDL cholesterol. However, only mice treated with the antisense inhibitor showed an enhanced effect on macrophage reverse cholesterol transport, presumably due to differences in HDL apolipoprotein composition and decreases in plasma triglyceride. Additionally, the ASO-mediated reductions in CETP mRNA were associated with less accumulation of aortic cholesterol. These preliminary findings suggest that CETP ASOs may represent an alternative means to inhibit that target and to support their continued development as a treatment for cardiovascular disease in man.

Regulation of mRNA abundance by polypyrimidine tract-binding protein-controlled alternate 5' splice site choice.

PubMed

Hamid, Fursham M; Makeyev, Eugene V

2014-11-01

Alternative splicing (AS) provides a potent mechanism for increasing protein diversity and modulating gene expression levels. How alternate splice sites are selected by the splicing machinery and how AS is integrated into gene regulation networks remain important questions of eukaryotic biology. Here we report that polypyrimidine tract-binding protein 1 (Ptbp1/PTB/hnRNP-I) controls alternate 5' and 3' splice site (5'ss and 3'ss) usage in a large set of mammalian transcripts. A top scoring event identified by our analysis was the choice between competing upstream and downstream 5'ss (u5'ss and d5'ss) in the exon 18 of the Hps1 gene. Hps1 is essential for proper biogenesis of lysosome-related organelles and loss of its function leads to a disease called type 1 Hermansky-Pudlak Syndrome (HPS). We show that Ptbp1 promotes preferential utilization of the u5'ss giving rise to stable mRNAs encoding a full-length Hps1 protein, whereas bias towards d5'ss triggered by Ptbp1 down-regulation generates transcripts susceptible to nonsense-mediated decay (NMD). We further demonstrate that Ptbp1 binds to pyrimidine-rich sequences between the u5'ss and d5'ss and activates the former site rather than repressing the latter. Consistent with this mechanism, u5'ss is intrinsically weaker than d5'ss, with a similar tendency observed for other genes with Ptbp1-induced u5'ss bias. Interestingly, the brain-enriched Ptbp1 paralog Ptbp2/nPTB/brPTB stimulated the u5'ss utilization but with a considerably lower efficiency than Ptbp1. This may account for the tight correlation between Hps1 with Ptbp1 expression levels observed across mammalian tissues. More generally, these data expand our understanding of AS regulation and uncover a post-transcriptional strategy ensuring co-expression of a subordinate gene with its master regulator through an AS-NMD tracking mechanism.

cis-acting intron mutations that affect the efficiency of avian retroviral RNA splicing: implication for mechanisms of control.

PubMed Central

Katz, R A; Kotler, M; Skalka, A M

1988-01-01

The full-length retroviral RNA transcript serves as (i) mRNA for the gag and pol gene products, (ii) genomic RNA that is assembled into progeny virions, and (iii) a pre-mRNA for spliced subgenomic mRNAs. Therefore, a balance of spliced and unspliced RNA is required to generate the appropriate levels of protein and RNA products for virion production. We have introduced an insertion mutation near the avian sarcoma virus env splice acceptor site that results in a significant increase in splicing to form functional env mRNA. The mutant virus is replication defective, but phenotypic revertant viruses that have acquired second-site mutations near the splice acceptor site can be isolated readily. Detailed analysis of one of these viruses revealed that a single nucleotide change at -20 from the splice acceptor site, within the original mutagenic insert, was sufficient to restore viral growth and significantly decrease splicing efficiency compared with the original mutant and wild-type viruses. Thus, minor sequence alterations near the env splice acceptor site can produce major changes in the balance of spliced and unspliced RNAs. Our results suggest a mechanism of control in which splicing is modulated by cis-acting sequences at the env splice acceptor site. Furthermore, this retroviral system provides a powerful genetic method for selection and analysis of mutations that affect splicing control. Images PMID:2839694

Genome-Wide Analysis of Alternative Splicing Landscapes Modulated during Plant-Virus Interactions in Brachypodium distachyon

PubMed Central

Scholthof, Karen-Beth G.

2015-01-01

In eukaryotes, alternative splicing (AS) promotes transcriptome and proteome diversity. The extent of genome-wide AS changes occurring during a plant-microbe interaction is largely unknown. Here, using high-throughput, paired-end RNA sequencing, we generated an isoform-level spliceome map of Brachypodium distachyon infected with Panicum mosaic virus and its satellite virus. Overall, we detected ∼44,443 transcripts in B. distachyon, ∼30% more than those annotated in the reference genome. Expression of ∼28,900 transcripts was ≥2 fragments per kilobase of transcript per million mapped fragments, and ∼42% of multi-exonic genes were alternatively spliced. Comparative analysis of AS patterns in B. distachyon, rice (Oryza sativa), maize (Zea mays), sorghum (Sorghum bicolor), Arabidopsis thaliana, potato (Solanum tuberosum), Medicago truncatula, and poplar (Populus trichocarpa) revealed conserved ratios of the AS types between monocots and dicots. Virus infection quantitatively altered AS events in Brachypodium with little effect on the AS ratios. We discovered AS events for >100 immune-related genes encoding receptor-like kinases, NB-LRR resistance proteins, transcription factors, RNA silencing, and splicing-associated proteins. Cloning and molecular characterization of SCL33, a serine/arginine-rich splicing factor, identified multiple novel intron-retaining splice variants that are developmentally regulated and modulated during virus infection. B. distachyon SCL33 splicing patterns are also strikingly conserved compared with a distant Arabidopsis SCL33 ortholog. This analysis provides new insights into AS landscapes conserved among monocots and dicots and uncovered AS events in plant defense-related genes. PMID:25634987

Antisense Oligonucleotide-mediated Exon Skipping as a Systemic Therapeutic Approach for Recessive Dystrophic Epidermolysis Bullosa.

PubMed

Bremer, Jeroen; Bornert, Olivier; Nyström, Alexander; Gostynski, Antoni; Jonkman, Marcel F; Aartsma-Rus, Annemieke; van den Akker, Peter C; Pasmooij, Anna Mg

2016-10-18

The "generalized severe" form of recessive dystrophic epidermolysis bullosa (RDEB-gen sev) is caused by bi-allelic null mutations in COL7A1, encoding type VII collagen. The absence of type VII collagen leads to blistering of the skin and mucous membranes upon the slightest trauma. Because most patients carry exonic point mutations or small insertions/deletions, most exons of COL7A1 are in-frame, and low levels of type VII collagen already drastically improve the disease phenotype, this gene seems a perfect candidate for antisense oligonucleotide (AON)-mediated exon skipping. In this study, we examined the feasibility of AON-mediated exon skipping in vitro in primary cultured keratinocytes and fibroblasts, and systemically in vivo using a human skin-graft mouse model. We show that treatment with AONs designed against exon 105 leads to in-frame exon 105 skipping at the RNA level and restores type VII collagen protein production in vitro. Moreover, we demonstrate that systemic delivery in vivo induces de novo expression of type VII collagen in skin grafts generated from patient cells. Our data demonstrate strong proof-of-concept for AON-mediated exon skipping as a systemic therapeutic strategy for RDEB.

Genome-wide analysis of alternative splicing during dendritic cell response to a bacterial challenge.

PubMed

Rodrigues, Raquel; Grosso, Ana Rita; Moita, Luís

2013-01-01

The immune system relies on the plasticity of its components to produce appropriate responses to frequent environmental challenges. Dendritic cells (DCs) are critical initiators of innate immunity and orchestrate the later and more specific adaptive immunity. The generation of diversity in transcriptional programs is central for effective immune responses. Alternative splicing is widely considered a key generator of transcriptional and proteomic complexity, but its role has been rarely addressed systematically in immune cells. Here we used splicing-sensitive arrays to assess genome-wide gene- and exon-level expression profiles in human DCs in response to a bacterial challenge. We find widespread alternative splicing events and splicing factor transcriptional signatures induced by an E. coli challenge to human DCs. Alternative splicing acts in concert with transcriptional modulation, but these two mechanisms of gene regulation affect primarily distinct functional gene groups. Alternative splicing is likely to have an important role in DC immunobiology because it affects genes known to be involved in DC development, endocytosis, antigen presentation and cell cycle arrest.

Antisense Oligonucleotide-Mediated Transcript Knockdown in Zebrafish.

PubMed

Pauli, Andrea; Montague, Tessa G; Lennox, Kim A; Behlke, Mark A; Schier, Alexander F

2015-01-01

Antisense oligonucleotides (ASOs) are synthetic, single-strand RNA-DNA hybrids that induce catalytic degradation of complementary cellular RNAs via RNase H. ASOs are widely used as gene knockdown reagents in tissue culture and in Xenopus and mouse model systems. To test their effectiveness in zebrafish, we targeted 20 developmental genes and compared the morphological changes with mutant and morpholino (MO)-induced phenotypes. ASO-mediated transcript knockdown reproduced the published loss-of-function phenotypes for oep, chordin, dnd, ctnnb2, bmp7a, alk8, smad2 and smad5 in a dosage-sensitive manner. ASOs knocked down both maternal and zygotic transcripts, as well as the long noncoding RNA (lncRNA) MALAT1. ASOs were only effective within a narrow concentration range and were toxic at higher concentrations. Despite this drawback, quantitation of knockdown efficiency and the ability to degrade lncRNAs make ASOs a useful knockdown reagent in zebrafish.

SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

PubMed Central

Pagliarini, Vittoria; Pelosi, Laura; Bustamante, Maria Blaire; Nobili, Annalisa; Berardinelli, Maria Grazia; D’Amelio, Marcello; Musarò, Antonio

2015-01-01

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. The almost identical SMN2 gene is unable to compensate for this deficiency because of the skipping of exon 7 during pre–messenger RNA (mRNA) processing. Although several splicing factors can modulate SMN2 splicing in vitro, the physiological regulators of this disease-causing event are unknown. We found that knockout of the splicing factor SAM68 partially rescued body weight and viability of SMAΔ7 mice. Ablation of SAM68 function promoted SMN2 splicing and expression in SMAΔ7 mice, correlating with amelioration of SMA-related defects in motor neurons and skeletal muscles. Mechanistically, SAM68 binds to SMN2 pre-mRNA, favoring recruitment of the splicing repressor hnRNP A1 and interfering with that of U2AF65 at the 3′ splice site of exon 7. These findings identify SAM68 as the first physiological regulator of SMN2 splicing in an SMA mouse model. PMID:26438828

High-throughput sequence analysis of Ciona intestinalis SL trans-spliced mRNAs: alternative expression modes and gene function correlates.

PubMed

Matsumoto, Jun; Dewar, Ken; Wasserscheid, Jessica; Wiley, Graham B; Macmil, Simone L; Roe, Bruce A; Zeller, Robert W; Satou, Yutaka; Hastings, Kenneth E M

2010-05-01

Pre-mRNA 5' spliced-leader (SL) trans-splicing occurs in some metazoan groups but not in others. Genome-wide characterization of the trans-spliced mRNA subpopulation has not yet been reported for any metazoan. We carried out a high-throughput analysis of the SL trans-spliced mRNA population of the ascidian tunicate Ciona intestinalis by 454 Life Sciences (Roche) pyrosequencing of SL-PCR-amplified random-primed reverse transcripts of tailbud embryo RNA. We obtained approximately 250,000 high-quality reads corresponding to 8790 genes, approximately 58% of the Ciona total gene number. The great depth of this data revealed new aspects of trans-splicing, including the existence of a significant class of "infrequently trans-spliced" genes, accounting for approximately 28% of represented genes, that generate largely non-trans-spliced mRNAs, but also produce trans-spliced mRNAs, in part through alternative promoter use. Thus, the conventional qualitative dichotomy of trans-spliced versus non-trans-spliced genes should be supplanted by a more accurate quantitative view recognizing frequently and infrequently trans-spliced gene categories. Our data include reads representing approximately 80% of Ciona frequently trans-spliced genes. Our analysis also revealed significant use of closely spaced alternative trans-splice acceptor sites which further underscores the mechanistic similarity of cis- and trans-splicing and indicates that the prevalence of +/-3-nt alternative splicing events at tandem acceptor sites, NAGNAG, is driven by spliceosomal mechanisms, and not nonsense-mediated decay, or selection at the protein level. The breadth of gene representation data enabled us to find new correlations between trans-splicing status and gene function, namely the overrepresentation in the frequently trans-spliced gene class of genes associated with plasma/endomembrane system, Ca(2+) homeostasis, and actin cytoskeleton.

CD82 suppresses CD44 alternative splicing-dependent melanoma metastasis by mediating U2AF2 ubiquitination and degradation

PubMed Central

Fu, Ailing; Zhu, Huifeng; Ren, Qiao; Wang, Bochu; Xu, Xingran; Bai, Huiyuan; Dong, Cheng

2016-01-01

Melanoma is one of the most lethal forms of skin cancer due to its early metastatic spread. The variant form of CD44 (CD44v), a cell surface glycoprotein, is highly expressed on metastatic melanoma. The mechanisms of regulation of CD44 alternative splicing in melanoma and its pathogenic contributions are so far poorly understood. Here, we investigated the expression level of CD44 in a large set of melanocytic lesions at different stages. We found that the expression of CD44v8-10 and a splicing factor, U2AF2, is significantly increased during melanoma progression, while CD82/KAI1, a tetraspanin family of tumor suppressor, is reduced in metastatic melanoma. CD44v8-10 and U2AF2 expressions which are negatively correlated with CD82 levels are dramatically elevated in primary melanoma compared with dysplastic nevi and further increased in metastatic melanoma. We also showed that patients with higher CD44v8-10 and U2AF2 expression levels tended to have shorter survival. By using both in vivo and in vitro assays, we demonstrated that CD82 inhibits the production of CD44v8-10 on melanoma. Mechanistically, U2AF2 is a downstream target of CD82 and in malignant melanoma facilitates CD44v8-10 alternative splicing. U2AF2-mediated CD44 isoform switch is required for melanoma migration in vitro and lung and liver metastasis in vivo. Notably, overexpression of CD82 suppresses U2AF2 activity by inducing U2AF2 ubiquitination. In addition, our data suggested that enhancement of melanoma migration by U2AF2-dependent CD44v8-10 splicing is mediated by Src/FAK/RhoA activation and formation of stress fibers as well as CD44-E-selectin binding reinforcement. These findings uncovered a hitherto unappreciated function of CD82 in severing the linkage between U2AF2-mediated CD44 alternative splicing and cancer aggressiveness, with potential prognostic and therapeutic implications in melanoma. PMID:27041584

Regulation of an antisense RNA with the transition of neonatal to IIb myosin heavy chain during postnatal development and hypothyroidism in rat skeletal muscle

PubMed Central

Jiang, Weihua; Qin, Anqi X.; Bodell, Paul W.; Baldwin, Kenneth M.; Haddad, Fadia

2012-01-01

Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development. PMID:22262309

Regulation of an antisense RNA with the transition of neonatal to IIb myosin heavy chain during postnatal development and hypothyroidism in rat skeletal muscle.

PubMed

Pandorf, Clay E; Jiang, Weihua; Qin, Anqi X; Bodell, Paul W; Baldwin, Kenneth M; Haddad, Fadia

2012-04-01

Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development.

Gene Silencing by Gold Nanoshell-Mediated Delivery and Laser-Triggered Release of Antisense Oligonucleotide and siRNA

PubMed Central

Huschka, Ryan; Barhoumi, Aoune; Liu, Qing; Roth, Jack A.; Ji, Lin; Halas, Naomi J.

2013-01-01

The approach of RNA interference (RNAi)- using antisense DNA or RNA oligonucleotides to silence activity of a specific pathogenic gene transcript and reduce expression of the encoded protein- is very useful in dissecting genetic function and holds significant promise as a molecular therapeutic. A major obstacle in achieving gene silencing with RNAi technology is the systemic delivery of therapeutic oligonucleotides. Here we demonstrate an engineered gold nanoshell (NS)-based therapeutic oligonucleotide delivery vehicle, designed to release its cargo on demand upon illumination with a near-infrared (NIR) laser. A poly(L)lysine peptide (PLL) epilayer covalently attached to the NS surface (NS-PLL) is used to capture intact, single-stranded antisense DNA oligonucleotides, or alternatively, double-stranded short-interfering RNA (siRNA) molecules. Controlled release of the captured therapeutic oligonucleotides in each case is accomplished by continuous wave NIR laser irradiation at 800 nm, near the resonance wavelength of the nanoshell. Fluorescently tagged oligonucleotides were used to monitor the time-dependent release process and light-triggered endosomal release. A green fluorescent protein (GFP)-expressing human lung cancer H1299 cell line was used to determine cellular uptake and gene silencing mediated by the NS-PLL carrying GFP gene-specific single-stranded DNA antisense oligonucleotide (AON-GFP), or a double-stranded siRNA (siRNA-GFP), in vitro. Light-triggered delivery resulted in ∼ 47% and ∼49% downregulation of the targeted GFP expression by AON-GFP and siRNA-GFP, respectively. Cytotoxicity induced by both the NS-PLL delivery vector and by laser irradiation is minimal, as demonstrated by a XTT cell proliferation assay. PMID:22862291

Gene delivery to the neurulating embryo during culture

EPA Science Inventory

Modulating expression of specific genes during embryogenesis will help elucidate their role in development. Transient overexpression of specific genes can be accomplished by adding additional copies, or else antisense transcripts can be used to block expression. Manipulation of g...

SPF45-related SPLICING FACTOR FOR PHYTOCHROME SIGNALING promotes photomorphogensis by regulating pre-mRNA splicing in arabidopsis

USDA-ARS?s Scientific Manuscript database

Light signals regulate plant growth and development by controlling a plethora of gene expression changes. Post-transcriptional regulation, especially pre-mRNA processing, is a key modulator of gene expression; however, the molecular mechanisms linking pre-mRNA processing and light signaling are not ...

Regulatory RNA binding proteins contribute to the transcriptome-wide splicing alterations in human cellular senescence.

PubMed

Dong, Qiongye; Wei, Lei; Zhang, Michael Q; Wang, Xiaowo

2018-06-24

Dysregulation of mRNA splicing has been observed in certain cellular senescence process. However, the common splicing alterations on the whole transcriptome shared by various types of senescence are poorly understood. In order to systematically identify senescence-associated transcriptomic changes in genome-wide scale, we collected RNA sequencing datasets of different human cell types with a variety of senescence-inducing methods from public databases and performed meta-analysis. First, we discovered that a group of RNA binding proteins were consistently down-regulated in diverse senescent samples and identified 406 senescence-associated common differential splicing events. Then, eight differentially expressed RNA binding proteins were predicted to regulate these senescence-associated splicing alterations through an enrichment analysis of their RNA binding information, including motif scanning and enhanced cross-linking immunoprecipitation data. In addition, we constructed the splicing regulatory modules that might contribute to senescence-associated biological processes. Finally, it was confirmed that knockdown of the predicted senescence-associated potential splicing regulators through shRNAs in HepG2 cell line could result in senescence-like splicing changes. Taken together, our work demonstrated a broad range of common changes in mRNA splicing switches and detected their central regulatory RNA binding proteins during senescence. These findings would help to better understand the coordinating splicing alterations in cellular senescence.

A zebrafish sox9 gene required for cartilage morphogenesis.

PubMed

Yan, Yi-Lin; Miller, Craig T; Nissen, Robert M; Singer, Amy; Liu, Dong; Kirn, Anette; Draper, Bruce; Willoughby, John; Morcos, Paul A; Amsterdam, Adam; Chung, Bon-Chu; Westerfield, Monte; Haffter, Pascal; Hopkins, Nancy; Kimmel, Charles; Postlethwait, John H; Nissen, Robert

2002-11-01

The molecular genetic mechanisms of cartilage construction are incompletely understood. Zebrafish embryos homozygous for jellyfish (jef) mutations show craniofacial defects and lack cartilage elements of the neurocranium, pharyngeal arches, and pectoral girdle similar to humans with campomelic dysplasia. We show that two alleles of jef contain mutations in sox9a, one of two zebrafish orthologs of the human transcription factor SOX9. A mutation induced by ethyl nitrosourea changed a conserved nucleotide at a splice junction and severely reduced splicing of sox9a transcript. A retrovirus insertion into sox9a disrupted its DNA-binding domain. Inhibiting splicing of the sox9a transcript in wild-type embryos with splice site-directed morpholino antisense oligonucleotides produced a phenotype like jef mutant larvae, and caused sox9a transcript to accumulate in the nucleus; this accumulation can serve as an assay for the efficacy of a morpholino independent of phenotype. RNase-protection assays showed that in morpholino-injected animals, the percent of splicing inhibition decreased from 80% at 28 hours post fertilization to 45% by 4 days. Homozygous mutant embryos had greatly reduced quantities of col2a1 message, the major collagen of cartilage. Analysis of dlx2 expression showed that neural crest specification and migration was normal in jef (sox9a) embryos. Confocal images of living embryos stained with BODIPY-ceramide revealed at single-cell resolution the formation of precartilage condensations in mutant embryos. Besides the lack of overt cartilage differentiation, pharyngeal arch condensations in jef (sox9a) mutants lacked three specific morphogenetic behaviors: the stacking of chondrocytes into orderly arrays, the individuation of pharyngeal cartilage organs and the proper shaping of individual cartilages. Despite the severe reduction of cartilages, analysis of titin expression showed normal muscle patterning in jef (sox9a) mutants. Likewise, calcein labeling revealed that early bone formation was largely unaffected in jef (sox9a) mutants. These studies show that jef (sox9a) is essential for both morphogenesis of condensations and overt cartilage differentiation.

Identifying RNA splicing factors using IFT genes in Chlamydomonas reinhardtii.

PubMed

Lin, Huawen; Zhang, Zhengyan; Iomini, Carlo; Dutcher, Susan K

2018-03-01

Intraflagellar transport moves proteins in and out of flagella/cilia and it is essential for the assembly of these organelles. Using whole-genome sequencing, we identified splice site mutations in two IFT genes, IFT81 ( fla9 ) and IFT121 ( ift121-2 ), which lead to flagellar assembly defects in the unicellular green alga Chlamydomonas reinhardtii The splicing defects in these ift mutants are partially corrected by mutations in two conserved spliceosome proteins, DGR14 and FRA10. We identified a dgr14 deletion mutant, which suppresses the 3' splice site mutation in IFT81 , and a frameshift mutant of FRA10 , which suppresses the 5' splice site mutation in IFT121 Surprisingly, we found dgr14-1 and fra10 mutations suppress both splice site mutations. We suggest these two proteins are involved in facilitating splice site recognition/interaction; in their absence some splice site mutations are tolerated. Nonsense mutations in SMG1 , which is involved in nonsense-mediated decay, lead to accumulation of aberrant transcripts and partial restoration of flagellar assembly in the ift mutants. The high density of introns and the conservation of noncore splicing factors, together with the ease of scoring the ift mutant phenotype, make Chlamydomonas an attractive organism to identify new proteins involved in splicing through suppressor screening. © 2018 The Authors.

Functional Properties of a Newly Identified C-terminal Splice Variant of Cav1.3 L-type Ca2+ Channels*

PubMed Central

Bock, Gabriella; Gebhart, Mathias; Scharinger, Anja; Jangsangthong, Wanchana; Busquet, Perrine; Poggiani, Chiara; Sartori, Simone; Mangoni, Matteo E.; Sinnegger-Brauns, Martina J.; Herzig, Stefan; Striessnig, Jörg; Koschak, Alexandra

2011-01-01

An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Cav1.3 L-type Ca2+ channels (Cav1.3L) is a major determinant of their voltage- and Ca2+-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Cav1.342A channels that activate at a more negative voltage range and exhibit more pronounced Ca2+-dependent inactivation. Here we describe the discovery of a novel short splice variant (Cav1.343S) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Cav1.342A, still contains PCRD. When expressed together with α2δ1 and β3 subunits in tsA-201 cells, Cav1.343S also activated at more negative voltages like Cav1.342A but Ca2+-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Cav1.3L. The presence of the proximal C terminus in Cav1.343S channels preserved their modulation by distal C terminus-containing Cav1.3- and Cav1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca2+ influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Cav1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca2+ channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca2+ accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca2+-induced neurodegenerative processes. PMID:21998310

Transcriptomic insights into the alternative splicing-mediated adaptation of the entomopathogenic fungus Beauveria bassiana to host niches: autophagy-related gene 8 as an example.

PubMed

Dong, Wei-Xia; Ding, Jin-Li; Gao, Yang; Peng, Yue-Jin; Feng, Ming-Guang; Ying, Sheng-Hua

2017-10-01

Alternative splicing (AS) regulates various biological processes in fungi by extending the cellular proteome. However, comprehensive studies investigating AS in entomopathogenic fungi are lacking. Based on transcriptome data obtained via dual RNA-seq, the first overview of AS events was developed for Beauveria bassiana growing in an insect haemocoel. The AS was demonstrated for 556 of 8840 expressed genes, accounting for 5.4% of the total genes in B. bassiana. Intron retention was the most abundant type of AS, accounting for 87.1% of all splicing events and exon skipping events were rare, only accounting for 2.0% of all events. Functional distribution analysis indicated an association between alternatively spliced genes and several physiological processes. Notably, B. bassiana autophagy-related gene 8 (BbATG8), an indispensable gene for autophagy, was spliced at an alternative 5' splice site to generate two transcripts (BbATG8-α and BbATG8-β). The BbATG8-α transcript was necessary for fungal autophagy and oxidation tolerance, while the BbATG8-β transcript was not. These two transcripts differentially contributed to the formation of conidia or blastospores as well as fungal virulence. Thus, AS acts as a powerful post-transcriptional regulatory strategy in insect mycopathogens and significantly mediates fungal transcriptional adaption to host niches. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Development of Neuroendocrine Prostate Cancers by the Ser/Arg Repetitive Matrix 4-Mediated RNA Splicing Network.

PubMed

Lee, Ahn R; Che, Nicole; Lovnicki, Jessica M; Dong, Xuesen

2018-01-01

While the use of next-generation androgen receptor pathway inhibition (ARPI) therapy has significantly increased the survival of patients with metastatic prostate adenocarcinoma (AdPC), several groups have reported a treatment-resistant mechanism, whereby cancer cells can become androgen receptor (AR) indifferent and gain a neuroendocrine (NE)-like phenotype. This subtype of castration-resistant prostate cancer has been termed "treatment-induced castration-resistant neuroendocrine prostate cancer" (CRPC-NE). Recent reports indicate that the overall genomic landscapes of castration-resistant tumors with AdPC phenotypes and CRPC-NE are not significantly altered. However, CRPC-NE tumors have been found to contain a NE-specific pattern throughout their epigenome and splicing transcriptome, which are significantly modified. The molecular mechanisms by which CRPC-NE develops remain unclear, but several factors have been implicated in the progression of the disease. Recently, Ser/Arg repetitive matrix 4 (SRRM4), a neuronal-specific RNA splicing factor that is upregulated in CRPC-NE tumors, has been shown to establish a CRPC-NE-unique splicing transcriptome, to induce a NE-like morphology in AdPC cells, and, most importantly, to transform AdPC cells into CRPC-NE xenografts under ARPI. Moreover, the SRRM4-targeted splicing genes are highly enriched in various neuronal processes, suggesting their roles in facilitating a CRPC-NE program. This article will address the importance of SRRM4-mediated alternative RNA splicing in reprogramming translated proteins to facilitate NE differentiation, survival, and proliferation of cells to establish CRPC-NE tumors. In addition, we will discuss the potential roles of SRRM4 in conjunction with other known pathways and factors important for CRPC-NE development, such as the AR pathway, TP53 and RB1 genes, the FOXA family of proteins, and environmental factors. This study aims to explore the multifaceted functions of SRRM4 and SRRM4-mediated splicing in driving a CRPC-NE program as a coping mechanism for therapy resistance, as well as define future SRRM4-targeted therapeutic approaches for treating CRPC-NE or mitigating its development.

Development of Neuroendocrine Prostate Cancers by the Ser/Arg Repetitive Matrix 4-Mediated RNA Splicing Network

PubMed Central

Lee, Ahn R.; Che, Nicole; Lovnicki, Jessica M.; Dong, Xuesen

2018-01-01

While the use of next-generation androgen receptor pathway inhibition (ARPI) therapy has significantly increased the survival of patients with metastatic prostate adenocarcinoma (AdPC), several groups have reported a treatment-resistant mechanism, whereby cancer cells can become androgen receptor (AR) indifferent and gain a neuroendocrine (NE)-like phenotype. This subtype of castration-resistant prostate cancer has been termed “treatment-induced castration-resistant neuroendocrine prostate cancer” (CRPC-NE). Recent reports indicate that the overall genomic landscapes of castration-resistant tumors with AdPC phenotypes and CRPC-NE are not significantly altered. However, CRPC-NE tumors have been found to contain a NE-specific pattern throughout their epigenome and splicing transcriptome, which are significantly modified. The molecular mechanisms by which CRPC-NE develops remain unclear, but several factors have been implicated in the progression of the disease. Recently, Ser/Arg repetitive matrix 4 (SRRM4), a neuronal-specific RNA splicing factor that is upregulated in CRPC-NE tumors, has been shown to establish a CRPC-NE-unique splicing transcriptome, to induce a NE-like morphology in AdPC cells, and, most importantly, to transform AdPC cells into CRPC-NE xenografts under ARPI. Moreover, the SRRM4-targeted splicing genes are highly enriched in various neuronal processes, suggesting their roles in facilitating a CRPC-NE program. This article will address the importance of SRRM4-mediated alternative RNA splicing in reprogramming translated proteins to facilitate NE differentiation, survival, and proliferation of cells to establish CRPC-NE tumors. In addition, we will discuss the potential roles of SRRM4 in conjunction with other known pathways and factors important for CRPC-NE development, such as the AR pathway, TP53 and RB1 genes, the FOXA family of proteins, and environmental factors. This study aims to explore the multifaceted functions of SRRM4 and SRRM4-mediated splicing in driving a CRPC-NE program as a coping mechanism for therapy resistance, as well as define future SRRM4-targeted therapeutic approaches for treating CRPC-NE or mitigating its development. PMID:29666783

Sugar and Glycerol Transport in Saccharomyces cerevisiae.

PubMed

Bisson, Linda F; Fan, Qingwen; Walker, Gordon A

2016-01-01

In Saccharomyces cerevisiae the process of transport of sugar substrates into the cell comprises a complex network of transporters and interacting regulatory mechanisms. Members of the large family of hexose (HXT) transporters display uptake efficiencies consistent with their environmental expression and play physiological roles in addition to feeding the glycolytic pathway. Multiple glucose-inducing and glucose-independent mechanisms serve to regulate expression of the sugar transporters in yeast assuring that expression levels and transporter activity are coordinated with cellular metabolism and energy needs. The expression of sugar transport activity is modulated by other nutritional and environmental factors that may override glucose-generated signals. Transporter expression and activity is regulated transcriptionally, post-transcriptionally and post-translationally. Recent studies have expanded upon this suite of regulatory mechanisms to include transcriptional expression fine tuning mediated by antisense RNA and prion-based regulation of transcription. Much remains to be learned about cell biology from the continued analysis of this dynamic process of substrate acquisition.

The excludon: a new concept in bacterial antisense RNA-mediated gene regulation.

PubMed

Sesto, Nina; Wurtzel, Omri; Archambaud, Cristel; Sorek, Rotem; Cossart, Pascale

2013-02-01

In recent years, non-coding RNAs have emerged as key regulators of gene expression. Among these RNAs, the antisense RNAs (asRNAs) are particularly abundant, but in most cases the function and mechanism of action for a particular asRNA remains elusive. Here, we highlight a recently discovered paradigm termed the excludon, which defines a genomic locus encoding an unusually long asRNA that spans divergent genes or operons with related or opposing functions. Because these asRNAs can inhibit the expression of one operon while functioning as an mRNA for the adjacent operon, they act as fine-tuning regulatory switches in bacteria.

Suppression of cell division by pKi-67 antisense-RNA and recombinant protein.

PubMed

Duchrow, M; Schmidt, M H; Zingler, M; Anemüller, S; Bruch, H P; Broll, R

2001-01-01

The human antigen defined by the monoclonal antibody Ki-67 (pKi-67) is a human nuclear protein strongly associated with cell proliferation and found in all tissues studied. It is widely used as a marker of proliferating cells, yet its function is unknown. To investigate its function we suppressed pKi-67 expression by antisense RNA and overexpressed a partial structure of pKi-67 in HeLa cells. A BrdU-incorporation assay showed a significant decrease in DNA synthesis after antisense inhibition. Cell cycle analysis indicated a higher proportion of cells in G1 phase and a lower proportion of cells in S phase while the number of G(2)/M phase cells remained constant. Overexpression of a recombinant protein encoding three of the repetitive elements from exon 13 of pKi-67 had a similar effect to that obtained by antisense inhibition. The similarity of the effect of expressing 'Ki-67 repeats' and pKi-67 antisense RNA could be explained by a negative effect on the folding of the endogenous protein in the endoplasmatic reticulum. Furthermore excessive self-association of pKi-67 via the repeat structure could inhibit its nuclear transport, preventing it from getting to its presumptive site of action. We conclude that the Ki-67 protein has an important role in the regulation of the cell cycle, which is mediated in part by its repetitive elements. Copyright 2001 S. Karger AG, Basel

Spliceosomal protein U1A is involved in alternative splicing and salt stress tolerance in Arabidopsis thaliana

PubMed Central

Gu, Jinbao; Xia, Zhiqiang; Luo, Yuehua; Jiang, Xingyu; Qian, Bilian; Xie, He; Zhu, Jian-Kang; Xiong, Liming; Zhu, Jianhua; Wang, Zhen-Yu

2018-01-01

Abstract Soil salinity is a significant threat to sustainable agricultural production worldwide. Plants must adjust their developmental and physiological processes to cope with salt stress. Although the capacity for adaptation ultimately depends on the genome, the exceptional versatility in gene regulation provided by the spliceosome-mediated alternative splicing (AS) is essential in these adaptive processes. However, the functions of the spliceosome in plant stress responses are poorly understood. Here, we report the in-depth characterization of a U1 spliceosomal protein, AtU1A, in controlling AS of pre-mRNAs under salt stress and salt stress tolerance in Arabidopsis thaliana. The atu1a mutant was hypersensitive to salt stress and accumulated more reactive oxygen species (ROS) than the wild-type under salt stress. RNA-seq analysis revealed that AtU1A regulates AS of many genes, presumably through modulating recognition of 5′ splice sites. We showed that AtU1A is associated with the pre-mRNA of the ROS detoxification-related gene ACO1 and is necessary for the regulation of ACO1 AS. ACO1 is important for salt tolerance because ectopic expression of ACO1 in the atu1a mutant can partially rescue its salt hypersensitive phenotype. Our findings highlight the critical role of AtU1A as a regulator of pre-mRNA processing and salt tolerance in plants. PMID:29228330

pPKCδ activates SC35 splicing factor during H9c2 myoblastic differentiation.

PubMed

Zara, Susi; Falconi, Mirella; Rapino, Monica; Zago, Michela; Orsini, Giovanna; Mazzotti, Giovanni; Cataldi, Amelia; Teti, Gabriella

2011-01-01

Although Protein Kinase C (PKC) isoforms' role in the neonatal and adult cardiac tissue development and ageing has been widely described "in vivo", the interaction of such enzymes with specific nuclear substrates needs to be investigated. The aim of our research has been the study of the expression, localization and interaction with the splicing factor SC35 of PKC isoforms (α, δ, ε, ζ) and their potential role in modulating the transcription machinery. H9c2 cells induced to myoblast differentiation in the presence of 1% Horse Serum (HS) have represented our experimental model. The expression of PKC isoforms, their distribution and interaction with SC35 have been evaluated by western blotting, co-immunoprecipitation and double gold immunolabeling for transmission and scanning electron microscopy. Our results show PKCδ as the most expressed isoform in differentiated cells. Surprisingly, the distribution of PKCδ and SC35 does not show any significant modification between 10%FBS and 1%HS treated samples and no co-localization is observed. Moreover the interaction between the phosphorylated form of PKCδ (pPKCδ) and SC35 increases, is distributed and co-localizes within the nucleus of differentiated H9c2. These data represent reasonable evidence of pPKCδ mediated SC35 splicing factor activation, suggesting its direct effect on transcription via interaction with the transcription machinery. Furthermore, this co-localization represents a crucial event resulting in downstream changes in transcription of components which determine the morphological modifications related to cardiomyoblast differentiated phenotype.

Dysferlin rescue by spliceosome-mediated pre-mRNA trans-splicing targeting introns harbouring weakly defined 3' splice sites.

PubMed

Philippi, Susanne; Lorain, Stéphanie; Beley, Cyriaque; Peccate, Cécile; Précigout, Guillaume; Spuler, Simone; Garcia, Luis

2015-07-15

The modification of the pre-mRNA cis-splicing process employing a pre-mRNA trans-splicing molecule (PTM) is an attractive strategy for the in situ correction of genes whose careful transcription regulation and full-length expression is determinative for protein function, as it is the case for the dysferlin (DYSF, Dysf) gene. Loss-of-function mutations of DYSF result in different types of muscular dystrophy mainly manifesting as limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi muscular dystrophy 1 (MMD1). We established a 3' replacement strategy for mutated DYSF pre-mRNAs induced by spliceosome-mediated pre-mRNA trans-splicing (SmaRT) by the use of a PTM. In contrast to previously established SmaRT strategies, we particularly focused on the identification of a suitable pre-mRNA target intron other than the optimization of the PTM design. By targeting DYSF pre-mRNA introns harbouring differentially defined 3' splice sites (3' SS), we found that target introns encoding weakly defined 3' SSs were trans-spliced successfully in vitro in human LGMD2B myoblasts as well as in vivo in skeletal muscle of wild-type and Dysf(-/-) mice. For the first time, we demonstrate rescue of Dysf protein by SmaRT in vivo. Moreover, we identified concordant qualities among the successfully targeted Dysf introns and targeted endogenous introns in previously reported SmaRT approaches that might facilitate a selective choice of target introns in future SmaRT strategies. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The delivery of therapeutic oligonucleotides

PubMed Central

Juliano, Rudolph L.

2016-01-01

The oligonucleotide therapeutics field has seen remarkable progress over the last few years with the approval of the first antisense drug and with promising developments in late stage clinical trials using siRNA or splice switching oligonucleotides. However, effective delivery of oligonucleotides to their intracellular sites of action remains a major issue. This review will describe the biological basis of oligonucleotide delivery including the nature of various tissue barriers and the mechanisms of cellular uptake and intracellular trafficking of oligonucleotides. It will then examine a variety of current approaches for enhancing the delivery of oligonucleotides. This includes molecular scale targeted ligand-oligonucleotide conjugates, lipid- and polymer-based nanoparticles, antibody conjugates and small molecules that improve oligonucleotide delivery. The merits and liabilities of these approaches will be discussed in the context of the underlying basic biology. PMID:27084936

Biological activity and biotechnological aspects of locked nucleic acids.

PubMed

Lundin, Karin E; Højland, Torben; Hansen, Bo R; Persson, Robert; Bramsen, Jesper B; Kjems, Jørgen; Koch, Troels; Wengel, Jesper; Smith, C I Edvard

2013-01-01

Locked nucleic acid (LNA) is one of the most promising new nucleic acid analogues that has been produced under the past two decades. In this chapter, we have tried to cover many of the different areas, where this molecule has been used to improve the function of synthetic oligonucleotides (ONs). The use of LNA in antisense ONs, including gapmers, splice-switching ONs, and siLNA, as well as antigene ONs, is reviewed. Pharmacokinetics as well as pharmacodynamics of LNA ONs and a description of selected compounds in, or close to, clinical testing are described. In addition, new LNA modifications and the adaptation of enzymes for LNA incorporation are reviewed. Such enzymes may become important for the development of stabilized LNA-containing aptamers. Copyright © 2013 Elsevier Inc. All rights reserved.

Effects of PTCs on nonsense-mediated mRNA decay are dependent on PTC location.

PubMed

Moon, Heegyum; Zheng, Xuexiu; Loh, Tiing Jen; Jang, Ha Na; Liu, Yongchao; Jung, Da-Woon; Williams, Darren R; Shen, Haihong

2017-03-01

The récepteur d'origine nantais (RON) gene is a proto-oncogene that is responsible for encoding the human macrophage-stimulating protein (MSP) 1 receptor. MSP activation induces RON-mediated cell dissociation, migration and matrix invasion. Isoforms of RON that exclude exons 5 and 6 encode the RONΔ160 protein, which promotes cell transformation in vitro and tumor metastasis in vivo . Premature termination codons (PTCs) in exons activate the nonsense-mediated mRNA decay (NMD) signaling pathway. The present study demonstrated that PTCs at various locations in the alternative exons 5 and 6 could induce NMD of the majority of the spliced, or partially spliced, isoforms. However, the isoforms that excluded exon 6 or exons 5 and 6 were markedly increased when produced from mutated minigenes with inserted PTCs. Furthermore, the unspliced isoform of intron 5 was not observed to be decreased by the presence of PTCs. Notably, these effects may be dependent on the location of the PTCs. The current study demonstrated a novel mechanism underlying the regulation of NMD in alternative splicing.

Conditional Toxin Splicing Using a Split Intein System.

PubMed

Alford, Spencer C; O'Sullivan, Connor; Howard, Perry L

2017-01-01

Protein toxin splicing mediated by split inteins can be used as a strategy for conditional cell ablation. The approach requires artificial fragmentation of a potent protein toxin and tethering each toxin fragment to a split intein fragment. The toxin-intein fragments are, in turn, fused to dimerization domains, such that addition of a dimerizing agent reconstitutes the split intein. These chimeric toxin-intein fusions remain nontoxic until the dimerizer is added, resulting in activation of intein splicing and ligation of toxin fragments to form an active toxin. Considerations for the engineering and implementation of conditional toxin splicing (CTS) systems include: choice of toxin split site, split site (extein) chemistry, and temperature sensitivity. The following method outlines design criteria and implementation notes for CTS using a previously engineered system for splicing a toxin called sarcin, as well as for developing alternative CTS systems.

The human ubiquitin-conjugating enzyme Cdc34 controls cellular proliferation through regulation of p27{sup Kip1} protein levels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Butz, Nicole; Ruetz, Stephan; Natt, Francois

2005-02-15

Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27{sup Kip1} was shown to be required for the activation of key cyclin-dependent kinases, thereby triggering the onset of DNA replication and cell cycle progression. Although the SCF{sup Skp2} ubiquitin ligase has been reported to mediate p27{sup Kip1} degradation, the nature of the human ubiquitin-conjugating enzyme involved in this process has not yet been determined at the cellular level. Here, we show that antisense oligonucleotides targeting the human ubiquitin-conjugating enzyme Cdc34 downregulate its expression, inhibit the degradation of p27{sup Kip1}, and prevent cellular proliferation. Elevation of p27{sup Kip1} protein level is found tomore » be the sole requirement for the inhibition of cellular proliferation induced upon downregulation of Cdc34. Indeed, reducing the expression of p27{sup Kip1} with a specific antisense oligonucleotide is sufficient to reverse the anti-proliferative phenotype elicited by the Cdc34 antisense. Furthermore, downregulation of Cdc34 is found to specifically increase the abundance of the SCF{sup Skp2} ubiquitin ligase substrate p27{sup Kip1}, but has no concomitant effect on the level of IkB{alpha} and {beta}-catenin, which are known substrates of a closely related SCF ligase.« less

The Role of Mesopontine NGF in Sleep and Wakefulness

PubMed Central

Ramos, Oscar V.; Torterolo, Pablo; Lim, Vincent; Chase, Michael H.; Sampogna, Sharon; Yamuy, Jack

2011-01-01

The microinjection of nerve growth factor (NGF) into the cat pontine tegmentum rapidly induces rapid eye movement (REM) sleep. To determine if NGF is involved in naturally-occurring REM sleep, we examined whether it is present in mesopontine cholinergic structures that promote the initiation of REM sleep, and whether the blockade of NGF production in these structures suppresses REM sleep. We found that cholinergic neurons in the cat dorsolateral mesopontine tegmentum exhibited NGF-like immunoreactivity. In addition, the microinjection of an oligodeoxyribonucleotide (OD) directed against cat NGF mRNA into this region resulted in a reduction in the time spent in REM sleep in conjunction with an increase in the time spent in wakefulness. Sleep and wakefulness returned to baseline conditions 2 to 5 days after antisense OD administration. The preceding antisense OD-induced effects occurred in conjunction with the suppression of NGF-like immunoreactivity within the site of antisense OD injection. These data support the hypothesis that NGF is involved in the modulation of naturally-occurring sleep and wakefulness. PMID:21840513

Alternative Splicing of hTERT Pre-mRNA: A Potential Strategy for the Regulation of Telomerase Activity.

PubMed

Liu, Xuewen; Wang, Yuchuan; Chang, Guangming; Wang, Feng; Wang, Fei; Geng, Xin

2017-03-07

The activation of telomerase is one of the key events in the malignant transition of cells, and the expression of human telomerase reverse transcriptase (hTERT) is indispensable in the process of activating telomerase. The pre-mRNA alternative splicing of hTERT at the post-transcriptional level is one of the mechanisms for the regulation of telomerase activity. Shifts in splicing patterns occur in the development, tumorigenesis, and response to diverse stimuli in a tissue-specific and cell type-specific manner. Despite the regulation of telomerase activity, the alternative splicing of hTERT pre-mRNA may play a role in other cellular functions. Modulating the mode of hTERT pre-mRNA splicing is providing a new precept of therapy for cancer and aging-related diseases. This review focuses on the patterns of hTERT pre-mRNA alternative splicing and their biological functions, describes the potential association between the alternative splicing of hTERT pre-mRNA and telomerase activity, and discusses the possible significance of the alternative splicing of the hTERT pre-mRNA in the diagnosis, therapy, and prognosis of cancer and aging-related diseases.

Modulation of ColE1-like Plasmid Replication for Recombinant Gene Expression

PubMed Central

Camps, Manel

2010-01-01

ColE1-like plasmids constitute the most popular vectors for recombinant protein expression. ColE1 plasmid replication is tightly controlled by an antisense RNA mechanism that is highly dynamic, tuning plasmid metabolic burden to the physiological state of the host. Plasmid homeostasis is upset upon induction of recombinant protein expression because of non-physiological levels of expression and because of the frequently biased amino acid composition of recombinant proteins. Disregulation of plasmid replication is the main cause of collapse of plasmid-based expression systems because of a simultaneous increase in the metabolic burden (due to increased average copy number) and in the probability of generation of plasmid-free cells (due to increased copy number variation). Interference between regulatory elements of co-resident plasmids causes comparable effects on plasmid stability (plasmid incompatibility). Modulating plasmid copy number for recombinant gene expression aims at achieving a high gene dosage while preserving the stability of the expression system. Here I present strategies targeting plasmid replication for optimizing recombinant gene expression. Specifically, I review approaches aimed at modulating the antisense regulatory system (as well as their implications for plasmid incompatibility) and innovative strategies involving modulation of host factors, of R-loop formation, and of the timing of recombinant gene expression. PMID:20218961

FOX-2 Dependent Splicing of Ataxin-2 Transcript Is Affected by Ataxin-1 Overexpression

PubMed Central

Welzel, Franziska; Kaehler, Christian; Isau, Melanie; Hallen, Linda; Lehrach, Hans; Krobitsch, Sylvia

2012-01-01

Alternative splicing is a fundamental posttranscriptional mechanism for controlling gene expression, and splicing defects have been linked to various human disorders. The splicing factor FOX-2 is part of a main protein interaction hub in a network related to human inherited ataxias, however, its impact remains to be elucidated. Here, we focused on the reported interaction between FOX-2 and ataxin-1, the disease-causing protein in spinocerebellar ataxia type 1. In this line, we further evaluated this interaction by yeast-2-hybrid analyses and co-immunoprecipitation experiments in mammalian cells. Interestingly, we discovered that FOX-2 localization and splicing activity is affected in the presence of nuclear ataxin-1 inclusions. Moreover, we observed that FOX-2 directly interacts with ataxin-2, a protein modulating spinocerebellar ataxia type 1 pathogenesis. Finally, we provide evidence that splicing of pre-mRNA of ataxin-2 depends on FOX-2 activity, since reduction of FOX-2 levels led to increased skipping of exon 18 in ataxin-2 transcripts. Most striking, we observed that ataxin-1 overexpression has an effect on this splicing event as well. Thus, our results demonstrate that FOX-2 is involved in splicing of ataxin-2 transcripts and that this splicing event is altered by overexpression of ataxin-1. PMID:22666429

A Challenging Pie to Splice: Drugging the Spliceosome.

PubMed

León, Brian; Kashyap, Manoj K; Chan, Warren C; Krug, Kelsey A; Castro, Januario E; La Clair, James J; Burkart, Michael D

2017-09-25

Since its discovery in 1977, the study of alternative RNA splicing has revealed a plethora of mechanisms that had never before been documented in nature. Understanding these transitions and their outcome at the level of the cell and organism has become one of the great frontiers of modern chemical biology. Until 2007, this field remained in the hands of RNA biologists. However, the recent identification of natural product and synthetic modulators of RNA splicing has opened new access to this field, allowing for the first time a chemical-based interrogation of RNA splicing processes. Simultaneously, we have begun to understand the vital importance of splicing in disease, which offers a new platform for molecular discovery and therapy. As with many natural systems, gaining clear mechanistic detail at the molecular level is key towards understanding the operation of any biological machine. This minireview presents recent lessons learned in this emerging field of RNA splicing chemistry and chemical biology. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

RBFox1-mediated RNA splicing regulates cardiac hypertrophy and heart failure.

PubMed

Gao, Chen; Ren, Shuxun; Lee, Jae-Hyung; Qiu, Jinsong; Chapski, Douglas J; Rau, Christoph D; Zhou, Yu; Abdellatif, Maha; Nakano, Astushi; Vondriska, Thomas M; Xiao, Xinshu; Fu, Xiang-Dong; Chen, Jau-Nian; Wang, Yibin

2016-01-01

RNA splicing is a major contributor to total transcriptome complexity; however, the functional role and regulation of splicing in heart failure remain poorly understood. Here, we used a total transcriptome profiling and bioinformatic analysis approach and identified a muscle-specific isoform of an RNA splicing regulator, RBFox1 (also known as A2BP1), as a prominent regulator of alternative RNA splicing during heart failure. Evaluation of developing murine and zebrafish hearts revealed that RBFox1 is induced during postnatal cardiac maturation. However, we found that RBFox1 is markedly diminished in failing human and mouse hearts. In a mouse model, RBFox1 deficiency in the heart promoted pressure overload-induced heart failure. We determined that RBFox1 is a potent regulator of RNA splicing and is required for a conserved splicing process of transcription factor MEF2 family members that yields different MEF2 isoforms with differential effects on cardiac hypertrophic gene expression. Finally, induction of RBFox1 expression in murine pressure overload models substantially attenuated cardiac hypertrophy and pathological manifestations. Together, this study identifies regulation of RNA splicing by RBFox1 as an important player in transcriptome reprogramming during heart failure that influence pathogenesis of the disease.

RBFox1-mediated RNA splicing regulates cardiac hypertrophy and heart failure

PubMed Central

Gao, Chen; Ren, Shuxun; Lee, Jae-Hyung; Qiu, Jinsong; Chapski, Douglas J.; Rau, Christoph D.; Zhou, Yu; Abdellatif, Maha; Nakano, Astushi; Vondriska, Thomas M.; Xiao, Xinshu; Fu, Xiang-Dong; Chen, Jau-Nian; Wang, Yibin

2015-01-01

RNA splicing is a major contributor to total transcriptome complexity; however, the functional role and regulation of splicing in heart failure remain poorly understood. Here, we used a total transcriptome profiling and bioinformatic analysis approach and identified a muscle-specific isoform of an RNA splicing regulator, RBFox1 (also known as A2BP1), as a prominent regulator of alternative RNA splicing during heart failure. Evaluation of developing murine and zebrafish hearts revealed that RBFox1 is induced during postnatal cardiac maturation. However, we found that RBFox1 is markedly diminished in failing human and mouse hearts. In a mouse model, RBFox1 deficiency in the heart promoted pressure overload–induced heart failure. We determined that RBFox1 is a potent regulator of RNA splicing and is required for a conserved splicing process of transcription factor MEF2 family members that yields different MEF2 isoforms with differential effects on cardiac hypertrophic gene expression. Finally, induction of RBFox1 expression in murine pressure overload models substantially attenuated cardiac hypertrophy and pathological manifestations. Together, this study identifies regulation of RNA splicing by RBFox1 as an important player in transcriptome reprogramming during heart failure that influence pathogenesis of the disease. PMID:26619120

Splicing of goose parvovirus pre-mRNA influences cytoplasmic translation of the processed mRNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Long; Pintel, David J., E-mail: pinteld@missouri.edu

2012-04-25

Translation of goose parvovirus (GPV) 72 kDa Rep 1 is initiated from unspliced P9-generated mRNAs in ORF1 from the first in-frame AUG (537 AUG); however, this AUG is bypassed in spliced P9-generated RNA: translation of the 52 kDa Rep 2 protein from spliced RNA is initiated in ORF2 at the next AUG downstream (650 AUG). Usage of the 537 AUG was restored in spliced RNA when the GPV intron was replaced with a chimeric SV40 intron, or following specific mutations of the GPV intron which did not appear in the final spliced mRNA. Additionally, 650 AUG usage was gained inmore » unspliced RNA when the GPV intron splice sites were debilitated. Splicing-dependent regulation of translation initiation was mediated in cis by GPV RNA surrounding the target AUGs. Thus, nuclear RNA processing of GPV P9-generated pre-mRNAs has a complex, but significant, effect on alternative translation initiation of the GPV Rep proteins.« less

A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection

NASA Astrophysics Data System (ADS)

Elliott, David J.; Bourgeois, Cyril F.; Klink, Albrecht; Stévenin, James; Cooke, Howard J.

2000-05-01

RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.

Targeting RNA structure in SMN2 reverses spinal muscular atrophy molecular phenotypes.

PubMed

Garcia-Lopez, Amparo; Tessaro, Francesca; Jonker, Hendrik R A; Wacker, Anna; Richter, Christian; Comte, Arnaud; Berntenis, Nikolaos; Schmucki, Roland; Hatje, Klas; Petermann, Olivier; Chiriano, Gianpaolo; Perozzo, Remo; Sciarra, Daniel; Konieczny, Piotr; Faustino, Ignacio; Fournet, Guy; Orozco, Modesto; Artero, Ruben; Metzger, Friedrich; Ebeling, Martin; Goekjian, Peter; Joseph, Benoît; Schwalbe, Harald; Scapozza, Leonardo

2018-05-23

Modification of SMN2 exon 7 (E7) splicing is a validated therapeutic strategy against spinal muscular atrophy (SMA). However, a target-based approach to identify small-molecule E7 splicing modifiers has not been attempted, which could reveal novel therapies with improved mechanistic insight. Here, we chose as a target the stem-loop RNA structure TSL2, which overlaps with the 5' splicing site of E7. A small-molecule TSL2-binding compound, homocarbonyltopsentin (PK4C9), was identified that increases E7 splicing to therapeutic levels and rescues downstream molecular alterations in SMA cells. High-resolution NMR combined with molecular modelling revealed that PK4C9 binds to pentaloop conformations of TSL2 and promotes a shift to triloop conformations that display enhanced E7 splicing. Collectively, our study validates TSL2 as a target for small-molecule drug discovery in SMA, identifies a novel mechanism of action for an E7 splicing modifier, and sets a precedent for other splicing-mediated diseases where RNA structure could be similarly targeted.

Bipartite functions of the CREB co-activators selectively direct alternative splicing or transcriptional activation

PubMed Central

Amelio, Antonio L; Caputi, Massimo; Conkright, Michael D

2009-01-01

The CREB regulated transcription co-activators (CRTCs) regulate many biological processes by integrating and converting environmental inputs into transcriptional responses. Although the mechanisms by which CRTCs sense cellular signals are characterized, little is known regarding how CRTCs contribute to the regulation of cAMP inducible genes. Here we show that these dynamic regulators, unlike other co-activators, independently direct either pre-mRNA splice-site selection or transcriptional activation depending on the cell type or promoter context. Moreover, in other scenarios, the CRTC co-activators coordinately regulate transcription and splicing. Mutational analyses showed that CRTCs possess distinct functional domains responsible for regulating either pre-mRNA splicing or transcriptional activation. Interestingly, the CRTC1–MAML2 oncoprotein lacks the splicing domain and is incapable of altering splice-site selection despite robustly activating transcription. The differential usage of these distinct domains allows CRTCs to selectively mediate multiple facets of gene regulation, indicating that co-activators are not solely restricted to coordinating alternative splicing with increase in transcriptional activity. PMID:19644446

Antisense inhibition of proprotein convertase subtilisin/kexin type 9 reduces serum LDL in hyperlipidemic mice.

PubMed

Graham, Mark J; Lemonidis, Kristina M; Whipple, Charles P; Subramaniam, Amuthakannan; Monia, Brett P; Crooke, Stanley T; Crooke, Rosanne M

2007-04-01

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of a family of proteases that is thought to promote the degradation of the low density lipoprotein receptor (LDLR) through an as yet undefined mechanism. We developed second generation antisense oligonucleotide (ASO) inhibitors targeting murine PCSK9 to determine their potential as lipid-lowering agents. Administration of a PCSK9 ASO to high fat-fed mice for 6 weeks reduced total cholesterol and LDL by 53% and 38%, respectively. Moreover, inhibition of PCSK9 expression resulted in a 2-fold increase in hepatic LDLR protein levels. This phenotype closely resembles that reported previously in Pcsk9-deficient mice. The absence of cholesterol lowering in Ldlr-deficient mice effectively demonstrated a critical role for this receptor in mediating the lipid-lowering effects of PCSK9 inhibition. Antisense inhibition of PCSK9 is an attractive and novel therapeutic approach for treating hypercholesterolemia in human.

Cellular RNA binding proteins NS1-BP and hnRNP K regulate influenza A virus RNA splicing.

PubMed

Tsai, Pei-Ling; Chiou, Ni-Ting; Kuss, Sharon; García-Sastre, Adolfo; Lynch, Kristen W; Fontoura, Beatriz M A

2013-01-01

Influenza A virus is a major human pathogen with a genome comprised of eight single-strand, negative-sense, RNA segments. Two viral RNA segments, NS1 and M, undergo alternative splicing and yield several proteins including NS1, NS2, M1 and M2 proteins. However, the mechanisms or players involved in splicing of these viral RNA segments have not been fully studied. Here, by investigating the interacting partners and function of the cellular protein NS1-binding protein (NS1-BP), we revealed novel players in the splicing of the M1 segment. Using a proteomics approach, we identified a complex of RNA binding proteins containing NS1-BP and heterogeneous nuclear ribonucleoproteins (hnRNPs), among which are hnRNPs involved in host pre-mRNA splicing. We found that low levels of NS1-BP specifically impaired proper alternative splicing of the viral M1 mRNA segment to yield the M2 mRNA without affecting splicing of mRNA3, M4, or the NS mRNA segments. Further biochemical analysis by formaldehyde and UV cross-linking demonstrated that NS1-BP did not interact directly with viral M1 mRNA but its interacting partners, hnRNPs A1, K, L, and M, directly bound M1 mRNA. Among these hnRNPs, we identified hnRNP K as a major mediator of M1 mRNA splicing. The M1 mRNA segment generates the matrix protein M1 and the M2 ion channel, which are essential proteins involved in viral trafficking, release into the cytoplasm, and budding. Thus, reduction of NS1-BP and/or hnRNP K levels altered M2/M1 mRNA and protein ratios, decreasing M2 levels and inhibiting virus replication. Thus, NS1-BP-hnRNPK complex is a key mediator of influenza A virus gene expression.

Large-scale identification and characterization of alternative splicing variants of human gene transcripts using 56 419 completely sequenced and manually annotated full-length cDNAs

PubMed Central

Takeda, Jun-ichi; Suzuki, Yutaka; Nakao, Mitsuteru; Barrero, Roberto A.; Koyanagi, Kanako O.; Jin, Lihua; Motono, Chie; Hata, Hiroko; Isogai, Takao; Nagai, Keiichi; Otsuki, Tetsuji; Kuryshev, Vladimir; Shionyu, Masafumi; Yura, Kei; Go, Mitiko; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Wiemann, Stefan; Nomura, Nobuo; Sugano, Sumio; Gojobori, Takashi; Imanishi, Tadashi

2006-01-01

We report the first genome-wide identification and characterization of alternative splicing in human gene transcripts based on analysis of the full-length cDNAs. Applying both manual and computational analyses for 56 419 completely sequenced and precisely annotated full-length cDNAs selected for the H-Invitational human transcriptome annotation meetings, we identified 6877 alternative splicing genes with 18 297 different alternative splicing variants. A total of 37 670 exons were involved in these alternative splicing events. The encoded protein sequences were affected in 6005 of the 6877 genes. Notably, alternative splicing affected protein motifs in 3015 genes, subcellular localizations in 2982 genes and transmembrane domains in 1348 genes. We also identified interesting patterns of alternative splicing, in which two distinct genes seemed to be bridged, nested or having overlapping protein coding sequences (CDSs) of different reading frames (multiple CDS). In these cases, completely unrelated proteins are encoded by a single locus. Genome-wide annotations of alternative splicing, relying on full-length cDNAs, should lay firm groundwork for exploring in detail the diversification of protein function, which is mediated by the fast expanding universe of alternative splicing variants. PMID:16914452

Control of calcitonin/calcitonin gene-related peptide pre-mRNA processing by constitutive intron and exon elements.

PubMed Central

Yeakley, J M; Hedjran, F; Morfin, J P; Merillat, N; Rosenfeld, M G; Emeson, R B

1993-01-01

The calcitonin/calcitonin gene-related peptide (CGRP) primary transcript is alternatively spliced in thyroid C cells and neurons, resulting in the tissue-specific production of calcitonin and CGRP mRNAs. Analyses of mutated calcitonin/CGRP transcription units in permanently transfected cell lines have indicated that alternative splicing is regulated by a differential capacity to utilize the calcitonin-specific splice acceptor. The analysis of an extensive series of mutations suggests that tissue-specific regulation of calcitonin mRNA production does not depend on the presence of a single, unique cis-active element but instead appears to be a consequence of suboptimal constitutive splicing signals. While only those mutations that altered constitutive splicing signals affected splice choices, the action of multiple regulatory sequences cannot be formally excluded. Further, we have identified a 13-nucleotide purine-rich element from a constitutive exon that, when placed in exon 4, entirely switches splice site usage in CGRP-producing cells. These data suggest that specific exon recruitment sequences, in combination with other constitutive elements, serve an important function in exon recognition. These results are consistent with the hypothesis that tissue-specific alternative splicing of the calcitonin/CGRP primary transcript is mediated by cell-specific differences in components of the constitutive splicing machinery. Images PMID:8413203

Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides

PubMed Central

Wang, Shiyu; Allen, Nickolas; Vickers, Timothy A; Revenko, Alexey S; Sun, Hong; Liang, Xue-hai; Crooke, Stanley T

2018-01-01

Abstract Chemically modified antisense oligonucleotides (ASOs) with phosphorothioate (PS) linkages have been extensively studied as research and therapeutic agents. PS-ASOs can enter the cell and trigger cleavage of complementary RNA by RNase H1 even in the absence of transfection reagent. A number of cell surface proteins have been identified that bind PS-ASOs and mediate their cellular uptake; however, the mechanisms that lead to productive internalization of PS-ASOs are not well understood. Here, we characterized the interaction between PS-ASOs and epidermal growth factor receptor (EGFR). We found that PS-ASOs trafficked together with EGF and EGFR into clathrin-coated pit structures. Their co-localization was also observed at early endosomes and inside enlarged late endosomes. Reduction of EGFR decreased PS-ASO activity without affecting EGF-mediated signaling pathways and overexpression of EGFR increased PS-ASO activity in cells. Furthermore, reduction of EGFR delays PS-ASO trafficking from early to late endosomes. Thus, EGFR binds to PS-ASOs at the cell surface and mediates essential steps for active (productive) cellular uptake of PS-ASOs through its cargo-dependent trafficking processes which migrate PS-ASOs from early to late endosomes. This EGFR-mediated process can also serve as an additional model to better understand the mechanism of intracellular uptake and endosomal release of PS-ASOs. PMID:29514240

On the path to genetic novelties: insights from programmed DNA elimination and RNA splicing.

PubMed

Catania, Francesco; Schmitz, Jürgen

2015-01-01

Understanding how genetic novelties arise is a central goal of evolutionary biology. To this end, programmed DNA elimination and RNA splicing deserve special consideration. While programmed DNA elimination reshapes genomes by eliminating chromatin during organismal development, RNA splicing rearranges genetic messages by removing intronic regions during transcription. Small RNAs help to mediate this class of sequence reorganization, which is not error-free. It is this imperfection that makes programmed DNA elimination and RNA splicing excellent candidates for generating evolutionary novelties. Leveraging a number of these two processes' mechanistic and evolutionary properties, which have been uncovered over the past years, we present recently proposed models and empirical evidence for how splicing can shape the structure of protein-coding genes in eukaryotes. We also chronicle a number of intriguing similarities between the processes of programmed DNA elimination and RNA splicing, and highlight the role that the variation in the population-genetic environment may play in shaping their target sequences. © 2015 Wiley Periodicals, Inc.

Hippocampal A-type current and Kv4.2 channel modulation by the sulfonylurea compound NS5806.

PubMed

Witzel, Katrin; Fischer, Paul; Bähring, Robert

2012-12-01

We examined the effects of the sulfonylurea compound NS5806 on neuronal A-type channel function. Using whole-cell patch-clamp we studied the effects of NS5806 on the somatodendritic A-type current (I(SA)) in cultured hippocampal neurons and the currents mediated by Kv4.2 channels coexpressed with different auxiliary β-subunits, including both Kv channel interacting proteins (KChIPs) and dipeptidyl aminopeptidase-related proteins (DPPs), in HEK 293 cells. The amplitude of the I(SA) component in hippocampal neurons was reduced in the presence of 20 μM NS5806. I(SA) decay kinetics were slowed and the recovery kinetics accelerated, but the voltage dependence of steady-state inactivation was shifted to more negative potentials by NS5806. The peak amplitudes of currents mediated by ternary Kv4.2 channel complexes, associated with DPP6-S (short splice-variant) and either KChIP2, KChIP3 or KChIP4, were potentiated and their macroscopic inactivation slowed by NS5806, whereas the currents mediated by binary Kv4.2 channels, associated only with DPP6-S, were suppressed, and the NS5806-mediated slowing of macroscopic inactivation was less pronounced. Neither potentiation nor suppression and no effect on current decay kinetics in the presence of NS5806 were observed for Kv4.2 channels associated with KChIP3 and the N-type inactivation-conferring DPP6a splice-variant. For all recombinant channel complexes, NS5806 slowed the recovery from inactivation and shifted the voltage dependence of steady-state inactivation to more negative potentials. Our results demonstrate the activity of NS5806 on native I(SA) and possible molecular correlates in the form of recombinant Kv4.2 channels complexed with different KChIPs and DPPs, and they shed some light on the mechanism of NS5806 action. Copyright © 2012 Elsevier Ltd. All rights reserved.

From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing.

PubMed

Marinov, Georgi K; Williams, Brian A; McCue, Ken; Schroth, Gary P; Gertz, Jason; Myers, Richard M; Wold, Barbara J

2014-03-01

Single-cell RNA-seq mammalian transcriptome studies are at an early stage in uncovering cell-to-cell variation in gene expression, transcript processing and editing, and regulatory module activity. Despite great progress recently, substantial challenges remain, including discriminating biological variation from technical noise. Here we apply the SMART-seq single-cell RNA-seq protocol to study the reference lymphoblastoid cell line GM12878. By using spike-in quantification standards, we estimate the absolute number of RNA molecules per cell for each gene and find significant variation in total mRNA content: between 50,000 and 300,000 transcripts per cell. We directly measure technical stochasticity by a pool/split design and find that there are significant differences in expression between individual cells, over and above technical variation. Specific gene coexpression modules were preferentially expressed in subsets of individual cells, including one enriched for mRNA processing and splicing factors. We assess cell-to-cell variation in alternative splicing and allelic bias and report evidence of significant differences in splice site usage that exceed splice variation in the pool/split comparison. Finally, we show that transcriptomes from small pools of 30-100 cells approach the information content and reproducibility of contemporary RNA-seq from large amounts of input material. Together, our results define an experimental and computational path forward for analyzing gene expression in rare cell types and cell states.

JMJD6 and U2AF65 co-regulate alternative splicing in both JMJD6 enzymatic activity dependent and independent manner

PubMed Central

Yi, Jia; Shen, Hai-Feng; Qiu, Jin-Song; Huang, Ming-Feng; Zhang, Wen-Juan; Ding, Jian-Cheng; Zhu, Xiao-Yan; Zhou, Yu

2017-01-01

Abstract JMJD6, a jumonji C (Jmj C) domain-containing protein demethylase and hydroxylase, has been implicated in an array of biological processes. It has been shown that JMJD6 interacts with and hydroxylates multiple serine/arginine-rich (SR) proteins and SR related proteins, including U2AF65, all of which are known to function in alternative splicing regulation. However, whether JMJD6 is widely involved in alternative splicing and the molecular mechanism underlying JMJD6-regulated alternative splicing have remained incompletely understood. Here, by using RASL-Seq, we investigated the functional impact of RNA-dependent interaction between JMJD6 and U2AF65, revealing that JMJD6 and U2AF65 co-regulated a large number of alternative splicing events. We further demonstrated the JMJD6 function in alternative splicing in jmjd6 knockout mice. Mechanistically, we showed that the enzymatic activity of JMJD6 was required for a subset of JMJD6-regulated splicing, and JMJD6-mediated lysine hydroxylation of U2AF65 could account for, at least partially, their co-regulated alternative splicing events, suggesting both JMJD6 enzymatic activity-dependent and independent control of alternative splicing. These findings reveal an intimate link between JMJD6 and U2AF65 in alternative splicing regulation, which has important implications in development and disease processes. PMID:27899633

Cocaine alters Homer1 natural antisense transcript in the nucleus accumbens.

PubMed

Sartor, Gregory C; Powell, Samuel K; Velmeshev, Dmitry; Lin, David Y; Magistri, Marco; Wiedner, Hannah J; Malvezzi, Andrea M; Andrade, Nadja S; Faghihi, Mohammad A; Wahlestedt, Claes

2017-12-01

Natural antisense transcripts (NATs) are an abundant class of long noncoding RNAs that have recently been shown to be key regulators of chromatin dynamics and gene expression in nervous system development and neurological disorders. However, it is currently unclear if NAT-based mechanisms also play a role in drug-induced neuroadaptations. Aberrant regulation of gene expression is one critical factor underlying the long-lasting behavioral abnormalities that characterize substance use disorder, and it is possible that some drug-induced transcriptional responses are mediated, in part, by perturbations in NAT activity. To test this hypothesis, we used an automated algorithm that mines the NCBI AceView transcriptomics database to identify NAT overlapping genes linked to addiction. We found that 22% of the genes examined contain NATs and that expression of Homer1 natural antisense transcript (Homer1-AS) was altered in the nucleus accumbens (NAc) of mice 2h and 10days following repeated cocaine administration. In in vitro studies, depletion of Homer1-AS lead to an increase in the corresponding sense gene expression, indicating a potential regulatory mechanisms of Homer1 expression by its corresponding antisense transcript. Future in vivo studies are needed to definitely determine a role for Homer1-AS in cocaine-induced behavioral and molecular adaptations. Copyright © 2017 Elsevier Inc. All rights reserved.

cis-antisense RNA, another level of gene regulation in bacteria.

PubMed

Georg, Jens; Hess, Wolfgang R

2011-06-01

A substantial amount of antisense transcription is a hallmark of gene expression in eukaryotes. However, antisense transcription was first demonstrated in bacteria almost 50 years ago. The transcriptomes of bacteria as different as Helicobacter pylori, Bacillus subtilis, Escherichia coli, Synechocystis sp. strain PCC6803, Mycoplasma pneumoniae, Sinorhizobium meliloti, Geobacter sulfurreducens, Vibrio cholerae, Chlamydia trachomatis, Pseudomonas syringae, and Staphylococcus aureus have now been reported to contain antisense RNA (asRNA) transcripts for a high percentage of genes. Bacterial asRNAs share functional similarities with trans-acting regulatory RNAs, but in addition, they use their own distinct mechanisms. Among their confirmed functional roles are transcription termination, codegradation, control of translation, transcriptional interference, and enhanced stability of their respective target transcripts. Here, we review recent publications indicating that asRNAs occur as frequently in simple unicellular bacteria as they do in higher organisms, and we provide a comprehensive overview of the experimentally confirmed characteristics of asRNA actions and intimately linked quantitative aspects. Emerging functional data suggest that asRNAs in bacteria mediate a plethora of effects and are involved in far more processes than were previously anticipated. Thus, the functional impact of asRNAs should be considered when developing new strategies against pathogenic bacteria and when optimizing bacterial strains for biotechnology.

cis-Antisense RNA, Another Level of Gene Regulation in Bacteria

PubMed Central

Georg, Jens; Hess, Wolfgang R.

2011-01-01

Summary: A substantial amount of antisense transcription is a hallmark of gene expression in eukaryotes. However, antisense transcription was first demonstrated in bacteria almost 50 years ago. The transcriptomes of bacteria as different as Helicobacter pylori, Bacillus subtilis, Escherichia coli, Synechocystis sp. strain PCC6803, Mycoplasma pneumoniae, Sinorhizobium meliloti, Geobacter sulfurreducens, Vibrio cholerae, Chlamydia trachomatis, Pseudomonas syringae, and Staphylococcus aureus have now been reported to contain antisense RNA (asRNA) transcripts for a high percentage of genes. Bacterial asRNAs share functional similarities with trans-acting regulatory RNAs, but in addition, they use their own distinct mechanisms. Among their confirmed functional roles are transcription termination, codegradation, control of translation, transcriptional interference, and enhanced stability of their respective target transcripts. Here, we review recent publications indicating that asRNAs occur as frequently in simple unicellular bacteria as they do in higher organisms, and we provide a comprehensive overview of the experimentally confirmed characteristics of asRNA actions and intimately linked quantitative aspects. Emerging functional data suggest that asRNAs in bacteria mediate a plethora of effects and are involved in far more processes than were previously anticipated. Thus, the functional impact of asRNAs should be considered when developing new strategies against pathogenic bacteria and when optimizing bacterial strains for biotechnology. PMID:21646430

The reduction in small ribosomal subunit abundance in ethanol-stressed cells of Bacillus subtilis is mediated by a SigB-dependent antisense RNA.

PubMed

Mars, Ruben A T; Mendonça, Karoline; Denham, Emma L; van Dijl, Jan Maarten

2015-10-01

One of the best-characterized general stress responses in bacteria is the σB-mediated stress response of the Gram-positive soil bacterium Bacillus subtilis. The σB regulon contains approximately 200 protein-encoding genes and 136 putative regulatory RNAs. One of these σB-dependent RNAs, named S1136-S1134, was recently mapped as being transcribed from the S1136 promoter on the opposite strand of the essential rpsD gene, which encodes the ribosomal primary-binding protein S4. Accordingly, S1136-S1134 transcription results in an rpsD-overlapping antisense RNA (asRNA). Upon exposure of B. subtilis to ethanol, the S1136 promoter was found to be induced, while rpsD transcription was downregulated. By quantitative PCR, we show that the activation of transcription from the S1136 promoter is directly responsible for the downregulation of rpsD upon ethanol exposure. We also show that this downregulation of rpsD leads to a reduced level of the small (30S) ribosomal subunit upon ethanol stress. The activation of the S1136 promoter thus represents the first example of antisense transcription-mediated regulation in the general stress response of B. subtilis and implicates the reduction of ribosomal protein abundance as a new aspect in the σB-dependent stress response. We propose that the observed reduction in the level of the small ribosomal subunit, which contains the ribosome-decoding center, may protect B. subtilis cells against misreading and spurious translation of possibly toxic aberrant peptides under conditions of ethanol stress. Copyright © 2015 Elsevier B.V. All rights reserved.

Splicing Modulation as a Potential Treatment for Vemurafenib-Resistant Melanoma | Center for Cancer Research

Cancer.gov

Over half of melanomas contain mutations in the serine/threonine kinase BRAF. The most common mutation, BRAF(V600E), leads to excessive activation of the MAPK proliferation pathway. Vemurafenib is a potent kinase inhibitor with remarkable clinical activity in BRAF(V600E)-positive melanoma tumors. Patients initially respond to treatment with vemurafenib, but inevitably develop resistance. One known resistance mechanism is aberrant splicing of the BRAF RNA. To understand the molecule mechanism of BRAF mis-splicing, Tom Misteli, Ph.D., and Maayan Salton, Ph.D., of CCR’s Laboratory of Receptor Biology and Gene Expression, and NCI colleagues set out on a molecular investigation to identify the mechanism behind generation of the vemurafenib-resistant BRAF isoforms. Their results led to insight into the molecular mechanism of BRAF splicing in vemurafenib resistance and point to splicing inhibitors as a novel therapeutic strategy to overcome vemurafenib resistance.

Strategies to identify natural antisense transcripts.

PubMed

Sun, Yulong; Li, Dijie; Zhang, Ru; Peng, Shang; Zhang, Ge; Yang, Tuanmin; Qian, Airong

2017-01-01

Natural antisense transcripts, originally considered as transcriptional noises arising from so-called "junk DNA″, are recently recognized as important modulators for gene regulation. They are prevalent in nearly all realms of life and have been found to modulate gene expression positively or negatively. By affecting almost all stages of gene expression range from pre-transcriptional, transcriptional and post-transcriptional to translation, NATs are fundamentally involved in various biological processes. However, compared to increasing huge data from transcriptional analysis especially high-throughput sequencing technologies (such as RNA-seq), limited functional NATs (around 70) are so far reported, which hinder our advanced comprehensive understanding for this field. Hence, efficient strategies for identifying NATs are urgently desired. In this review, we discussed the current strategies for identifying NATs, with a focus on the advantages, disadvantages, and applications of methods isolating functional NATs. Moreover, publicly available databases for NATs were also discussed. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

ChloroSeq, an optimized chloroplast RNA-Seq bioinformatic pipeline, reveals remodeling of the organellar transcriptome under heat stress

DOE PAGES

Castandet, Benoît; Hotto, Amber M.; Strickler, Susan R.; ...

2016-07-06

Although RNA-Seq has revolutionized transcript analysis, organellar transcriptomes are rarely assessed even when present in published datasets. Here, we describe the development and application of a rapid and convenient method, ChloroSeq, to delineate qualitative and quantitative features of chloroplast RNA metabolism from strand-specific RNA-Seq datasets, including processing, editing, splicing, and relative transcript abundance. The use of a single experiment to analyze systematically chloroplast transcript maturation and abundance is of particular interest due to frequent pleiotropic effects observed in mutants that affect chloroplast gene expression and/or photosynthesis. To illustrate its utility, ChloroSeq was applied to published RNA-Seq datasets derived from Arabidopsismore » thaliana grown under control and abiotic stress conditions, where the organellar transcriptome had not been examined. The most appreciable effects were found for heat stress, which induces a global reduction in splicing and editing efficiency, and leads to increased abundance of chloroplast transcripts, including genic, intergenic, and antisense transcripts. Moreover, by concomitantly analyzing nuclear transcripts that encode chloroplast gene expression regulators from the same libraries, we demonstrate the possibility of achieving a holistic understanding of the nucleus-organelle system. In conclusion, ChloroSeq thus represents a unique method for streamlining RNA-Seq data interpretation of the chloroplast transcriptome and its regulators.« less

ChloroSeq, an optimized chloroplast RNA-Seq bioinformatic pipeline, reveals remodeling of the organellar transcriptome under heat stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Castandet, Benoît; Hotto, Amber M.; Strickler, Susan R.

Although RNA-Seq has revolutionized transcript analysis, organellar transcriptomes are rarely assessed even when present in published datasets. Here, we describe the development and application of a rapid and convenient method, ChloroSeq, to delineate qualitative and quantitative features of chloroplast RNA metabolism from strand-specific RNA-Seq datasets, including processing, editing, splicing, and relative transcript abundance. The use of a single experiment to analyze systematically chloroplast transcript maturation and abundance is of particular interest due to frequent pleiotropic effects observed in mutants that affect chloroplast gene expression and/or photosynthesis. To illustrate its utility, ChloroSeq was applied to published RNA-Seq datasets derived from Arabidopsismore » thaliana grown under control and abiotic stress conditions, where the organellar transcriptome had not been examined. The most appreciable effects were found for heat stress, which induces a global reduction in splicing and editing efficiency, and leads to increased abundance of chloroplast transcripts, including genic, intergenic, and antisense transcripts. Moreover, by concomitantly analyzing nuclear transcripts that encode chloroplast gene expression regulators from the same libraries, we demonstrate the possibility of achieving a holistic understanding of the nucleus-organelle system. In conclusion, ChloroSeq thus represents a unique method for streamlining RNA-Seq data interpretation of the chloroplast transcriptome and its regulators.« less

Complexity of the Alternative Splicing Landscape in Plants[C][W][OPEN

PubMed Central

Reddy, Anireddy S.N.; Marquez, Yamile; Kalyna, Maria; Barta, Andrea

2013-01-01

Alternative splicing (AS) of precursor mRNAs (pre-mRNAs) from multiexon genes allows organisms to increase their coding potential and regulate gene expression through multiple mechanisms. Recent transcriptome-wide analysis of AS using RNA sequencing has revealed that AS is highly pervasive in plants. Pre-mRNAs from over 60% of intron-containing genes undergo AS to produce a vast repertoire of mRNA isoforms. The functions of most splice variants are unknown. However, emerging evidence indicates that splice variants increase the functional diversity of proteins. Furthermore, AS is coupled to transcript stability and translation through nonsense-mediated decay and microRNA-mediated gene regulation. Widespread changes in AS in response to developmental cues and stresses suggest a role for regulated splicing in plant development and stress responses. Here, we review recent progress in uncovering the extent and complexity of the AS landscape in plants, its regulation, and the roles of AS in gene regulation. The prevalence of AS in plants has raised many new questions that require additional studies. New tools based on recent technological advances are allowing genome-wide analysis of RNA elements in transcripts and of chromatin modifications that regulate AS. Application of these tools in plants will provide significant new insights into AS regulation and crosstalk between AS and other layers of gene regulation. PMID:24179125

Human osteopontin splicing isoforms: known roles, potential clinical applications and activated signaling pathways.

PubMed

Gimba, E R; Tilli, T M

2013-04-30

Human osteopontin is subject to alternative splicing, which generates three isoforms, termed OPNa, OPNb and OPNc. These variants show specific expression and roles in different cell contexts. We present an overview of current knowledge of the expression profile of human OPN splicing isoforms (OPN-SIs), their tissue-specific roles, and the pathways mediating their functional properties in different pathophysiological conditions. We also describe their putative application as biomarkers, and their potential use as therapeutic targets by using antibodies, oligonucleotides or siRNA molecules. This synthesis provides new clues for a better understanding of human OPN splice variants, their roles in normal and pathological conditions, and their possible clinical applications. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Large-scale remodeling of a repressed exon ribonucleoprotein to an exon definition complex active for splicing

PubMed Central

Wongpalee, Somsakul Pop; Vashisht, Ajay; Sharma, Shalini; Chui, Darryl; Wohlschlegel, James A; Black, Douglas L

2016-01-01

Polypyrimidine-tract binding protein PTBP1 can repress splicing during the exon definition phase of spliceosome assembly, but the assembly steps leading to an exon definition complex (EDC) and how PTBP1 might modulate them are not clear. We found that PTBP1 binding in the flanking introns allowed normal U2AF and U1 snRNP binding to the target exon splice sites but blocked U2 snRNP assembly in HeLa nuclear extract. Characterizing a purified PTBP1-repressed complex, as well as an active early complex and the final EDC by SILAC-MS, we identified extensive PTBP1-modulated changes in exon RNP composition. The active early complex formed in the absence of PTBP1 proceeded to assemble an EDC with the eviction of hnRNP proteins, the late recruitment of SR proteins, and binding of the U2 snRNP. These results demonstrate that during early stages of splicing, exon RNP complexes are highly dynamic with many proteins failing to bind during PTBP1 arrest. DOI: http://dx.doi.org/10.7554/eLife.19743.001 PMID:27882870

Complex organisation and structure of the ghrelin antisense strand gene GHRLOS, a candidate non-coding RNA gene

PubMed Central

Seim, Inge; Carter, Shea L; Herington, Adrian C; Chopin, Lisa K

2008-01-01

Background The peptide hormone ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH) release, appetite regulation, gut motility and proliferation of cancer cells. We previously identified a gene on the opposite strand of the ghrelin gene, ghrelinOS (GHRLOS), which spans the promoter and untranslated regions of the ghrelin gene (GHRL). Here we further characterise GHRLOS. Results We have described GHRLOS mRNA isoforms that extend over 1.4 kb of the promoter region and 106 nucleotides of exon 4 of the ghrelin gene, GHRL. These GHRLOS transcripts initiate 4.8 kb downstream of the terminal exon 4 of GHRL and are present in the 3' untranslated exon of the adjacent gene TATDN2 (TatD DNase domain containing 2). Interestingly, we have also identified a putative non-coding TATDN2-GHRLOS chimaeric transcript, indicating that GHRLOS RNA biogenesis is extremely complex. Moreover, we have discovered that the 3' region of GHRLOS is also antisense, in a tail-to-tail fashion to a novel terminal exon of the neighbouring SEC13 gene, which is important in protein transport. Sequence analyses revealed that GHRLOS is riddled with stop codons, and that there is little nucleotide and amino-acid sequence conservation of the GHRLOS gene between vertebrates. The gene spans 44 kb on 3p25.3, is extensively spliced and harbours multiple variable exons. We have also investigated the expression of GHRLOS and found evidence of differential tissue expression. It is highly expressed in tissues which are emerging as major sites of non-coding RNA expression (the thymus, brain, and testis), as well as in the ovary and uterus. In contrast, very low levels were found in the stomach where sense, GHRL derived RNAs are highly expressed. Conclusion GHRLOS RNA transcripts display several distinctive features of non-coding (ncRNA) genes, including 5' capping, polyadenylation, extensive splicing and short open reading frames. The gene is also non-conserved, with differential and tissue-restricted expression. The overlapping genomic arrangement of GHRLOS with the ghrelin gene indicates that it is likely to have interesting regulatory and functional roles in the ghrelin axis. PMID:18954468

Complex organisation and structure of the ghrelin antisense strand gene GHRLOS, a candidate non-coding RNA gene.

PubMed

Seim, Inge; Carter, Shea L; Herington, Adrian C; Chopin, Lisa K

2008-10-28

The peptide hormone ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH) release, appetite regulation, gut motility and proliferation of cancer cells. We previously identified a gene on the opposite strand of the ghrelin gene, ghrelinOS (GHRLOS), which spans the promoter and untranslated regions of the ghrelin gene (GHRL). Here we further characterise GHRLOS. We have described GHRLOS mRNA isoforms that extend over 1.4 kb of the promoter region and 106 nucleotides of exon 4 of the ghrelin gene, GHRL. These GHRLOS transcripts initiate 4.8 kb downstream of the terminal exon 4 of GHRL and are present in the 3' untranslated exon of the adjacent gene TATDN2 (TatD DNase domain containing 2). Interestingly, we have also identified a putative non-coding TATDN2-GHRLOS chimaeric transcript, indicating that GHRLOS RNA biogenesis is extremely complex. Moreover, we have discovered that the 3' region of GHRLOS is also antisense, in a tail-to-tail fashion to a novel terminal exon of the neighbouring SEC13 gene, which is important in protein transport. Sequence analyses revealed that GHRLOS is riddled with stop codons, and that there is little nucleotide and amino-acid sequence conservation of the GHRLOS gene between vertebrates. The gene spans 44 kb on 3p25.3, is extensively spliced and harbours multiple variable exons. We have also investigated the expression of GHRLOS and found evidence of differential tissue expression. It is highly expressed in tissues which are emerging as major sites of non-coding RNA expression (the thymus, brain, and testis), as well as in the ovary and uterus. In contrast, very low levels were found in the stomach where sense, GHRL derived RNAs are highly expressed. GHRLOS RNA transcripts display several distinctive features of non-coding (ncRNA) genes, including 5' capping, polyadenylation, extensive splicing and short open reading frames. The gene is also non-conserved, with differential and tissue-restricted expression. The overlapping genomic arrangement of GHRLOS with the ghrelin gene indicates that it is likely to have interesting regulatory and functional roles in the ghrelin axis.

Characterization and Modulation of Proteins Involved in Sulfur Mustard Vesication

DTIC Science & Technology

2000-06-01

PARP staining was present throughout the nucleus, the DBD showed a more localized punctate pattern in the region of the nucleolus and throughout the...34 oligonucleotide is synthesized that is identical in base composition to the antisense, but had a randomly generated sequence. This is an important control...reversed this inhibitory effect. The roles of PARP in modulating the composition and enzyme activities of the DNA synthesome were further investigated by

An EMT–Driven Alternative Splicing Program Occurs in Human Breast Cancer and Modulates Cellular Phenotype

PubMed Central

Flytzanis, Nicholas C.; Balsamo, Michele; Condeelis, John S.; Oktay, Maja H.; Burge, Christopher B.; Gertler, Frank B.

2011-01-01

Epithelial-mesenchymal transition (EMT), a mechanism important for embryonic development, plays a critical role during malignant transformation. While much is known about transcriptional regulation of EMT, alternative splicing of several genes has also been correlated with EMT progression, but the extent of splicing changes and their contributions to the morphological conversion accompanying EMT have not been investigated comprehensively. Using an established cell culture model and RNA–Seq analyses, we determined an alternative splicing signature for EMT. Genes encoding key drivers of EMT–dependent changes in cell phenotype, such as actin cytoskeleton remodeling, regulation of cell–cell junction formation, and regulation of cell migration, were enriched among EMT–associated alternatively splicing events. Our analysis suggested that most EMT–associated alternative splicing events are regulated by one or more members of the RBFOX, MBNL, CELF, hnRNP, or ESRP classes of splicing factors. The EMT alternative splicing signature was confirmed in human breast cancer cell lines, which could be classified into basal and luminal subtypes based exclusively on their EMT–associated splicing pattern. Expression of EMT–associated alternative mRNA transcripts was also observed in primary breast cancer samples, indicating that EMT–dependent splicing changes occur commonly in human tumors. The functional significance of EMT–associated alternative splicing was tested by expression of the epithelial-specific splicing factor ESRP1 or by depletion of RBFOX2 in mesenchymal cells, both of which elicited significant changes in cell morphology and motility towards an epithelial phenotype, suggesting that splicing regulation alone can drive critical aspects of EMT–associated phenotypic changes. The molecular description obtained here may aid in the development of new diagnostic and prognostic markers for analysis of breast cancer progression. PMID:21876675

[The influence of HOXB2 anti-sense oligodeoxynucleotides on the proliferation and expression of human umbilical vein endothelial cells].

PubMed

Zhang, X; Liu, X; Liu, L

2001-12-01

To explore the effects of HOXB2 anti-sense oligodeoxynucleotides (asodn) on the proliferation and the expression of human umbilical vein endothelial cells (HUVECs). Various concentrations of HOXB2 ASODN modified by thiophosphate were transfected into HUVECs by liposome mediation. MTT and RT-PCR methods were employed to determine the influence of different concentrations of ASODN on endothelial proliferation and the expression level of HOXB2 mRNA. After the transfection of HOXB2 ASODN, the endothelial proliferation was inhibited in dose-dependent manner. Simultaneously, the expression level of HOXB2 mRNA decreased significantly. HOXB2 might play important roles in the proliferation of endothelial cells.

A HLA class I cis-regulatory element whose activity can be modulated by hormones.

PubMed

Sim, B C; Hui, K M

1994-12-01

To elucidate the basis of the down-regulation in major histocompatibility complex (MHC) class I gene expression and to identify possible DNA-binding regulatory elements that have the potential to interact with class I MHC genes, we have studied the transcriptional regulation of class I HLA genes in human breast carcinoma cells. A 9 base pair (bp) negative cis-regulatory element (NRE) has been identified using band-shift assays employing DNA sequences derived from the 5'-flanking region of HLA class I genes. This 9-bp element, GTCATGGCG, located within exon I of the HLA class I gene, can potently inhibit the expression of a heterologous thymidine kinase (TK) gene promoter and the HLA enhancer element. Furthermore, this regulatory element can exert its suppressive function in either the sense or anti-sense orientation. More interestingly, NRE can suppress dexamethasone-mediated gene activation in the context of the reported glucocorticoid-responsive element (GRE) in MCF-7 cells but has no influence on the estrogen-mediated transcriptional activation of MCF-7 cells in the context of the reported estrogen-responsive element (ERE). Furthermore, the presence of such a regulatory element within the HLA class I gene whose activity can be modulated by hormones correlates well with our observation that the level of HLA class I gene expression can be down-regulated by hormones in human breast carcinoma cells. Such interactions between negative regulatory elements and specific hormone trans-activators are novel and suggest a versatile form of transcriptional control.

RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smialowska, Agata, E-mail: smialowskaa@gmail.com; School of Life Sciences, Södertörn Högskola, Huddinge 141-89; Djupedal, Ingela

Highlights: • Protein coding genes accumulate anti-sense sRNAs in fission yeast S. pombe. • RNAi represses protein-coding genes in S. pombe. • RNAi-mediated gene repression is post-transcriptional. - Abstract: RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its rolemore » in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe.« less

Regulation of mRNA Abundance by Polypyrimidine Tract-Binding Protein-Controlled Alternate 5′ Splice Site Choice

PubMed Central

Hamid, Fursham M.; Makeyev, Eugene V.

2014-01-01

Alternative splicing (AS) provides a potent mechanism for increasing protein diversity and modulating gene expression levels. How alternate splice sites are selected by the splicing machinery and how AS is integrated into gene regulation networks remain important questions of eukaryotic biology. Here we report that polypyrimidine tract-binding protein 1 (Ptbp1/PTB/hnRNP-I) controls alternate 5′ and 3′ splice site (5′ss and 3′ss) usage in a large set of mammalian transcripts. A top scoring event identified by our analysis was the choice between competing upstream and downstream 5′ss (u5′ss and d5′ss) in the exon 18 of the Hps1 gene. Hps1 is essential for proper biogenesis of lysosome-related organelles and loss of its function leads to a disease called type 1 Hermansky-Pudlak Syndrome (HPS). We show that Ptbp1 promotes preferential utilization of the u5′ss giving rise to stable mRNAs encoding a full-length Hps1 protein, whereas bias towards d5′ss triggered by Ptbp1 down-regulation generates transcripts susceptible to nonsense-mediated decay (NMD). We further demonstrate that Ptbp1 binds to pyrimidine-rich sequences between the u5′ss and d5′ss and activates the former site rather than repressing the latter. Consistent with this mechanism, u5′ss is intrinsically weaker than d5′ss, with a similar tendency observed for other genes with Ptbp1-induced u5′ss bias. Interestingly, the brain-enriched Ptbp1 paralog Ptbp2/nPTB/brPTB stimulated the u5′ss utilization but with a considerably lower efficiency than Ptbp1. This may account for the tight correlation between Hps1 with Ptbp1 expression levels observed across mammalian tissues. More generally, these data expand our understanding of AS regulation and uncover a post-transcriptional strategy ensuring co-expression of a subordinate gene with its master regulator through an AS-NMD tracking mechanism. PMID:25375251

Melanoma Differentiation-associated Gene 7/IL-24 Exerts Cytotoxic Effects by Altering the Alternative Splicing of Bcl-x Pre-mRNA via the SRC/PKCδ Signaling Axis*

PubMed Central

Shapiro, Brian A.; Vu, Ngoc T.; Shultz, Michael D.; Shultz, Jacqueline C.; Mietla, Jennifer A.; Gouda, Mazen M.; Yacoub, Adly; Dent, Paul; Fisher, Paul B.; Park, Margaret A.; Chalfant, Charles E.

2016-01-01

Melanoma differentiation-associated gene 7 (MDA-7/IL-24) exhibits cytotoxic effects on tumor cells while sparing untransformed cells, and Bcl-x(L) is reported to efficiently block the induction of cell death by MDA-7/IL-24. The expression of Bcl-x(L) is regulated at the level of RNA splicing via alternative 5′ splice site selection within exon 2 to produce either the pro-apoptotic Bcl-x(s) or the anti-apoptotic Bcl-x(L). Our laboratory previously reported that Bcl-x RNA splicing is dysregulated in a large percentage of human non-small cell lung cancer (NSCLC) tumors. Therefore, we investigated whether the alternative RNA splicing of Bcl-x pre-mRNA was modulated by MDA-7/IL-24, which would suggest that specific NSCLC tumors are valid targets for this cytokine therapy. Adenovirus-delivered MDA-7/IL-24 (Ad.mda-7) reduced the viability of NSCLC cells of varying oncogenotypes, which was preceded by a decrease in the ratio of Bcl-x(L)/Bcl-x(s) mRNA and Bcl-x(L) protein expression. Importantly, both the expression of Bcl-x(L) and the loss of cell viability were “rescued” in Ad.mda-7-treated cells incubated with Bcl-x(s) siRNA. In addition, NSCLC cells ectopically expressing Bcl-x(s) exhibited significantly reduced Bcl-x(L) expression, which was again restored by Bcl-x(s) siRNA, suggesting the existence of a novel mechanism by which Bcl-x(s) mRNA restrains the expression of Bcl-x(L). In additional mechanistic studies, inhibition of SRC and PKCδ completely ablated the ability of MDA-7/IL-24 to reduce the Bcl-x(L)/(s) mRNA ratio and cell viability. These findings show that Bcl-x(s) expression is an important mediator of MDA-7/IL-24-induced cytotoxicity requiring the SRC/PKCδ signaling axis in NSCLC cells. PMID:27519412

Design of Multi-core Fiber Patch Panel for Space Division Multiplexing Implementations

NASA Astrophysics Data System (ADS)

González, Luz E.; Morales, Alvaro; Rommel, Simon; Jørgensen, Bo F.; Porras-Montenegro, N.; Tafur Monroy, Idelfonso

2018-03-01

A multi-core fiber (MCF) patch panel was designed, allowing easy coupling of individual signals to and from a 7-core MCF. The device was characterized, measuring insertion loss and cross talk, finding highest insertion loss and lowest crosstalk at 1300 nm with values of 9.7 dB and -36.5 dB respectively, while at 1600 nm insertion loss drops to 4.8 dB and crosstalk increases to -24.1 dB. Two MCF splices between the fan-in module, the MCF, and the fan-out module are included in the characterization, and splicing parameters are discussed.

Transcriptome analysis reveals the complexity of alternative splicing regulation in the fungus Verticillium dahliae.

PubMed

Jin, Lirong; Li, Guanglin; Yu, Dazhao; Huang, Wei; Cheng, Chao; Liao, Shengjie; Wu, Qijia; Zhang, Yi

2017-02-06

Alternative splicing (AS) regulation is extensive and shapes the functional complexity of higher organisms. However, the contribution of alternative splicing to fungal biology is not well studied. This study provides sequences of the transcriptomes of the plant wilt pathogen Verticillium dahliae, using two different strains and multiple methods for cDNA library preparations. We identified alternatively spliced mRNA isoforms in over a half of the multi-exonic fungal genes. Over one-thousand isoforms involve TopHat novel splice junction; multiple types of combinatory alternative splicing patterns were identified. We showed that one Verticillium gene could use four different 5' splice sites and two different 3' donor sites to produce up to five mature mRNAs, representing one of the most sophisticated alternative splicing model in eukaryotes other than animals. Hundreds of novel intron types involving a pair of new splice sites were identified in the V. dahliae genome. All the types of AS events were validated by using RT-PCR. Functional enrichment analysis showed that AS genes are involved in most known biological functions and enriched in ATP biosynthesis, sexual/asexual reproduction, morphogenesis, signal transduction etc., predicting that the AS regulation modulates mRNA isoform output and shapes the V. dahliae proteome plasticity of the pathogen in response to the environmental and developmental changes. These findings demonstrate the comprehensive alternative splicing mechanisms in a fungal plant pathogen, which argues the importance of this fungus in developing complicate genome regulation strategies in eukaryotes.

Mechanisms of splicing-dependent trans-synaptic adhesion by PTPδ-IL1RAPL1/IL-1RAcP for synaptic differentiation

NASA Astrophysics Data System (ADS)

Yamagata, Atsushi; Yoshida, Tomoyuki; Sato, Yusuke; Goto-Ito, Sakurako; Uemura, Takeshi; Maeda, Asami; Shiroshima, Tomoko; Iwasawa-Okamoto, Shiho; Mori, Hisashi; Mishina, Masayoshi; Fukai, Shuya

2015-04-01

Synapse formation is triggered through trans-synaptic interaction between pairs of pre- and postsynaptic adhesion molecules, the specificity of which depends on splice inserts known as `splice-insert signaling codes'. Receptor protein tyrosine phosphatase δ (PTPδ) can bidirectionally induce pre- and postsynaptic differentiation of neurons by trans-synaptically binding to interleukin-1 receptor accessory protein (IL-1RAcP) and IL-1RAcP-like-1 (IL1RAPL1) in a splicing-dependent manner. Here, we report crystal structures of PTPδ in complex with IL1RAPL1 and IL-1RAcP. The first immunoglobulin-like (Ig) domain of IL1RAPL1 directly recognizes the first splice insert, which is critical for binding to IL1RAPL1. The second splice insert functions as an adjustable linker that positions the Ig2 and Ig3 domains of PTPδ for simultaneously interacting with the Ig1 domain of IL1RAPL1 or IL-1RAcP. We further identified the IL1RAPL1-specific interaction, which appears coupled to the first-splice-insert-mediated interaction. Our results thus reveal the decoding mechanism of splice-insert signaling codes for synaptic differentiation induced by trans-synaptic adhesion between PTPδ and IL1RAPL1/IL-1RAcP.

Alternative splice variants of AID are not stoichiometrically present at the protein level in chronic lymphocytic leukemia

PubMed Central

Rebhandl, Stefan; Huemer, Michael; Zaborsky, Nadja; Gassner, Franz Josef; Catakovic, Kemal; Felder, Thomas Klaus; Greil, Richard; Geisberger, Roland

2014-01-01

Activation-induced deaminase (AID) is a DNA-mutating enzyme that mediates class-switch recombination as well as somatic hypermutation of antibody genes in B cells. Due to off-target activity, AID is implicated in lymphoma development by introducing genome-wide DNA damage and initiating chromosomal translocations such as c-myc/IgH. Several alternative splice transcripts of AID have been reported in activated B cells as well as malignant B cells such as chronic lymphocytic leukemia (CLL). As most commercially available antibodies fail to recognize alternative splice variants, their abundance in vivo, and hence their biological significance, has not been determined. In this study, we assessed the protein levels of AID splice isoforms by introducing an AID splice reporter construct into cell lines and primary CLL cells from patients as well as from WT and TCL1tg C57BL/6 mice (where TCL1 is T-cell leukemia/lymphoma 1). The splice construct is 5′-fused to a GFP-tag, which is preserved in all splice isoforms and allows detection of translated protein. Summarizing, we show a thorough quantification of alternatively spliced AID transcripts and demonstrate that the corresponding protein abundances, especially those of splice variants AID-ivs3 and AID-ΔE4, are not stoichiometrically equivalent. Our data suggest that enhanced proteasomal degradation of low-abundance proteins might be causative for this discrepancy. PMID:24668151

Diversification of the muscle proteome through alternative splicing.

PubMed

Nakka, Kiran; Ghigna, Claudia; Gabellini, Davide; Dilworth, F Jeffrey

2018-03-06

Skeletal muscles express a highly specialized proteome that allows the metabolism of energy sources to mediate myofiber contraction. This muscle-specific proteome is partially derived through the muscle-specific transcription of a subset of genes. Surprisingly, RNA sequencing technologies have also revealed a significant role for muscle-specific alternative splicing in generating protein isoforms that give specialized function to the muscle proteome. In this review, we discuss the current knowledge with respect to the mechanisms that allow pre-mRNA transcripts to undergo muscle-specific alternative splicing while identifying some of the key trans-acting splicing factors essential to the process. The importance of specific splicing events to specialized muscle function is presented along with examples in which dysregulated splicing contributes to myopathies. Though there is now an appreciation that alternative splicing is a major contributor to proteome diversification, the emergence of improved "targeted" proteomic methodologies for detection of specific protein isoforms will soon allow us to better appreciate the extent to which alternative splicing modifies the activity of proteins (and their ability to interact with other proteins) in the skeletal muscle. In addition, we highlight a continued need to better explore the signaling pathways that contribute to the temporal control of trans-acting splicing factor activity to ensure specific protein isoforms are expressed in the proper cellular context. An understanding of the signal-dependent and signal-independent events driving muscle-specific alternative splicing has the potential to provide us with novel therapeutic strategies to treat different myopathies.

JMJD6 and U2AF65 co-regulate alternative splicing in both JMJD6 enzymatic activity dependent and independent manner.

PubMed

Yi, Jia; Shen, Hai-Feng; Qiu, Jin-Song; Huang, Ming-Feng; Zhang, Wen-Juan; Ding, Jian-Cheng; Zhu, Xiao-Yan; Zhou, Yu; Fu, Xiang-Dong; Liu, Wen

2017-04-07

JMJD6, a jumonji C (Jmj C) domain-containing protein demethylase and hydroxylase, has been implicated in an array of biological processes. It has been shown that JMJD6 interacts with and hydroxylates multiple serine/arginine-rich (SR) proteins and SR related proteins, including U2AF65, all of which are known to function in alternative splicing regulation. However, whether JMJD6 is widely involved in alternative splicing and the molecular mechanism underlying JMJD6-regulated alternative splicing have remained incompletely understood. Here, by using RASL-Seq, we investigated the functional impact of RNA-dependent interaction between JMJD6 and U2AF65, revealing that JMJD6 and U2AF65 co-regulated a large number of alternative splicing events. We further demonstrated the JMJD6 function in alternative splicing in jmjd6 knockout mice. Mechanistically, we showed that the enzymatic activity of JMJD6 was required for a subset of JMJD6-regulated splicing, and JMJD6-mediated lysine hydroxylation of U2AF65 could account for, at least partially, their co-regulated alternative splicing events, suggesting both JMJD6 enzymatic activity-dependent and independent control of alternative splicing. These findings reveal an intimate link between JMJD6 and U2AF65 in alternative splicing regulation, which has important implications in development and disease processes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Targeting GH-1 splicing as a novel pharmacological strategy for growth hormone deficiency type II.

PubMed

Miletta, Maria Consolata; Flück, Christa E; Mullis, Primus-E

2017-01-15

Isolated growth hormone deficiency type II (IGHD II) is a rare genetic splicing disorder characterized by reduced growth hormone (GH) secretion and short stature. It is mainly caused by autosomal dominant-negative mutations within the growth hormone gene (GH-1) which results in missplicing at the mRNA level and the subsequent loss of exon 3, producing the 17.5-kDa GH isoform: a mutant and inactive GH protein that reduces the stability and the secretion of the 22-kDa GH isoform, the main biologically active GH form. At present, patients suffering from IGHD II are treated with daily injections of recombinant human GH (rhGH) in order to reach normal height. However, this type of replacement therapy, although effective in terms of growth, does not prevent the toxic effects of the 17.5-kDa mutant on the pituitary gland, which may eventually lead to other hormonal deficiencies. As the severity of the disease inversely correlates with the 17.5-kDa/22-kDa ratio, increasing the inclusion of exon 3 is expected to ameliorate disease symptoms. This review focuses on the recent advances in experimental and therapeutic strategies applicable to treat IGHD II in clinical and preclinical contexts. Several avenues for alternative IGHD II therapy will be discussed including the use of small interfering RNA (siRNA) and short hairpin RNA (shRNA) constructs that specifically target the exon 3-deleted transcripts as well as the application of histone deacetylase inhibitors (HDACi) and antisense oligonucleotides (AONs) to enhance full-length GH-1 transcription, correct GH-1 exon 3 splicing and manipulate GH pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

Expression and function of Allergin-1 on human primary mast cells.

PubMed

Nagai, Kei; Tahara-Hanaoka, Satoko; Morishima, Yuko; Tokunaga, Takahiro; Imoto, Yoshimasa; Noguchi, Emiko; Kanemaru, Kazumasa; Imai, Masamichi; Shibayama, Shiro; Hizawa, Nobuyuki; Fujieda, Shigeharu; Yamagata, Kunihiro; Shibuya, Akira

2013-01-01

Mast cells (MC) play an important role in allergic and non-allergic immune responses. Activation of human MC is modulated by several cell surface inhibitory receptors, including recently identified Allergin-1 expressed on both human and mouse MC. Although Allergin-1 suppresses IgE-mediated, mast cell-dependent anaphylaxis in mice, the expression profile and function of Allergin-1 on human primary MC remains undetermined. Here, we established a seven-color flow cytometry method for assessing expression and function of a very small number of human primary MC. We show that Allergin-1S1, a splicing isoform of Allergin-1, is predominantly expressed on human primary MC in both bronchoalveolar lavage (BAL) fluid and nasal scratching specimens. Moreover, Allergin-1S1 inhibits IgE-mediated activation from human primary MC in BAL fluid. These results indicate that Allergin-1 on human primary MC exhibits similar characteristics as mouse Allergin-1 in the expression profile and function.

Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels.

PubMed

Bock, Gabriella; Gebhart, Mathias; Scharinger, Anja; Jangsangthong, Wanchana; Busquet, Perrine; Poggiani, Chiara; Sartori, Simone; Mangoni, Matteo E; Sinnegger-Brauns, Martina J; Herzig, Stefan; Striessnig, Jörg; Koschak, Alexandra

2011-12-09

An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Ca(v)1.3 L-type Ca(2+) channels (Ca(v)1.3(L)) is a major determinant of their voltage- and Ca(2+)-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Ca(v)1.3(42A) channels that activate at a more negative voltage range and exhibit more pronounced Ca(2+)-dependent inactivation. Here we describe the discovery of a novel short splice variant (Ca(v)1.3(43S)) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Ca(v)1.3(42A), still contains PCRD. When expressed together with α2δ1 and β3 subunits in tsA-201 cells, Ca(v)1.3(43S) also activated at more negative voltages like Ca(v)1.3(42A) but Ca(2+)-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Ca(v)1.3(L). The presence of the proximal C terminus in Ca(v)1.3(43S) channels preserved their modulation by distal C terminus-containing Ca(v)1.3- and Ca(v)1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca(2+) influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Ca(v)1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca(2+) channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca(2+) accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca(2+)-induced neurodegenerative processes.

Expanding the action of duplex RNAs into the nucleus: redirecting alternative splicing

PubMed Central

Liu, Jing; Hu, Jiaxin; Corey, David R.

2012-01-01

Double-stranded RNAs are powerful agents for silencing gene expression in the cytoplasm of mammalian cells. The potential for duplex RNAs to control expression in the nucleus has received less attention. Here, we investigate the ability of small RNAs to redirect splicing. We identify RNAs targeting an aberrant splice site that restore splicing and production of functional protein. RNAs can target sequences within exons or introns and affect the inclusion of exons within SMN2 and dystrophin, genes responsible for spinal muscular atrophy and Duchenne muscular dystrophy, respectively. Duplex RNAs recruit argonaute 2 (AGO2) to pre-mRNA transcripts and altered splicing requires AGO2 expression. AGO2 promotes transcript cleavage in the cytoplasm, but recruitment of AGO2 to pre-mRNAs does not reduce transcript levels, exposing a difference between cytoplasmic and nuclear pathways. Involvement of AGO2 in splicing, a classical nuclear process, reinforces the conclusion from studies of RNA-mediated transcriptional silencing that RNAi pathways can be adapted to function in the mammalian nucleus. These data provide a new strategy for controlling splicing and expand the reach of small RNAs within the nucleus of mammalian cells. PMID:21948593

A novel method for size uniform 200nm particles: multimetallic particles and in vitro gene delivery

NASA Astrophysics Data System (ADS)

Mair, Lamar; Ford, Kris; Superfine, Richard

2008-10-01

We report on the fabrication of arrays of mono- and multimetallic particles via metal evaporation onto lithographically patterned posts. Metal particles evaporated on cylindrical structures 0.20μm in diameter and 0.33μm tall are released via photoresist dissolution, resulting in freely suspended, shape defined particles. These Post-Particles have highly tunable composition, as demonstrated by our deposition of five different multimetallic particle blends. We calculate the susceptibility and magnetization of 200nm Fe particles in an applied 0.081T magnetic field. In order to evaluate their usefulness as magnetofection agents an antisense oligonucleotide designed to correct the aberrant splicing of enhanced green fluorescent protein mRNA was successfully attached to Fe Post-Particles via a polyethyleneimine linker and transfected into a modified HeLa cell line.

Systemic Delivery of Morpholinos to Skip Multiple Exons in a Dog Model of Duchenne Muscular Dystrophy.

PubMed

Maruyama, Rika; Echigoya, Yusuke; Caluseriu, Oana; Aoki, Yoshitsugu; Takeda, Shin'ichi; Yokota, Toshifumi

2017-01-01

Exon-skipping therapy is an emerging approach that uses synthetic DNA-like molecules called antisense oligonucleotides (AONs) to splice out frame-disrupting parts of mRNA, restore the reading frame, and produce truncated yet functional proteins. Multiple exon skipping utilizing a cocktail of AONs can theoretically treat 80-90% of patients with Duchenne muscular dystrophy (DMD). The success of multiple exon skipping by the systemic delivery of a cocktail of AONs called phosphorodiamidate morpholino oligomers (PMOs) in a DMD dog model has made a significant impact on the development of therapeutics for DMD, leading to clinical trials of PMO-based drugs. Here, we describe the systemic delivery of a cocktail of PMOs to skip multiple exons in dystrophic dogs and the evaluation of the efficacies and toxicity in vivo.

Sex-dependent Association of a Common Low Density Lipoprotein Receptor Polymorphism with RNA Splicing Efficiency in the Brain and Alzheimers Disease

PubMed Central

Zou, Fanggeng; Gopalraj, Rangaraj K.; Lok, Johann; Zhu, Haiyan; Ling, I-Fang; Simpson, James F.; Tucker, H. Michael; Kelly, Jeremiah F.; Younkin, Samuel G.; Dickson, Dennis W.; Petersen, Ronald C; Graff-Radford, Neill R.; Bennett, David A.; Crook, Julia E.; G.Younkin, Steven; Estus, Steven

2008-01-01

Since apoE allele status is the predominant Alzheimers disease (AD) genetic risk factor, functional single nucleotide polymorphisms (SNP)s in brain apoE receptors represent excellent candidates for association with AD. Recently, we identified a SNP, rs688, as modulating the splicing efficiency of low-density lipoprotein receptor (LDLR) exon 12 in the female human liver and in minigene transfected HepG2 cells. Moreover, the rs688T minor allele associated with significantly higher LDL and total cholesterol in women in the Framingham Offspring Study. Since LDLR is a major apoE receptor in the brain, we hypothesized that rs688 modulates LDLR splicing in neural tissues and associates with AD. To evaluate this hypothesis, we first transfected LDLR minigenes into SH-SY5Y neuroblastoma cells and found that rs688T reduces exon 12 inclusion in this neural model. We then evaluated rs688 association with exon 12 splicing efficiency in vivo by quantifying LDLR splicing in human anterior cingulate tissue obtained at autopsy; the rs688T allele associated with decreased LDLR exon 12 splicing efficiency in aged men but not women. Lastly, we evaluated whether rs688 associates with AD by genotyping DNA from 1,457 men and 2,055 women drawn from three case-control series. The rs688T/T genotype was associated with increased AD odds in males (recessive model, odds ratio (OR) of 1.49, 95% confidence interval (CI) of 1.13−1.97, uncorrected p=0.005), but not in females. In summary, these studies identify a functional apoE receptor SNP that is associated with AD in a sex-dependent fashion. PMID:18065781

The human endonuclease III enzyme is a relevant target to potentiate cisplatin cytotoxicity in Y-box-binding protein-1 overexpressing tumor cells.

PubMed

Guay, David; Garand, Chantal; Reddy, Shanti; Schmutte, Chris; Lebel, Michel

2008-04-01

Y-box-binding protein-1 (YB-1) is a multifunctional protein involved in the regulation of transcription, translation, and mRNA splicing. In recent years, several laboratories have demonstrated that YB-1 is directly involved in the cellular response to genotoxic stress. Importantly, YB-1 is increased in tumor cell lines resistant to cisplatin, and the level of nuclear expression of YB-1 is predictive of drug resistance and patient outcome in breast tumors, ovarian cancers, and synovial sarcomas. YB-1 binds to several DNA repair enzymes in vitro including human endonuclease III (hNTH1). Human NTH1 is a bifunctional DNA glycosylase/apurinic/apyrimidinic lyase involved in base excision repair. In this study, we show that YB-1 binds specifically to the auto-inhibitory domain of hNTH1, providing a mechanism by which YB-1 stimulates hNTH1 activity. Indeed, YB-1 strongly stimulates in vitro the activity of hNTH1 toward DNA duplex probes containing oxidized bases, lesions prone to be present in cisplatin treated cells. We also observed an increase in YB-1/hNTH1 complex formation in the mammary adenocarcinoma MCF7 cell line treated with UV light and cisplatin. Such an increase was not observed with mitomycin C or the topoisomerase I inhibitor camptothecin. Accordingly, antisense RNAs against either YB-1 or hNTH1 increased cellular sensitivity to UV and cisplatin but not to mitomycin C. An antisense RNA against YB-1 increased camptothecin sensitivity. In contrast, an antisense against hNTH1 did not. Finally, siRNA against hNTH1 re-established cytotoxicity in otherwise cisplatin-resistant YB-1 overexpressing MCF7 cells. These data indicate that hNTH1 is a relevant target to potentiate cisplatin cytotoxicity in YB-1 overexpressing tumor cells.

Lunar Module Wiring Design Considerations and Failure Modes

NASA Technical Reports Server (NTRS)

Interbartolo, Michael

2009-01-01

This slide presentation reviews the considerations for the design of wiring for the Lunar Module. Included are a review of the choice of conductors and insulations, the wire splicing (i.e., crimping, and soldering), the wire connectors, and the fabrication of the wire harnesses. The problems in fabrication include the wires being the wrong length, the damage due to the sharp edges, the requried use of temproary protective covers and inadequate training. The problems in the wire harness installation include damge from sharp eges, work on adjacent harnesses, connector damage, and breaking wires. Engineering suggestions from the Apollo-era in reference to the conductors that are reviewed include: the use of plated conductors, and the use of alloys for stronger wiring. In refernce to insulation, the suggestions from Apollo era include the use of polymer tape-wrap wire insulation due to the light weight, however, other types of modern insulation might be more cost-effective. In reference to wire splices and terminal boards the suggestions from the Apollo Era include the use of crimp splices as superior to solder splices, joining multiple wire to a common point using modular plug-ins might be more reliable, but are heavier than crimp splicing. For connectors, the lessons from the Apollo era indicate that a rear environmental seal that does not require additional potting is preferred, and pins should be crimped or welded to the incoming wires and be removable from the rear of the connector.

Controlled Enhancemnt of Long-Term Memory by Modulating Neuronal miRNA Function

DTIC Science & Technology

2012-09-20

Faraday Ave. Carlsbad CA 92008 Prepare antisense oligonucleotides 8/1/2007 12:00:00AM 2/1/2009 12:00:00AM Sub Contractor Numbers (c): Patent Clause...with ampakines. However, ampakines that accomplish positive modulation have shown undesired clinical side effects . Another approach is to improve...of times that a mouse touches objects introduced into the cage in a fixed period of time. Objects are either “old” (prior exposure) or “new” (not

Functional Implications of Novel Human Acid Sphingomyelinase Splice Variants

PubMed Central

Rhein, Cosima; Tripal, Philipp; Seebahn, Angela; Konrad, Alice; Kramer, Marcel; Nagel, Christine; Kemper, Jonas; Bode, Jens; Mühle, Christiane; Gulbins, Erich; Reichel, Martin; Becker, Cord-Michael; Kornhuber, Johannes

2012-01-01

Background Acid sphingomyelinase (ASM) hydrolyses sphingomyelin and generates the lipid messenger ceramide, which mediates a variety of stress-related cellular processes. The pathological effects of dysregulated ASM activity are evident in several human diseases and indicate an important functional role for ASM regulation. We investigated alternative splicing as a possible mechanism for regulating cellular ASM activity. Methodology/Principal Findings We identified three novel ASM splice variants in human cells, termed ASM-5, -6 and -7, which lack portions of the catalytic- and/or carboxy-terminal domains in comparison to full-length ASM-1. Differential expression patterns in primary blood cells indicated that ASM splicing might be subject to regulatory processes. The newly identified ASM splice variants were catalytically inactive in biochemical in vitro assays, but they decreased the relative cellular ceramide content in overexpression studies and exerted a dominant-negative effect on ASM activity in physiological cell models. Conclusions/Significance These findings indicate that alternative splicing of ASM is of functional significance for the cellular stress response, possibly representing a mechanism for maintaining constant levels of cellular ASM enzyme activity. PMID:22558155

Arrest is a regulator of fiber-specific alternative splicing in the indirect flight muscles of Drosophila

PubMed Central

Oas, Sandy T.

2014-01-01

Drosophila melanogaster flight muscles are distinct from other skeletal muscles, such as jump muscles, and express several uniquely spliced muscle-associated transcripts. We sought to identify factors mediating splicing differences between the flight and jump muscle fiber types. We found that the ribonucleic acid–binding protein Arrest (Aret) is expressed in flight muscles: in founder cells, Aret accumulates in a novel intranuclear compartment that we termed the Bruno body, and after the onset of muscle differentiation, Aret disperses in the nucleus. Down-regulation of the aret gene led to ultrastructural changes and functional impairment of flight muscles, and transcripts of structural genes expressed in the flight muscles became spliced in a manner characteristic of jump muscles. Aret also potently promoted flight muscle splicing patterns when ectopically expressed in jump muscles or tissue culture cells. Genetically, aret is located downstream of exd (extradenticle), hth (homothorax), and salm (spalt major), transcription factors that control fiber identity. Our observations provide insight into a transcriptional and splicing regulatory network for muscle fiber specification. PMID:25246617

A Major Facilitator Superfamily Transporter Plays a Dual Role in Polar Auxin Transport and Drought Stress Tolerance in Arabidopsis[W

PubMed Central

Remy, Estelle; Cabrito, Tânia R.; Baster, Pawel; Batista, Rita A.; Teixeira, Miguel C.; Friml, Jiri; Sá-Correia, Isabel; Duque, Paula

2013-01-01

Many key aspects of plant development are regulated by the polarized transport of the phytohormone auxin. Cellular auxin efflux, the rate-limiting step in this process, has been shown to rely on the coordinated action of PIN-formed (PIN) and B-type ATP binding cassette (ABCB) carriers. Here, we report that polar auxin transport in the Arabidopsis thaliana root also requires the action of a Major Facilitator Superfamily (MFS) transporter, Zinc-Induced Facilitator-Like 1 (ZIFL1). Sequencing, promoter-reporter, and fluorescent protein fusion experiments indicate that the full-length ZIFL1.1 protein and a truncated splice isoform, ZIFL1.3, localize to the tonoplast of root cells and the plasma membrane of leaf stomatal guard cells, respectively. Using reverse genetics, we show that the ZIFL1.1 transporter regulates various root auxin-related processes, while the ZIFL1.3 isoform mediates drought tolerance by regulating stomatal closure. Auxin transport and immunolocalization assays demonstrate that ZIFL1.1 indirectly modulates cellular auxin efflux during shootward auxin transport at the root tip, likely by regulating plasma membrane PIN2 abundance. Finally, heterologous expression in yeast revealed that ZIFL1.1 and ZIFL1.3 share H+-coupled K+ transport activity. Thus, by determining the subcellular and tissue distribution of two isoforms, alternative splicing dictates a dual function for the ZIFL1 transporter. We propose that this MFS carrier regulates stomatal movements and polar auxin transport by modulating potassium and proton fluxes in Arabidopsis cells. PMID:23524662

Regulation of the LDL receptor gene expression by hormones.

PubMed

Streicher, R; Kotzka, J; Müller-Wieland, D; Krone, W

1998-01-01

Promoter activity of the LDL receptor gene is stimulated by insulin and estradiol and mediated by SRE-1, which acts as a hormone sensitive cis-elemente. Using the antisense technique we reveal that SREBP-1 is selectively involved in the signal transduction pathway of insulin and IGF-I.

PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice.

PubMed

Zinker, Bradley A; Rondinone, Cristina M; Trevillyan, James M; Gum, Rebecca J; Clampit, Jill E; Waring, Jeffrey F; Xie, Nancy; Wilcox, Denise; Jacobson, Peer; Frost, Leigh; Kroeger, Paul E; Reilly, Regina M; Koterski, Sandra; Opgenorth, Terry J; Ulrich, Roger G; Crosby, Seth; Butler, Madeline; Murray, Susan F; McKay, Robert A; Bhanot, Sanjay; Monia, Brett P; Jirousek, Michael R

2002-08-20

The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA(1C). Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50alpha, were increased and PI3-kinase p85alpha expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes.

PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice

PubMed Central

Zinker, Bradley A.; Rondinone, Cristina M.; Trevillyan, James M.; Gum, Rebecca J.; Clampit, Jill E.; Waring, Jeffrey F.; Xie, Nancy; Wilcox, Denise; Jacobson, Peer; Frost, Leigh; Kroeger, Paul E.; Reilly, Regina M.; Koterski, Sandra; Opgenorth, Terry J.; Ulrich, Roger G.; Crosby, Seth; Butler, Madeline; Murray, Susan F.; McKay, Robert A.; Bhanot, Sanjay; Monia, Brett P.; Jirousek, Michael R.

2002-01-01

The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA1C. Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50α, were increased and PI3-kinase p85α expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes. PMID:12169659

Corepressors: custom tailoring and alterations while you wait

PubMed Central

Goodson, Michael; Jonas, Brian A.; Privalsky, Martin A.

2005-01-01

A diverse cadre of metazoan transcription factors mediate repression by recruiting protein complexes containing the SMRT (silencing mediator of retinoid and thyroid hormone receptor) or N-CoR (nuclear receptor corepressor) corepressors. SMRT and N-CoR nucleate the assembly of still larger corepressor complexes that perform the specific molecular incantations necessary to confer transcriptional repression. Although SMRT and N-CoR are paralogs and possess similar molecular architectures and mechanistic strategies, they nonetheless exhibit distinct molecular and biological properties. It is now clear that the functions of both SMRT and N-CoR are further diversified through alternative mRNA splicing, yielding a series of corepressor protein variants that participate in distinctive transcription factor partnerships and display distinguishable repression properties. This review will discuss what is known about the structure and actions of SMRT, N-CoR, and their splicing variants, and how alternative splicing may allow the functions of these corepressors to be adapted and tailored to different cells and to different developmental stages. PMID:16604171

Improved silencing properties using small internally segmented interfering RNAs

PubMed Central

Bramsen, Jesper B.; Laursen, Maria B.; Damgaard, Christian K.; Lena, Suzy W.; Ravindra Babu, B.; Wengel, Jesper; Kjems, Jørgen

2007-01-01

RNA interference is mediated by small interfering RNAs (siRNAs) that upon incorporation into the RNA-induced silencing complex (RISC) can target complementary mRNA for degradation. Standard siRNA design usually feature a 19–27 base pair contiguous double-stranded region that is believed to be important for RISC incorporation. Here, we describe a novel siRNA design composed of an intact antisense strand complemented with two shorter 10–12 nt sense strands. This three-stranded construct, termed small internally segmented interfering RNA (sisiRNA), is highly functional demonstrating that an intact sense strand is not a prerequisite for RNA interference. Moreover, when using the sisiRNA design only the antisense strand is functional in activated RISC thereby completely eliminating unintended mRNA targeting by the sense strand. Interestingly, the sisiRNA design supports the function of chemically modified antisense strands, which are non-functional within the context of standard siRNA designs. This suggests that the sisiRNA design has a clear potential of improving the pharmacokinetic properties of siRNA in vivo. PMID:17726057

Small-interfering RNAs from natural antisense transcripts derived from a cellulose synthase gene modulate cell wall biosynthesis in barley

PubMed Central

Held, Michael A.; Penning, Bryan; Brandt, Amanda S.; Kessans, Sarah A.; Yong, Weidong; Scofield, Steven R.; Carpita, Nicholas C.

2008-01-01

Small-interfering RNAs (siRNAs) from natural cis-antisense pairs derived from the 3′-coding region of the barley (Hordeum vulgare) CesA6 cellulose synthase gene substantially increase in abundance during leaf elongation. Strand-specific RT-PCR confirmed the presence of an antisense transcript of HvCesA6 that extends ≥1230 bp from the 3′ end of the CesA-coding sequence. The increases in abundance of the CesA6 antisense transcript and the 21-nt and 24-nt siRNAs derived from the transcript are coincident with the down-regulation of primary wall CesAs, several Csl genes, and GT8 glycosyl transferase genes, and are correlated with the reduction in rates of cellulose and (1 → 3),(1 → 4)-β-D-glucan synthesis. Virus induced gene silencing using unique target sequences derived from HvCesA genes attenuated expression not only of the HvCesA6 gene, but also of numerous nontarget Csls and the distantly related GT8 genes and reduced the incorporation of D-14C-Glc into cellulose and into mixed-linkage (1 → 3),(1 → 4)-β-D-glucans of the developing leaves. Unique target sequences for CslF and CslH conversely silenced the same genes and lowered rates of cellulose and (1 → 3),(1 → 4)-β-D-glucan synthesis. Our results indicate that the expression of individual members of the CesA/Csl superfamily and glycosyl transferases share common regulatory control points, and siRNAs from natural cis-antisense pairs derived from the CesA/Csl superfamily could function in this global regulation of cell-wall synthesis. PMID:19075248

RBFOX2 protein domains and cellular activities.

PubMed

Arya, Anurada D; Wilson, David I; Baralle, Diana; Raponi, Michaela

2014-08-01

RBFOX2 (RNA-binding protein, Fox-1 homologue 2)/RBM9 (RNA-binding-motif protein 9)/RTA (repressor of tamoxifen action)/HNRBP2 (hexaribonucleotide-binding protein 2) encodes an RNA-binding protein involved in tissue specific alternative splicing regulation and steroid receptors transcriptional activity. Its ability to regulate specific splicing profiles depending on context has been related to different expression levels of the RBFOX2 protein itself and that of other splicing regulatory proteins involved in the shared modulation of specific genes splicing. However, this cannot be the sole explanation as to why RBFOX2 plays a widespread role in numerous cellular mechanisms from development to cell survival dependent on cell/tissue type. RBFOX2 isoforms with altered protein domains exist. In the present article, we describe the main RBFOX2 protein domains, their importance in the context of splicing and transcriptional regulation and we propose that RBFOX2 isoform distribution may play a fundamental role in RBFOX2-specific cellular effects.

Activity-induced histone modifications govern Neurexin-1 mRNA splicing and memory preservation.

PubMed

Ding, Xinlu; Liu, Sanxiong; Tian, Miaomiao; Zhang, Wenhao; Zhu, Tao; Li, Dongdong; Wu, Jiawei; Deng, HaiTeng; Jia, Yichang; Xie, Wei; Xie, Hong; Guan, Ji-Song

2017-05-01

Epigenetic mechanisms regulate the formation, consolidation and reconsolidation of memories. However, the signaling path from neuronal activation to epigenetic modifications within the memory-related brain circuit remains unknown. We report that learning induces long-lasting histone modifications in hippocampal memory-activated neurons to regulate memory stability. Neuronal activity triggers a late-onset shift in Nrxn1 splice isoform choice at splicing site 4 by accumulating a repressive histone marker, H3K9me3, to modulate the splicing process. Activity-dependent phosphorylation of p66α via AMP-activated protein kinase recruits HDAC2 and Suv39h1 to establish repressive histone markers and changes the connectivity of the activated neurons. Removal of Suv39h1 abolished the activity-dependent shift in Nrxn1 splice isoform choice and reduced the stability of established memories. We uncover a cell-autonomous process for memory preservation in which memory-related neurons initiate a late-onset reduction of their rewiring capacities through activity-induced histone modifications.

In Situ Observation of Modulated Light Emission of Fiber Fuse Synchronized with Void Train over Hetero-Core Splice Point

PubMed Central

Todoroki, Shin-ichi

2008-01-01

Background Fiber fuse is a process of optical fiber destruction under the action of laser radiation, found 20 years ago. Once initiated, opical discharge runs along the fiber core region to the light source and leaves periodic voids whose shape looks like a bullet pointing the direction of laser beam. The relation between damage pattern and propagation mode of optical discharge is still unclear even after the first in situ observation three years ago. Methodology/Principal Findings Fiber fuse propagation over hetero-core splice point (Corning SMF-28e and HI 1060) was observed in situ. Sequential photographs obtained at intervals of 2.78 µs recorded a periodic emission at the tail of an optical discharge pumped by 1070 nm and 9 W light. The signal stopped when the discharge ran over the splice point. The corresponding damage pattern left in the fiber core region included a segment free of periodicity. Conclusions The spatial modulation pattern of the light emission agreed with the void train formed over the hetero-core splice point. Some segments included a bullet-shaped void pointing in the opposite direction to the laser beam propagation although the sequential photographs did not reveal any directional change in the optical discharge propagation. PMID:18815621

Identification of a chemical inhibitor for nuclear speckle formation: Implications for the function of nuclear speckles in regulation of alternative pre-mRNA splicing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kurogi, Yutaro; Matsuo, Yota; Mihara, Yuki

2014-03-28

Highlights: • We identified tubercidin as a compound inducing aberrant formation of the speckles. • Tubercidin causes delocalization of poly (A){sup +}RNAs from nuclear speckles. • Tubercidin induces dispersion of splicing factors from nuclear speckles. • Tubercidin affects alternative pre-mRNA splicing. • Nuclear speckles play a role in regulation of alternative pre-mRNA splicing. - Abstract: Nuclear speckles are subnuclear structures enriched with RNA processing factors and poly (A){sup +} RNAs comprising mRNAs and poly (A){sup +} non-coding RNAs (ncRNAs). Nuclear speckles are thought to be involved in post-transcriptional regulation of gene expression, such as pre-mRNA splicing. By screening 3585 culturemore » extracts of actinomycetes with in situ hybridization using an oligo dT probe, we identified tubercidin, an analogue of adenosine, as an inhibitor of speckle formation, which induces the delocalization of poly (A){sup +} RNA and dispersion of splicing factor SRSF1/SF2 from nuclear speckles in HeLa cells. Treatment with tubercidin also decreased steady-state MALAT1 long ncRNA, thought to be involved in the retention of SRSF1/SF2 in nuclear speckles. In addition, we found that tubercidin treatment promoted exon skipping in the alternative splicing of Clk1 pre-mRNA. These results suggest that nuclear speckles play a role in modulating the concentration of splicing factors in the nucleoplasm to regulate alternative pre-mRNA splicing.« less

PASTA: splice junction identification from RNA-Sequencing data

PubMed Central

2013-01-01

Background Next generation transcriptome sequencing (RNA-Seq) is emerging as a powerful experimental tool for the study of alternative splicing and its regulation, but requires ad-hoc analysis methods and tools. PASTA (Patterned Alignments for Splicing and Transcriptome Analysis) is a splice junction detection algorithm specifically designed for RNA-Seq data, relying on a highly accurate alignment strategy and on a combination of heuristic and statistical methods to identify exon-intron junctions with high accuracy. Results Comparisons against TopHat and other splice junction prediction software on real and simulated datasets show that PASTA exhibits high specificity and sensitivity, especially at lower coverage levels. Moreover, PASTA is highly configurable and flexible, and can therefore be applied in a wide range of analysis scenarios: it is able to handle both single-end and paired-end reads, it does not rely on the presence of canonical splicing signals, and it uses organism-specific regression models to accurately identify junctions. Conclusions PASTA is a highly efficient and sensitive tool to identify splicing junctions from RNA-Seq data. Compared to similar programs, it has the ability to identify a higher number of real splicing junctions, and provides highly annotated output files containing detailed information about their location and characteristics. Accurate junction data in turn facilitates the reconstruction of the splicing isoforms and the analysis of their expression levels, which will be performed by the remaining modules of the PASTA pipeline, still under development. Use of PASTA can therefore enable the large-scale investigation of transcription and alternative splicing. PMID:23557086

Retroviral gene transfer of an antisense construct against membrane type 1 matrix metalloproteinase reduces the invasiveness of rheumatoid arthritis synovial fibroblasts.

PubMed

Rutkauskaite, Edita; Volkmer, Dagmar; Shigeyama, Yukio; Schedel, Jörg; Pap, Geza; Müller-Ladner, Ulf; Meinecke, Ingmar; Alexander, Dorothea; Gay, Renate E; Drynda, Susanne; Neumann, Wolfram; Michel, Beat A; Aicher, Wilhelm K; Gay, Steffen; Pap, Thomas

2005-07-01

Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed prominently in rheumatoid arthritis synovial fibroblasts (RASFs), but the specific contribution of MT1-MMP to fibroblast-mediated destruction of articular cartilage is incompletely understood. This study used gene transfer of an antisense expression construct to assess the effects of MT1-MMP inhibition on the invasiveness of RASFs. Retroviral gene transfer of a pLXIN vector-based antisense RNA expression construct (MT1-MMPalphaS) to MT1-MMP was used to stably transduce RASFs. Levels of MT1-MMP RNA and protein were determined by quantitative polymerase chain reaction, Western blotting, and immunocytochemistry in MT1-MMPalphaS-transduced RASFs as well as in control cells, with monitoring for 60 days. The effects of MT1-MMPalphaS on the invasiveness of RASFs were analyzed in the SCID mouse co-implantation model of RA. MT1-MMPalphaS-transduced RASFs produced high levels of antisense RNA that exceeded endogenous levels of MT1-MMP messenger RNA by 15-fold and resulted in a down-regulation of MT1-MMP at the protein level. Inhibition of MT1-MMP production was maintained for 60 days and significantly reduced the invasiveness of RASFs in the SCID mouse model. Whereas prominent invasion into cartilage by non-transduced and mock-transduced RASFs was observed (mean invasion scores 3.0 and 3.1, respectively), MT1-MMPalphaS-transduced cells showed only moderate invasiveness (mean invasion score 1.8; P < 0.05). The data demonstrate that an antisense RNA expression construct against MT1-MMP can be generated and expressed in RASFs for at least 60 days. Inhibition of MT1-MMP significantly reduces the cartilage degradation by RASFs.

C-fos mediates antipsychotic-induced neurotensin gene expression in the rodent striatum.

PubMed

Robertson, G S; Tetzlaff, W; Bedard, A; St-Jean, M; Wigle, N

1995-07-01

The ubiquitous inducibility of the immediate-early gene c-fos in the central nervous system has led to the search for downstream genes which are regulated by its product, Fos. Recent evidence suggests that c-fos induction by a single injection of the classical antipsychotic haloperidol may contribute to the subsequent increase in neurotensin gene expression in the rodent striatum. Consistent with this proposal, in the present study haloperidol-induced Fos-like immunoreactivity and neurotensin/neuromedin N messenger RNA were found to be expressed by the same population of striatal neurons. Moreover, inhibition of haloperidol-induced c-fos expression by intrastriatal injection of antisense phosphorothioate oligodeoxynucleotides complimentary either to bases 109-126 or 127-144 of c-fos attenuated the subsequent increase in neurotensin/neuromedin N messenger RNA. However, injection of a sense phosphorothioate oligodeoxynucleotide corresponding to bases 127-144 of c-fos did not reduce haloperidol-induced c-fos or neurotensin/neuromedin N expression. Furthermore, constitutive expression of Jun-like immunoreactivity in the striatum was not reduced by either the sense or antisense phosphorothioate oligodeoxynucleotides. Similarly, the sense and antisense phosphorothioate oligodeoxynucleotide failed to reduce proenkephalin messenger RNA, which is located in the same striatal neurons that express haloperidol-induced neurotensin/neuromedin N messenger RNA, which is located in the same striatal neurons that express haloperidol-induced neurotensin/neuromedin N messenger RNA. Lastly, haloperidol-induced increases in nerve growth factor I-A-, JunB- and FosB-like immunoreactivity and fosB messenger RNA were not decreased by intrastriatal injection of either the sense or antisense phosphorothioate oligodeoxynucleotides. These results indicate that the antisense phosphorothioate oligodeoxynucleotides attenuated haloperidol-induced neurotensin/neuromedin N expression by selectively reducing c-fos expression and emphasize the potential importance of immediate-early gene induction in the mechanism of action of this antipsychotic drug.

Analysis of Autoregulation at the Level of Pre-mRNA Splicing of the Suppressor-of-White-Apricot Gene in Drosophila

PubMed Central

Zachar, Z.; Chou, T. B.; Kramer, J.; Mims, I. P.; Bingham, P. M.

1994-01-01

The Drosophila suppressor-of-white-apricot [su(w(a))] protein regulates/modulates at least two somatic RNA processing events. It is a potent regulator of its own expression. We report here new studies of this autoregulatory circuit. Among other things, our studies show the following. First, new evidence that su(w(a)) expression is autoregulated at the level of pre-mRNA splicing is reported. su(w(a)) protein represses accumulation of the fully spliced su(w(a)) mRNA encoding it and promotes accumulation of high levels of incompletely spliced su(w(a)) pre-mRNA. Second, the fully spliced su(w(a)) mRNA is sufficient for all known su(w(a)) genetic functions indicating that it encodes the sole su(w(a)) protein. Third, the incompletely spliced su(w(a)) pre-mRNAs resulting from autoregulation are not translated (probably as a result of nuclear retention) and apparently represent nonfunctional by-products. Fourth, the special circumstances of su(w(a)) expression during oogenesis allows maternal deposition exclusively of fully spliced su(w(a)) mRNA. Fifth, su(w(a)) protein immunolocalizes to nuclei consistent with its being a direct regulator of pre-mRNA processing. We discuss the implications of our results for mechanisms of splicing regulation and for developmental control of su(w(a)) expression. PMID:8056305

Long-range RNA pairings contribute to mutually exclusive splicing

PubMed Central

Yue, Yuan; Yang, Yun; Dai, Lanzhi; Cao, Guozheng; Chen, Ran; Hong, Weiling; Liu, Baoping; Shi, Yang; Meng, Yijun; Shi, Feng; Xiao, Mu; Jin, Yongfeng

2016-01-01

Mutually exclusive splicing is an important means of increasing the protein repertoire, by which the Down's syndrome cell adhesion molecule (Dscam) gene potentially generates 38,016 different isoforms in Drosophila melanogaster. However, the regulatory mechanisms remain obscure due to the complexity of the Dscam exon cluster. Here, we reveal a molecular model for the regulation of the mutually exclusive splicing of the serpent pre-mRNA based on competition between upstream and downstream RNA pairings. Such dual RNA pairings confer fine tuning of the inclusion of alternative exons. Moreover, we demonstrate that the splicing outcome of alternative exons is mediated in relative pairing strength-correlated mode. Combined comparative genomics analysis and experimental evidence revealed similar bidirectional structural architectures in exon clusters 4 and 9 of the Dscam gene. Our findings provide a novel mechanistic framework for the regulation of mutually exclusive splicing and may offer potentially applicable insights into long-range RNA–RNA interactions in gene regulatory networks. PMID:26554032

Long-range RNA pairings contribute to mutually exclusive splicing.

PubMed

Yue, Yuan; Yang, Yun; Dai, Lanzhi; Cao, Guozheng; Chen, Ran; Hong, Weiling; Liu, Baoping; Shi, Yang; Meng, Yijun; Shi, Feng; Xiao, Mu; Jin, Yongfeng

2016-01-01

Mutually exclusive splicing is an important means of increasing the protein repertoire, by which the Down's syndrome cell adhesion molecule (Dscam) gene potentially generates 38,016 different isoforms in Drosophila melanogaster. However, the regulatory mechanisms remain obscure due to the complexity of the Dscam exon cluster. Here, we reveal a molecular model for the regulation of the mutually exclusive splicing of the serpent pre-mRNA based on competition between upstream and downstream RNA pairings. Such dual RNA pairings confer fine tuning of the inclusion of alternative exons. Moreover, we demonstrate that the splicing outcome of alternative exons is mediated in relative pairing strength-correlated mode. Combined comparative genomics analysis and experimental evidence revealed similar bidirectional structural architectures in exon clusters 4 and 9 of the Dscam gene. Our findings provide a novel mechanistic framework for the regulation of mutually exclusive splicing and may offer potentially applicable insights into long-range RNA-RNA interactions in gene regulatory networks. © 2015 Yue et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Corticotropin (ACTH) regulates alternative RNA splicing in Y1 mouse adrenocortical tumor cells.

PubMed

Schimmer, Bernard P; Cordova, Martha

2015-06-15

The stimulatory effect of ACTH on gene expression is well documented and is thought to be a major mechanism by which ACTH maintains the functional and structural integrity of the gland. Previously, we showed that ACTH regulates the accumulation of over 1200 transcripts in Y1 adrenal cells, including a cluster with functions in alternative splicing of RNA. On this basis, we postulated that some of the effects of ACTH on the transcription landscape of Y1 cells are mediated by alternative splicing. In this study, we demonstrate that ACTH regulates the alternative splicing of four transcripts - Gnas, Cd151, Dab2 and Tia1. Inasmuch as alternative splicing potentially affects transcripts from more than two-thirds of the mouse genome, we suggest that these findings are representative of a genome-wide effect of ACTH that impacts on the mRNA and protein composition of the adrenal cortex. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Alternative RNA splicing in the endothelium mediated in part by Rbfox2 regulates the arterial response to low flow

PubMed Central

Butty, Vincent L; Boutz, Paul L; Begum, Shahinoor; Kimble, Amy L; Sharp, Phillip A; Burge, Christopher B

2018-01-01

Low and disturbed blood flow drives the progression of arterial diseases including atherosclerosis and aneurysms. The endothelial response to flow and its interactions with recruited platelets and leukocytes determine disease progression. Here, we report widespread changes in alternative splicing of pre-mRNA in the flow-activated murine arterial endothelium in vivo. Alternative splicing was suppressed by depletion of platelets and macrophages recruited to the arterial endothelium under low and disturbed flow. Binding motifs for the Rbfox-family are enriched adjacent to many of the regulated exons. Endothelial deletion of Rbfox2, the only family member expressed in arterial endothelium, suppresses a subset of the changes in transcription and RNA splicing induced by low flow. Our data reveal an alternative splicing program activated by Rbfox2 in the endothelium on recruitment of platelets and macrophages and demonstrate its relevance in transcriptional responses during flow-driven vascular inflammation. PMID:29293084

TANK-Binding Kinase 1 (TBK1) Isoforms Negatively Regulate Type I Interferon Induction by Inhibiting TBK1-IRF3 Interaction and IRF3 Phosphorylation.

PubMed

Hu, Yi Wei; Zhang, Jie; Wu, Xiao Man; Cao, Lu; Nie, Pin; Chang, Ming Xian

2018-01-01

TANK-binding kinase 1 (TBK1) is an important serine/threonine-protein kinase that mediates phosphorylation and nuclear translocation of IRF3, which contributes to induction of type I interferons (IFNs) in the innate antiviral response. In mammals, TBK1 spliced isoform negatively regulates the virus-triggered IFN-β signaling pathway by disrupting the interaction between retinoic acid-inducible gene I (RIG-I) and mitochondria antiviral-signaling protein (MAVS). However, it is still unclear whether alternative splicing patterns and the function of TBK1 isoform(s) exist in teleost fish. In this study, we identify two alternatively spliced isoforms of TBK1 from zebrafish, termed TBK1_tv1 and TBK1_tv2. Both TBK1_tv1 and TBK1_tv2 contain an incomplete STKc_TBK1 domain. Moreover, the UBL_TBK1_like domain is also missing for TBK1_tv2. TBK1_tv1 and TBK1_tv2 are expressed in zebrafish larvae. Overexpression of TBK1_tv1 and TBK1_tv2 inhibits RIG-I-, MAVS-, TBK1-, and IRF3-mediated activation of IFN promoters in response to spring viremia of carp virus infection. Also, TBK1_tv1 and TBK1_tv2 inhibit expression of IFNs and IFN-stimulated genes induced by MAVS and TBK1 . Mechanistically, TBK1_tv1 and TBK1_tv2 competitively associate with TBK1 and IRF3 to disrupt the formation of a functional TBK1-IRF3 complex, impeding the phosphorylation of IRF3 mediated by TBK1. Collectively, these results demonstrate that TBK1 spliced isoforms are dominant negative regulators in the RIG-I/MAVS/TBK1/IRF3 antiviral pathway by targeting the functional TBK1-IRF3 complex formation. Identification and functional characterization of piscine TBK1 spliced isoforms may contribute to understanding the role of TBK1 expression in innate antiviral response.

Expression of REST4 in human gliomas in vivo and influence of pioglitazone on REST in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, Huan; Institute of Clinical Pharmacology, Central South University, Hunan Key Laboratory of Pharmacogenetics, Changsha 410078; Gao, Zhangfeng

The repressor element-1 (RE1) silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) has an irreplaceable role during the differentiation of neurons. REST has multiple splice variants which link to various types of cancer. Previous work had highlighted the role of REST in glioma, where the expression of REST is enhanced. But whether alternative splicing of REST is expressed in glioma has not been described. Here, we show that a specific isoform REST4 is expressed in glioma specimens, and will influence the mRNA level of REST in vivo. Peroxisome proliferator-activated receptor-γ (PPARγ) agonists have a role of antineoplastic in various tumor cells, which includingmore » glioma cells. Moreover, study indicated that PPARγ agonist pioglitazone can promote alternative splicing of REST pre-mRNA. In this study, we selected pioglitazone as a tool drug to explore whether the role of pioglitazone in anti-glioma is mediated by regulating REST expression or promoting alternative splicing of REST in glioma cells. Results show that pioglitazone can inhibit proliferation and induce apoptosis of glioma cell in vitro, which may be mediated by down-regulating REST mRNA level but not by inducing alternative splicing of REST pre-mRNA. Our study firstly reports the expression of REST4 in glioma tissue samples. And we recommend that pioglitazone, which can reduce the expression level of REST, represents a promising drug for therapy of glioma. - Highlights: • A specific isoform REST4 is expressed in glioma specimens in vivo. • REST4 will influence the mRNA level of REST in vivo. • Pioglitazone can inhibit proliferation and induce apoptosis of glioma cells. • The role of pioglitazone in anti-glioma may be mediated by down-regulating REST.« less

ABERRANT SPLICING OF A BRAIN-ENRICHED ALTERNATIVE EXON ELIMINATES TUMOR SUPPRESSOR FUNCTION AND PROMOTES ONCOGENE FUNCTION DURING BRAIN TUMORIGENESIS

PubMed Central

Bredel, Markus; Ferrarese, Roberto; Harsh, Griffith R.; Yadav, Ajay K.; Bug, Eva; Maticzka, Daniel; Reichardt, Wilfried; Masilamani, Anie P.; Dai, Fangping; Kim, Hyunsoo; Hadler, Michael; Scholtens, Denise M.; Yu, Irene L.Y.; Beck, Jürgen; Srinivasasainagendra, Vinodh; Costa, Fabrizio; Baxan, Nicoleta; Pfeifer, Dietmar; Elverfeldt, Dominik v.; Backofen, Rolf; Weyerbrock, Astrid; Duarte, Christine W.; He, Xiaolin; Prinz, Marco; Chandler, James P.; Vogel, Hannes; Chakravarti, Arnab; Rich, Jeremy N.; Carro, Maria S.

2014-01-01

BACKGROUND: Tissue-specific alternative splicing is known to be critical to emergence of tissue identity during development, yet its role in malignant transformation is undefined. Tissue-specific splicing involves evolutionary-conserved, alternative exons, which represent only a minority of total alternative exons. Many, however, have functional features that influence activity in signaling pathways to profound biological effect. Given that tissue-specific splicing has a determinative role in brain development and the enrichment of genes containing tissue-specific exons for proteins with roles in signaling and development, it is thus plausible that changes in such exons could rewire normal neurogenesis towards malignant transformation. METHODS: We used integrated molecular genetic and cell biology analyses, computational biology, animal modeling, and clinical patient profiles to characterize the effect of aberrant splicing of a brain-enriched alternative exon in the membrane-binding tumor suppressor Annexin A7 (ANXA7) on oncogene regulation and brain tumorigenesis. RESULTS: We show that aberrant splicing of a tissue-specific cassette exon in ANXA7 diminishes endosomal targeting and consequent termination of the signal of the EGFR oncoprotein during brain tumorigenesis. Splicing of this exon is mediated by the ribonucleoprotein Polypyrimidine Tract-Binding Protein 1 (PTBP1), which is normally repressed during brain development but, we find, is excessively expressed in glioblastomas through either gene amplification or loss of a neuron-specific microRNA, miR-124. Silencing of PTBP1 attenuates both malignancy and angiogenesis in a stem cell-derived glioblastoma animal model characterized by a high native propensity to generate tumor endothelium or vascular pericytes to support tumor growth. We show that EGFR amplification and PTBP1 overexpression portend a similarly poor clinical outcome, further highlighting the importance of PTBP1-mediated activation of EGFR. CONCLUSIONS: Our data illustrate how anomalous splicing of a tissue-regulated exon in a constituent of an oncogenic signaling pathway eliminates its tumor suppressor function and promotes tumorigenesis. This paradigm of malignant glial transformation as a consequence of tissue-specific alternative exon splicing in a tumor suppressor, may have widespread applicability in explaining how changes in critical tissue-specific regulatory mechanisms reprogram normal development to oncogenesis. SECONDARY CATEGORY: n/a.

The fitness cost of mis-splicing is the main determinant of alternative splicing patterns.

PubMed

Saudemont, Baptiste; Popa, Alexandra; Parmley, Joanna L; Rocher, Vincent; Blugeon, Corinne; Necsulea, Anamaria; Meyer, Eric; Duret, Laurent

2017-10-30

Most eukaryotic genes are subject to alternative splicing (AS), which may contribute to the production of protein variants or to the regulation of gene expression via nonsense-mediated messenger RNA (mRNA) decay (NMD). However, a fraction of splice variants might correspond to spurious transcripts and the question of the relative proportion of splicing errors to functional splice variants remains highly debated. We propose a test to quantify the fraction of AS events corresponding to errors. This test is based on the fact that the fitness cost of splicing errors increases with the number of introns in a gene and with expression level. We analyzed the transcriptome of the intron-rich eukaryote Paramecium tetraurelia. We show that in both normal and in NMD-deficient cells, AS rates strongly decrease with increasing expression level and with increasing number of introns. This relationship is observed for AS events that are detectable by NMD as well as for those that are not, which invalidates the hypothesis of a link with the regulation of gene expression. Our results show that in genes with a median expression level, 92-98% of observed splice variants correspond to errors. We observed the same patterns in human transcriptomes and we further show that AS rates correlate with the fitness cost of splicing errors. These observations indicate that genes under weaker selective pressure accumulate more maladaptive substitutions and are more prone to splicing errors. Thus, to a large extent, patterns of gene expression variants simply reflect the balance between selection, mutation, and drift.

RNA Structure Design Improves Activity and Specificity of trans-Splicing-Triggered Cell Death in a Suicide Gene Therapy Approach.

PubMed

Poddar, Sushmita; Loh, Pei She; Ooi, Zi Hao; Osman, Farhana; Eul, Joachim; Patzel, Volker

2018-06-01

Spliceosome-mediated RNA trans-splicing enables correction or labeling of pre-mRNA, but therapeutic applications are hampered by issues related to the activity and target specificity of trans-splicing RNA (tsRNA). We employed computational RNA structure design to improve both on-target activity and specificity of tsRNA in a herpes simplex virus thymidine kinase/ganciclovir suicide gene therapy approach targeting alpha fetoprotein (AFP), a marker of hepatocellular carcinoma (HCC) or human papillomavirus type 16 (HPV-16) pre-mRNA. While unstructured, mismatched target binding domains significantly improved 3' exon replacement (3'ER), 5' exon replacement (5'ER) correlated with the thermodynamic stability of the tsRNA 3' end. Alternative on-target trans-splicing was found to be a prevalent event. The specificity of trans-splicing with the intended target splice site was improved 10-fold by designing tsRNA that harbors secondary target binding domains shielding alternative on-target and blinding off-target splicing events. Such rationally designed suicide RNAs efficiently triggered death of HPV-16-transduced or hepatoblastoma-derived human tissue culture cells without evidence for off-target cell killing. Highest cell death activities were observed with novel dual-targeting tsRNAs programmed for trans-splicing toward AFP and a second HCC pre-mRNA biomarker. Our observations suggest trans-splicing represents a promising approach to suicide gene therapy. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Integration of a splicing regulatory network within the meiotic gene expression program of Saccharomyces cerevisiae

PubMed Central

Munding, Elizabeth M.; Igel, A. Haller; Shiue, Lily; Dorighi, Kristel M.; Treviño, Lisa R.; Ares, Manuel

2010-01-01

Splicing regulatory networks are essential components of eukaryotic gene expression programs, yet little is known about how they are integrated with transcriptional regulatory networks into coherent gene expression programs. Here we define the MER1 splicing regulatory network and examine its role in the gene expression program during meiosis in budding yeast. Mer1p splicing factor promotes splicing of just four pre-mRNAs. All four Mer1p-responsive genes also require Nam8p for splicing activation by Mer1p; however, other genes require Nam8p but not Mer1p, exposing an overlapping meiotic splicing network controlled by Nam8p. MER1 mRNA and three of the four Mer1p substrate pre-mRNAs are induced by the transcriptional regulator Ume6p. This unusual arrangement delays expression of Mer1p-responsive genes relative to other genes under Ume6p control. Products of Mer1p-responsive genes are required for initiating and completing recombination and for activation of Ndt80p, the activator of the transcriptional network required for subsequent steps in the program. Thus, the MER1 splicing regulatory network mediates the dependent relationship between the UME6 and NDT80 transcriptional regulatory networks in the meiotic gene expression program. This study reveals how splicing regulatory networks can be interlaced with transcriptional regulatory networks in eukaryotic gene expression programs. PMID:21123654

De Novo Mutations in SON Disrupt RNA Splicing of Genes Essential for Brain Development and Metabolism, Causing an Intellectual-Disability Syndrome.

PubMed

Kim, Jung-Hyun; Shinde, Deepali N; Reijnders, Margot R F; Hauser, Natalie S; Belmonte, Rebecca L; Wilson, Gregory R; Bosch, Daniëlle G M; Bubulya, Paula A; Shashi, Vandana; Petrovski, Slavé; Stone, Joshua K; Park, Eun Young; Veltman, Joris A; Sinnema, Margje; Stumpel, Connie T R M; Draaisma, Jos M; Nicolai, Joost; Yntema, Helger G; Lindstrom, Kristin; de Vries, Bert B A; Jewett, Tamison; Santoro, Stephanie L; Vogt, Julie; Bachman, Kristine K; Seeley, Andrea H; Krokosky, Alyson; Turner, Clesson; Rohena, Luis; Hempel, Maja; Kortüm, Fanny; Lessel, Davor; Neu, Axel; Strom, Tim M; Wieczorek, Dagmar; Bramswig, Nuria; Laccone, Franco A; Behunova, Jana; Rehder, Helga; Gordon, Christopher T; Rio, Marlène; Romana, Serge; Tang, Sha; El-Khechen, Dima; Cho, Megan T; McWalter, Kirsty; Douglas, Ganka; Baskin, Berivan; Begtrup, Amber; Funari, Tara; Schoch, Kelly; Stegmann, Alexander P A; Stevens, Servi J C; Zhang, Dong-Er; Traver, David; Yao, Xu; MacArthur, Daniel G; Brunner, Han G; Mancini, Grazia M; Myers, Richard M; Owen, Laurie B; Lim, Ssang-Taek; Stachura, David L; Vissers, Lisenka E L M; Ahn, Eun-Young Erin

2016-09-01

The overall understanding of the molecular etiologies of intellectual disability (ID) and developmental delay (DD) is increasing as next-generation sequencing technologies identify genetic variants in individuals with such disorders. However, detailed analyses conclusively confirming these variants, as well as the underlying molecular mechanisms explaining the diseases, are often lacking. Here, we report on an ID syndrome caused by de novo heterozygous loss-of-function (LoF) mutations in SON. The syndrome is characterized by ID and/or DD, malformations of the cerebral cortex, epilepsy, vision problems, musculoskeletal abnormalities, and congenital malformations. Knockdown of son in zebrafish resulted in severe malformation of the spine, brain, and eyes. Importantly, analyses of RNA from affected individuals revealed that genes critical for neuronal migration and cortex organization (TUBG1, FLNA, PNKP, WDR62, PSMD3, and HDAC6) and metabolism (PCK2, PFKL, IDH2, ACY1, and ADA) are significantly downregulated because of the accumulation of mis-spliced transcripts resulting from erroneous SON-mediated RNA splicing. Our data highlight SON as a master regulator governing neurodevelopment and demonstrate the importance of SON-mediated RNA splicing in human development. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Functional analysis of alternative transcripts of the soybean Rj2 gene that restricts nodulation with specific rhizobial strains.

PubMed

Tang, F; Yang, S; Zhu, H

2016-05-01

The Rj2 gene is a TIR-NBS-LRR-type resistance gene in soybean (Glycine max) that restricts root nodule symbiosis with a group of Bradyrhizobium japonicum strains including USDA122. Rj2 generates two distinct transcript variants in its expression profile through alternative splicing. Alternative splicing of Rj2 is caused by the retention of the 86-bp intron 4. Inclusion of intron 4 in mature mRNA introduces an in-frame stop codon; as such, the alternative transcript is predicted to encode a truncated protein consisting of the entire portion of the TIR, NBS and LRR domains but missing the C-terminal domain of the full-length Rj2 protein encoded by the regular transcript. Since alternative splicing has been shown to be essential for full activity of several plant R genes, we attempted to test whether the alternative splicing is required for Rj2-mediated nodulation restriction. Here we demonstrated that the Rj2-mediated nodulation restriction does not require the combined presence of the regular and alternative transcripts, and the expression of the regular transcript alone is sufficient to confer nodulation restriction. © 2016 German Botanical Society and The Royal Botanical Society of the Netherlands.

Random Splicing of Several Exons Caused by a Single Base Change in the Target Exon of CRISPR/Cas9 Mediated Gene Knockout.

PubMed

Kapahnke, Marcel; Banning, Antje; Tikkanen, Ritva

2016-12-14

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated sequence 9 (CRISPR/Cas9) system is widely used for genome editing purposes as it facilitates an efficient knockout of a specific gene in, e.g. cultured cells. Targeted double-strand breaks are introduced to the target sequence of the guide RNAs, which activates the cellular DNA repair mechanism for non-homologous-end-joining, resulting in unprecise repair and introduction of small deletions or insertions. Due to this, sequence alterations in the coding region of the target gene frequently cause frame-shift mutations, facilitating degradation of the mRNA. We here show that such CRISPR/Cas9-mediated alterations in the target exon may also result in altered splicing of the respective pre-mRNA, most likely due to mutations of splice-regulatory sequences. Using the human FLOT-1 gene as an example, we demonstrate that such altered splicing products also give rise to aberrant protein products. These may potentially function as dominant-negative proteins and thus interfere with the interpretation of the data generated with these cell lines. Since most researchers only control the consequences of CRISPR knockout at genomic and protein level, our data should encourage to also check the alterations at the mRNA level.

Interplay between estrogen receptor and AKT in Estradiol-induced alternative splicing

PubMed Central

2013-01-01

Background Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown. Methods MCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ERα binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively. Results We identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ERα cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ERα binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ERα and splicing factors, influenced ERα:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ERα-dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ERα-dependent isoform switching, which correlated with altered response to KGF. Conclusion E2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens. PMID:23758675

Emerging functions of alternative splicing coupled with nonsense-mediated decay.

PubMed

Hamid, Fursham M; Makeyev, Eugene V

2014-08-01

Higher eukaryotes rely on AS (alternative splicing) of pre-mRNAs (mRNA precursors) to generate more than one protein product from a single gene and to regulate mRNA stability and translational activity. An important example of the latter function involves an interplay between AS and NMD (nonsense-mediated decay), a cytoplasmic quality control mechanism eliminating mRNAs containing PTCs (premature translation termination codons). Although originally identified as an error surveillance process, AS-NMD additionally provides an efficient strategy for deterministic regulation of gene expression outputs. In this review, we discuss recently published examples of AS-NMD and delineate functional contexts where recurrent use of this mechanism orchestrates expression of important genes.

Crosstalk between vertical and horizontal gene transfer: plasmid replication control by a conjugative relaxase

PubMed Central

Lorenzo-Díaz, Fabián; Fernández-López, Cris; Lurz, Rudi

2017-01-01

Abstract Horizontal gene transfer is a key process in the evolution of bacteria and also represents a source of genetic variation in eukaryotes. Among elements participating in gene transfer, thousands of small (<10 kb) mobile bacterial plasmids that replicate by the rolling circle mechanism represent a driving force in the spread of antibiotic resistances. In general, these plasmids are built as genetic modules that encode a replicase, an antibiotic-resistance determinant, and a relaxase that participates in their conjugative mobilization. Further, they control their relatively high copy number (∼30 copies per genome equivalent) by antisense RNAs alone or combined with a repressor protein. We report here that the MobM conjugative relaxase encoded by the promiscuous plasmid pMV158 participates in regulation of the plasmid copy number by transcriptional repression of the antisense RNA, thus increasing the number of plasmid molecules ready to be horizontally transferred (mobilization) and/or vertically inherited (replication). This type of crosstalk between genetic modules involved in vertical and horizontal gene flow has not been reported before. PMID:28525572

Therapeutic gene targeting approaches for the treatment of dyslipidemias and atherosclerosis.

PubMed

Mäkinen, Petri I; Ylä-Herttuala, Seppo

2013-04-01

Despite improved therapies, cardiovascular diseases are the leading cause of morbidity and mortality worldwide. Therefore, new therapeutic approaches are still needed. In the gene therapy field, RNA interference (RNAi) and regulation of microRNAs (miRNAs) have gained a lot of attention in addition to traditional overexpression based strategies. Here, recent findings in therapeutic gene silencing and modulation of small RNA expression related to atherogenesis and dyslipidemia are summarized. Novel gene therapy approaches for the treatment of hyperlipidemia have been addressed. Antisense oligonucleotide and RNAi-based therapies against apolipoprotein B100 and proprotein convertase subtilisin/kexin type 9 have shown already efficacy in preclinical and clinical trials. In addition, several miRNAs dysregulated in atherosclerotic lesions and regulating cholesterol homeostasis have been found, which may represent novel targets for future therapies. New therapies for lowering lipid levels are now being tested in clinical trials, and both antisense oligonucleotide and RNAi-based therapies have shown promising results in lowering cholesterol levels. However, the modulation of inflammatory component in atherosclerosis by gene therapy and targeting of the effects to plaques are still difficult challenges.

Noncanonical ATM Activation and Signaling in Response to Transcription-Blocking DNA Damage.

PubMed

Marteijn, Jurgen A; Vermeulen, Wim; Tresini, Maria

2017-01-01

Environmental genotoxins and metabolic byproducts generate DNA lesions that can cause genomic instability and disrupt tissue homeostasis. To ensure genomic integrity, cells employ mechanisms that convert signals generated by stochastic DNA damage into organized responses, including activation of repair systems, cell cycle checkpoints, and apoptotic mechanisms. DNA damage response (DDR) signaling pathways coordinate these responses and determine cellular fates in part, by transducing signals that modulate RNA metabolism. One of the master DDR coordinators, the Ataxia Telangiectasia Mutated (ATM) kinase, has a fundamental role in mediating DNA damage-induced changes in mRNA synthesis. ATM acts by modulating a variety of RNA metabolic pathways including nascent RNA splicing, a process catalyzed by the spliceosome. Interestingly, ATM and the spliceosome influence each other's activity in a reciprocal manner by a pathway that initiates when transcribing RNA polymerase II (RNAPII) encounters DNA lesions that prohibit forward translocation. In response to stalling of RNAPII assembly of late-stage spliceosomes is disrupted resulting in increased splicing factor mobility. Displacement of spliceosomes from lesion-arrested RNA polymerases facilitates formation of R-loops between the nascent RNA and DNA adjacent to the transcription bubble. R-loops signal for noncanonical ATM activation which in quiescent cells occurs in absence of detectable dsDNA breaks. In turn, activated ATM signals to regulate spliceosome dynamics and AS genome wide.This chapter describes the use of fluorescence microscopy methods that can be used to evaluate noncanonical ATM activation by transcription-blocking DNA damage. First, we present an immunofluorescence-detection method that can be used to evaluate ATM activation by autophosphorylation, in fixed cells. Second, we present a protocol for Fluorescence Recovery After Photobleaching (FRAP) of GFP-tagged splicing factors, a highly sensitive and reproducible readout to measure in living cells, the ATM influence on the spliceosome. These approaches have been extensively used in our laboratory for a number of cell lines of various origins and are particularly informative when used in primary cells that can be synchronized in quiescence, to avoid generation of replication stress-induced dsDNA breaks and consequent ATM activation through its canonical pathway.

Generation and Characterization of a Transgenic Mouse Carrying a Functional Human β-Globin Gene with the IVSI-6 Thalassemia Mutation

PubMed Central

Mancini, Irene; Lampronti, Ilaria; Salvatori, Francesca; Fabbri, Enrica; Zuccato, Cristina; Cosenza, Lucia C.; Montagner, Giulia; Borgatti, Monica; Altruda, Fiorella; Fagoonee, Sharmila; Carandina, Gianni; Aiello, Vincenzo; Breda, Laura; Rivella, Stefano; Gambari, Roberto

2015-01-01

Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the β-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece) are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-β-IVSI-6) carrying the IVSI-6 thalassemia point mutation within the human β-globin gene. In the TG-β-IVSI-6 mouse (a) the transgenic integration region is located in mouse chromosome 7; (b) the expression of the transgene is tissue specific; (c) as expected, normally spliced human β-globin mRNA is produced, giving rise to β-globin production and formation of a human-mouse tetrameric chimeric hemoglobin mu α-globin2/hu β-globin2 and, more importantly, (d) the aberrant β-globin-IVSI-6 RNAs are present in blood cells. The TG-β-IVSI-6 mouse reproduces the molecular features of IVSI-6 β-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced β-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for β-thalassemia. PMID:26097845

Transcriptome interrogation of human myometrium identifies differentially expressed sense-antisense pairs of protein-coding and long non-coding RNA genes in spontaneous labor at term.

PubMed

Romero, Roberto; Tarca, Adi L; Chaemsaithong, Piya; Miranda, Jezid; Chaiworapongsa, Tinnakorn; Jia, Hui; Hassan, Sonia S; Kalita, Cynthia A; Cai, Juan; Yeo, Lami; Lipovich, Leonard

2014-09-01

To identify differentially expressed long non-coding RNA (lncRNA) genes in human myometrium in women with spontaneous labor at term. Myometrium was obtained from women undergoing cesarean deliveries who were not in labor (n = 19) and women in spontaneous labor at term (n = 20). RNA was extracted and profiled using an Illumina® microarray platform. We have used computational approaches to bound the extent of long non-coding RNA representation on this platform, and to identify co-differentially expressed and correlated pairs of long non-coding RNA genes and protein-coding genes sharing the same genomic loci. We identified co-differential expression and correlation at two genomic loci that contain coding-lncRNA gene pairs: SOCS2-AK054607 and LMCD1-NR_024065 in women in spontaneous labor at term. This co-differential expression and correlation was validated by qRT-PCR, an experimental method completely independent of the microarray analysis. Intriguingly, one of the two lncRNA genes differentially expressed in term labor had a key genomic structure element, a splice site, that lacked evolutionary conservation beyond primates. We provide, for the first time, evidence for coordinated differential expression and correlation of cis-encoded antisense lncRNAs and protein-coding genes with known as well as novel roles in pregnancy in the myometrium of women in spontaneous labor at term.

Antisense Oligonucleotides Used to Target the DUX4 mRNA as Therapeutic Approaches in FaciosScapuloHumeral Muscular Dystrophy (FSHD)

PubMed Central

Ansseau, Eugénie; Vanderplanck, Céline; Wauters, Armelle; Harper, Scott Q.; Coppée, Frédérique; Belayew, Alexandra

2017-01-01

FacioScapuloHumeral muscular Dystrophy (FSHD) is one of the most prevalent hereditary myopathies and is generally characterized by progressive muscle atrophy affecting the face, scapular fixators; upper arms and distal lower legs. The FSHD locus maps to a macrosatellite D4Z4 repeat array on chromosome 4q35. Each D4Z4 unit contains a DUX4 gene; the most distal of which is flanked by a polyadenylation site on FSHD-permissive alleles, which allows for production of stable DUX4 mRNAs. In addition, an open chromatin structure is required for DUX4 gene transcription. FSHD thus results from a gain of function of the toxic DUX4 protein that normally is only expressed in germ line and stem cells. Therapeutic strategies are emerging that aim to decrease DUX4 expression or toxicity in FSHD muscle cells. We review here the heterogeneity of DUX4 mRNAs observed in muscle and stem cells; and the use of antisense oligonucleotides (AOs) targeting the DUX4 mRNA to interfere either with transcript cleavage/polyadenylation or intron splicing. We show in primary cultures that DUX4-targeted AOs suppress the atrophic FSHD myotube phenotype; but do not improve the disorganized FSHD myotube phenotype which could be caused by DUX4c over-expression. Thus; DUX4c might constitute another therapeutic target in FSHD. PMID:28273791

Osteopontin-c mediates the upregulation of androgen responsive genes in LNCaP cells through PI3K/Akt and androgen receptor signaling.

PubMed

Tilli, Tatiana Martins; Ferreira, Luciana Bueno; Gimba, Etel Rodrigues Pereira

2015-04-01

Androgen receptor (AR) signaling is a key pathway modulating prostate cancer (PCa) progression. Several steps in this pathway have been investigated in order to propose novel treatment strategies for advanced PCa. Total osteopontin (OPN) has been described as a biomarker for PCa, in addition to its role in activating the progression of this tumor. Based on the known effects of the OPNc splice variant on PCa progression, the present study investigated whether this isoform can also modulate AR signaling. In order to test this, an in vitro model was used in which LNCaP cells were cultured in the presence of conditioned medium (CM) secreted by PCa cells overexpressing OPNc (OPNc-CM). The activation of AR signaling was evaluated by measuring the expression levels of AR-responsive genes (ARGs) using quantitative polymerase chain reaction and specific oligonucleotides. The data demonstrated that all nine tested ARGs ( Fgf8 , TMPRSS2 , Greb1 , Cdk2 , Ndrg1 , Cdk1 , Pmepa1 , Psa and Ar ) are significantly upregulated in response to OPNc-CM compared with LNCaP cells cultured in CM secreted by control cells transfected with empty expression vector. The specific involvement of OPNc was demonstrated by depleting OPNc from OPNc-CM using an anti-OPNc neutralizing antibody. In addition, by using a phosphoinositide 3-kinase (PI3K)-specific inhibitor and AR antagonists, such as flutamide and bicalutamide, it was also observed that upregulation of ARGs in response to OPNc-CM involves PI3K signaling and depends on the AR. In conclusion, these data indicated that OPNc is able to activate AR signaling through the PI3K pathway and the AR. These data further corroborate our previous data, revealing the OPNc splice variant to be a key molecule that is able to modulate key signaling pathways involved in PCa progression.

Osteopontin-c mediates the upregulation of androgen responsive genes in LNCaP cells through PI3K/Akt and androgen receptor signaling

PubMed Central

TILLI, TATIANA MARTINS; FERREIRA, LUCIANA BUENO; GIMBA, ETEL RODRIGUES PEREIRA

2015-01-01

Androgen receptor (AR) signaling is a key pathway modulating prostate cancer (PCa) progression. Several steps in this pathway have been investigated in order to propose novel treatment strategies for advanced PCa. Total osteopontin (OPN) has been described as a biomarker for PCa, in addition to its role in activating the progression of this tumor. Based on the known effects of the OPNc splice variant on PCa progression, the present study investigated whether this isoform can also modulate AR signaling. In order to test this, an in vitro model was used in which LNCaP cells were cultured in the presence of conditioned medium (CM) secreted by PCa cells overexpressing OPNc (OPNc-CM). The activation of AR signaling was evaluated by measuring the expression levels of AR-responsive genes (ARGs) using quantitative polymerase chain reaction and specific oligonucleotides. The data demonstrated that all nine tested ARGs (Fgf8, TMPRSS2, Greb1, Cdk2, Ndrg1, Cdk1, Pmepa1, Psa and Ar) are significantly upregulated in response to OPNc-CM compared with LNCaP cells cultured in CM secreted by control cells transfected with empty expression vector. The specific involvement of OPNc was demonstrated by depleting OPNc from OPNc-CM using an anti-OPNc neutralizing antibody. In addition, by using a phosphoinositide 3-kinase (PI3K)-specific inhibitor and AR antagonists, such as flutamide and bicalutamide, it was also observed that upregulation of ARGs in response to OPNc-CM involves PI3K signaling and depends on the AR. In conclusion, these data indicated that OPNc is able to activate AR signaling through the PI3K pathway and the AR. These data further corroborate our previous data, revealing the OPNc splice variant to be a key molecule that is able to modulate key signaling pathways involved in PCa progression. PMID:25789054

Viral and bacterial septicaemic infections modulate the expression of PACAP splicing variants and VIP/PACAP receptors in brown trout immune organs.

PubMed

Gorgoglione, Bartolomeo; Carpio, Yamila; Secombes, Christopher J; Taylor, Nick G H; Lugo, Juana María; Estrada, Mario Pablo

2015-12-01

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and PACAP-Related Peptide (PRP) are structurally similar peptides encoded in the same transcripts. Their transcription has been detected not only in the brain but also in a wide range of peripheral tissues, even including organs of the immune system. PACAP exerts pleiotropic activities through G-protein coupled membrane receptors: the PACAP-specific PAC-1 and the VPAC-1 and VPAC-2 receptors that exhibit similar affinities for the Vasoactive Intestinal Peptide (VIP) and PACAP. Recent findings added PACAP and its receptors to the growing list of mediators that allow cross-talk between the nervous, endocrine and immune systems in fish. In this study the expression of genes encoding for PACAP and PRP, as well as VIP/PACAP receptors was studied in laboratory-reared brown trout (Salmo trutta) after septicaemic infections. Respectively Viral Haemorrhagic Septicaemia Virus (VHSV-Ia) or the Gram-negative bacterium Yersinia ruckeri (ser. O1 - biot. 2) were used in infection challenges. Kidney and spleen, the teleost main lymphopoietic organs, were sampled during the first two weeks post-infection. RT-qPCR analysis assessed specific pathogens burden and gene expression levels. PACAP and PRP transcription in each organ was positively correlated to the respective pathogen burden, assessed targeting the VHSV-glycoprotein or Y. ruckeri 16S rRNA. Results showed as the transcription of PACAP splicing variants and VIP/PACAP receptors is modulated in these organs during an acute viral and bacterial septicaemic infections in brown trout. These gene expression results provide clues as to how the PACAP system is modulated in fish, confirming an involvement during active immune responses elicited by both viral and bacterial aetiological agents. However, further experimental evidence is still required to fully elucidate and characterize the role of PACAP and PRP for an efficient immune response against pathogens. Copyright © 2015 Elsevier Ltd. All rights reserved.

LY404187: a novel positive allosteric modulator of AMPA receptors.

PubMed

Quirk, Jennifer C; Nisenbaum, Eric S

2002-01-01

LY404187 is a selective, potent and centrally active positive allosteric modulator of AMPA receptors. LY404187 preferentially acts at recombinant human homomeric GluR2 and GluR4 versus GluR1 and GluR3 AMPA receptors. In addition, LY404187 potentiates the flip splice variant of these AMPA receptors to a greater degree than the flop splice variant. In both recombinant and native AMPA receptors, potentiation by LY404187 displays a unique time-dependent growth that appears to involve a suppression of the desensitization process of these ion channels. LY404187 has been shown to enhance glutamatergic synaptic transmission both in vitro and in vivo. This augmentation of synaptic activity is due to the direct potentiation of AMPA receptor function, as well as an indirect recruitment of voltage-dependent NMDA receptor activity. Enhanced calcium influx through NMDA receptors is known to be a critical step in initiating long-term modifications in synaptic function (e.g., long-term potentiation, LTP). These modifications in synaptic function may be substrates for certain forms of memory encoding. Consistent with a recruitment of NMDA receptor activity, LY404187 has been shown to enhance performance in animal models of cognitive function requiring different mnemonic processes. These data suggest that AMPA receptor potentiators may be therapeutically beneficial for treating cognitive deficits in a variety of disorders, particularly those that are associated with reduced glutamatergic signaling such as schizophrenia. In addition, LY404187 has been demonstrated to be efficacious in animal models of behavioral despair that possess considerable predictive validity for antidepressant activity. Although the therapeutic efficacy of AMPA receptor potentiators in these and other diseases will ultimately be determined in the clinic, evidence suggests that the benefit of these compounds will be mediated by multiple mechanisms of action. These mechanisms include direct enhancement of AMPA receptor function, secondary mobilization of intracellular signaling cascades, and prolonged modulation of gene expression.

Expression of cathepsin S antisense transcripts by adenovirus in retinal pigment epithelial cells.

PubMed

Rakoczy, P E; Lai, M C; Baines, M G; Spilsbury, K; Constable, I J

1998-10-01

To show the production of sense or antisense transcripts by recombinant adenoviruses, to investigate whether the transcripts produced were suitable for downregulating the expression of the targeted gene, cathepsin S (CatS), and to examine the effect of antisense transcript production on the biologic function of retinal pigment epithelial (RPE) cells, including the regulation of endogenous aspartic protease expression. Ad.MLP.CatSAS, Ad.RSV.CatSAS, and Ad.MLP.CatSS recombinant viruses were produced by homologous recombination. The recombinant viruses were tested by restriction enzyme digestion to confirm the orientation of the inserts. The expression of antisense transcripts was tested by northern blot analysis. Western blot analysis was used to study the regulation of the endogenous CatS protein in ARPE19 cells. The biologic effect of CatS downregulation in ARPE19 cells was tested by proliferation and phagocytosis assays, de novo cathepsin D (CatD) synthesis, and measurement of aspartic protease activity. After characterization of the recombinant adenovirus constructs, the production of antisense and sense CatS transcripts was shown in ARPE19 cells. The transcripts appeared at approximately 1.9 kb 48 hours after transduction, and the expression of the antisense transcripts was similar in constructs carrying either the MLP or the RSV promoter. Western blot analysis showed that ARPE19 cells transduced with Ad.MLP.CatSAS and Ad.RSV.CatSAS had no detectable CatS. In contrast, there was a strong signal appearing at 24 kDa in ARPE19 cells transduced with Ad.MLP.CatSS. ARPE19 cells were transduced to a high level. The transduction of ARPE19 cells with the recombinant adenoviruses did not affect the morphologic appearance of the cells, their proliferation, or their phagocytosing ability. However, ARPE19 cells transduced by Ad.MLP.CatSAS recombinant adenovirus showed a significant downregulation of de novo CatD synthesis and a twofold decrease in aspartic protease activity. Recombinant adenoviruses were shown to be suitable for producing antisense CatS transcripts to modulate endogenous CatS expression in RPE cells. It is proposed that CatS may play an important role, directly or indirectly, in the lysosomal digestion of outer segments through the regulation of other lysosomal enzyme activity, such as the expression of CatD.

Structure and function of splice variants of the cardiac voltage-gated sodium channel Na(v)1.5.

PubMed

Schroeter, Annett; Walzik, Stefan; Blechschmidt, Steve; Haufe, Volker; Benndorf, Klaus; Zimmer, Thomas

2010-07-01

Voltage-gated sodium channels mediate the rapid upstroke of the action potential in excitable tissues. The tetrodotoxin (TTX) resistant isoform Na(v)1.5, encoded by the SCN5A gene, is the predominant isoform in the heart. This channel plays a key role for excitability of atrial and ventricular cardiomyocytes and for rapid impulse propagation through the specific conduction system. During recent years, strong evidence has been accumulated in support of the expression of several Na(v)1.5 splice variants in the heart, and in various other tissues and cell lines including brain, dorsal root ganglia, breast cancer cells and neuronal stem cell lines. This review summarizes our knowledge on the structure and putative function of nine Na(v)1.5 splice variants detected so far. Attention will be paid to the distinct biophysical properties of the four functional splice variants, to the pronounced tissue- and species-specific expression, and to the developmental regulation of Na(v)1.5 splicing. The implications of alternative splicing for SCN5A channelopathies, and for a better understanding of genotype-phenotype correlations, are discussed. Copyright 2010 Elsevier Ltd. All rights reserved.

The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing.

PubMed

Makeyev, Eugene V; Zhang, Jiangwen; Carrasco, Monica A; Maniatis, Tom

2007-08-03

Both microRNAs and alternative pre-mRNA splicing have been implicated in the development of the nervous system (NS), but functional interactions between these two pathways are poorly understood. We demonstrate that the neuron-specific microRNA miR-124 directly targets PTBP1 (PTB/hnRNP I) mRNA, which encodes a global repressor of alternative pre-mRNA splicing in nonneuronal cells. Among the targets of PTBP1 is a critical cassette exon in the pre-mRNA of PTBP2 (nPTB/brPTB/PTBLP), an NS-enriched PTBP1 homolog. When this exon is skipped, PTBP2 mRNA is subject to nonsense-mediated decay (NMD). During neuronal differentiation, miR-124 reduces PTBP1 levels, leading to the accumulation of correctly spliced PTBP2 mRNA and a dramatic increase in PTBP2 protein. These events culminate in the transition from non-NS to NS-specific alternative splicing patterns. We also present evidence that miR-124 plays a key role in the differentiation of progenitor cells to mature neurons. Thus, miR-124 promotes NS development, at least in part by regulating an intricate network of NS-specific alternative splicing.

Cystic Fibrosis Gene Encodes a cAMP-Dependent Chloride Channel in Heart

NASA Astrophysics Data System (ADS)

Hart, Padraig; Warth, John D.; Levesque, Paul C.; Collier, Mei Lin; Geary, Yvonne; Horowitz, Burton; Hume, Joseph R.

1996-06-01

cAMP-dependent chloride channels in heart contribute to autonomic regulation of action potential duration and membrane potential and have been inferred to be due to cardiac expression of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In this report, a cDNA from rabbit ventricle was isolated and sequenced, which encodes an exon 5 splice variant (exon 5-) of CFTR, with >90% identity to human CFTR cDNA present in epithelial cells. Expression of this cDNA in Xenopus oocytes gave rise to robust cAMP-activated chloride currents that were absent in control water-injected oocytes. Antisense oligodeoxynucleotides directed against CFTR significnatly reduced the density of cAMP-dependent chloride currents in acutely cultured myocytes, thereby establishing a direct functional link between cardiac expression of CFTR protein and an endogenous chloride channel in native cardiac myocytes.

A Novel Bcl-x Isoform Connected to the T Cell Receptor Regulates Apoptosis in T Cells

PubMed Central

Yang, Xiao-Feng; Weber, Georg F.

2014-01-01

Summary We define a novel Bcl-x isoform, Bcl-xγ, that is generated by alternative splicing and characterized by a unique 47 amino acid C-terminus. Bcl-xγ is expressed primarily in thymocytes, where it may depend on an interaction between the TCR and host MHC products, and in mature T cells, where its expression is associated with ligation of the T cell receptor. Overexpression of Bcl-xγ in T cells inhibits activation-induced apoptosis; inhibition of Bcl-xγ, after stable expression of Bcl-xγ antisense cDNA, enhances activation-induced apoptosis. In contrast to other Bcl-x isoforms, cells that fail to express Bcl-xγ after CD3 ligation undergo programmed cell death, while activated T cells that express Bcl-xγ are spared. Identification of Bcl-xγ helps provide amolecular explanation of T cell activation and death after antigen engagement. PMID:9390687

A Novel Collection of snRNA-Like Promoters with Tissue-Specific Transcription Properties

PubMed Central

Garritano, Sonia; Gigoni, Arianna; Costa, Delfina; Malatesta, Paolo; Florio, Tullio; Cancedda, Ranieri; Pagano, Aldo

2012-01-01

We recently identified a novel dataset of snRNA-like trascriptional units in the human genome. The investigation of a subset of these elements showed that they play relevant roles in physiology and/or pathology. In this work we expand our collection of small RNAs taking advantage of a newly developed algorithm able to identify genome sequence stretches with RNA polymerase (pol) III type 3 promoter features thus constituting putative pol III binding sites. The bioinformatic analysis of a subset of these elements that map in introns of protein-coding genes in antisense configuration suggest their association with alternative splicing, similarly to other recently characterized small RNAs. Interestingly, the analysis of the transcriptional activity of these novel promoters shows that they are active in a cell-type specific manner, in accordance with the emerging body of evidence of a tissue/cell-specific activity of pol III. PMID:23109855

A novel collection of snRNA-like promoters with tissue-specific transcription properties.

PubMed

Garritano, Sonia; Gigoni, Arianna; Costa, Delfina; Malatesta, Paolo; Florio, Tullio; Cancedda, Ranieri; Pagano, Aldo

2012-01-01

We recently identified a novel dataset of snRNA-like trascriptional units in the human genome. The investigation of a subset of these elements showed that they play relevant roles in physiology and/or pathology. In this work we expand our collection of small RNAs taking advantage of a newly developed algorithm able to identify genome sequence stretches with RNA polymerase (pol) III type 3 promoter features thus constituting putative pol III binding sites. The bioinformatic analysis of a subset of these elements that map in introns of protein-coding genes in antisense configuration suggest their association with alternative splicing, similarly to other recently characterized small RNAs. Interestingly, the analysis of the transcriptional activity of these novel promoters shows that they are active in a cell-type specific manner, in accordance with the emerging body of evidence of a tissue/cell-specific activity of pol III.

Genetic validation of whole-transcriptome sequencing for mapping expression affected by cis-regulatory variation.

PubMed

Babak, Tomas; Garrett-Engele, Philip; Armour, Christopher D; Raymond, Christopher K; Keller, Mark P; Chen, Ronghua; Rohl, Carol A; Johnson, Jason M; Attie, Alan D; Fraser, Hunter B; Schadt, Eric E

2010-08-13

Identifying associations between genotypes and gene expression levels using microarrays has enabled systematic interrogation of regulatory variation underlying complex phenotypes. This approach has vast potential for functional characterization of disease states, but its prohibitive cost, given hundreds to thousands of individual samples from populations have to be genotyped and expression profiled, has limited its widespread application. Here we demonstrate that genomic regions with allele-specific expression (ASE) detected by sequencing cDNA are highly enriched for cis-acting expression quantitative trait loci (cis-eQTL) identified by profiling of 500 animals in parallel, with up to 90% agreement on the allele that is preferentially expressed. We also observed widespread noncoding and antisense ASE and identified several allele-specific alternative splicing variants. Monitoring ASE by sequencing cDNA from as little as one sample is a practical alternative to expression genetics for mapping cis-acting variation that regulates RNA transcription and processing.

Latency of Epstein-Barr virus is stabilized by antisense-mediated control of the viral immediate-early gene BZLF-1.

PubMed

Prang, N; Wolf, H; Schwarzmann, F

1999-12-01

The ability of the Epstein-Barr virus (EBV) to avoid lytic replication and to establish a latent infection in B-lymphocytes is fundamental for its lifelong persistence and the pathogenesis of various EBV-associated diseases. The viral immediate-early gene BZLF-1 plays a key role for the induction of lytic replication and its activity is strictly regulated on different levels of gene expression. Recently, it was demonstrated that BZLF-1 is also controlled by a posttranscriptional mechanism. Transient synthesis of a mutated competitor RNA saturated this mechanism and caused both expression of the BZLF-1 protein and the induction of lytic viral replication. Using short overlapping fragments of the competitor, it is shown that this control acts on the unspliced primary transcript. RT-PCR demonstrated unspliced BZLF-1 RNA in latently infected B-lymphocytes in the absence of BZLF-1 protein. Due to the complementarity of the gene BZLF-1 and the latency-associated gene EBNA-1 on the opposite strand of the genome, we propose an antisense-mediated mechanism. RNase protection assays demonstrated transcripts in antisense orientation to the BZLF-1 transcript during latency, which comprise a comparable constellation to other herpesviruses. A combined RNAse protection/RT-PCR assay detected the double-stranded hybrid RNA, consisting of the unspliced BZLF-1 transcript and a noncoding intron of the EBNA-1 gene. Binding of BZLF-1 transcripts is suggested to be an important backup control mechanism in addition to transcriptional regulation, stabilizing latency and preventing inappropriate lytic viral replication in vivo. Copyright 1999 Wiley-Liss, Inc.

RNA splicing and its connection with other regulatory layers in somatic cell reprogramming.

PubMed

Zavolan, Mihaela; Kanitz, Alexander

2018-06-01

Understanding how cell identity is established and maintained is one of the most exciting challenges of molecular biology today. Recent work has added a conserved layer of RNA splicing and other post-transcriptional regulatory processes to the transcriptional and epigenetic networks already known to cooperate in the establishment and maintenance of cell identity. Here we summarize these findings, highlighting specifically the multitude of splicing factors that can modulate the efficiency of somatic cell reprogramming. Distinct patterns of gene expression dynamics of these factors during reprogramming suggest that further improvements in efficiency could be obtained through optimal timing of overexpression or knockdown of individual regulators. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genome-wide mapping of alternative splicing in Arabidopsis thaliana

PubMed Central

Filichkin, Sergei A.; Priest, Henry D.; Givan, Scott A.; Shen, Rongkun; Bryant, Douglas W.; Fox, Samuel E.; Wong, Weng-Keen; Mockler, Todd C.

2010-01-01

Alternative splicing can enhance transcriptome plasticity and proteome diversity. In plants, alternative splicing can be manifested at different developmental stages, and is frequently associated with specific tissue types or environmental conditions such as abiotic stress. We mapped the Arabidopsis transcriptome at single-base resolution using the Illumina platform for ultrahigh-throughput RNA sequencing (RNA-seq). Deep transcriptome sequencing confirmed a majority of annotated introns and identified thousands of novel alternatively spliced mRNA isoforms. Our analysis suggests that at least ∼42% of intron-containing genes in Arabidopsis are alternatively spliced; this is significantly higher than previous estimates based on cDNA/expressed sequence tag sequencing. Random validation confirmed that novel splice isoforms empirically predicted by RNA-seq can be detected in vivo. Novel introns detected by RNA-seq were substantially enriched in nonconsensus terminal dinucleotide splice signals. Alternative isoforms with premature termination codons (PTCs) comprised the majority of alternatively spliced transcripts. Using an example of an essential circadian clock gene, we show that intron retention can generate relatively abundant PTC+ isoforms and that this specific event is highly conserved among diverse plant species. Alternatively spliced PTC+ isoforms can be potentially targeted for degradation by the nonsense mediated mRNA decay (NMD) surveillance machinery or regulate the level of functional transcripts by the mechanism of regulated unproductive splicing and translation (RUST). We demonstrate that the relative ratios of the PTC+ and reference isoforms for several key regulatory genes can be considerably shifted under abiotic stress treatments. Taken together, our results suggest that like in animals, NMD and RUST may be widespread in plants and may play important roles in regulating gene expression. PMID:19858364

Engineering the expression level of cytosolic nucleoside diphosphate kinase in transgenic Solanum tuberosum roots alters growth, respiration and carbon metabolism.

PubMed

Dorion, Sonia; Clendenning, Audrey; Rivoal, Jean

2017-03-01

Nucleoside diphosphate kinase (NDPK) is a ubiquitous enzyme that catalyzes the transfer of the γ-phosphate from a donor nucleoside triphosphate to an acceptor nucleoside diphosphate. In this study we used a targeted metabolomic approach and measurement of physiological parameters to report the effects of the genetic manipulation of cytosolic NDPK (NDPK1) expression on physiology and carbon metabolism in potato (Solanum tuberosum) roots. Sense and antisense NDPK1 constructs were introduced in potato using Agrobacterium rhizogenes to generate a population of root clones displaying a 40-fold difference in NDPK activity. Root growth, O 2 uptake, flux of carbon between sucrose and CO 2 , levels of reactive oxygen species and some tricarboxylic acid cycle intermediates were positively correlated with levels of NDPK1 expression. In addition, NDPK1 levels positively affected UDP-glucose and cellulose contents. The activation state of ADP-glucose pyrophosphorylase, a key enzyme in starch synthesis, was higher in antisense roots than in roots overexpressing NDPK1. Further analyses demonstrated that ADP-glucose pyrophosphorylase was more oxidized, and therefore less active, in sense clones than antisense clones. Consequently, antisense NDPK1 roots accumulated more starch and the starch to cellulose ratio was negatively affected by the level of NDPK1. These data support the idea that modulation of NDPK1 affects the distribution of carbon between starch and cellulose biosynthetic pathways. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels

PubMed Central

Scholl, Elizabeth Storer; Pirone, Antonella; Cox, Daniel H; Duncan, R Keith; Jacob, Michele H

2014-01-01

Small conductance Ca2+-sensitive potassium (SK2) channels are voltage-independent, Ca2+-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca2+ permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3′ terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca2+ and Ca2+-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca2+ influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses. PMID:24394769

A novel splice variant of the Fas gene in patients with cutaneous T-cell lymphoma.

PubMed

van Doorn, Remco; Dijkman, Remco; Vermeer, Maarten H; Starink, Theo M; Willemze, Rein; Tensen, Cornelis P

2002-10-01

Defective apoptosis signaling has been implicated in the pathogenesis of primary cutaneous T-cell lymphomas (CTCLs), a group of malignancies derived from skin-homing T cells. An important mediator of apoptosis in T cells is the Fas receptor. We identified a novel splice variant of the Fas gene that displays retention of intron 5 and encodes a dysfunctional Fas protein in 13 of 22 patients (59%) in both early and advanced CTCL. Impairment of Fas-induced apoptosis resulting from aberrant splicing potentially contributes to the development and progression of CTCL by allowing continued clonal expansion of activated T cells and by reducing susceptibility to antitumor immune responses.

Genome-Wide Survey of Cold Stress Regulated Alternative Splicing in Arabidopsis thaliana with Tiling Microarray

PubMed Central

Leviatan, Noam; Alkan, Noam; Leshkowitz, Dena; Fluhr, Robert

2013-01-01

Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Alternative splicing is often regulated in a tissue-specific and stress-responsive manner. Cold stress, which adversely affects plant growth and development, regulates the transcription and splicing of plant splicing factors. This can affect the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions. A novel algorithm was used for detection of statistically relevant changes in intron expression within a transcript between control and cold growth conditions. A reverse transcription polymerase chain reaction (RT-PCR) analysis of a number of randomly selected genes confirmed the changes in splicing patterns under cold stress predicted by tiling array. Our analysis revealed new types of cold responsive genes. While their expression level remains relatively unchanged under cold stress their splicing pattern shows detectable changes in the relative abundance of isoforms. The majority of cold regulated alternative splicing introduced a premature termination codon (PTC) into the transcripts creating potential targets for degradation by the nonsense mediated mRNA decay (NMD) process. A number of these genes were analyzed in NMD-defective mutants by RT-PCR and shown to evade NMD. This may result in new and truncated proteins with altered functions or dominant negative effects. The results indicate that cold affects both quantitative and qualitative aspects of gene expression. PMID:23776682

Misregulation of Alternative Splicing in a Mouse Model of Rett Syndrome

PubMed Central

Li, Ronghui; Dong, Qiping; Yuan, Xinni; Zeng, Xin; Gao, Yu; Li, Hongda; Keles, Sunduz; Wang, Zefeng; Chang, Qiang

2016-01-01

Mutations in the human MECP2 gene cause Rett syndrome (RTT), a severe neurodevelopmental disorder that predominantly affects girls. Despite decades of work, the molecular function of MeCP2 is not fully understood. Here we report a systematic identification of MeCP2-interacting proteins in the mouse brain. In addition to transcription regulators, we found that MeCP2 physically interacts with several modulators of RNA splicing, including LEDGF and DHX9. These interactions are disrupted by RTT causing mutations, suggesting that they may play a role in RTT pathogenesis. Consistent with the idea, deep RNA sequencing revealed misregulation of hundreds of splicing events in the cortex of Mecp2 knockout mice. To reveal the functional consequence of altered RNA splicing due to the loss of MeCP2, we focused on the regulation of the splicing of the flip/flop exon of Gria2 and other AMPAR genes. We found a significant splicing shift in the flip/flop exon toward the flop inclusion, leading to a faster decay in the AMPAR gated current and altered synaptic transmission. In summary, our study identified direct physical interaction between MeCP2 and splicing factors, a novel MeCP2 target gene, and established functional connection between a specific RNA splicing change and synaptic phenotypes in RTT mice. These results not only help our understanding of the molecular function of MeCP2, but also reveal potential drug targets for future therapies. PMID:27352031

The splicing regulator Rbfox1 (A2BP1) controls neuronal excitation in the mammalian brain

PubMed Central

Gehman, Lauren T.; Stoilov, Peter; Maguire, Jamie; Damianov, Andrey; Lin, Chia-Ho; Shiue, Lily; Ares, Manuel; Mody, Istvan; Black, Douglas L.

2011-01-01

The Rbfox family of RNA binding proteins regulates alternative splicing of many important neuronal transcripts but their role in neuronal physiology is not clear1. We show here that central nervous system (CNS)-specific deletion of the Rbfox1 gene results in heightened susceptibility to spontaneous and kainic acid-induced seizures. Electrophysiological recording reveals a corresponding increase in neuronal excitability in the dentate gyrus of the knockout mice. Whole transcriptome analyses identify multiple splicing changes in the Rbfox1−/− brain with few changes in overall transcript abundance. These splicing changes alter proteins that mediate synaptic transmission and membrane excitation, some of which are implicated in human epilepsy. Thus, Rbfox1 directs a genetic program required in the prevention of neuronal hyperexcitation and seizures. The Rbfox1 knockout mice provide a new model to study the post-transcriptional regulation of synaptic function. PMID:21623373

The RNA recognition motif domains of RBM5 are required for RNA binding and cancer cell proliferation inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Lei; Zhang, Qing; Yang, Yu

Highlights: • RNA recognition motif domains of RBM5 are essential for cell proliferation inhibition. • RNA recognition motif domains of RBM5 are essential for apoptosis induction. • RNA recognition motif domains of RBM5 are essential for RNA binding. • RNA recognition motif domains of RBM5 are essential for caspase-2 alternative splicing. - Abstract: RBM5 is a known putative tumor suppressor gene that has been shown to function in cell growth inhibition by modulating apoptosis. RBM5 also plays a critical role in alternative splicing as an RNA binding protein. However, it is still unclear which domains of RBM5 are required formore » RNA binding and related functional activities. We hypothesized the two putative RNA recognition motif (RRM) domains of RBM5 spanning from amino acids 98–178 and 231–315 are essential for RBM5-mediated cell growth inhibition, apoptosis regulation, and RNA binding. To investigate this hypothesis, we evaluated the activities of the wide-type and mutant RBM5 gene transfer in low-RBM5 expressing A549 cells. We found that, unlike wild-type RBM5 (RBM5-wt), a RBM5 mutant lacking the two RRM domains (RBM5-ΔRRM), is unable to bind RNA, has compromised caspase-2 alternative splicing activity, lacks cell proliferation inhibition and apoptosis induction function in A549 cells. These data provide direct evidence that the two RRM domains of RBM5 are required for RNA binding and the RNA binding activity of RBM5 contributes to its function on apoptosis induction and cell growth inhibition.« less

Morpholino-mediated Knockdown of DUX4 Toward Facioscapulohumeral Muscular Dystrophy Therapeutics.

PubMed

Chen, Jennifer Cj; King, Oliver D; Zhang, Yuanfan; Clayton, Nicholas P; Spencer, Carrie; Wentworth, Bruce M; Emerson, Charles P; Wagner, Kathryn R

2016-08-01

Derepression of DUX4 in skeletal muscle has emerged as a likely cause of pathology in facioscapulohumeral muscular dystrophy (FSHD). Here we report on the use of antisense phosphorodiamidate morpholino oligonucleotides to suppress DUX4 expression and function in FSHD myotubes and xenografts. The most effective was phosphorodiamidate morpholino oligonucleotide FM10, which targets the polyadenylation signal of DUX4. FM10 had no significant cell toxicity, and RNA-seq analyses of FSHD and control myotubes revealed that FM10 down-regulated many transcriptional targets of DUX4, without overt off-target effects. Electroporation of FM10 into FSHD patient muscle xenografts in mice also down-regulated DUX4 and DUX4 targets. These findings demonstrate the potential of antisense phosphorodiamidate morpholino oligonucleotides as an FSHD therapeutic option.

Modulation of Tau Isoforms Imbalance Precludes Tau Pathology and Cognitive Decline in a Mouse Model of Tauopathy.

PubMed

Espíndola, Sonia Lorena; Damianich, Ana; Alvarez, Rodrigo Javier; Sartor, Manuela; Belforte, Juan Emilio; Ferrario, Juan Esteban; Gallo, Jean-Marc; Avale, María Elena

2018-04-17

The microtubule-associated protein tau regulates myriad neuronal functions, such as microtubule dynamics, axonal transport and neurite outgrowth. Tauopathies are neurodegenerative disorders characterized by the abnormal metabolism of tau, which accumulates as insoluble neuronal deposits. The adult human brain contains equal amounts of tau isoforms with three (3R) or four (4R) repeats of microtubule-binding domains, derived from the alternative splicing of exon 10 (E10) in the tau transcript. Several tauopathies are associated with imbalances of tau isoforms, due to splicing deficits. Here, we used a trans-splicing strategy to shift the inclusion of E10 in a mouse model of tauopathy that produces abnormal excess of 3R tau. Modulating the 3R/4R ratio in the prefrontal cortex led to a significant reduction of pathological tau accumulation concomitant with improvement of neuronal firing and reduction of cognitive impairments. Our results suggest promising potential for the use of RNA reprogramming in human neurodegenerative diseases. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Human DBR1 modulates the recycling of snRNPs to affect alternative RNA splicing and contributes to the suppression of cancer development.

PubMed

Han, B; Park, H K; Ching, T; Panneerselvam, J; Wang, H; Shen, Y; Zhang, J; Li, L; Che, R; Garmire, L; Fei, P

2017-09-21

The contribution of RNA processing to tumorigenesis is understudied. Here, we report that the human RNA debranching enzyme (hDBR1), when inappropriately regulated, induces oncogenesis by causing RNA processing defects, for example, splicing defects. We found that wild-type p53 and hypoxia-inducible factor 1 co-regulate hDBR1 expression, and insufficient hDBR1 leads to a higher rate of exon skipping. Transcriptomic sequencing confirmed the effect of hDBR1 on RNA splicing, and metabolite profiling supported the observation that neoplasm is triggered by a decrease in hDBR1 expression both in vitro and in vivo. Most importantly, when modulating the expression of hDBR1, which was found to be generally low in malignant human tissues, higher expression of hDBR1 only affected exon-skipping activity in malignant cells. Together, our findings demonstrate previously unrecognized regulation and functions of hDBR1, with immediate clinical implications regarding the regulation of hDBR1 as an effective strategy for combating human cancer.

Adenovirus-mediated transfer of HPV 16 E6/E7 antisense RNA combined with cisplatin inhibits cellular growth and induces apoptosis in HPV-positive head and neck cancer cells.

PubMed

Kojima, Yasutaka; Otsuki, Naoki; Kubo, Mie; Kitamoto, Junko; Takata, Eri; Saito, Hiroki; Kosaka, Kyoko; Morishita, Naoya; Uehara, Natsumi; Shirakawa, Toshiro; Nibu, Ken-Ich

2018-05-24

Human papillomavirus (HPV) infection has been identified as an etiologic factor of head and neck cancers (HNCs). We explored the potential use of antisense HPV RNA transcripts for gene therapy and its effect in combination with cisplatin (CDDP) for HPV-positive HNCs. We introduced the antisense RNA transcripts of the E6 and E7 genes of HPV type 16 into UM-SCC-47 cells harboring HPV 16 and YCU-T892 cells that were HPV-negative using a recombinant adenoviral vector, Ad-E6/E7-AS. We then analyzed the effects of the introduction of Ad-E7-AS on cell and tumor growth and the synergistic effect with CDDP in vitro and in vivo. After infection of Ad-E6/E7-AS, the cellular growth of UM-SCC-47 cells were suppressed, but not that of YCU-T892 cells. E7 protein expression was suppressed, and p53 and pRb protein expression increased after infection of Ad-E7-AS. Cell growth and tumorigenicity were greatly suppressed in combination with CDDP compared with Ad-E7-AS or CDDP treatment alone in vitro. Ad-E7-AS combined with CDDP treatment significantly reduced the volumes of established subcutaneous tumors. Transfection with HPV 16 E7 antisense RNA combined with CDDP treatment might be a potentially useful approach to the therapy of HPV 16-positive HNC.

Dissecting the hybridization of oligonucleotides to structured complementary sequences.

PubMed

Peracchi, Alessio

2016-06-01

When oligonucleotides hybridize to long target molecules, the process is slowed by the secondary structure in the targets. The phenomenon has been analyzed in several previous studies, but many details remain poorly understood. I used a spectrofluorometric strategy, focusing on the formation/breaking of individual base pairs, to study the kinetics of association between a DNA hairpin and >20 complementary oligonucleotides ('antisenses'). Hybridization rates differed by over three orders of magnitude. Association was toehold-mediated, both for antisenses binding to the target's ends and for those designed to interact with the loop. Binding of these latter, besides being consistently slower, was affected to variable, non-uniform extents by the asymmetric loop structure. Divalent metal ions accelerated hybridization, more pronouncedly when nucleation occurred at the loop. Incorporation of locked nucleic acid (LNA) residues in the antisenses substantially improved the kinetics only when LNAs participated to the earliest hybridization steps. The effects of individual LNAs placed along the antisense indicated that the reaction transition state occurred after invading at least the first base pair of the stem. The experimental approach helps dissect hybridization reactions involving structured nucleic acids. Toehold-dependent, nucleation-invasion models appear fully appropriate for describing such reactions. Estimating the stability of nucleation complexes formed at internal toeholds is the major hurdle for the quantitative prediction of hybridization rates. While analyzing the mechanisms of a fundamental biochemical process (hybridization), this work also provides suggestions for the improvement of technologies that rely on such process. Copyright © 2016 Elsevier B.V. All rights reserved.

Splicing factor NSSR1 reduces neuronal injury after mouse transient global cerebral ischemia.

PubMed

Qi, Yao; Li, Ya; Cui, Shi-Chao; Zhao, Jing-Jing; Liu, Xiao-Yan; Ji, Chun-Xia; Sun, Feng-Yan; Xu, Ping; Chen, Xian-Hua

2015-05-01

This study focuses on the function of NSSR1, a splicing factor, in neuronal injury in the ischemic mouse brain using the transient global cerebral ischemic mouse model and the cultured cells treated with oxygen-glucose deprivation (OGD). The results showed that the cerebral ischemia triggers the expression of NSSR1 in hippocampal astrocytes, predominantly the dephosphorylated NSSR1 proteins, and the Exon3 inclusive NCAM-L1 variant and the Exon4 inclusive CREB variant. While in the hippocampus of astrocyte-specific NSSR1 conditional knockdown (cKD) mice, where cerebral ischemia no longer triggers NSSR1 expression in astrocytes, the expression of Exon3 inclusive NCAM-L1 variant and Exon4 inclusive CREB variant were no longer triggered as well. In addition, the injury of hippocampal neurons was more severe in astrocyte-specific NSSR1 cKD mice compared with in wild-type mice after brain ischemia. Of note, the culture media harvested from the astrocytes with overexpression of NSSR1 or the Exon3 inclusive NCAM-L1 variant, or Exon4 inclusive CREB variant were all able to reduce the neuronal injury induced by OGD. The results provide the evidence demonstrating that: (1) Splicing factor NSSR1 is a new factor involved in reducing ischemic injury. (2) Ischemia induces NSSR1 expression in astrocytes, not in neurons. (3) NSSR1-mediated pathway in astrocytes is required for reducing ischemic neuronal injury. (4) NCAM-L1 and CREB are probably mediators in NSSR1-mediated pathway. In conclusion, our results suggest for the first time that NSSR1 may provide a novel mechanism for reducing neuronal injury after ischemia, probably through regulation on alternative splicing of NCAM-L1 and CREB in astrocytes. © 2014 Wiley Periodicals, Inc.

Proteasome-Mediated Proteolysis of SRSF5 Splicing Factor Intriguingly Co-occurs with SRSF5 mRNA Upregulation during Late Erythroid Differentiation

PubMed Central

Breig, Osman; Baklouti, Faouzi

2013-01-01

SR proteins exhibit diverse functions ranging from their role in constitutive and alternative splicing, to virtually all aspects of mRNA metabolism. These findings have attracted growing interest in deciphering the regulatory mechanisms that control the tissue-specific expression of these SR proteins. In this study, we show that SRSF5 protein decreases drastically during erythroid cell differentiation, contrasting with a concomitant upregulation of SRSF5 mRNA level. Proteasome chemical inhibition provided strong evidence that endogenous SRSF5 protein, as well as protein deriving from stably transfected SRSF5 cDNA, are both targeted to proteolysis as the cells undergo terminal differentiation. Consistently, functional experiments show that overexpression of SRSF5 enhances a specific endogenous pre-mRNA splicing event in proliferating cells, but not in differentiating cells, due to proteasome-mediated targeting of both endogenous and transfection-derived SRSF5. Further investigation of the relationship between SRSF5 structure and its post-translation regulation and function, suggested that the RNA recognition motifs of SRSF5 are sufficient to activate pre-mRNA splicing, whereas proteasome-mediated proteolysis of SRSF5 requires the presence of the C-terminal RS domain of the protein. Phosphorylation of SR proteins is a key post-translation regulation that promotes their activity and subcellular availability. We here show that inhibition of the CDC2-like kinase (CLK) family and mutation of the AKT phosphorylation site Ser86 on SRSF5, have no effect on SRSF5 stability. We reasoned that at least AKT and CLK signaling pathways are not involved in proteasome-induced turnover of SRSF5 during late erythroid development. PMID:23536862

Structural insights into alternative splicing-mediated desensitization of jasmonate signaling.

PubMed

Zhang, Feng; Ke, Jiyuan; Zhang, Li; Chen, Rongzhi; Sugimoto, Koichi; Howe, Gregg A; Xu, H Eric; Zhou, Mingguo; He, Sheng Yang; Melcher, Karsten

2017-02-14

Jasmonate ZIM-domain (JAZ) transcriptional repressors play a key role in regulating jasmonate (JA) signaling in plants. Below a threshold concentration of jasmonoyl isoleucine (JA-Ile), the active form of JA, the C-terminal Jas motif of JAZ proteins binds MYC transcription factors to repress JA signaling. With increasing JA-Ile concentration, the Jas motif binds to JA-Ile and the COI1 subunit of the SCF COI1 E3 ligase, which mediates ubiquitination and proteasomal degradation of JAZ repressors, resulting in derepression of MYC transcription factors. JA signaling subsequently becomes desensitized, in part by feedback induction of JAZ splice variants that lack the C-terminal Jas motif but include an N-terminal cryptic MYC-interaction domain (CMID). The CMID sequence is dissimilar to the Jas motif and is incapable of recruiting SCF COI1 , allowing CMID-containing JAZ splice variants to accumulate in the presence of JA and to re-repress MYC transcription factors as an integral part of reestablishing signal homeostasis. The mechanism by which the CMID represses MYC transcription factors remains elusive. Here we describe the crystal structure of the MYC3-CMID JAZ10 complex. In contrast to the Jas motif, which forms a single continuous helix when bound to MYC3, the CMID adopts a loop-helix-loop-helix architecture with modular interactions with both the Jas-binding groove and the backside of the Jas-interaction domain of MYC3. This clamp-like interaction allows the CMID to bind MYC3 tightly and block access of MED25 (a subunit of the Mediator coactivator complex) to the MYC3 transcriptional activation domain, shedding light on the enigmatic mechanism by which JAZ splice variants desensitize JA signaling.

Role of the thrombin receptor in restenosis and atherosclerosis.

PubMed

Baykal, D; Schmedtje, J F; Runge, M S

1995-02-23

Thrombus generation is central to thrombosis at vascular lesion sites, including post-PCTA acute reocclusion and chronic restenosis. Thrombin stimulates platelet activation, monocyte and neutrophil chemotaxis, and endothelial production of prothrombotic factors. The varied physiologic effects of thrombin are due to the widespread presence of thrombin receptors in many cell types. The receptor is uniquely activated: thrombin binds to the receptor at the thrombin anion-binding exosite, the receptor ligand ("tethered ligand") apparently being a sequence of 6 amino acids (SFLLRN). Thus, peptides corresponding to the sequence of the tethered ligand can stimulate almost all functions of native thrombin itself. Several intracellular signaling pathways have been identified as important in the restenosis process: the G protein-related pathway, cyclic adenosine monophosphate (cAMP) mediator pathway, and tyrosine kinase activation pathway. In situ hybridization has demonstrated an increase in thrombin receptor mRNA throughout the period of neointimal and vascular lesion development. The mechanism of this increase is unknown, but may be mediated by multiple inflammatory modulators. Several strategies have been tested in animal models for inhibiting thrombin: (1) Hirudin not only prevents thrombin from cleaving fibrinogen, but also prevents thrombin receptor activation. (2) Thrombin receptor antagonist peptides block platelet aggregation effects of thrombin. (3) Mono- and polyclonal antibodies inhibit thrombin receptor activation. (4) Antisense oligonucleotides block thrombin receptor expression.

Differential agonist sensitivity of glycine receptor α2 subunit splice variants

PubMed Central

Miller, Paul S; Harvey, Robert J; Smart, Trevor G

2004-01-01

The glycine receptor (GlyR) α2A and α2B splice variants differ by a dual, adjacent amino acid substitution from α2AV58,T59 to α2BI58,A59 in the N-terminal extracellular domain. Comparing the effects of the GlyR agonists, glycine, β-alanine and taurine, on the GlyR α2 isoforms, revealed a significant increase in potency for all three agonists at the α2B variant. The sensitivities of the splice variants to the competitive antagonist, strychnine, and to the biphasic modulator Zn2+, were comparable. In contrast, the allosteric inhibitor picrotoxin was more potent on GlyR α2A compared to GlyR α2B receptors. Coexpression of α2A or α2B subunits with the GlyR β subunit revealed that the higher agonist potencies observed with the α2B homomer were retained for the α2Bβ heteromer. The identical sensitivity to strychnine combined with a reduction in the maximum current induced by the partial agonist taurine at the GlyR α2A homomer, suggested that the changed sensitivity to agonists is in accordance with a modulation of agonist efficacy rather than agonist affinity. An effect on agonist efficacy was also supported by using a structural model of the GlyR, localising the region of splice variation to the proposed docking region between GlyR loop 2 and the TM2-3 loop, an area associated with channel activation. The existence of a spasmodic mouse phenotype linked to a GlyR α1A52S mutation, the equivalent position to the source of the α2 splice variation, raises the possibility that the GlyR α2 splice variants may be responsible for distinct roles in neuronal function. PMID:15302677

An Alternative Splice Product of IκB Kinase (IKKγ), IKKγ-Δ, Differentially Mediates Cytokine and Human T-Cell Leukemia Virus Type 1 Tax-Induced NF-κB Activation

PubMed Central

Hai, Tao; Yeung, Man-Lung; Wood, Thomas G.; Wei, Yuanfen; Yamaoka, Shoji; Gatalica, Zoran; Jeang, Kuan-Teh; Brasier, Allan R.

2006-01-01

NF-κB is an inducible transcription factor mediating innate immune responses whose activity is controlled by the multiprotein IκB kinase (IKK) “signalsome”. The core IKK consists of two catalytic serine kinases, IKKα and IKKβ, and a noncatalytic subunit, IKKγ. IKKγ is required for IKK activity by mediating kinase oligomerization and serving to couple the core catalytic subunits to upstream mitogen-activated protein 3-kinase cascades. We have discovered an alternatively spliced IKKγ mRNA isoform, encoding an in-frame deletion of exon 5, termed IKKγ-Δ. Using a specific reverse transcription-PCR assay, we find that IKKγ-Δ is widely expressed in cultured human cells and normal human tissues. Because IKKγ-Δ protein is lacking a critical coiled-coil domain important in protein-protein interactions, we sought to determine its signaling properties by examining its ability to self associate, couple to activators of the canonical pathway, and mediate human T-cell leukemia virus type 1 (HTLV-1) Tax-induced NF-κB activity. Coimmunoprecipitation and confocal colocalization assays indicate IKKγ-Δ has strong homo- and heterotypic association with wild-type (WT) IKKγ and, like IKKγ WT, associates with the IKKβ kinase. Similarly, IKKγ-Δ mediates IKK kinase activity and downstream NF-κB-dependent transcription in response to tumor necrosis factor (TNF) and the NF-κB-inducing kinase-IKKα signaling pathway. Surprisingly, however, in contrast to IKKγ WT, IKKγ-Δ is not able to mediate HTLV-1 Tax-induced NF-κB-dependent transcription, even though IKKγ-Δ binds and colocalizes with Tax. These observations suggest that IKKγ-Δ is a functionally distinct alternatively spliced mRNA product differentially mediating TNF-induced, but not Tax-induced, signals converging on the IKK signalsome. Differing levels of IKKγ-Δ expression, therefore, may affect signal transduction cascades coupling to IKK. PMID:16611882

Evidence for a major role of antisense RNAs in cyanobacterial gene regulation

PubMed Central

Georg, Jens; Voß, Björn; Scholz, Ingeborg; Mitschke, Jan; Wilde, Annegret; Hess, Wolfgang R

2009-01-01

Information on the numbers and functions of naturally occurring antisense RNAs (asRNAs) in eubacteria has thus far remained incomplete. Here, we screened the model cyanobacterium Synechocystis sp. PCC 6803 for asRNAs using four different methods. In the final data set, the number of known noncoding RNAs rose from 6 earlier identified to 60 and of asRNAs from 1 to 73 (28 were verified using at least three methods). Among these, there are many asRNAs to housekeeping, regulatory or metabolic genes, as well as to genes encoding electron transport proteins. Transferring cultures to high light, carbon-limited conditions or darkness influenced the expression levels of several asRNAs, suggesting their functional relevance. Examples include the asRNA to rpl1, which accumulates in a light-dependent manner and may be required for processing the L11 r-operon and the SyR7 noncoding RNA, which is antisense to the murF 5′ UTR, possibly modulating murein biosynthesis. Extrapolated to the whole genome, ∼10% of all genes in Synechocystis are influenced by asRNAs. Thus, chromosomally encoded asRNAs may have an important function in eubacterial regulatory networks. PMID:19756044

Evidence for a major role of antisense RNAs in cyanobacterial gene regulation.

PubMed

Georg, Jens; Voss, Björn; Scholz, Ingeborg; Mitschke, Jan; Wilde, Annegret; Hess, Wolfgang R

2009-01-01

Information on the numbers and functions of naturally occurring antisense RNAs (asRNAs) in eubacteria has thus far remained incomplete. Here, we screened the model cyanobacterium Synechocystis sp. PCC 6803 for asRNAs using four different methods. In the final data set, the number of known noncoding RNAs rose from 6 earlier identified to 60 and of asRNAs from 1 to 73 (28 were verified using at least three methods). Among these, there are many asRNAs to housekeeping, regulatory or metabolic genes, as well as to genes encoding electron transport proteins. Transferring cultures to high light, carbon-limited conditions or darkness influenced the expression levels of several asRNAs, suggesting their functional relevance. Examples include the asRNA to rpl1, which accumulates in a light-dependent manner and may be required for processing the L11 r-operon and the SyR7 noncoding RNA, which is antisense to the murF 5' UTR, possibly modulating murein biosynthesis. Extrapolated to the whole genome, approximately 10% of all genes in Synechocystis are influenced by asRNAs. Thus, chromosomally encoded asRNAs may have an important function in eubacterial regulatory networks.

Glucose-dependent insulinotropic peptide stimulates thymidine incorporation in endothelial cells: role of endothelin-1

NASA Technical Reports Server (NTRS)

Ding, Ke-Hong; Zhong, Qing; Isales, Carlos M.; Iscules, C. M. (Principal Investigator)

2003-01-01

We have previously characterized the receptor for glucose-dependent insulinotropic polypeptide (GIPR) in vascular endothelial cells (EC). Different EC types were found to contain distinct GIPR splice variants. To determine whether activation of the GIPR splice variants resulted in different cellular responses, we examined GIP effects on human umbilical vein endothelial cells (HUVEC), which contain two GIPR splice variants, and compared them with a spontaneously transformed human umbilical vein EC line, ECV 304, which contains four GIPR splice variants. GIP dose-dependently stimulated HUVEC and ECV 304 proliferation as measured by [3H]thymidine incorporation. GIP increased endothelin-1 (ET-1) secretion from HUVEC but not from ECV 304. Use of the endothelin B receptor blocker BQ-788 resulted in an inhibition of [3H]thymidine incorporation in HUVEC but not in ECV 304. These findings suggest that, although GIP increases [3H]thymidine incorporation in both HUVEC and ECV 304, this proliferative response is mediated by ET-1 only in HUVEC. These differences in cellular response to GIP may be related to differences in activation of GIPR splice variants.

Lineage-specific splicing of a brain-enriched alternative exon promotes glioblastoma progression

PubMed Central

Ferrarese, Roberto; Harsh, Griffith R.; Yadav, Ajay K.; Bug, Eva; Maticzka, Daniel; Reichardt, Wilfried; Dombrowski, Stephen M.; Miller, Tyler E.; Masilamani, Anie P.; Dai, Fangping; Kim, Hyunsoo; Hadler, Michael; Scholtens, Denise M.; Yu, Irene L.Y.; Beck, Jürgen; Srinivasasainagendra, Vinodh; Costa, Fabrizio; Baxan, Nicoleta; Pfeifer, Dietmar; von Elverfeldt, Dominik; Backofen, Rolf; Weyerbrock, Astrid; Duarte, Christine W.; He, Xiaolin; Prinz, Marco; Chandler, James P.; Vogel, Hannes; Chakravarti, Arnab; Rich, Jeremy N.; Carro, Maria S.; Bredel, Markus

2014-01-01

Tissue-specific alternative splicing is critical for the emergence of tissue identity during development, yet the role of this process in malignant transformation is undefined. Tissue-specific splicing involves evolutionarily conserved, alternative exons that represent only a minority of the total alternative exons identified. Many of these conserved exons have functional features that influence signaling pathways to profound biological effect. Here, we determined that lineage-specific splicing of a brain-enriched cassette exon in the membrane-binding tumor suppressor annexin A7 (ANXA7) diminishes endosomal targeting of the EGFR oncoprotein, consequently enhancing EGFR signaling during brain tumor progression. ANXA7 exon splicing was mediated by the ribonucleoprotein PTBP1, which is normally repressed during neuronal development. PTBP1 was highly expressed in glioblastomas due to loss of a brain-enriched microRNA (miR-124) and to PTBP1 amplification. The alternative ANXA7 splicing trait was present in precursor cells, suggesting that glioblastoma cells inherit the trait from a potential tumor-initiating ancestor and that these cells exploit this trait through accumulation of mutations that enhance EGFR signaling. Our data illustrate that lineage-specific splicing of a tissue-regulated alternative exon in a constituent of an oncogenic pathway eliminates tumor suppressor functions and promotes glioblastoma progression. This paradigm may offer a general model as to how tissue-specific regulatory mechanisms can reprogram normal developmental processes into oncogenic ones. PMID:24865424

Evidence that C9ORF72 Dipeptide Repeat Proteins Associate with U2 snRNP to Cause Mis-splicing in ALS/FTD Patients.

PubMed

Yin, Shanye; Lopez-Gonzalez, Rodrigo; Kunz, Ryan C; Gangopadhyay, Jaya; Borufka, Carl; Gygi, Steven P; Gao, Fen-Biao; Reed, Robin

2017-06-13

Hexanucleotide repeat expansion in the C9ORF72 gene results in production of dipeptide repeat (DPR) proteins that may disrupt pre-mRNA splicing in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. At present, the mechanisms underlying this mis-splicing are not understood. Here, we show that addition of proline-arginine (PR) and glycine-arginine (GR) toxic DPR peptides to nuclear extracts blocks spliceosome assembly and splicing, but not other types of RNA processing. Proteomic and biochemical analyses identified the U2 small nuclear ribonucleoprotein particle (snRNP) as a major interactor of PR and GR peptides. In addition, U2 snRNP, but not other splicing factors, mislocalizes from the nucleus to the cytoplasm both in C9ORF72 patient induced pluripotent stem cell (iPSC)-derived motor neurons and in HeLa cells treated with the toxic peptides. Bioinformatic studies support a specific role for U2-snRNP-dependent mis-splicing in C9ORF72 patient brains. Together, our data indicate that DPR-mediated dysfunction of U2 snRNP could account for as much as ∼44% of the mis-spliced cassette exons in C9ORF72 patient brains. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Drug discovery with an RBM20 dependent titin splice reporter identifies cardenolides as lead structures to improve cardiac filling.

PubMed

Liss, Martin; Radke, Michael H; Eckhard, Jamina; Neuenschwander, Martin; Dauksaite, Vita; von Kries, Jens-Peter; Gotthardt, Michael

2018-01-01

Diastolic dysfunction is increasingly prevalent in our ageing society and an important contributor to heart failure. The giant protein titin could serve as a therapeutic target, as its elastic properties are a main determinant of cardiac filling in diastole. This study aimed to develop a high throughput pharmacological screen to identify small molecules that affect titin isoform expression through differential inclusion of exons encoding the elastic PEVK domains. We used a dual luciferase splice reporter assay that builds on the titin splice factor RBM20 to screen ~34,000 small molecules and identified several compounds that inhibit the exclusion of PEVK exons. These compounds belong to the class of cardenolides and affect RBM20 dependent titin exon exclusion but did not affect RBFOX1 mediated splicing of FMNL3. We provide evidence that cardenolides do not bind to the RNA interacting domain of RBM20, but reduce RBM20 protein levels and alter transcription of select splicing factors that interact with RBM20. Cardenolides affect titin isoform expression. Understanding their mode of action and harnessing the splice effects through chemical modifications that suppress the effects on ion homeostasis and more selectively affect cardiac splicing has the potential to improve cardiac filling and thus help patients with diastolic heart failure, for which currently no targeted therapy exists.

Nuclear poly(A)-binding protein aggregates misplace a pre-mRNA outside of SC35 speckle causing its abnormal splicing

PubMed Central

Klein, Pierre; Oloko, Martine; Roth, Fanny; Montel, Valérie; Malerba, Alberto; Jarmin, Susan; Gidaro, Teresa; Popplewell, Linda; Perie, Sophie; Lacau St Guily, Jean; de la Grange, Pierre; Antoniou, Michael N.; Dickson, George; Butler-Browne, Gillian; Bastide, Bruno; Mouly, Vincent; Trollet, Capucine

2016-01-01

A short abnormal polyalanine expansion in the polyadenylate-binding protein nuclear-1 (PABPN1) protein causes oculopharyngeal muscular dystrophy (OPMD). Mutated PABPN1 proteins accumulate as insoluble intranuclear aggregates in muscles of OPMD patients. While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice have been established, the molecular mechanisms which trigger pathological defects in OPMD and the role of aggregates remain to be determined. Using exon array, for the first time we have identified several splicing defects in OPMD. In particular, we have demonstrated a defect in the splicing regulation of the muscle-specific Troponin T3 (TNNT3) mutually exclusive exons 16 and 17 in OPMD samples compared to controls. This splicing defect is directly linked to the SC35 (SRSF2) splicing factor and to the presence of nuclear aggregates. As reported here, PABPN1 aggregates are able to trap TNNT3 pre-mRNA, driving it outside nuclear speckles, leading to an altered SC35-mediated splicing. This results in a decreased calcium sensitivity of muscle fibers, which could in turn plays a role in muscle pathology. We thus report a novel mechanism of alternative splicing deregulation that may play a role in various other diseases with nuclear inclusions or foci containing an RNA binding protein. PMID:27507886

Reflections on protein splicing: structures, functions and mechanisms

PubMed Central

Anraku, Yasuhiro; Satow, Yoshinori

2009-01-01

Twenty years ago, evidence that one gene produces two enzymes via protein splicing emerged from structural and expression studies of the VMA1 gene in Saccharomyces cerevisiae. VMA1 consists of a single open reading frame and contains two independent genetic information for Vma1p (a catalytic 70-kDa subunit of the vacuolar H+-ATPase) and VDE (a 50-kDa DNA endonuclease) as an in-frame spliced insert in the gene. Protein splicing is a posttranslational cellular process, in which an intervening polypeptide termed as the VMA1 intein is self-catalytically excised out from a nascent 120-kDa VMA1 precursor and two flanking polypeptides of the N- and C-exteins are ligated to produce the mature Vma1p. Subsequent studies have demonstrated that protein splicing is not unique to the VMA1 precursor and there are many operons in nature, which implement genetic information editing at protein level. To elucidate its structure-directed chemical mechanisms, a series of biochemical and crystal structural studies has been carried out with the use of various VMA1 recombinants. This article summarizes a VDE-mediated self-catalytic mechanism for protein splicing that is triggered and terminated solely via thiazolidine intermediates with tetrahedral configurations formed within the splicing sites where proton ingress and egress are driven by balanced protonation and deprotonation. PMID:19907126

Achieving large dynamic range control of gene expression with a compact RNA transcription–translation regulator

PubMed Central

2017-01-01

Abstract RNA transcriptional regulators are emerging as versatile components for genetic network construction. However, these regulators suffer from incomplete repression in their OFF state, making their dynamic range less than that of their protein counterparts. This incomplete repression causes expression leak, which impedes the construction of larger synthetic regulatory networks as leak propagation can interfere with desired network function. To address this, we demonstrate how naturally derived antisense RNA-mediated transcriptional regulators can be configured to regulate both transcription and translation in a single compact RNA mechanism that functions in Escherichia coli. Using in vivo gene expression assays, we show that a combination of transcriptional termination and ribosome binding site sequestration increases repression from 85% to 98%, or activation from 10-fold to over 900-fold, in response to cognate antisense RNAs. We also show that orthogonal repressive versions of this mechanism can be created through engineering minimal antisense RNAs. Finally, to demonstrate the utility of this mechanism, we use it to reduce network leak in an RNA-only cascade. We anticipate these regulators will find broad use as synthetic biology moves beyond parts engineering to the design and construction of more sophisticated regulatory networks. PMID:28387839

Evidence that "brain-specific" FOX-1, FOX-2, and nPTB alternatively spliced isoforms are produced in the lens.

PubMed

Bitel, Claudine L; Nathan, Rachel; Wong, Patrick; Kuppasani, Sunil; Matsushita, Masafumi; Kanazawa, Hrioshi; Frederikse, Peter H

2011-04-01

Alternative RNA splicing is essential in development and more rapid physiological processes that include disease mechanisms. Studies over the last 20 years demonstrated that RNA binding protein families, which mediate the alternative splicing of a large percentage of genes in mammals, contain isoforms with mutually exclusive expression in non-neural and neural progenitor cells vs. post-mitotic neurons, and regulate the comprehensive reprogramming of alternative splicing during neurogenesis. Polypyrimidine tract binding (PTB) proteins and Fox-1 proteins also undergo mutually exclusive alternative splicing in neural and non-neural cells that regulates their tissue-specific expression and splicing activities. Over the past 50 years, striking morphological similarities noted between lens fiber cells and neurons suggested that cell biology processes and gene expression profiles may be shared as well. Here, we examined mouse and rat lenses to determine if alternative splicing of neuronal nPTB and Fox-1/Fox-2 isoforms also occurs in lenses. Immunoblot, immunofluorescence, and RT-PCR were used to examine expression and alternative splicing of transcripts in lens and brain. We demonstrated that exon 10 is predominantly included in nPTB transcripts consistent with nPTB protein in lenses, and that alternatively spliced Fox-1/-2 lens transcripts contain exons that have been considered neuron-specific. We identified a 3' alternative Fox-1 exon in lenses that encodes a nuclear localization signal consistent with its protein distribution detected in fiber cells. Neuronal alternative splicing of kinesin KIF1Bβ2 has been associated with PTB/nPTB and Fox-2, and we found that two 'neuron-specific' exons are also included in lenses. The present study provides evidence that alternative neuronal nPTB and Fox-1/Fox-2 isoforms are also produced in lenses. These findings raise questions regarding the extent these factors contribute to a similar reprogramming of alternative splicing during lens differentiation, and the degree that alternative gene transcripts produced during neurogenesis are also expressed in the lens.

Cold-dependent alternative splicing of a Jumonji C domain-containing gene MtJMJC5 in Medicago truncatula.

PubMed

Shen, Yingfang; Wu, Xiaopei; Liu, Demei; Song, Shengjing; Liu, Dengcai; Wang, Haiqing

2016-05-27

Histone methylation is an epigenetic modification mechanism that regulates gene expression in eukaryotic cells. Jumonji C domain-containing demethylases are involved in removal of methyl groups at lysine or arginine residues. The JmjC domain-only member, JMJ30/JMJD5 of Arabidopsis, is a component of the plant circadian clock. Although some plant circadian clock genes undergo alternative splicing in response to external cues, there is no evidence that JMJ30/JMJD5 is regulated by alternative splicing. In this study, the expression of an Arabidopsis JMJ30/JMJD5 ortholog in Medicago truncatula, MtJMJC5, in response to circadian clock and abiotic stresses were characterized. The results showed that MtJMJC5 oscillates with a circadian rhythm, and undergoes cold specifically induced alternative splicing. The cold-induced alternative splicing could be reversed after ambient temperature returning to the normal. Sequencing results revealed four alternative splicing RNA isoforms including a full-length authentic protein encoding variant, and three premature termination condon-containing variants due to alternative 3' splice sites at the first and second intron. Under cold treatment, the variants that share a common 3' alternative splicing site at the second intron were intensively up-regulated while the authentic protein encoding variant and the premature termination condon-containing variant only undergoing a 3' alternative splicing at the first intron were down regulated. Although all the premature termination condon-harboring alternative splicing variants were sensitive to nonsense-mediated decay, the premature termination codon-harboring alternative splicing variants sharing the 3' alternative splicing site at the second intron showed less sensitivity than the one only containing the 3' alternative slicing site at the first intron under cold treatment. These results suggest that the cold-dependent alternative splicing of MtJMJC5 is likely a species or genus-specific mechanism of gene expression regulation on RNA levels, and might play a role in epigenetic regulation of the link between the circadian clock and ambient temperature fluctuation in Medicago. Copyright © 2016 Elsevier Inc. All rights reserved.

RNA splicing and splicing regulator changes in prostate cancer pathology.

PubMed

Munkley, Jennifer; Livermore, Karen; Rajan, Prabhakar; Elliott, David J

2017-09-01

Changes in mRNA splice patterns have been associated with key pathological mechanisms in prostate cancer progression. The androgen receptor (abbreviated AR) transcription factor is a major driver of prostate cancer pathology and activated by androgen steroid hormones. Selection of alternative promoters by the activated AR can critically alter gene function by switching mRNA isoform production, including creating a pro-oncogenic isoform of the normally tumour suppressor gene TSC2. A number of androgen-regulated genes generate alternatively spliced mRNA isoforms, including a prostate-specific splice isoform of ST6GALNAC1 mRNA. ST6GALNAC1 encodes a sialyltransferase that catalyses the synthesis of the cancer-associated sTn antigen important for cell mobility. Genetic rearrangements occurring early in prostate cancer development place ERG oncogene expression under the control of the androgen-regulated TMPRSS2 promoter to hijack cell behaviour. This TMPRSS2-ERG fusion gene shows different patterns of alternative splicing in invasive versus localised prostate cancer. Alternative AR mRNA isoforms play a key role in the generation of prostate cancer drug resistance, by providing a mechanism through which prostate cancer cells can grow in limited serum androgen concentrations. A number of splicing regulator proteins change expression patterns in prostate cancer and may help drive key stages of disease progression. Up-regulation of SRRM4 establishes neuronal splicing patterns in neuroendocrine prostate cancer. The splicing regulators Sam68 and Tra2β increase expression in prostate cancer. The SR protein kinase SRPK1 that modulates the activity of SR proteins is up-regulated in prostate cancer and has already given encouraging results as a potential therapeutic target in mouse models.

RNA editing in nascent RNA affects pre-mRNA splicing

PubMed Central

Hsiao, Yun-Hua Esther; Bahn, Jae Hoon; Yang, Yun; Lin, Xianzhi; Tran, Stephen; Yang, Ei-Wen; Quinones-Valdez, Giovanni

2018-01-01

In eukaryotes, nascent RNA transcripts undergo an intricate series of RNA processing steps to achieve mRNA maturation. RNA editing and alternative splicing are two major RNA processing steps that can introduce significant modifications to the final gene products. By tackling these processes in isolation, recent studies have enabled substantial progress in understanding their global RNA targets and regulatory pathways. However, the interplay between individual steps of RNA processing, an essential aspect of gene regulation, remains poorly understood. By sequencing the RNA of different subcellular fractions, we examined the timing of adenosine-to-inosine (A-to-I) RNA editing and its impact on alternative splicing. We observed that >95% A-to-I RNA editing events occurred in the chromatin-associated RNA prior to polyadenylation. We report about 500 editing sites in the 3′ acceptor sequences that can alter splicing of the associated exons. These exons are highly conserved during evolution and reside in genes with important cellular function. Furthermore, we identified a second class of exons whose splicing is likely modulated by RNA secondary structures that are recognized by the RNA editing machinery. The genome-wide analyses, supported by experimental validations, revealed remarkable interplay between RNA editing and splicing and expanded the repertoire of functional RNA editing sites. PMID:29724793

RNA editing in nascent RNA affects pre-mRNA splicing.

PubMed

Hsiao, Yun-Hua Esther; Bahn, Jae Hoon; Yang, Yun; Lin, Xianzhi; Tran, Stephen; Yang, Ei-Wen; Quinones-Valdez, Giovanni; Xiao, Xinshu

2018-06-01

In eukaryotes, nascent RNA transcripts undergo an intricate series of RNA processing steps to achieve mRNA maturation. RNA editing and alternative splicing are two major RNA processing steps that can introduce significant modifications to the final gene products. By tackling these processes in isolation, recent studies have enabled substantial progress in understanding their global RNA targets and regulatory pathways. However, the interplay between individual steps of RNA processing, an essential aspect of gene regulation, remains poorly understood. By sequencing the RNA of different subcellular fractions, we examined the timing of adenosine-to-inosine (A-to-I) RNA editing and its impact on alternative splicing. We observed that >95% A-to-I RNA editing events occurred in the chromatin-associated RNA prior to polyadenylation. We report about 500 editing sites in the 3' acceptor sequences that can alter splicing of the associated exons. These exons are highly conserved during evolution and reside in genes with important cellular function. Furthermore, we identified a second class of exons whose splicing is likely modulated by RNA secondary structures that are recognized by the RNA editing machinery. The genome-wide analyses, supported by experimental validations, revealed remarkable interplay between RNA editing and splicing and expanded the repertoire of functional RNA editing sites. © 2018 Hsiao et al.; Published by Cold Spring Harbor Laboratory Press.

Physical and Functional Interaction of NCX1 and EAAC1 Transporters Leading to Glutamate-Enhanced ATP Production in Brain Mitochondria

PubMed Central

Arcangeli, Sara; Nasti, Annamaria Assunta; Giordano, Antonio; Amoroso, Salvatore

2012-01-01

Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na+-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production. PMID:22479505

Functions for fission yeast splicing factors SpSlu7 and SpPrp18 in alternative splice-site choice and stress-specific regulated splicing.

PubMed

Melangath, Geetha; Sen, Titash; Kumar, Rakesh; Bawa, Pushpinder; Srinivasan, Subha; Vijayraghavan, Usha

2017-01-01

Budding yeast spliceosomal factors ScSlu7 and ScPrp18 interact and mediate intron 3'ss choice during second step pre-mRNA splicing. The fission yeast genome with abundant multi-intronic transcripts, degenerate splice signals and SR proteins is an apt unicellular fungal model to deduce roles for core spliceosomal factors in alternative splice-site choice, intron retention and to study the cellular implications of regulated splicing. From our custom microarray data we deduce a stringent reproducible subset of S. pombe alternative events. We examined the role of factors SpSlu7 or SpPrp18 for these splice events and investigated the relationship to growth phase and stress. Wild-type log and stationary phase cells showed ats1+ exon 3 skipped and intron 3 retained transcripts. Interestingly the non-consensus 5'ss in ats1+ intron 3 caused SpSlu7 and SpPrp18 dependent intron retention. We validated the use of an alternative 5'ss in dtd1+ intron 1 and of an upstream alternative 3'ss in DUF3074 intron 1. The dtd1+ intron 1 non-canonical 5'ss yielded an alternative mRNA whose levels increased in stationary phase. Utilization of dtd1+ intron 1 sub-optimal 5' ss required functional SpPrp18 and SpSlu7 while compromise in SpSlu7 function alone hampered the selection of the DUF3074 intron 1 non canonical 3'ss. We analysed the relative abundance of these splice isoforms during mild thermal, oxidative and heavy metal stress and found stress-specific splice patterns for ats1+ and DUF3074 intron 1 some of which were SpSlu7 and SpPrp18 dependent. By studying ats1+ splice isoforms during compromised transcription elongation rates in wild-type, spslu7-2 and spprp18-5 mutant cells we found dynamic and intron context-specific effects in splice-site choice. Our work thus shows the combinatorial effects of splice site strength, core splicing factor functions and transcription elongation kinetics to dictate alternative splice patterns which in turn serve as an additional recourse of gene regulation in fission yeast.

Functions for fission yeast splicing factors SpSlu7 and SpPrp18 in alternative splice-site choice and stress-specific regulated splicing

PubMed Central

Kumar, Rakesh; Bawa, Pushpinder; Srinivasan, Subha

2017-01-01

Budding yeast spliceosomal factors ScSlu7 and ScPrp18 interact and mediate intron 3’ss choice during second step pre-mRNA splicing. The fission yeast genome with abundant multi-intronic transcripts, degenerate splice signals and SR proteins is an apt unicellular fungal model to deduce roles for core spliceosomal factors in alternative splice-site choice, intron retention and to study the cellular implications of regulated splicing. From our custom microarray data we deduce a stringent reproducible subset of S. pombe alternative events. We examined the role of factors SpSlu7 or SpPrp18 for these splice events and investigated the relationship to growth phase and stress. Wild-type log and stationary phase cells showed ats1+ exon 3 skipped and intron 3 retained transcripts. Interestingly the non-consensus 5’ss in ats1+ intron 3 caused SpSlu7 and SpPrp18 dependent intron retention. We validated the use of an alternative 5’ss in dtd1+ intron 1 and of an upstream alternative 3’ss in DUF3074 intron 1. The dtd1+ intron 1 non-canonical 5’ss yielded an alternative mRNA whose levels increased in stationary phase. Utilization of dtd1+ intron 1 sub-optimal 5’ ss required functional SpPrp18 and SpSlu7 while compromise in SpSlu7 function alone hampered the selection of the DUF3074 intron 1 non canonical 3’ss. We analysed the relative abundance of these splice isoforms during mild thermal, oxidative and heavy metal stress and found stress-specific splice patterns for ats1+ and DUF3074 intron 1 some of which were SpSlu7 and SpPrp18 dependent. By studying ats1+ splice isoforms during compromised transcription elongation rates in wild-type, spslu7-2 and spprp18-5 mutant cells we found dynamic and intron context-specific effects in splice-site choice. Our work thus shows the combinatorial effects of splice site strength, core splicing factor functions and transcription elongation kinetics to dictate alternative splice patterns which in turn serve as an additional recourse of gene regulation in fission yeast. PMID:29236736

Loss of nuclear TDP-43 in amyotrophic lateral sclerosis (ALS) causes altered expression of splicing machinery and widespread dysregulation of RNA splicing in motor neurones.

PubMed

Highley, J Robin; Kirby, Janine; Jansweijer, Joeri A; Webb, Philip S; Hewamadduma, Channa A; Heath, Paul R; Higginbottom, Adrian; Raman, Rohini; Ferraiuolo, Laura; Cooper-Knock, Johnathan; McDermott, Christopher J; Wharton, Stephen B; Shaw, Pamela J; Ince, Paul G

2014-10-01

Loss of nuclear TDP-43 characterizes sporadic and most familial forms of amyotrophic lateral sclerosis (ALS). TDP-43 (encoded by TARDBP) has multiple roles in RNA processing. We aimed to determine whether (1) RNA splicing dysregulation is present in lower motor neurones in ALS and in a motor neurone-like cell model; and (2) TARDBP mutations (mtTARDBP) are associated with aberrant RNA splicing using patient-derived fibroblasts. Affymetrix exon arrays were used to study mRNA expression and splicing in lower motor neurones obtained by laser capture microdissection of autopsy tissue from individuals with sporadic ALS and TDP-43 proteinopathy. Findings were confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in NSC34 motor neuronal cells following shRNA-mediated TDP-43 depletion. Exon arrays and immunohistochemistry were used to study mRNA splicing and TDP-43 expression in fibroblasts from patients with mtTARDBP-associated, sporadic and mutant SOD1-associated ALS. We found altered expression of spliceosome components in motor neurones and widespread aberrations of mRNA splicing that specifically affected genes involved in ribonucleotide binding. This was confirmed in TDP-43-depleted NSC34 cells. Fibroblasts with mtTARDBP showed loss of nuclear TDP-43 protein and demonstrated similar changes in splicing and gene expression, which were not present in fibroblasts from patients with sporadic or SOD1-related ALS. Loss of nuclear TDP-43 is associated with RNA processing abnormalities in ALS motor neurones, patient-derived cells with mtTARDBP, and following artificial TDP-43 depletion, suggesting that splicing dysregulation directly contributes to disease pathogenesis. Key functional pathways affected include those central to RNA metabolism. © 2014 British Neuropathological Society.

Intron-mediated alternative splicing of WOOD-ASSOCIATED NAC TRANSCRIPTION FACTOR1B regulates cell wall thickening during fiber development in Populus species.

PubMed

Zhao, Yunjun; Sun, Jiayan; Xu, Peng; Zhang, Rui; Li, Laigeng

2014-02-01

Alternative splicing is an important mechanism involved in regulating the development of multicellular organisms. Although many genes in plants undergo alternative splicing, little is understood of its significance in regulating plant growth and development. In this study, alternative splicing of black cottonwood (Populus trichocarpa) wood-associated NAC domain transcription factor (PtrWNDs), PtrWND1B, is shown to occur exclusively in secondary xylem fiber cells. PtrWND1B is expressed with a normal short-transcript PtrWND1B-s as well as its alternative long-transcript PtrWND1B-l. The intron 2 structure of the PtrWND1B gene was identified as a critical sequence that causes PtrWND1B alternative splicing. Suppression of PtrWND1B expression specifically inhibited fiber cell wall thickening. The two PtrWND1B isoforms play antagonistic roles in regulating cell wall thickening during fiber cell differentiation in Populus spp. PtrWND1B-s overexpression enhanced fiber cell wall thickening, while overexpression of PtrWND1B-l repressed fiber cell wall thickening. Alternative splicing may enable more specific regulation of processes such as fiber cell wall thickening during wood formation.

Intron-Mediated Alternative Splicing of WOOD-ASSOCIATED NAC TRANSCRIPTION FACTOR1B Regulates Cell Wall Thickening during Fiber Development in Populus Species1[W

PubMed Central

Zhao, Yunjun; Sun, Jiayan; Xu, Peng; Zhang, Rui; Li, Laigeng

2014-01-01

Alternative splicing is an important mechanism involved in regulating the development of multicellular organisms. Although many genes in plants undergo alternative splicing, little is understood of its significance in regulating plant growth and development. In this study, alternative splicing of black cottonwood (Populus trichocarpa) wood-associated NAC domain transcription factor (PtrWNDs), PtrWND1B, is shown to occur exclusively in secondary xylem fiber cells. PtrWND1B is expressed with a normal short-transcript PtrWND1B-s as well as its alternative long-transcript PtrWND1B-l. The intron 2 structure of the PtrWND1B gene was identified as a critical sequence that causes PtrWND1B alternative splicing. Suppression of PtrWND1B expression specifically inhibited fiber cell wall thickening. The two PtrWND1B isoforms play antagonistic roles in regulating cell wall thickening during fiber cell differentiation in Populus spp. PtrWND1B-s overexpression enhanced fiber cell wall thickening, while overexpression of PtrWND1B-l repressed fiber cell wall thickening. Alternative splicing may enable more specific regulation of processes such as fiber cell wall thickening during wood formation. PMID:24394777

The MicroRNA miR-124 Promotes Neuronal Differentiation by Triggering Brain-Specific Alternative Pre-mRNA Splicing

PubMed Central

Makeyev, Eugene V.; Zhang, Jiangwen; Carrasco, Monica A.; Maniatis, Tom

2011-01-01

SUMMARY Both microRNAs and alternative pre-mRNA splicing have been implicated in the development of the nervous system (NS), but functional interactions between these two pathways are poorly understood. We demonstrate that the neuron-specific microRNA miR-124 directly targets PTBP1 (PTB/hnRNP I) mRNA, which encodes a global repressor of alternative pre-mRNA splicing in nonneuronal cells. Among the targets of PTBP1 is a critical cassette exon in the pre-mRNA of PTBP2 (nPTB/brPTB/PTBLP), an NS-enriched PTBP1 homolog. When this exon is skipped, PTBP2 mRNA is subject to nonsense-mediated decay (NMD). During neuronal differentiation, miR-124 reduces PTBP1 levels, leading to the accumulation of correctly spliced PTBP2 mRNA and a dramatic increase in PTBP2 protein. These events culminate in the transition from non-NS to NS-specific alternative splicing patterns. We also present evidence that miR-124 plays a key role in the differentiation of progenitor cells to mature neurons. Thus, miR-124 promotes NS development, at least in part by regulating an intricate network of NS-specific alternative splicing. PMID:17679093

Early Clinical Diagnosis of PC1/3 Deficiency in a Patient With a Novel Homozygous PCSK1 Splice-Site Mutation.

PubMed

Härter, Bettina; Fuchs, Irene; Müller, Thomas; Akbulut, Ulas Emre; Cakir, Murat; Janecke, Andreas R

2016-04-01

Autosomal recessive proprotein convertase 1/3 (PC1/3) deficiency, caused by mutations in the PCSK1 gene, is characterized by severe congenital malabsorptive diarrhea, early-onset obesity, and certain endocrine abnormalities. We suspected PC1/3 deficiency in a 4-month-old girl based on the presence of congenital diarrhea and polyuria. Sequencing the whole coding region and splice sites detected a novel homozygous PCSK1 splice-site mutation, c.544-2A>G, in the patient. The mutation resulted in the skipping of exon 5, the generation of a premature termination codon, and nonsense-mediated PCSK1 messenger ribonucleic acid decay, which was demonstrated in complementary DNA derived from fibroblasts.

Pressure-Mediated Oligonucleotide Transfection of Rat and Human Cardiovascular Tissues

NASA Astrophysics Data System (ADS)

Mann, Michael J.; Gibbons, Gary H.; Hutchinson, Howard; Poston, Robert S.; Hoyt, E. Grant; Robbins, Robert C.; Dzau, Victor J.

1999-05-01

The application of gene therapy to human disease is currently restricted by the relatively low efficiency and potential hazards of methods of oligonucleotide or gene delivery. Antisense or transcription factor decoy oligonucleotides have been shown to be effective at altering gene expression in cell culture expreriments, but their in vivo application is limited by the efficiency of cellular delivery, the intracellular stability of the compounds, and their duration of activity. We report herein the development of a highly efficient method for naked oligodeoxynucleotide (ODN) transfection into cardiovascular tissues by using controlled, nondistending pressure without the use of viral vectors, lipid formulations, or exposure to other adjunctive, potentially hazardous substances. In this study, we have documented the ability of ex vivo, pressure-mediated transfection to achieve nuclear localization of fluorescent (FITC)-labeled ODN in approximately 90% and 50% of cells in intact human saphenous vein and rat myocardium, respectively. We have further documented that pressure-mediated delivery of antisense ODN can functionally inhibited target gene expression in both of these tissues in a sequence-specific manner at the mRNA and protein levels. This oligonucleotide transfection system may represent a safe means of achieving the intraoperative genetic engineering of failure-resistant human bypass grafts and may provide an avenue for the genetic manipulation of cardiac allograft rejection, allograft vasculopathy, or other transplant diseases.

Temporal and Spatial Post-Transcriptional Regulation of Zebrafish Tie1 mRNA by Long Noncoding RNA During Brain Vascular Assembly.

PubMed

Chowdhury, Tamjid A; Koceja, Chris; Eisa-Beygi, Shahram; Kleinstiver, Benjamin P; Kumar, Suresh N; Lin, Chien-Wei; Li, Keguo; Prabhudesai, Shubhangi; Joung, J Keith; Ramchandran, Ramani

2018-05-03

Tie1 (tyrosine kinase containing immunoglobulin and epidermal growth factor homology 1), an endothelial and hematopoietic cell-specific receptor tyrosine kinase, is an important regulator of angiogenesis and critical for maintaining vascular integrity. The post-transcriptional regulation of tie1 mRNA expression is not understood, but it might partly explain Tie1's differential expression pattern in endothelium. Following up on our previous work that identified natural antisense transcripts from the tie1 locus- tie1 antisense ( tie1AS ), which regulates tie1 mRNA levels in zebrafish-we attempted to identify the mechanism of this regulation. Through in vitro and in vivo ribonucleoprotein binding studies, we demonstrated that tie1AS long noncoding RNA interacts with an RNA binding protein-embryonic lethal and abnormal vision Drosophila-like 1 (Elavl1)-that regulates tie1 mRNA levels. When we disrupted the interaction between tie1AS and Elavl1 by using constitutively active antisense morpholino oligonucleotides or photoactivatable morpholino oligonucleotides, tie1 mRNA levels increased between 26 and 31 hours post-fertilization, particularly in the head. This increase correlated with dilation of primordial midbrain channels, smaller eyes, and reduced ventricular space. We also observed these phenotypes when we used CRISPR (clustered regularly interspaced short palindromic repeats)-mediated CRISPRi (CRISPR-mediated interference) to knock down tie1AS . Treatment of the morpholino oligonucleotide-injected embryos with a small molecule that decreased tie1 mRNA levels rescued all 3 abnormal phenotypes. We identified a novel mode of temporal and spatial post-transcriptional regulation of tie1 mRNA. It involves long noncoding RNA, tie1AS, and Elavl1 (an interactor of tie1AS ). © 2018 American Heart Association, Inc.

Multitasking of the piRNA Silencing Machinery: Targeting Transposable Elements and Foreign Genes in the Bdelloid Rotifer Adineta vaga.

PubMed

Rodriguez, Fernando; Arkhipova, Irina R

2016-05-01

RNA-mediated silencing processes play a key role in silencing of transposable elements, especially in the germ line, where piwi-interacting RNAs (piRNAs) are responsible for suppressing transposon mobility and maintaining genome integrity. We previously reported that the genome of Adineta vaga, the first sequenced representative of the phylum Rotifera (class Bdelloidea), is characterized by massive levels of horizontal gene transfer, by unusually low transposon content, and by highly diversified RNA-mediated silencing machinery. Here, we investigate genome-wide distribution of pi-like small RNAs, which in A. vaga are 25-31 nucleotides in length and have a strong 5'-uridine bias, while lacking ping-pong amplification signatures. In agreement with expectations, 71% of mapped reads corresponded to annotated transposons, with 93% of these reads being in the antisense orientation. Unexpectedly, a significant fraction of piRNAs originate from predicted coding regions corresponding to genes of putatively foreign origin. The distribution of piRNAs across foreign genes is not biased toward 3'-UTRs, instead resembling transposons in uniform distribution pattern throughout the gene body, and in predominantly antisense orientation. We also find that genes with small RNA coverage, including a number of genes of metazoan origin, are characterized by higher occurrence of telomeric repeats in the surrounding genomic regions, and by higher density of transposons in the vicinity, which have the potential to promote antisense transcription. Our findings highlight the complex interplay between RNA-based silencing processes and acquisition of genes at the genome periphery, which can result either in their loss or eventual domestication and integration into the host genome. Copyright © 2016 by the Genetics Society of America.

Evidence from immunoneutralization and antisense studies that the inhibitory actions of glucocorticoids on growth hormone release in vitro require annexin 1 (lipocortin 1)

PubMed Central

Taylor, A D; Christian, H C; Morris, J F; Flower, R J; Buckingham, J C

2000-01-01

Our previous studies have identified a role for annexin 1 as a mediator of glucocorticoid action in the neuroendocrine system. The present study centred on growth hormone (GH) and exploited antisense and immunoneutralization strategies to examine in vitro the potential role of annexin 1 in effecting the regulatory actions of glucocorticoids on the secretion of this pituitary hormone. Rat anterior pituitary tissue responded in vitro to growth hormone releasing hormone, forskolin, 8-Bromo-cyclic adenosine 3′5′-monophosphate (8-Br-cyclic AMP) and an L-Ca2+ channel opener (BAY K8644) with concentration-dependent increases GH release which were readily inhibited by corticosterone and dexamethasone. The inhibitory actions of the steroids on GH release elicited by the above secretagogues were effectively reversed by an annexin 1 antisense oligodeoxynucleotide (ODN), but not by control (sense or scrambled) ODNs, as also were the glucocorticoid-induced increases in annexin 1. Similarly, a specific anti-annexin 1 monoclonal antibody quenched the corticosterone-induced suppression of secretagogue-evoked GH release while an isotype matched control antibody was without effect. Transmission electron micrographs showed that the integrity and ultrastructural morphology of the pituitary cells were well preserved at the end of the incubation and unaffected by exposure to the ODNs, antibodies, steroids or secretagogues. The results provide novel evidence for a role for annexin 1 as a mediator of the inhibitory actions of glucocorticoids on the secretion of GH by the anterior pituitary gland and suggest that its actions are effected at a point distal to the formation of cyclic AMP and Ca2+ entry. PMID:11090102

Evidence from immunoneutralization and antisense studies that the inhibitory actions of glucocorticoids on growth hormone release in vitro require annexin 1 (lipocortin 1).

PubMed

Taylor, A D; Christian, H C; Morris, J F; Flower, R J; Buckingham, J C

2000-12-01

1. Our previous studies have identified a role for annexin 1 as a mediator of glucocorticoid action in the neuroendocrine system. The present study centred on growth hormone (GH) and exploited antisense and immunoneutralization strategies to examine in vitro the potential role of annexin 1 in effecting the regulatory actions of glucocorticoids on the secretion of this pituitary hormone. 2. Rat anterior pituitary tissue responded in vitro to growth hormone releasing hormone, forskolin, 8-Bromo-cyclic adenosine 3'5'-monophosphate (8-Br-cyclic AMP) and an L-Ca(2+) channel opener (BAY K8644) with concentration-dependent increases GH release which were readily inhibited by corticosterone and dexamethasone. 3. The inhibitory actions of the steroids on GH release elicited by the above secretagogues were effectively reversed by an annexin 1 antisense oligodeoxynucleotide (ODN), but not by control (sense or scrambled) ODNs, as also were the glucocorticoid-induced increases in annexin 1. Similarly, a specific anti-annexin 1 monoclonal antibody quenched the corticosterone-induced suppression of secretagogue-evoked GH release while an isotype matched control antibody was without effect. 4. Transmission electron micrographs showed that the integrity and ultrastructural morphology of the pituitary cells were well preserved at the end of the incubation and unaffected by exposure to the ODNs, antibodies, steroids or secretagogues. 5. The results provide novel evidence for a role for annexin 1 as a mediator of the inhibitory actions of glucocorticoids on the secretion of GH by the anterior pituitary gland and suggest that its actions are effected at a point distal to the formation of cyclic AMP and Ca(2+) entry.

Multitasking of the piRNA Silencing Machinery: Targeting Transposable Elements and Foreign Genes in the Bdelloid Rotifer Adineta vaga

PubMed Central

Rodriguez, Fernando; Arkhipova, Irina R.

2016-01-01

RNA-mediated silencing processes play a key role in silencing of transposable elements, especially in the germ line, where piwi-interacting RNAs (piRNAs) are responsible for suppressing transposon mobility and maintaining genome integrity. We previously reported that the genome of Adineta vaga, the first sequenced representative of the phylum Rotifera (class Bdelloidea), is characterized by massive levels of horizontal gene transfer, by unusually low transposon content, and by highly diversified RNA-mediated silencing machinery. Here, we investigate genome-wide distribution of pi-like small RNAs, which in A. vaga are 25–31 nucleotides in length and have a strong 5′-uridine bias, while lacking ping-pong amplification signatures. In agreement with expectations, 71% of mapped reads corresponded to annotated transposons, with 93% of these reads being in the antisense orientation. Unexpectedly, a significant fraction of piRNAs originate from predicted coding regions corresponding to genes of putatively foreign origin. The distribution of piRNAs across foreign genes is not biased toward 3′-UTRs, instead resembling transposons in uniform distribution pattern throughout the gene body, and in predominantly antisense orientation. We also find that genes with small RNA coverage, including a number of genes of metazoan origin, are characterized by higher occurrence of telomeric repeats in the surrounding genomic regions, and by higher density of transposons in the vicinity, which have the potential to promote antisense transcription. Our findings highlight the complex interplay between RNA-based silencing processes and acquisition of genes at the genome periphery, which can result either in their loss or eventual domestication and integration into the host genome. PMID:27017627

Pseudomonas fluorescens WCS374r-Induced Systemic Resistance in Rice against Magnaporthe oryzae Is Based on Pseudobactin-Mediated Priming for a Salicylic Acid-Repressible Multifaceted Defense Response1[C][OA

PubMed Central

De Vleesschauwer, David; Djavaheri, Mohammad; Bakker, Peter A.H.M.; Höfte, Monica

2008-01-01

Selected strains of nonpathogenic rhizobacteria can reduce disease in foliar tissues through the induction of a defense state known as induced systemic resistance (ISR). Compared with the large body of information on ISR in dicotyledonous plants, little is known about the mechanisms underlying rhizobacteria-induced resistance in cereal crops. Here, we demonstrate the ability of Pseudomonas fluorescens WCS374r to trigger ISR in rice (Oryza sativa) against the leaf blast pathogen Magnaporthe oryzae. Using salicylic acid (SA)-nonaccumulating NahG rice, an ethylene-insensitive OsEIN2 antisense line, and the jasmonate-deficient mutant hebiba, we show that this WCS374r-induced resistance is regulated by an SA-independent but jasmonic acid/ethylene-modulated signal transduction pathway. Bacterial mutant analysis uncovered a pseudobactin-type siderophore as the crucial determinant responsible for ISR elicitation. Root application of WCS374r-derived pseudobactin (Psb374) primed naive leaves for accelerated expression of a pronounced multifaceted defense response, consisting of rapid recruitment of phenolic compounds at sites of pathogen entry, concerted expression of a diverse set of structural defenses, and a timely hyperinduction of hydrogen peroxide formation putatively driving cell wall fortification. Exogenous SA application alleviated this Psb374-modulated defense priming, while Psb374 pretreatment antagonized infection-induced transcription of SA-responsive PR genes, suggesting that the Psb374- and SA-modulated signaling pathways are mutually antagonistic. Interestingly, in sharp contrast to WCS374r-mediated ISR, chemical induction of blast resistance by the SA analog benzothiadiazole was independent of jasmonic acid/ethylene signaling and involved the potentiation of SA-responsive gene expression. Together, these results offer novel insights into the signaling circuitry governing induced resistance against M. oryzae and suggest that rice is endowed with multiple blast-effective resistance pathways. PMID:18945932

ISIS 301012 gene therapy for hypercholesterolemia: sense, antisense, or nonsense?

PubMed

Ito, Matthew K

2007-10-01

To present an overview of antisense technology and to review and assess available literature on the chemistry, pharmacology, pharmacokinetics, drug interactions, preclinical and clinical studies, dosing, and adverse events of ISIS 301012 in the treatment of hyperlipidemia. PubMed database searches were conducted from 1966 to May 2007 using the search terms ISIS 301012, antisense, oligonucleotide, hypercholesterolemia, hyperlipidemia, and apolipoprotein B. Bibliographies of relevant review articles and information from the manufacturer were reviewed for additional references. Available English-language literature, including abstracts, preclinical, and clinical trials, review articles, and scientific presentations were examined. Apolipoprotein B is an important structural protein on the surface of atherogenic lipoproteins such as remnant very-low-density lipoprotein and low-density lipoprotein and facilitates the clearance of these particles from the circulation by binding to the low-density lipoprotein receptor. Overproduction of apolipoprotein B or reduced receptor-mediated clearance of lipoproteins leads to elevated serum cholesterol levels and premature atherosclerosis. ISIS 301012 is an antisense oligonucleotide that inhibits apolipoprotein B production by binding directly to and reducing the expression of apolipoprotein B messenger RNA. In a clinical trial, ISIS 301012 50-400 mg administered weekly via subcutaneous injection for 4 weeks reduced apolipoprotein B by 14.3-47.4% and low-density lipoprotein cholesterol by 5.9-40% at 55 days. The most frequent adverse event was injection-site erythema that resolved spontaneously. Studies are ongoing to further define the safety, efficacy, and pharmacokinetics of ISIS 301012 as add-on therapy in patients with heterozygous and homozygous familial hypercholesterolemia. No pharmacokinetic interactions have been demonstrated with ezetimibe and simvastatin. ISIS 301012 is the first agent to enter clinical trials utilizing an antisense mechanism for reducing the production of apolipoprotein B. Further studies are needed to verify its safety, efficacy, and position of therapy in the dyslipidemic patient.

Long Non-Coding RNA and Alternative Splicing Modulations in Parkinson's Leukocytes Identified by RNA Sequencing

PubMed Central

Soreq, Lilach; Guffanti, Alessandro; Salomonis, Nathan; Simchovitz, Alon; Israel, Zvi; Bergman, Hagai; Soreq, Hermona

2014-01-01

The continuously prolonged human lifespan is accompanied by increase in neurodegenerative diseases incidence, calling for the development of inexpensive blood-based diagnostics. Analyzing blood cell transcripts by RNA-Seq is a robust means to identify novel biomarkers that rapidly becomes a commonplace. However, there is lack of tools to discover novel exons, junctions and splicing events and to precisely and sensitively assess differential splicing through RNA-Seq data analysis and across RNA-Seq platforms. Here, we present a new and comprehensive computational workflow for whole-transcriptome RNA-Seq analysis, using an updated version of the software AltAnalyze, to identify both known and novel high-confidence alternative splicing events, and to integrate them with both protein-domains and microRNA binding annotations. We applied the novel workflow on RNA-Seq data from Parkinson's disease (PD) patients' leukocytes pre- and post- Deep Brain Stimulation (DBS) treatment and compared to healthy controls. Disease-mediated changes included decreased usage of alternative promoters and N-termini, 5′-end variations and mutually-exclusive exons. The PD regulated FUS and HNRNP A/B included prion-like domains regulated regions. We also present here a workflow to identify and analyze long non-coding RNAs (lncRNAs) via RNA-Seq data. We identified reduced lncRNA expression and selective PD-induced changes in 13 of over 6,000 detected leukocyte lncRNAs, four of which were inversely altered post-DBS. These included the U1 spliceosomal lncRNA and RP11-462G22.1, each entailing sequence complementarity to numerous microRNAs. Analysis of RNA-Seq from PD and unaffected controls brains revealed over 7,000 brain-expressed lncRNAs, of which 3,495 were co-expressed in the leukocytes including U1, which showed both leukocyte and brain increases. Furthermore, qRT-PCR validations confirmed these co-increases in PD leukocytes and two brain regions, the amygdala and substantia-nigra, compared to controls. This novel workflow allows deep multi-level inspection of RNA-Seq datasets and provides a comprehensive new resource for understanding disease transcriptome modifications in PD and other neurodegenerative diseases. PMID:24651478

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli.

PubMed

Bernheim, Aude G; Libis, Vincent K; Lindner, Ariel B; Wintermute, Edwin H

2016-03-20

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Characterization of Ether-à-go-go Channels Present in Photoreceptors Reveals Similarity to IKx, a K+ Current in Rod Inner Segments

PubMed Central

Frings, Stephan; Brüll, Nicole; Dzeja, Claudia; Angele, Albert; Hagen, Volker; Kaupp, U. Benjamin; Baumann, Arnd

1998-01-01

In this study, we describe two splice variants of an ether-à-go-go (EAG) K+ channel cloned from bovine retina: bEAG1 and bEAG2. The bEAG2 polypeptide contains an additional insertion of 27 amino acids in the extracellular linker between transmembrane segments S3 and S4. The heterologously expressed splice variants differ in their activation kinetics and are differently modulated by extracellular Mg2+. Cooperativity of modulation by Mg2+ suggests that each subunit of the putative tetrameric channel binds a Mg2+ ion. The channels are neither permeable to Ca2+ ions nor modulated by cyclic nucleotides. In situ hybridization localizes channel transcripts to photoreceptors and retinal ganglion cells. Comparison of EAG currents with IKx, a noninactivating K+ current in the inner segment of rod photoreceptors, reveals an intriguing similarity, suggesting that EAG polypeptides are involved in the formation of Kx channels. PMID:9524140

Intelligent Therapeutics and Metabolic Programming Through Tailormade, Ligand-Controlled RNA Switches

DTIC Science & Technology

2007-02-05

lines. Three regulatory mechanisms have been examined in our laboratory: antisense inhibition, ribozyme cleavage, and RNA interference (RNAi...cell lines. However, the latter two regulatory mechanisms, ribozyme -based inactivation and RNAi-mediated silencing, demonstrated significant activity...in these cell lines as is briefly described below. Microswitches responsive to the small molecule theophylline and targeting GFP based on a ribozyme

SKIP Is a Component of the Spliceosome Linking Alternative Splicing and the Circadian Clock in Arabidopsis[W

PubMed Central

Wang, Xiaoxue; Wu, Fangming; Xie, Qiguang; Wang, Huamei; Wang, Ying; Yue, Yanling; Gahura, Ondrej; Ma, Shuangshuang; Liu, Lei; Cao, Ying; Jiao, Yuling; Puta, Frantisek; McClung, C. Robertson; Xu, Xiaodong; Ma, Ligeng

2012-01-01

Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for clock function. However, little is known about the molecular mechanisms linking RNA metabolism to the circadian clock network. Here, we report that a conserved SNW/Ski-interacting protein (SKIP) domain protein, SKIP, a splicing factor and component of the spliceosome, is involved in posttranscriptional regulation of circadian clock genes in Arabidopsis thaliana. Mutation in SKIP lengthens the circadian period in a temperature-sensitive manner and affects light input and the sensitivity of the clock to light resetting. SKIP physically interacts with the spliceosomal splicing factor Ser/Arg-rich protein45 and associates with the pre-mRNA of clock genes, such as PSEUDORESPONSE REGULATOR7 (PRR7) and PRR9, and is necessary for the regulation of their alternative splicing and mRNA maturation. Genome-wide investigations reveal that SKIP functions in regulating alternative splicing of many genes, presumably through modulating recognition or cleavage of 5′ and 3′ splice donor and acceptor sites. Our study addresses a fundamental question on how the mRNA splicing machinery contributes to circadian clock function at a posttranscriptional level. PMID:22942380

Alternative splicing: the pledge, the turn, and the prestige : The key role of alternative splicing in human biological systems.

PubMed

Gallego-Paez, L M; Bordone, M C; Leote, A C; Saraiva-Agostinho, N; Ascensão-Ferreira, M; Barbosa-Morais, N L

2017-09-01

Alternative pre-mRNA splicing is a tightly controlled process conducted by the spliceosome, with the assistance of several regulators, resulting in the expression of different transcript isoforms from the same gene and increasing both transcriptome and proteome complexity. The differences between alternative isoforms may be subtle but enough to change the function or localization of the translated proteins. A fine control of the isoform balance is, therefore, needed throughout developmental stages and adult tissues or physiological conditions and it does not come as a surprise that several diseases are caused by its deregulation. In this review, we aim to bring the splicing machinery on stage and raise the curtain on its mechanisms and regulation throughout several systems and tissues of the human body, from neurodevelopment to the interactions with the human microbiome. We discuss, on one hand, the essential role of alternative splicing in assuring tissue function, diversity, and swiftness of response in these systems or tissues, and on the other hand, what goes wrong when its regulatory mechanisms fail. We also focus on the possibilities that splicing modulation therapies open for the future of personalized medicine, along with the leading techniques in this field. The final act of the spliceosome, however, is yet to be fully revealed, as more knowledge is needed regarding the complex regulatory network that coordinates alternative splicing and how its dysfunction leads to disease.

Rhythmic Behavior Is Controlled by the SRm160 Splicing Factor in Drosophila melanogaster.

PubMed

Beckwith, Esteban J; Hernando, Carlos E; Polcowñuk, Sofía; Bertolin, Agustina P; Mancini, Estefania; Ceriani, M Fernanda; Yanovsky, Marcelo J

2017-10-01

Circadian clocks organize the metabolism, physiology, and behavior of organisms throughout the day-night cycle by controlling daily rhythms in gene expression at the transcriptional and post-transcriptional levels. While many transcription factors underlying circadian oscillations are known, the splicing factors that modulate these rhythms remain largely unexplored. A genome-wide assessment of the alterations of gene expression in a null mutant of the alternative splicing regulator SR-related matrix protein of 160 kDa (SRm160) revealed the extent to which alternative splicing impacts on behavior-related genes. We show that SRm160 affects gene expression in pacemaker neurons of the Drosophila brain to ensure proper oscillations of the molecular clock. A reduced level of SRm160 in adult pacemaker neurons impairs circadian rhythms in locomotor behavior, and this phenotype is caused, at least in part, by a marked reduction in period ( per ) levels. Moreover, rhythmic accumulation of the neuropeptide PIGMENT DISPERSING FACTOR in the dorsal projections of these neurons is abolished after SRm160 depletion. The lack of rhythmicity in SRm160-downregulated flies is reversed by a fully spliced per construct, but not by an extra copy of the endogenous locus, showing that SRm160 positively regulates per levels in a splicing-dependent manner. Our findings highlight the significant effect of alternative splicing on the nervous system and particularly on brain function in an in vivo model. Copyright © 2017 by the Genetics Society of America.

Pre-mRNA splicing repression triggers abiotic stress signaling in plants.

PubMed

Ling, Yu; Alshareef, Sahar; Butt, Haroon; Lozano-Juste, Jorge; Li, Lixin; Galal, Aya A; Moustafa, Ahmed; Momin, Afaque A; Tashkandi, Manal; Richardson, Dale N; Fujii, Hiroaki; Arold, Stefan; Rodriguez, Pedro L; Duque, Paula; Mahfouz, Magdy M

2017-01-01

Alternative splicing (AS) of precursor RNAs enhances transcriptome plasticity and proteome diversity in response to diverse growth and stress cues. Recent work has shown that AS is pervasive across plant species, with more than 60% of intron-containing genes producing different isoforms. Mammalian cell-based assays have discovered various inhibitors of AS. Here, we show that the macrolide pladienolide B (PB) inhibits constitutive splicing and AS in plants. Also, our RNA sequencing (RNA-seq) data revealed that PB mimics abiotic stress signals including salt, drought and abscisic acid (ABA). PB activates the abiotic stress- and ABA-responsive reporters RD29A::LUC and MAPKKK18::uidA in Arabidopsis thaliana and mimics the effects of ABA on stomatal aperture. Genome-wide analysis of AS by RNA-seq revealed that PB perturbs the splicing machinery and leads to a striking increase in intron retention and a reduction in other forms of AS. Interestingly, PB treatment activates the ABA signaling pathway by inhibiting the splicing of clade A PP2C phosphatases while still maintaining to some extent the splicing of ABA-activated SnRK2 kinases. Taken together, our data establish PB as an inhibitor and modulator of splicing and a mimic of abiotic stress signals in plants. Thus, PB reveals the molecular underpinnings of the interplay between stress responses, ABA signaling and post-transcriptional regulation in plants. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Antisense pre-treatment increases gene therapy efficacy in dystrophic muscles.

PubMed

Peccate, Cécile; Mollard, Amédée; Le Hir, Maëva; Julien, Laura; McClorey, Graham; Jarmin, Susan; Le Heron, Anita; Dickson, George; Benkhelifa-Ziyyat, Sofia; Piétri-Rouxel, France; Wood, Matthew J; Voit, Thomas; Lorain, Stéphanie

2016-08-15

In preclinical models for Duchenne muscular dystrophy, dystrophin restoration during adeno-associated virus (AAV)-U7-mediated exon-skipping therapy was shown to decrease drastically after six months in treated muscles. This decline in efficacy is strongly correlated with the loss of the therapeutic AAV genomes, probably due to alterations of the dystrophic myofiber membranes. To improve the membrane integrity of the dystrophic myofibers at the time of AAV-U7 injection, mdx muscles were pre-treated with a single dose of the peptide-phosphorodiamidate morpholino (PPMO) antisense oligonucleotides that induced temporary dystrophin expression at the sarcolemma. The PPMO pre-treatment allowed efficient maintenance of AAV genomes in mdx muscles and enhanced the AAV-U7 therapy effect with a ten-fold increase of the protein level after 6 months. PPMO pre-treatment was also beneficial to AAV-mediated gene therapy with transfer of micro-dystrophin cDNA into muscles. Therefore, avoiding vector genome loss after AAV injection by PPMO pre-treatment would allow efficient long-term restoration of dystrophin and the use of lower and thus safer vector doses for Duchenne patients. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Asialoglycoprotein receptor 1 mediates productive uptake of N-acetylgalactosamine-conjugated and unconjugated phosphorothioate antisense oligonucleotides into liver hepatocytes

PubMed Central

Hettrick, Lisa; Revenko, Alexey; Kinberger, Garth A.; Prakash, Thazha P.; Seth, Punit P.

2017-01-01

Abstract Antisense oligonucleotide (ASO) therapeutics show tremendous promise for the treatment of previously intractable human diseases but to exert their effects on cellular RNA processing they must first cross the plasma membrane by endocytosis. The conjugation of ASOs to a receptor ligand can dramatically increase their entry into certain cells and tissues, as demonstrated by the implementation of N-acetylgalactosamine (GalNAc)-conjugated ASOs for Asialoglycoprotein Receptor (ASGR)-mediated uptake into liver hepatocytes. We compared the internalization and activity of GalNAc-conjugated ASOs and their parents in endogenous ASGR-expressing cells and were able to recapitulate hepatocyte ASO uptake and activity in cells engineered to heterologously express the receptor. We found that the minor receptor subunit, ASGR2, is not required for effective in vitro or in vivo uptake of GalNAc-conjugated ASO and that the major subunit, ASGR1, plays a small but significant role in the uptake of unconjugated phosphorothioate ASOs into hepatocytes. Moreover, our data demonstrates there is a large excess capacity of liver ASGR for the effective uptake of GalNAc–ASO conjugates, suggesting broad opportunities to exploit receptors with relatively moderate levels of expression. PMID:29069408

Antithrombotic Effect of Antisense Factor XI Oligonucleotide Treatment in Primates

PubMed Central

Crosby, Jeffrey R.; Marzec, Ulla; Revenko, Alexey S.; Zhao, Chenguang; Gao, Dacao; Matafonov, Anton; Gailani, David; MacLeod, A. Robert; Tucker, Erik I.; Gruber, Andras; Hanson, Stephen R.; Monia, Brett P.

2013-01-01

Objective During coagulation, factor IX (FIX) is activated by two distinct mechanisms mediated by the active proteases of either factors VII (FVIIa) or XI (FXIa). Both coagulation factors may contribute to thrombosis; factor XI, however, plays only a limited role in the arrest of bleeding. Therefore, therapeutic targeting of FXI may produce an antithrombotic effect with relatively low hemostatic risk. Approach and Results We have reported that reducing FXI levels with FXI antisense oligonucleotides (ASOs) produces antithrombotic activity in mice, and that administration of FXI ASOs to primates decreases circulating FXI levels and activity in a dose- and time-dependent manner. Here we evaluated the relationship between FXI plasma levels and thrombogenicity in an established baboon model of thrombosis and hemostasis. In previous studies with this model, antibody-induced inhibition of FXI produced potent antithrombotic effects. In the present report, ASO-mediated reduction of FXI plasma levels by ≥50% resulted in a demonstrable and sustained antithrombotic effect without an increased risk of bleeding. Conclusion These results indicate that reducing FXI levels using ASOs is a promising alternative to direct FXI inhibition, and that targeting FXI may be potentially safer than conventional antithrombotic therapies that can markedly impair primary hemostasis. PMID:23559626

Widespread antisense transcription of Populus genome under drought.

PubMed

Yuan, Yinan; Chen, Su

2018-06-06

Antisense transcription is widespread in many genomes and plays important regulatory roles in gene expression. The objective of our study was to investigate the extent and functional relevance of antisense transcription in forest trees. We employed Populus, a model tree species, to probe the antisense transcriptional response of tree genome under drought, through stranded RNA-seq analysis. We detected nearly 48% of annotated Populus gene loci with antisense transcripts and 44% of them with co-transcription from both DNA strands. Global distribution of reads pattern across annotated gene regions uncovered that antisense transcription was enriched in untranslated regions while sense reads were predominantly mapped in coding exons. We further detected 1185 drought-responsive sense and antisense gene loci and identified a strong positive correlation between the expression of antisense and sense transcripts. Additionally, we assessed the antisense expression in introns and found a strong correlation between intronic expression and exonic expression, confirming antisense transcription of introns contributes to transcriptional activity of Populus genome under drought. Finally, we functionally characterized drought-responsive sense-antisense transcript pairs through gene ontology analysis and discovered that functional groups including transcription factors and histones were concordantly regulated at both sense and antisense transcriptional level. Overall, our study demonstrated the extensive occurrence of antisense transcripts of Populus genes under drought and provided insights into genome structure, regulation pattern and functional significance of drought-responsive antisense genes in forest trees. Datasets generated in this study serve as a foundation for future genetic analysis to improve our understanding of gene regulation by antisense transcription.

Antisense targeting of 3' end elements involved in DUX4 mRNA processing is an efficient therapeutic strategy for facioscapulohumeral dystrophy: a new gene-silencing approach.

PubMed

Marsollier, Anne-Charlotte; Ciszewski, Lukasz; Mariot, Virginie; Popplewell, Linda; Voit, Thomas; Dickson, George; Dumonceaux, Julie

2016-04-15

Defects in mRNA 3'end formation have been described to alter transcription termination, transport of the mRNA from the nucleus to the cytoplasm, stability of the mRNA and translation efficiency. Therefore, inhibition of polyadenylation may lead to gene silencing. Here, we choose facioscapulohumeral dystrophy (FSHD) as a model to determine whether or not targeting key 3' end elements involved in mRNA processing using antisense oligonucleotide drugs can be used as a strategy for gene silencing within a potentially therapeutic context. FSHD is a gain-of-function disease characterized by the aberrant expression of the Double homeobox 4 (DUX4) transcription factor leading to altered pathogenic deregulation of multiple genes in muscles. Here, we demonstrate that targeting either the mRNA polyadenylation signal and/or cleavage site is an efficient strategy to down-regulate DUX4 expression and to decrease the abnormally high-pathological expression of genes downstream of DUX4. We conclude that targeting key functional 3' end elements involved in pre-mRNA to mRNA maturation with antisense drugs can lead to efficient gene silencing and is thus a potentially effective therapeutic strategy for at least FSHD. Moreover, polyadenylation is a crucial step in the maturation of almost all eukaryotic mRNAs, and thus all mRNAs are virtually eligible for this antisense-mediated knockdown strategy. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Structure, dynamics and RNA binding of the multi-domain splicing factor TIA-1

PubMed Central

Wang, Iren; Hennig, Janosch; Jagtap, Pravin Kumar Ankush; Sonntag, Miriam; Valcárcel, Juan; Sattler, Michael

2014-01-01

Alternative pre-messenger ribonucleic acid (pre-mRNA) splicing is an essential process in eukaryotic gene regulation. The T-cell intracellular antigen-1 (TIA-1) is an apoptosis-promoting factor that modulates alternative splicing of transcripts, including the pre-mRNA encoding the membrane receptor Fas. TIA-1 is a multi-domain ribonucleic acid (RNA) binding protein that recognizes poly-uridine tract RNA sequences to facilitate 5′ splice site recognition by the U1 small nuclear ribonucleoprotein (snRNP). Here, we characterize the RNA interaction and conformational dynamics of TIA-1 by nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC) and small angle X-ray scattering (SAXS). Our NMR-derived solution structure of TIA-1 RRM2–RRM3 (RRM2,3) reveals that RRM2 adopts a canonical RNA recognition motif (RRM) fold, while RRM3 is preceded by an non-canonical helix α0. NMR and SAXS data show that all three RRMs are largely independent structural modules in the absence of RNA, while RNA binding induces a compact arrangement. RRM2,3 binds to pyrimidine-rich FAS pre-mRNA or poly-uridine (U9) RNA with nanomolar affinities. RRM1 has little intrinsic RNA binding affinity and does not strongly contribute to RNA binding in the context of RRM1,2,3. Our data unravel the role of binding avidity and the contributions of the TIA-1 RRMs for recognition of pyrimidine-rich RNAs. PMID:24682828

A Novel Morpholino Oligomer Targeting ISS-N1 Improves Rescue of Severe Spinal Muscular Atrophy Transgenic Mice

PubMed Central

Janghra, Narinder; Mitrpant, Chalermchai; Dickinson, Rachel L.; Anthony, Karen; Price, Loren; Eperon, Ian C.; Wilton, Stephen D.; Morgan, Jennifer

2013-01-01

Abstract In the search for the most efficacious antisense oligonucleotides (AOs) aimed at inducing SMN2 exon 7 inclusion, we systematically assessed three AOs, PMO25 (−10, −34), PMO18 (−10, −27), and PMO20 (−10, −29), complementary to the SMN2 intron 7 splicing silencer (ISS-N1). PMO25 was the most efficacious in augmenting exon 7 inclusion in vitro in spinal muscular atrophy (SMA) patient fibroblasts and in vitro splicing assays. PMO25 and PMO18 were compared further in a mouse model of severe SMA. After a single intracerebroventricular (ICV) injection in neonatal mice, PMO25 increased the life span of severe SMA mice up to 30-fold, with average survival greater by 3-fold compared with PMO18 at a dose of 20 μg/g and 2-fold at 40 μg/g. Exon 7 inclusion was increased in the CNS but not in peripheral tissues. Systemic delivery of PMO25 at birth achieved a similar outcome and produced increased exon 7 inclusion both in the CNS and peripherally. Systemic administration of a 10-μg/g concentration of PMO25 conjugated to an octaguanidine dendrimer (VMO25) increased the life span only 2-fold in neonatal type I SMA mice, although it prevented tail necrosis in mild SMA mice. Higher doses and ICV injection of VMO25 were associated with toxicity. We conclude that (1) the 25-mer AO is more efficient than the 18-mer and 20-mer in modifying SMN2 splicing in vitro; (2) it is more efficient in prolonging survival in SMA mice; and (3) naked Morpholino oligomers are more efficient and safer than the Vivo-Morpholino and have potential for future SMA clinical applications. PMID:23339722

Novel splice site mutation in the growth hormone receptor gene in Turkish patients with Laron-type dwarfism.

PubMed

Arman, Ahmet; Ozon, Alev; Isguven, Pinar S; Coker, Ajda; Peker, Ismail; Yordam, Nursen

2008-01-01

Growth hormone (GH) is involved in growth, and fat and carbohydrate metabolism. Interaction of GH with the GH receptor (GHR) is necessary for systemic and local production of insulin-like growth factor-I (IGF-I) which mediates GH actions. Mutations in the GHR cause severe postnatal growth failure; the disorder is an autosomal recessive genetic disease resulting in GH insensitivity, called Laron syndrome. It is characterized by dwarfism with elevated serum GH and low levels of IGF-I. We analyzed the GHR gene for mutations and polymorphisms in eight patients with Laron-type dwarfism from six families. We found three missense mutations (S40L, V125A, I526L), one nonsense mutation (W157X), and one splice site mutation in the extracellular domain of GHR. Furthermore, G168G and exon 3 deletion polymorphisms were detected in patients with Laron syndrome. The splice site mutation, which is a novel mutation, was located at the donor splice site of exon 2/ intron 2 within GHR. Although this mutation changed the highly conserved donor splice site consensus sequence GT to GGT by insertion of a G residue, the intron splicing between exon 2 and exon 3 was detected in the patient. These results imply that the splicing occurs arthe GT site in intron 2, leaving the extra inserted G residue at the end of exon 2, thus changing the open reading frame of GHR resulting in a premature termination codon in exon 3.

Motor Neuron Gene Therapy: Lessons from Spinal Muscular Atrophy for Amyotrophic Lateral Sclerosis

PubMed Central

Tosolini, Andrew P.; Sleigh, James N.

2017-01-01

Spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) are severe nervous system diseases characterized by the degeneration of lower motor neurons. They share a number of additional pathological, cellular, and genetic parallels suggesting that mechanistic and clinical insights into one disorder may have value for the other. While there are currently no clinical ALS gene therapies, the splice-switching antisense oligonucleotide, nusinersen, was recently approved for SMA. This milestone was achieved through extensive pre-clinical research and patient trials, which together have spawned fundamental insights into motor neuron gene therapy. We have thus tried to distil key information garnered from SMA research, in the hope that it may stimulate a more directed approach to ALS gene therapy. Not only must the type of therapeutic (e.g., antisense oligonucleotide vs. viral vector) be sensibly selected, but considerable thought must be applied to the where, which, what, and when in order to enhance treatment benefit: to where (cell types and tissues) must the drug be delivered and how can this be best achieved? Which perturbed pathways must be corrected and can they be concurrently targeted? What dosing regime and concentration should be used? When should medication be administered? These questions are intuitive, but central to identifying and optimizing a successful gene therapy. Providing definitive solutions to these quandaries will be difficult, but clear thinking about therapeutic testing is necessary if we are to have the best chance of developing viable ALS gene therapies and improving upon early generation SMA treatments. PMID:29270111

Recapitulating muscle disease phenotypes with myotonic dystrophy 1 iPS cells: a tool for disease modeling and drug discovery.

PubMed

Mondragon-Gonzalez, Ricardo; Perlingeiro, Rita C R

2018-06-13

Myotonic Dystrophy 1 (DM1) is a multi-system disorder primarily affecting the central nervous system, heart and skeletal muscle. It is caused by an expansion of the CTG trinucleotide repeats in the 3' untranslated region of the DMPK gene. Although patient myoblasts have been used for studying the disease in vitro , the invasiveness as well as the low accessibility to muscle biopsies motivate the development of alternative reliable myogenic models. Here, we established two DM1 iPS cell lines from patient-derived fibroblasts, and using the PAX7 conditional expression system, differentiated these into myogenic progenitors, and subsequently, terminally differentiated myotubes. Both DM1 myogenic progenitors and myotubes were found to express the intranuclear RNA foci exhibiting sequestration of MBNL1. Moreover, we found the DM1-related mis-splicing, namely BIN1 exon 11 in DM1 myotubes. We use this model to test a specific therapy, antisense oligonucleotide treatment, and find that this efficiently abolished RNA foci and rescued BIN1 mis-splicing in DM1 iPS cell-derived myotubes. Together, our results demonstrate that myotubes derived from DM1 iPS cells recapitulate the critical molecular features of DM1 and are sensitive to ASO treatment, confirming that these cells can be used for in vitro disease modeling and candidate drug testing or screening. © 2018. Published by The Company of Biologists Ltd.

Abscisic Acid (ABA) Regulation of Arabidopsis SR Protein Gene Expression

PubMed Central

Cruz, Tiago M. D.; Carvalho, Raquel F.; Richardson, Dale N.; Duque, Paula

2014-01-01

Serine/arginine-rich (SR) proteins are major modulators of alternative splicing, a key generator of proteomic diversity and flexible means of regulating gene expression likely to be crucial in plant environmental responses. Indeed, mounting evidence implicates splicing factors in signal transduction of the abscisic acid (ABA) phytohormone, which plays pivotal roles in the response to various abiotic stresses. Using real-time RT-qPCR, we analyzed total steady-state transcript levels of the 18 SR and two SR-like genes from Arabidopsis thaliana in seedlings treated with ABA and in genetic backgrounds with altered expression of the ABA-biosynthesis ABA2 and the ABA-signaling ABI1 and ABI4 genes. We also searched for ABA-responsive cis elements in the upstream regions of the 20 genes. We found that members of the plant-specific SC35-Like (SCL) Arabidopsis SR protein subfamily are distinctively responsive to exogenous ABA, while the expression of seven SR and SR-related genes is affected by alterations in key components of the ABA pathway. Finally, despite pervasiveness of established ABA-responsive promoter elements in Arabidopsis SR and SR-like genes, their expression is likely governed by additional, yet unidentified cis-acting elements. Overall, this study pinpoints SR34, SR34b, SCL30a, SCL28, SCL33, RS40, SR45 and SR45a as promising candidates for involvement in ABA-mediated stress responses. PMID:25268622

Dehydration-responsive nuclear proteome landscape of chickpea (Cicer arietinum L.) reveals phosphorylation-mediated regulation of stress response.

PubMed

Barua, Pragya; Lande, Nilesh Vikram; Subba, Pratigya; Gayen, Dipak; Pinto, Sneha; Prasad, T S Keshav; Chakraborty, Subhra; Chakraborty, Niranjan

2018-05-10

Non-availability of water or dehydration remains recurring climatic disorder affecting yield of major food crops, legumes in particular. Nuclear proteins (NP) and phosphoproteins (NPPs) execute crucial cellular functions that form the regulatory hub for coordinated stress response. Phosphoproteins hold enormous influence over cellular signalling. Four-week-old seedlings of a grain legume, chickpea, were subjected to gradual dehydration and nuclear proteins were extracted from unstressed control as well as from 72 and 144 h stressed tissues. We identified 4832 NPs and 478 phosphosites, corresponding to 299 unique NPPs involved in multivariate cellular processes including protein modification and gene expression regulation, among others. The identified proteins included several novel kinases, phosphatases and transcription factors, besides 660 uncharacterised proteins. Spliceosome complex and splicing related proteins were dominant among differentially regulated NPPs, indicating their dehydration modulated regulation. Phospho-motif analysis revealed stress-induced enrichment of proline-directed serine phosphorylation. Association mapping of NPPs revealed predominance of differential phosphorylation of spliceosome and splicing associated proteins. Also, regulatory proteins of key processes viz., protein degradation, regulation of flowering time and circadian clock were observed to undergo dehydration-induced dephosphorylation. The characterization of novel regulatory proteins would provide new insights into stress adaptation and enable directed genetic manipulations for developing climate-resilient crops. This article is protected by copyright. All rights reserved.

MITA/STING and Its Alternative Splicing Isoform MRP Restrict Hepatitis B Virus Replication

PubMed Central

Liu, Shuhui; Zhao, Kaitao; Su, Xi; Lu, Lu; Zhao, He; Zhang, Xianwen; Wang, Yun; Wu, Chunchen; Chen, Jizheng; Zhou, Yuan; Hu, Xue; Wang, Yanyi; Lu, Mengji; Chen, Xinwen; Pei, Rongjuan

2017-01-01

An efficient clearance of hepatitis B virus (HBV) requires the coordinated work of both the innate and adaptive immune responses. MITA/STING, an adapter protein of the innate immune signaling pathways, plays a key role in regulating innate and adaptive immune responses to DNA virus infection. Previously, we identified an alternatively spliced isoform of MITA/STING, called MITA-related protein (MRP), and found that MRP could specifically block MITA-mediated interferon (IFN) induction while retaining the ability to activate NF-κB. Here, we asked whether MITA/STING and MRP were able to control the HBV replication. Both MITA/STING and MRP significantly inhibited HBV replication in vitro. MITA overexpression stimulated IRF3-IFN pathway; while MRP overexpression activated NF-κB pathway, suggesting these two isoforms may inhibit HBV replication through different ways. Using a hydrodynamic injection (HI) mouse model, we found that HBV replication was reduced following MITA/STING and MRP expression vectors in mice and was enhanced by the knockout of MITA/STING (MITA/STING-/-). The HBV specific humoral and CD8+ T cell responses were impaired in MITA/STING deficient mice, suggesting the participation of MITA/STING in the initiation of host adaptive immune responses. In summary, our data suggest that MITA/STING and MRP contribute to HBV control via modulation of the innate and adaptive responses. PMID:28056087

MITA/STING and Its Alternative Splicing Isoform MRP Restrict Hepatitis B Virus Replication.

PubMed

Liu, Shuhui; Zhao, Kaitao; Su, Xi; Lu, Lu; Zhao, He; Zhang, Xianwen; Wang, Yun; Wu, Chunchen; Chen, Jizheng; Zhou, Yuan; Hu, Xue; Wang, Yanyi; Lu, Mengji; Chen, Xinwen; Pei, Rongjuan

2017-01-01

An efficient clearance of hepatitis B virus (HBV) requires the coordinated work of both the innate and adaptive immune responses. MITA/STING, an adapter protein of the innate immune signaling pathways, plays a key role in regulating innate and adaptive immune responses to DNA virus infection. Previously, we identified an alternatively spliced isoform of MITA/STING, called MITA-related protein (MRP), and found that MRP could specifically block MITA-mediated interferon (IFN) induction while retaining the ability to activate NF-κB. Here, we asked whether MITA/STING and MRP were able to control the HBV replication. Both MITA/STING and MRP significantly inhibited HBV replication in vitro. MITA overexpression stimulated IRF3-IFN pathway; while MRP overexpression activated NF-κB pathway, suggesting these two isoforms may inhibit HBV replication through different ways. Using a hydrodynamic injection (HI) mouse model, we found that HBV replication was reduced following MITA/STING and MRP expression vectors in mice and was enhanced by the knockout of MITA/STING (MITA/STING-/-). The HBV specific humoral and CD8+ T cell responses were impaired in MITA/STING deficient mice, suggesting the participation of MITA/STING in the initiation of host adaptive immune responses. In summary, our data suggest that MITA/STING and MRP contribute to HBV control via modulation of the innate and adaptive responses.

A mechanism underlying position-specific regulation of alternative splicing

PubMed Central

Hamid, Fursham M.

2017-01-01

Abstract Many RNA-binding proteins including a master regulator of splicing in developing brain and muscle, polypyrimidine tract-binding protein 1 (PTBP1), can either activate or repress alternative exons depending on the pre-mRNA recruitment position. When bound upstream or within regulated exons PTBP1 tends to promote their skipping, whereas binding to downstream sites often stimulates inclusion. How this switch is orchestrated at the molecular level is poorly understood. Using bioinformatics and biochemical approaches we show that interaction of PTBP1 with downstream intronic sequences can activate natural cassette exons by promoting productive docking of the spliceosomal U1 snRNP to a suboptimal 5′ splice site. Strikingly, introducing upstream PTBP1 sites to this circuitry leads to a potent splicing repression accompanied by the assembly of an exonic ribonucleoprotein complex with a tightly bound U1 but not U2 snRNP. Our data suggest a molecular mechanism underlying the transition between a better-known repressive function of PTBP1 and its role as a bona fide splicing activator. More generally, we argue that the functional outcome of individual RNA contacts made by an RNA-binding protein is subject to extensive context-specific modulation.

Comprehensive proteomic analysis of the human spliceosome

NASA Astrophysics Data System (ADS)

Zhou, Zhaolan; Licklider, Lawrence J.; Gygi, Steven P.; Reed, Robin

2002-09-01

The precise excision of introns from pre-messenger RNA is performed by the spliceosome, a macromolecular machine containing five small nuclear RNAs and numerous proteins. Much has been learned about the protein components of the spliceosome from analysis of individual purified small nuclear ribonucleoproteins and salt-stable spliceosome `core' particles. However, the complete set of proteins that constitutes intact functional spliceosomes has yet to be identified. Here we use maltose-binding protein affinity chromatography to isolate spliceosomes in highly purified and functional form. Using nanoscale microcapillary liquid chromatography tandem mass spectrometry, we identify ~145 distinct spliceosomal proteins, making the spliceosome the most complex cellular machine so far characterized. Our spliceosomes comprise all previously known splicing factors and 58 newly identified components. The spliceosome contains at least 30 proteins with known or putative roles in gene expression steps other than splicing. This complexity may be required not only for splicing multi-intronic metazoan pre-messenger RNAs, but also for mediating the extensive coupling between splicing and other steps in gene expression.

Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing.

PubMed

Zhang, Li; Tran, Ngoc-Tung; Su, Hairui; Wang, Rui; Lu, Yuheng; Tang, Haiping; Aoyagi, Sayura; Guo, Ailan; Khodadadi-Jamayran, Alireza; Zhou, Dewang; Qian, Kun; Hricik, Todd; Côté, Jocelyn; Han, Xiaosi; Zhou, Wenping; Laha, Suparna; Abdel-Wahab, Omar; Levine, Ross L; Raffel, Glen; Liu, Yanyan; Chen, Dongquan; Li, Haitao; Townes, Tim; Wang, Hengbin; Deng, Haiteng; Zheng, Y George; Leslie, Christina; Luo, Minkui; Zhao, Xinyang

2015-11-17

RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood. We demonstrate that RBM15 is methylated by protein arginine methyltransferase 1 (PRMT1) at residue R578, leading to its degradation via ubiquitylation by an E3 ligase (CNOT4). Overexpression of PRMT1 in acute megakaryocytic leukemia cell lines blocks megakaryocyte terminal differentiation by downregulation of RBM15 protein level. Restoring RBM15 protein level rescues megakaryocyte terminal differentiation blocked by PRMT1 overexpression. At the molecular level, RBM15 binds to pre-messenger RNA intronic regions of genes important for megakaryopoiesis such as GATA1, RUNX1, TAL1 and c-MPL. Furthermore, preferential binding of RBM15 to specific intronic regions recruits the splicing factor SF3B1 to the same sites for alternative splicing. Therefore, PRMT1 regulates alternative RNA splicing via reducing RBM15 protein concentration. Targeting PRMT1 may be a curative therapy to restore megakaryocyte differentiation for acute megakaryocytic leukemia.

Domain-based prediction of the human isoform interactome provides insights into the functional impact of alternative splicing.

PubMed

Ghadie, Mohamed Ali; Lambourne, Luke; Vidal, Marc; Xia, Yu

2017-08-01

Alternative splicing is known to remodel protein-protein interaction networks ("interactomes"), yet large-scale determination of isoform-specific interactions remains challenging. We present a domain-based method to predict the isoform interactome from the reference interactome. First, we construct the domain-resolved reference interactome by mapping known domain-domain interactions onto experimentally-determined interactions between reference proteins. Then, we construct the isoform interactome by predicting that an isoform loses an interaction if it loses the domain mediating the interaction. Our prediction framework is of high-quality when assessed by experimental data. The predicted human isoform interactome reveals extensive network remodeling by alternative splicing. Protein pairs interacting with different isoforms of the same gene tend to be more divergent in biological function, tissue expression, and disease phenotype than protein pairs interacting with the same isoforms. Our prediction method complements experimental efforts, and demonstrates that integrating structural domain information with interactomes provides insights into the functional impact of alternative splicing.

Domain-based prediction of the human isoform interactome provides insights into the functional impact of alternative splicing

PubMed Central

Lambourne, Luke; Vidal, Marc

2017-01-01

Alternative splicing is known to remodel protein-protein interaction networks (“interactomes”), yet large-scale determination of isoform-specific interactions remains challenging. We present a domain-based method to predict the isoform interactome from the reference interactome. First, we construct the domain-resolved reference interactome by mapping known domain-domain interactions onto experimentally-determined interactions between reference proteins. Then, we construct the isoform interactome by predicting that an isoform loses an interaction if it loses the domain mediating the interaction. Our prediction framework is of high-quality when assessed by experimental data. The predicted human isoform interactome reveals extensive network remodeling by alternative splicing. Protein pairs interacting with different isoforms of the same gene tend to be more divergent in biological function, tissue expression, and disease phenotype than protein pairs interacting with the same isoforms. Our prediction method complements experimental efforts, and demonstrates that integrating structural domain information with interactomes provides insights into the functional impact of alternative splicing. PMID:28846689

Genomic Context Analysis of de Novo STXBP1 Mutations Identifies Evidence of Splice Site DNA-Motif Associated Hotspots.

PubMed

Uddin, Mohammed; Woodbury-Smith, Marc; Chan, Ada J S; Albanna, Ammar; Minassian, Berge; Boelman, Cyrus; Scherer, Stephen W

2018-03-28

Mutations within STXBP1 have been associated with a range of neurodevelopmental disorders implicating the pleotropic impact of this gene. Although the frequency of de novo mutations within STXBP1 for selective cohorts with early onset epileptic encephalopathy is more than 1%, there is no evidence for a hotspot within the gene. In this study, we analyzed the genomic context of de novo STXBP1 mutations to examine whether certain motifs indicated a greater risk of mutation. Through a comprehensive context analysis of 136 de novo /rare mutation (SNV/Indels) sites in this gene, strikingly 26.92% of all SNV mutations occurred within 5bp upstream or downstream of a 'GTA' motif ( P < 0.0005). This implies a genomic context modulated mutagenesis. Moreover, 51.85% (14 out of 27) of the 'GTA' mutations are splicing compared to 14.70% (20 out of 136) of all reported mutations within STXBP1 We also noted that 11 of these 14 'GTA' associated mutations are de novo in origin. Our analysis provides strong evidence of DNA motif modulated mutagenesis for STXBP1 de novo splicing mutations. Copyright © 2018 Uddin et al.

Unfolded protein response transducer IRE1-mediated signaling independent of XBP1 mRNA splicing is not required for growth and development of medaka fish

PubMed Central

Ishikawa, Tokiro; Kashima, Makoto; Nagano, Atsushi J; Ishikawa-Fujiwara, Tomoko; Kamei, Yasuhiro; Todo, Takeshi

2017-01-01

When activated by the accumulation of unfolded proteins in the endoplasmic reticulum, metazoan IRE1, the most evolutionarily conserved unfolded protein response (UPR) transducer, initiates unconventional splicing of XBP1 mRNA. Unspliced and spliced mRNA are translated to produce pXBP1(U) and pXBP1(S), respectively. pXBP1(S) functions as a potent transcription factor, whereas pXBP1(U) targets pXBP1(S) to degradation. In addition, activated IRE1 transmits two signaling outputs independent of XBP1, namely activation of the JNK pathway, which is initiated by binding of the adaptor TRAF2 to phosphorylated IRE1, and regulated IRE1-dependent decay (RIDD) of various mRNAs in a relatively nonspecific manner. Here, we conducted comprehensive and systematic genetic analyses of the IRE1-XBP1 branch of the UPR using medaka fish and found that the defects observed in XBP1-knockout or IRE1-knockout medaka were fully rescued by constitutive expression of pXBP1(S). Thus, the JNK and RIDD pathways are not required for the normal growth and development of medaka. The unfolded protein response sensor/transducer IRE1-mediated splicing of XBP1 mRNA encoding its active downstream transcription factor to maintain the homeostasis of the endoplasmic reticulum is sufficient for growth and development of medaka fish. PMID:28952924

Nuclear degradation of Wilms tumor 1-associating protein and survivin splice variant switching underlie IGF-1-mediated survival.

PubMed

Small, Theodore W; Pickering, J Geoffrey

2009-09-11

WTAP (Wilms tumor 1-associating protein) is a recently identified nuclear protein that is essential for mouse embryo development. The Drosophila homolog of WTAP, Fl(2)d, regulates pre-mRNA splicing; however, the role of WTAP in mammalian cells is uncertain. To elucidate a context for WTAP action, we screened growth and survival factors for their effects on WTAP expression in vascular smooth muscle cells (SMCs), a cell type previously found to express WTAP dynamically. This revealed that insulin-like growth factor-1 (IGF-1) uniquely reduced WTAP abundance. This decline in WTAP proved to be necessary for IGF-1 to confer its antiapoptotic properties, which were blocked by transducing the WTAP gene into SMCs. WTAP down-regulation by IGF-1 was mediated by an IGF-1 receptor-phosphatidylinositol 3-kinase-Akt signaling axis that directed WTAP degradation via a nuclear 26 S proteasome. Moreover, by promoting the degradation of WTAP, IGF-1 shifted the pre-mRNA splicing program for the survival factor, survivin, to reduce expression of survivin-2B, which is proapoptotic, and increase expression of survivin, which is antiapoptotic. Knockdown of survivin-2B rescued the ability of IGF-1 to promote survival when WTAP was overexpressed. These data uncover a novel regulatory cascade for human SMC survival based on adjusting the nuclear abundance of WTAP to define the splice variant balance among survivin isoforms.

HnRNP L and L-like cooperate in multiple-exon regulation of CD45 alternative splicing

PubMed Central

Preußner, Marco; Schreiner, Silke; Hung, Lee-Hsueh; Porstner, Martina; Jäck, Hans-Martin; Benes, Vladimir; Rätsch, Gunnar; Bindereif, Albrecht

2012-01-01

CD45 encodes a trans-membrane protein-tyrosine phosphatase expressed in diverse cells of the immune system. By combinatorial use of three variable exons 4–6, isoforms are generated that differ in their extracellular domain, thereby modulating phosphatase activity and immune response. Alternative splicing of these CD45 exons involves two heterogeneous ribonucleoproteins, hnRNP L and its cell-type specific paralog hnRNP L-like (LL). To address the complex combinatorial splicing of exons 4–6, we investigated hnRNP L/LL protein expression in human B-cells in relation to CD45 splicing patterns, applying RNA-Seq. In addition, mutational and RNA-binding analyses were carried out in HeLa cells. We conclude that hnRNP LL functions as the major CD45 splicing repressor, with two CA elements in exon 6 as its primary target. In exon 4, one element is targeted by both hnRNP L and LL. In contrast, exon 5 was never repressed on its own and only co-regulated with exons 4 and 6. Stable L/LL interaction requires CD45 RNA, specifically exons 4 and 6. We propose a novel model of combinatorial alternative splicing: HnRNP L and LL cooperate on the CD45 pre-mRNA, bridging exons 4 and 6 and looping out exon 5, thereby achieving full repression of the three variable exons. PMID:22402488

Gene silencing by siRNAs and antisense oligonucleotides in the laboratory and the clinic

PubMed Central

Watts, Jonathan K.; Corey, David R.

2014-01-01

Synthetic nucleic acids are commonly used laboratory tools for modulating gene expression and have the potential to be widely used in the clinic. Progress towards nucleic acid drugs, however, has been slow and many challenges remain to be overcome before their full impact on patient care can be understood. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are the two most widely used strategies for silencing gene expression. We first describe these two approaches and contrast their relative strengths and weaknesses for laboratory applications. We then review the choices faced during development of clinical candidates and the current state of clinical trials. Attitudes towards clinical development of nucleic acid silencing strategies have repeatedly swung from optimism to depression during the past twenty years. Our goal is to provide the information needed to design robust studies with oligonucleotides, making use of the strengths of each oligonucleotide technology. PMID:22069063

Structure and Function of the Two Tandem WW Domains of the Pre-mRNA Splicing Factor FBP21 (Formin-binding Protein 21)*

PubMed Central

Huang, Xiaojuan; Beullens, Monique; Zhang, Jiahai; Zhou, Yi; Nicolaescu, Emilia; Lesage, Bart; Hu, Qi; Wu, Jihui; Bollen, Mathieu; Shi, Yunyu

2009-01-01

Human FBP21 (formin-binding protein 21) contains a matrin-type zinc finger and two tandem WW domains. It is a component of the spliceosomes and interacts with several established splicing factors. Here we demonstrate for the first time that FBP21 is an activator of pre-mRNA splicing in vivo and that its splicing activation function and interaction with the splicing factor SIPP1 (splicing factor that interacts with PQBP1 and PP1) are both mediated by the two tandem WW domains of group III. We determined the solution structure of the tandem WW domains of FBP21 and found that the WW domains recognize peptide ligands containing either group II (PPLP) or group III (PPR) motifs. The binding interfaces involve both the XP and XP2 grooves of the two WW domains. Significantly, the tandem WW domains of FBP21 are connected by a highly flexible region, enabling their simultaneous interaction with two proline-rich motifs of SIPP1. The strong interaction between SIPP1 and FBP21 can be explained by the conjugation of two low affinity interactions with the tandem WW domains. Our study provides a structural basis for understanding the molecular mechanism underlying the functional implication of FBP21 and the biological specificity of tandem WW domains. PMID:19592703

Partial androgen insensitivity syndrome caused by a deep intronic mutation creating an alternative splice acceptor site of the AR gene.

PubMed

Ono, Hiroyuki; Saitsu, Hirotomo; Horikawa, Reiko; Nakashima, Shinichi; Ohkubo, Yumiko; Yanagi, Kumiko; Nakabayashi, Kazuhiko; Fukami, Maki; Fujisawa, Yasuko; Ogata, Tsutomu

2018-02-02

Although partial androgen insensitivity syndrome (PAIS) is caused by attenuated responsiveness to androgens, androgen receptor gene (AR) mutations on the coding regions and their splice sites have been identified only in <25% of patients with a diagnosis of PAIS. We performed extensive molecular studies including whole exome sequencing in a Japanese family with PAIS, identifying a deep intronic variant beyond the branch site at intron 6 of AR (NM_000044.4:c.2450-42 G > A). This variant created the splice acceptor motif that was accompanied by pyrimidine-rich sequence and two candidate branch sites. Consistent with this, reverse transcriptase (RT)-PCR experiments for cycloheximide-treated lymphoblastoid cell lines revealed a relatively large amount of aberrant mRNA produced by the newly created splice acceptor site and a relatively small amount of wildtype mRNA produced by the normal splice acceptor site. Furthermore, most of the aberrant mRNA was shown to undergo nonsense mediated decay (NMD) and, if a small amount of aberrant mRNA may have escaped NMD, such mRNA was predicted to generate a truncated AR protein missing some functional domains. These findings imply that the deep intronic mutation creating an alternative splice acceptor site resulted in the production of a relatively small amount of wildtype AR mRNA, leading to PAIS.

Exon Specific U1 snRNAs improve ELP1 exon 20 definition and rescue ELP1 protein expression in a Familial Dysautonomia mouse model.

PubMed

Donadon, Irving; Pinotti, Mirko; Rajkowska, Katarzyna; Pianigiani, Giulia; Barbon, Elena; Morini, Elisabetta; Motaln, Helena; Rogelj, Boris; Mingozzi, Federico; Slaugenhaupt, Susan A; Pagani, Franco

2018-04-25

Familial dysautonomia (FD) is a rare genetic disease with no treatment, caused by an intronic point mutation (c.2204 + 6T>C) that negatively affects the definition of exon 20 in the Elongator complex protein 1 gene (ELP1 also known as IKBKAP). This substitution modifies the 5' splice site and, in combination with regulatory splicing factors, induces different levels of exon 20 skipping, in various tissues. Here, we evaluated the therapeutic potential of a novel class of U1 snRNA molecules, Exon-Specific U1s (ExSpeU1s), in correcting ELP1 exon 20 recognition. Lentivirus-mediated expression of ELP1-ExSpeU1 in FD fibroblasts improved ELP1 splicing and protein levels. We next focused on a transgenic mouse model that recapitulates the same tissue-specific mis-splicing seen in FD patients. Intraperitoneal delivery of ELP1-ExSpeU1s-adeno-associated virus particles successfully increased the production of full-length human ELP1 transcript and protein. This splice-switching class of molecules is the first to specifically correct the ELP1 exon 20 splicing defect. Our data provide proof of principle of ExSpeU1s-adeno-associated virus particles as a novel therapeutic strategy for FD.

Gene-breaking: A new paradigm for human retrotransposon-mediated gene evolution

PubMed Central

Wheelan, Sarah J.; Aizawa, Yasunori; Han, Jeffrey S.; Boeke, Jef D.

2005-01-01

The L1 retrotransposon is the most highly successful autonomous retrotransposon in mammals. This prolific genome parasite may on occasion benefit its host through genome rearrangements or adjustments of host gene expression. In examining possible effects of L1 elements on host gene expression, we investigated whether a full-length L1 element inserted in the antisense orientation into an intron of a cellular gene may actually split the gene's transcript into two smaller transcripts: (1) a transcript containing the upstream exons and terminating in the major antisense polyadenylation site (MAPS) of the L1, and (2) a transcript derived from the L1 antisense promoter (ASP) that includes the downstream exons of the gene. Bioinformatic analysis and experimental follow-up provide evidence for this L1 “gene-breaking” hypothesis. We identified three human genes apparently “broken” by L1 elements, as well as 12 more candidate genes. Most of the inserted L1 elements in our 15 candidate genes predate the human/chimp divergence. If indeed split, the transcripts of these genes may in at least one case encode potentially interacting proteins, and in another case may encode novel proteins. Gene-breaking represents a new mechanism through which L1 elements remodel mammalian genomes. PMID:16024818

Using in-cell SHAPE-Seq and simulations to probe structure–function design principles of RNA transcriptional regulators

PubMed Central

Takahashi, Melissa K.; Watters, Kyle E.; Gasper, Paul M.; Abbott, Timothy R.; Carlson, Paul D.; Chen, Alan A.

2016-01-01

Antisense RNA-mediated transcriptional regulators are powerful tools for controlling gene expression and creating synthetic gene networks. RNA transcriptional repressors derived from natural mechanisms called attenuators are particularly versatile, though their mechanistic complexity has made them difficult to engineer. Here we identify a new structure–function design principle for attenuators that enables the forward engineering of new RNA transcriptional repressors. Using in-cell SHAPE-Seq to characterize the structures of attenuator variants within Escherichia coli, we show that attenuator hairpins that facilitate interaction with antisense RNAs require interior loops for proper function. Molecular dynamics simulations of these attenuator variants suggest these interior loops impart structural flexibility. We further observe hairpin flexibility in the cellular structures of natural RNA mechanisms that use antisense RNA interactions to repress translation, confirming earlier results from in vitro studies. Finally, we design new transcriptional attenuators in silico using an interior loop as a structural requirement and show that they function as desired in vivo. This work establishes interior loops as an important structural element for designing synthetic RNA gene regulators. We anticipate that the coupling of experimental measurement of cellular RNA structure and function with computational modeling will enable rapid discovery of structure–function design principles for a diverse array of natural and synthetic RNA regulators. PMID:27103533

REM sleep enhancement and behavioral cataplexy following orexin (hypocretin)-II receptor antisense perfusion in the pontine reticular formation.

PubMed

Thakkar, M M; Ramesh, V; Cape, E G; Winston, S; Strecker, R E; McCarley, R W

1999-01-01

Orexin (hypocretin)-containing neurons of the hypothalamus project to brainstem sites that are involved in the neural control of REM sleep, including the locus coeruleus, the dorsal raphe nucleus, the cholinergic zone of the mesopontine tegmentum, and the pontine reticular formation (PRF). Orexin knockout mice exhibit narcolepsy/cataplexy, and a mutant and defective gene for the orexin type II receptor is present in dogs with an inherited form of narcolepsy/cataplexy. However, the physiological systems mediating these effects have not been described. We reasoned that, since the effector neurons for the majority of REM sleep signs, including muscle atonia, were located in the PRF, this region was likely implicated in the production of these orexin-related abnormalities. To test this possibility, we used microdialysis perfusion of orexin type II receptor antisense in the PRF of rats. Ten to 24 hours after antisense perfusion, REM sleep increased two- to three-fold during both the light period (quiescent phase) and the dark period (active phase), and infrared video showed episodes of behavioral cataplexy. Moreover, preliminary data indicated no REM-related effects following perfusion with nonsense DNA, or when perfusion sites were outside the PRF. More work is needed to provide precise localization of the most effective site of orexin-induced inhibition of REM sleep phenomena.

Inhibition of connective tissue growth factor (CTGF/CCN2) expression decreases the survival and myogenic differentiation of human rhabdomyosarcoma cells.

PubMed

Croci, Stefania; Landuzzi, Lorena; Astolfi, Annalisa; Nicoletti, Giordano; Rosolen, Angelo; Sartori, Francesca; Follo, Matilde Y; Oliver, Noelynn; De Giovanni, Carla; Nanni, Patrizia; Lollini, Pier-Luigi

2004-03-01

Connective tissue growth factor (CTGF/CCN2), a cysteine-rich protein of the CCN (Cyr61, CTGF, Nov) family of genes, emerged from a microarray screen of genes expressed by human rhabdomyosarcoma cells. Rhabdomyosarcoma is a soft tissue sarcoma of childhood deriving from skeletal muscle cells. In this study, we investigated the role of CTGF in rhabdomyosarcoma. Human rhabdomyosarcoma cells of the embryonal (RD/12, RD/18, CCA) and the alveolar histotype (RMZ-RC2, SJ-RH4, SJ-RH30), rhabdomyosarcoma tumor specimens, and normal skeletal muscle cells expressed CTGF. To determine the function of CTGF, we treated rhabdomyosarcoma cells with a CTGF antisense oligonucleotide or with a CTGF small interfering RNA (siRNA). Both treatments inhibited rhabdomyosarcoma cell growth, suggesting the existence of a new autocrine loop based on CTGF. CTGF antisense oligonucleotide-mediated growth inhibition was specifically due to a significant increase in apoptosis, whereas cell proliferation was unchanged. CTGF antisense oligonucleotide induced a strong decrease in the level of myogenic differentiation of rhabdomyosarcoma cells, whereas the addition of recombinant CTGF significantly increased the proportion of myosin-positive cells. CTGF emerges as a survival and differentiation factor and could be a new therapeutic target in human rhabdomyosarcoma.

Antisense reduction of tau in adult mice protects against seizures.

PubMed

DeVos, Sarah L; Goncharoff, Dustin K; Chen, Guo; Kebodeaux, Carey S; Yamada, Kaoru; Stewart, Floy R; Schuler, Dorothy R; Maloney, Susan E; Wozniak, David F; Rigo, Frank; Bennett, C Frank; Cirrito, John R; Holtzman, David M; Miller, Timothy M

2013-07-31

Tau, a microtubule-associated protein, is implicated in the pathogenesis of Alzheimer's Disease (AD) in regard to both neurofibrillary tangle formation and neuronal network hyperexcitability. The genetic ablation of tau substantially reduces hyperexcitability in AD mouse lines, induced seizure models, and genetic in vivo models of epilepsy. These data demonstrate that tau is an important regulator of network excitability. However, developmental compensation in the genetic tau knock-out line may account for the protective effect against seizures. To test the efficacy of a tau reducing therapy for disorders with a detrimental hyperexcitability profile in adult animals, we identified antisense oligonucleotides that selectively decrease endogenous tau expression throughout the entire mouse CNS--brain and spinal cord tissue, interstitial fluid, and CSF--while having no effect on baseline motor or cognitive behavior. In two chemically induced seizure models, mice with reduced tau protein had less severe seizures than control mice. Total tau protein levels and seizure severity were highly correlated, such that those mice with the most severe seizures also had the highest levels of tau. Our results demonstrate that endogenous tau is integral for regulating neuronal hyperexcitability in adult animals and suggest that an antisense oligonucleotide reduction of tau could benefit those with epilepsy and perhaps other disorders associated with tau-mediated neuronal hyperexcitability.

Antisense transcriptional interference mediates condition-specific gene repression in budding yeast.

PubMed

Nevers, Alicia; Doyen, Antonia; Malabat, Christophe; Néron, Bertrand; Kergrohen, Thomas; Jacquier, Alain; Badis, Gwenael

2018-05-18

Pervasive transcription generates many unstable non-coding transcripts in budding yeast. The transcription of such noncoding RNAs, in particular antisense RNAs (asRNAs), has been shown in a few examples to repress the expression of the associated mRNAs. Yet, such mechanism is not known to commonly contribute to the regulation of a given class of genes. Using a mutant context that stabilized pervasive transcripts, we observed that the least expressed mRNAs during the exponential phase were associated with high levels of asRNAs. These asRNAs also overlapped their corresponding gene promoters with a much higher frequency than average. Interrupting antisense transcription of a subset of genes corresponding to quiescence-enriched mRNAs restored their expression. The underlying mechanism acts in cis and involves several chromatin modifiers. Our results convey that transcription interference represses up to 30% of the 590 least expressed genes, which includes 163 genes with quiescence-enriched mRNAs. We also found that pervasive transcripts constitute a higher fraction of the transcriptome in quiescence relative to the exponential phase, consistent with gene expression itself playing an important role to suppress pervasive transcription. Accordingly, the HIS1 asRNA, normally only present in quiescence, is expressed in exponential phase upon HIS1 mRNA transcription interruption.

Different effects of antisense RelA p65 and NF-kappaB1 p50 oligonucleotides on the nuclear factor-kappaB mediated expression of ICAM-1 in human coronary endothelial and smooth muscle cells.

PubMed

Voisard, R; Huber, N; Baur, R; Susa, M; Ickrath, O; Both, A; Koenig, W; Hombach, V

2001-01-01

Activation of nuclear factor-kappaB (NF-kappaB) is one of the key events in early atherosclerosis and restenosis. We hypothesized that tumor necrosis factor-alpha (TNF-alpha) induced and NF-kappaB mediated expression of intercellular adhesion molecule-1 (ICAM-1) can be inhibited by antisense RelA p65 and NF-kappaB1 p50 oligonucleotides (RelA p65 and NF-kappaB1 p50). Smooth muscle cells (SMC) from human coronary plaque material (HCPSMC, plaque material of 52 patients), SMC from the human coronary media (HCMSMC), human endothelial cells (EC) from umbilical veins (HUVEC), and human coronary EC (HCAEC) were successfully isolated (HCPSMC, HUVEC), identified and cultured (HCPSMC, HCMSMC, HUVEC, HCAEC). 12 hrs prior to TNF-alpha stimulus (20 ng/mL, 6 hrs) RelA p65 and NF-kappaB1 p50 (1, 2, 4, 10, 20, and 30 microM) and controls were added for a period of 18 hrs. In HUVEC and HCAEC there was a dose dependent inhibition of ICAM-1 expression after adding of both RelA p65 and NF-kappaB1 p50. No inhibitory effect was seen after incubation of HCMSMC with RelA p65 and NF-kappaB1 p50. A moderate inhibition of ICAM-1 expression was found after simultaneous addition of RelA p65 and NF-kappaB1 p50 to HCPSMC, no inhibitory effect was detected after individual addition of RelA p65 and NF-kappaB1 p50. The data point out that differences exist in the NF-kappaB mediated expression of ICAM-1 between EC and SMC. Experimental antisense strategies directed against RelA p65 and NF-kappaB1 p50 in early atherosclerosis and restenosis are promising in HCAEC but will be confronted with redundant pathways in HCMSMC and HCPSMC.

mTORβ Splicing Isoform Promotes Cell Proliferation and Tumorigenesis*

PubMed Central

Panasyuk, Ganna; Nemazanyy, Ivan; Zhyvoloup, Aleksander; Filonenko, Valeriy; Davies, Derek; Robson, Mathew; Pedley, R. Barbara; Waterfield, Michael; Gout, Ivan

2009-01-01

The mTOR (mammalian target of rapamycin) promotes growth in response to nutrients and growth factors and is deregulated in numerous pathologies, including cancer. The mechanisms by which mTOR senses and regulates energy metabolism and cell growth are relatively well understood, whereas the molecular events underlining how it mediates survival and proliferation remain to be elucidated. Here, we describe the existence of the mTOR splicing isoform, TORβ, which, in contrast to the full-length protein (mTORα), has the potential to regulate the G1 phase of the cell cycle and to stimulate cell proliferation. mTORβ is an active protein kinase that mediates downstream signaling through complexing with Rictor and Raptor proteins. Remarkably, overexpression of mTORβ transforms immortal cells and is tumorigenic in nude mice and therefore could be a proto-oncogene. PMID:19726679

Strawberry: Fast and accurate genome-guided transcript reconstruction and quantification from RNA-Seq.

PubMed

Liu, Ruolin; Dickerson, Julie

2017-11-01

We propose a novel method and software tool, Strawberry, for transcript reconstruction and quantification from RNA-Seq data under the guidance of genome alignment and independent of gene annotation. Strawberry consists of two modules: assembly and quantification. The novelty of Strawberry is that the two modules use different optimization frameworks but utilize the same data graph structure, which allows a highly efficient, expandable and accurate algorithm for dealing large data. The assembly module parses aligned reads into splicing graphs, and uses network flow algorithms to select the most likely transcripts. The quantification module uses a latent class model to assign read counts from the nodes of splicing graphs to transcripts. Strawberry simultaneously estimates the transcript abundances and corrects for sequencing bias through an EM algorithm. Based on simulations, Strawberry outperforms Cufflinks and StringTie in terms of both assembly and quantification accuracies. Under the evaluation of a real data set, the estimated transcript expression by Strawberry has the highest correlation with Nanostring probe counts, an independent experiment measure for transcript expression. Strawberry is written in C++14, and is available as open source software at https://github.com/ruolin/strawberry under the MIT license.

The antiphasic regulatory module comprising CDF5 and its antisense RNA FLORE links the circadian clock to photoperiodic flowering.

PubMed

Henriques, Rossana; Wang, Huan; Liu, Jun; Boix, Marc; Huang, Li-Fang; Chua, Nam-Hai

2017-11-01

Circadian rhythms of gene expression are generated by the combinatorial action of transcriptional and translational feedback loops as well as chromatin remodelling events. Recently, long noncoding RNAs (lncRNAs) that are natural antisense transcripts (NATs) to transcripts encoding central oscillator components were proposed as modulators of core clock function in mammals (Per) and fungi (frq/qrf). Although oscillating lncRNAs exist in plants, their functional characterization is at an initial stage. By screening an Arabidopsis thaliana lncRNA custom-made array we identified CDF5 LONG NONCODING RNA (FLORE), a circadian-regulated lncRNA that is a NAT of CDF5. Quantitative real-time RT-PCR confirmed the circadian regulation of FLORE, whereas GUS-staining and flowering time evaluation were used to determine its biological function. FLORE and CDF5 antiphasic expression reflects mutual inhibition in a similar way to frq/qrf. Moreover, whereas the CDF5 protein delays flowering by directly repressing FT transcription, FLORE promotes it by repressing several CDFs (CDF1, CDF3, CDF5) and increasing FT transcript levels, indicating both cis and trans function. We propose that the CDF5/FLORE NAT pair constitutes an additional circadian regulatory module with conserved (mutual inhibition) and unique (function in trans) features, able to fine-tune its own circadian oscillation, and consequently, adjust the onset of flowering to favourable environmental conditions. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

A Predictive Model of Intein Insertion Site for Use in the Engineering of Molecular Switches

PubMed Central

Apgar, James; Ross, Mary; Zuo, Xiao; Dohle, Sarah; Sturtevant, Derek; Shen, Binzhang; de la Vega, Humberto; Lessard, Philip; Lazar, Gabor; Raab, R. Michael

2012-01-01

Inteins are intervening protein domains with self-splicing ability that can be used as molecular switches to control activity of their host protein. Successfully engineering an intein into a host protein requires identifying an insertion site that permits intein insertion and splicing while allowing for proper folding of the mature protein post-splicing. By analyzing sequence and structure based properties of native intein insertion sites we have identified four features that showed significant correlation with the location of the intein insertion sites, and therefore may be useful in predicting insertion sites in other proteins that provide native-like intein function. Three of these properties, the distance to the active site and dimer interface site, the SVM score of the splice site cassette, and the sequence conservation of the site showed statistically significant correlation and strong predictive power, with area under the curve (AUC) values of 0.79, 0.76, and 0.73 respectively, while the distance to secondary structure/loop junction showed significance but with less predictive power (AUC of 0.54). In a case study of 20 insertion sites in the XynB xylanase, two features of native insertion sites showed correlation with the splice sites and demonstrated predictive value in selecting non-native splice sites. Structural modeling of intein insertions at two sites highlighted the role that the insertion site location could play on the ability of the intein to modulate activity of the host protein. These findings can be used to enrich the selection of insertion sites capable of supporting intein splicing and hosting an intein switch. PMID:22649521

CYP3A5 mRNA degradation by nonsense-mediated mRNA decay.

PubMed

Busi, Florent; Cresteil, Thierry

2005-09-01

The total CYP3A5 mRNA level is significantly greater in carriers of the CYP3A5*1 allele than in CYP3A5*3 homozygotes. Most of the CYP3A5*3 mRNA includes an intronic sequence (exon 3B) containing premature termination codons (PTCs) between exons 3 and 4. Two models were used to investigate the degradation of CYP3A5 mRNA: a CYP3A5 minigene consisting of CYP3A5 exons and introns 3 to 6 transfected into MCF7 cells, and the endogenous CYP3A5 gene expressed in HepG2 cells. The 3'-untranslated region g.31611C>T mutation has no effect on CYP3A5 mRNA decay. Splice variants containing exon 3B were more unstable than wild-type (wt) CYP3A5 mRNA. Cycloheximide prevents the recognition of PTCs by ribosomes: in transfected MCF7 and HepG2 cells, cycloheximide slowed down the degradation of exon 3B-containing splice variants, suggesting the participation of nonsense-mediated decay (NMD). When PTCs were removed from pseudoexon 3B or when UPF1 small interfering RNA was used to impair the NMD mechanism, the decay of the splice variant was reduced, confirming the involvement of NMD in the degradation of CYP3A5 splice variants. Induction could represent a source of variability for CYP3A5 expression and could modify the proportion of splice variants. The extent of CYP3A5 induction was investigated after exposure to barbiturates or steroids: CYP3A4 was markedly induced in a pediatric population compared with untreated neonates. However, no effect could be detected in either the total CYP3A5 RNA, the proportion of splice variant RNA, or the protein level. Therefore, in these carriers, induction is unlikely to switch on the phenotypic CYP3A5 expression in carriers of CYP3A5*3/*3.

Inducible knock-down of GNOM during root formation reveals tissue-specific response to auxin transport and its modulation of local auxin biosynthesis

PubMed Central

Sun, Meng-Xiang

2014-01-01

In plants, active transport of auxin plays an essential role in root development. Localization of the PIN1 auxin transporters to the basal membrane of cells directs auxin flow and depends on the trafficking mediator GNOM. GNOM-dependent auxin transport is vital for root development and thus offers a useful tool for the investigation of a possible tissue-specific response to dynamic auxin transport. To avoid pleiotropic effects, DEX-inducible expression of GNOM antisense RNA was used to disrupt GNOM expression transiently or persistently during embryonic root development. It was found that the elongation zone and the pericycle layer are the most sensitive to GNOM-dependent auxin transport variations, which is shown by the phenotypes in cell elongation and the initiation of lateral root primordia, respectively. This suggests that auxin dynamics is critical to cell differentiation and cell fate transition, but not to cell division. The results also reveal that GNOM-dependent auxin transport could affect local auxin biosynthesis. This suggests that local auxin biosynthesis may also contribute to the establishment of GNOM-dependent auxin gradients in specific tissues, and that auxin transport and local auxin biosynthesis may function together in the regulatory network for initiation and development of lateral root primordia. Thus, the data reveal a tissue-specific response to auxin transport and modulation of local auxin biosynthesis by auxin transport. PMID:24453227

Characterization of prmt7alpha and beta isozymes from Chinese hamster cells sensitive and resistant to topoisomerase II inhibitors.

PubMed

Gros, Laurent; Renodon-Cornière, Axelle; de Saint Vincent, Bruno Robert; Feder, Marcin; Bujnicki, Janusz M; Jacquemin-Sablon, Alain

2006-11-01

By selection of genetic suppressor elements (GSEs) conferring resistance to topoisomerase II inhibitors in Chinese hamster cells (DC-3F), we identified a gene encoding two proteins of 78 and 82 kDa which belong to the protein arginine methyltransferase (PRMT) family. Down-regulation of these enzymes (named PRMT7alpha and beta), either induced by an antisense GSE or as observed in the 9-OH-ellipticine (9-OH-E) resistant mutant DC-3F/9-OH-E, was responsible for cell resistance to various DNA damaging agents. Alternative splicing alterations in the 5'-terminal region and changes of the polyadenylation site of PRMT7 mRNAs were observed in these resistant mutant cells. PRMT7alpha and beta are isoforms of a highly conserved protein containing two copies of a module common to all PRMTs, comprising a Rossmann-fold domain and a beta-barrel domain. The C-terminal repeat appears to be degenerate and catalytically inactive. PRMT7alpha and beta form homo- and hetero-dimers but differ by their sub-cellular localization and in vitro recognize different substrates. PRMT7beta was only observed in Chinese hamster cells while mouse 10T1/2 fibroblasts only contain PRMT7alpha. Surprisingly, in human cells the anti-PRMT7 antibody essentially recognized an approximately 37 kDa peptide, which is not formed during extraction, and a faint band at 78 kDa. Analysis of in vitro and in vivo methylation patterns in cell lines under- or over-expressing PRMT7alpha and beta detected a discrete number of proteins which methylation and/or expression are under the control of these enzymes.

Tribolium castaneum Transformer-2 regulates sex determination and development in both males and females

PubMed Central

Shukla, Jayendra Nath; Palli, Subba Reddy

2014-01-01

Tribolium castaneum Transformer (TcTra) is essential for female sex determination and maintenance through the regulation of sex-specific splicing of doublesex (dsx) pre-mRNA. In females, TcTra also regulates the sex-specific splicing of its own pre-mRNA to ensure continuous production of functional Tra protein. Transformer protein is absent in males and hence dsx pre-mRNA is spliced in a default mode. The mechanisms by which males inhibit the production of functional Tra protein are not known. Here, we report on functional characterization of transformer-2 (tra-2) gene (an ortholog of Drosophila transformer-2) in T. castaneum. RNA interference-mediated knockdown in the expression of gene coding for tra-2 in female pupae or adults resulted in the production of male-specific isoform of dsx and both female and male isoforms of tra suggesting that Tra-2 is essential for the female-specific splicing of tra and dsx pre-mRNAs. Interestingly, knockdown of tra-2 in males did not affect the splicing of dsx but resulted in the production of both female and male isoforms of tra suggesting that Tra-2 suppresses female-specific splicing of tra pre-mRNA in males. This dual regulation of sex-specific splicing of tra pre-mRNA ensures a tight regulation of sex determination and maintenance. These data suggest a critical role for Tra-2 in suppression of female sex determination cascade in males. In addition, RNAi studies showed that Tra-2 is also required for successful embryonic and larval development in both sexes. PMID:24056158

Long Noncoding RNA HOTAIR Modulates MiR-206-mediated Bcl-w Signaling to Facilitate Cell Proliferation in Breast Cancer.

PubMed

Ding, Wei; Ren, Jin; Ren, Hui; Wang, Dan

2017-12-08

LncRNA HOX transcript antisense RNA (HOTAIR) is involved in lots of cancers. The pro-survival protein Bcl-w is frequently found in cancer development. However, the effect of HOTAIR on Bcl-w in breast cancer is not well documented. In this study, we first evaluated the correlation between HOTAIR level and Bcl-w expression in clinical breast cancer tissues. We observed that the expression levels of Bcl-w were much higher in the breast cancer samples than that in their paired noncancerous tissues. Moreover, the levels of HOTAIR were positively associated with those of Bcl-w in clinical breast cancer samples. As expected, we observed that HOTAIR was able to up-regulate the expression of Bcl-w in breast cancer cells. Mechanistically, we found that miR-206 was capable of inhibiting the expression of Bcl-w by directly binding to the 3'UTR of Bcl-w mRNA. Interestingly, HOTAIR could increase the expression of Bcl-w through sequestering miR-206 at post-transcriptional level. Functionally, our data showed that HOTAIR-induced Bcl-w by miR-206 facilitated the proliferation of breast cancer cells. Thus, we conclude that HOTAIR up-regulates Bcl-w to enhance cell proliferation through sequestering miR-206 in breast cancer. Our finding provides new insights into the mechanism of breast cancer mediated by HOTAIR.

The Antisense RNA Approach: a New Application for In Vivo Investigation of the Stress Response of Oenococcus oeni, a Wine-Associated Lactic Acid Bacterium

PubMed Central

Darsonval, Maud; Msadek, Tarek; Alexandre, Hervé

2015-01-01

Oenococcus oeni is a wine-associated lactic acid bacterium mostly responsible for malolactic fermentation in wine. In wine, O. oeni grows in an environment hostile to bacterial growth (low pH, low temperature, and ethanol) that induces stress response mechanisms. To survive, O. oeni is known to set up transitional stress response mechanisms through the synthesis of heat stress proteins (HSPs) encoded by the hsp genes, notably a unique small HSP named Lo18. Despite the availability of the genome sequence, characterization of O. oeni genes is limited, and little is known about the in vivo role of Lo18. Due to the lack of genetic tools for O. oeni, an efficient expression vector in O. oeni is still lacking, and deletion or inactivation of the hsp18 gene is not presently practicable. As an alternative approach, with the goal of understanding the biological function of the O. oeni hsp18 gene in vivo, we have developed an expression vector to produce antisense RNA targeting of hsp18 mRNA. Recombinant strains were exposed to multiple stresses inducing hsp18 gene expression: heat shock and acid shock. We showed that antisense attenuation of hsp18 affects O. oeni survival under stress conditions. These results confirm the involvement of Lo18 in heat and acid tolerance of O. oeni. Results of anisotropy experiments also confirm a membrane-protective role for Lo18, as previous observations had already suggested. This study describes a new, efficient tool to demonstrate the use of antisense technology for modulating gene expression in O. oeni. PMID:26452552

Genetic validation of whole-transcriptome sequencing for mapping expression affected by cis-regulatory variation

PubMed Central

2010-01-01

Background Identifying associations between genotypes and gene expression levels using microarrays has enabled systematic interrogation of regulatory variation underlying complex phenotypes. This approach has vast potential for functional characterization of disease states, but its prohibitive cost, given hundreds to thousands of individual samples from populations have to be genotyped and expression profiled, has limited its widespread application. Results Here we demonstrate that genomic regions with allele-specific expression (ASE) detected by sequencing cDNA are highly enriched for cis-acting expression quantitative trait loci (cis-eQTL) identified by profiling of 500 animals in parallel, with up to 90% agreement on the allele that is preferentially expressed. We also observed widespread noncoding and antisense ASE and identified several allele-specific alternative splicing variants. Conclusion Monitoring ASE by sequencing cDNA from as little as one sample is a practical alternative to expression genetics for mapping cis-acting variation that regulates RNA transcription and processing. PMID:20707912

Motor neuron cell-nonautonomous rescue of spinal muscular atrophy phenotypes in mild and severe transgenic mouse models

PubMed Central

Liu, Ying Hsiu; Sahashi, Kentaro; Rigo, Frank; Bennett, C. Frank

2015-01-01

Survival of motor neuron (SMN) deficiency causes spinal muscular atrophy (SMA), but the pathogenesis mechanisms remain elusive. Restoring SMN in motor neurons only partially rescues SMA in mouse models, although it is thought to be therapeutically essential. Here, we address the relative importance of SMN restoration in the central nervous system (CNS) versus peripheral tissues in mouse models using a therapeutic splice-switching antisense oligonucleotide to restore SMN and a complementary decoy oligonucleotide to neutralize its effects in the CNS. Increasing SMN exclusively in peripheral tissues completely rescued necrosis in mild SMA mice and robustly extended survival in severe SMA mice, with significant improvements in vulnerable tissues and motor function. Our data demonstrate a critical role of peripheral pathology in the mortality of SMA mice and indicate that peripheral SMN restoration compensates for its deficiency in the CNS and preserves motor neurons. Thus, SMA is not a cell-autonomous defect of motor neurons in SMA mice. PMID:25583329

PRAPI: post-transcriptional regulation analysis pipeline for Iso-Seq.

PubMed

Gao, Yubang; Wang, Huiyuan; Zhang, Hangxiao; Wang, Yongsheng; Chen, Jinfeng; Gu, Lianfeng

2018-05-01

The single-molecule real-time (SMRT) isoform sequencing (Iso-Seq) based on Pacific Bioscience (PacBio) platform has received increasing attention for its ability to explore full-length isoforms. Thus, comprehensive tools for Iso-Seq bioinformatics analysis are extremely useful. Here, we present a one-stop solution for Iso-Seq analysis, called PRAPI to analyze alternative transcription initiation (ATI), alternative splicing (AS), alternative cleavage and polyadenylation (APA), natural antisense transcripts (NAT), and circular RNAs (circRNAs) comprehensively. PRAPI is capable of combining Iso-Seq full-length isoforms with short read data, such as RNA-Seq or polyadenylation site sequencing (PAS-seq) for differential expression analysis of NAT, AS, APA and circRNAs. Furthermore, PRAPI can annotate new genes and correct mis-annotated genes when gene annotation is available. Finally, PRAPI generates high-quality vector graphics to visualize and highlight the Iso-Seq results. The Dockerfile of PRAPI is available at http://www.bioinfor.org/tool/PRAPI. lfgu@fafu.edu.cn.