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1

Aberrant Alternative Splicing Is Another Hallmark of Cancer  

PubMed Central

The vast majority of human genes are alternatively spliced. Not surprisingly, aberrant alternative splicing is increasingly linked to cancer. Splice isoforms often encode proteins that have distinct and even antagonistic properties. The abnormal expression of splice factors and splice factor kinases in cancer changes the alternative splicing of critically important pre-mRNAs. Aberrant alternative splicing should be added to the growing list of cancer hallmarks.

Ladomery, Michael

2013-01-01

2

Aberrant splicing in neurological diseases.  

PubMed

Splicing of precursor messenger RNA (pre-mRNA) removes the intervening sequences (introns) and joins the expressed regions (exons) in the nucleus, before an intron-containing eukaryotic mRNA transcript can be exported and translated into proteins in the cytoplasm. While some sequences are always included or excluded (constitutive splicing), others can be selectively used (alternative splicing) in this process. Particularly by alternative splicing, up to tens of thousands of variant transcripts can be produced from a single gene, which contributes greatly to the proteomic diversity for such complex cellular functions as 'wiring' neurons in the nervous system. Disruption of this process leads to aberrant splicing, which accounts for the defects of up to 50% of mutations that cause certain human genetic diseases. In this review, we describe the different mechanisms of aberrant splicing that cause or have been associated with neurological diseases. WIREs RNA 2013, 4:631-649. doi: 10.1002/wrna.1184 For further resources related to this article, please visit the WIREs website. Conflict of interest: The authors have declared no conflicts of interest for this article. PMID:23821330

Feng, Dairong; Xie, Jiuyong

2013-07-02

3

Lentiviral vector integration in the human genome induces alternative splicing and generates aberrant transcripts.  

PubMed

Retroviral vectors integrate in genes and regulatory elements and may cause transcriptional deregulation of gene expression in target cells. Integration into transcribed genes also has the potential to deregulate gene expression at the posttranscriptional level by interfering with splicing and polyadenylation of primary transcripts. To examine the impact of retroviral vector integration on transcript splicing, we transduced primary human cells or cultured cells with HIV-derived vectors carrying a reporter gene or a human ?-globin gene under the control of a reduced-size locus-control region (LCR). Cells were randomly cloned and integration sites were determined in individual clones. We identified aberrantly spliced, chimeric transcripts in more than half of the targeted genes in all cell types. Chimeric transcripts were generated through the use of constitutive and cryptic splice sites in the HIV 5? long terminal repeat and gag gene as well as in the ?-globin gene and LCR. Compared with constitutively spliced transcripts, most aberrant transcripts accumulated at a low level, at least in part as a consequence of nonsense-mediated mRNA degradation. A limited set of cryptic splice sites caused the majority of aberrant splicing events, providing a strategy for recoding lentiviral vector backbones and transgenes to reduce their potential posttranscriptional genotoxicity. PMID:22523069

Moiani, Arianna; Paleari, Ylenia; Sartori, Daniela; Mezzadra, Riccardo; Miccio, Annarita; Cattoglio, Claudia; Cocchiarella, Fabienne; Lidonnici, Maria Rosa; Ferrari, Giuliana; Mavilio, Fulvio

2012-04-23

4

Alternative splicing in the human cytochrome P450IIB6 gene generates a high level of aberrant messages.  

PubMed Central

Polymorphisms within the human cytochrome P450 system can have severe clinical consequences and have been associated with adverse drug side effects and susceptibility to environmentally linked diseases such as cancer. Aberrant splicing of cytochrome P450 mRNA has been proposed as a potential mechanism for these polymorphisms. We have isolated aberrantly, as well as normally, spliced mRNAs (cDNAs) from the human P450IIB6 gene which either contain part of intron 5 and lack exon 8 or which contain a 58-bp fragment (exon 8A) instead of exon 8. Sequence analysis of the P450IIB6 gene demonstrates the presence of cryptic splice sites in intron 8 which will account for the generation of exon 8A. The mRNAs were therefore generated by alternative splicing. These data gain significance as the mRNAs will not encode a functional P450 enzyme and appear to represent a high proportion of the P450IIB6 mRNA population. Analysis of mRNA from fifteen individual human livers and cDNA libraries constructed from a variety of human tissues using the polymerase chain reaction shows that the aberrant splicing occurs in all cells and all individuals tested. This suggests a high level of infidelity in the processing of P450IIB6 mRNAs and demonstrates that the presence of abnormal transcripts does not imply the presence of a functionally inactive gene. Images

Miles, J S; McLaren, A W; Wolf, C R

1989-01-01

5

Aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy  

PubMed Central

Myotonic dystrophy (DM1) is associated with expression of expanded CTG DNA repeats as RNA (CUGexp RNA). To test whether CUGexp RNA creates a global splicing defect, we compared skeletal muscle of two mouse DM1 models, one expressing a CTGexp transgene, and another homozygous for a defective Mbnl1 gene. Strong correlation in splicing changes for ~100 new Mbnl1-regulated exons indicates loss of Mbnl1 explains >80% of the splicing pathology due to CUGexp RNA. In contrast, only about half of mRNA level changes can be attributed to loss of Mbnl1, indicating CUGexp RNA has Mbnl1-independent effects, particularly on mRNAs for extracellular matrix (ECM) proteins. We propose that CUGexp RNA causes two separate effects: loss of Mbnl1 function, disrupting splicing, and loss of another function that disrupts ECM mRNA regulation, possibly mediated by MBNL2. These findings reveal unanticipated similarities between DM1 and other muscular dystrophies.

Du, Hongqing; Cline, Melissa S.; Osborne, Robert J.; Tuttle, Daniel L.; Clark, Tyson A.; Donohue, John Paul; Hall, Megan P.; Shiue, Lily; Swanson, Maurice S.; Thornton, Charles A.; Ares, Manuel

2009-01-01

6

Alternative splicing in ascomycetes.  

PubMed

Alternative splicing is a complex and regulated process, which results in mRNA with different coding capacities from a single gene. Extend and types of alternative splicing vary greatly among eukaryotes. In this review, I focus on alternative splicing in ascomycetes, which in general have significant lower extend of alternative splicing than mammals. Yeast-like species have low numbers of introns and consequently alternative splicing is lower compared to filamentous fungi. Several examples from single studies as well as from genomic scale analysis are presented, including a survey of alternative splicing in Neurospora crassa. Another focus is regulation by riboswitch RNA and alternative splicing in a heterologous system, along with putative protein factors involved in regulation. PMID:23515838

Kempken, Frank

2013-03-21

7

Alternative splicing and muscular dystrophy  

PubMed Central

Alternative splicing of pre-mRNAs is a major contributor to proteomic diversity and to the control of gene expression in higher eukaryotic cells. For this reasons, alternative splicing is tightly regulated in different tissues and developmental stages and its disruption can lead to a wide range of human disorders. The aim of this review is to focus on the relevance of alternative splicing for muscle function and muscle disease. We begin by giving a brief overview of alternative splicing, muscle-specific gene expression and muscular dystrophy. Next, to illustrate these concepts we focus on two muscular dystrophy, myotonic muscular dystrophy and facioscapulohumeral muscular dystrophy, both associated to disruption of splicing regulation in muscle.

Pistoni, Mariaelena; Ghigna, Claudia; Gabellini, Davide

2013-01-01

8

The splice of life: Alternative splicing and neurological disease  

Microsoft Academic Search

Splicing of pre-messenger RNA is regulated differently in the brain compared with other tissues. Recognition of aberrations in splicing events that are associated with neurological disease has contributed to our understanding of disease pathogenesis in some cases. Neuron-specific proteins involved in RNA splicing and metabolism are also affected in several neurological disorders. These findings have begun to bridge what we

B. Kate Dredge; Alexandros D. Polydorides; Robert B. Darnell

2001-01-01

9

Calculation of Splicing Potential from the Alternative Splicing Mutation Database  

Microsoft Academic Search

BACKGROUND: The Alternative Splicing Mutation Database (ASMD) presents a collection of all known mutations inside human exons which affect splicing enhancers and silencers and cause changes in the alternative splicing pattern of the corresponding genes. FINDINGS: An algorithm was developed to derive a Splicing Potential (SP) table from the ASMD information. This table characterizes the influence of each oligonucleotide on

Jason M Bechtel; Preeti Rajesh; Irina Ilikchyan; Ying Deng; Pankaj K Mishra; Qi Wang; Xiaochun Wu; Kirill A Afonin; William E Grose; Ye Wang; Sadik Khuder; Alexei Fedorov

2008-01-01

10

Characterization and prediction of alternative splice sites  

Microsoft Academic Search

Human alternative isoform, cryptic, skipped, and constitutive splice sites from the ALTEXTRON database were analysed regarding splice site strength, composition, GC content, position and binding site strength of polypyrimidine tract and branch site. Several features were identified which distinguish alternative isoform and cryptic splice sites, but not skipped splice sites from constitutive ones. These include splice site strength, introns GC

Magnus Wang; Antonio Marín

2006-01-01

11

Regulation of Alternative Splicing by Histone Modifications  

Microsoft Academic Search

Alternative splicing of pre-mRNA is a prominent mechanism to generate protein diversity, yet its regulation is poorly understood. We demonstrated a direct role for histone modifications in alternative splicing. We found distinctive histone modification signatures that correlate with the splicing outcome in a set of human genes, and modulation of histone modifications causes splice site switching. Histone marks affect splicing

Reini F. Luco; Qun Pan; Kaoru Tominaga; Benjamin J. Blencowe; Olivia M. Pereira-Smith; Tom Misteli

2010-01-01

12

Kiwifruit floral gene APETALA2 is alternatively spliced and accumulates in aberrant indeterminate flowers in the absence of miR172.  

PubMed

In Arabidopsis, the identity of perianth and reproductive organs are specified by antagonistic action of two floral homeotic genes, APETALA2 (AP2) and AGAMOUS (AG). AP2 is also negatively regulated by an evolutionary conserved interaction with a microRNA, miR172, and has additional roles in general plant development. A kiwifruit gene with high levels of homology to AP2 and AP2-like genes from other plant species was identified. The transcript was abundant in the kiwifruit flower, particularly petal, suggesting a role in floral organ identity. Splice variants were identified, all containing both AP2 domains, including a variant that potentially produces a shorter transcript without the miRNA172 targeting site. Increased AP2 transcript accumulation was detected in the aberrant flowers of the mutant ‘Pukekohe dwarf’ with multiple perianth whorls and extended petaloid features. In contrast to normal kiwifruit flowers, the aberrant flowers failed to accumulate miR172 in the developing whorls, although accumulation was detected at the base of the flower. An additional role during dormancy in kiwifruit was proposed based on AP2 transcript accumulation in axillary buds before and after budbreak. PMID:22290408

Varkonyi-Gasic, Erika; Lough, Robyn H; Moss, Sarah M A; Wu, Rongmei; Hellens, Roger P

2012-03-01

13

Disturbed Expression of Splicing Factors in Renal Cancer Affects Alternative Splicing of Apoptosis Regulators, Oncogenes, and Tumor Suppressors  

Microsoft Academic Search

BackgroundClear cell renal cell carcinoma (ccRCC) is the most common type of renal cancer. One of the processes disturbed in this cancer type is alternative splicing, although phenomena underlying these disturbances remain unknown. Alternative splicing consists of selective removal of introns and joining of residual exons of the primary transcript, to produce mRNA molecules of different sequence. Splicing aberrations may

Agnieszka Piekielko-Witkowska; Hanna Wiszomirska; Anna Wojcicka; Piotr Poplawski; Joanna Boguslawska; Zbigniew Tanski; Alicja Nauman; Juan Valcarcel

2010-01-01

14

Understanding alternative splicing: towards a cellular code  

Microsoft Academic Search

In violation of the 'one gene, one polypeptide' rule, alternative splicing allows individual genes to produce multiple protein isoforms — thereby playing a central part in generating complex proteomes. Alternative splicing also has a largely hidden function in quantitative gene control, by targeting RNAs for nonsense-mediated decay. Traditional gene-by-gene investigations of alternative splicing mechanisms are now being complemented by global

Arianne J. Matlin; Francis Clark; Christopher W. J. Smith

2005-01-01

15

Regulation of Alternative Splicing by Histone Modifications  

PubMed Central

Alternative splicing of pre-mRNA is a prominent mechanism to generate protein diversity, yet its regulation is poorly understood. We demonstrated a direct role for histone modifications in alternative splicing. We found distinctive histone modification signatures that correlate with the splicing outcome in a set of human genes, and modulation of histone modifications causes splice site switching. Histone marks affect splicing outcome by influencing the recruitment of splicing regulators via a chromatin-binding protein. These results outline an adaptor system for the reading of histone marks by the pre-mRNA splicing machinery.

Luco, Reini F.; Pan, Qun; Tominaga, Kaoru; Blencowe, Benjamin J.; Pereira-Smith, Olivia M.; Misteli, Tom

2010-01-01

16

Regulation of alternative splicing by histone modifications.  

PubMed

Alternative splicing of pre-mRNA is a prominent mechanism to generate protein diversity, yet its regulation is poorly understood. We demonstrated a direct role for histone modifications in alternative splicing. We found distinctive histone modification signatures that correlate with the splicing outcome in a set of human genes, and modulation of histone modifications causes splice site switching. Histone marks affect splicing outcome by influencing the recruitment of splicing regulators via a chromatin-binding protein. These results outline an adaptor system for the reading of histone marks by the pre-mRNA splicing machinery. PMID:20133523

Luco, Reini F; Pan, Qun; Tominaga, Kaoru; Blencowe, Benjamin J; Pereira-Smith, Olivia M; Misteli, Tom

2010-02-04

17

Directing alternative splicing: cast and scenarios  

Microsoft Academic Search

Recent progress in the study of alternative RNA splicing indicates that the interaction of RNA-binding proteins with specific target elements modulates splice site recognition and spliceosome assembly. The identity of splicing signals, the presence of modulating elements and differences in the distribution of RNA-binding proteins are key determinants involved in the tissue-specific regulation of splice site selection.

Benoit Chabot

1996-01-01

18

Evolution of alternative splicing after gene duplication  

PubMed Central

Alternative splicing and gene duplication are two major sources of proteomic function diversity. Here, we study the evolutionary trend of alternative splicing after gene duplication by analyzing the alternative splicing differences between duplicate genes. We observed that duplicate genes have fewer alternative splice (AS) forms than single-copy genes, and that a negative correlation exists between the mean number of AS forms and the gene family size. Interestingly, we found that the loss of alternative splicing in duplicate genes may occur shortly after the gene duplication. These results support the subfunctionization model of alternative splicing in the early stage after gene duplication. Further analysis of the alternative splicing distribution in human duplicate pairs showed the asymmetric evolution of alternative splicing after gene duplications; i.e., the AS forms between duplicates may differ dramatically. We therefore conclude that alternative splicing and gene duplication may not evolve independently. In the early stage after gene duplication, young duplicates may take over a certain amount of protein function diversity that previously was carried out by the alternative splicing mechanism. In the late stage, the gain and loss of alternative splicing seem to be independent between duplicates.

Su, Zhixi; Wang, Jianmin; Yu, Jun; Huang, Xiaoqiu; Gu, Xun

2006-01-01

19

Bioinformatics of alternative splicing and its regulation  

Microsoft Academic Search

The sequencing of the human genome and ensuing wave of data generation have brought new light upon the extent and importance of alternative splicing as an RNA regulatory mechanism. Alternative splicing could potentially explain the complexity of protein repertoire during evolution, and defects in the splicing mechanism are responsible for diseases as complex as cancer. Among the challenges that rise

Liliana Florea

2006-01-01

20

Regulation of alternative splicing within the supraspliceosome  

PubMed Central

Alternative splicing is a fundamental feature in regulating the eukaryotic transcriptome, as ~95% of multi-exon human Pol II transcripts are subject to this process. Regulated splicing operates through the combinatorial interplay of positive and negative regulatory signals present in the pre-mRNA, which are recognized by trans-acting factors. All these RNA and protein components are assembled in a gigantic, 21 MDa, ribonucleoprotein splicing machine – the supraspliceosome. Because most alternatively spliced mRNA isoforms vary between different cell and tissue types, the ability to perform alternative splicing is expected to be an integral part of the supraspliceosome, which constitutes the splicing machine in vivo. Here we show that both the constitutively and alternatively spliced mRNAs of the endogenous human pol II transcripts: hnRNP A/B, survival of motor neuron (SMN) and ADAR2 are predominantly found in supraspliceosomes. This finding is consistent with our observations that the splicing regulators hnRNP G as well as all phosphorylated SR proteins are predominantly associated with supraspliceosomes. We further show that changes in alternative splicing of hnRNP A/B, affected by up regulation of SRSF5 (SRp40) or by treatment with C6-ceramide, occur within supraspliceosomes. These observations support the proposed role of the supraspliceosome in splicing regulation and alternative splicing.

Sebbag-Sznajder, Naama; Raitskin, Oleg; Angenitzki, Minna; Sato, Taka-Aki; Sperling, Joseph; Sperling, Ruth

2012-01-01

21

Regulated functional alternative splicing in Drosophila  

PubMed Central

Alternative splicing expands the coding capacity of metazoan genes, and it was largely genetic studies in the fruit-fly Drosophila melanogaster that established the principle that regulated alternative splicing results in tissue- and stage-specific protein isoforms with different functions in development. Alternative splicing is particularly prominent in germ cells, muscle and the central nervous system where it modulates the expression of various proteins including cell-surface molecules and transcription factors. Studies in flies have given us numerous insights into alternative splicing in terms of upstream regulation, the exquisite diversity of their forms and the key differential cellular functions of alternatively spliced gene products. The current inundation of transcriptome sequencing data from Drosophila provides an unprecedented opportunity to gain a comprehensive view of alternative splicing.

Venables, Julian P.; Tazi, Jamal; Juge, Francois

2012-01-01

22

Alternative Splicing: Therapeutic Target and Tool  

Microsoft Academic Search

Alternative splicing swells the coding capacity of the human genome, expanding the pharmacoproteome, the proteome that provides targets for ther- apy. Splicing, both constitutive and regulated forms, can itself be targeted by conventional and molecular therapies. This review focuses on splicing as a therapeutic target with a particular emphasis on molecular approaches. The review looks at the use of antisense

Mariano A. Garcia-Blanco

23

Alternative splicing in bone following mechanical loading  

Microsoft Academic Search

It is estimated that more than 90% of human genes express multiple mRNA transcripts due to alternative splicing. Consequently, the proteins produced by different splice variants will likely have different functions and expression levels. Several genes with splice variants are known in bone, with functions that affect osteoblast function and bone formation. The primary goal of this study was to

Sara M. Mantila Roosa; Yunlong Liu; Charles H. Turner

2011-01-01

24

Selecting for Functional Alternative Splices in ESTs  

Microsoft Academic Search

The expressed sequence tag (EST) collection in dbEST provides an extensive resource for detecting alternative splicing on a genomic scale. Using genomically aligned ESTs,a computatio nal tool (TAP) was used to identify alternative splice patterns for 6400 known human genes from the RefSeq database. With sufficient EST coverage,one or more alternatively spliced forms could be detected for ne arly all

Zhengyan Kan; David States; Warren Gish

2002-01-01

25

Aberrant Splice Variants of HAS1 (Hyaluronan Synthase 1) Multimerize with and Modulate Normally Spliced HAS1 Protein  

PubMed Central

Most human genes undergo alternative splicing, but aberrant splice forms are hallmarks of many cancers, usually resulting from mutations initiating abnormal exon skipping, intron retention, or the introduction of a new splice sites. We have identified a family of aberrant splice variants of HAS1 (the hyaluronan synthase 1 gene) in some B lineage cancers, characterized by exon skipping and/or partial intron retention events that occur either together or independently in different variants, apparently due to accumulation of inherited and acquired mutations. Cellular, biochemical, and oncogenic properties of full-length HAS1 (HAS1-FL) and HAS1 splice variants Va, Vb, and Vc (HAS1-Vs) are compared and characterized. When co-expressed, the properties of HAS1-Vs are dominant over those of HAS1-FL. HAS1-FL appears to be diffusely expressed in the cell, but HAS1-Vs are concentrated in the cytoplasm and/or Golgi apparatus. HAS1-Vs synthesize detectable de novo HA intracellularly. Each of the HAS1-Vs is able to relocalize HAS1-FL protein from diffuse cytoskeleton-anchored locations to deeper cytoplasmic spaces. This HAS1-Vs-mediated relocalization occurs through strong molecular interactions, which also serve to protect HAS1-FL from its otherwise high turnover kinetics. In co-transfected cells, HAS1-FL and HAS1-Vs interact with themselves and with each other to form heteromeric multiprotein assemblies. HAS1-Vc was found to be transforming in vitro and tumorigenic in vivo when introduced as a single oncogene to untransformed cells. The altered distribution and half-life of HAS1-FL, coupled with the characteristics of the HAS1-Vs suggest possible mechanisms whereby the aberrant splicing observed in human cancer may contribute to oncogenesis and disease progression.

Ghosh, Anirban; Kuppusamy, Hemalatha; Pilarski, Linda M.

2009-01-01

26

The Application of Alternative Splicing Graphs in Quantitative Analysis of Alternative Splicing Form from EST Database  

Microsoft Academic Search

Alternative splicing of a single pre-mRNA can give rise to different mRNA transcripts. Alternative splicing of pre-messenger RNA is an important layer of gene expression regulation in eukaryotic cell. Consequently, alternative splicing is an important mechanism for generating protein diversity from a single gene. Although alternative splicing is an important biological process, standard molecular biology techniques have only identified several

Hsun-chang Chang; Po-shun Yu; Tze-wei Huang; Yaw-ling Lin; Fang-rong Hsu

2004-01-01

27

Highlights of Alternative Splicing Regulation Session: Yes, No, Maybe--A History of Paradigm Shifts  

NSDL National Science Digital Library

Cooper summarizes the discussions and presentations from the session entitled "Control of Splice Site Selection" held at the Sixth Annual Meeting of the RNA Society. Paradigms are shifting as experiments show that some of the proteins involved in regulating splicing can act as splicing enhancers or repressors, depending on the cellular context. The complex interactions among the ribonucleoproteins (RNPs) and proteins, and the role of cis elements, in controlling cell-specific splicing are highlighted. The importance of properly regulated splicing is emphasized by examples of disease pathologies in which alternative splicing is aberrant.

Thomas A. Cooper (Baylor College of Medicine;Departments of Pathology and Molecular and Cellular Biology REV)

2001-10-23

28

Titin Diversity--Alternative Splicing Gone Wild  

PubMed Central

Titin is an extremely large protein found in highest concentrations in heart and skeletal muscle. The single mammalian gene is expressed in multiple isoforms as a result of alternative splicing. Although titin isoform expression is controlled developmentally and in a tissue specific manner, the vast number of potential splicing pathways far exceeds those described in any other alternatively spliced gene. Over 1 million human splice pathways for a single individual can be potentially derived from the PEVK region alone. A new splicing pattern for the human cardiac N2BA isoform type has been found in which the PEVK region includes only the N2B type exons. The alterations in splicing and titin isoform expression in human heart disease provide impetus for future detailed study of the splicing mechanisms for this giant protein.

Guo, Wei; Bharmal, Sheila J.; Esbona, Karla; Greaser, Marion L.

2010-01-01

29

Analysis of SRrp86-regulated alternative splicing  

PubMed Central

Previous work led to the hypothesis that SRrp86, a related member of the SR protein superfamily, can interact with and modulate the activity of other SR proteins. Here, we sought to test this hypothesis by examining the effect of changing SRrp86 concentrations on overall alternative splicing patterns. SpliceArrays were used to examine 9,854 splicing events in wild-type cells, cells overexpressing SRrp86, and cells treated with siRNAs to knockdown SRrp86. From among the 500 splicing events exhibiting altered splicing under these conditions, the splicing of c-Jun and I?B? were validated as being regulated by SRrp86 resulting in altered regulation of their downstream targets. In both cases, functionally distinct isoforms were generated that demonstrate the role SRrp86 plays in controlling alternative splicing.

Solis, Amanda S

2010-01-01

30

Mechano-Regulation of Alternative Splicing  

PubMed Central

Alternative splicing contributes to the complexity of proteome by producing multiple mRNAs from a single gene. Affymetrix exon arrays and experiments in vivo or in vitro demonstrated that alternative splicing was regulated by mechanical stress. Expression of mechano-growth factor (MGF) which is the splicing isoform of insulin-like growth factor 1(IGF-1) and vascular endothelial growth factor (VEGF) splicing variants such as VEGF121, VEGF165, VEGF206, VEGF189, VEGF165 and VEGF145 are regulated by mechanical stress. However, the mechanism of this process is not yet clear. Increasing evidences showed that the possible mechanism is related to Ca2+ signal pathway and phosphorylation signal pathway. This review proposes possible mechanisms of mechanical splicing regulation. This will contribute to the biomechanical study of alternative splicing.

Liu, Huan; Tang, Liling

2013-01-01

31

Splicing and alternative splicing in rice and humans.  

PubMed

Rice is a monocot gramineous crop, and one of the most important staple foods. Rice is considered a model species for most gramineous crops. Extensive research on rice has provided critical guidance for other crops, such as maize and wheat. In recent years, climate change and exacerbated soil degradation have resulted in a variety of abiotic stresses, such as greenhouse effects, lower temperatures, drought, floods, soil salinization and heavy metal pollution. As such, there is an extremely high demand for additional research, in order to address these negative factors. Studies have shown that the alternative splicing of many genes in rice is affected by stress conditions, suggesting that manipulation of the alternative splicing of specific genes may be an effective approach for rice to adapt to abiotic stress. With the advancement of microarrays, and more recently, next generation sequencing technology, several studies have shown that more than half of the genes in the rice genome undergo alternative splicing. This mini-review summarizes the latest progress in the research of splicing and alternative splicing in rice, compared to splicing in humans. Furthermore, we discuss how additional studies may change the landscape of investigation of rice functional genomics and genetically improved rice. [BMB Reports 2013; 46(9): 439-447]. PMID:24064058

E, Zhiguo; Wang, Lei; Zhou, Jianhua

2013-09-01

32

An Alu-derived intronic splicing enhancer facilitates intronic processing and modulates aberrant splicing in ATM.  

PubMed

We have previously reported a natural GTAA deletion within an intronic splicing processing element (ISPE) of the ataxia telangiectasia mutated (ATM) gene that disrupts a non-canonical U1 snRNP interaction and activates the excision of the upstream portion of the intron. The resulting pre-mRNA splicing intermediate is then processed to a cryptic exon, whose aberrant inclusion in the final mRNA is responsible for ataxia telangiectasia. We show here that the last 40 bases of a downstream intronic antisense Alu repeat are required for the activation of the cryptic exon by the ISPE deletion. Evaluation of the pre-mRNA splicing intermediate by a hybrid minigene assay indicates that the identified intronic splicing enhancer represents a novel class of enhancers that facilitates processing of splicing intermediates possibly by recruiting U1 snRNP to defective donor sites. In the absence of this element, the splicing intermediate accumulates and is not further processed to generate the cryptic exon. Our results indicate that Alu-derived sequences can provide intronic splicing regulatory elements that facilitate pre-mRNA processing and potentially affect the severity of disease-causing splicing mutations. PMID:19773425

Pastor, Tibor; Talotti, Gabriele; Lewandowska, Marzena Anna; Pagani, Franco

2009-11-01

33

Depolarization-Mediated Regulation of Alternative Splicing  

PubMed Central

Alternative splicing in eukaryotes plays an important role in regulating gene expression by selectively including alternative exons. A wealth of information has been accumulated that explains how alternative exons are selected in a developmental stage- or tissue-specific fashion. However, our knowledge of how cells respond to environmental changes to alter alternative splicing is very limited. For example, although a number of alternative exons have been shown to be regulated by calcium level alterations, the underlying mechanisms are not well understood. As calcium signaling in neurons plays a crucial role in essential neuronal functions such as learning and memory formation, it is important to understand how this process is regulated at every level in gene expression. The significance of the dynamic control of alternative splicing in response to changes of calcium levels has been largely unappreciated. In this communication, we will summarize the recent advances in calcium signaling-mediated alternative splicing that have provided some insights into the important regulatory mechanisms. In addition to describing the cis-acting RNA elements on the pre-mRNA molecules that respond to changes of intracellular calcium levels, we will summarize how splicing regulators change and affect alternative splicing in this process. We will also discuss a novel mode of calcium-mediated splicing regulation at the level of chromatin structure and transcription.

Sharma, Alok; Lou, Hua

2011-01-01

34

Alternative Splicing in Bone Following Mechanical Loading  

PubMed Central

It is estimated that more than 90% of human genes express multiple mRNA transcripts due to alternative splicing. Consequently, the proteins produced by different splice variants will likely have different functions and expression levels. Several genes with splice variants are known in bone, with functions that affect osteoblast function and bone formation. The primary goal of this study was to evaluate the extent of alternative splicing in a bone subjected to mechanical loading and subsequent bone formation. We used the rat forelimb loading model, in which the right forelimb was loaded axially for 3 minutes, while the left forearm served as a non-loaded control. Animals were subjected to loading sessions every day, with 24 hours between sessions. Ulnae were sampled at 11 time points, from 4 hours to 32 days after beginning loading. RNA was isolated and mRNA abundance was measured at each time point using Affymetrix exon arrays (GeneChip® Rat Exon 1.0 ST Arrays). An ANOVA model was used to identify potential alternatively spliced genes across the time course, and five alternatively spliced genes were validated with qPCR: Akap12, Fn1, Pcolce, Sfrp4, and Tpm1. The number of alternatively spliced genes varied with time, ranging from a low of 68 at 12h to a high of 992 at 16d. We identified genes across the time course that encoded proteins with known functions in bone formation, including collagens, matrix proteins, and components of the Wnt/?-catenin and TGF-? signaling pathways. We also identified alternatively spliced genes encoding cytokines, ion channels, muscle-related genes, and solute carriers that do not have a known function in bone formation and represent potentially novel findings. In addition, a functional characterization was performed to categorize the global functions of the alternatively spliced genes in our data set. In conclusion, mechanical loading induces alternative splicing in bone, which may play an important role in the response of bone to mechanical loading.

Mantila Roosa, Sara M.; Liu, Yunlong; Turner, Charles H.

2010-01-01

35

Aberrant splicing, hyaluronan synthases and intracellular hyaluronan as drivers of oncogenesis and potential drug targets.  

PubMed

Current evidence suggests a significant role of aberrant splicing in the development and maintenance of malignancy. This multistep, tightly regulated epigenetic process leads to the production of abnormal proteins with abnormal functions contributing to underlying mechanisms of malignant transformation. Splicing patterns in malignant cells can be altered not only by the mutations detected on the aberrantly spliced gene, but also by the mutations detected on the genes encoding splicing factors. For example, aberrant pre-mRNA splicing, leading to intracellular or extracellular HA synthesis by HASs, contributes to the initiation and progression of various types of cancer. The influence of intracellular HA appears to be particularly significant and is promoted by aberrant splicing. In this review we report a model describing oncogenic potential of aberrant splicing, with a focus on HAS1 and intracellular HA. We also suggest that the influence of splicing mutations on malignant disease is likely multifactorial. For the triple axis of HA, HAS1 and RHAMM, mutations in HAS1 provide an indicator that these aberrations contribute to the events that lead to malignancy through increased risk and predisposition. Here, we also summarize the impact of splicing abnormalities on cancer and the possible oncogenic impact of aberrantly spliced HAS1. In conclusion, we emphasize that specific gene splice variants and the splicing process itself offer potential targets for novel drug treatment strategies. PMID:23517594

Adamia, Sophia; Pilarski, Patrick M; Belch, Andrew R; Pilarski, Linda M

2013-05-01

36

Alternative splicing in disease and therapy  

Microsoft Academic Search

Alternative splicing is the major source of proteome diversity in humans and thus is highly relevant to disease and therapy. For example, recent work suggests that the long-sought-after target of the analgesic acetaminophen is a neural-specific, alternatively spliced isoform of cyclooxygenase 1 (COX-1). Several important diseases, such as cystic fibrosis, have been linked with mutations or variations in either cis-acting

Andrew P Baraniak; Erika L Lasda; Mariano A Garcia-Blanco

2004-01-01

37

Phosphorylation-Mediated Regulation of Alternative Splicing in Cancer  

PubMed Central

Alternative splicing (AS) is one of the key processes involved in the regulation of gene expression in eukaryotic cells. AS catalyzes the removal of intronic sequences and the joining of selected exons, thus ensuring the correct processing of the primary transcript into the mature mRNA. The combinatorial nature of AS allows a great expansion of the genome coding potential, as multiple splice-variants encoding for different proteins may arise from a single gene. Splicing is mediated by a large macromolecular complex, the spliceosome, whose activity needs a fine regulation exerted by cis-acting RNA sequence elements and trans-acting RNA binding proteins (RBP). The activity of both core spliceosomal components and accessory splicing factors is modulated by their reversible phosphorylation. The kinases and phosphatases involved in these posttranslational modifications significantly contribute to AS regulation and to its integration in the complex regulative network that controls gene expression in eukaryotic cells. Herein, we will review the major canonical and noncanonical splicing factor kinases and phosphatases, focusing on those whose activity has been implicated in the aberrant splicing events that characterize neoplastic transformation.

Sette, Claudio

2013-01-01

38

Identification of alternative 5?/3? splice sites based on the mechanism of splice site competition  

PubMed Central

Alternative splicing plays an important role in regulating gene expression. Currently, most efficient methods use expressed sequence tags or microarray analysis for large-scale detection of alternative splicing. However, it is difficult to detect all alternative splice events with them because of their inherent limitations. Previous computational methods for alternative splicing prediction could only predict particular kinds of alternative splice events. Thus, it would be highly desirable to predict alternative 5?/3? splice sites with various splicing levels using genomic sequences alone. Here, we introduce the competition mechanism of splice sites selection into alternative splice site prediction. This approach allows us to predict not only rarely used but also frequently used alternative splice sites. On a dataset extracted from the AltSplice database, our method correctly classified ?70% of the splice sites into alternative and constitutive, as well as ?80% of the locations of real competitors for alternative splice sites. It outperforms a method which only considers features extracted from the splice sites themselves. Furthermore, this approach can also predict the changes in activation level arising from mutations in flanking cryptic splice sites of a given splice site. Our approach might be useful for studying alternative splicing in both computational and molecular biology.

Xia, Huiyu; Bi, Jianning; Li, Yanda

2006-01-01

39

The Alternative Splicing Mutation Database: a hub for investigations of alternative splicing using mutational evidence  

PubMed Central

Background Some mutations in the internal regions of exons occur within splicing enhancers and silencers, influencing the pattern of alternative splicing in the corresponding genes. To understand how these sequence changes affect splicing, we created a database of these mutations. Findings The Alternative Splicing Mutation Database (ASMD) serves as a repository for all exonic mutations not associated with splicing junctions that measurably change the pattern of alternative splicing. In this initial published release (version 1.2), only human sequences are present, but the ASMD will grow to include other organisms, (see Availability and requirements section for the ASMD web address). This relational database allows users to investigate connections between mutations and features of the surrounding sequences, including flanking sequences, RNA secondary structures and strengths of splice junctions. Splicing effects of the mutations are quantified by the relative presence of alternative mRNA isoforms with and without a given mutation. This measure is further categorized by the accuracy of the experimental methods employed. The database currently contains 170 mutations in 66 exons, yet these numbers increase regularly. We developed an algorithm to derive a table of oligonucleotide Splicing Potential (SP) values from the ASMD dataset. We present the SP concept and tools in detail in our corresponding article. Conclusion The current data set demonstrates that mutations affecting splicing are located throughout exons and might be enriched within local RNA secondary structures. Exons from the ASMD have below average splicing junction strength scores, but the difference is small and is judged not to be significant.

Bechtel, Jason M; Rajesh, Preeti; Ilikchyan, Irina; Deng, Ying; Mishra, Pankaj K; Wang, Qi; Wu, Xiaochun; Afonin, Kirill A; Grose, William E; Wang, Ye; Khuder, Sadik; Fedorov, Alexei

2008-01-01

40

Identification of alternative 50\\/30 splice sites based on the mechanism of splice site competition  

Microsoft Academic Search

Alternative splicing plays an important role in regu- lating gene expression. Currently, most efficient methods use expressed sequence tags or microar- ray analysis for large-scale detection of alternative splicing. However, it is difficult to detect all alterna- tive splice events with them because of their in- herent limitations. Previous computational methods for alternative splicing prediction could only predict particular kinds

Huiyu Xia; Jianning Bi; Yanda Li

2006-01-01

41

Rbm20 regulates titin alternative splicing as a splicing repressor  

PubMed Central

Titin, a sarcomeric protein expressed primarily in striated muscles, is responsible for maintaining the structure and biomechanical properties of muscle cells. Cardiac titin undergoes developmental size reduction from 3.7 megadaltons in neonates to primarily 2.97 megadaltons in the adult. This size reduction results from gradually increased exon skipping between exons 50 and 219 of titin mRNA. Our previous study reported that Rbm20 is the splicing factor responsible for this process. In this work, we investigated its molecular mechanism. We demonstrate that Rbm20 mediates exon skipping by binding to titin pre-mRNA to repress the splicing of some regions; the exons/introns in these Rbm20-repressed regions are ultimately skipped. Rbm20 was also found to mediate intron retention and exon shuffling. The two Rbm20 speckles found in nuclei from muscle tissues were identified as aggregates of Rbm20 protein on the partially processed titin pre-mRNAs. Cooperative repression and alternative 3? splice site selection were found to be used by Rbm20 to skip different subsets of titin exons, and the splicing pathway selected depended on the ratio of Rbm20 to other splicing factors that vary with tissue type and developmental age.

Li, Shijun; Guo, Wei; Dewey, Colin N.; Greaser, Marion L.

2013-01-01

42

Alternative Splicing Regulation During C. elegans Development: Splicing Factors as Regulated Targets  

Microsoft Academic Search

Alternative splicing generates protein diversity and allows for post-transcriptional gene regulation. Estimates suggest that 10% of the genes in Caenorhabditis elegans undergo alternative splicing. We constructed a splicing-sensitive microarray to detect alternative splicing for 352 cassette exons and tested for changes in alternative splicing of these genes during development. We found that the microarray data predicted that 62\\/352 (?18%) of

Sergio Barberan-Soler; Alan M. Zahler

2008-01-01

43

EuSplice: a unified resource for the analysis of splice signals and alternative splicing in eukaryotic genes  

Microsoft Academic Search

Motivation: Despite increased availability of genome annotation data, a comprehensive resource for in-depth analysis of splice signal distributions and alternative splicing (AS) patterns in eukaryote genomes is still lacking. To meet this need, we have developed EuSplice—a unique splice-centric database which provides reliable splice signal and AS information for 23 eukaryotes. Results: The EuSplice database contains 95822 AS events and

Ashwini Bhasi; Ram Vinay Pandey; Suriya Prabha Utharasamy; Periannan Senapathy

2007-01-01

44

Variation in alternative splicing across human tissues  

PubMed Central

Background Alternative pre-mRNA splicing (AS) is widely used by higher eukaryotes to generate different protein isoforms in specific cell or tissue types. To compare AS events across human tissues, we analyzed the splicing patterns of genomically aligned expressed sequence tags (ESTs) derived from libraries of cDNAs from different tissues. Results Controlling for differences in EST coverage among tissues, we found that the brain and testis had the highest levels of exon skipping. The most pronounced differences between tissues were seen for the frequencies of alternative 3' splice site and alternative 5' splice site usage, which were about 50 to 100% higher in the liver than in any other human tissue studied. Quantifying differences in splice junction usage, the brain, pancreas, liver and the peripheral nervous system had the most distinctive patterns of AS. Analysis of available microarray expression data showed that the liver had the most divergent pattern of expression of serine-arginine protein and heterogeneous ribonucleoprotein genes compared to the other human tissues studied, possibly contributing to the unusually high frequency of alternative splice site usage seen in liver. Sequence motifs enriched in alternative exons in genes expressed in the brain, testis and liver suggest specific splicing factors that may be important in AS regulation in these tissues. Conclusions This study distinguishes the human brain, testis and liver as having unusually high levels of AS, highlights differences in the types of AS occurring commonly in different tissues, and identifies candidate cis-regulatory elements and trans-acting factors likely to have important roles in tissue-specific AS in human cells.

Yeo, Gene; Holste, Dirk; Kreiman, Gabriel; Burge, Christopher B

2004-01-01

45

Prediction of Alternative Splice Sites in Human Genes  

Microsoft Academic Search

This thesis addresses the problem of predicting alternative splice sites in human genes. The most common way to identify alternative splice sites are the use of expressed sequence tags and microarray data. Since genes only produce alternative proteins under certain conditions, these methods are limited to detecting only alternative splice sites in genes whose alternative protein forms are expressed under

Douglas Simmons

2007-01-01

46

Gene and alternative splicing annotation with AIR  

PubMed Central

Designing effective and accurate tools for identifying the functional and structural elements in a genome remains at the frontier of genome annotation owing to incompleteness and inaccuracy of the data, limitations in the computational models, and shifting paradigms in genomics, such as alternative splicing. We present a methodology for the automated annotation of genes and their alternatively spliced mRNA transcripts based on existing cDNA and protein sequence evidence from the same species or projected from a related species using syntenic mapping information. At the core of the method is the splice graph, a compact representation of a gene, its exons, introns, and alternatively spliced isoforms. The putative transcripts are enumerated from the graph and assigned confidence scores based on the strength of sequence evidence, and a subset of the high-scoring candidates are selected and promoted into the annotation. The method is highly selective, eliminating the unlikely candidates while retaining 98% of the high-quality mRNA evidence in well-formed transcripts, and produces annotation that is measurably more accurate than some evidence-based gene sets. The process is fast, accurate, and fully automated, and combines the traditionally distinct gene annotation and alternative splicing detection processes in a comprehensive and systematic way, thus considerably aiding in the ensuing manual curation efforts.

Florea, Liliana; Di Francesco, Valentina; Miller, Jason; Turner, Russell; Yao, Alison; Harris, Michael; Walenz, Brian; Mobarry, Clark; Merkulov, Gennady V.; Charlab, Rosane; Dew, Ian; Deng, Zuoming; Istrail, Sorin; Li, Peter; Sutton, Granger

2005-01-01

47

Gene and alternative splicing annotation with AIR.  

PubMed

Designing effective and accurate tools for identifying the functional and structural elements in a genome remains at the frontier of genome annotation owing to incompleteness and inaccuracy of the data, limitations in the computational models, and shifting paradigms in genomics, such as alternative splicing. We present a methodology for the automated annotation of genes and their alternatively spliced mRNA transcripts based on existing cDNA and protein sequence evidence from the same species or projected from a related species using syntenic mapping information. At the core of the method is the splice graph, a compact representation of a gene, its exons, introns, and alternatively spliced isoforms. The putative transcripts are enumerated from the graph and assigned confidence scores based on the strength of sequence evidence, and a subset of the high-scoring candidates are selected and promoted into the annotation. The method is highly selective, eliminating the unlikely candidates while retaining 98% of the high-quality mRNA evidence in well-formed transcripts, and produces annotation that is measurably more accurate than some evidence-based gene sets. The process is fast, accurate, and fully automated, and combines the traditionally distinct gene annotation and alternative splicing detection processes in a comprehensive and systematic way, thus considerably aiding in the ensuing manual curation efforts. PMID:15632090

Florea, Liliana; Di Francesco, Valentina; Miller, Jason; Turner, Russell; Yao, Alison; Harris, Michael; Walenz, Brian; Mobarry, Clark; Merkulov, Gennady V; Charlab, Rosane; Dew, Ian; Deng, Zuoming; Istrail, Sorin; Li, Peter; Sutton, Granger

2005-01-01

48

Quantitative and evolutionary biology of alternative splicing: how changing the mix of alternative transcripts affects phenotypic plasticity and reaction norms  

Microsoft Academic Search

Alternative splicing (AS) of pre-messenger RNA is a common phenomenon that creates different transcripts from a single gene, and these alternative transcripts affect phenotypes. The majority of AS research has examined tissue and developmental specificity of expression of particular AS transcripts, how this specificity affects cell function, and how aberrant AS is related to disease. Few studies have examined quantitative

J H Marden

2008-01-01

49

Alternative Splicing for Diseases, Cancers, Drugs, and Databases  

PubMed Central

Alternative splicing is a major diversification mechanism in the human transcriptome and proteome. Several diseases, including cancers, have been associated with dysregulation of alternative splicing. Thus, correcting alternative splicing may restore normal cell physiology in patients with these diseases. This paper summarizes several alternative splicing-related diseases, including cancers and their target genes. Since new cancer drugs often target spliceosomes, several clinical drugs and natural products or their synthesized derivatives were analyzed to determine their effects on alternative splicing. Other agents known to have modulating effects on alternative splicing during therapeutic treatment of cancer are also discussed. Several commonly used bioinformatics resources are also summarized.

Lee, Jin-Ching; Hou, Ming-Feng; Wang, Chun-Lin; Chen, Chien-Chi; Huang, Hurng-Wern

2013-01-01

50

Model-based detection of alternative splicing signals  

PubMed Central

Motivation: Transcripts from ?95% of human multi-exon genes are subject to alternative splicing (AS). The growing interest in AS is propelled by its prominent contribution to transcriptome and proteome complexity and the role of aberrant AS in numerous diseases. Recent technological advances enable thousands of exons to be simultaneously profiled across diverse cell types and cellular conditions, but require accurate identification of condition-specific splicing changes. It is necessary to accurately identify such splicing changes to elucidate the underlying regulatory programs or link the splicing changes to specific diseases. Results: We present a probabilistic model tailored for high-throughput AS data, where observed isoform levels are explained as combinations of condition-specific AS signals. According to our formulation, given an AS dataset our tasks are to detect common signals in the data and identify the exons relevant to each signal. Our model can incorporate prior knowledge about underlying AS signals, measurement quality and gene expression level effects. Using a large-scale multi-tissue AS dataset, we demonstrate the advantage of our method over standard alternative approaches. In addition, we describe newly found tissue-specific AS signals which were verified experimentally, and discuss associated regulatory features. Contact: yoseph@psi.utoronto.ca; frey@psi.utoronto.ca Supplementary information: Supplementary data are available at Bioinformatics online.

Barash, Yoseph; Blencowe, Benjamin J.; Frey, Brendan J.

2010-01-01

51

Global analysis of alternative splicing differences between humans and chimpanzees  

Microsoft Academic Search

Alternative splicing is a powerful mechanism affording extensive proteomic and regulatory diversity from a limited repertoire of genes. However, the extent to which alternative splicing has contributed to the evolution of primate species-specific characteristics has not been assessed previously. Using comparative genomics and quantitative microarray profiling, we performed the first global analysis of alternative splicing differences between humans and chimpanzees.

John A. Calarco; Yi Xing; M. Caceres; Joseph P. Calarco; Xinshu Xiao; Qun Pan; Christopher Lee; Todd M. Preuss; Benjamin J. Blencowe

2007-01-01

52

Identification of cells deficient in signaling-induced alternative splicing by use of somatic cell genetics.  

PubMed Central

In recent years, a growing number of mammalian genes have been shown to undergo alternative splicing in response to extracellular stimuli. However, the factors and pathways involved in such signal-induced alternative splicing are almost entirely unknown. Here we describe a novel method for identifying candidate trans-acting factors that are involved in regulating mammalian alternative splicing, using the activation-induced alternative splicing of the human CD45 gene in T cells as a model system. We generated a cell line that stably expresses a CD45 minigene-based GFP reporter construct, such that the levels of green-fluorescent protein (GFP) expressed in the cell reflect the splicing state of the endogenous CD45 gene. Following mutagenesis of this cell line, and multiple rounds of selection for cells that displayed aberrant levels of GFP expression, we isolated several cell lines that are at least partially defective in their ability to support regulated alternative splicing of endogenous CD45 pre-mRNA in response to cell stimulation. Thus we have successfully isolated mutants in a mammalian alternative splicing pathway through use of a somatic cell-based genetic screen. This study clearly demonstrates the feasibility of using genetic screens to further our understanding of the regulation of mammalian splicing, particularly as it occurs in response to environmental cues.

Sheives, Paul; Lynch, Kristen W

2002-01-01

53

Alternative splicing at the right time.  

PubMed

Alternative splicing (AS) allows the production of multiple mRNA variants from a single gene, which contributes to increase the complexity of the proteome. There is evidence that AS is regulated not only by auxiliary splicing factors, but also by components of the core spliceosomal machinery, as well as through epigenetic modifications. However, to what extent these different mechanisms contribute to the regulation of AS in response to endogenous or environmental stimuli is still unclear. Circadian clocks allow organisms to adjust physiological processes to daily changes in environmental conditions. Here we review recent evidence linking circadian clock and AS, and discuss the role of Protein Arginine Methyltransferase 5 (PRMT5) in these processes. We propose that the interactions between daily oscillations in AS and circadian rhythms in the expression of splicing factors and epigenetic regulators offer a great opportunity to dissect the contribution of these mechanisms to the regulation of AS in a physiologically relevant context. PMID:21941124

Sanchez, Sabrina E; Petrillo, Ezequiel; Kornblihtt, Alberto R; Yanovsky, Marcelo J

2011-11-01

54

Co-transcriptional splicing of pre-messenger RNAs: considerations for the mechanism of alternative splicing  

Microsoft Academic Search

Nascent transcripts are the true substrates for many splicing events in mammalian cells. In this review we discuss transcription, splicing, and alternative splicing in the context of co-transcriptional processing of pre-mRNA. The realization that splicing occurs co-transcriptionally requires two important considerations: First, the cis-acting elements in the splicing substrate are synthesized at different times in a 5? to 3? direction.

Aaron C Goldstrohm; Arno L Greenleaf; Mariano A Garcia-Blanco

2001-01-01

55

Integrating alternative splicing detection into gene prediction  

PubMed Central

Background Alternative splicing (AS) is now considered as a major actor in transcriptome/proteome diversity and it cannot be neglected in the annotation process of a new genome. Despite considerable progresses in term of accuracy in computational gene prediction, the ability to reliably predict AS variants when there is local experimental evidence of it remains an open challenge for gene finders. Results We have used a new integrative approach that allows to incorporate AS detection into ab initio gene prediction. This method relies on the analysis of genomically aligned transcript sequences (ESTs and/or cDNAs), and has been implemented in the dynamic programming algorithm of the graph-based gene finder EuGÈNE. Given a genomic sequence and a set of aligned transcripts, this new version identifies the set of transcripts carrying evidence of alternative splicing events, and provides, in addition to the classical optimal gene prediction, alternative optimal predictions (among those which are consistent with the AS events detected). This allows for multiple annotations of a single gene in a way such that each predicted variant is supported by a transcript evidence (but not necessarily with a full-length coverage). Conclusions This automatic combination of experimental data analysis and ab initio gene finding offers an ideal integration of alternatively spliced gene prediction inside a single annotation pipeline.

Foissac, Sylvain; Schiex, Thomas

2005-01-01

56

DBASS3 and DBASS5: databases of aberrant 3'- and 5'-splice sites.  

PubMed

DBASS3 and DBASS5 provide comprehensive repositories of new exon boundaries that were induced by pathogenic mutations in human disease genes. Aberrant 5'- and 3'-splice sites were activated either by mutations in the consensus sequences of natural exon-intron junctions (cryptic sites) or elsewhere ('de novo' sites). DBASS3 and DBASS5 currently contain approximately 900 records of cryptic and de novo 3'- and 5'-splice sites that were produced by over a thousand different mutations in approximately 360 genes. DBASS3 and DBASS5 data can be searched by disease phenotype, gene, mutation, location of aberrant splice sites in introns and exons and their distance from authentic counterparts, by bibliographic references and by the splice-site strength estimated with several prediction algorithms. The user can also retrieve reference sequences of both aberrant and authentic splice sites with the underlying mutation. These data will facilitate identification of introns or exons frequently involved in aberrant splicing, mutation analysis of human disease genes and study of germline or somatic mutations that impair RNA processing. Finally, this resource will be useful for fine-tuning splice-site prediction algorithms, better definition of auxiliary splicing signals and design of new reporter assays. DBASS3 and DBASS5 are freely available at http://www.dbass.org.uk/. PMID:20929868

Buratti, Emanuele; Chivers, Martin; Hwang, Gyulin; Vorechovsky, Igor

2010-10-06

57

EGCG corrects aberrant splicing of IKAP mRNA in cells from patients with familial dysautonomia  

Microsoft Academic Search

Familial dysautonomia (FD) is an autosomal recessive neurodegenerative disorder. The most prevalent causative mutation is a T?C transition in a donor splice site of the IKBKAP transcript, resulting in aberrant splicing and a truncated protein. The mutation’s position and leaky nature suggested that its impact might be moderated by altering the level of splice-regulating proteins. The reported ability of (?)-epigallocatechin

Sylvia L. Anderson; Jinsong Qiu; Berish Y. Rubin

2003-01-01

58

Validation of Human Alternative Splice Forms Using the EASED Platform and Multiple Splice Site Discriminating Features  

Microsoft Academic Search

We have shown for a dataset of computationally predicted alternative splice sites how inherent information can be utilized\\u000a to validate the predictions by applying statistics on different features typical for splice sites. As a promising splice site\\u000a feature we investigated the frequencies of binding motifs in the context of exonic and intronic splice site flanks and between\\u000a the alternative and

Ralf Bortfeldt; Alexander Herrmann; Heike Pospisil; Stefan Schuster

59

The importance of being divisible by three in alternative splicing  

Microsoft Academic Search

Alternative splicing events that are conserved in orthologous genes in different species are commonly viewed as reliable evidence of authentic, functionally significant alternative splicing events. Several recent bioinformatic analyses have shown that con- served alternative exons possess several features that distinguish them from alternative exons that are species-specific. One of the most striking differ- ences between conserved and species-specific alternative

Alon Magen; Gil Ast

2005-01-01

60

Alternative splicing: decoding an expansive regulatory layer.  

PubMed

Alternative splicing (AS) is the process by which splice sites in precursor (pre)-mRNA are differentially selected to produce multiple mRNA and protein isoforms. During the past few years the application of genome-wide profiling technologies coupled with bioinformatic approaches has transformed our understanding of AS complexity and regulation. These studies are further driving research directed at elucidating the functions of networks of regulated AS events in the context of normal physiology and disease. Major strides have also been made in understanding how AS is functionally integrated with- and coupled to- gene regulation at the level of chromatin and transcription. Particularly intriguing is the discovery of new AS 'switches' that control transcriptional networks required for animal development and behavior. PMID:22465326

Irimia, Manuel; Blencowe, Benjamin J

2012-03-30

61

Alternative Splicing and Subfunctionalization Generates Functional Diversity in Fungal Proteomes  

PubMed Central

Alternative splicing is commonly used by the Metazoa to generate more than one protein from a gene. However, such diversification of the proteome by alternative splicing is much rarer in fungi. We describe here an ancient fungal alternative splicing event in which these two proteins are generated from a single alternatively spliced ancestral SKI7/HBS1 gene retained in many species in both the Ascomycota and Basidiomycota. While the ability to express two proteins from a single SKI7/HBS1 gene is conserved in many fungi, the exact mechanism by which they achieve this varies. The alternative splicing was lost in Saccharomyces cerevisiae following the whole-genome duplication event as these two genes subfunctionalized into the present functionally distinct HBS1 and SKI7 genes. When expressed in yeast, the single gene from Lachancea kluyveri generates two functionally distinct proteins. Expression of one of these proteins complements hbs1, but not ski7 mutations, while the other protein complements ski7, but not hbs1. This is the first known case of subfunctionalization by loss of alternative splicing in yeast. By coincidence, the ancestral alternatively spliced gene was also duplicated in Schizosaccharomyces pombe with subsequent subfunctionalization and loss of splicing. Similar subfunctionalization by loss of alternative splicing in fungi also explains the presence of two PTC7 genes in the budding yeast Tetrapisispora blattae, suggesting that this is a common mechanism to preserve duplicate alternatively spliced genes.

Jimenez-Lopez, Claudia; Lorenz, Michael C.; van Hoof, Ambro

2013-01-01

62

Alternative splicing and trans-splicing events revealed by analysis of the Bombyx mori transcriptome  

PubMed Central

Alternative splicing and trans-splicing events have not been systematically studied in the silkworm Bombyx mori. Here, the silkworm transcriptome was analyzed by RNA-seq. We identified 320 novel genes, modified 1140 gene models, and found thousands of alternative splicing and 58 trans-splicing events. Studies of three SR proteins show that both their alternative splicing patterns and mRNA products are conserved from insect to human, and one isoform of Srsf6 with a retained intron is expressed sex-specifically in silkworm gonads. Trans-splicing of mod(mdg4) in silkworm was experimentally confirmed. We identified integrations from a common 5?-gene with 46 newly identified alternative 3?-exons that are located on both DNA strands over a 500-kb region. Other trans-splicing events in B. mori were predicted by bioinformatic analysis, in which 12 events were confirmed by RT-PCR, six events were further validated by chimeric SNPs, and two events were confirmed by allele-specific RT-PCR in F1 hybrids from distinct silkworm lines of JS and L10, indicating that trans-splicing is more widespread in insects than previously thought. Analysis of the B. mori transcriptome by RNA-seq provides valuable information of regulatory alternative splicing events. The conservation of splicing events across species and newly identified trans-splicing events suggest that B. mori is a good model for future studies.

Shao, Wei; Zhao, Qiong-Yi; Wang, Xiu-Ye; Xu, Xin-Yan; Tang, Qing; Li, Muwang; Li, Xuan; Xu, Yong-Zhen

2012-01-01

63

A study of alternative splicing in the pig  

Microsoft Academic Search

BACKGROUND: Since at least half of the genes in mammalian genomes are subjected to alternative splicing, alternative pre-mRNA splicing plays an important contribution to the complexity of the mammalian proteome. Expressed sequence tags (ESTs) provide evidence of a great number of possible alternative isoforms. With the EST resource for the domestic pig now containing more than one million porcine ESTs,

Ann-Britt Nygard; Susanna Cirera; Michael J Gilchrist; Jan Gorodkin; Claus B Jørgensen; Merete Fredholm

2010-01-01

64

Aberrant splicing caused by a MLH1 splice donor site mutation found in a young Japanese patient with Lynch syndrome.  

PubMed

Lynch syndrome, also known as hereditary non-polyposis colorectal cancer, characterized by predisposition to colorectal cancer and other associated cancers, is an autosomal-dominant disorder mainly caused by germline mutations in DNA mismatch repair (MMR) genes such as MLH1, MSH2, and MSH6. Some mutations that disrupt splice donor or acceptor sites cause aberrant mRNA splicing. These mutations are generally considered as pathogenic ones, however, it is sometimes uneasy to accurately predict their pathogenicity without functional assays, particularly when the mutation is a single nucleotide substitution. In this report, we describe a 25-year-old patient with Lynch syndrome who carries a germline variant in a splice donor site of the MLH1 gene (c.790 + 5 G > T), which was first detected among Asian populations. The immunohistochemical analysis revealed loss of MLH1 protein expression in the tumor. Our splicing assay confirmed that the intronic MLH1 variant actually caused aberrant splicing, supporting its pathogenic effect. Our data accumulate more information on the genotype-phenotype relationships in patients with Lynch syndrome. PMID:22766992

Takahashi, Masanobu; Furukawa, Yoichi; Shimodaira, Hideki; Sakayori, Masato; Moriya, Takuya; Moriya, Yoshihiro; Nakamura, Yusuke; Ishioka, Chikashi

2012-12-01

65

Neuronal Signaling through Alternative Splicing: Some Exons CaRRE...  

NSDL National Science Digital Library

Alternative splicing represents a mechanism by which a single gene can be used to create proteins with different functions. Neurons use alternative splicing to produce channels with different sequences and biophysical or regulatory properties. O'Donovan and Darnell discuss a mechanism by which neurons can alter channel splicing in response to neuronal activity through a signal generated by calcium and calcium/calmodulin-dependent kinase activity.

Kevin J. O'Donovan (The Rockefeller University;Laboratory of Molecular Neuro-Oncology REV); Robert B. Darnell (The Rockefeller University;Laboratory of Molecular Neuro-Oncology REV)

2001-08-07

66

Alternative splicing: increasing diversity in the proteomic world  

Microsoft Academic Search

How can the genome of Drosophila melanogaster contain fewer genes than the undoubtedly simpler organism Caenorhabditis elegans? The answer must lie within their proteomes. It is becoming clear that alternative splicing has an extremely important role in expanding protein diversity and might therefore partially underlie the apparent discrepancy between gene number and organismal complexity. Alternative splicing can generate more transcripts

Brenton R. Graveley

2001-01-01

67

Alternative splicing and biological heterogeneity in prostate cancer  

Microsoft Academic Search

The biological diversity of prostate cancer confounds standardization of therapy. Advances in molecular profiling suggest that differences in the genetic composition of tumors significantly contribute to the complexity of the disease. Alternative pre-mRNA splicing is a key genetic process underlying biological diversity. During alternative splicing, coding and noncoding regions of a single gene are rearranged to generate several messenger RNA

David J. Elliott; Craig N. Robson; Hing Y. Leung; Prabhakar Rajan

2009-01-01

68

A Unique, Consistent Identifier for Alternatively Spliced Transcript Variants  

Microsoft Academic Search

BackgroundAs research into alternative splicing reveals the fundamental importance of this phenomenon in the genome expression of higher organisms, there is an increasing need for a standardized, consistent and unique identifier for alternatively spliced isoforms. Such an identifier would be useful to eliminate ambiguities in references to gene isoforms, and would allow for the reliable comparison of isoforms from different

Alberto Riva; Graziano Pesole; Juan Valcarcel

2009-01-01

69

Intron mis-splicing: no alternative?  

PubMed Central

A recent report reveals widespread mis-splicing of RNA transcripts in eukaryotes, with mis-spliced RNA destroyed by nonsense-mediated mRNA decay. This striking inefficiency deepens the mystery of the proliferation and persistence of introns.

Roy, Scott William; Irimia, Manuel

2008-01-01

70

Conditional knockout mice to study alternative splicing in vivo.  

PubMed

Analysis of genomes has revealed that the total number of human genes is comparable to those of simpler organisms, and thus, the number of genes does not correlate with the complexity and functional diversity of different organisms. Multiple mechanisms, including alternative splicing, are believed to contribute to the molecular complexity in higher eukaryotes. Given the fact that more than half of human genes undergo alternative splicing, however, little is known about the biological relevance of most alternative splicing events and their regulatory mechanisms. Recent work has highlighted the power of reverse genetic approaches in addressing regulated splicing in animal models. Here, we focus on the conditional knockout approach adapted for splicing research with the intention to provide a general guide to the generation of mouse models to study regulated splicing in development and disease. PMID:16314268

Xu, Xiangdong; Fu, Xiang-Dong

2005-12-01

71

Alternative 5' splice site selection induced by heat shock.  

PubMed Central

The mouse HSP47 gene consists of six exons separated by five introns. Three HSP47 cDNAs differing only in their 5' noncoding regions have been reported. One of these alternatively spliced mRNAs was detected only after heat shock, which caused an alternative 5' splice donor site selection. Other stress inducers, including an amino acid analog and sodium arsenite, had no effect on the alternative splicing. The alternatively spliced mRNA, which was 169 nucleotides longer in the 5' noncoding region compared to mRNA transcribed in non-heat shock conditions, was efficiently translated under heat shock conditions. This novel finding that alternative splicing is caused by artificial treatment like heat shock will provide a useful in vivo model for understanding the exon-intron recognition mechanism as well as heat shock-induced alterations in gene expression. Images

Takechi, H; Hosokawa, N; Hirayoshi, K; Nagata, K

1994-01-01

72

Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged  

PubMed Central

Alternative splicing of mRNA precursors is a nearly ubiquitous and extremely flexible point of gene control in humans. It provides cells with the opportunity to create protein isoforms of differing, even opposing, functions from a single gene. Cancer cells often take advantage of this flexibility to produce proteins that promote growth and survival. Many of the isoforms produced in this manner are developmentally regulated and are preferentially re-expressed in tumors. Emerging insights into this process indicate that pathways that are frequently deregulated in cancer often play important roles in promoting aberrant splicing, which in turn contributes to all aspects of tumor biology.

David, Charles J.; Manley, James L.

2010-01-01

73

Identical Splicing of Aberrant Epidermal Growth Factor Receptor Transcripts from Amplified Rearranged Genes in Human Glioblastomas  

Microsoft Academic Search

The epidermal growth factor receptor gene has been found to be amplified and rearranged in human glioblastomas in vivo. Here we present the sequence across a splice junction of aberrant epidermal growth factor receptor transcripts derived from corresponding and uniquely rearranged genes that are coamplified and coexpressed with non-rearranged epidermal growth factor receptor genes in six primary human glioblastomas. Each

Noriaki Sugawa; A. Jonas Ekstrand; C. David James; V. Peter Collins

1990-01-01

74

BIPASS: BioInformatics Pipeline Alternative Splicing Services  

PubMed Central

BioInformatics Pipeline Alternative Splicing Services (BIPASS) offer support to scientists interested in gathering information related to alternative splicing (AS) events. The service BIPAS–SpliceDB provides access to AS information that has been extracted a priori from various public databases and stored in a data warehouse. In contrast, the BIPAS–Align&Splice service allows scientists to submit their own sequences and genome to compute AS analysis results. BIPAS services offer various user-friendly ways to navigate through the results. AS results are organized at different conceptual levels (clusters and sequences), and are displayed in graphs or summarized in tables that can be downloaded in XML or text format. The two BIPAS services SpliceDB and Align&Splice are available online at http://bip.umiacs.umd.edu:8080/.

Lacroix, Zoe; Legendre, Christophe; Raschid, Louiqa; Snyder, Ben

2007-01-01

75

Genome-wide analysis of alternative splicing in Chlamydomonas reinhardtii  

PubMed Central

Background Genome-wide computational analysis of alternative splicing (AS) in several flowering plants has revealed that pre-mRNAs from about 30% of genes undergo AS. Chlamydomonas, a simple unicellular green alga, is part of the lineage that includes land plants. However, it diverged from land plants about one billion years ago. Hence, it serves as a good model system to study alternative splicing in early photosynthetic eukaryotes, to obtain insights into the evolution of this process in plants, and to compare splicing in simple unicellular photosynthetic and non-photosynthetic eukaryotes. We performed a global analysis of alternative splicing in Chlamydomonas reinhardtii using its recently completed genome sequence and all available ESTs and cDNAs. Results Our analysis of AS using BLAT and a modified version of the Sircah tool revealed AS of 498 transcriptional units with 611 events, representing about 3% of the total number of genes. As in land plants, intron retention is the most prevalent form of AS. Retained introns and skipped exons tend to be shorter than their counterparts in constitutively spliced genes. The splice site signals in all types of AS events are weaker than those in constitutively spliced genes. Furthermore, in alternatively spliced genes, the prevalent splice form has a stronger splice site signal than the non-prevalent form. Analysis of constitutively spliced introns revealed an over-abundance of motifs with simple repetitive elements in comparison to introns involved in intron retention. In almost all cases, AS results in a truncated ORF, leading to a coding sequence that is around 50% shorter than the prevalent splice form. Using RT-PCR we verified AS of two genes and show that they produce more isoforms than indicated by EST data. All cDNA/EST alignments and splice graphs are provided in a website at http://combi.cs.colostate.edu/as/chlamy. Conclusions The extent of AS in Chlamydomonas that we observed is much smaller than observed in land plants, but is much higher than in simple unicellular heterotrophic eukaryotes. The percentage of different alternative splicing events is similar to flowering plants. Prevalence of constitutive and alternative splicing in Chlamydomonas, together with its simplicity, many available public resources, and well developed genetic and molecular tools for this organism make it an excellent model system to elucidate the mechanisms involved in regulated splicing in photosynthetic eukaryotes.

2010-01-01

76

Safer, silencing-resistant lentiviral vectors: optimization of the ubiquitous chromatin-opening element through elimination of aberrant splicing.  

PubMed

Gammaretroviral and lentiviral vectors have been used successfully in several clinical gene therapy trials, although powerful enhancer elements have caused insertional mutagenesis and clonal dysregulation. Self-inactivating vectors with internal heterologous regulatory elements have been developed as potentially safer and more effective alternatives. Lentiviral vectors containing a ubiquitous chromatin opening element from the human HNRPA2B1-CBX3 locus (A2UCOE), which allows position-independent, long-term transgene expression, are particularly promising. In a recently described assay, aberrantly spliced mRNA transcripts initiated in the vector A2UCOE sequence were found to lead to upregulation of growth hormone receptor gene (Ghr) expression in transduced murine Bcl-15 cells. Aberrant hybrid mRNA species formed between A2UCOE and a number of other cellular genes were also detected in transduced human PLB-985 myelomonocytic cells. Modification of the A2UCOE by mutation or deletion of recognized and potential cryptic splice donor sites was able to abrogate these splicing events and hybrid mRNA formation in Bcl-15 cells. This modification did not compromise A2UCOE regulatory activity in terms of resistance to CpG methylation and gene silencing in murine P19 embryonic carcinoma cells. These refined A2UCOE regulatory elements are likely to improve intrinsic biosafety and may be particularly useful for a number of clinical applications where robust gene expression is desirable. PMID:22696657

Knight, Sean; Zhang, Fang; Mueller-Kuller, Uta; Bokhoven, Marieke; Gupta, Abhinav; Broughton, Thomas; Sha, Sha; Antoniou, Michael N; Brendel, Christian; Grez, Manuel; Thrasher, Adrian J; Collins, Mary; Takeuchi, Yasuhiro

2012-06-13

77

Safer, Silencing-Resistant Lentiviral Vectors: Optimization of the Ubiquitous Chromatin-Opening Element through Elimination of Aberrant Splicing  

PubMed Central

Gammaretroviral and lentiviral vectors have been used successfully in several clinical gene therapy trials, although powerful enhancer elements have caused insertional mutagenesis and clonal dysregulation. Self-inactivating vectors with internal heterologous regulatory elements have been developed as potentially safer and more effective alternatives. Lentiviral vectors containing a ubiquitous chromatin opening element from the human HNRPA2B1-CBX3 locus (A2UCOE), which allows position-independent, long-term transgene expression, are particularly promising. In a recently described assay, aberrantly spliced mRNA transcripts initiated in the vector A2UCOE sequence were found to lead to upregulation of growth hormone receptor gene (Ghr) expression in transduced murine Bcl-15 cells. Aberrant hybrid mRNA species formed between A2UCOE and a number of other cellular genes were also detected in transduced human PLB-985 myelomonocytic cells. Modification of the A2UCOE by mutation or deletion of recognized and potential cryptic splice donor sites was able to abrogate these splicing events and hybrid mRNA formation in Bcl-15 cells. This modification did not compromise A2UCOE regulatory activity in terms of resistance to CpG methylation and gene silencing in murine P19 embryonic carcinoma cells. These refined A2UCOE regulatory elements are likely to improve intrinsic biosafety and may be particularly useful for a number of clinical applications where robust gene expression is desirable.

Knight, Sean; Zhang, Fang; Mueller-Kuller, Uta; Bokhoven, Marieke; Gupta, Abhinav; Broughton, Thomas; Sha, Sha; Antoniou, Michael N.; Brendel, Christian; Grez, Manuel; Thrasher, Adrian J.; Collins, Mary

2012-01-01

78

An Alternative Splicing Network Links Cell Cycle Control to Apoptosis  

PubMed Central

Summary Alternative splicing is a vast source of biological regulation and diversity that is misregulated in cancer and other diseases. To investigate global control of alternative splicing in human cells, we analyzed splicing of mRNAs encoding Bcl2-family apoptosis factors in a genome-wide siRNA screen. The screen identified many novel regulators of Bcl-x and Mcl1 splicing, notably an extensive network of cell cycle factors linked to aurora kinase A. Drugs or siRNAs that induce mitotic arrest promoted pro-apoptotic splicing of Bcl-x, Mcl1, and caspase-9, and altered splicing of other apoptotic transcripts. This response preceded mitotic arrest, indicating coordinated upregulation of pro-death splice variants that promotes apoptosis in arrested cells. These shifts corresponded to post-translational turnover of splicing regulator ASF/SF2, which directly binds and regulates these target mRNAs and globally regulates apoptosis. Broadly, our results reveal an alternative splicing network linking cell cycle control to apoptosis.

Moore, Michael J.; Wang, Qingqing; Kennedy, Caleb J.; Silver, Pamela A.

2010-01-01

79

Aberrant splicing induced by missense mutations in BRCA1: clues from a humanized mouse model.  

PubMed

Numerous missense mutations in human BRCA1 gene have been linked to predisposition to breast cancer. However, the functional significance of the majority of these mutations remains unknown. We have examined the molecular basis for three such cancer-causing mutations. The first mutation, a T-->G transversion in codon 64, is predicted to change a conserved cysteine residue to glycine in the RING finger domain of the 1863 amino acid BRCA1 protein. Using a humanized mouse model we demonstrate that this missense mutation actually results in a functionally null protein. This striking result occurs because the single base alteration generates a new 5' splice site in exon 5 and also disrupts a putative exonic splicing enhancer motif. Consequently, the normal splice donor site is disrupted and an internal cryptic splice site is activated. This results in a 22-nucleotide deletion and the aberrant transcript is predicted to encode a severely truncated protein consisting of only 63 amino acids. To identify other missense mutations in BRCA1 that may result in aberrant splicing, we screened various mutations using the Genscan program. We demonstrate that at least two other missense mutations in codons 1495 and 1823 result in aberrant splicing due to the possible disruption of cis-acting splicing regulatory elements. In conclusion, our study demonstrates for the first time the application of a humanized mouse model for functional analysis of human mutations in mice and also shows the need for a careful examination of the functional consequences of single base alterations and single nucleotide polymorphisms identified in human disease-causing genes. PMID:12915465

Yang, Yongping; Swaminathan, Srividya; Martin, Betty K; Sharan, Shyam K

2003-07-08

80

Mammalian tissues defective in nonsense-mediated mRNA decay display highly aberrant splicing patterns  

PubMed Central

Background Nonsense-mediated mRNA decay (NMD) affects the outcome of alternative splicing by degrading mRNA isoforms with premature termination codons. Splicing regulators constitute important NMD targets; however, the extent to which loss of NMD causes extensive deregulation of alternative splicing has not previously been assayed in a global, unbiased manner. Here, we combine mouse genetics and RNA-seq to provide the first in vivo analysis of the global impact of NMD on splicing patterns in two primary mouse tissues ablated for the NMD factor UPF2. Results We developed a bioinformatic pipeline that maps RNA-seq data to a combinatorial exon database, predicts NMD-susceptibility for mRNA isoforms and calculates the distribution of major splice isoform classes. We present a catalog of NMD-regulated alternative splicing events, showing that isoforms of 30% of all expressed genes are upregulated in NMD-deficient cells and that NMD targets all major splicing classes. Importantly, NMD-dependent effects are not restricted to premature termination codon+ isoforms but also involve an abundance of splicing events that do not generate premature termination codons. Supporting their functional importance, the latter events are associated with high intronic conservation. Conclusions Our data demonstrate that NMD regulates alternative splicing outcomes through an intricate web of splicing regulators and that its loss leads to the deregulation of a panoply of splicing events, providing novel insights into its role in core- and tissue-specific regulation of gene expression. Thus, our study extends the importance of NMD from an mRNA quality pathway to a regulator of several layers of gene expression.

2012-01-01

81

Modulating alternative splicing by cotranscriptional cleavage of nascent intronic RNA  

PubMed Central

Cotranscriptional cleavage mediated by a hammerhead ribozyme can affect alternative splicing if interposed between an exon and its intronic regulatory elements. This has been demonstrated using two different alternative splicing systems based on ?-tropomyosin and fibronectin genes. We suggest that there is a requirement for intronic regulatory elements to be covalently attached to exons that are in turn tethered to the elongating polymerase. In the case of the alternatively spliced EDA exon of the fibronectin gene, we demonstrate that the newly identified intronic downstream regulatory element is associated with the splicing regulatory protein SRp20. Our results suggest that targeted hammerhead ribozyme cleavage within introns can be used as a tool to define splicing regulatory elements.

Gromak, Natalia; Talotti, Gabriele; Proudfoot, Nicholas J.; Pagani, Franco

2008-01-01

82

Intermolecular domain swapping induces intein-mediated protein alternative splicing.  

PubMed

Protein sequences are diversified on the DNA level by recombination and mutation and can be further increased on the RNA level by alternative RNA splicing, involving introns that have important roles in many biological processes. The protein version of introns (inteins), which catalyze protein splicing, were first reported in the 1990s. The biological roles of protein splicing still remain elusive because inteins neither provide any clear benefits nor have an essential role in their host organisms. We now report protein alternative splicing, in which new protein sequences can be produced by protein recombination by intermolecular domain swapping of inteins, as elucidated by NMR spectroscopy and crystal structures. We demonstrate that intein-mediated protein alternative splicing could be a new strategy to increase protein diversity (that is, functions) without any modification in genetic backgrounds. We also exploited it as a post-translational protein conformation-driven switch of protein functions (for example, as highly specific protein interference). PMID:23974115

Aranko, A Sesilja; Oeemig, Jesper S; Kajander, Tommi; Iwaï, Hideo

2013-08-25

83

Alternative splicing and evolution: diversification, exon definition and function  

Microsoft Academic Search

Over the past decade, it has been shown that alternative splicing (AS) is a major mechanism for the enhancement of transcriptome and proteome diversity, particularly in mammals. Splicing can be found in species from bacteria to humans, but its prevalence and characteristics vary considerably. Evolutionary studies are helping to address questions that are fundamental to understanding this important process: how

Galit Lev-Maor; Gil Ast; Hadas Keren

2010-01-01

84

Alternative splicing in angiogenesis: the vascular endothelial growth factor paradigm.  

PubMed

Alternative splicing, first discovered in the 1970s, has emerged as one of the key generators of proteomic diversity. Not surprisingly, alternative splicing is increasingly linked to the etiology of cancer. This is illustrated by vascular endothelial growth factor (VEGF), the dominant angiogenic factor. Recently, an antiangiogenic family of VEGF isoforms was discovered, and termed VEGF(xxx)b. VEGF(xxx)b isoforms arise from an alternative 3' splice site in exon 8, and differ by a mere six amino acids at the C-terminus. These alternative six amino acids radically change the functional properties of VEGF. VEGF(xxx)b isoform expression is regulated in human tissues and development, and disregulated in many pathological states including cancer. Understanding what regulates VEGF(xxx)b alternative splicing, and therefore the balance of pro- and antiangiogenic isoforms is of great importance and will be explored in detail over the next few years. PMID:17027147

Ladomery, Michael R; Harper, Steven J; Bates, David O

2006-10-05

85

Genome-wide analysis of alternative splicing in Caenorhabditis elegans.  

PubMed

Alternative splicing (AS) plays a crucial role in the diversification of gene function and regulation. Consequently, the systematic identification and characterization of temporally regulated splice variants is of critical importance to understanding animal development. We have used high-throughput RNA sequencing and microarray profiling to analyze AS in C. elegans across various stages of development. This analysis identified thousands of novel splicing events, including hundreds of developmentally regulated AS events. To make these data easily accessible and informative, we constructed the C. elegans Splice Browser, a web resource in which researchers can mine AS events of interest and retrieve information about their relative levels and regulation across development. The data presented in this study, along with the Splice Browser, provide the most comprehensive set of annotated splice variants in C. elegans to date, and are therefore expected to facilitate focused, high resolution in vivo functional assays of AS function. PMID:21177968

Ramani, Arun K; Calarco, John A; Pan, Qun; Mavandadi, Sepand; Wang, Ying; Nelson, Andrew C; Lee, Leo J; Morris, Quaid; Blencowe, Benjamin J; Zhen, Mei; Fraser, Andrew G

2010-12-22

86

Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF  

PubMed Central

CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200tr) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200.

Chen, Zhiqi; Ma, Xuezhong; Zhang, Jianhua; Hu, Jim; Gorczynski, Reginald M.

2010-01-01

87

Recognition of Unknown Conserved Alternatively Spliced Exons  

PubMed Central

The split structure of most mammalian protein-coding genes allows for the potential to produce multiple different mRNA and protein isoforms from a single gene locus through the process of alternative splicing (AS). We propose a computational approach called UNCOVER based on a pair hidden Markov model to discover conserved coding exonic sequences subject to AS that have so far gone undetected. Applying UNCOVER to orthologous introns of known human and mouse genes predicts skipped exons or retained introns present in both species, while discriminating them from conserved noncoding sequences. The accuracy of the model is evaluated on a curated set of genes with known conserved AS events. The prediction of skipped exons in the ~1% of the human genome represented by the ENCODE regions leads to more than 50 new exon candidates. Five novel predicted AS exons were validated by RT-PCR and sequencing analysis of 15 introns with strong UNCOVER predictions and lacking EST evidence. These results imply that a considerable number of conserved exonic sequences and associated isoforms are still completely missing from the current annotation of known genes. UNCOVER also identifies a small number of candidates for conserved intron retention.

Ohler, Uwe; Shomron, Noam; Burge, Christopher B

2005-01-01

88

Spliced Leader Trapping Reveals Widespread Alternative Splicing Patterns in the Highly Dynamic Transcriptome of Trypanosoma brucei  

PubMed Central

Trans-splicing of leader sequences onto the 5?ends of mRNAs is a widespread phenomenon in protozoa, nematodes and some chordates. Using parallel sequencing we have developed a method to simultaneously map 5?splice sites and analyze the corresponding gene expression profile, that we term spliced leader trapping (SLT). The method can be applied to any organism with a sequenced genome and trans-splicing of a conserved leader sequence. We analyzed the expression profiles and splicing patterns of bloodstream and insect forms of the parasite Trypanosoma brucei. We detected the 5? splice sites of 85% of the annotated protein-coding genes and, contrary to previous reports, found up to 40% of transcripts to be differentially expressed. Furthermore, we discovered more than 2500 alternative splicing events, many of which appear to be stage-regulated. Based on our findings we hypothesize that alternatively spliced transcripts present a new means of regulating gene expression and could potentially contribute to protein diversity in the parasite. The entire dataset can be accessed online at TriTrypDB or through: http://splicer.unibe.ch/.

Nilsson, Daniel; Gunasekera, Kapila; Mani, Jan; Osteras, Magne; Farinelli, Laurent; Baerlocher, Loic; Roditi, Isabel; Ochsenreiter, Torsten

2010-01-01

89

Deep Intron Elements Mediate Nested Splicing Events at Consecutive AG Dinucleotides To Regulate Alternative 3? Splice Site Choice in Vertebrate 4.1 Genes  

PubMed Central

Distal intraexon (iE) regulatory elements in 4.1R pre-mRNA govern 3? splice site choice at exon 2 (E2) via nested splicing events, ultimately modulating expression of N-terminal isoforms of cytoskeletal 4.1R protein. Here we explored intrasplicing in other normal and disease gene contexts and found conservation of intrasplicing through vertebrate evolution. In the paralogous 4.1B gene, we identified ?120 kb upstream of E2 an ultradistal intraexon, iEB, that mediates intrasplicing by promoting two intricately coupled splicing events that ensure selection of a weak distal acceptor at E2 (E2dis) by prior excision of the competing proximal acceptor (E2prox). Mutating iEB in minigene splicing reporters abrogated intrasplicing, as did blocking endogenous iEB function with antisense morpholinos in live mouse and zebrafish animal models. In a human elliptocytosis patient with a mutant 4.1R gene lacking E2 through E4, we showed that aberrant splicing is consistent with iER-mediated intrasplicing at the first available exons downstream of iER, namely, alternative E5 and constitutive E6. Finally, analysis of heterologous acceptor contexts revealed a strong preference for nested 3? splice events at consecutive pairs of AG dinucleotides. Distal regulatory elements may control intrasplicing at a subset of alternative 3? splice sites in vertebrate pre-mRNAs to generate proteins with functional diversity.

Parra, Marilyn K.; Gallagher, Thomas L.; Amacher, Sharon L.; Mohandas, Narla

2012-01-01

90

Coupling of signal transduction to alternative pre-mRNA splicing by a composite splice regulator.  

PubMed Central

Alternative splicing of pre-mRNA is a fundamental mechanism of differential gene expression in that it can give rise to functionally distinct proteins from a single gene, according to the developmental or physiological state of cells in multicellular organisms. In the pre-mRNA of the cell surface molecule CD44, the inclusion of up to 10 variant exons (v1-v10) is regulated during development, upon activation of lymphocytes and dendritic cells, and during tumour progression. Using minigene constructs containing CD44 exon v5, we have discovered exonic RNA elements that couple signal transduction to alternative splicing. They form a composite splice regulator encompassing an exon recognition element and splice silencer elements. Both type of elements are necessary to govern cell type-specific inclusion of the exon as well as inducible inclusion in T cells after stimulation by concanavalin A, by Ras signalling or after activation of protein kinase C by phorbol ester. Inducible splicing does not depend on de novo protein synthesis. The coupling of signal transduction to alternative splicing by such elements probably represents the mechanism whereby splice patterns of genes are established during development and can be changed under physiological and pathological conditions.

Konig, H; Ponta, H; Herrlich, P

1998-01-01

91

Alternative splice variants of the human PD1 gene  

Microsoft Academic Search

PD-1 is an immunoregulatory receptor expressed on the surface of activated T cells, B cells, and monocytes. We describe four alternatively spliced PD-1 mRNA transcripts (PD-1?ex2, PD-1?ex3, PD-1?ex2,3, and PD-1?ex2,3,4) in addition to the full length isoform. PD-1?ex2 and PD-1?ex3 are generated by alternative splicing where exon 2 (extracellular IgV-like domain) and exon 3 (transmembrane domain) respectively are spliced out.

Christian Nielsen; Line Ohm-Laursen; Torben Barington; Steffen Husby; Søren T. Lillevang

2005-01-01

92

Regulation of telomerase alternative splicing: a target for chemotherapy.  

PubMed

Telomerase is present in human cancer cells but absent in most somatic tissues. The messenger RNA of human telomerase (hTERT) is alternatively spliced into mostly nonfunctional products. We sought to understand splicing so that we could decrease functional splice isoforms to reduce telomerase activity in order to complement direct enzyme inhibition. Unexpectedly, minigenes containing hTERT exons 5-10 flanked by 150-300 bp intronic sequences did not produce alternative splicing. A 1.1 kb region of 38 bp repeats ~2 kb from the exon 6/intron junction restored the exclusion of exons 7 and 8. An element within intron 8, also >1 kb from intron/exon junctions, modulated this effect. Transducing an oligonucleotide complementary to this second element increased nonfunctional hTERT messenger RNA from endogenous telomerase. These results demonstrate the potential of manipulating hTERT splicing for both chemotherapy and regenerative medicine and provide specific sequences deep within introns that regulate alternative splicing in mammalian cells by mechanisms other than the introduction of cryptic splice sites. PMID:23562158

Wong, Mandy S; Chen, Ling; Foster, Christopher; Kainthla, Radhika; Shay, Jerry W; Wright, Woodring E

2013-04-04

93

Alternative splicing networks regulated by signaling in human T cells.  

PubMed

The formation and execution of a productive immune response requires the maturation of competent T cells and a robust change in cellular activity upon antigen challenge. Such changes in cellular function depend on regulated alterations to protein expression. Previous research has focused on defining transcriptional changes that regulate protein expression during T-cell maturation and antigen stimulation. Here, we globally analyze another critical process in gene regulation during T-cell stimulation, alternative splicing. Specifically, we use RNA-seq profiling to identify 178 exons in 168 genes that exhibit robust changes in inclusion in response to stimulation of a human T-cell line. Supporting an important role for the global coordination of alternative splicing following T-cell stimulation, these signal-responsive exons are significantly enriched in genes with functional annotations specifically related to immune response. The vast majority of these genes also exhibit differential alternative splicing between naive and activated primary T cells. Comparison of the responsiveness of splicing to various stimuli in the cultured and primary T cells further reveals at least three distinct networks of signal-induced alternative splicing events. Importantly, we find that each regulatory network is specifically associated with distinct sequence features, suggesting that they are controlled by independent regulatory mechanisms. These results thus provide a basis for elucidating mechanisms of signal pathway-specific regulation of alternative splicing during T-cell stimulation. PMID:22454538

Martinez, Nicole M; Pan, Qun; Cole, Brian S; Yarosh, Christopher A; Babcock, Grace A; Heyd, Florian; Zhu, William; Ajith, Sandya; Blencowe, Benjamin J; Lynch, Kristen W

2012-03-27

94

Alternative splicing networks regulated by signaling in human T cells  

PubMed Central

The formation and execution of a productive immune response requires the maturation of competent T cells and a robust change in cellular activity upon antigen challenge. Such changes in cellular function depend on regulated alterations to protein expression. Previous research has focused on defining transcriptional changes that regulate protein expression during T-cell maturation and antigen stimulation. Here, we globally analyze another critical process in gene regulation during T-cell stimulation, alternative splicing. Specifically, we use RNA-seq profiling to identify 178 exons in 168 genes that exhibit robust changes in inclusion in response to stimulation of a human T-cell line. Supporting an important role for the global coordination of alternative splicing following T-cell stimulation, these signal-responsive exons are significantly enriched in genes with functional annotations specifically related to immune response. The vast majority of these genes also exhibit differential alternative splicing between naive and activated primary T cells. Comparison of the responsiveness of splicing to various stimuli in the cultured and primary T cells further reveals at least three distinct networks of signal-induced alternative splicing events. Importantly, we find that each regulatory network is specifically associated with distinct sequence features, suggesting that they are controlled by independent regulatory mechanisms. These results thus provide a basis for elucidating mechanisms of signal pathway–specific regulation of alternative splicing during T-cell stimulation.

Martinez, Nicole M.; Pan, Qun; Cole, Brian S.; Yarosh, Christopher A.; Babcock, Grace A.; Heyd, Florian; Zhu, William; Ajith, Sandya; Blencowe, Benjamin J.; Lynch, Kristen W.

2012-01-01

95

Alternative Splicing of SLC39A14 in Colorectal Cancer is Regulated by the Wnt Pathway*  

PubMed Central

Alternative splicing is a crucial step in the generation of protein diversity and its misregulation is observed in many human cancer types. By analyzing 143 colorectal samples using exon arrays, SLC39A14, a divalent cation transporter, was identified as being aberrantly spliced in tumor samples. SLC39A14 contains two mutually exclusive exons 4A and 4B and the exon 4A/4B ratio was significantly altered in adenomas (p = 3.6 × 10?10) and cancers (p = 9.4 × 10?11), independent of microsatellite stability status. The findings were validated in independent exon array data sets and by quantitative real-time reverse-transcription PCR (qRT-PCR). Aberrant Wnt signaling is a hallmark of colorectal tumorigenesis and is characterized by nuclear ?-catenin. Experimental inactivation of Wnt signaling in DLD1 and Ls174T cells by knockdown of ?-catenin or overexpression of dominant negative TCFs (TCF1 and TCF4) altered the 4A/4B ratio, indicating that SLC39A14 splicing is regulated by the Wnt pathway. An altered 4A/4B ratio was also observed in gastric and lung cancer where Wnt signaling is also known to be aberrantly activated. The splicing factor SRSF1 and its regulator, the kinase SRPK1, were found to be deregulated upon Wnt inactivation in colorectal carcinoma cells. SRPK1 was also found up-regulated in both adenoma samples (p = 1.5 × 10?5) and cancer samples (p = 5 × 10?4). In silico splicing factor binding analysis predicted SRSF1 to bind predominantly to the cancer associated exon 4B, hence, it was hypothesized that SRPK1 activates SRSF1 through phosphorylation, followed by SRSF1 binding to exon 4B and regulation of SLC39A14 splicing. Indeed, siRNA-mediated knockdown of SRPK1 and SRSF1 in DLD1 and SW480 colorectal cancer cells led to a change in the 4A/4B isoform ratio, supporting a role of these factors in the regulation of SLC39A14 splicing. In conclusion, alternative splicing of SLC39A14 was identified in colorectal tumors and found to be regulated by the Wnt pathway, most likely through regulation of SRPK1 and SRSF1.

Thorsen, Kasper; Mansilla, Francisco; Schepeler, Troels; ?ster, Bodil; Rasmussen, Mads H.; Dyrskj?t, Lars; Karni, Rotem; Akerman, Martin; Krainer, Adrian R.; Laurberg, S?ren; Andersen, Claus L.; ?rntoft, Torben F.

2011-01-01

96

Alternative splicing of SLC39A14 in colorectal cancer is regulated by the Wnt pathway.  

PubMed

Alternative splicing is a crucial step in the generation of protein diversity and its misregulation is observed in many human cancer types. By analyzing 143 colorectal samples using exon arrays, SLC39A14, a divalent cation transporter, was identified as being aberrantly spliced in tumor samples. SLC39A14 contains two mutually exclusive exons 4A and 4B and the exon 4A/4B ratio was significantly altered in adenomas (p = 3.6 × 10(-10)) and cancers (p = 9.4 × 10(-11)), independent of microsatellite stability status. The findings were validated in independent exon array data sets and by quantitative real-time reverse-transcription PCR (qRT-PCR). Aberrant Wnt signaling is a hallmark of colorectal tumorigenesis and is characterized by nuclear ?-catenin. Experimental inactivation of Wnt signaling in DLD1 and Ls174T cells by knockdown of ?-catenin or overexpression of dominant negative TCFs (TCF1 and TCF4) altered the 4A/4B ratio, indicating that SLC39A14 splicing is regulated by the Wnt pathway. An altered 4A/4B ratio was also observed in gastric and lung cancer where Wnt signaling is also known to be aberrantly activated. The splicing factor SRSF1 and its regulator, the kinase SRPK1, were found to be deregulated upon Wnt inactivation in colorectal carcinoma cells. SRPK1 was also found up-regulated in both adenoma samples (p = 1.5 × 10(-5)) and cancer samples (p = 5 × 10(-4)). In silico splicing factor binding analysis predicted SRSF1 to bind predominantly to the cancer associated exon 4B, hence, it was hypothesized that SRPK1 activates SRSF1 through phosphorylation, followed by SRSF1 binding to exon 4B and regulation of SLC39A14 splicing. Indeed, siRNA-mediated knockdown of SRPK1 and SRSF1 in DLD1 and SW480 colorectal cancer cells led to a change in the 4A/4B isoform ratio, supporting a role of these factors in the regulation of SLC39A14 splicing. In conclusion, alternative splicing of SLC39A14 was identified in colorectal tumors and found to be regulated by the Wnt pathway, most likely through regulation of SRPK1 and SRSF1. PMID:20938052

Thorsen, Kasper; Mansilla, Francisco; Schepeler, Troels; Øster, Bodil; Rasmussen, Mads H; Dyrskjøt, Lars; Karni, Rotem; Akerman, Martin; Krainer, Adrian R; Laurberg, Søren; Andersen, Claus L; Ørntoft, Torben F

2010-10-11

97

Position-dependent FUS-RNA interactions regulate alternative splicing events and transcriptions  

PubMed Central

FUS is an RNA-binding protein that regulates transcription, alternative splicing, and mRNA transport. Aberrations of FUS are causally associated with familial and sporadic ALS/FTLD. We analyzed FUS-mediated transcriptions and alternative splicing events in mouse primary cortical neurons using exon arrays. We also characterized FUS-binding RNA sites in the mouse cerebrum with HITS-CLIP. We found that FUS-binding sites tend to form stable secondary structures. Analysis of position-dependence of FUS-binding sites disclosed scattered binding of FUS to and around the alternatively spliced exons including those associated with neurodegeneration such as Mapt, Camk2a, and Fmr1. We also found that FUS is often bound to the antisense RNA strand at the promoter regions. Global analysis of these FUS-tags and the expression profiles disclosed that binding of FUS to the promoter antisense strand downregulates transcriptions of the coding strand. Our analysis revealed that FUS regulates alternative splicing events and transcriptions in a position-dependent manner.

Ishigaki, Shinsuke; Masuda, Akio; Fujioka, Yusuke; Iguchi, Yohei; Katsuno, Masahisa; Shibata, Akihide; Urano, Fumihiko; Sobue, Gen; Ohno, Kinji

2012-01-01

98

The evolutionary landscape of alternative splicing in vertebrate species.  

PubMed

How species with similar repertoires of protein-coding genes differ so markedly at the phenotypic level is poorly understood. By comparing organ transcriptomes from vertebrate species spanning ~350 million years of evolution, we observed significant differences in alternative splicing complexity between vertebrate lineages, with the highest complexity in primates. Within 6 million years, the splicing profiles of physiologically equivalent organs diverged such that they are more strongly related to the identity of a species than they are to organ type. Most vertebrate species-specific splicing patterns are cis-directed. However, a subset of pronounced splicing changes are predicted to remodel protein interactions involving trans-acting regulators. These events likely further contributed to the diversification of splicing and other transcriptomic changes that underlie phenotypic differences among vertebrate species. PMID:23258890

Barbosa-Morais, Nuno L; Irimia, Manuel; Pan, Qun; Xiong, Hui Y; Gueroussov, Serge; Lee, Leo J; Slobodeniuc, Valentina; Kutter, Claudia; Watt, Stephen; Colak, Recep; Kim, TaeHyung; Misquitta-Ali, Christine M; Wilson, Michael D; Kim, Philip M; Odom, Duncan T; Frey, Brendan J; Blencowe, Benjamin J

2012-12-21

99

Functional Manipulations of Acetylcholinesterase Splice Variants Highlight Alternative Splicing Contributions to Murine Neocortical Development  

Microsoft Academic Search

Proliferation and differentiation of mammalian central nervous system progenitor cells involve concertedly controlled transcrip- tional and alternative splicing modulations. Searching for the developmental implications of this programming, we manipulated specific acetylcholinesterase (AChE) splice variants in the embry- onic mouse brain. In wild type mice, 'synaptic' AChE-S appeared in migrating neurons, whereas the C-terminus cleaved off the stress-induced AChE-R variant associated

Amir Dori; Jonathan Cohen; William F. Silverman

2005-01-01

100

A structured RNA in hepatitis B virus post-transcriptional regulatory element represses alternative splicing in a sequence-independent and position-dependent manner.  

PubMed

Hepatitis B virus (HBV) transcripts are subjected to multiple splicing decisions, but the mechanism of splicing regulation remains poorly understood. In this study, we used a well-investigated alternative splicing reporter to dissect splicing regulatory elements residing in the post-transcriptional regulatory element (PRE) of HBV. A strong intronic splicing silencer (ISS) with a minimal functional element of 105 nucleotides (referred to as PRE-ISS) was identified and, interestingly, both the sense and antisense strands of the element were found to strongly suppress alternative splicing in multiple human cell lines. PRE-ISS folds into a double-hairpin structure, in which substitution mutations disrupting the double-hairpin structure abolish the splicing silencer activity. Although it harbors two previously identified binding sites for polypyrimidine tract binding protein, PRE-ISS represses splicing independent of this protein. The silencing function of PRE-ISS exhibited a strong position dependence, decreasing with the distance from affected splice sites. PRE-ISS does not belong to the intronic region of any HBV splicing variants identified thus far, preventing the testing of this intronic silencer function in the regulation of HBV splicing. These findings, together with the identification of multiple sense-antisense ISSs in the HBV genome, support the hypothesis that a sequence-independent and structure-dependent regulatory mechanism may have evolved to repress cryptic splice sites in HBV transcripts, thereby preventing their aberrant splicing during viral replication in the host. PMID:21371260

Huang, Chen; Xie, Mao-Hua; Liu, Wei; Yang, Bo; Yang, Fan; Huang, Jingang; Huang, Jie; Wu, Qijia; Fu, Xiang-Dong; Zhang, Yi

2011-04-01

101

TassDB2 - A comprehensive database of subtle alternative splicing events  

Microsoft Academic Search

BACKGROUND: Subtle alternative splicing events involving tandem splice sites separated by a short (2-12 nucleotides) distance are frequent and evolutionarily widespread in eukaryotes, and a major contributor to the complexity of transcriptomes and proteomes. However, these events have been either omitted altogether in databases on alternative splicing, or only the cases of experimentally confirmed alternative splicing have been reported. Thus,

Rileen Sinha; Thorsten Lenser; Niels Jahn; Ulrike Gausmann; Swetlana Friedel; Karol Szafranski; Klaus Huse; Philip Rosenstiel; Jochen Hampe; Stefan Schuster; Michael Hiller; Rolf Backofen; Matthias Platzer

2010-01-01

102

Splicing aberrations caused by constitutional RB1 gene mutations in retinoblastoma.  

PubMed

Analysis of RB1 mRNA from blood leukocytes of patients with retinoblastoma identified the effects of mutations involving consensus splice site, exonic substitution and whole-exon deletions identified in genomic DNA of these patients. In addition, this study identified mutations in cases in which no mutations were detectable in the genomic DNA. One proband had mutation at the canonical splice site at +5 position of IVS22, and analysis of the transcripts in this family revealed skipping of exon 22 in three members of this family. In one proband, a missense substitution of c.652T greater than G (g.56897T greater than G; Leu218Val) in exon 7 led to splicing aberrations involving deletions of exons 7 and 8, suggesting the formation of a cryptic splice site. In two probands with no detectable changes in the genomic DNA upon screening of RB1 exons and flanking intronic sequences, transcripts were found to have deletions of exon 6 in one, and exons 21 and 22 in another family. In two probands, RNA analysis confirmed genomic deletions involving one or more exons. This study reveals novel effects of RB1 mutations on splicing and suggests the utility of RNA analysis as an adjunct to mutational screening of genomic DNA in retinoblastoma. PMID:21654082

Parsam, Vidya Latha; Ali, Mohammed Javed; Honavar, Santosh G; Vemuganti, Geeta K; Kannabiran, Chitra

2011-06-01

103

The Caenorhabditis elegans Gene mfap-1 Encodes a Nuclear Protein That Affects Alternative Splicing  

Microsoft Academic Search

RNA splicing is a major regulatory mechanism for controlling eukaryotic gene expression. By generating various splice isoforms from a single pre–mRNA, alternative splicing plays a key role in promoting the evolving complexity of metazoans. Numerous splicing factors have been identified. However, the in vivo functions of many splicing factors remain to be understood. In vivo studies are essential for understanding

Long Ma; Xiaoyang Gao; Jintao Luo; Liange Huang; Yanling Teng; H. Robert Horvitz

2012-01-01

104

Estimation of alternative splicing variability in human populations  

PubMed Central

DNA arrays have been widely used to perform transcriptome-wide analysis of gene expression, and many methods have been developed to measure gene expression variability and to compare gene expression between conditions. Because RNA-seq is also becoming increasingly popular for transcriptome characterization, the possibility exists for further quantification of individual alternative transcript isoforms, and therefore for estimating the relative ratios of alternative splice forms within a given gene. Changes in splicing ratios, even without changes in overall gene expression, may have important phenotypic effects. Here we have developed statistical methodology to measure variability in splicing ratios within conditions, to compare it between conditions, and to identify genes with condition-specific splicing ratios. Furthermore, we have developed methodology to deconvolute the relative contribution of variability in gene expression versus variability in splicing ratios to the overall variability of transcript abundances. As a proof of concept, we have applied this methodology to estimates of transcript abundances obtained from RNA-seq experiments in lymphoblastoid cells from Caucasian and Yoruban individuals. We have found that protein-coding genes exhibit low splicing variability within populations, with many genes exhibiting constant ratios across individuals. When comparing these two populations, we have found that up to 10% of the studied protein-coding genes exhibit population-specific splicing ratios. We estimate that ?60% of the total variability observed in the abundance of transcript isoforms can be explained by variability in transcription. A large fraction of the remaining variability can likely result from variability in splicing. Finally, we also detected that variability in splicing is uncommon without variability in transcription.

Gonzalez-Porta, Mar; Calvo, Miquel; Sammeth, Michael; Guigo, Roderic

2012-01-01

105

Alternative splicing caused by lentiviral integration in the human genome.  

PubMed

Gene transfer vectors derived from murine oncoretroviruses or human lentiviruses are widely used in human gene therapy. Integration of these vectors in the human genome may, however, have genotoxic effects, caused by deregulation of gene expression at the transcriptional or posttranscriptional level. In particular, integration of lentiviral vectors within transcribed genes has a significant potential to affect their expression by interfering with splicing and polyadenylation of primary transcripts. Aberrant splicing is caused by the usage of both constitutive and cryptic splice sites located in the retroviral backbone as well as in the gene expression cassettes. We describe a set of simple methods that allow the identification of chimeric transcripts generated by the insertion of a lentiviral vector within genes and the evaluation of their relative abundance. Identification of the splice sites, either constitutive or cryptic, that are frequently used by the cell splicing machinery within a given vector provides a useful resource to attempt recoding of the vector with the objective of reducing its potential genotoxicity in a clinical context. PMID:22365773

Moiani, Arianna; Mavilio, Fulvio

2012-01-01

106

Alternative splicing produces structural and functional changes in CUGBP2  

PubMed Central

Background CELF/Bruno-like proteins play multiple roles, including the regulation of alternative splicing and translation. These RNA-binding proteins contain two RNA recognition motif (RRM) domains at the N-terminus and another RRM at the C-terminus. CUGBP2 is a member of this family of proteins that possesses several alternatively spliced exons. Results The present study investigated the expression of exon 14, which is an alternatively spliced exon and encodes the first half of the third RRM of CUGBP2. The ratio of exon 14 skipping product (R3?) to its inclusion was reduced in neuronal cells induced from P19 cells and in the brain. Although full length CUGBP2 and the CUGBP2 R3? isoforms showed a similar effect on the inclusion of the smooth muscle (SM) exon of the ACTN1 gene, these isoforms showed an opposite effect on the skipping of exon 11 in the insulin receptor gene. In addition, examination of structural changes in these isoforms by molecular dynamics simulation and NMR spectrometry suggested that the third RRM of R3? isoform was flexible and did not form an RRM structure. Conclusion Our results suggest that CUGBP2 regulates the splicing of ACTN1 and insulin receptor by different mechanisms. Alternative splicing of CUGBP2 exon 14 contributes to the regulation of the splicing of the insulin receptor. The present findings specifically show how alternative splicing events that result in three-dimensional structural changes in CUGBP2 can lead to changes in its biological activity.

2012-01-01

107

Sporadic ALS has compartment-specific aberrant exon splicing and altered cell-matrix adhesion biology  

PubMed Central

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive weakness from loss of motor neurons. The fundamental pathogenic mechanisms are unknown and recent evidence is implicating a significant role for abnormal exon splicing and RNA processing. Using new comprehensive genomic technologies, we studied exon splicing directly in 12 sporadic ALS and 10 control lumbar spinal cords acquired by a rapid autopsy system that processed nervous systems specifically for genomic studies. ALS patients had rostral onset and caudally advancing disease and abundant residual motor neurons in this region. We created two RNA pools, one from motor neurons collected by laser capture microdissection and one from the surrounding anterior horns. From each, we isolated RNA, amplified mRNA, profiled whole-genome exon splicing, and applied advanced bioinformatics. We employed rigorous quality control measures at all steps and validated findings by qPCR. In the motor neuron enriched mRNA pool, we found two distinct cohorts of mRNA signals, most of which were up-regulated: 148 differentially expressed genes (P ? 10?3) and 411 aberrantly spliced genes (P ? 10?5). The aberrantly spliced genes were highly enriched in cell adhesion (P ? 10?57), especially cell–matrix as opposed to cell–cell adhesion. Most of the enriching genes encode transmembrane or secreted as opposed to nuclear or cytoplasmic proteins. The differentially expressed genes were not biologically enriched. In the anterior horn enriched mRNA pool, we could not clearly identify mRNA signals or biological enrichment. These findings, perturbed and up-regulated cell–matrix adhesion, suggest possible mechanisms for the contiguously progressive nature of motor neuron degeneration. Data deposition: GeneChip raw data (CEL-files) have been deposited for public access in the Gene Expression Omnibus (GEO), www.ncbi.nlm.nih.gov/geo, accession number GSE18920.

Rabin, Stuart J.; Kim, Jae Mun 'Hugo'; Baughn, Michael; Libby, Ryan T.; Kim, Young Joo; Fan, Yuxin; Libby, Randell T.; La Spada, Albert; Stone, Brad; Ravits, John

2010-01-01

108

Iron availability modulates aberrant splicing of ferrochelatase through the iron- and 2-oxoglutarate dependent dioxygenase Jmjd6 and U2AF(65.).  

PubMed

Erythropoietic protoporphyria (EPP) results from partial deficiency of ferrochelatase (FECH). Genetically, EPP patients differ from asymptomatic mutation carriers at the unmutated FECH allele, the expression of which is modulated by single nucleotide polymorphism IVS3-48C/T. The IVS3-48C genotype, which is present among patients, leads to correct splicing of 60% of the pre-mRNA and to alternative splicing of 40%, the latter mRNA-product being destroyed by nonsense-mediated decay. An IVS3-48T genotype generates 80% correct and 20% aberrant products. Our study demonstrated that under iron deficient conditions, the aberrant splice product was increased to 56% and 50% of total FECH mRNA in erythroleukemic K562 and lymphoblastoid cell lines, respectively, both being homozygous for IVS3-48T. Concomitantly, FECH protein was decreased. Iron deficiency had less effect on the FECH splice ratio in an IVS3-48C/C lymphoblastoid cell line. Effects similar to iron deficiency were generated by siRNA knockdown of either splicing factor U2AF(65) or Fe(II)- and 2-oxoglutarate-dependent dioxygenase Jumonji domain-containing protein 6 (Jmjd6), which interacts with U2AF(65) by lysyl-hydroxylation. Based on these results, we propose that the availability of iron, a co-factor of Jmjd6, modulates U2AF(65)-lysyl-hydroxylation. This in turn, influences the relative amounts of correct and aberrant FECH mRNA splice products and thus, regulates the FECH enzyme activity. PMID:23787363

Barman-Aksözen, Jasmin; Béguin, Chantal; Dogar, Afzal M; Schneider-Yin, Xiaoye; Minder, Elisabeth I

2013-06-18

109

Cell-to-cell variability of alternative RNA splicing  

PubMed Central

Heterogeneity in the expression levels of mammalian genes is large even in clonal populations and has phenotypic consequences. Alternative splicing is a fundamental aspect of gene expression, yet its contribution to heterogeneity is unknown. Here, we use single-molecule imaging to characterize the cell-to-cell variability in mRNA isoform ratios for two endogenous genes, CAPRIN1 and MKNK2. We show that isoform variability in non-transformed, diploid cells is remarkably close to the minimum possible given the stochastic nature of individual splicing events, while variability in HeLa cells is considerably higher. Analysis of the potential sources of isoform ratio heterogeneity indicates that a difference in the control over splicing factor activity is one origin of this increase. Our imaging approach also visualizes non-alternatively spliced mRNA and active transcription sites, and yields spatial information regarding the relationship between splicing and transcription. Together, our work demonstrates that mammalian cells minimize fluctuations in mRNA isoform ratios by tightly regulating the splicing machinery.

Waks, Zeev; Klein, Allon M; Silver, Pamela A

2011-01-01

110

Identification of three mouse ?-opioid receptor (MOR) gene ( Oprm1) splice variants containing a newly identified alternatively spliced exon  

Microsoft Academic Search

The mouse ?-opioid receptor gene, Oprm1, is recognized currently to contain 17 alternatively spliced exons that generate 24 splice variants encoding at least 11 morphine-binding isoforms of the receptor. Here, we identify three new MOR splice variants that contain a previously undescribed exon, exon 18, and provide evidence that they are expressed in two mouse strains. The transcripts containing the

Glenn A. Doyle; X. Rebecca Sheng; Sharon S. J. Lin; Dorothy E. Grice; Russell J. Buono; Thomas N. Ferraro; Wade H. Berrettini

2007-01-01

111

Alternating phase-shifting mask design for low aberration sensitivity  

Microsoft Academic Search

Abstract. The aberration present in the lenses of exposure,systems,can cause,placement,errors to the images,produced,by alternating phase- shifting masks (PSMs). In reality, when the aberration signature varies from one lens to another, the magnitude of placement error also varies. It remains a question of how the alternating PSM should be designed, so that the image placement error, on average, can be minimized.

Giuseppe Y. Mak; Alfred K. Wong; Edmund Y. Lam

2005-01-01

112

Alternative Splicing Regulates Targeting of Malate Dehydrogenase in Yarrowia lipolytica  

PubMed Central

Alternative pre-mRNA splicing is a major mechanism contributing to the proteome complexity of most eukaryotes, especially mammals. In less complex organisms, such as yeasts, the numbers of genes that contain introns are low and cases of alternative splicing (AS) with functional implications are rare. We report the first case of AS with functional consequences in the yeast Yarrowia lipolytica. The splicing pattern was found to govern the cellular localization of malate dehydrogenase, an enzyme of the central carbon metabolism. This ubiquitous enzyme is involved in the tricarboxylic acid cycle in mitochondria and in the glyoxylate cycle, which takes place in peroxisomes and the cytosol. In Saccharomyces cerevisiae, three genes encode three compartment-specific enzymes. In contrast, only two genes exist in Y. lipolytica. One gene (YlMDH1, YALI0D16753g) encodes a predicted mitochondrial protein, whereas the second gene (YlMDH2, YALI0E14190g) generates the cytosolic and peroxisomal forms through the alternative use of two 3?-splice sites in the second intron. Both splicing variants were detected in cDNA libraries obtained from cells grown under different conditions. Mutants expressing the individual YlMdh2p isoforms tagged with fluorescent proteins confirmed that they localized to either the cytosolic or the peroxisomal compartment.

Kabran, Philomene; Rossignol, Tristan; Gaillardin, Claude; Nicaud, Jean-Marc; Neuveglise, Cecile

2012-01-01

113

Autistic-like phenotypes in Cadps2-knockout mice and aberrant CADPS2 splicing in autistic patients  

PubMed Central

Autism, characterized by profound impairment in social interactions and communicative skills, is the most common neurodevelopmental disorder, and its underlying molecular mechanisms remain unknown. Ca2+-dependent activator protein for secretion 2 (CADPS2; also known as CAPS2) mediates the exocytosis of dense-core vesicles, and the human CADPS2 is located within the autism susceptibility locus 1 on chromosome 7q. Here we show that Cadps2-knockout mice not only have impaired brain-derived neurotrophic factor release but also show autistic-like cellular and behavioral phenotypes. Moreover, we found an aberrant alternatively spliced CADPS2 mRNA that lacks exon 3 in some autistic patients. Exon 3 was shown to encode the dynactin 1–binding domain and affect axonal CADPS2 protein distribution. Our results suggest that a disturbance in CADPS2-mediated neurotrophin release contributes to autism susceptibility.

Sadakata, Tetsushi; Washida, Miwa; Iwayama, Yoshimi; Shoji, Satoshi; Sato, Yumi; Ohkura, Takeshi; Katoh-Semba, Ritsuko; Nakajima, Mizuho; Sekine, Yukiko; Tanaka, Mika; Nakamura, Kazuhiko; Iwata, Yasuhide; Tsuchiya, Kenji J.; Mori, Norio; Detera-Wadleigh, Sevilla D.; Ichikawa, Hironobu; Itohara, Shigeyoshi; Yoshikawa, Takeo; Furuichi, Teiichi

2007-01-01

114

Regulation of alternative splicing by the core spliceosomal machinery.  

PubMed

Alternative splicing (AS) plays a major role in the generation of proteomic diversity and in gene regulation. However, the role of the basal splicing machinery in regulating AS remains poorly understood. Here we show that the core snRNP (small nuclear ribonucleoprotein) protein SmB/B' self-regulates its expression by promoting the inclusion of a highly conserved alternative exon in its own pre-mRNA that targets the spliced transcript for nonsense-mediated mRNA decay (NMD). Depletion of SmB/B' in human cells results in reduced levels of snRNPs and a striking reduction in the inclusion levels of hundreds of additional alternative exons, with comparatively few effects on constitutive exon splicing levels. The affected alternative exons are enriched in genes encoding RNA processing and other RNA-binding factors, and a subset of these exons also regulate gene expression by activating NMD. Our results thus demonstrate a role for the core spliceosomal machinery in controlling an exon network that appears to modulate the levels of many RNA processing factors. PMID:21325135

Saltzman, Arneet L; Pan, Qun; Blencowe, Benjamin J

2011-02-15

115

Regulation of alternative splicing by the core spliceosomal machinery  

PubMed Central

Alternative splicing (AS) plays a major role in the generation of proteomic diversity and in gene regulation. However, the role of the basal splicing machinery in regulating AS remains poorly understood. Here we show that the core snRNP (small nuclear ribonucleoprotein) protein SmB/B? self-regulates its expression by promoting the inclusion of a highly conserved alternative exon in its own pre-mRNA that targets the spliced transcript for nonsense-mediated mRNA decay (NMD). Depletion of SmB/B? in human cells results in reduced levels of snRNPs and a striking reduction in the inclusion levels of hundreds of additional alternative exons, with comparatively few effects on constitutive exon splicing levels. The affected alternative exons are enriched in genes encoding RNA processing and other RNA-binding factors, and a subset of these exons also regulate gene expression by activating NMD. Our results thus demonstrate a role for the core spliceosomal machinery in controlling an exon network that appears to modulate the levels of many RNA processing factors.

Saltzman, Arneet L.; Pan, Qun; Blencowe, Benjamin J.

2011-01-01

116

A procedure for identifying homologous alternative splicing events  

Microsoft Academic Search

Background: The study of the functional role of alternative splice isoforms of a gene is a very active area of research in biology. The difficulty of the experimental approach (in particular, in its high-throughput version) leaves ample room for the development of bioinformatics tools that can provide a useful first picture of the problem. Among the possible approaches, one of

David Talavera; Modesto Orozco; Xavier De La Cruz

2007-01-01

117

AsMamDB: an alternative splice database of mammals  

PubMed Central

The objective of database AsMamDB is to facilitate the systematic study of alternatively spliced genes of mammals. Version 1.0 of AsMamDB contains 1563 alternatively spliced genes of human, mouse and rat, each associated with a cluster of nucleotide sequences. The main information provided by AsMamDB includes gene alternative splicing patterns, gene structures, locations in chromosomes, products of genes and tissues where they express. Alternative splicing patterns are represented by multiple alignments of various gene transcripts and by graphs of their topological structures. Gene structures are illustrated by exon, intron and various regulatory elements distributions. There are 4204 DNAs, 3977 mRNAs, 8989 CDSs and 126 931 ESTs in the current database. More than 130 000 GenBank entries are covered and 4443 MEDLINE records are linked. DNA, mRNA, exon, intron and relevant regulatory element sequences are provided in FASTA format. More information can be obtained by using the web-based multiple alignment tool Asalign and various category lists. AsMamDB can be accessed at http://166.111.30.6 5/ASMAM DB.html.

Ji, Hongkai; Zhou, Qing; Wen, Fang; Xia, Huiyu; Lu, Xin; Li, Yanda

2001-01-01

118

Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene  

SciTech Connect

In the protein 4.1R gene, alternative first exons splice differentially to alternative 3' splice sites far downstream in exon 2'/2 (E2'/2). We describe a novel intrasplicing mechanism by which exon 1A (E1A) splices exclusively to the distal E2'/2 acceptor via two nested splicing reactions regulated by novel properties of exon 1B (E1B). E1B behaves as an exon in the first step, using its consensus 5' donor to splice to the proximal E2'/2 acceptor. A long region of downstream intron is excised, juxtaposing E1B with E2'/2 to generate a new composite acceptor containing the E1B branchpoint/pyrimidine tract and E2 distal 3' AG-dinucleotide. Next, the upstream E1A splices over E1B to this distal acceptor, excising the remaining intron plus E1B and E2' to form mature E1A/E2 product. We mapped branch points for both intrasplicing reactions and demonstrated that mutation of the E1B 5' splice site or branchpoint abrogates intrasplicing. In the 4.1R gene, intrasplicing ultimately determines N-terminal protein structure and function. More generally, intrasplicing represents a new mechanism whereby alternative promoters can be coordinated with downstream alternative splicing.

Conboy, John G.; Parra, Marilyn K.; Tan, Jeff S.; Mohandas, Narla; Conboy, John G.

2008-11-07

119

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

Microsoft Academic Search

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

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

2007-01-01

120

Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing  

Microsoft Academic Search

Alternative splicing of pre-messenger RNAs diversifies gene products in eukaryotes and is guided by factors that enable spliceosomes to recognize particular splice sites. Here we report that alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA is promoted by the conserved ubiquitin-like protein Hub1. Structural and biochemical data show that Hub1 binds non-covalently to a conserved element termed HIND, which is present

Shravan Kumar Mishra; Tim Ammon; Grzegorz M. Popowicz; Marcin Krajewski; Roland J. Nagel; Manuel Ares; Tad A. Holak; Stefan Jentsch

2011-01-01

121

Alternating phase-shifting mask design for low aberration sensitivity  

NASA Astrophysics Data System (ADS)

Theories are developed to optimize the mask structure of alternating phase-shifting masks (PSMs) to minimize the average image placement error towards aberration under coherent imaging. The constraint of the optimization is a given mean value of RMS aberration, which corresponds to infinitely many sets of random Zernike coefficients. To begin the analysis, the image placement error is expressed as a function of the mask spectrum and the wave aberration. Monte Carlo analysis on the Zernike coefficients is then performed, which assures us that a global minimum of average image placement error is likely to occur at low phase widths. This result is confirmed by analytically considering the expected value of the square of the image placement error. By Golden Section Search, the optimal phase width is found to be 0.3707 (?/NA) at 0.07 ? RMS aberration. This result is applicable to the design of all alternating PSMs.

Mak, Giuseppe Y.; Wong, Alfred K.; Lam, Edmund Y.

2004-05-01

122

Therapeutic Potential of Splice-Switching Oligonucleotides  

PubMed Central

Alternative splicing enables a single pre-messenger RNA transcript to yield multiple protein isoforms, making it a major contributor to the diversity of the proteome. While this process is essential for normal development, aberrations in alternative splicing are the cause of a multitude of human diseases. Methods for manipulating alternative splicing would thus be of therapeutic value. Chemically modified antisense oligonucleotides that alter alternative splicing by directing splice site selection have been developed to achieve this end. These splice-switching oligonucleotides (SSOs) have been applied to correct aberrant splicing, induce expression of a therapeutic splice variant, or induce expression of a novel therapeutic splice variant in a number of disease-relevant genes. Recently, in vivo efficacy of SSOs has been reported using animal disease models, as well as in results from the first clinical trial.

Bauman, John; Jearawiriyapaisarn, Natee

2009-01-01

123

Splice-Junction Elements and Intronic Sequences Regulate Alternative Splicing of the Drosophila Myosin Heavy Chain Gene Transcript  

PubMed Central

The Drosophila muscle myosin heavy chain (Mhc) gene primary transcript contains five alternatively spliced exon groups (exons 3, 7, 9, 11 and 15), each of which contains two to five mutually exclusive members. Individual muscles typically select a specific alternative exon from each group for incorporation into the processed message. We report here on the cis-regulatory mechanisms that direct the processing of alternative exons in Mhc exon 11 in individual muscles using transgenic reporter constructs, RT-PCR and directed mutagenesis. The 6.0-kilobase exon 11 domain is sufficient to direct the correct processing of exon 11 alternatives, demonstrating that the alternative splicing cis-regulatory elements are local to Mhc exon 11. Mutational analysis of Mhc exon 11 reveals that the alternative exon nonconsensus 5'-splice donors are essential for alternative splicing regulation in general, but do not specify alternative exons for inclusion in individual muscles. Rather, we show, through exon substitutions and deletion analyses, that a 360-nucleotide intronic domain precisely directs the normal processing of one exon, Mhc exon 11e, in the indirect flight muscle. These and other data indicate that alternative exons are regulated in appropriate muscles through interactions between intronic alternative splice-specificity elements, nonconsensus exon 11 splice donors and, likely, novel exon-specific alternative splicing factors.

Standiford, D. M.; Davis, M. B.; Sun, W.; Emerson-Jr., C. P.

1997-01-01

124

ASPMF: A new approach for identifying alternative splicing isoforms using peptide mass fingerprinting  

Microsoft Academic Search

Alternative splicing is generally accepted as a mechanism that explains the discrepancy between the number of genes and proteins. We used peptide mass fingerprinting with a theoretical database and scoring method to discover and identify alternative splicing isoforms. Our theoretical database was built using published alternative splicing databases such as ECgene, H-DBAS, and TISA. According to our theoretical database of

Seung-Won Lee; Jae-Pil Choi; Hyun-Jin Kim; Ji-Man Hong; Cheol-Goo Hur

2008-01-01

125

Leukocyte adhesion deficiency. Aberrant splicing of a conserved integrin sequence causes a moderate deficiency phenotype.  

PubMed

Leukocyte adhesion deficiency (LAD) is a heritable deficiency of the LFA-1, Mac-1, p150,95 family of leukocyte alpha beta heterodimers (the leukocyte integrins). We have studied the defect in patients who synthesize an aberrantly small form of the beta subunit common to all three proteins. S1 nuclease protection showed the presence of a 90-nucleotide mismatch in RNA from patients and relatives, correlating with inheritance of the disease. Use of the Taq polymerase chain reaction to amplify this region of RNA after first strand cDNA synthesis and sequencing showed an in-frame deletion of 90 nucleotides in the extracellular domain. Thus, this highly conserved region, 63% and 53% identical in amino acid sequence to two other beta subunits of the integrin family, is required for association of the beta subunit with alpha subunits. The 90-nucleotide region corresponds to a single exon present in both the normal and patient genome. The patient DNA has a single G to C substitution in the 5' splice site. This results in the direct joining of nonconsecutive exons in an unusual type of abnormal RNA splicing. A small amount of normally spliced message, detected by S1 nuclease protection and Taq polymerase chain reaction, encodes a normal sized beta subunit which is surface-expressed and accounts for the low levels of leukocyte integrin expression observed in these patients, and hence the moderate phenotype. PMID:2464599

Kishimoto, T K; O'Conner, K; Springer, T A

1989-02-25

126

Technologies for the global discovery and analysis of alternative splicing.  

PubMed

During the past approximately 20 years, studies on alternative splicing (AS) have largely been directed at the identification and characterization of factors and mecha nisms responsible for the control of splice site selection, using model substrates and on a case by case basis. These studies have provided a wealth of information on the factors and interactions that control formation of the spliceosome. However, relatively little is known about the global regulatory properties of AS. Important questions that need to be addressed are: which exons are alternatively spliced and under which cellular contexts, what are the functional roles of AS events in different cellular contexts, and how are AS events controlled and coordinated with each other and with other levels of gene regulation to achieve cell- and development-specific functions. During the past several years, new technologies and experimental strategies have provided insight into these questions. For example, custom microarrays and data analysis tools are playing a prominent role in the discovery and analysis of splicing regulation. Moreover, several non-microarray-based technologies are emerging that will likely further fuel progress in this area. This review focuses on recent advances made in the development and application of high-throughput methods to study AS. PMID:18380341

Calarco, John A; Saltzman, Arneet L; Ip, Joanna Y; Blencowe, Benjamin J

2007-01-01

127

Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing  

PubMed Central

Alternative splicing of pre-messenger RNAs diversifies gene products in eukaryotes and is guided by factors that enable spliceosomes to recognize particular splice sites. Here we report that alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA is promoted by the conserved ubiquitin-like protein Hub1. Structural and biochemical data show that Hub1 binds non-covalently to a conserved element termed HIND, which is present in the spliceosomal protein Snu66 in yeast and mammals, and Prp38 in plants. Hub1 binding mildly alters spliceosomal protein interactions and barely affects general splicing in S. cerevisiae. However, spliceosomes that lack Hub1, or are defective in Hub1–HIND interaction, cannot use certain non-canonical 5? splice sites and are defective in alternative SRC1 splicing. Hub1 confers alternative splicing not only when bound to HIND, but also when experimentally fused to Snu66, Prp38, or even the core splicing factor Prp8. Our study indicates a novel mechanism for splice site utilization that is guided by non-covalent modification of the spliceosome by an unconventional ubiquitin-like modifier.

Mishra, Shravan Kumar; Ammon, Tim; Popowicz, Grzegorz M.; Krajewski, Marcin; Nagel, Roland J.; Ares, Manuel; Holak, Tad A.; Jentsch, Stefan

2013-01-01

128

Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing.  

PubMed

Alternative splicing of pre-messenger RNAs diversifies gene products in eukaryotes and is guided by factors that enable spliceosomes to recognize particular splice sites. Here we report that alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA is promoted by the conserved ubiquitin-like protein Hub1. Structural and biochemical data show that Hub1 binds non-covalently to a conserved element termed HIND, which is present in the spliceosomal protein Snu66 in yeast and mammals, and Prp38 in plants. Hub1 binding mildly alters spliceosomal protein interactions and barely affects general splicing in S. cerevisiae. However, spliceosomes that lack Hub1, or are defective in Hub1-HIND interaction, cannot use certain non-canonical 5' splice sites and are defective in alternative SRC1 splicing. Hub1 confers alternative splicing not only when bound to HIND, but also when experimentally fused to Snu66, Prp38, or even the core splicing factor Prp8. Our study indicates a novel mechanism for splice site utilization that is guided by non-covalent modification of the spliceosome by an unconventional ubiquitin-like modifier. PMID:21614000

Mishra, Shravan Kumar; Ammon, Tim; Popowicz, Grzegorz M; Krajewski, Marcin; Nagel, Roland J; Ares, Manuel; Holak, Tad A; Jentsch, Stefan

2011-05-25

129

A splicing silencer that regulates smooth muscle specific alternative splicing is active in multiple cell types  

Microsoft Academic Search

Alternative splicing of a-tropomyosin (a-TM) involves mutually exclusive selection of exons 2 and 3. Selection of exon 2 in smooth muscle (SM) cells is due to inhibition of exon 3, which requires both binding sites for polypyrimidine tract-binding protein as well as UGC (or CUG) repeat elements on both sides of exon 3. Point mutations or substitu- tions of the

Natalia Gromak; Christopher W. J. Smith

2002-01-01

130

Alternative splicing of BRAF transcripts and characterization of C-terminally truncated B-Raf isoforms in colorectal cancer.  

PubMed

The BRAF proto-oncogene is mutated in a subset of human tumors, including colorectal cancer. A splicing variant lacking exons 14 and 15 (BRAF del E14/15) has been described recently. However, the frequency of the variant, the kinase activity of the protein isoform, its biological function, and which allele it is derived from remains unknown. BRAF mRNA from colorectal cancer cell lines and colonic epithelium was reversely transcribed, subcloned, and screened for alternative splicing. New transcript variants and allelic origin of alternatively spliced transcripts were analyzed by DNA sequencing. Kinase activity of the B-Raf isoforms was determined by Western blotting after transfections with expression constructs of the different BRAF variants. Four additional BRAF transcript variants resulting in C-terminal truncation of the gene product were found. Alternative splicing was found at frequencies from 4.7 to 16.7% in normal and neoplastic colorectal cells. Alternative transcripts were shown to be derived from both wild-type and V600E alleles. All nonconsensus B-Raf protein variants were found to be kinase-dead and failed to coactivate full-length B-Raf. In conclusion, we present a highly sensitive method for the detection of aberrantly spliced transcripts. Alternative splicing of exons 14, 15, 15b, 16b and 16c occurs in a considerable fraction of BRAF mRNA in normal colon and colorectal cancer cells and is independent of the V600E mutational status of the parental allele. Splicing of nonfunctional transcripts affects overall cellular B-Raf activity and might represent a mechanism to decrease sensitivity to growth signals. PMID:23354951

Hirschi, Benjamin; Kolligs, Frank T

2013-02-27

131

Deciphering the Plant Splicing Code: Experimental and Computational Approaches for Predicting Alternative Splicing and Splicing Regulatory Elements  

PubMed Central

Extensive alternative splicing (AS) of precursor mRNAs (pre-mRNAs) in multicellular eukaryotes increases the protein-coding capacity of a genome and allows novel ways to regulate gene expression. In flowering plants, up to 48% of intron-containing genes exhibit AS. However, the full extent of AS in plants is not yet known, as only a few high-throughput RNA-Seq studies have been performed. As the cost of obtaining RNA-Seq reads continues to fall, it is anticipated that huge amounts of plant sequence data will accumulate and help in obtaining a more complete picture of AS in plants. Although it is not an onerous task to obtain hundreds of millions of reads using high-throughput sequencing technologies, computational tools to accurately predict and visualize AS are still being developed and refined. This review will discuss the tools to predict and visualize transcriptome-wide AS in plants using short-reads and highlight their limitations. Comparative studies of AS events between plants and animals have revealed that there are major differences in the most prevalent types of AS events, suggesting that plants and animals differ in the way they recognize exons and introns. Extensive studies have been performed in animals to identify cis-elements involved in regulating AS, especially in exon skipping. However, few such studies have been carried out in plants. Here, we review the current state of research on splicing regulatory elements (SREs) and briefly discuss emerging experimental and computational tools to identify cis-elements involved in regulation of AS in plants. The availability of curated alternative splice forms in plants makes it possible to use computational tools to predict SREs involved in AS regulation, which can then be verified experimentally. Such studies will permit identification of plant-specific features involved in AS regulation and contribute to deciphering the splicing code in plants.

Reddy, Anireddy S. N.; Rogers, Mark F.; Richardson, Dale N.; Hamilton, Michael; Ben-Hur, Asa

2012-01-01

132

The dystrophin gene is alternatively spliced throughout its coding sequence.  

PubMed

We have analysed splicing patterns in the human dystrophin gene region encoding the rod and cysteine-rich domains in normal skeletal muscle, brain and heart tissues. Sixteen novel alternative transcripts were identified, the majority of them being present in all three tissues. Tissue-specific variants were also identified, suggesting a functional role of transcriptional diversity. Transcript analysis in dystrophinopathic autoptic and bioptic specimens revealed that pre-mRNAs secondary structure formation and relative strength of exon/exon association play little or no role in directing alternative splicing events. This analysis also showed that independent deletion events leading to the loss of the same exons may be associated with transcriptional variability. PMID:12062429

Sironi, M; Cagliani, R; Pozzoli, U; Bardoni, A; Comi, G P; Giorda, R; Bresolin, N

2002-04-24

133

Intracellular calcium activates TRPM2 and its alternative spliced isoforms  

PubMed Central

Melastatin-related transient receptor potential channel 2 (TRPM2) is a Ca2+-permeable, nonselective cation channel that is involved in oxidative stress-induced cell death and inflammation processes. Although TRPM2 can be activated by ADP-ribose (ADPR) in vitro, it was unknown how TRPM2 is gated in vivo. Moreover, several alternative spliced isoforms of TRPM2 identified recently are insensitive to ADPR, and their gating mechanisms remain unclear. Here, we report that intracellular Ca2+ ([Ca2+]i) can activate TRPM2 as well as its spliced isoforms. We demonstrate that TRPM2 mutants with disrupted ADPR-binding sites can be activated readily by [Ca2+]i, indicating that [Ca2+]i gating of TRPM2 is independent of ADPR. The mechanism by which [Ca2+]i activates TRPM2 is via a calmodulin (CaM)-binding domain in the N terminus of TRPM2. Whereas Ca2+-mediated TRPM2 activation is independent of ADPR and ADPR-binding sites, both [Ca2+]i and the CaM-binding motif are required for ADPR-mediated TRPM2 gating. Importantly, we demonstrate that intracellular Ca2+ release activates both recombinant and endogenous TRPM2 in intact cells. Moreover, receptor activation-induced Ca2+ release is capable of activating TRPM2. These results indicate that [Ca2+]i is a key activator of TRPM2 and the only known activator of the spliced isoforms of TRPM2. Our findings suggest that [Ca2+]i-mediated activation of TRPM2 and its alternative spliced isoforms may represent a major gating mechanism in vivo, therefore conferring important physiological and pathological functions of TRPM2 and its spliced isoforms in response to elevation of [Ca2+]i.

Du, Jianyang; Xie, Jia; Yue, Lixia

2009-01-01

134

Microenvironment Changes (in pH) Affect VEGF Alternative Splicing  

Microsoft Academic Search

Vascular endothelial growth factor-A (VEGF-A) has several isoforms, which differ in their capacity to bind extracellular matrix\\u000a proteins and also in their affinity for VEGF receptors. Although the relative contribution of the VEGF isoforms has been studied\\u000a in tumor angiogenesis, little is known about the mechanisms that regulate the alternative splicing process. Here, we tested\\u000a microenvironment cues that might regulate

Ana Paula Elias; Sergio Dias

2008-01-01

135

Ca 2+ Signaling, Alternative Splicing and Endoplasmic Reticulum Stress Responses  

Microsoft Academic Search

Ca2+-signaling, alternative splicing, and stress responses by the endoplasmic reticulum are three important cellular activities\\u000a which can be strongly interconnected to alter the expression of protein isoforms in a tissue dependent manner or during development\\u000a depending on the environmental conditions. This integrated network of signaling pathways permits a high degree of versatility\\u000a and adaptation to metabolic, developmental and stress processes.

Joachim Krebs; Jody Groenendyk; Marek Michalak

2011-01-01

136

A sensitive procedure to detect alternatively spliced mRNA in pooled-tissue samples  

Microsoft Academic Search

One important goal of genomics is to explore the extent of alternative splicing in the transcriptome and generate a comprehensive catalog of splice forms. New computational and experimental approaches have led to an increase in the number of predicted alternatively spliced tran- scripts; however, validation of these predictions has not kept pace. In this work, we systematically explore different methods

German Gaston Leparc; Robi David Mitra

2007-01-01

137

The Alternative Splicing Gallery (ASG): bridging the gap between genome and transcriptome  

Microsoft Academic Search

Alternative splicing essentially increases the divers- ity of the transcriptome and has important implica- tions for physiology, development and the genesis of diseases. Conventionally, alternative splicing is investigated in a case-by-case fashion, but this becomes cumbersome and error prone if genes show a huge abundance of different splice variants. We use a different approach and integrate all tran- scripts derived

Jeremy Leipzig; Pavel Pevzner; Steffen Heber

2004-01-01

138

The PTB interacting protein raver1 regulates ?-tropomyosin alternative splicing  

PubMed Central

Regulated switching of the mutually exclusive exons 2 and 3 of ?-tropomyosin (TM) involves repression of exon 3 in smooth muscle cells. Polypyrimidine tract-binding protein (PTB) is necessary but not sufficient for regulation of TM splicing. Raver1 was identified in two-hybrid screens by its interactions with the cytoskeletal proteins actinin and vinculin, and was also found to interact with PTB. Consistent with these interactions raver1 can be localized in either the nucleus or cytoplasm. Here we show that raver1 is able to promote the smooth muscle-specific alternative splicing of TM by enhancing PTB-mediated repression of exon 3. This activity of raver1 is dependent upon characterized PTB-binding regulatory elements and upon a region of raver1 necessary for interaction with PTB. Heterologous recruitment of raver1, or just its C-terminus, induced very high levels of exon 3 skipping, bypassing the usual need for PTB binding sites downstream of exon 3. This suggests a novel mechanism for PTB-mediated splicing repression involving recruitment of raver1 as a potent splicing co-repressor.

Gromak, Natalia; Rideau, Alexis; Southby, Justine; Scadden, A.D.J.; Gooding, Clare; Huttelmaier, Stefan; Singer, Robert H.; Smith, Christopher W.J.

2003-01-01

139

Alternative splice acceptor utilization during human immunodeficiency virus type 1 infection of cultured cells.  

PubMed Central

The utilization of alternative splice acceptors for excision of the 5' major intron of human immunodeficiency virus type 1 RNA was observed after infection in vitro. Specific splice events were monitored by a cDNA-polymerase chain reaction. These splice events shared a common splice donor but utilized several alternative splice acceptors. In addition to identifying the previously documented splice acceptors for tat and nef (S. K. Arya, C. Guo, S. F. Josephs, and F. Wong-Staal, Science 229:69-73, 1985), nucleotide sequence analysis of cDNA-polymerase chain reaction fragments also revealed the following: (i) two splice acceptors 15 and 9 nucleotides upstream from the rev start codon, which are utilized to create transcripts suitable for specific rev expression; and (ii) use of the splice acceptor previously attributed to nef to generate a singly spliced, env-encoding transcript. Hybridization signals representing the nef/env, tat, and rev splice events increased in intensity between 6 and 12 h after infection of CEM cells with the LAV-1BRU strain of human immunodeficiency virus type 1. In contrast, the signal for utilization of the nef/env splice acceptor for the singly spliced env transcript appeared first at 12 h and increased to maximum intensity by 24 h. The nef/env splice acceptor was dominant at all time points examined. We propose that this dominance ensures efficient downstream splicing proximal to the env initiation codon in singly spliced transcripts. However, early after infection, the dominance of the nef/env splice acceptor appears to divert primary transcripts away from tat- and rev-specific processing paths. The relative proportions of hybridization signals representing these alternative splice events remained constant throughout the viral replicative cycle. This result suggests that trans-acting factors that might influence splice choices are not induced during infection, but rather that cis-acting, sequence-specific splice preferences determine the relative efficiency of alternative acceptor utilization. Images

Guatelli, J C; Gingeras, T R; Richman, D D

1990-01-01

140

The cardiotonic steroid digitoxin regulates alternative splicing through depletion of the splicing factors SRSF3 and TRA2B  

PubMed Central

Modulation of alternative pre-mRNA splicing is a potential approach to therapeutic targeting for a variety of human diseases. We investigated the mechanism by which digitoxin, a member of the cardiotonic steroid class of drugs, regulates alternative splicing. Transcriptome-wide analysis identified a large set of alternative splicing events that change after digitoxin treatment. Within and adjacent to these regulated exons, we identified enrichment of potential binding sites for the splicing factors SRp20 (SRSF3/SFRS3) and Tra2-? (SFRS10/TRA2B). We further find that both of these proteins are depleted from cells by digitoxin treatment. Characterization of SRp20 and Tra2-? splicing targets revealed that many, but not all, digitoxin-induced splicing changes can be attributed to the depletion of one or both of these factors. Re-expression of SRp20 or Tra2-? after digitoxin treatment restores normal splicing of their targets, indicating that the digitoxin effect is directly due to these factors. These results demonstrate that cardiotonic steroids, long prescribed in the clinical treatment of heart failure, have broad effects on the cellular transcriptome through these and likely other RNA binding proteins. The approach described here can be used to identify targets of other potential therapeutics that act as alternative splicing modulators.

Anderson, Erik S.; Lin, Chia-Ho; Xiao, Xinshu; Stoilov, Peter; Burge, Christopher B.; Black, Douglas L.

2012-01-01

141

Gene Array Analyzer: alternative usage of gene arrays to study alternative splicing events  

PubMed Central

Exon arrays are regularly used to analyze differential splicing events. GeneChip Gene 1.0 ST Arrays (gene arrays) manufactured by Affymetrix, Inc. are primarily used to determine expression levels of transcripts, although their basic design is rather similar to GeneChip Exon 1.0 ST Arrays (exon arrays). Here, we show that the newly developed Gene Array Analyzer (GAA), which evolved from our previously published Exon Array Analyzer (EAA), enables economic and user-friendly analysis of alternative splicing events using gene arrays. To demonstrate the applicability of GAA, we profiled alternative splicing events during embryonic heart development. In addition, we found that numerous developmental splicing events are also activated under pathological conditions. We reason that the usage of GAA considerably expands the analysis of gene expression based on gene arrays and supplies an additional level of information without further costs and with only little effort.

Jenniches, Katharina; De Gaspari, Piera; John, David; grosse Kreymborg, Karsten; Braun, Thomas; Uchida, Shizuka

2012-01-01

142

Aberrant splicing of proteolipid protein mRNA in the dysmyelinating jimpy mutant mouse.  

PubMed Central

cDNA clones encoding proteolipid protein (PLP) were isolated from a mouse brain library and sequenced. We describe two transcripts arising from the PLP locus by alternative splicing: the major one encodes the 277-amino acid PLP protein and the minor one corresponds to the DM-20 protein, a PLP-like protein of 20,000 Mr that shares both amino and carboxyl regions with PLP. These two transcripts lack approximately 70 bases in PLP mRNA from the dysmyelinating jimpy mutant. The deletion spans amino acids 208-232; however, this region is present in the jimpy PLP-encoding gene. We propose that the jimpy mutant suffers a point mutation or the deletion of a few bases in the PLP gene that alters the normal splicing pattern and generates partially deleted PLP transcripts. Images

Hudson, L D; Berndt, J A; Puckett, C; Kozak, C A; Lazzarini, R A

1987-01-01

143

A General Definition and Nomenclature for Alternative Splicing Events  

PubMed Central

Understanding the molecular mechanisms responsible for the regulation of the transcriptome present in eukaryotic cells is one of the most challenging tasks in the postgenomic era. In this regard, alternative splicing (AS) is a key phenomenon contributing to the production of different mature transcripts from the same primary RNA sequence. As a plethora of different transcript forms is available in databases, a first step to uncover the biology that drives AS is to identify the different types of reflected splicing variation. In this work, we present a general definition of the AS event along with a notation system that involves the relative positions of the splice sites. This nomenclature univocally and dynamically assigns a specific “AS code” to every possible pattern of splicing variation. On the basis of this definition and the corresponding codes, we have developed a computational tool (AStalavista) that automatically characterizes the complete landscape of AS events in a given transcript annotation of a genome, thus providing a platform to investigate the transcriptome diversity across genes, chromosomes, and species. Our analysis reveals that a substantial part—in human more than a quarter—of the observed splicing variations are ignored in common classification pipelines. We have used AStalavista to investigate and to compare the AS landscape of different reference annotation sets in human and in other metazoan species and found that proportions of AS events change substantially depending on the annotation protocol, species-specific attributes, and coding constraints acting on the transcripts. The AStalavista system therefore provides a general framework to conduct specific studies investigating the occurrence, impact, and regulation of AS.

Guigo, Roderic

2008-01-01

144

A novel protein factor is required for use of distal alternative 5' splice sites in vitro.  

PubMed Central

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites. Images

Harper, J E; Manley, J L

1991-01-01

145

Co-transcriptional regulation of alternative pre-mRNA splicing  

PubMed Central

While studies of alternative pre-mRNA splicing regulation have typically focused on RNA-binding proteins and their target sequences within nascent message, it is becoming increasingly evident that mRNA splicing, RNA polymerase II (pol II) elongation and chromatin structure are intricately intertwined. The majority of introns in higher eukaryotes are excised prior to transcript release in a manner that is dependent on transcription through pol II. As a result of co-transcriptional splicing, variations in pol II elongation influence alternative splicing patterns, wherein a slower elongation rate is associated with increased inclusion of alternative exons within mature mRNA. Physiological barriers to pol II elongation, such as repressive chromatin structure, can thereby similarly impact splicing decisions. Surprisingly, pre-mRNA splicing can reciprocally influence pol II elongation and chromatin structure. Here, we highlight recent advances in co-transcriptional splicing that reveal an extensive network of coupling between splicing, transcription and chromatin remodeling complexes.

Shukla, Sanjeev; Oberdoerffer, Shalini

2012-01-01

146

Functional premature polyadenylation signals and aberrant splicing within a recombinant protein coding sequence limit expression.  

PubMed

Recombinant glycoproteins can be produced at high levels in permanently transfected mammalian cells using expression vectors with strong viral promoters. CHO-K1 cell lines developed to produce the recombinant complement activator blocking protein, CAB-2 (a fusion of membrane co-factor protein, MCP, and decay accelerating factor, DAF), showed unexpectedly low expression. Northern blot analysis revealed that in addition to the expected 2300 base CAB-2 mRNA species, these cell lines expressed 790 and 1500 base mRNA species accounting for ?50% and ?10% of the total CAB-2 mRNA, respectively. RT-PCR studies established that the 1500 base species resulted from aberrant splicing from within the DAF region of the CAB-2 coding sequence to a site within the 3' untranslated region. 3' RACE analysis confirmed that the 790 base species resulted from premature polyadenylation at an AATAAA site within the MCP coding region of CAB-2. Another prematurely polyadenylated species, not observed on Northern blots, was observed in the DAF region by 3' RACE. Analysis of human tissues and cell lines revealed that these internal polyadenylation signals in native MCP and DAF coding regions also generated prematurely polyadenylated mRNAs. Genetic modification of these functional RNA processing elements within the CAB-2 gene eliminated the aberrant mRNA species and significantly increased recombinant CAB-2 expression. These results illustrate that protein expression can be limited by aberrant mRNA processing and demonstrate the importance of identifying and eliminating these mRNA processing signals from within coding DNA to maximize recombinant protein expression. PMID:23994311

Wijesuriya, Sujeewa D; Cotter, Robyn L; Horwitz, Arnold H

2013-08-28

147

Identification of five mouse ?-opioid receptor (MOR) gene ( Oprm1) splice variants containing a newly identified alternatively spliced exon  

Microsoft Academic Search

The mouse ?-opioid receptor gene, Oprm1, currently contains 18 recognized alternatively spliced exons [Doyle, G.A., Sheng, X.R., Lin, S.S.J., Press, D.M., Grice, D.E., Buono, R.J., Ferraro, T.N., Berrettini, W.H., 2007. Identification of three mouse ?-opioid receptor (MOR) gene (Oprm1) splice variants containing a newly identified alternatively spliced exon. Gene 388 (1–2) 135–147, in press (doi:10.1016\\/j.gene.2006.10.017). Electronic publication 2006 November 1

Glenn A. Doyle; X. Rebecca Sheng; Sharon S. J. Lin; Dorothy E. Grice; Russell J. Buono; Thomas N. Ferraro; Wade H. Berrettini

2007-01-01

148

Distinctive Features of Drosophila Alternative Splicing Factor RS Domain: Implication for Specific Phosphorylation, Shuttling, and Splicing Activation  

PubMed Central

The human splicing factor 2, also called human alternative splicing factor (hASF), is the prototype of the highly conserved SR protein family involved in constitutive and regulated splicing of metazoan mRNA precursors. Here we report that the Drosophila homologue of hASF (dASF) lacks eight repeating arginine-serine dipeptides at its carboxyl-terminal region (RS domain), previously shown to be important for both localization and splicing activity of hASF. While this difference has no effect on dASF localization, it impedes its capacity to shuttle between the nucleus and cytoplasm and abolishes its phosphorylation by SR protein kinase 1 (SRPK1). dASF also has an altered splicing activity. While being competent for the regulation of 5? alternative splice site choice and activation of specific splicing enhancers, dASF fails to complement S100-cytoplasmic splicing-deficient extracts. Moreover, targeted overexpression of dASF in transgenic flies leads to higher deleterious developmental defects than hASF overexpression, supporting the notion that the distinctive structural features at the RS domain between the two proteins are likely to be functionally relevant in vivo.

Allemand, Eric; Gattoni, Renata; Bourbon, Henri-Marc; Stevenin, James; Caceres, Javier F.; Soret, Johann; Tazi, Jamal

2001-01-01

149

Alternative splicing and the progesterone receptor in breast cancer  

PubMed Central

Progesterone receptor status is a marker for hormone responsiveness and disease prognosis in breast cancer. Progesterone receptor negative tumours have generally been shown to have a poorer prognosis than progesterone receptor positive tumours. The observed loss of progesterone receptor could be through a range of mechanisms, including the generation of alternatively spliced progesterone receptor variants that are not detectable by current screening methods. Many progesterone receptor mRNA variants have been described with deletions of various whole, multiple or partial exons that encode differing protein functional domains. These variants may alter the progestin responsiveness of a tissue and contribute to the abnormal growth associated with breast cancer. Absence of specific functional domains from these spliced variants may also make them undetectable or indistinguishable from full length progesterone receptor by conventional antibodies. A comprehensive investigation into the expression profile and activity of progesterone receptor spliced variants in breast cancer is required to advance our understanding of tumour hormone receptor status. This, in turn, may aid the development of new biomarkers of disease prognosis and improve adjuvant treatment decisions.

Cork, David MW; Lennard, Thomas WJ; Tyson-Capper, Alison J

2008-01-01

150

APPRIS: annotation of principal and alternative splice isoforms  

PubMed Central

Here, we present APPRIS (http://appris.bioinfo.cnio.es), a database that houses annotations of human splice isoforms. APPRIS has been designed to provide value to manual annotations of the human genome by adding reliable protein structural and functional data and information from cross-species conservation. The visual representation of the annotations provided by APPRIS for each gene allows annotators and researchers alike to easily identify functional changes brought about by splicing events. In addition to collecting, integrating and analyzing reliable predictions of the effect of splicing events, APPRIS also selects a single reference sequence for each gene, here termed the principal isoform, based on the annotations of structure, function and conservation for each transcript. APPRIS identifies a principal isoform for 85% of the protein-coding genes in the GENCODE 7 release for ENSEMBL. Analysis of the APPRIS data shows that at least 70% of the alternative (non-principal) variants would lose important functional or structural information relative to the principal isoform.

Rodriguez, Jose Manuel; Maietta, Paolo; Ezkurdia, Iakes; Pietrelli, Alessandro; Wesselink, Jan-Jaap; Lopez, Gonzalo; Valencia, Alfonso; Tress, Michael L.

2013-01-01

151

Finding signals that regulate alternative splicing in the post-genomic era  

PubMed Central

Alternative splicing of pre-mRNAs is central to the generation of diversity from the relatively small number of genes in metazoan genomes. Auxiliary cis elements and trans-acting factors are required for the recognition of constitutive and alternatively spliced exons and their inclusion in pre-mRNA. Here, we discuss the regulatory elements that direct alternative splicing and how genome-wide analyses can aid in their identification.

Ladd, Andrea N; Cooper, Thomas A

2002-01-01

152

Identifying Alternative Hyper-Splicing Signatures in MG-Thymoma by Exon Arrays  

Microsoft Academic Search

BackgroundThe vast majority of human genes (>70%) are alternatively spliced. Although alternative pre-mRNA processing is modified in multiple tumors, alternative hyper-splicing signatures specific to particular tumor types are still lacking. Here, we report the use of Affymetrix Human Exon Arrays to spot hyper-splicing events characteristic of myasthenia gravis (MG)-thymoma, thymic tumors which develop in patients with MG and discriminate them

Lilach Soreq; Adi Gilboa-Geffen; Sonia Berrih-Aknin; Paul Lacoste; Ariel Darvasi; Eyal Soreq; Hagai Bergman; Hermona Soreq; Stefan Maas

2008-01-01

153

Identifying Alternative Hyper-Splicing Signatures in MG-Thymoma by Exon Arrays  

Microsoft Academic Search

Abstract Background:,The vast majority,of human,genes,(.70%) are alternatively spliced. Although,alternative pre-mRNA processing is modified in multiple tumors, alternative hyper-splicing signatures specific to particular tumor types are still lacking. Here, we report the use of Affymetrix Human Exon Arrays to spot hyper-splicing events characteristic of myasthenia gravis (MG)-thymoma, thymic tumors which develop in patients with MG and discriminate them from colon cancer

Lilach Soreq; Adi Gilboa-Geffen; Sonia Berrih-Aknin; Paul Lacoste; Ariel Darvasi; Eyal Soreq; Hagai Bergman; Hermona Soreq

2008-01-01

154

Real-time imaging of cotranscriptional splicing reveals a kinetic model that reduces noise: implications for alternative splicing regulation.  

PubMed

Splicing is a key process that expands the coding capacity of genomes. Its kinetics remain poorly characterized, and the distribution of splicing time caused by the stochasticity of single splicing events is expected to affect regulation efficiency. We conducted a small-scale survey on 40 introns in human cells and observed that most were spliced cotranscriptionally. Consequently, we constructed a reporter system that splices cotranscriptionally and can be monitored in live cells and in real time through the use of MS2-GFP. All small nuclear ribonucleoproteins (snRNPs) are loaded on nascent pre-mRNAs, and spliceostatin A inhibits splicing but not snRNP recruitment. Intron removal occurs in minutes and is best described by a model where several successive steps are rate limiting. Each pre-mRNA molecule is predicted to require a similar time to splice, reducing kinetic noise and improving the regulation of alternative splicing. This model is relevant to other kinetically controlled processes acting on few molecules. PMID:21624952

Schmidt, Ute; Basyuk, Eugenia; Robert, Marie-Cécile; Yoshida, Minoru; Villemin, Jean-Philippe; Auboeuf, Didier; Aitken, Stuart; Bertrand, Edouard

2011-05-30

155

An alternatively spliced form of Met receptor is tumorigenic.  

PubMed

The Met tyrosine kinase receptor is a widely expressed molecule, which mediates pleiotropic cellular responses following activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF). Previously, one of the authors identified an alternatively spliced form of Met (Met-SM) that lacked a single exon of a 47-amino-acid segment in the juxtamembrane domain. Here we report that Met-SM is a potent transforming gene in NIH3T3 mouse fibroblast cells. Met-SM-transfected NIH3T3 cells show stronger foci-forming activity than wild type- Met-transfected ones. In addition, Met-SM-transfected NIH3T3 cells form colonies in soft agar and are tumorigenic in athymic nu/nu mice. Furthermore, HGF/SF significantly increases the focus-forming activity of Met-SM comparing to wild type Met. The amount of protein and of tyrosine kinase activity of Met-SM accumulates to a high level following HGF/SF treatment. The accumulation of Met-SM correlated well with its delayed ubiquitination and increased stability. These results are consistent with the important role of the juxtamembrane domain in protein stability of Met receptor and suggest that the alternatively-spliced form may contribute to the development and progression of human cancer. PMID:17079873

Lee, Jae-Ho; Gao, Chong Feng; Lee, Chong Chou; Kim, Myung Deok; Vande Woude, George F

2006-10-31

156

Induced HMGA1a expression causes aberrant splicing of Presenilin-2 pre-mRNA in sporadic Alzheimer's disease  

Microsoft Academic Search

The aberrant splicing isoform (PS2V), generated by exon 5 skipping of the Presenilin-2 (PS2) gene transcript, is a diagnostic feature of sporadic Alzheimer's disease (AD). We found PS2V is hypoxia-inducible in human neuroblastoma SK-N-SH cells. We purified a responsible trans-acting factor based on its binding to an exon 5 fragment. The factor was identified as the high mobility group A1a

T Manabe; T Katayama; N Sato; F Gomi; J Hitomi; T Yanagita; T Kudo; A Honda; Y Mori; S Matsuzaki; K Imaizumi; A Mayeda; M Tohyama

2003-01-01

157

U7 snRNA-mediated correction of aberrant splicing caused by activation of cryptic splice sites  

Microsoft Academic Search

A considerable fraction of mutations associated with hereditary disorders and cancers affect splicing. Some of them cause\\u000a exon skipping or the inclusion of an additional exon, whereas others lead to the inclusion of intronic sequences or deletion\\u000a of exonic sequences through the activation of cryptic splice sites. We focused on the latter cases and have designed a series\\u000a of vectors

Hideki Uchikawa; Katsunori Fujii; Yoichi Kohno; Noriyuki Katsumata; Kazuaki Nagao; Masao Yamada; Toshiyuki Miyashita

2007-01-01

158

SAM68 regulates neuronal activity-dependent alternative splicing of Neurexin-1  

PubMed Central

Summary The assembly of synapses and neuronal circuits relies on an array of molecular recognition events and their modification by neuronal activity. Neurexins are a highly polymorphic family of synaptic receptors diversified by extensive alternative splicing. Neurexin variants exhibit distinct isoform-specific biochemical interactions and synapse assembly functions but the mechanisms governing splice isoform choice are not understood. We demonstrate that Nrxn1 alternative splicing is temporally and spatially controlled in the mouse brain. Neuronal activity triggers a shift in Nrxn1 splice isoform choice via calcium/calmodulin-dependent kinase IV signaling. Activity-dependent alternative splicing of Nrxn1 requires the KH-domain RNA binding protein SAM68 which associates with RNA response elements in the Nrxn1 pre-mRNA. Our findings uncover SAM68 as a key regulator of dynamic control of Nrxn1 molecular diversity and activity-dependent alternative splicing in the central nervous system.

Iijima, Takatoshi; Wu, Karen; Witte, Harald; Hanno-Iijima, Yoko; Glatter, Timo; Richard, Stephane; Scheiffele, Peter

2011-01-01

159

CD28 Costimulation Regulates Genome-Wide Effects on Alternative Splicing  

PubMed Central

CD28 is the major costimulatory receptor required for activation of naïve T cells, yet CD28 costimulation affects the expression level of surprisingly few genes over those altered by TCR stimulation alone. Alternate splicing of genes adds diversity to the proteome and contributes to tissue-specific regulation of genes. Here we demonstrate that CD28 costimulation leads to major changes in alternative splicing during activation of naïve T cells, beyond the effects of TCR alone. CD28 costimulation affected many more genes through modulation of alternate splicing than by modulation of transcription. Different families of biological processes are over-represented among genes alternatively spliced in response to CD28 costimulation compared to those genes whose transcription is altered, suggesting that alternative splicing regulates distinct biological effects. Moreover, genes dependent upon hnRNPLL, a global regulator of splicing in activated T cells, were enriched in T cells activated through TCR plus CD28 as compared to TCR alone. We show that hnRNPLL expression is dependent on CD28 signaling, providing a mechanism by which CD28 can regulate splicing in T cells and insight into how hnRNPLL can influence signal-induced alternative splicing in T cells. The effects of CD28 on alternative splicing provide a newly appreciated means by which CD28 can regulate T cell responses.

Jesneck, Jonathan; Keir, Mary E.; Haining, W. Nicholas; Sharpe, Arlene H.

2012-01-01

160

Periostin shows increased evolutionary plasticity in its alternatively spliced region  

PubMed Central

Background Periostin (POSTN) is a secreted extracellular matrix protein of poorly defined function that has been related to bone and heart development as well as to cancer. In human and mouse, it is known to undergo alternative splicing in its C-terminal region, which is devoid of known protein domains. Differential expression of periostin, sometimes of specific splicing isoforms, is observed in a broad range of human cancers, including breast, pancreatic, and colon cancer. Here, we combine genomic and transcriptomic sequence data from vertebrate organisms to study the evolution of periostin and particularly of its C-terminal region. Results We found that the C-terminal part of periostin is markedly more variable among vertebrates than the rest of periostin in terms of exon count, length, and splicing pattern, which we interpret as a consequence of neofunctionalization after the split between periostin and its paralog transforming growth factor, beta-induced (TGFBI). We also defined periostin's sequential 13-amino acid repeat units - well conserved in teleost fish, but more obscure in higher vertebrates - whose secondary structure is predicted to be consecutive beta strands. We suggest that these beta strands may mediate binding interactions with other proteins through an extended beta-zipper in a manner similar to the way repeat units in bacterial cell wall proteins have been reported to bind human fibronectin. Conclusions Our results, obtained with the help of the increasingly large collection of complete vertebrate genomes, document the evolutionary plasticity of periostin's C-terminal region, and for the first time suggest a basis for its functional role.

2010-01-01

161

Regulation of Alternative Splicing by the ATP-Dependent DEAD-Box RNA Helicase p72  

Microsoft Academic Search

Although a number of ATP-dependent RNA helicases are important for constitutive RNA splicing, no helicases have been implicated in alternative RNA splicing. Here, we show that the abundant DEAD-box RNA helicase p72, but not its close relative p68, affects the splicing of alternative exons containing AC-rich exon enhancer elements. The effect of p72 was tested by using mini-genes that undergo

Arnd Honig; Didier Auboeuf; Marjorie M. Parker; Bert W. O'Malley; Susan M. Berget

2002-01-01

162

Identification of embryo specific human isoforms using a database of predicted alternative splice forms  

Microsoft Academic Search

Alternative splicing is one of the most important mechanisms to generate a large number of mRNA and protein isoforms from a small number of genes. Its study became one of the hot topics in computational genome analysis. The repository EASED (Extended Alternatively Spliced EST Database, http:\\/\\/eased.bioinf.mdc-berlin.de\\/) stores a large collection of splice variants predicted from comparing the human genome against

Heike Pospisil

2006-01-01

163

In Vivo Regulation of Alternative Pre-mRNA Splicing by the Clk1 Protein Kinase  

Microsoft Academic Search

truncated inactive polypeptides (Clk1 and Clk1T, respectively). We present evidence that Clk1 and Clk1T proteins regulate the splicing of Clk1 and adenovirus pre-mRNAs in vivo. The peptide domain encoded by the alternatively spliced exon of Clk1 is essential for the regulatory activity of the Clk1 kinase. This is the first direct demonstration of an in vivo link between alternative splicing

PETER I. DUNCAN; DAVID F. STOJDL; RICARDO M. MARIUS; JOHN C. BELL

1997-01-01

164

A functional alternative splicing mutation in human tryptophan hydroxylase-2  

PubMed Central

The brain serotonergic system has an essential role in the physiological functions of the central nervous system and dysregulation of serotonin (5-HT) homeostasis has been implicated in many neuropsychiatric disorders. The tryptophan hydroxylase-2 (TPH2) gene is the rate-limiting enzyme in brain 5-HT synthesis, and thus is an ideal candidate gene for understanding the role of dysregulation of brain serotonergic homeostasis. Here, we characterized a common, but functional single-nucleotide polymorphism (SNP rs1386493) in the TPH2 gene, which decreases efficiency of normal RNA splicing, resulting in a truncated TPH2 protein (TPH2-TR) by alternative splicing. TPH2-TR, which lacks TPH2 enzyme activity, dominant-negatively affects full-length TPH2 function, causing reduced 5-HT production. The predicted mRNA for TPH2-TR is present in postmortem brain of rs1386493 carriers. The rs13864923 variant does not appear to be overrepresented in either global or multiplex depression cohorts. However, in combination with other gene variants linked to 5-HT homeostasis, this variant may exhibit important epistatic influences.

Zhang, X; Nicholls, P J; Laje, G; Sotnikova, T D; Gainetdinov, R R; Albert, P R; Rajkowska, G; Stockmeier, C A; Speer, M C; Steffens, D C; Austin, M C; McMahon, F J; Krishnan, K R R; Garcia-Blanco, M A; Caron, M G

2011-01-01

165

Circular dystrophin RNAs consisting of exons that were skipped by alternative splicing  

Microsoft Academic Search

Exon skipping by alternative splicing and circular RNA formation are proposed to be interrelated events. Since multiple patterns of alternative splicing have been demonstrated in both the 54 and 34 regions of the dystrophin gene, the dystrophin transcript in skeletal muscle cells provides a model system in which this idea is tested. Nine circular RNAs that were expected to result

Agus Surono; Yasuhiro Takeshima; Tri Wibawa; Makoto Ikezawa; Ikuya Nonaka; Masafumi Matsuo

1999-01-01

166

Alternatively spliced isoforms of TRIP8b differentially control h channel trafficking and function.  

PubMed

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (h channels) are the molecular basis for the current, I(h), which contributes crucially to intrinsic neuronal excitability. The subcellular localization and biophysical properties of h channels govern their function, but the mechanisms controlling these characteristics, and especially the potential role of auxiliary subunits or other binding proteins, remain unclear. We focused on TRIP8b, an h channel-interacting protein that colocalizes with HCN1 in cortical and hippocampal pyramidal neuron dendrites, and found that it exists in multiple alternative splice variants with distinct effects on h channel trafficking and function. The developmentally regulated splice variants of TRIP8b all shared dual, C terminus-located interaction sites with HCN1. When coexpressed with HCN1 in heterologous cells individual TRIP8b isoforms similarly modulated gating of I(h), causing a hyperpolarizing shift in voltage dependence of channel activation, but differentially upregulated or downregulated I(h) current density and HCN1 surface expression. In hippocampal neurons, coexpression of TRIP8b isoforms with HCN1 produced isoform-specific changes of HCN1 localization. Interestingly, the TRIP8b isoforms most abundant in the brain are those predicted to enhance h channel surface expression. Indeed, shRNA knockdown of TRIP8b in hippocampal neurons significantly reduced native I(h). Thus, although TRIP8b exists in multiple splice isoforms, our data suggest that the predominant role of this protein in brain is to promote h channel surface expression and enhance I(h). Because I(h) expression is altered in models of several diseases, including temporal lobe epilepsy, TRIP8b may play a role in both normal neuronal function and in aberrant neuronal excitability associated with neurological disease. PMID:19439603

Lewis, Alan S; Schwartz, Emily; Chan, C Savio; Noam, Yoav; Shin, Minyoung; Wadman, Wytse J; Surmeier, D James; Baram, Tallie Z; Macdonald, Robert L; Chetkovich, Dane M

2009-05-13

167

Alternatively spliced isoforms of TRIP8b differentially control h channel trafficking and function  

PubMed Central

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (h channels) are the molecular basis for the current, Ih, which contributes crucially to intrinsic neuronal excitability. The subcellular localization and biophysical properties of h channels govern their function, but the mechanisms controlling these characteristics, and especially the potential role of auxiliary subunits or other binding proteins remain unclear. We focused on TRIP8b, an h channel-interacting protein that colocalizes with HCN1 in cortical and hippocampal pyramidal neuron dendrites, and found that it exists in multiple alternative splice variants with distinct effects on h channel trafficking and function. The developmentally-regulated splice variants of TRIP8b all shared dual, C-terminus-located interaction sites with HCN1. When coexpressed with HCN1 in heterologous cells individual TRIP8b isoforms similarly modulated gating of Ih, causing a hyperpolarizing shift in voltage-dependence of channel activation, but differentially upregulated or downregulated Ih current density and HCN1 surface expression. In hippocampal neurons, coexpression of TRIP8b isoforms with HCN1 produced isoform-specific changes of HCN1 localization. Interestingly, the TRIP8b isoforms most abundant in the brain are those predicted to enhance h channel surface expression. Indeed, shRNA knockdown of TRIP8b in hippocampal neurons significantly reduced native Ih. Thus, although TRIP8b exists in multiple splice isoforms, our data suggest that the predominant role of this protein in brain is to promote h channel surface expression and enhance Ih. Because Ih expression is altered in models of several diseases, including temporal lobe epilepsy, TRIP8b may play a role in both normal neuronal function and in aberrant neuronal excitability associated with neurological disease.

Lewis, Alan S.; Schwartz, Emily; Chan, C. Savio; Noam, Yoav; Shin, Minyoung; Wadman, Wytse J.; Surmeier, D. James; Baram, Tallie Z.; Macdonald, Robert L.; Chetkovich, Dane M.

2009-01-01

168

The Conserved Splicing Factor SUA Controls Alternative Splicing of the Developmental Regulator ABI3 in Arabidopsis[W][OA  

PubMed Central

ABSCISIC ACID INSENSITIVE3 (ABI3) is a major regulator of seed maturation in Arabidopsis thaliana. We detected two ABI3 transcripts, ABI3-? and ABI3-?, which encode full-length and truncated proteins, respectively. Alternative splicing of ABI3 is developmentally regulated, and the ABI3-? transcript accumulates at the end of seed maturation. The two ABI3 transcripts differ by the presence of a cryptic intron in ABI3-?, which is spliced out in ABI3-?. The suppressor of abi3-5 (sua) mutant consistently restores wild-type seed features in the frameshift mutant abi3-5 but does not suppress other abi3 mutant alleles. SUA is a conserved splicing factor, homologous to the human protein RBM5, and reduces splicing of the cryptic ABI3 intron, leading to a decrease in ABI3-? transcript. In the abi3-5 mutant, ABI3-? codes for a functional ABI3 protein due to frameshift restoration.

Sugliani, Matteo; Brambilla, Vittoria; Clerkx, Emile J.M.; Koornneef, Maarten; Soppe, Wim J.J.

2010-01-01

169

Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR).  

PubMed

Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery. PMID:23474544

Solomon, Oz; Oren, Shirley; Safran, Michal; Deshet-Unger, Naamit; Akiva, Pinchas; Jacob-Hirsch, Jasmine; Cesarkas, Karen; Kabesa, Reut; Amariglio, Ninette; Unger, Ron; Rechavi, Gideon; Eyal, Eran

2013-03-08

170

Evolution of exon-intron structure and alternative splicing.  

PubMed

Despite significant advances in high-throughput DNA sequencing, many important species remain understudied at the genome level. In this study we addressed a question of what can be predicted about the genome-wide characteristics of less studied species, based on the genomic data from completely sequenced species. Using NCBI databases we performed a comparative genome-wide analysis of such characteristics as alternative splicing, number of genes, gene products and exons in 36 completely sequenced model species. We created statistical regression models to fit these data and applied them to loblolly pine (Pinus taeda L.), an example of an important species whose genome has not been completely sequenced yet. Using these models, the genome-wide characteristics, such as total number of genes and exons, can be roughly predicted based on parameters estimated from available limited genomic data, e.g. exon length and exon/gene ratio. PMID:21464961

Koralewski, Tomasz E; Krutovsky, Konstantin V

2011-03-25

171

Prediction of alternatively skipped exons and splicing enhancers from exon junction arrays  

PubMed Central

Background Alternative splicing of exons in a pre-mRNA transcript is an important mechanism which contributes to protein diversity in human. Arrays for detecting alternative splicing are available using several different probe designs, including those based on exon-junctions. In this work, we introduce a new method for predicting alternatively skipped exons from exon-junction arrays. Predictions based on our method are compared against controls and their sequences are analyzed to identify motifs important for regulating alternative splicing. Results Our comparison of several alternative methods shows that an exon-skipping score based on neighboring junctions best discriminates between positive and negative controls. Sequence analysis of our predicted exons confirms the presence of known splicing regulatory sequences. In addition, we also derive a set of development-related alternatively spliced genes based on fetal versus adult tissue comparisons and find that our predictions are consistent with their functional annotations. Ab initio motif finding algorithms are applied to identify several motifs that may be relevant for splicing during development. Conclusion This work describes a new method for analyzing exon-junction arrays, identifies sequence motifs that are specific for alternative and constitutive splicing and suggests a role for several known splicing factors and their motifs in developmental regulation.

Kechris, Katerina; Yang, Yee Hwa; Yeh, Ru-Fang

2008-01-01

172

Diverging Alternative Splicing Fingerprints in the Transforming Growth Factor-? Signaling Pathway Identified in Thoracic Aortic Aneurysms  

PubMed Central

Impaired regulation of the transforming growth factor-? (TGF?) signaling pathway has been linked to thoracic aortic aneurysm (TAA). Previous work has indicated that differential splicing is a common phenomenon, potentially influencing the function of proteins. In the present study we investigated the occurrence of differential splicing in the TGF? pathway associated with TAA in patients with bicuspid aortic valve (BAV) and tricuspid aortic valve (TAV). Affymetrix human exon arrays were applied to 81 intima/media tissue samples from dilated (n = 51) and nondilated (n = 30) aortas of TAV and BAV patients. To analyze the occurrence of alternative splicing in the TGF? pathway, multivariate techniques, including principal component analysis and OPLS-DA (orthogonal partial least squares to latent structures discriminant analysis), were applied on all exons (n = 614) of the TGF? pathway. The scores plot, based on the splice index of individual exons, showed separate clusters of patients with both dilated and nondilated aorta, thereby illustrating the potential importance of alternative splicing in TAA. In total, differential splicing was detected in 187 exons. Furthermore, the pattern of alternative splicing is clearly differs between TAV and BAV patients. Differential splicing was specific for BAV and TAV patients in 40 and 86 exons, respectively, and splicings of 61 exons were shared between the two phenotypes. The occurrence of differential splicing was demonstrated in selected genes by reverse transcription–polymerase chain reaction. In summary, alternative splicing is a common feature of TAA formation. Our results suggest that dilatation in TAV and BAV patients has different alternative splicing fingerprints in the TGF? pathway.

Kurtovic, Sanela; Paloschi, Valentina; Folkersen, Lasse; Gottfries, Johan; Franco-Cereceda, Anders; Eriksson, Per

2011-01-01

173

Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB  

Microsoft Academic Search

To gain global insights into the role of the well-known repressive splicing regulator PTB, we analyzed the consequences of PTB knockdown in HeLa cells using high-density oligonucleotide splice-sensitive microarrays. The major class of identified PTB-regulated splicing event was PTB-repressed cassette exons, but there was also a substantial number of PTB-activated splicing events. PTB-repressed and PTB-activated exons showed a distinct arrangement

Miriam Llorian; Schraga Schwartz; Tyson A Clark; Dror Hollander; Lit-Yeen Tan; Rachel Spellman; Adele Gordon; Anthony C Schweitzer; Pierre de la Grange; Gil Ast; Christopher W J Smith

2010-01-01

174

Integrative analysis of tissue-specific methylation and alternative splicing identifies conserved transcription factor binding motifs  

PubMed Central

The exact role of intragenic DNA methylation in regulating tissue-specific gene regulation is unclear. Recently, the DNA-binding protein CTCF has been shown to participate in the regulation of alternative splicing in a DNA methylation-dependent manner. To globally evaluate the relationship between DNA methylation and tissue-specific alternative splicing, we performed genome-wide DNA methylation profiling of mouse retina and brain. In protein-coding genes, tissue-specific differentially methylated regions (T-DMRs) were preferentially located in exons and introns. Gene ontology and evolutionary conservation analysis suggest that these T-DMRs are likely to be biologically relevant. More than 14% of alternatively spliced genes were associated with a T-DMR. T-DMR-associated genes were enriched for developmental genes, suggesting that a specific set of alternatively spliced genes may be regulated through DNA methylation. Novel DNA sequences motifs overrepresented in T-DMRs were identified as being associated with positive and/or negative regulation of alternative splicing in a position-dependent context. The majority of these evolutionarily conserved motifs contain a CpG dinucleotide. Some transcription factors, which recognize these motifs, are known to be involved in splicing. Our results suggest that DNA methylation-dependent alternative splicing is widespread and lay the foundation for further mechanistic studies of the role of DNA methylation in tissue-specific splicing regulation.

Wan, Jun; Oliver, Verity F.; Zhu, Heng; Zack, Donald J.; Qian, Jiang; Merbs, Shannath L.

2013-01-01

175

EST comparison indicates 38% of human mRNAs contain possible alternative splice forms.  

PubMed

Expressed sequence tag (EST) databases represent a large volume of information on expressed genes including tissue type, expression profile and exon structure. In this study we create an extensive data set of human alternative splicing. We report the analysis of 7867 non-redundant mRNAs, 3011 of which contained alternative splice forms (38% of all mRNAs analysed). From a total of 12572 ESTs 4560 different possible alternative splice forms were detected. Interestingly, 70% of the alternative splice forms correspond to exon deletion events with only 30% exonic insertions. We experimentally verified 19 different splice forms from 16 genes in a total subset of 20 studied; all of the respective genes are of medical relevance. PMID:10828456

Brett, D; Hanke, J; Lehmann, G; Haase, S; Delbrück, S; Krueger, S; Reich, J; Bork, P

2000-05-26

176

Alternative splicing of the FMR1 gene in human fetal brain neurons  

SciTech Connect

The alternative splicing expression of the FMR1 gene was reported in several human and mouse tissues. Five regions of FMR1 gene can be alternatively spliced, but the combination of them has not been investigated fully. We reported here the analysis of alternative splicing pattern of the FMR1 gene in cultured fetal human neurons, using a RT-PCR and cloning strategy. Eleven splicing types were cloned and different isoforms were not equally represented. The dominant isoform represents nearly 40%, and the other isoforms were relatively rare. One isoform has a different carboxyl-terminus. Most of the alternative spliced regions appear hydrophilic; thus, they may locate on the surface of the FMR1 protein. 16 refs., 2 figs.

Tao Huang; Yan Shen; Xue-bin Qin; Guan-Yun Wu [Chinese Academy of Medical Sciences, Beijing (China)] [and others

1996-08-09

177

Workshop: Using a transcript catalog and paired-end RNA-Seq data to identify differential alternative splicing  

Microsoft Academic Search

Alternative splicing is one of the major contributors to transcript and protein diversity in many higher eukaryotes including humans, but so far the extent and impact of alternative splicing in plants has not been thoroughly investigated [1]. The purpose of our NSF-Eager grant is to use high-throughput RNA sequencing of the transcriptome to assess the role of alternative splicing in

Brian E. Howard; Xiaoping Tan; Paola Veronese; Steffen Heber

2011-01-01

178

Alternative splicing of a Drosophila GABA receptor subunit gene identifies determinants of agonist potency  

Microsoft Academic Search

Alternative splicing of the Drosophila melanogaster Rdl gene yields four ionotropic GABA receptor subunits. The two Rdl splice variants cloned to date, RDLac and RDLbd (DRC17-1-2), differ in their apparent agonist affinity. Here, we report the cloning of a third splice variant of Rdl, RDLad. Two-electrode voltage clamp electrophysiology was used to investigate agonist pharmacology of this expressed subunit following

A. M Hosie; S. D Buckingham; J. K Presnail; D. B Sattelle

2001-01-01

179

Fine-Scale Variation and Genetic Determinants of Alternative Splicing across Individuals  

Microsoft Academic Search

Recently, thanks to the increasing throughput of new technologies, we have begun to explore the full extent of alternative pre–mRNA splicing (AS) in the human transcriptome. This is unveiling a vast layer of complexity in isoform-level expression differences between individuals. We used previously published splicing sensitive microarray data from lymphoblastoid cell lines to conduct an in-depth analysis on splicing efficiency

Jasmin Coulombe-Huntington; Kevin C. L. Lam; Christel Dias; Jacek Majewski

2009-01-01

180

REMAS: a new regression model to identify alternative splicing events from exon array data  

Microsoft Academic Search

BACKGROUND: Alternative splicing (AS) is an important regulatory mechanism for gene expression and protein diversity in eukaryotes. Previous studies have demonstrated that it can be causative for, or specific to splicing-related diseases. Understanding the regulation of AS will be helpful for diagnostic efforts and drug discoveries on those splicing-related diseases. As a novel exon-centric microarray platform, exon array enables a

Hao Zheng; Xingyi Hang; Ji Zhu; Minping Qian; Wubin Qu; Chenggang Zhang; Minghua Deng

2009-01-01

181

Copy Number Variations in Alternative Splicing Gene Networks Impact Lifespan  

PubMed Central

Longevity has a strong genetic component evidenced by family-based studies. Lipoprotein metabolism, FOXO proteins, and insulin/IGF-1 signaling pathways in model systems have shown polygenic variations predisposing to shorter lifespan. To test the hypothesis that rare variants could influence lifespan, we compared the rates of CNVs in healthy children (0–18 years of age) with individuals 67 years or older. CNVs at a significantly higher frequency in the pediatric cohort were considered risk variants impacting lifespan, while those enriched in the geriatric cohort were considered longevity protective variants. We performed a whole-genome CNV analysis on 7,313 children and 2,701 adults of European ancestry genotyped with 302,108 SNP probes. Positive findings were evaluated in an independent cohort of 2,079 pediatric and 4,692 geriatric subjects. We detected 8 deletions and 10 duplications that were enriched in the pediatric group (P?=?3.33×10?8–1.6×10?2 unadjusted), while only one duplication was enriched in the geriatric cohort (P?=?6.3×10?4). Population stratification correction resulted in 5 deletions and 3 duplications remaining significant (P?=?5.16×10?5–4.26×10?2) in the replication cohort. Three deletions and four duplications were significant combined (combined P?=?3.7×10?4?3.9×10?2). All associated loci were experimentally validated using qPCR. Evaluation of these genes for pathway enrichment demonstrated ?50% are involved in alternative splicing (P?=?0.0077 Benjamini and Hochberg corrected). We conclude that genetic variations disrupting RNA splicing could have long-term biological effects impacting lifespan.

Glessner, Joseph T.; Smith, Albert Vernon; Panossian, Saarene; Kim, Cecilia E.; Takahashi, Nagahide; Thomas, Kelly A.; Wang, Fengxiang; Seidler, Kallyn; Harris, Tamara B.; Launer, Lenore J.; Keating, Brendan; Connolly, John; Sleiman, Patrick M. A.; Buxbaum, Joseph D.; Grant, Struan F. A.; Gudnason, Vilmundur; Hakonarson, Hakon

2013-01-01

182

Unproductive alternative splicing and nonsense mRNAs: A widespread phenomenon among plant circadian clock genes  

PubMed Central

Background Recent mapping of eukaryotic transcriptomes and spliceomes using massively parallel RNA sequencing (RNA-seq) has revealed that the extent of alternative splicing has been considerably underestimated. Evidence also suggests that many pre-mRNAs undergo unproductive alternative splicing resulting in incorporation of in-frame premature termination codons (PTCs). The destinies and potential functions of the PTC-harboring mRNAs remain poorly understood. Unproductive alternative splicing in circadian clock genes presents a special case study because the daily oscillations of protein expression levels require rapid and steep adjustments in mRNA levels. Results We conducted a systematic survey of alternative splicing of plant circadian clock genes using RNA-seq and found that many Arabidopsis thaliana circadian clock-associated genes are alternatively spliced. Results were confirmed using reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR), and/or Sanger sequencing. Intron retention events were frequently observed in mRNAs of the CCA1/LHY-like subfamily of MYB transcription factors. In contrast, the REVEILLE2 (RVE2) transcript was alternatively spliced via inclusion of a "poison cassette exon" (PCE). The PCE type events introducing in-frame PTCs are conserved in some mammalian and plant serine/arginine-rich splicing factors. For some circadian genes such as CCA1 the ratio of the productive isoform (i.e., a representative splice variant encoding the full-length protein) to its PTC counterpart shifted sharply under specific environmental stress conditions. Conclusions Our results demonstrate that unproductive alternative splicing is a widespread phenomenon among plant circadian clock genes that frequently generates mRNA isoforms harboring in-frame PTCs. Because LHY and CCA1 are core components of the plant central circadian oscillator, the conservation of alternatively spliced variants between CCA1 and LHY and for CCA1 across phyla [2] indicates a potential role of nonsense transcripts in regulation of circadian rhythms. Most of the alternatively spliced isoforms harbor in-frame PTCs that arise from full or partial intron retention events. However, a PTC in the RVE2 transcript is introduced through a PCE event. The conservation of AS events and modulation of the relative abundance of nonsense isoforms by environmental and diurnal conditions suggests possible regulatory roles for these alternatively spliced transcripts in circadian clock function. The temperature-dependent expression of the PTC transcripts among members of CCA1/LHY subfamily indicates that alternative splicing may be involved in regulation of the clock temperature compensation mechanism. Reviewers This article was reviewed by Dr. Eugene Koonin, Dr. Chungoo Park (nominated by Dr. Kateryna Makova), and Dr. Marcelo Yanovsky (nominated by Dr. Valerian Dolja).

2012-01-01

183

Rapid-Response Splicing Reporter Screens Identify Differential Regulators of Constitutive and Alternative Splicing? † ‡  

PubMed Central

Bioactive compounds have been invaluable for dissecting the mechanisms, regulation, and functions of cellular processes. However, very few such reagents have been described for pre-mRNA splicing. To facilitate their systematic discovery, we developed a high-throughput cell-based assay that measures pre-mRNA splicing by utilizing a quantitative reporter system with advantageous features. The reporter, consisting of a destabilized, intron-containing luciferase expressed from a short-lived mRNA, allows rapid screens (<4 h), thereby obviating the potential toxicity of splicing inhibitors. We describe three inhibitors (out of >23,000 screened), all pharmacologically active: clotrimazole, flunarizine, and chlorhexidine. Interestingly, none was a general splicing inhibitor. Rather, each caused distinct splicing changes of numerous genes. We further discovered the target of action of chlorhexidine and show that it is a selective inhibitor of specific Cdc2-like kinases (Clks) that phosphorylate serine-arginine-rich (SR) protein splicing factors. Our findings reveal unexpected activities of clinically used drugs in splicing and uncover differential regulation of constitutively spliced introns.

Younis, Ihab; Berg, Michael; Kaida, Daisuke; Dittmar, Kimberly; Wang, Congli; Dreyfuss, Gideon

2010-01-01

184

Quantitative and evolutionary biology of alternative splicing: how changing the mix of alternative transcripts affects phenotypic plasticity and reaction norms.  

PubMed

Alternative splicing (AS) of pre-messenger RNA is a common phenomenon that creates different transcripts from a single gene, and these alternative transcripts affect phenotypes. The majority of AS research has examined tissue and developmental specificity of expression of particular AS transcripts, how this specificity affects cell function, and how aberrant AS is related to disease. Few studies have examined quantitative between-individual variation in AS within a cell or tissue type, or in relation to phenotypes, but the results are compelling: quantitative variation in AS affects plastic traits such as stress, anxiety, fear, egg production, muscle performance, energetics and plant growth. Genomic analyses of AS are also at a nascent stage, but have revealed a number of significant evolutionary patterns. Growing knowledge of upstream genes and kinases that regulate AS provides the as-yet little explored potential to examine how these genes and pathways respond to environmental and genotype variables. Research in this area can provide glimpses of a labyrinth of genetic architectures that have rarely been considered in evolutionary and organismal biology, or in quantitative genetics. The scarcity of contribution to knowledge about AS from these fields is illustrated by the fact that heritability of quantitative variation in AS has not yet been determined for any gene in any organism. New research tactics that incorporate quantitative analyses of AS will allow organismal and evolutionary biologists to attain a fuller mechanistic understanding of many of the traits they study, and may lead to more rapid discovery of functionally important polymorphisms. PMID:17006532

Marden, J H

2006-09-27

185

Sporadic ALS has compartment-specific aberrant exon splicing and altered cell-matrix adhesion biology  

Microsoft Academic Search

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive weak- ness from loss of motor neurons. The fundamental pathogenic mechanisms are unknown and recent evidence is implicating a significant role for abnormal exon splicing and RNA processing. Using new comprehensive genomic technologies, we studied exon splicing directly in 12 sporadic ALS and 10 control lumbar spinal cords

Stuart J. Rabin; Jae Mun; Michael Baughn; Ryan T. Libby; Young Joo Kim; Yuxin Fan; Randell T. Libby; Albert La Spada; Brad Stone; John Ravits

2009-01-01

186

Alternative splicing of Alu exons--two arms are better than one  

Microsoft Academic Search

Alus, primate-specific retroelements, are the most abundant repetitive elements in the human genome. They are composed of two related but distinct monomers, left and right arms. Intronic Alu ele- ments may acquire mutations that generate func- tional splice sites, a process called exonization. Most exonizations occur in right arms of antisense Alu elements, and are alternatively spliced. Here we show

Nurit Gal-Mark; Schraga Schwartz; Gil Ast

2008-01-01

187

Alternative Splicing Modulation by a LAMMER Kinase Impinges on Developmental and Transcriptome Expression  

Microsoft Academic Search

Alternative splicing is a major contributor to genome complexity, playing a significant role in various cellular functions, in- cluding signal transduction, immunity, and development. The spliceosomal machinery is responsible for the processing of nuclear RNA. Several splicing factors associated with this complex are phosphorylated by kinases that possess a con- served LAMMER motif. We demonstrate in BY-2 tobacco cells a

Sigal Savaldi-Goldstein; Dvora Aviv; Olga Davydov; Robert Fluhr

2003-01-01

188

Multiple cardiovascular defects caused by the absence of alternatively spliced segments of fibronectin  

Microsoft Academic Search

Alternatively spliced variants of fibronectin (FN) containing exons EIIIA and EIIIB are expressed around newly forming vessels in development and disease but are downregulated in mature vasculature. The sequences and patterns of expression of these splice variants are highly conserved among vertebrates, suggestive of their biological importance; however the functions of EIIIA and EIIIB-containing FNs are unknown. To understand the

Sophie Astrof; Denise Crowley; Richard O. Hynes

2007-01-01

189

Inferring global levels of alternative splicing isoforms using a generative model of microarray data  

Microsoft Academic Search

Motivation: Alternative splicing (AS) is a frequent step in metozoan gene expression whereby the exons of genes are spliced in different combinations to generate multiple isoforms of mature mRNA. AS func- tions to enrich an organism's proteomic complexity and regulates gene expression. Despite its importance, the mechanisms underlying AS and its regulation are not well understood, especially in the context

Ofer Shai; Quaid Morris; Benjamin J. Blencowe; Brendan J. Frey

2006-01-01

190

Splicing Reporter Mice Revealed the Evolutionally Conserved Switching Mechanism of Tissue-Specific Alternative Exon Selection  

PubMed Central

Since alternative splicing of pre-mRNAs is essential for generating tissue-specific diversity in proteome, elucidating its regulatory mechanism is indispensable to understand developmental process or tissue-specific functions. We have been focusing on tissue-specific regulation of mutually exclusive selection of alternative exons because this implies the typical molecular mechanism of alternative splicing regulation and also can be good examples to elicit general rule of “splice code”. So far, mutually exclusive splicing regulation has been explained by the outcome from the balance of multiple regulators that enhance or repress either of alternative exons discretely. However, this “balance” model is open to questions of how to ensure the selection of only one appropriate exon out of several candidates and how to switch them. To answer these questions, we generated an original bichromatic fluorescent splicing reporter system for mammals using fibroblast growth factor-receptor 2 (FGFR2) gene as model. By using this splicing reporter, we demonstrated that FGFR2 gene is regulated by the “switch-like” mechanism, in which key regulators modify the ordered splice-site recognition of two mutually exclusive exons, eventually ensure single exon selection and their distinct switching. Also this finding elucidated the evolutionally conserved “splice code,” in which combination of tissue-specific and broadly expressed RNA binding proteins regulate alternative splicing of specific gene in a tissue-specific manner. These findings provide the significant cue to understand how a number of spliced genes are regulated in various tissue-specific manners by a limited number of regulators, eventually to understand developmental process or tissue-specific functions.

Takeuchi, Akihide; Hosokawa, Motoyasu; Nojima, Takayuki; Hagiwara, Masatoshi

2010-01-01

191

Structures of alternatively spliced isoforms of human ketohexokinase.  

PubMed

A molecular understanding of the unique aspects of dietary fructose metabolism may be the key to understanding and controlling the current epidemic of fructose-related obesity, diabetes and related adverse metabolic states in Western populations. Fructose catabolism is initiated by its phosphorylation to fructose 1-phosphate, which is performed by ketohexokinase (KHK). Here, the crystal structures of the two alternatively spliced isoforms of human ketohexokinase, hepatic KHK-C and the peripheral isoform KHK-A, and of the ternary complex of KHK-A with the substrate fructose and AMP-PNP are reported. The structure of the KHK-A ternary complex revealed an active site with both the substrate fructose and the ATP analogue in positions ready for phosphorylation following a reaction mechanism similar to that of the pfkB family of carbohydrate kinases. Hepatic KHK deficiency causes the benign disorder essential fructosuria. The effects of the disease-causing mutations (Gly40Arg and Ala43Thr) have been modelled in the context of the KHK structure. PMID:19237742

Trinh, Chi H; Asipu, Aruna; Bonthron, David T; Phillips, Simon E V

2009-02-20

192

Targeting RNA splicing for disease therapy.  

PubMed

Splicing of pre-messenger RNA into mature messenger RNA is an essential step for the expression of most genes in higher eukaryotes. Defects in this process typically affect cellular function and can have pathological consequences. Many human genetic diseases are caused by mutations that cause splicing defects. Furthermore, a number of diseases are associated with splicing defects that are not attributed to overt mutations. Targeting splicing directly to correct disease-associated aberrant splicing is a logical approach to therapy. Splicing is a favorable intervention point for disease therapeutics, because it is an early step in gene expression and does not alter the genome. Significant advances have been made in the development of approaches to manipulate splicing for therapy. Splicing can be manipulated with a number of tools including antisense oligonucleotides, modified small nuclear RNAs (snRNAs), trans-splicing, and small molecule compounds, all of which have been used to increase specific alternatively spliced isoforms or to correct aberrant gene expression resulting from gene mutations that alter splicing. Here we describe clinically relevant splicing defects in disease states, the current tools used to target and alter splicing, specific mutations and diseases that are being targeted using splice-modulating approaches, and emerging therapeutics. PMID:23512601

Havens, Mallory A; Duelli, Dominik M; Hastings, Michelle L

2013-03-19

193

smg Mutants Affect the Expression of Alternatively Spliced SR Protein mRNAs in Caenorhabditis elegans  

Microsoft Academic Search

The expression of alternatively spliced mRNAs from genes is an ubiquitous phenomenon in metazoa. A screen for trans-acting factors that alter the expression of alternatively spliced mRNAs reveals that the smg genes of Caenorhabditis elegans participate in this process. smg genes have been proposed to function in degradation of nonsense mutant mRNAs. Here we show that smg genes affect normal

Mike Morrison; Kevin S. Harris; Mark B. Roth

1997-01-01

194

Regulation of Vertebrate Nervous System Alternative Splicing and Development by an SR-Related Protein  

Microsoft Academic Search

SUMMARY Alternative splicing is a key process underlying the evolution of increased proteomic and functional complexity and is especially prevalent in the mam- malian nervous system. However, the factors and mechanisms governing nervous system-specific alternative splicing are not well understood. Through a genome-wide computational and expression pro- filing strategy, we have identified a tissue- and verte- brate-restricted Ser\\/Arg (SR) repeat

John A. Calarco; Simone Superina; Dave O'Hanlon; Mathieu Gabut; Bushra Raj; Qun Pan; Ursula Skalska; Laura Clarke; Danielle Gelinas; Derek van der Kooy; Mei Zhen; Brian Ciruna; Benjamin J. Blencowe

2009-01-01

195

Auto and Cross-Regulation of the hnRNP L Proteins by Alternative Splicing  

Microsoft Academic Search

We recently characterized human hnRNP L as a global regulator of alternative splicing, binding to CA- repeat and CA-rich elements. Here we report that hnRNP L autoregulates its own expression on the level of alternative splicing. Intron 6 of the human hnRNP L gene contains a short exon that, if used, introduces a premature termination codon, resulting in nonsense-mediated decay

Oliver Rossbach; Lee-Hsueh Hung; Silke Schreiner; Inna Grishina; Monika Heiner; Jingyi Hui; Albrecht Bindereif

2009-01-01

196

Discovery of Gene Families and Alternatively Spliced Variants by RecA-Mediated Cloning  

Microsoft Academic Search

Probing the functional complexity of the human genome will require new gene cloning techniques, not only to discover intraspecies gene homologs and interspecies gene orthologs, but also to identify alternatively spliced gene variants. We report homologous cDNA cloning methods that allow cloning of gene family members, genes from different species, and alternatively spliced gene variants. We cloned human 14-3-3 gene

Hong Zeng; Elizabeth Allen; Chris W. Lehman; R. Geoffrey Sargent; Sushma Pati; David A. Zarling

2002-01-01

197

Dynamic regulation of alternative splicing and chromatin structure in Drosophila gonads revealed by RNA-seq  

Microsoft Academic Search

Both transcription and post-transcriptional processes, such as alternative splicing, play crucial roles in controlling developmental programs in metazoans. Recently emerged RNA-seq method has brought our understanding of eukaryotic transcriptomes to a new level, because it can resolve both gene expression level and alternative splicing events simultaneously. To gain a better understanding of cellular differentiation in gonads, we analyzed mRNA profiles

Qiang Gan; Iouri Chepelev; Gang Wei; Lama Tarayrah; Kairong Cui; Keji Zhao; Xin Chen

2010-01-01

198

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

PubMed

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'. PMID:23357783

Yap, Karen; Makeyev, Eugene V

2013-01-26

199

Extensive relationship between antisense transcription and alternative splicing in the human genome.  

PubMed

To analyze the relationship between antisense transcription and alternative splicing, we developed a computational approach for the detection of antisense-correlated exon splicing events using Affymetrix exon array data. Our analysis of expression data from 176 lymphoblastoid cell lines revealed that the majority of expressed sense-antisense genes exhibited alternative splicing events that were correlated to the expression of the antisense gene. Most of these events occurred in areas of sense-antisense (SAS) gene overlap, which were significantly enriched in both exons and nucleosome occupancy levels relative to nonoverlapping regions of the same genes. Nucleosome occupancy was highly correlated with Pol II abundance across overlapping regions and with concomitant increases in local alternative exon usage. These results are consistent with an antisense transcription-mediated mechanism of splicing regulation in normal human cells. A comparison of the prevalence of antisense-correlated splicing events between individuals of Mormon versus African descent revealed population-specific events that may indicate the continued evolution of new SAS loci. Furthermore, the presence of antisense transcription was correlated to alternative splicing across multiple metazoan species, suggesting that it may be a conserved mechanism contributing to splicing regulation. PMID:21719572

Morrissy, A Sorana; Griffith, Malachi; Marra, Marco A

2011-06-30

200

Extensive relationship between antisense transcription and alternative splicing in the human genome  

PubMed Central

To analyze the relationship between antisense transcription and alternative splicing, we developed a computational approach for the detection of antisense-correlated exon splicing events using Affymetrix exon array data. Our analysis of expression data from 176 lymphoblastoid cell lines revealed that the majority of expressed sense–antisense genes exhibited alternative splicing events that were correlated to the expression of the antisense gene. Most of these events occurred in areas of sense–antisense (SAS) gene overlap, which were significantly enriched in both exons and nucleosome occupancy levels relative to nonoverlapping regions of the same genes. Nucleosome occupancy was highly correlated with Pol II abundance across overlapping regions and with concomitant increases in local alternative exon usage. These results are consistent with an antisense transcription-mediated mechanism of splicing regulation in normal human cells. A comparison of the prevalence of antisense-correlated splicing events between individuals of Mormon versus African descent revealed population-specific events that may indicate the continued evolution of new SAS loci. Furthermore, the presence of antisense transcription was correlated to alternative splicing across multiple metazoan species, suggesting that it may be a conserved mechanism contributing to splicing regulation.

Morrissy, A. Sorana; Griffith, Malachi; Marra, Marco A.

2011-01-01

201

New Way of Regulating Alternative Splicing in Retroviruses: the Promoter Makes a Difference?  

PubMed Central

Alternative splicing has been recognized as a major mechanism for creating proteomic diversity from a limited number of genes. However, not all determinants regulating this process have been characterized. Using subviral human immunodeficiency virus (HIV) env constructs we observed an enhanced splicing of the RNA when expression was under control of the cytomegalovirus (CMV) promoter instead of the HIV long terminal repeat (LTR). We extended these observations to LTR- or CMV-driven murine leukemia proviruses, suggesting that retroviral LTRs are adapted to inefficient alternative splicing at most sites in order to maintain balanced gene expression.

Bohne, Jens; Schambach, Axel; Zychlinski, Daniela

2007-01-01

202

Structural determinants of allosteric regulation in alternatively spliced AMPA receptors  

Microsoft Academic Search

The flip and flop splice variants of AMPA receptors show strikingly different sensitivity to allosteric regulation by cyclothiazide; heteromers assembled from GluR-A and GIuR-B also exhibit splice variant-dependent differences in efficacy for activation by glutamate and kainate. The sensitivity for attenuation of desensitization by cyclothiazide for homomeric GIuR-A was solely dependent upon exchange of Ser-750 (flip) and Asn-750 (flop), and

Kathryn M. Partin; Derek Bowie; Mark L. Mayer

1995-01-01

203

The evolution of alternative splicing in the Pax family: the view from the Basal chordate amphioxus.  

PubMed

Pax genes encode transcription factors critical for metazoan development. Large-scale gene duplication with subsequent gene losses during vertebrate evolution has resulted in two human genes for each of the Pax1/9, Pax3/7, and Pax4/6 subfamilies and three for the Pax2/5/8 subfamily, compared to one each in the cephalochordate amphioxus. In addition, alternative splicing occurs in vertebrate Pax transcripts from all four subfamilies, and many splice forms are known to have functional importance. To better understand the evolution of alternative splicing within the Pax family, we systematically surveyed transcripts of the four amphioxus Pax genes. We have found alternative splicing in every gene. Comparisons with vertebrates suggest that the number of alternative splicing events per gene has not decreased following duplication; there are comparable levels in the four amphioxus Pax genes as in each gene of the equivalent vertebrate families. Thus, the total number of isoforms for the nine vertebrate genes is considerably higher than for the four amphioxus genes. Most alternative splicing events appear to have arisen since the divergence of amphioxus and vertebrate lineages, suggesting that differences in alternative splicing could account for divergent functions of the highly conserved Pax genes in both lineages. However, several events predicted to dramatically alter known functional domains are conserved between amphioxus and vertebrates, suggestive of a common chordate function. Our results, together with previous studies of vertebrate Pax genes, support the theory that alternative splicing impacts functional motifs more than gene duplication followed by divergence. PMID:18473110

Short, Stephen; Holland, Linda Z

2008-05-14

204

Intron Retention in the Alternatively Spliced Region of RON Results from Weak 3' Splice Site Recognition  

PubMed Central

The RON gene encodes a tyrosine kinase receptor for macrophage-stimulating protein. A constitutively active isoform that arises by skipping of exon 11 is expressed in carcinomas and contributes to an invasive phenotype. However, a high proportion of the mRNA expressed from the endogenous gene, or from transfected minigenes, appears to retain introns 10 and 11. It is not known whether this represents specific repression or the presence of weak splicing signals. We have used chimeric pre-mRNAs spliced in vitro to investigate the reason for intron retention. A systematic test showed that, surprisingly, the exon sequences known to modulate exon 11 skipping were not limiting, but the 3’ splice site regions adjacent to exons 11 and 12 were too weak to support splicing when inserted into a globin intron. UV-crosslinking experiments showed binding of hnRNP F/H just 5’ of these regions, but the hnRNP F/H target sequences did not mediate inhibition. Instead, the failure of splicing is linked to weak binding of U2AF65, and spliceosome assembly stalls prior to formation of any of the ATP-dependent complexes. We discuss mechanisms by which U2AF65 binding is facilitated in vivo.

Smith, Lindsay D.; Lucas, Christian M.; Eperon, Ian C.

2013-01-01

205

A novel computational method for the identification of plant alternative splice sites.  

PubMed

Alternative splicing (AS) increases protein diversity by generating multiple transcript isoforms from a single gene in higher eukaryotes. Up to 48% of plant genes exhibit alternative splicing, which has proven to be involved in some important plant functions such as the stress response. A hybrid feature extraction approach which combing the position weight matrix (PWM) with the increment of diversity (ID) was proposed to represent the base conservative level (BCL) near splice sites and the similarity level of two datasets, respectively. Using the extracted features, the support vector machine (SVM) was applied to classify alternative and constitutive splice sites. By the proposed algorithm, 80.8% of donor sites and 85.4% of acceptor sites were correctly classified. It is anticipated that the novel computational method is promising for the identification of AS sites in plants. PMID:23313482

Cui, Ying; Han, Jiuqiang; Zhong, Dexing; Liu, Ruiling

2013-01-09

206

Analysis of alternative splicing of cassette exons at single-cell level using two fluorescent proteins  

PubMed Central

Alternative splicing plays a major role in increasing proteome complexity and regulating gene expression. Here, we developed a new fluorescent protein-based approach to quantitatively analyze the alternative splicing of a target cassette exon (skipping or inclusion), which results in an open-reading frame shift. A fragment of a gene of interest is cloned between red and green fluorescent protein (RFP and GFP)-encoding sequences in such a way that translation of the normally spliced full-length transcript results in expression of both RFP and GFP. In contrast, alternative exon skipping results in the synthesis of RFP only. Green and red fluorescence intensities can be used to estimate the proportions of normal and alternative transcripts in each cell. The new method was successfully tested for human PIG3 (p53-inducible gene 3) cassette exon 4. Expected pattern of alternative splicing of PIG3 minigene was observed, including previously characterized effects of UV light irradiation and specific mutations. Interestingly, we observed a broad distribution of normal to alternative transcript ratio in individual cells with at least two distinct populations with ?45% and >95% alternative transcript. We believe that this method is useful for fluorescence-based quantitative analysis of alternative splicing of target genes in a variety of biological models.

Gurskaya, Nadya G.; Staroverov, Dmitry B.; Zhang, Lijuan; Fradkov, Arkady F.; Markina, Nadezhda M.; Pereverzev, Anton P.; Lukyanov, Konstantin A.

2012-01-01

207

A 6-bp deletion at the splice donor site of the first intron resulted in aberrant splicing using a cryptic splice site within exon 1 in a patient with succinyl-CoA: 3Ketoacid CoA transferase (SCOT) deficiency  

Microsoft Academic Search

Succinyl-CoA: 3-ketoacid-CoA transferase (SCOT; locus symbol OXCT, EC 2.8.3.5) deficiency is a rare genetic disorder affecting ketone body utilization in extra-hepatic tissues. A 6-bp deletion at the splice donor site of intron 1 resulted in the absence of a full-length mature SCOT mRNA with faint amounts of aberrantly spliced transcripts using a cryptic splice donor site within exon 1, which

Toshiyuki Fukao; Satomi Sakurai; Marie-Odile Rolland; Marie-Therese Zabot; Andreas Schulze; Keitaro Yamada; Naomi Kondo

2006-01-01

208

Proteomic Characterization of Novel Alternative Splice Variant Proteins in HER2/neu-induced Breast Cancers  

PubMed Central

Multifaceted alternative splicing in cancer cells greatly diversifies protein structure independently of genome changes, but characterization of cancer-associated splice variants is quite limited. In this study, we used mass spectrometric data to interrogate a custom-built database created with three-frame translations of mRNA sequences from Ensembl and ECgene to find alternative splice variant proteins. In mass spectrometric files from LC-MS/MS analyses of normal mouse mammary glands or mammary tumors derived from MMTV-Her-2/neu transgenic mice, we identified a total of 608 alternative splice variants, of which peptides from 216 proteins were found only in the tumor sample. Among the 608 splice variants were 68 novel proteins that were not completely matched to any known protein sequence in mice, for which we found known functional motifs. Biological process enrichment analysis of the splice variants identified suggested involvement of these proteins especially in cell motility and translation initiation. The cancer-associated differentially-expressed splice variant proteins offer novel biomarker candidates that may function in breast cancer progression or metastasis.

Menon, Rajasree; Omenn, Gilbert S.

2010-01-01

209

Comprehensive exon array data processing method for quantitative analysis of alternative spliced variants  

PubMed Central

Alternative splicing of pre-mRNA generates protein diversity. Dysfunction of splicing machinery and expression of specific transcripts has been linked to cancer progression and drug response. Exon microarray technology enables genome-wide quantification of expression levels of the majority of exons and facilitates the discovery of alternative splicing events. Analysis of exon array data is more challenging than the analysis of gene expression data and there is a need for reliable quantification of exons and alternatively spliced variants. We introduce a novel, computationally efficient methodology, Multiple Exon Array Preprocessing (MEAP), for exon array data pre-processing, analysis and visualization. We compared MEAP with existing pre-processing methods, and validation of six exons and two alternatively spliced variants with qPCR corroborated MEAP expression estimates. Analysis of exon array data from head and neck squamous cell carcinoma (HNSCC) cell lines revealed several transcripts associated with 11q13 amplification, which is related with decreased survival and metastasis in HNSCC patients. Our results demonstrate that MEAP produces reliable expression values at exon, alternatively spliced variant and gene levels, which allows generating novel experimentally testable predictions.

Chen, Ping; Lepikhova, Tatiana; Hu, Yizhou; Monni, Outi; Hautaniemi, Sampsa

2011-01-01

210

Expanding the action of duplex RNAs into the nucleus: redirecting alternative splicing.  

PubMed

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

Liu, Jing; Hu, Jiaxin; Corey, David R

2011-09-23

211

Genome-Wide Analysis of Alternative Splicing during Dendritic Cell Response to a Bacterial Challenge  

PubMed Central

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.

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

2013-01-01

212

Proteins Associated with the Exon Junction Complex Also Control the Alternative Splicing of Apoptotic Regulators  

PubMed Central

Several apoptotic regulators, including Bcl-x, are alternatively spliced to produce isoforms with opposite functions. We have used an RNA interference strategy to map the regulatory landscape controlling the expression of the Bcl-x splice variants in human cells. Depleting proteins known as core (Y14 and eIF4A3) or auxiliary (RNPS1, Acinus, and SAP18) components of the exon junction complex (EJC) improved the production of the proapoptotic Bcl-xS splice variant. This effect was not seen when we depleted EJC proteins that typically participate in mRNA export (UAP56, Aly/Ref, and TAP) or that associate with the EJC to enforce nonsense-mediated RNA decay (MNL51, Upf1, Upf2, and Upf3b). Core and auxiliary EJC components modulated Bcl-x splicing through different cis-acting elements, further suggesting that this activity is distinct from the established EJC function. In support of a direct role in splicing control, recombinant eIF4A3, Y14, and Magoh proteins associated preferentially with the endogenous Bcl-x pre-mRNA, interacted with a model Bcl-x pre-mRNA in early splicing complexes, and specifically shifted Bcl-x alternative splicing in nuclear extracts. Finally, the depletion of Y14, eIF4A3, RNPS1, SAP18, and Acinus also encouraged the production of other proapoptotic splice variants, suggesting that EJC-associated components are important regulators of apoptosis acting at the alternative splicing level.

Michelle, Laetitia; Cloutier, Alexandre; Toutant, Johanne; Shkreta, Lulzim; Thibault, Philippe; Durand, Mathieu; Garneau, Daniel; Gendron, Daniel; Lapointe, Elvy; Couture, Sonia; Le Hir, Herve; Klinck, Roscoe; Elela, Sherif Abou; Prinos, Panagiotis

2012-01-01

213

U2AF-homology motif interactions are required for alternative splicing regulation by SPF45.  

PubMed

The U2AF-homology motif (UHM) mediates protein-protein interactions between factors involved in constitutive RNA splicing. Here we report that the splicing factor SPF45 regulates alternative splicing of the apoptosis regulatory gene FAS (also called CD95). The SPF45 UHM is necessary for this activity and binds UHM-ligand motifs (ULMs) present in the 3' splice site-recognizing factors U2AF65, SF1 and SF3b155. We describe a 2.1-A crystal structure of SPF45-UHM in complex with a ULM peptide from SF3b155. Features distinct from those of previously described UHM-ULM structures allowed the design of mutations in the SPF45 UHM that selectively impair binding to individual ULMs. Splicing assays using the ULM-selective SPF45 variants demonstrate that individual UHM-ULM interactions are required for FAS splicing regulation by SPF45 in vivo. Our data suggest that networks of UHM-ULM interactions are involved in regulating alternative splicing. PMID:17589525

Corsini, Lorenzo; Bonnal, Sophie; Bonna, Sophie; Basquin, Jerome; Hothorn, Michael; Scheffzek, Klaus; Valcárcel, Juan; Sattler, Michael

2007-06-24

214

Alternatively spliced lysyl oxidase-like 4 isoforms have a pro-metastatic role in cancer.  

PubMed

We previously found LOXL4 to be alternatively spliced in an anatomic site-specific manner in tumors involving the serosal cavities. LOXL4 splice variants were predominantly or exclusively expressed in effusion specimens from ovarian and breast carcinoma patients, and were absent in primary carcinomas. In the present study, LOXL4 full-length or splice variants were overexpressed in ES-2 and MDA-MB-231 cells and their invasive and metastatic potential and microRNA expression profile were evaluated. ES-2 cells were further injected into SCID mice ovaries and the extent of tumor progression and metastases formation were compared. We show that both splice variants have a positive effect on the metastatic potential of cells in vitro and on tumor progression in vivo. In contrast, full-length LOXL4 is not pro-metastatic, and may even be considered as a tumor suppressor. In addition, we show that LOXL4 is a possible splicing target of the oncogenic splicing factors SRSF1 and hnRNP A1. In conclusion, our results point to a significant role for LOXL4 alternative splicing in tumor progression. PMID:22806361

Sebban, Shulamit; Golan-Gerstl, Regina; Karni, Rotem; Vaksman, Olga; Davidson, Ben; Reich, Reuven

2012-07-18

215

Alternative Splicing of the Amelogenin Gene in a Caudate Amphibian, Plethodoncinereus  

PubMed Central

As the major enamel matrix protein contributing to tooth development, amelogenin has been demonstrated to play a crucial role in tooth enamel formation. Previous studies have revealed amelogenin alternative splicing as a mechanism for amelogenin heterogeneous expression in mammals. While amelogenin and its splicing forms in mammalian vertebrates have been characterized, splicing variants of amelogenin gene still remains largely unknown in non-mammalian species. Here, using PCR and sequence analysis we discovered two novel amelogenin transcript variants in tooth organ extracts from a caudate amphibian, the salamander Plethodoncinereus. The one was shorter -S- (416 nucleotides including untranslated regions, 5 exons) and the other larger -L- (851 nt, 7 exons) than the previously published “normal” gene in this species -M- (812 nucleotides, 6 exons). This is the first report demonstrating the amelogenin alternative splicing in amphibian, revealing a unique exon 2b and two novel amelogenin gene transcripts in Plethodoncinereus.

Wang, Xinping; Xing, Zeli; Zhang, Xichen; Zhu, Lisai; Diekwisch, Thomas G. H.

2013-01-01

216

SPACE: an algorithm to predict and quantify alternatively spliced isoforms using microarrays  

PubMed Central

Exon and exon+junction microarrays are promising tools for studying alternative splicing. Current analytical tools applied to these arrays lack two relevant features: the ability to predict unknown spliced forms and the ability to quantify the concentration of known and unknown isoforms. SPACE is an algorithm that has been developed to (1) estimate the number of different transcripts expressed under several conditions, (2) predict the precursor mRNA splicing structure and (3) quantify the transcript concentrations including unknown forms. The results presented here show its robustness and accuracy for real and simulated data.

Anton, Miguel A; Gorostiaga, Dorleta; Guruceaga, Elizabeth; Segura, Victor; Carmona-Saez, Pedro; Pascual-Montano, Alberto; Pio, Ruben; Montuenga, Luis M; Rubio, Angel

2008-01-01

217

A mutation in a rare type of intron in a sodium-channel gene results in aberrant splicing and causes myotonia.  

PubMed

Many mutations in the skeletal-muscle sodium-channel gene SCN4A have been associated with myotonia and/or periodic paralysis, but so far all of these mutations are located in exons. We found a patient with myotonia caused by a deletion/insertion located in intron 21 of SCN4A, which is an AT-AC type II intron. This is a rare class of introns that, despite having AT-AC boundaries, are spliced by the major or U2-type spliceosome. The patient's skeletal muscle expressed aberrantly spliced SCN4A mRNA isoforms generated by activation of cryptic splice sites. In addition, genetic suppression experiments using an SCN4A minigene showed that the mutant 5' splice site has impaired binding to the U1 and U6 snRNPs, which are the cognate factors for recognition of U2-type 5' splice sites. One of the aberrantly spliced isoforms encodes a channel with a 35-amino acid insertion in the cytoplasmic loop between domains III and IV of Nav1.4. The mutant channel exhibited a marked disruption of fast inactivation, and a simulation in silico showed that the channel defect is consistent with the patient's myotonic symptoms. This is the first report of a disease-associated mutation in an AT-AC type II intron, and also the first intronic mutation in a voltage-gated ion channel gene showing a gain-of-function defect. PMID:21412952

Kubota, Tomoya; Roca, Xavier; Kimura, Takashi; Kokunai, Yosuke; Nishino, Ichizo; Sakoda, Saburo; Krainer, Adrian R; Takahashi, Masanori P

2011-04-28

218

Aberrant splicing and transcription termination caused by P element insertion into the intron of a Drosophila gene  

SciTech Connect

Insertional mutagenesis screens using the P[lacZ, rosy{sup +}] (PZ) transposable element have provided thousands of mutant lines for analyzing genes of varied function in the fruitfly, Drosophila melanogaster. As has been observed with other P elements, many of the PZ-induced mutations result from insertion of the P element into the promoter or 5{prime} untranslated regions of the affected gene. We document here a novel mechanism for mutagenesis by this element. We show that sequences present within the element direct aberrant splicing and termination events that produce an mRNA composed of 5{prime} sequences from the mutated gene (in this case, pipsqueak) and 3{prime} sequences from within the P[lacZ, rosy{sup +}] element. These truncated RNAs could yield proteins with dominant mutant effects. 43 refs., 4 figs.

Horowitz, H.; Berg, C.A. [Univ. of Washington, Seattle, WA (United States)

1995-01-01

219

Alternative pre-mRNA splicing and proteome expansion in metazoans  

Microsoft Academic Search

The protein coding sequences of most eukaryotic messenger RNA precursors (pre-mRNAs) are interrupted by non-coding sequences called introns. Pre-mRNA splicing is the process by which introns are removed and the protein coding elements assembled into mature mRNAs. Alternative pre-mRNA splicing selectively joins different protein coding elements to form mRNAs that encode proteins with distinct functions, and is therefore an important

Tom Maniatis; Bosiljka Tasic

2002-01-01

220

Anatomic Localization of Alternatively Spliced Leptin Receptors (Ob-R) in Mouse Brain and other Tissues  

Microsoft Academic Search

Leptin's effects are mediated by interactions with a receptor that is alternatively spliced, resulting in at least five different murine forms: Ob-Ra, Ob-Rb, Ob-Rc, Ob-Rd, and Ob-Re. A mutation in one splice form, Ob-Rb, results in obesity in mice. Northern blots, RNase protection assays, and PCR indicate that Ob-Rb is expressed at a relatively high level in hypothalamus and low

Hong Fei; Hirotaka J. Okano; Cai Li; Gwo-Hwa Lee; Connie Zhao; Robert Darnell; Jeffrey M. Friedman

1997-01-01

221

Revealing Global Regulatory Features of Mammalian Alternative Splicing Using a Quantitative Microarray Platform  

Microsoft Academic Search

We describe the application of a microarray platform, which combines information from exon body and splice-junction probes, to perform a quantitative analysis of tissue-specific alternative splicing (AS) for thousands of exons in mammalian cells. Through this system, we have analyzed global features of AS in major mouse tissues. The results provide numerous inferences for the functions of tissue-specific AS, insights

Qun Pan; Ofer Shai; Christine Misquitta; Wen Zhang; Arneet L. Saltzman; Naveed Mohammad; Tomas Babak; Henry Siu; Timothy R. Hughes; Quaid D. Morris; Brendan J. Frey; Benjamin J. Blencowe

2004-01-01

222

Identification of alternatively spliced Act1 and implications for its roles in oncogenesis  

Microsoft Academic Search

Act1 (also called CIKS) is a recently identified molecule, which activates NF-?B and AP-1. Here, we identified alternatively spliced Act1 that lacked the exon 2 encoding the first nine amino acids in the amino terminus of the protein. Compared to full-length Act1, this truncated Act1 appeared to be equally active. We demonstrated further that only the spliced Act1 was detected

Yi-Feng Xia; Yi-Dan Li; Xiaoxia Li; Jian-Guo Geng

2002-01-01

223

Comprehensive Analysis of Alternative Splicing and Functionality in Neuronal Differentiation of P19 Cells  

PubMed Central

Background Alternative splicing, which produces multiple mRNAs from a single gene, occurs in most human genes and contributes to protein diversity. Many alternative isoforms are expressed in a spatio-temporal manner, and function in diverse processes, including in the neural system. Methodology/Principal Findings The purpose of the present study was to comprehensively investigate neural-splicing using P19 cells. GeneChip Exon Array analysis was performed using total RNAs purified from cells during neuronal cell differentiation. To efficiently and readily extract the alternative exon candidates, 9 filtering conditions were prepared, yielding 262 candidate exons (236 genes). Semiquantitative RT-PCR results in 30 randomly selected candidates suggested that 87% of the candidates were differentially alternatively spliced in neuronal cells compared to undifferentiated cells. Gene ontology and pathway analyses suggested that many of the candidate genes were associated with neural events. Together with 66 genes whose functions in neural cells or organs were reported previously, 47 candidate genes were found to be linked to 189 events in the gene-level profile of neural differentiation. By text-mining for the alternative isoform, distinct functions of the isoforms of 9 candidate genes indicated by the result of Exon Array were confirmed. Conclusions/Significance Alternative exons were successfully extracted. Results from the informatics analyses suggested that neural events were primarily governed by genes whose expression was increased and whose transcripts were differentially alternatively spliced in the neuronal cells. In addition to known functions in neural cells or organs, the uninvestigated alternative splicing events of 11 genes among 47 candidate genes suggested that cell cycle events are also potentially important. These genes may help researchers to differentiate the roles of alternative splicing in cell differentiation and cell proliferation.

Suzuki, Hitoshi; Osaki, Ken; Sano, Kaori; Alam, A. H. M. Khurshid; Nakamura, Yuichiro; Ishigaki, Yasuhito; Kawahara, Kozo; Tsukahara, Toshifumi

2011-01-01

224

Emetine regulates the alternative splicing of caspase 9 in tumor cells.  

PubMed

Exons 3 to 6 in the caspase 9 gene undergo alternative splicing in which the larger caspase 9 splice variant promotes apoptosis, in contrast to the dominant negative anti-apoptotic splice variant, the smaller caspase 9b. In this study, the regulation of the alternative splicing of caspase 9 pre-mRNA was examined in response to Emetine. Treatment of C33A cells, breast cancer MCF-7 cells and MCF-7/Adr cells with Emetine dihydrochloride upregulated the level of smaller caspase 9b mRNA and concomitantly decreased the mRNA level of larger caspase 9 in a dose- and time-dependent manner, indicating that Emetine desensitizes C33A, MCF-7 and MCF-7/Adr to cell death. In contrast, treatment of PC3 cells, a prostate cancer cell line, manifested an opposite effect: a greater production of the larger caspase 9 mRNA with a concomitant decrease of caspase 9b mRNA. Pretreatment with calyculin A, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) blocked Emetine-induced alternative splicing in cells, in contrast to okadaic acid, a specific inhibitor of PP2A, demonstrating a PP1-mediated mechanism. These results suggest that the various splicing patterns of the caspase 9 gene that are regulated by chemotherapy reagents may contribute to the resistance or sensitization of the tumors to other cell death inducers. PMID:22848307

Pan, Danmin; Boon-Unge, Kritsanapol; Govitrapong, Piyarat; Zhou, Jianhua

2011-08-29

225

Alternative pre-mRNA splicing switches modulate gene expression in late erythropoiesis  

SciTech Connect

Differentiating erythroid cells execute a unique gene expression program that insures synthesis of the appropriate proteome at each stage of maturation. Standard expression microarrays provide important insight into erythroid gene expression but cannot detect qualitative changes in transcript structure, mediated by RNA processing, that alter structure and function of encoded proteins. We analyzed stage-specific changes in the late erythroid transcriptome via use of high-resolution microarrays that detect altered expression of individual exons. Ten differentiation-associated changes in erythroblast splicing patterns were identified, including the previously known activation of protein 4.1R exon 16 splicing. Six new alternative splicing switches involving enhanced inclusion of internal cassette exons were discovered, as well as 3 changes in use of alternative first exons. All of these erythroid stage-specific splicing events represent activated inclusion of authentic annotated exons, suggesting they represent an active regulatory process rather than a general loss of splicing fidelity. The observation that 3 of the regulated transcripts encode RNA binding proteins (SNRP70, HNRPLL, MBNL2) may indicate significant changes in the RNA processing machinery of late erythroblasts. Together, these results support the existence of a regulated alternative pre-mRNA splicing program that is critical for late erythroid differentiation.

Yamamoto, Miki L.; Clark, Tyson A.; Gee, Sherry L.; Kang, Jeong-Ah; Schweitzer, Anthony C.; Wickrema, Amittha; Conboy, John G.

2009-02-03

226

The polypyrimidine tract binding protein regulates desaturase alternative splicing and PUFA composition.  

PubMed

The ?6 desaturase, encoded by FADS2, plays a crucial role in omega-3 and omega-6 fatty acid synthesis. These fatty acids are essential components of the central nervous system, and they act as precursors for eicosanoid signaling molecules and as direct modulators of gene expression. The polypyrimidine tract binding protein (PTB or hnRNP I) is a splicing factor that regulates alternative pre-mRNA splicing. Here, PTB is shown to bind an exonic splicing silencer element and repress alternative splicing of FADS2 into FADS2 AT1. PTB and FADS2AT1 were inversely correlated in neonatal baboon tissues, implicating PTB as a major regulator of tissue-specific FADS2 splicing. In HepG2 cells, PTB knockdown modulated alternative splicing of FADS2, as well as FADS3, a putative desaturase of unknown function. Omega-3 fatty acids decreased by nearly one half relative to omega-6 fatty acids in PTB knockdown cells compared with controls, with a particularly strong decrease in eicosapentaenoic acid (EPA) concentration and its ratio to arachidonic acid (ARA). This is a rare demonstration of a mechanism specifically altering the cellular omega-3 to omega-6 fatty acid ratio without any change in diet/media. These findings reveal a novel role for PTB, regulating availability of membrane components and eicosanoid precursors for cell signaling. PMID:21980057

Reardon, Holly T; Park, Woo Jung; Zhang, Jimmy; Lawrence, Peter; Kothapalli, Kumar S D; Brenna, J Thomas

2011-10-06

227

Presynaptic neurexin-3 alternative splicing trans-synaptically controls postsynaptic AMPA receptor trafficking.  

PubMed

Neurexins are essential presynaptic cell adhesion molecules that are linked to schizophrenia and autism and are subject to extensive alternative splicing. Here, we used a genetic approach to test the physiological significance of neurexin alternative splicing. We generated knockin mice in which alternatively spliced sequence #4 (SS4) of neuexin-3 is constitutively included but can be selectively excised by cre-recombination. SS4 of neurexin-3 was chosen because it is highly regulated and controls neurexin binding to neuroligins, LRRTMs, and other ligands. Unexpectedly, constitutive inclusion of SS4 in presynaptic neurexin-3 decreased postsynaptic AMPA, but not NMDA receptor levels, and enhanced postsynaptic AMPA receptor endocytosis. Moreover, constitutive inclusion of SS4 in presynaptic neurexin-3 abrogated postsynaptic AMPA receptor recruitment during NMDA receptor-dependent LTP. These phenotypes were fully rescued by constitutive excision of SS4 in neurexin-3. Thus, alternative splicing of presynaptic neurexin-3 controls postsynaptic AMPA receptor trafficking, revealing an unanticipated alternative splicing mechanism for trans-synaptic regulation of synaptic strength and long-term plasticity. PMID:23827676

Aoto, Jason; Martinelli, David C; Malenka, Robert C; Tabuchi, Katsuhiko; Südhof, Thomas C

2013-07-01

228

MBNL proteins repress ES-cell-specific alternative splicing and reprogramming.  

PubMed

Previous investigations of the core gene regulatory circuitry that controls the pluripotency of embryonic stem (ES) cells have largely focused on the roles of transcription, chromatin and non-coding RNA regulators. Alternative splicing represents a widely acting mode of gene regulation, yet its role in regulating ES-cell pluripotency and differentiation is poorly understood. Here we identify the muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved and direct negative regulators of a large program of cassette exon alternative splicing events that are differentially regulated between ES cells and other cell types. Knockdown of MBNL proteins in differentiated cells causes switching to an ES-cell-like alternative splicing pattern for approximately half of these events, whereas overexpression of MBNL proteins in ES cells promotes differentiated-cell-like alternative splicing patterns. Among the MBNL-regulated events is an ES-cell-specific alternative splicing switch in the forkhead family transcription factor FOXP1 that controls pluripotency. Consistent with a central and negative regulatory role for MBNL proteins in pluripotency, their knockdown significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells during somatic cell reprogramming. PMID:23739326

Han, Hong; Irimia, Manuel; Ross, P Joel; Sung, Hoon-Ki; Alipanahi, Babak; David, Laurent; Golipour, Azadeh; Gabut, Mathieu; Michael, Iacovos P; Nachman, Emil N; Wang, Eric; Trcka, Dan; Thompson, Tadeo; O'Hanlon, Dave; Slobodeniuc, Valentina; Barbosa-Morais, Nuno L; Burge, Christopher B; Moffat, Jason; Frey, Brendan J; Nagy, Andras; Ellis, James; Wrana, Jeffrey L; Blencowe, Benjamin J

2013-06-05

229

Temperature-modulated Alternative Splicing and Promoter Use in the Circadian Clock Gene frequencyD?  

PubMed Central

The expression of FREQUENCY, a central component of the circadian clock in Neurospora crassa, shows daily cycles that are exquisitely sensitive to the environment. Two forms of FRQ that differ in length by 99 amino acids, LFRQ and SFRQ, are synthesized from alternative initiation codons and the change in their ratio as a function of temperature contributes to robust rhythmicity across a range of temperatures. We have found frq expression to be surprisingly complex, despite our earlier prediction of a simple transcription unit based on limited cDNA sequencing. Two distinct environmentally regulated major promoters drive primary transcripts whose environmentally influenced alternative splicing gives rise to six different major mRNA species as well as minor forms. Temperature-sensitive alternative splicing determines AUG choice and, as a consequence, the ratio of LFRQ to SFRQ. Four of the six upstream ORFs are spliced out of the vast majority of frq mRNA species. Alternative splice site choice in the 5? UTR and relative use of two major promoters are also influenced by temperature, and the two promoters are differentially regulated by light. Evolutionary comparisons with the Sordariaceae reveal conservation of 5? UTR sequences, as well as significant conservation of the alternative splicing events, supporting their relevance to proper regulation of clock function.

Colot, Hildur V.; Loros, Jennifer J.; Dunlap, Jay C.

2005-01-01

230

Species-specific regulation of alternative splicing in the C-terminal region of the p53 tumor suppressor gene  

PubMed Central

Alternative splicing occurs in the C-terminal region of the p53 tumor suppressor gene between two alternative 3? splice sites in intron 10. This alternative splicing event has been detected in murine cells, but not in rat or human tissues. In this paper, we have characterized the pattern of p53 alternative splicing in cell lines from five different species. Our results confirm that p53 alternative splicing is species-specific, being detected only in cell lines of rodent origin. Using transient transfection assays, we have established that the rat p53 gene undergoes efficient alternative splicing in both mouse and rat cell lines, thus demonstrating that it has all the necessary cis-acting sequences to be alternatively spliced. In contrast, we were unable to detect any usage of the human alternative 3? splice site under the same experimental conditions. Thus, the low levels or absence of alternatively spliced p53 mRNA in rat and human cell lines seems to be the result of different mechanisms. Our results support the hypothesis that there are species-specific mechanisms implicated in the regulation of p53 activity.

Laverdiere, M.; Beaudoin, J.; Lavigueur, Alain

2000-01-01

231

Internal Polyadenylation of the Parvovirus B19 Precursor mRNA Is Regulated by Alternative Splicing*  

PubMed Central

Alternative processing of parvovirus B19 (B19V) pre-mRNA is critical to generating appropriate levels of B19V mRNA transcripts encoding capsid proteins and small nonstructural proteins. Polyadenylation of the B19V pre-mRNA at the proximal polyadenylation site ((pA)p), which prevents generation of full-length capsid proteins encoding mRNA transcripts, has been suggested as a step that blocks B19V permissiveness. We report here that efficient splicing of the B19V pre-mRNA within the first intron (upstream of the (pA)p site) stimulated the polyadenylation; in contrast, splicing of the B19V pre-mRNA within the second intron (in which the (pA)p site resides) interfered with the polyadenylation, leading to the generation of a sufficient number of B19V mRNA transcripts polyadenylated at the distal polyadenylation site ((pA)d). We also found that splicing within the second intron and polyadenylation at the (pA)p site compete during processing of the B19V pre-mRNA. Furthermore, we discovered that the U1 RNA that binds to the 5? splice donor site of the second intron is fully responsible for inhibiting polyadenylation at the (pA)p site, whereas actual splicing, and perhaps assembly of the functional spliceosome, is not required. Finally, we demonstrated that inhibition of B19V pre-mRNA splicing within the second intron by targeting an intronic splicing enhancer using a Morpholino antisense oligonucleotide prevented B19V mRNA transcripts polyadenylated at the (pA)d site during B19V infection of human erythroid progenitors. Thus, our study reveals the mechanism by which alternative splicing coordinates alternative polyadenylation to generate full-length B19V mRNA transcripts at levels sufficient to support productive B19V infection.

Guan, Wuxiang; Huang, Qinfeng; Cheng, Fang; Qiu, Jianming

2011-01-01

232

The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation.  

PubMed

Alternative splicing (AS) of pre-mRNA is utilized by higher eukaryotes to achieve increased transcriptome and proteomic complexity. The serine/arginine (SR) splicing factors regulate tissue- or cell-type-specific AS in a concentration- and phosphorylation-dependent manner. However, the mechanisms that modulate the cellular levels of active SR proteins remain to be elucidated. In the present study, we provide evidence for a role for the long nuclear-retained regulatory RNA (nrRNA), MALAT1 in AS regulation. MALAT1 interacts with SR proteins and influences the distribution of these and other splicing factors in nuclear speckle domains. Depletion of MALAT1 or overexpression of an SR protein changes the AS of a similar set of endogenous pre-mRNAs. Furthermore, MALAT1 regulates cellular levels of phosphorylated forms of SR proteins. Taken together, our results suggest that MALAT1 regulates AS by modulating the levels of active SR proteins. Our results further highlight the role for an nrRNA in the regulation of gene expression. PMID:20797886

Tripathi, Vidisha; Ellis, Jonathan D; Shen, Zhen; Song, David Y; Pan, Qun; Watt, Andrew T; Freier, Susan M; Bennett, C Frank; Sharma, Alok; Bubulya, Paula A; Blencowe, Benjamin J; Prasanth, Supriya G; Prasanth, Kannanganattu V

2010-09-24

233

Alternative splicing modulation by a LAMMER kinase impinges on developmental and transcriptome expression.  

PubMed

Alternative splicing is a major contributor to genome complexity, playing a significant role in various cellular functions, including signal transduction, immunity, and development. The spliceosomal machinery is responsible for the processing of nuclear RNA. Several splicing factors associated with this complex are phosphorylated by kinases that possess a conserved LAMMER motif. We demonstrate in BY-2 tobacco cells a novel role for the LAMMER motif in the maintenance of proper subnuclear localization. Furthermore, high expression of the LAMMER kinase in Arabidopsis plants modulated the alternative splicing of specific endogenous genes and resulted in abnormal plant development and a novel transcriptome profile. A prominent feature was the upregulation of genes that play a role in protein turnover, suggesting a moderating function for these gene products in the control of alternative splicing events. Together, these results demonstrate alternative splicing modulation as a result of phosphorylation activity, providing an opportunity to study its global effect on the plasticity of plant development and gene expression at the organism level. PMID:12671088

Savaldi-Goldstein, Sigal; Aviv, Dvora; Davydov, Olga; Fluhr, Robert

2003-04-01

234

SKIP is a component of the spliceosome linking alternative splicing and the circadian clock in Arabidopsis.  

PubMed

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

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

2012-08-31

235

Computational Analysis of an Evolutionarily Conserved VertebrateMuscle Alternative Splicing Program  

SciTech Connect

A novel exon microarray format that probes gene expression with single exon resolution was employed to elucidate critical features of a vertebrate muscle alternative splicing program. A dataset of 56 microarray-defined, muscle-enriched exons and their flanking introns were examined computationally in order to investigate coordination of the muscle splicing program. Candidate intron regulatory motifs were required to meet several stringent criteria: significant over-representation near muscle-enriched exons, correlation with muscle expression, and phylogenetic conservation among genomes of several vertebrate orders. Three classes of regulatory motifs were identified in the proximal downstream intron, within 200nt of the target exons: UGCAUG, a specific binding site for Fox-1 related splicing factors; ACUAAC, a novel branchpoint-like element; and UG-/UGC-rich elements characteristic of binding sites for CELF splicing factors. UGCAUG was remarkably enriched, being present in nearly one-half of all cases. These studies suggest that Fox and CELF splicing factors play a major role in enforcing the muscle-specific alternative splicing program, facilitating expression of a set of unique isoforms of cytoskeletal proteins that are critical to muscle cell differentiation. Supplementary materials: There are four supplementary tables and one supplementary figure. The tables provide additional detailed information concerning the muscle-enriched datasets, and about over-represented oligonucleotide sequences in the flanking introns. The supplementary figure shows RT-PCR data confirming the muscle-enriched expression of exons predicted from the microarray analysis.

Das, Debopriya; Clark, Tyson A.; Schweitzer, Anthony; Marr,Henry; Yamamoto, Miki L.; Parra, Marilyn K.; Arribere, Josh; Minovitsky,Simon; Dubchak, Inna; Blume, John E.; Conboy, John G.

2006-06-15

236

Dynamic regulation of alternative splicing and chromatin structure in Drosophila gonads revealed by RNA-seq.  

PubMed

Both transcription and post-transcriptional processes, such as alternative splicing, play crucial roles in controlling developmental programs in metazoans. Recently emerged RNA-seq method has brought our understanding of eukaryotic transcriptomes to a new level, because it can resolve both gene expression level and alternative splicing events simultaneously. To gain a better understanding of cellular differentiation in gonads, we analyzed mRNA profiles from Drosophila testes and ovaries using RNA-seq. We identified a set of genes that have sex-specific isoforms in wild-type (WT) gonads, including several transcription factors. We found that differentiation of sperms from undifferentiated germ cells induced a dramatic downregulation of RNA splicing factors. Our data confirmed that RNA splicing events are significantly more frequent in the undifferentiated cell-enriched bag of marbles (bam) mutant testis, but downregulated upon differentiation in WT testis. Consistent with this, we showed that genes required for meiosis and terminal differentiation in WT testis were mainly regulated at the transcriptional level, but not by alternative splicing. Unexpectedly, we observed an increase in expression of all families of chromatin remodeling factors and histone modifying enzymes in the undifferentiated cell-enriched bam testis. More interestingly, chromatin regulators and histone modifying enzymes with opposite enzymatic activities are coenriched in undifferentiated cells in testis, suggesting that these cells may possess dynamic chromatin architecture. Finally, our data revealed many new features of the Drosophila gonadal transcriptomes, and will lead to a more comprehensive understanding of how differential gene expression and splicing regulate gametogenesis in Drosophila. Our data provided a foundation for the systematic study of gene expression and alternative splicing in many interesting areas of germ cell biology in Drosophila, such as the molecular basis for sexual dimorphism and the regulation of the proliferation vs terminal differentiation programs in germline stem cell lineages. The GEO accession number for the raw and analyzed RNA-seq data is GSE16960. PMID:20440302

Gan, Qiang; Chepelev, Iouri; Wei, Gang; Tarayrah, Lama; Cui, Kairong; Zhao, Keji; Chen, Xin

2010-05-04

237

Sex Determination in Insects: a binary decision based on alternative splicing  

PubMed Central

The gene regulatory networks that control sex determination vary between species. Despite these differences, comparative studies in insects have found that alternative splicing is reiteratively used in evolution to control expression of the key sex determining genes. Sex determination is best understood in Drosophila where activation of the RNA binding protein encoding gene Sex-lethal is the central female-determining event. Sex-lethal serves as a genetic switch because once activated it controls its own expression by a positive feedback splicing mechanism. Sex fate choice in is also maintained by self-sustaining positive feedback splicing mechanisms in other dipteran and hymenopteran insects, although different RNA binding protein encoding genes function as the binary switch. Studies exploring the mechanisms of sex-specific splicing have revealed the extent to which sex determination is integrated with other developmental regulatory networks.

Salz, Helen K.

2011-01-01

238

Fine-Scale Variation and Genetic Determinants of Alternative Splicing across Individuals  

PubMed Central

Recently, thanks to the increasing throughput of new technologies, we have begun to explore the full extent of alternative pre–mRNA splicing (AS) in the human transcriptome. This is unveiling a vast layer of complexity in isoform-level expression differences between individuals. We used previously published splicing sensitive microarray data from lymphoblastoid cell lines to conduct an in-depth analysis on splicing efficiency of known and predicted exons. By combining publicly available AS annotation with a novel algorithm designed to search for AS, we show that many real AS events can be detected within the usually unexploited, speculative majority of the array and at significance levels much below standard multiple-testing thresholds, demonstrating that the extent of cis-regulated differential splicing between individuals is potentially far greater than previously reported. Specifically, many genes show subtle but significant genetically controlled differences in splice-site usage. PCR validation shows that 42 out of 58 (72%) candidate gene regions undergo detectable AS, amounting to the largest scale validation of isoform eQTLs to date. Targeted sequencing revealed a likely causative SNP in most validated cases. In all 17 incidences where a SNP affected a splice-site region, in silico splice-site strength modeling correctly predicted the direction of the micro-array and PCR results. In 13 other cases, we identified likely causative SNPs disrupting predicted splicing enhancers. Using Fst and REHH analysis, we uncovered significant evidence that 2 putative causative SNPs have undergone recent positive selection. We verified the effect of five SNPs using in vivo minigene assays. This study shows that splicing differences between individuals, including quantitative differences in isoform ratios, are frequent in human populations and that causative SNPs can be identified using in silico predictions. Several cases affected disease-relevant genes and it is likely some of these differences are involved in phenotypic diversity and susceptibility to complex diseases.

Coulombe-Huntington, Jasmin; Lam, Kevin C. L.; Dias, Christel; Majewski, Jacek

2009-01-01

239

Emerging Roles of Alternative Pre-mRNA Splicing Regulation in Neuronal Development and Function  

PubMed Central

Alternative pre-mRNA splicing has the potential to greatly diversify the repertoire of transcripts in multicellular organisms. Increasing evidence suggests that this expansive layer of gene regulation plays a particularly important role in the development and function of the nervous system, one of the most complex organ systems found in nature. In this review, we highlight recent studies that continue to emphasize the influence and contribution of alternative splicing regulation to various aspects of neuronal development in addition to its role in the mature nervous system.

Norris, Adam D.; Calarco, John A.

2012-01-01

240

Regulation of alternative splicing by short non-coding nuclear RNAs  

PubMed Central

Recent results from deep-sequencing and tiling array studies indicated the existence of a large number of short, metabolically stable, non-coding RNAs. Some of these short RNAs derive from known RNA classes like snoRNA or tRNAs. There are intriguing similarities between short non-coding nuclear RNAs and oligonucleotides used to change alternative splicing events, which usually target a disease-relevant RNA. We review the current knowledge of this emerging class of RNAs and discuss evidence that some of these short RNAs could function in alternative splice site selection.

Khanna, Amit

2010-01-01

241

Visualization and genetic analysis of alternative splicing regulation in vivo using fluorescence reporters in transgenic Caenorhabditis elegans  

Microsoft Academic Search

Transgenic multicolor fluorescence reporters enable the visualization of alternative splicing patterns at a single-cell resolution in living organisms and facilitate further genetic analyses to identify cis-elements and trans-acting factors involved in splicing regulation. In this paper, we describe a method of generating fluorescence alternative splicing reporters for the nematode Caenorhabditis elegans. We describe strategies for designing minigene reporters and methods

Genta Ohno; Hiroaki Sakane; Hiroyuki Maruoka; Hidehito Kuroyanagi; Masatoshi Hagiwara

2010-01-01

242

Genome-wide detection of condition-sensitive alternative splicing in Arabidopsis roots.  

PubMed

Iron (Fe) deficiency is a world-wide nutritional disorder in both plants and humans, resulting from its restricted bioavailability for plants and, subsequently, low Fe concentration in edible plant parts. Plants have evolved sophisticated mechanisms to alleviate Fe deficiency, with the aim of recalibrating metabolic fluxes and maintaining cellular Fe homeostasis. To analyze condition-sensitive changes in precursor mRNA (pre-mRNA) splicing pattern, we mapped the transcriptome of Fe-deficient and Fe-sufficient Arabidopsis (Arabidopsis thaliana) roots using the RNA sequencing technology and a newly developed software toolbox, the Read Analysis & Comparison Kit in Java (RACKJ). In alternatively spliced genes, stress-related Gene Ontology categories were overrepresented, while housekeeping cellular functions were mainly transcriptionally controlled. Fe deficiency increased the complexity of the splicing pattern and triggered the differential alternative splicing of 313 genes, the majority of which had differentially retained introns. Several genes with important functions in Fe acquisition and homeostasis were both differentially expressed and differentially alternatively spliced upon Fe deficiency, indicating a complex regulation of gene activity in Fe-deficient conditions. A comparison with a data set for phosphate-deficient plants suggests that changes in splicing patterns are nutrient specific and not or not chiefly caused by stochastic fluctuations. In sum, our analysis identified extensive posttranscriptional control, biasing the abundance and activity of proteins in a condition-dependent manner. The production of a mixture of functional and nonfunctional transcripts may provide a means to fine-tune the abundance of transcripts with critical importance in cellular Fe homeostasis. It is assumed that differential gene expression and nutrient deficiency-induced changes in pre-mRNA splicing represent parallel, but potentially interacting, regulatory mechanisms. PMID:23735510

Li, Wenfeng; Lin, Wen-Dar; Ray, Prasun; Lan, Ping; Schmidt, Wolfgang

2013-06-04

243

Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle function  

PubMed Central

Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates. Thus, conservation of intronic Rbfox binding motifs is a good predictor of Rbfox-regulated alternative splicing. Morphology and development of single morphant embryos was strikingly normal; however, muscle development in double morphants was severely disrupted. Defects in cardiac muscle were marked by reduced heart rate and in skeletal muscle by complete paralysis. The predominance of wavy myofibers and abnormal thick and thin filaments in skeletal muscle revealed that myofibril assembly is defective and disorganized in double morphants. Ultra-structural analysis revealed that although sarcomeres with electron dense M- and Z-bands are present in muscle fibers of rbfox1l/rbox2 morphants, they are substantially reduced in number and alignment. Importantly, splicing changes and morphological defects were rescued by expression of morpholino-resistant rbfox cDNA. Additionally, a target-blocking MO complementary to a single UGCAUG motif adjacent to an rbfox target exon of fxr1 inhibited inclusion in a similar manner to rbfox knockdown, providing evidence that Rbfox regulates the splicing of target exons via direct binding to intronic regulatory motifs. We conclude that Rbfox proteins regulate an alternative splicing program essential for vertebrate heart and skeletal muscle function.

Gallagher, Thomas L.; Arribere, Joshua A.; Geurts, Paul A.; Exner, Cameron R. T.; McDonald, Kent L.; Dill, Kariena K.; Marr, Henry L.; Adkar, Shaunak S.; Garnett, Aaron T.; Amacher, Sharon L.; Conboy, John G.

2012-01-01

244

Genome-Wide Landscape of Alternative Splicing Events in Brachypodium distachyon  

PubMed Central

Recently, Brachypodium distachyon has emerged as a model plant for studying monocot grasses and cereal crops. Using assembled expressed transcript sequences and subsequent mapping to the corresponding genome, we identified 1219 alternative splicing (AS) events spanning across 2021 putatively assembled transcripts generated from 941 genes. Approximately, 6.3% of expressed genes are alternatively spliced in B. distachyon. We observed that a majority of the identified AS events were related to retained introns (55.5%), followed by alternative acceptor sites (16.7%). We also observed a low percentage of exon skipping (5.0%) and alternative donor site events (8.8%). The ‘complex event’ that consists of a combination of two or more basic splicing events accounted for ?14.0%. Comparative AS transcript analysis revealed 163 and 39 homologous pairs between B. distachyon and Oryza sativa and between B. distachyon and Arabidopsis thaliana, respectively. In all, we found 16 AS transcripts to be conserved in all 3 species. AS events and related putative assembled transcripts annotation can be systematically browsed at Plant Alternative Splicing Database (http://proteomics.ysu.edu/altsplice/plant/).

Walters, Braden; Lum, Gengkon; Sablok, Gaurav; Min, Xiang Jia

2013-01-01

245

Multiple transcripts of the murine immunoglobulin ? membrane locus are generated by alternative splicing and differential usage of two polyadenylation sites  

Microsoft Academic Search

The human C? gene produces a number of alternatively spliced heavy chain transcripts of which some encode functional IgE isoforms. We now show that differentially processed ? mRNA variants also exist in the mouse and are generated by differential polyadenylation and alternative splicing of primary ? chain transcripts. The two poly(A) sites of the mouse membrane transcripts were identified in

Shubha Anand; Facundo D. Batista; Tatiana Tkach; Dimitar G. Efremov; Oscar R. Burrone

1997-01-01

246

PQBP1, a factor linked to intellectual disability, affects alternative splicing associated with neurite outgrowth  

PubMed Central

Polyglutamine-binding protein 1 (PQBP1) is a highly conserved protein associated with neurodegenerative disorders. Here, we identify PQBP1 as an alternative messenger RNA (mRNA) splicing (AS) effector capable of influencing splicing of multiple mRNA targets. PQBP1 is associated with many splicing factors, including the key U2 small nuclear ribonucleoprotein (snRNP) component SF3B1 (subunit 1 of the splicing factor 3B [SF3B] protein complex). Loss of functional PQBP1 reduced SF3B1 substrate mRNA association and led to significant changes in AS patterns. Depletion of PQBP1 in primary mouse neurons reduced dendritic outgrowth and altered AS of mRNAs enriched for functions in neuron projection development. Disease-linked PQBP1 mutants were deficient in splicing factor associations and could not complement neurite outgrowth defects. Our results indicate that PQBP1 can affect the AS of multiple mRNAs and indicate specific affected targets whose splice site determination may contribute to the disease phenotype in PQBP1-linked neurological disorders.

Wang, Qingqing; Moore, Michael J.; Adelmant, Guillaume; Marto, Jarrod A.; Silver, Pamela A.

2013-01-01

247

Insulin Promotes Neuronal Survival via the Alternatively Spliced Protein Kinase C?II Isoform*  

PubMed Central

Insulin signaling pathways in the brain regulate food uptake and memory and learning. Insulin and protein kinase C (PKC) pathways are integrated and function closely together. PKC activation in the brain is essential for learning and neuronal repair. Intranasal delivery of insulin to the central nervous system (CNS) has been shown to improve memory, reduce cerebral atrophy, and reverse neurodegeneration. However, the neuronal molecular mechanisms of these effects have not been studied in depth. PKC? plays a central role in cell survival. Its splice variants, PKC?I and PKC?II, are switches that determine cell survival and fate. PKC?I promotes apoptosis, whereas PKC?II promotes survival. Here, we demonstrate that insulin promotes alternative splicing of PKC?II isoform in HT22 cells. The expression of PKC?I splice variant remains unchanged. Insulin increases PKC?II alternative splicing via the PI3K pathway. We further demonstrate that Akt kinase mediates phosphorylation of the splicing factor SC35 to promote PKC?II alternative splicing. Using overexpression and knockdown assays, we demonstrate that insulin increases expression of Bcl2 and bcl-xL via PKC?II. We demonstrate increased cell proliferation and increased BrdU incorporation in insulin-treated cells as well as in HT22 cells overexpressing PKC?II. Finally, we demonstrate in vivo that intranasal insulin promotes cognitive function in mice with concomitant increases in PKC?II expression in the hippocampus. This is the first report of insulin, generally considered a growth or metabolic hormone, regulating the alternative isoform expression of a key signaling kinase in neuronal cells such that it results in increased neuronal survival.

Apostolatos, Andre; Song, Shijie; Acosta, Sandra; Peart, Mishka; Watson, James E.; Bickford, Paula; Cooper, Denise R.; Patel, Niketa A.

2012-01-01

248

Regulation of alternative RNA splicing by exon definition and exon sequences in viral and mammalian gene expression  

PubMed Central

Intron removal from a pre-mRNA by RNA splicing was once thought to be controlled mainly by intron splicing signals. However, viral and other eukaryotic RNA exon sequences have recently been found to regulate RNA splicing, polyadenylation, export, and nonsense-mediated RNA decay in addition to their coding function. Regulation of alternative RNA splicing by exon sequences is largely attributable to the presence of two major cis-acting elements in the regulated exons, the exonic splicing enhancer (ESE) and the suppressor or silencer (ESS). Two types of ESEs have been verified from more than 50 genes or exons: purine-rich ESEs, which are the more common, and non-purine-rich ESEs. In contrast, the sequences of ESSs identified in approximately 21 genes or exons are highly diverse and show little similarity to each other. Through interactions with cellular splicing factors, an ESE or ESS determines whether or not a regulated splice site, usually an upstream 3? splice site, will be used for RNA splicing. However, how these elements function precisely in selecting a regulated splice site is only partially understood. The balance between positive and negative regulation of splice site selection likely depends on the cis-element’s identity and changes in cellular splicing factors under physiological or pathological conditions.

Zheng, Zhi-Ming

2008-01-01

249

Differential alternative splicing activity of isoforms of polypyrimidine tract binding protein (PTB).  

PubMed Central

Polypyrimidine tract binding protein (PTB) is an RNA-binding protein that regulates splicing by repressing specific splicing events. It also has roles in 3'-end processing, internal initiation of translation, and RNA localization. PTB exists in three alternatively spliced isoforms, PTB1, PTB2, and PTB4, which differ by the insertion of 19 or 26 amino acids, respectively, between the second and third RNA recognition motif domains. Here we show that the PTB isoforms have distinct activities upon alpha-tropomyosin (TM) alternative splicing. PTB1 reduced the repression of TM exon 3 in transfected smooth muscle cells, whereas PTB4 enhanced TM exon 3 skipping in vivo and in vitro. PTB2 had an intermediate effect. The PTB4 > PTB2 > PTB1 repressive hierarchy was observed in all in vivo and in vitro assays with TM, but the isoforms were equally active in inducing skipping of alpha-actinin exons and showed the opposite hierarchy of activity when tested for activation of IRES-driven translation. These findings establish that the ratio of PTB isoforms could form part of a cellular code that in turn controls the splicing of various other pre-mRNAs.

Wollerton, M C; Gooding, C; Robinson, F; Brown, E C; Jackson, R J; Smith, C W

2001-01-01

250

Regulation of alternative splicing by SRrp86 through coactivation and repression of specific SR proteins.  

PubMed Central

SRrp86 is an 86-kDa member of the SR protein superfamily that is unique in that it can alter splice site selection by regulating the activity of other SR proteins. To study the function of SRrp86, inducible cell lines were created in which the concentration of SRrp86 could be varied and its effects on alternative splicing determined. Here, we show that SRrp86 can activate SRp20 and repress SC35 in a dose-dependent manner both in vitro and in vivo. These effects are apparently mediated through direct protein-protein interaction, as pull-down assays showed that SRrp86 interacts with both SRp20 and SC35. Consistent with the hypothesis that relatively modest changes in the concentration or activity of one or more splicing factors can combinatorially regulate overall splicing, protein expression patterns of SRrp86, SRp20, and SC35 reveal that each tissue maintains a unique ratio of these factors. Regulation of SR protein activity, coupled with regulated protein expression, suggest that SRrp86 may play a crucial role in determining tissue specific patterns of alternative splicing.

Barnard, Daron C; Li, Jun; Peng, Rui; Patton, James G

2002-01-01

251

Eukaryotic TPP riboswitch regulation of alternative splicing involving long-distance base pairing.  

PubMed

Thiamin pyrophosphate (TPP) riboswitches are found in organisms from all three domains of life. Examples in bacteria commonly repress gene expression by terminating transcription or by blocking ribosome binding, whereas most eukaryotic TPP riboswitches are predicted to regulate gene expression by modulating RNA splicing. Given the widespread distribution of eukaryotic TPP riboswitches and the diversity of their locations in precursor messenger RNAs (pre-mRNAs), we sought to examine the mechanism of alternative splicing regulation by a fungal TPP riboswitch from Neurospora crassa, which is mostly located in a large intron separating protein-coding exons. Our data reveal that this riboswitch uses a long-distance (?530-nt separation) base-pairing interaction to regulate alternative splicing. Specifically, a portion of the TPP-binding aptamer can form a base-paired structure with a conserved sequence element (?) located near a 5' splice site, which greatly increases use of this 5' splice site and promotes gene expression. Comparative sequence analyses indicate that many fungal species carry a TPP riboswitch with similar intron architecture, and therefore the homologous genes in these fungi are likely to use the same mechanism. Our findings expand the scope of genetic control mechanisms relying on long-range RNA interactions to include riboswitches. PMID:23376932

Li, Sanshu; Breaker, Ronald R

2013-02-01

252

Eukaryotic TPP riboswitch regulation of alternative splicing involving long-distance base pairing  

PubMed Central

Thiamin pyrophosphate (TPP) riboswitches are found in organisms from all three domains of life. Examples in bacteria commonly repress gene expression by terminating transcription or by blocking ribosome binding, whereas most eukaryotic TPP riboswitches are predicted to regulate gene expression by modulating RNA splicing. Given the widespread distribution of eukaryotic TPP riboswitches and the diversity of their locations in precursor messenger RNAs (pre-mRNAs), we sought to examine the mechanism of alternative splicing regulation by a fungal TPP riboswitch from Neurospora crassa, which is mostly located in a large intron separating protein-coding exons. Our data reveal that this riboswitch uses a long-distance (?530-nt separation) base-pairing interaction to regulate alternative splicing. Specifically, a portion of the TPP-binding aptamer can form a base-paired structure with a conserved sequence element (?) located near a 5? splice site, which greatly increases use of this 5? splice site and promotes gene expression. Comparative sequence analyses indicate that many fungal species carry a TPP riboswitch with similar intron architecture, and therefore the homologous genes in these fungi are likely to use the same mechanism. Our findings expand the scope of genetic control mechanisms relying on long-range RNA interactions to include riboswitches.

Li, Sanshu; Breaker, Ronald R.

2013-01-01

253

Alternative splicing of a Drosophila GABA receptor subunit gene identifies determinants of agonist potency.  

PubMed

Alternative splicing of the Drosophila melanogaster Rdl gene yields four ionotropic GABA receptor subunits. The two Rdl splice variants cloned to date, RDL(ac) and RDL(bd) (DRC17-1-2), differ in their apparent agonist affinity. Here, we report the cloning of a third splice variant of Rdl, RDL(ad). Two-electrode voltage clamp electrophysiology was used to investigate agonist pharmacology of this expressed subunit following cRNA injection into Xenopus laevis oocytes. The EC(so) values for GABA and its analogues isoguvacine, muscimol, isonipecotic acid and 3-amino sulphonic acid on the RDL(ad) homomeric receptor differed from those previously described for RDL(ac) and DRC17-1-2 receptors. In addition to providing a possible physiological role for the alternative splicing of Rdl, these data delineate a hitherto functionally unassigned region of the N-terminal domain of GABA receptor subunits, which affects agonist potency and aligns closely with known determinants of potency in nicotinic acetylcholine receptors. Thus, using expression in Xenopus oocytes, we have demonstrated differences in agonist potency for the neurotransmitter GABA (and four analogues) between splice variant products of the Drosophila melanogaster Rdl gene encoding homomer-forming GABA receptor subunits. PMID:11226707

Hosie, A M; Buckingham, S D; Presnail, J K; Sattelle, D B

2001-01-01

254

Alternative splicing of human insulin receptor gene (INSR) in type I and type II skeletal muscle fibers of patients with myotonic dystrophy type 1 and type 2.  

PubMed

INSR, one of those genes aberrantly expressed in myotonic dystrophy type 1 (DM1) and type 2 (DM2) due to a toxic RNA effect, encodes for the insulin receptor (IR). Its expression is regulated by alternative splicing generating two isoforms: IR-A, which predominates in embryonic tissue, and IR-B, which is highly expressed in adult, insulin-responsive tissues (skeletal muscle, liver, and adipose tissue). The aberrant INSR expression detected in DM1 and DM2 muscles tissues, characterized by a relative increase of IR-A versus IR-B, was pathogenically related to the insulin resistance occurring in DM patients. To assess if differences in the aberrant splicing of INSR could underlie the distinct fiber type involvement observed in DM1 and DM2 muscle tissues, we have used laser capture microdissection (LCM) and RT-PCR, comparing the alternative splicing of INSR in type I and type II muscle fibers isolated from muscle biopsies of DM1, DM2 patients and controls. In the controls, the relative amounts of IR-A and IR-B showed no obvious differences between type I and type II fibers, as in the whole muscle tissue. In DM1 and DM2 patients, both fiber types showed a similar, relative increase of IR-A versus IR-B, as also evident in the whole muscle tissue. Our data suggest that the distinct fiber type involvement in DM1 and DM2 muscle tissues would not be related to qualitative differences in the expression of INSR. LCM can represent a powerful tool to give a better understanding of the pathogenesis of myotonic dystrophies, as well as other myopathies. PMID:23666741

Santoro, Massimo; Masciullo, Marcella; Bonvissuto, Davide; Bianchi, Maria Laura Ester; Michetti, Fabrizio; Silvestri, Gabriella

2013-05-11

255

CLONING, SEQUENCING, AND CHARACTERIZATION OF ALTERNATIVELY SPLICED GLUTAREDOXIN CDNA AND ITS GENOMIC GENE  

Technology Transfer Automated Retrieval System (TEKTRAN)

Alternatively spliced human glutaredoxin (GRXas) cDNA was isolated from a neutrophil cDNA library using the 32P-labeled glutaredoxin cDNA probe in non-stringent conditions. The sequence of this GRXas cDNA indicates that the open reading frame (ORF) of the gene is identical to the ORF of the previou...

256

b Subunits Modulate Alternatively Spliced, Large Conductance, Calcium-Activated Potassium Channels of Avian Hair Cells  

Microsoft Academic Search

Electrical tuning confers frequency selectivity onto sensory hair cells in the auditory periphery of frogs, turtles, and chicks. The resonant frequency is determined in large part by the number and kinetics of large conductance, calcium-activated potas- sium (BK) channels. BK channels in hair cells are encoded by the alternatively spliced slo gene and may include an accessory b subunit. Here

Krishnan Ramanathan; Timothy H. Michael; Paul A. Fuchs

257

Adhesion Mediated by Fibronectin's Alternatively Spliced ED b(EIIIB) and Its Neighboring Type III Repeats  

Microsoft Academic Search

Adhesion functions of cellular fibronectin's (FN) alternatively spliced EDb(EIIIB) domain, as well as its neighboring type III repeats III7 and III8, were investigated with several cultured murine and human cell types. Minigene constructs encoding various permutations of these repeats and expressed in bacteria were used as shown previously in function studies of EDaand its neighboring repeats (P. Xia and L.

Wensheng Chen; Lloyd A. Culp

1996-01-01

258

PPS, a Large Multidomain Protein, Functions with Sex-Lethal to Regulate Alternative Splicing in Drosophila  

Microsoft Academic Search

Alternative splicing controls the expression of many genes, including the Drosophila sex determination gene Sex-lethal (Sxl). Sxl expression is controlled via a negative regulatory mechanism where inclusion of the translation-terminating male exon is blocked in females. Previous studies have shown that the mechanism leading to exon skipping is autoregulatory and requires the SXL protein to antagonize exon inclusion by interacting

Matthew L. Johnson; Alexis A. Nagengast; Helen K. Salz

2010-01-01

259

Regulation of vertebrate nervous system alternative splicing and development by an SR-related protein.  

PubMed

Alternative splicing is a key process underlying the evolution of increased proteomic and functional complexity and is especially prevalent in the mammalian nervous system. However, the factors and mechanisms governing nervous system-specific alternative splicing are not well understood. Through a genome-wide computational and expression profiling strategy, we have identified a tissue- and vertebrate-restricted Ser/Arg (SR) repeat splicing factor, the neural-specific SR-related protein of 100 kDa (nSR100). We show that nSR100 regulates an extensive network of brain-specific alternative exons enriched in genes that function in neural cell differentiation. nSR100 acts by increasing the levels of the neural/brain-enriched polypyrimidine tract binding protein and by interacting with its target transcripts. Disruption of nSR100 prevents neural cell differentiation in cell culture and in the developing zebrafish. Our results thus reveal a critical neural-specific alternative splicing regulator, the evolution of which has contributed to increased complexity in the vertebrate nervous system. PMID:19737518

Calarco, John A; Superina, Simone; O'Hanlon, Dave; Gabut, Mathieu; Raj, Bushra; Pan, Qun; Skalska, Ursula; Clarke, Laura; Gelinas, Danielle; van der Kooy, Derek; Zhen, Mei; Ciruna, Brian; Blencowe, Benjamin J

2009-09-01

260

Functional coordination of alternative splicing in the mammalian central nervous system  

Microsoft Academic Search

BACKGROUND: Alternative splicing (AS) functions to expand proteomic complexity and plays numerous important roles in gene regulation. However, the extent to which AS coordinates functions in a cell and tissue type specific manner is not known. Moreover, the sequence code that underlies cell and tissue type specific regulation of AS is poorly understood. RESULTS: Using quantitative AS microarray profiling, we

Matthew Fagnani; Yoseph Barash; Joanna Y Ip; Christine Misquitta; Qun Pan; Arneet L Saltzman; Ofer Shai; Leo Lee; Aviad Rozenhek; Naveed Mohammad; Sandrine Willaime-Morawek; Tomas Babak; Wen Zhang; Timothy R Hughes; Derek van der Kooy; Brendan J Frey; Benjamin J Blencowe

2007-01-01

261

Alternative Splice Variant of ?-Calmodulin-Dependent Protein Kinase II Alters Activation by Calmodulin  

Microsoft Academic Search

Calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous, multifunctional enzyme family involved in the regulation of a variety of Ca2+-signaling pathways. These family members are expressed from four highly homologous genes (?, ?, ?, and ?) with similar catalytic properties. Additional isoforms of each gene, created by alternative splicing of variable regions I–XI, are differentially expressed in various cell types.

Ann P. Kwiatkowski; James M. McGill

2000-01-01

262

Gene Selection, Alternative Splicing, and Posttranslational Processing Regulate Neuroligin Selectivity for ?-Neurexins †  

Microsoft Academic Search

Neuroligins 1-4 are postsynaptic transmembrane proteins capable of initiating presynaptic maturation via interactions with ‚-neurexin. Both neuroligins and ‚-neurexins have alternatively spliced inserts in their extracellular domains. Using analytical ultracentrifugation, we determined that the extracellular domains of the neuroligins sediment as dimers, whereas the extracellular domains of the ‚-neurexins appear monomeric. Sedimentation velocity experiments of titrated stoichiometry ratios of ‚-neurexin

Davide Comoletti; Robyn E. Flynn; Antony A. Boucard; Borries Demeler; Virgil Schirf; Jianxin Shi; Lori L. Jennings; Helen R. Newlin; Thomas C. Südhof; Palmer Taylor

2006-01-01

263

Pyrvinium pamoate changes alternative splicing of the serotonin receptor 2C by influencing its RNA structure.  

PubMed

The serotonin receptor 2C plays a central role in mood and appetite control. It undergoes pre-mRNA editing as well as alternative splicing. The RNA editing suggests that the pre-mRNA forms a stable secondary structure in vivo. To identify substances that promote alternative exons inclusion, we set up a high-throughput screen and identified pyrvinium pamoate as a drug-promoting exon inclusion without editing. Circular dichroism spectroscopy indicates that pyrvinium pamoate binds directly to the pre-mRNA and changes its structure. SHAPE (selective 2'-hydroxyl acylation analysed by primer extension) assays show that part of the regulated 5'-splice site forms intramolecular base pairs that are removed by this structural change, which likely allows splice site recognition and exon inclusion. Genome-wide analyses show that pyrvinium pamoate regulates >300 alternative exons that form secondary structures enriched in A-U base pairs. Our data demonstrate that alternative splicing of structured pre-mRNAs can be regulated by small molecules that directly bind to the RNA, which is reminiscent to an RNA riboswitch. PMID:23393189

Shen, Manli; Bellaousov, Stanislav; Hiller, Michael; de La Grange, Pierre; Creamer, Trevor P; Malina, Orit; Sperling, Ruth; Mathews, David H; Stoilov, Peter; Stamm, Stefan

2013-02-07

264

Alternative splicing of lola generates 19 transcription factors controlling axon guidance in Drosophila  

Microsoft Academic Search

The Drosophila melanogaster transcription factor Lola (longitudinals lacking) is a pivotal regulator of neural wiring that sets the precise expression levels of proteins that execute specific axon guidance decisions. Lola has a zinc finger DNA binding domain and a BTB (for Broad-complex, Tramtrack and Bric a brac) dimerization motif. We now show that alternative splicing of the lola gene creates

Scott Goeke; Elizabeth A. Greene; Paul K. Grant; Michael A. Gates; Daniel Crowner; Toshiro Aigaki; Edward Giniger

2003-01-01

265

Precise and Parallel Characterization of Coding Polymorphisms, Alternative Splicing, and Modifications in Human Proteins by  

Microsoft Academic Search

The human proteome is a highly complex extension of the genome wherein a single gene often produces distinct protein forms due to alternative splicing, RNA editing, polymorphisms, and posttranslational modifications. Such biological variation compounded by the high se- quence identity within gene families currently overwhelms the complete and routine characterization of mammalian proteins by MS. A new data base of

Mass Spectrometry; Michael J. Roth; Andrew J. Forbes; Michael T. Boyne II; Yong-Bin Kim; Dana E. Robinson; Neil L. Kelleher

266

Alternative splicing of the unique "PLUS" domain of chicken PG-M/versican is developmentally regulated.  

PubMed

We investigated the occurrence of alternatively spliced forms (V0, V1, V2, and V3) of PG-M/versican, a large chondroitin sulfate proteoglycan in developing chicken retinas, using the reverse transcription-polymerase chain reaction. We characterized the PLUS domain, which is apparently unique to the chicken molecule and is regulated by alternative splicing. PG-M in chicken retinas consisted of four forms with (V0, V1, V2, and V3) and two forms without (V1 and V3) the PLUS domain (PG-M+ and PG-M-, respectively). The four forms of PG-M+ were found in all samples examined, but the occurrence of the two PG-M- forms was regulated developmentally. Genomic analysis has revealed that the PLUS and CS-alpha domains are encoded by a single exon, and this exon has an internal alternative 5'-splice donor site, allowing alternative spliced forms that do not include the 3'-end of the exon. Sequences corresponding to the chicken PLUS domain (plus) were not found in mouse and human and may have disappeared during evolution. Sequence similarity suggests that the PLUS domain corresponds to the keratan sulfate attachment domain of aggrecan and that it has a distinct function in the chicken eye. PMID:9083069

Zako, M; Shinomura, T; Kimata, K

1997-04-01

267

A novel intronic mutation in SHOX causes short stature by disrupting a splice acceptor site: direct demonstration of aberrant splicing by expression of a minigene in HEK-293T cells.  

PubMed

SHOX, the short stature homeobox-containing gene, encodes a critical regulatory protein controlling long bone growth. We examined patients in one family, identified an intronic mutation, and expressed SHOX minigenes in HEK293T cells to characterize the effect on gene splicing. We identified a novel mutation at position -3 (c.-432-3C>A;g.6120C>A) of the intron 1 splice acceptor site; three short (height Z-score -2.4 to -1.7) children were heterozygous and the father (height Z-score -3.4) was homozygous. A wild-type minigene produced alternative transcripts; one utilized the normal splice site between intron 1 and exon 2, the other a cryptic splice site in exon 2. Mutant SHOX minigene generated only the smaller transcript. The exon 2 acceptor splice site is weak; an alternative transcript is normally produced using a downstream cryptic splice site. The c.-432-3C>A mutation causes further weakening, and the cryptic splice site is preferentially utilized, resulting in SHOX deficiency and short stature. PMID:23426818

Danzig, Jennifer; Levine, Michael A

2012-01-01

268

Cardiac glycosides correct aberrant splicing of IKBKAP-encoded mRNA in familial dysautonomia derived cells by suppressing expression of SRSF3.  

PubMed

The ability to modulate the production of the wild-type transcript in cells bearing the splice-altering familial dysautonomia (FD) causing mutation in the IKBKAP gene prompted a study of the impact of a panel of pharmaceuticals on the splicing of this transcript, which revealed the ability of the cardiac glycoside digoxin to increase the production of the wild-type, exon-20-containing, IKBKAP-encoded transcript and the full-length I?B-kinase-complex-associated protein in FD-derived cells. Characterization of the cis elements and trans factors involved in the digoxin-mediated effect on splicing reveals that this response is dependent on an SRSF3 binding site(s) located in the intron 5' of the alternatively spliced exon and that digoxin mediates its effect by suppressing the level of the SRSF3 protein. Characterization of the digoxin-mediated effect on the RNA splicing process was facilitated by the identification of several RNA splicing events in which digoxin treatment mediates the enhanced inclusion of exonic sequence. Moreover, we demonstrate the ability of digoxin to impact the splicing process in neuronal cells, a cell type profoundly impacted by FD. This study represents the first demonstration that digoxin possesses splice-altering capabilities that are capable of reversing the impact of the FD-causing mutation. These findings support the clinical evaluation of the impact of digoxin on the FD patient population. PMID:23711097

Liu, Bo; Anderson, Sylvia L; Qiu, Jinsong; Rubin, Berish Y

2013-06-18

269

Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery  

PubMed Central

The tight control of gene expression at the level of both transcription and post-transcriptional RNA processing is essential for mammalian development. We here investigate the role of protein arginine methyltransferase 5 (PRMT5), a putative splicing regulator and transcriptional cofactor, in mammalian development. We demonstrate that selective deletion of PRMT5 in neural stem/progenitor cells (NPCs) leads to postnatal death in mice. At the molecular level, the absence of PRMT5 results in reduced methylation of Sm proteins, aberrant constitutive splicing, and the alternative splicing of specific mRNAs with weak 5? donor sites. Intriguingly, the products of these mRNAs are, among others, several proteins regulating cell cycle progression. We identify Mdm4 as one of these key mRNAs that senses the defects in the spliceosomal machinery and transduces the signal to activate the p53 response, providing a mechanistic explanation of the phenotype observed in vivo. Our data demonstrate that PRMT5 is a master regulator of splicing in mammals and uncover a new role for the Mdm4 pre-mRNA, which could be exploited for anti-cancer therapy.

Bezzi, Marco; Teo, Shun Xie; Muller, Julius; Mok, Wei Chuen; Sahu, Sanjeeb Kumar; Vardy, Leah A.; Bonday, Zahid Q.; Guccione, Ernesto

2013-01-01

270

Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery.  

PubMed

The tight control of gene expression at the level of both transcription and post-transcriptional RNA processing is essential for mammalian development. We here investigate the role of protein arginine methyltransferase 5 (PRMT5), a putative splicing regulator and transcriptional cofactor, in mammalian development. We demonstrate that selective deletion of PRMT5 in neural stem/progenitor cells (NPCs) leads to postnatal death in mice. At the molecular level, the absence of PRMT5 results in reduced methylation of Sm proteins, aberrant constitutive splicing, and the alternative splicing of specific mRNAs with weak 5' donor sites. Intriguingly, the products of these mRNAs are, among others, several proteins regulating cell cycle progression. We identify Mdm4 as one of these key mRNAs that senses the defects in the spliceosomal machinery and transduces the signal to activate the p53 response, providing a mechanistic explanation of the phenotype observed in vivo. Our data demonstrate that PRMT5 is a master regulator of splicing in mammals and uncover a new role for the Mdm4 pre-mRNA, which could be exploited for anti-cancer therapy. PMID:24013503

Bezzi, Marco; Teo, Shun Xie; Muller, Julius; Mok, Wei Chuen; Sahu, Sanjeeb Kumar; Vardy, Leah A; Bonday, Zahid Q; Guccione, Ernesto

2013-09-01

271

Identification and expression analysis of alternative splice variants of the rat Hax-1 gene  

Microsoft Academic Search

Hax-1 protein, which has been studied in mice and humans, shows a potent anti-apoptotic activity and is involved in regulation of cell motility. Cloning of the rat Hax-1 cDNA has revealed seven alternative transcripts, which differ mostly in their 5? region. Alternative splicing concerns exon 1, skipped in 5 transcripts, intron 1 which is partially retained in these transcripts, exon

Ewa A. Grzybowska; El?bieta Sarnowska; Ryszard Konopi?ski; Anna Wilczy?ska; Tomasz J. Sarnowski; Janusz A. Siedlecki

2006-01-01

272

Regulation of the Ras-MAPK and PI3K-mTOR Signalling Pathways by Alternative Splicing in Cancer  

PubMed Central

Alternative splicing is a fundamental step in regulation of gene expression of many tumor suppressors and oncogenes in cancer. Signalling through the Ras-MAPK and PI3K-mTOR pathways is misregulated and hyperactivated in most types of cancer. However, the regulation of the Ras-MAPK and PI3K-mTOR signalling pathways by alternative splicing is less well established. Recent studies have shown the contribution of alternative splicing regulation of these signalling pathways which can lead to cellular transformation, cancer development, and tumor maintenance. This review will discuss findings in the literature which describe new modes of regulation of components of the Ras-MAPK and PI3K-mTOR signalling pathways by alternative splicing. We will also describe the mechanisms by which signals from extracellular stimuli can be communicated to the splicing machinery and to specific RNA-binding proteins that ultimately control exon definition events.

Bonomi, Serena

2013-01-01

273

AG-dependent 3?-splice sites are predisposed to aberrant splicing due to a mutation at the first nucleotide of an exon  

PubMed Central

In pre-mRNA splicing, a conserved AG/G at the 3?-splice site is recognized by U2AF35. A disease-causing mutation abrogating the G nucleotide at the first position of an exon (E+1) causes exon skipping in GH1, FECH and EYA1, but not in LPL or HEXA. Knockdown of U2AF35 enhanced exon skipping in GH1 and FECH. RNA-EMSA revealed that wild-type FECH requires U2AF35 but wild-type LPL does not. A series of artificial mutations in the polypyrimidine tracts of GH1, FECH, EYA1, LPL and HEXA disclosed that a stretch of at least 10–15 pyrimidines is required to ensure normal splicing in the presence of a mutation at E+1. Analysis of nine other disease-causing mutations at E+1 detected five splicing mutations. Our studies suggest that a mutation at the AG-dependent 3?-splice site that requires U2AF35 for spliceosome assembly causes exon skipping, whereas one at the AG-independent 3?-splice site that does not require U2AF35 gives rise to normal splicing. The AG-dependence of the 3?-splice site that we analyzed in disease-causing mutations at E+1 potentially helps identify yet unrecognized splicing mutations at E+1.

Fu, Yuan; Masuda, Akio; Ito, Mikako; Shinmi, Jun; Ohno, Kinji

2011-01-01

274

Alternative splicing is required for RCT1-mediated disease resistance in Medicago truncatula.  

PubMed

RCT1 is a TIR-NBS-LRR-type resistance (R) gene in Medicago truncatula that confers resistance to multiple races of Colletotrichum trifolii, a hemi-biotrophic fungal pathogen that causes anthracnose disease in Medicago and other closely related legumes. RCT1 undergoes alternative splicing at both coding and 3'-untranslated regions, thereby producing multiple transcript variants in its expression profile. Alternative splicing of RCT1 in the coding region results from the retention of intron 4. Because intron 4 lies downstream of the LRR-encoding exons and contains an in-frame stop codon, the alternative transcript is predicted to encode a truncated protein consisting of the entire portion of the TIR, NBS, and LRR domains but lacks the C-terminal domain of the full-length RCT1 protein encoded by the regular transcript. Here we provide evidence that the RCT1-mediated disease resistance requires the combined presence of the regular and alternative transcripts. Neither the regular nor the alternative RCT1 transcript alone is sufficient to confer resistance against the pathogen. This study, in addition to the reports on the tobacco N and Arabidopsis RPS4 genes, adds another significant example showing the involvement of alternative splicing in R gene-mediated plant immunity. PMID:23657790

Tang, Fang; Yang, Shengming; Gao, Muqiang; Zhu, Hongyan

2013-05-09

275

FIRMA: a method for detection of alternative splicing from exon array data  

PubMed Central

Motivation: Analyses of EST data show that alternative splicing is much more widespread than once thought. The advent of exon and tiling microarrays means that researchers now have the capacity to experimentally measure alternative splicing on a genome wide level. New methods are needed to analyze the data from these arrays. Results: We present a method, finding isoforms using robust multichip analysis (FIRMA), for detecting differential alternative splicing in exon array data. FIRMA has been developed for Affymetrix exon arrays, but could in principle be extended to other exon arrays, tiling arrays or splice junction arrays. We have evaluated the method using simulated data, and have also applied it to two datasets: a panel of 11 human tissues and a set of 10 pairs of matched normal and tumor colon tissue. FIRMA is able to detect exons in several genes confirmed by reverse transcriptase PCR. Availability: R code implementing our methods is contributed to the package aroma.affymetrix. Contact: epurdom@stat.berkeley.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Purdom, E.; Simpson, K. M.; Robinson, M. D.; Conboy, J. G.; Lapuk, A. V.; Speed, T.P.

2008-01-01

276

Complement receptor 2 polymorphisms associated with systemic lupus erythematosus modulate alternative splicing  

PubMed Central

Genetic factors influence susceptibility to systemic lupus erythematosus (SLE). A recent family-based analysis in Caucasian and Chinese populations provided evidence for association of single-nucleotide polymorphisms (SNPs) in the complement receptor 2 (CR2/CD21) gene with SLE. Here we confirmed this result in a case-control analysis of an independent European-derived population including 2084 patients with SLE and 2853 healthy controls. A haplotype formed by the minor alleles of three CR2 SNPs (rs1048971, rs17615, rs4308977) showed significant association with decreased risk of SLE (30.4% in cases vs. 32.6% in controls, P = 0.016, OR = 0.90 [0.82-0.98]). Two of these SNPs are in exon 10, directly 5? of an alternatively spliced exon preferentially expressed in follicular dendritic cells (FDC), and the third is in the alternatively spliced exon. Effects of these SNPs as well as a fourth SNP in exon 11 (rs17616) on alternative splicing were evaluated. We found that the minor alleles of these SNPs decreased splicing efficiency of exon 11 both in vitro and ex vivo. These findings further implicate CR2 in the pathogenesis of SLE and suggest that CR2 variants alter the maintenance of tolerance and autoantibody production in the secondary lymphoid tissues where B cells and FDCs interact.

Douglas, Katherine B.; Windels, Daniel C.; Zhao, Jian; Gadeliya, Agnessa V.; Wu, Hui; Kaufman, Kenneth M.; Harley, John B.; Merrill, Joan; Kimberly, Robert P.; Alarcon, Graciela S.; Brown, Elizabeth E.; Edberg, Jeffrey C.; Ramsey-Goldman, Rosalind; Petri, Michelle; Reveille, John D.; Vila, Luis M.; Gaffney, Patrick M.; James, Judith A.; Moser, Kathy L.; Alarcon-Riquelme, Marta E.; Vyse, Timothy J.; Gilkeson, Gary S.; Jacob, Chaim O.; Ziegler, Julie T.; Langefeld, Carl D.; Ulgiati, Daniela; Tsao, Betty P.; Boackle, Susan A.

2009-01-01

277

Alternative splicing of agrin regulates its binding to heparin alpha-dystroglycan, and the cell surface.  

PubMed Central

Agrin is a basal lamina molecule that directs key events in postsynaptic differentiation, most notably the aggregation of acetylcholine receptors (AChRs) on the muscle cell surface. Agrin's AChR clustering activity is regulated by alternative mRNA splicing. Agrin splice forms having inserts at two sites (y and z) in the C-terminal region are highly active, but isoforms lacking these inserts are weakly active. The biochemical consequences of this alternative splicing are unknown. Here, the binding of four recombinant agrin isoforms to heparin, to alpha-dystroglycan (a component of an agrin receptor), and to myoblasts was tested. The presence of a four-amino acid insert at the y site is necessary and sufficient to confer heparin binding ability to agrin. Moreover, the binding of agrin to alpha-dystroglycan is inhibited by heparin when this insert is present. Agrin binding to the cell surface showed analogous properties: heparin inhibits the binding of only those agrin isoforms containing this four-amino acid insert. The results show that alternative splicing of agrin regulates its binding to heparin and suggest that agrin's interaction with alpha-dystroglycan may be modulated by cell surface glycosaminoglycans in an isoform-dependent manner. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4

O'Toole, J J; Deyst, K A; Bowe, M A; Nastuk, M A; McKechnie, B A; Fallon, J R

1996-01-01

278

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

PubMed Central

Alternative pre-mRNA splicing predominates in the nervous systems of complex organisms including humans dramatically 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 CaV2.2 calcium channels in nociceptors. Using an exon replacement strategy in mice, we show that mutually exclusive splicing in the CaV2.2 gene modulates N-type channel function in nociceptors leading to a change in morphine analgesia. Exon 37a enhances ?-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 without influencing the basal response to noxious thermal stimuli. 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.

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

2010-01-01

279

Alternative splicing in spinal muscular atrophy underscores the role of an intron definition model  

PubMed Central

Humans have two nearly identical copies of the Survival Motor Neuron (SMN) gene: SMN1 and SMN2. The two SMN genes code for identical proteins; however, SMN2 predominantly generates a shorter transcript due to skipping of exon 7, the last coding exon. Skipping of SMN2 exon 7 leads to production of a truncated SMN protein that is highly unstable. The inability of SMN2 to compensate for the loss of SMN1 results in spinal muscular atrophy (SMA), the second most prevalent genetic cause of infant mortality. Since SMN2 is almost universally present in SMA patients, correction of SMN2 exon 7 splicing holds the promise for cure. Consistently, SMN2 exon 7 splicing has emerged as one of the best studied splicing systems in humans. The vast amount of recent literature provides a clue that SMN2 exon 7 splicing is regulated by an intron definition mechanism, which does not require cross-exon communication as prerequisite for exon inclusion. Our conclusion is based on the prominent role of intronic cis-elements, some of them have emerged as the frontrunners among potential therapeutic targets of SMA. Further, the widely expressed T-cell-restricted intracellular antigen-1 (TIA1), a member of the glutamine rich domain containing RNA-binding proteins, has recently been found to regulate SMN exon 7 splicing by binding to intron 7 sequences away from the 5? splice site (ss). These findings make a strong argument for an “intron definition model,” according to which regulatory sequences within a downstream intron are capable of enforcing exon inclusion even in the absence of a defined upstream 3? ss of an alternatively spliced exon.

Singh, Natalia N

2011-01-01

280

Exon array analysis reveals neuroblastoma tumors have distinct alternative splicing patterns according to stage and MYCN amplification status  

PubMed Central

Background Neuroblastoma (NB) tumors are well known for their pronounced clinical and molecular heterogeneity. The global gene expression and DNA copy number alterations have been shown to have profound differences in tumors of low or high stage and those with or without MYCN amplification. RNA splicing is an important regulatory mechanism of gene expression, and differential RNA splicing may be associated with the clinical behavior of a tumor. Methods In this study, we used exon array profiling to investigate global alternative splicing pattern of 47 neuroblastoma samples in stage 1 and stage 4 with normal or amplified MYCN copy number (stage 1-, 4- and 4+). The ratio of exon-level expression to gene-level expression was used to detect alternative splicing events, while the gene-level expression was applied to characterize whole gene expression change. Results Principal component analysis (PCA) demonstrated distinct splicing pattern in three groups of samples. Pairwise comparison identified genes with splicing changes and/or whole gene expression changes in high stage tumors. In stage 4- compared with stage 1- tumors, alternatively spliced candidate genes had little overlap with genes showing whole gene expression changes, and most of them were involved in different biological processes. In contrast, a larger number of genes exhibited either exon-level splicing, gene-level expression or both changes in stage 4+ versus stage 1- tumors. Those biological processes involved in stage 4- tumors were disrupted to a greater extent by both splicing and transcription regulations in stage 4+ tumors. Conclusions Our results demonstrated a significant role of alternative splicing in high stage neuroblastoma, and suggested a MYCN-associated splicing regulation pathway in stage 4+ tumors. The identification of differentially spliced genes and pathways in neuroblastoma tumors of different stages and molecular subtypes may be important to the understanding of cancer biology and the discovery of diagnostic markers or therapeutic targets in neuroblastoma.

2011-01-01

281

Alternative Splicing of the Pituitary Adenylate Cyclase-Activating Polypeptide Receptor PAC1: Mechanisms of Fine Tuning of Brain Activity  

PubMed Central

Alternative splicing of the precursor mRNA encoding for the neuropeptide receptor PAC1/ADCYAP1R1 generates multiple protein products that exhibit pleiotropic activities. Recent studies in mammals and zebrafish have implicated some of these splice isoforms in control of both cellular and body homeostasis. Here, we review the regulation of PAC1 splice variants and their underlying signal transduction and physiological processes in the nervous system.

Blechman, Janna; Levkowitz, Gil

2013-01-01

282

The Apoptosis-Promoting Factor TIA-1 Is a Regulator of Alternative Pre-mRNA Splicing  

Microsoft Academic Search

We report here that the apoptosis-promoting protein TIA-1 regulates alternative pre-mRNA splicing of the Drosophila melanogaster gene male-specific-lethal 2 and of the human apoptotic gene Fas. TIA-1 associates selectively with pre-mRNAs that contain 5? splice sites followed by U-rich sequences. TIA-1 binding to the U-rich stretches facilitates 5? splice site recognition by U1 snRNP. This activity is critical for activation

Patrik Förch; Oscar Puig; Nancy Kedersha; Concepción Martínez; Sander Granneman; Bertrand Séraphin; Paul Anderson; Juan Valcárcel

2000-01-01

283

Alternative splicing of neuroligin regulates the rate of presynaptic differentiation.  

PubMed

Neuroligins (NLGs) and Neurexins (NRXs) are important adhesion molecules that promote synapse formation. Multiple splice variants of NLG and NRX exist, but their specific functions are unclear. Here we report that a surrogate postsynaptic cell expressing full-length NLG-1 triggers slow presynaptic differentiation in a contacting axon. In contrast, a version of NLG-1, which lacks insert B (NLG-1DeltaB), induces rapid presynaptic differentiation, reaching the rate seen at native neuronal synapses. We show that this acceleration is attributed to the removal of the N-linked glycosylation site within insert B. NLG-1DeltaB also increases synaptic density at neuro-neuronal synapses more than does full-length NLG-1. Other postsynaptic adhesion proteins, such as N-cadherin, EphB2, and SynCAM-1, alone or in combination with full-length NLG-1, do not trigger fast differentiation, suggesting that rapid presynaptic differentiation depends on a unique interaction of NLG-1DeltaB with axonal proteins. Indeed, we find that NLG-1DeltaB recruits more axonal alpha-NRX. Our results suggest that the engagement of alpha-NRX is a key to rapid induction of synapses at new sites of axo-dendritic contact. PMID:20739565

Lee, Hanson; Dean, Camin; Isacoff, Ehud

2010-08-25

284

Sudemycins, novel small molecule analogues of FR901464, induce alternative gene splicing.  

PubMed

Two unrelated bacterial natural products, FR901464 and pladienolide B, have previously been shown to have significant antitumor activity in vivo. These compounds target the SF3b subunit of the spliceosome, with a derivative of pladienolide (E7107) entering clinical trials for cancer. However, due to the structural complexity of these molecules, their research and development has been significantly constrained. We have generated a set of novel analogues (Sudemycins) that possess the pharmacophore that is common to FR901464 and pladienolide, via a flexible enantioselective route, which allows for the production of gram quantities of drug. These compounds demonstrate cytotoxicity toward human tumor cell lines in culture and exhibit antitumor activity in a xenograft model. Here, we present evidence that Sudemycins are potent modulators of alternative splicing in human cells, both of endogenous genes and from minigene constructs. Furthermore, levels of alternative splicing are increased in tumor cells relative to normal cells, and these modifications can be observed in human tumor xenografts in vivo following exposure of animals to the drug. In addition, the change in the splicing pattern observed with the Sudemycins are similar to that observed with Spliceostatin A, a molecule known to interact with the SF3b subunit of the spliceosome. Hence, we conclude that Sudemycins can regulate the production of alternatively spliced RNA transcripts and these alterations are more prevalent in tumors, as compared to normal cells, following drug exposure. These studies suggest that modulation of alternative splicing may play a role in the antitumor activity of this class of agents. PMID:21344922

Fan, Liying; Lagisetti, Chandraiah; Edwards, Carol C; Webb, Thomas R; Potter, Philip M

2011-03-07

285

Sudemycins, novel small molecule analogues of FR901464, induce alternative gene splicing  

PubMed Central

Two unrelated bacterial natural products, FR901464 and pladienolide B, have previously been shown to have significant anti-tumor activity in vivo. These compounds target the SF3b subunit of the spliceosome, with a derivative of pladienolide (E7107) entering clinical trials for cancer. However, due to the structural complexity of these molecules, their research and development has been significantly constrained. We have generated a set of novel analogues (Sudemycins) that possess the pharmacophore that is common to FR901464 and pladienolide, via a flexible enantioselective route, and allows for the production of gram quantities of drug. These compounds demonstrate cytotoxicity towards human tumor cell lines in culture and exhibit antitumor activity in a xenograft model. Here, we present evidence that Sudemycins are potent modulators of alternative splicing in human cells, both of endogenous genes and from minigene constructs. Furthermore, levels of alternative splicing are increased in tumor cells relative to normal cells and these modifications can be observed in human tumor xenografts in vivo following exposure of animals to the drug. In addition, the change in the splicing pattern observed with the Sudemycins are similar to that observed with Spliceostatin A, a molecule known to interact with the SF3b subunit of the spliceosome. Hence, we conclude that Sudemycins can regulate the production of alternatively spliced RNA transcripts and these alterations are more prevalent in tumor, as compared to normal cells, following drug exposure. These studies suggest that modulation of alternative splicing may play a role in the antitumor activity of this class of agents.

Fan, Liying; Lagisetti, Chandraiah; Edwards, Carol C.; Webb, Thomas R.; Potter, Philip M.

2011-01-01

286

Emerging role of alternative splicing of CRF1 receptor in CRF signaling  

PubMed Central

Alternative splicing of mRNA is one of the most important mechanisms responsible for an increase of the genomic capacity. Thus the majority of human proteins including G protein-coupled receptors (GPCRs) possess several isoforms as a result of mRNA splicing. The corticotropin-releasing factor (CRF) and its receptors are the most proximal elements of hypothalamic-pituitary-adrenal axis (HPA) — the central machinery of stress response. Moreover, expression of CRF and regulated activity of CRF receptor type 1 (CRF1) can also play an important role in regulation of local stress response in peripheral tissues including skin, gastrointestinal tract or reproductive system. In humans, expression of at least eight variants of CRF1 mRNA (?, ?, c, d, e, f, g and h) was detected and alternative splicing was found to be regulated by diverse physiological and pathological factors including: growth conditions, onset of labor, during pregnancy or exposure to ultraviolet irradiation. The pattern of expression of CRF1 isoforms is cell type specific and recently has been linked to observed differences in responsiveness to CRF stimulation. In the proposed model of regulation of CRF-signaling, isoform CRF1? plays a central role. Other isoforms modulate its activity by oligomerization, leading to alteration in receptor trafficking, localization and function. Co-expression of CRF1 isoforms modulates sensitivity of cells to the ligands and influences downstream coupling to G-proteins. The other possible regulatory mechanisms include fast mRNA and/or protein turnover or decoy receptor function of CRF1 isoforms. Taken together, alternative splicing of CRF1 can represent another level of regulation of CRF-mediated stress responses at the central and peripheral levels. Chronic stress or malfunction of the HPA-axis have been linked to numerous human pathologies, suggesting that alternative splicing of CRF1 receptor could represent a promising target for drugs development.

Zmijewski, Michal A.; Slominski, Andrzej T.

2010-01-01

287

Regulation of neuron-specific alternative splicing of neurofibromatosis type 1 pre-mRNA.  

PubMed

Neurofibromatosis type 1 (NF1) is one of the most common heritable autosomal dominant disorders. Alternative splicing modulates the function of neurofibromin, the NF1 gene product, by inserting the in-frame exon 23a into the region of NF1 mRNA that encodes the GTPase-activating protein-related domain. This insertion, which is predominantly skipped in neurons, reduces the ability of neurofibromin to regulate Ras by 10-fold. Here, we report that the neuron-specific Hu proteins control the production of the short protein isoform by suppressing inclusion of NF1 exon 23a, while TIA-1/TIAR proteins promote inclusion of this exon. We identify two binding sites for Hu proteins, located upstream and downstream of the regulated exon, and provide biochemical evidence that Hu proteins specifically block exon definition by preventing binding of essential splicing factors. In vitro analyses using nuclear extracts show that at the downstream site, Hu proteins prevent binding of U1 and U6 snRNPs to the 5' splice site, while TIAR increases binding. Hu proteins also decrease U2AF binding at the 3' splice site located upstream of exon 23a. In addition to providing the first mechanistic insight into tissue-specific control of NF1 splicing, these studies establish a novel strategy whereby Hu proteins regulate RNA processing. PMID:18086893

Zhu, Hui; Hinman, Melissa N; Hasman, Robert A; Mehta, Priyesh; Lou, Hua

2007-12-17

288

Exon-level microarray analyses identify alternative splicing programs in breast cancer  

PubMed Central

Protein isoforms produced by alternative splicing (AS) of many genes have been implicated in several aspects of cancer genesis and progression. These observations motivated a genome-wide assessment of AS in breast cancer. We accomplished this by measuring exon level expression in 31 breast cancer and nonmalignant immortalized cell lines representing luminal, basal and claudin-low breast cancer subtypes using Affymetrix Human Junction Arrays (HJAY). We analyzed these data using a computational pipeline specifically designed to detect AS with a low false positive rate. This identified 181 splice events representing 156 genes as candidates for AS. RT-PCR validation of a subset of predicted AS events confirmed 90%. Approximately half of the AS events were associated with basal, luminal or claudin-low breast cancer subtypes. Exons involved in claudin-low subtype-specific AS were significantly associated with the presence of evolutionarily conserved binding motifs for the tissue-specific Fox2 splicing factor. siRNA knockdown of Fox2 confirmed the involvement of this splicing factor in subtype specific AS. The subtype specific AS detected in this study likely reflects the splicing pattern in the breast cancer progenitor cells in which the tumor arose and suggests the utility of assays for Fox-mediated AS in cancer subtype definition and early detection. These data also suggest the possibility of reducing the toxicity of protein-targeted breast cancer treatments by targeting protein isoforms that are not present in limiting normal tissues.

Lapuk, Anna; Marr, Henry; Jakkula, Lakshmi; Pedro, Helder; Bhattacharya, Sanchita; Purdom, Elizabeth; Hu, Zhi; Simpson, Ken; Pachter, Lior; Durinck, Steffen; Wang, Nicholas; Parvin, Bahram; Fontenay, Gerald; Speed, Terence; Garbe, James; Stampfer, Martha; Bayandorian, Hovig; Dorton, Shannon; Clark, Tyson A.; Schweitzer, Anthony; Wyrobek, Andrew; Feiler, Heidi; Spellman, Paul; Conboy, John; Gray, Joe W.

2010-01-01

289

Global profiling of alternative splicing events and gene expression regulated by hnRNPH/F.  

PubMed

In this study, we have investigated the global impact of heterogeneous nuclear Ribonuclear Protein (hnRNP) H/F-mediated regulation of splicing events and gene expression in oligodendrocytes. We have performed a genome-wide transcriptomic analysis at the gene and exon levels in Oli-neu cells treated with siRNA that targets hnRNPH/F compared to untreated cells using Affymetrix Exon Array. Gene expression levels and regulated exons were identified with the GenoSplice EASANA algorithm. Bioinformatics analyses were performed to determine the structural properties of G tracts that correlate with the function of hnRNPH/F as enhancers vs. repressors of exon inclusion. Different types of alternatively spliced events are regulated by hnRNPH/F. Intronic G tracts density, length and proximity to the 5' splice site correlate with the hnRNPH/F enhancer function. Additionally, 6% of genes are differently expressed upon knock down of hnRNPH/F. Genes that regulate the transition of oligodendrocyte progenitor cells to oligodendrocytes are differentially expressed in hnRNPH/F depleted Oli-neu cells, resulting in a decrease of negative regulators and an increase of differentiation-inducing regulators. The changes were confirmed in developing oligodendrocytes in vivo. This is the first genome wide analysis of splicing events and gene expression regulated by hnRNPH/F in oligodendrocytes and the first report that hnRNPH/F regulate genes that are involved in the transition from oligodendrocyte progenitor cells to oligodendrocytes. PMID:23284676

Wang, Erming; Aslanzadeh, Vahid; Papa, Filomena; Zhu, Haiyan; de la Grange, Pierre; Cambi, Franca

2012-12-17

290

Correction of alternative splicing of tau in frontotemporal dementia and parkinsonism linked to chromosome 17.  

PubMed

Mutations in the human tau gene cause frontotemporal dementia and Parkinsonism associated with chromosome 17 (FTDP-17). One of the major disease mechanisms in FTDP-17 is the increased inclusion of tau exon 10 during pre-mRNA splicing. Here we show that modified oligonucleotides directed against the tau exon 10 splice junctions suppress inclusion of tau exon 10. The effect is mediated by the formation of a stable pre-mRNA-oligonucleotide hybrid, which blocks access of the splicing machinery to the pre-mRNA. Correction of tau splicing occurs in a tau minigene system and in endogenous tau RNA in neuronal pheochromocytoma cells and is specific to exon 10 of the tau gene. Antisense oligonucleotide-mediated exclusion of exon 10 has a physiological effect by increasing the ratio of protein lacking the microtubule-binding domain encoded by exon 10. As a consequence, the microtubule cytoskeleton becomes destabilized and cell morphology is altered. Our results demonstrate that alternative splicing defects of tau as found in FTDP-17 patients can be corrected by application of antisense oligonucleotides. These findings provide a tool to study specific tau isoforms in vivo and might lead to a novel therapeutic strategy for FTDP-17. PMID:11560926

Kalbfuss, B; Mabon, S A; Misteli, T

2001-09-17

291

Alternative splicing regulates kv3.1 polarized targeting to adjust maximal spiking frequency.  

PubMed

Synaptic inputs received at dendrites are converted into digital outputs encoded by action potentials generated at the axon initial segment in most neurons. Here, we report that alternative splicing regulates polarized targeting of Kv3.1 voltage-gated potassium (Kv) channels to adjust the input-output relationship. The spiking frequency of cultured hippocampal neurons correlated with the level of endogenous Kv3 channels. Expression of axonal Kv3.1b, the longer form of Kv3.1 splice variants, effectively converted slow-spiking young neurons to fast-spiking ones; this was not the case for Kv1.2 or Kv4.2 channel constructs. Despite having identical biophysical properties as Kv3.1b, dendritic Kv3.1a was significantly less effective at increasing the maximal firing frequency. This suggests a possible role of channel targeting in regulating spiking frequency. Mutagenesis studies suggest the electrostatic repulsion between the Kv3.1b N/C termini, created by its C-terminal splice domain, unmasks the Kv3.1b axonal targeting motif. Kv3.1b axonal targeting increased the maximal spiking frequency in response to prolonged depolarization. This finding was further supported by the results of local application of channel blockers and computer simulations. Taken together, our studies have demonstrated that alternative splicing controls neuronal firing rates by regulating the polarized targeting of Kv3.1 channels. PMID:22105078

Gu, Yuanzheng; Barry, Joshua; McDougel, Robert; Terman, David; Gu, Chen

2011-11-21

292

Alternative Splicing and Extensive RNA Editing of Human TPH2 Transcripts  

PubMed Central

Brain serotonin (5-HT) neurotransmission plays a key role in the regulation of mood and has been implicated in a variety of neuropsychiatric conditions. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis of 5-HT. Recently, we discovered a second TPH isoform (TPH2) in vertebrates, including man, which is predominantly expressed in brain, while the previously known TPH isoform (TPH1) is primarly a non-neuronal enzyme. Overwhelming evidence now points to TPH2 as a candidate gene for 5-HT-related psychiatric disorders. To assess the role of TPH2 gene variability in the etiology of psychiatric diseases we performed cDNA sequence analysis of TPH2 transcripts from human post mortem amygdala samples obtained from individuals with psychiatric disorders (drug abuse, schizophrenia, suicide) and controls. Here we show that TPH2 exists in two alternatively spliced variants in the coding region, denoted TPH2a and TPH2b. Moreover, we found evidence that the pre-mRNAs of both splice variants are dynamically RNA-edited in a mutually exclusive manner. Kinetic studies with cell lines expressing recombinant TPH2 variants revealed a higher activity of the novel TPH2B protein compared with the previously known TPH2A, whereas RNA editing was shown to inhibit the enzymatic activity of both TPH2 splice variants. Therefore, our results strongly suggest a complex fine-tuning of central nervous system 5-HT biosynthesis by TPH2 alternative splicing and RNA editing. Finally, we present molecular and large-scale linkage data evidencing that deregulated alternative splicing and RNA editing is involved in the etiology of psychiatric diseases, such as suicidal behaviour.

Grohmann, Maik; Hammer, Paul; Walther, Maria; Paulmann, Nils; Buttner, Andreas; Eisenmenger, Wolfgang; Baghai, Thomas C.; Schule, Cornelius; Rupprecht, Rainer; Bader, Michael; Bondy, Brigitta; Zill, Peter

2010-01-01

293

Self-splicing of a group IIC intron: 5? exon recognition and alternative 5? splicing events implicate the stem-loop motif of a transcriptional terminator  

PubMed Central

Bacterial IIC introns are a newly recognized subclass of group II introns whose ribozyme properties have not been characterized in detail. IIC introns are typically located downstream of transcriptional terminator motifs (inverted repeat followed by T's) or other inverted repeats in bacterial genomes. Here we have characterized the self-splicing activity of a IIC intron, B.h.I1, from Bacillus halodurans. B.h.I1 self-splices in vitro through hydrolysis to produce linear intron, but interestingly, additional unexpected products were formed that were highly dependent on ionic conditions. These products were determined to represent alternative splicing events at the 5? junction and cleavages throughout the RNA transcript. The alternative splicing and cleavage events occurred at cryptic splice sites containing stem–loop and IBS1 motifs, suggesting that the 5? exon is recognized by both elements. These results provide the first example of a group II intron that uses 5? splice sites nonadjacent to the ribozyme structure. Furthermore, the data suggest that IIC introns differ from IIA and IIB introns with respect to 5? exon definition, and that the terminator stem–loop substitutes in part for the missing IBS2–EBS2 (intron and exon binding sites 2) interaction.

Toor, Navtej; Robart, Aaron R.; Christianson, Joshua; Zimmerly, Steven

2006-01-01

294

Species- and tissue-specific expression of the C-terminal alternatively spliced form of the tumor suppressor p53.  

PubMed Central

Alternative splicing of the p53 transcript which so far has been demonstrated only in the murine system has been proposed as a general regulatory mechanism for the generation of functionally different p53 proteins. We analyzed by RT-PCR the pattern of p53 mRNAs within the region spanning exons 10 and 11 of the p53 gene in 13 different tissues from two independent mouse strains, in 10 different rat tissues and in six different human tissues. PCR products of the expected sizes, corresponding to the normally spliced and the alternatively spliced p53 mRNAs, were detected in mice. Alternatively spliced mRNA was found at approximately 25-20% the level of the normally spliced p53 mRNA in most tissues analyzed. In spleen and kidney the proportion of alternatively spliced p53 mRNA was much lower. Surprisingly, examination of p53 mRNAs isolated from 10 different rat tissues and six human tissues within the same region of the p53 gene showed only products of normal size. Although a potential homologous alternative 3' splice site within intron 10 of the human p53 gene is present in the genomic sequence of human p53, the expected corresponding alternatively spliced p53 mRNA was undetectable. These findings imply that the generation of functionally different forms of p53 by alternative splicing of p53 transcripts is a species-specific event, possibly indicating species-specific mechanisms for regulating p53 activities. Images

Will, K; Warnecke, G; Bergmann, S; Deppert, W

1995-01-01

295

Tailoring of Membrane Proteins by Alternative Splicing of Pre-mRNA†  

PubMed Central

Alternative splicing (ASfootnote_1) of RNA is a key mechanism for diversification of the eukaryotic proteome. In this process, different mRNA transcripts can be produced through altered excision/inclusion of exons during processing of the pre-mRNA molecule. Since its discovery, AS has been shown to play roles in protein structure, function, and localization. Dysregulation of this process can result in disease phenotypes. Moreover, AS pathways are promising therapeutic targets for a number of diseases. Integral membrane proteins (MPs) represent a class of proteins that may be particularly amenable to regulation by alternative splicing due to the distinctive topological restraints associated with their folding, structure, trafficking, and function. Here, we review the impact of AS on MP form and function, and the roles of AS in MP-related disorders such as Alzheimer’s disease.

Mittendorf, Kathleen F.; Deatherage, Catherine L.; Ohi, Melanie D.; Sanders, Charles R.

2012-01-01

296

Tailoring of membrane proteins by alternative splicing of pre-mRNA.  

PubMed

Alternative splicing (AS) of RNA is a key mechanism for diversification of the eukaryotic proteome. In this process, different mRNA transcripts can be produced through altered excision and/or inclusion of exons during processing of the pre-mRNA molecule. Since its discovery, AS has been shown to play roles in protein structure, function, and localization. Dysregulation of this process can result in disease phenotypes. Moreover, AS pathways are promising therapeutic targets for a number of diseases. Integral membrane proteins (MPs) represent a class of proteins that may be particularly amenable to regulation by alternative splicing because of the distinctive topological restraints associated with their folding, structure, trafficking, and function. Here, we review the impact of AS on MP form and function and the roles of AS in MP-related disorders such as Alzheimer's disease. PMID:22708632

Mittendorf, Kathleen F; Deatherage, Catherine L; Ohi, Melanie D; Sanders, Charles R

2012-06-29

297

Minor fibrillar collagens; variable regions alternative splicing, intrinsic disorder, and tyrosine sulfation  

PubMed Central

Minor fibrillar collagen types V and XI, are those less abundant than the fibrillar collagens types I, II and III. The alpha chains share a high degree of similarity with respect to protein sequence in all domains except the variable region. Genomic variation and, in some cases, extensive alternative splicing contribute to the unique sequence characteristics of the variable region. While unique expression patterns in tissues exist, the functions and biological relevance of the variable regions have not been elucidated. In this review, we summarize the existing knowledge about expression patterns and biological functions of the collagen types V and XI alpha chains. Analysis of biochemical similarities among the peptides encoded by each exon of the variable region suggest the potential for shared function. The alternative splicing, conservation of biochemical characteristics in light of low sequence conservation, and evidence for intrinsic disorder, suggests modulation of binding events between the surface of collagen fibrils and surrounding extracellular molecules as a shared function.

Fang, Ming; Jacob, Reed; McDougal, Owen; Oxford, Julia Thom

2012-01-01

298

ASTALAVISTA: dynamic and flexible analysis of alternative splicing events in custom gene datasets  

PubMed Central

In the process of establishing more and more complete annotations of eukaryotic genomes, a constantly growing number of alternative splicing (AS) events has been reported over the last decade. Consequently, the increasing transcript coverage also revealed the real complexity of some variations in the exon–intron structure between transcript variants and the need for computational tools to address ‘complex’ AS events. ASTALAVISTA (alternative splicing transcriptional landscape visualization tool) employs an intuitive and complete notation system to univocally identify such events. The method extracts AS events dynamically from custom gene annotations, classifies them into groups of common types and visualizes a comprehensive picture of the resulting AS landscape. Thus, ASTALAVISTA can characterize AS for whole transcriptome data from reference annotations (GENCODE, REFSEQ, ENSEMBL) as well as for genes selected by the user according to common functional/structural attributes of interest: http://genome.imim.es/astalavista

Foissac, Sylvain; Sammeth, Michael

2007-01-01

299

Borrelidin modulates the alternative splicing of VEGF in favour of anti-angiogenic isoforms.  

PubMed

The polyketide natural product borrelidin 1 is a potent inhibitor of angiogenesis and spontaneous metastasis. Affinity biopanning of a phage display library of colon tumor cell cDNAs identified the tandem WW domains of spliceosome-associated protein formin binding protein 21 (FBP21) as a novel molecular target of borrelidin, suggesting that borrelidin may act as a modulator of alternative splicing. In support of this idea, 1, and its more selective analog 2, bound to purified recombinant WW domains of FBP21. They also altered the ratio of vascular endothelial growth factor (VEGF) isoforms in retinal pigmented endothelial (RPE) cells in favour of anti-angiogenic isoforms. Transfection of RPE cells with FBP21 altered the ratio in favour of pro-angiogenic VEGF isoforms, an effect inhibited by 2. These data implicate FBP21 in the regulation of alternative splicing and suggest the potential of borrelidin analogs as tools to deconvolute key steps of spliceosome function. PMID:22822423

Woolard, Jeanette; Vousden, William; Moss, Steven J; Krishnakumar, Arjun; Gammons, Melissa Vr; Nowak, David G; Dixon, Neil; Micklefield, Jason; Spannhoff, Astrid; Bedford, Mark T; Gregory, Matthew A; Martin, Christine J; Leadlay, Peter F; Zhang, Ming Q; Harper, Steven J; Bates, David O; Wilkinson, Barrie

2011-02-01

300

Borrelidin modulates the alternative splicing of VEGF in favour of anti-angiogenic isoforms  

PubMed Central

The polyketide natural product borrelidin 1 is a potent inhibitor of angiogenesis and spontaneous metastasis. Affinity biopanning of a phage display library of colon tumor cell cDNAs identified the tandem WW domains of spliceosome-associated protein formin binding protein 21 (FBP21) as a novel molecular target of borrelidin, suggesting that borrelidin may act as a modulator of alternative splicing. In support of this idea, 1, and its more selective analog 2, bound to purified recombinant WW domains of FBP21. They also altered the ratio of vascular endothelial growth factor (VEGF) isoforms in retinal pigmented endothelial (RPE) cells in favour of anti-angiogenic isoforms. Transfection of RPE cells with FBP21 altered the ratio in favour of pro-angiogenic VEGF isoforms, an effect inhibited by 2. These data implicate FBP21 in the regulation of alternative splicing and suggest the potential of borrelidin analogs as tools to deconvolute key steps of spliceosome function.

2012-01-01

301

Alternative splicing of the mRNA encoding the human cholesteryl ester transfer protein  

SciTech Connect

The plasma cholesteryl ester transfer protein (CETP) is known to facilitate the transfer of lipids between plasma lipoproteins. The human CETP gene is a complex locus encompassing 16 exons. The CETP mRNA is found in liver and small intestine as well as in a variety of peripheral tissues. While the CETP cDNA from human adipose tissue was being cloned, a variant CETP cDNA was discovered which excluded the complete sequence encoded by exon 9, but which was otherwise identical to the full-length CETP cDNA, suggesting modification of the CETP gene transcript by an alternative RNA splicing mechanism. RNase protection analysis of tissue RNA confirmed the presence of exon 9 deleted transcripts and showed that they represented a variable proportion of the total CETP mRNA in various human tissues including adipose tissue (25%), liver (33%), and spleen (46%). Transient expression of the exon 9 deleted cDNA in COS cells or stable expression in CHO cells showed that the protein encoded by the alternatively spliced transcript was inactive in neutral lipid transfer, smaller, and poorly secreted compared to the protein derived from the full-length cDNA. Endo H digestion suggested that the inactive, cell-associated protein was present within the endoplasmic reticulum. The experiments show that the expression of the human CETP gene is modified by alternative splicing of the ninth exon, in a tissue-specific fashion. The function of alternative splicing is unknown but could serve to produce a protein with a function other than plasma neutral lipid transfer, or as an on-off switch to regulate the local concentration of biologically active protein.

Inazu, Akihiro; Quinet, E.M.; Suke Wang; Brown, M.L.; Stevenson, S.; Barr, M.L.; Moulin, P.; Tall, A.R. (Columbia Univ., New York, NY (United States))

1992-03-03

302

fruitless alternative splicing and sex behaviour in insects: an ancient and unforgettable love story?  

Microsoft Academic Search

Courtship behaviours are common features of animal species that reproduce sexually. Typically, males are involved in courting\\u000a females. Insects display an astonishing variety of courtship strategies primarily based on innate stereotyped responses to\\u000a various external stimuli. In Drosophila melanogaster, male courtship requires proteins encoded by the fruitless (fru) gene that are produced in different sex-specific isoforms via alternative splicing. Drosophila

Marco Salvemini; Catello Polito; Giuseppe Saccone

2010-01-01

303

The Evolution of Alternative Splicing in the Pax Family: The View from the Basal Chordate Amphioxus  

Microsoft Academic Search

Pax genes encode transcription factors critical for metazoan development. Large-scale gene duplication with subsequent gene losses\\u000a during vertebrate evolution has resulted in two human genes for each of the Pax1\\/9, Pax3\\/7, and Pax4\\/6 subfamilies and three for the Pax2\\/5\\/8 subfamily, compared to one each in the cephalochordate amphioxus. In addition, alternative splicing occurs in vertebrate\\u000a Pax transcripts from all four

Stephen Short; Linda Z. Holland

2008-01-01

304

A novel alternative splice variant of nicastrin and its implication in Alzheimer disease  

Microsoft Academic Search

Nicastrin interacts with ?-secretase complex components predominantly via the N-terminal third of the transmembrane domain. The authentic transmembrane domain is critically required for the interaction with ?-secretase complex components and for formation of an active ?-secretase complex. In this study, we have identified a novel alternatively spliced transcript of nicastrin in human brain tissue. This transcript (NCSTN-?E16) lacks exon 16

Noriaki Mitsuda; Hidehisa D. Yamagata; Wangtao Zhong; Mamoru Aoto; Hiroyasu Akatsu; Natsuko Uekawa; Kouzin Kamino; Keiko Taguchi; Takayuki Yamamoto; Mitsuo Maruyama; Kenji Kosaka; Masatoshi Takeda; Ikuko Kondo; Tetsuro Miki

2006-01-01

305

Alternative splicing of hMSH2 in normal human tissues  

Microsoft Academic Search

hMSH2 is a homolog of bacterial mutS and yeast Msh2, a member of the group of mismatch repair genes whose products bind to mismatched regions of double-stranded DNA. We analyzed\\u000a expression of hMSH2 in normal human organs by the polymerase chain reaction coupled with reverse transcription and found two novel types of alternatively\\u000a spliced mRNAs that were expressed in normal

Yuriko Mori; Hiromi Shiwaku; Shinichi Fukushige; Shigeru Wakatsuki; Masami Sato; Toshihiro Nukiwa; A. Horii

1997-01-01

306

Estimation of Alternative Splicing isoform Frequencies from RNA-Seq Data  

Microsoft Academic Search

\\u000a In this paper we present a novel expectation-maximization algorithm for inference of alternative splicing isoform frequencies\\u000a from high-throughput transcriptome sequencing (RNA-Seq) data. Our algorithm exploits disambiguation information provided by\\u000a the distribution of insert sizes generated during sequencing library preparation, and takes advantage of base quality scores,\\u000a strand and read pairing information if available. Empirical experiments on synthetic datasets show that

Marius Nicolae; Serghei Mangul; Ion I. Mandoiu; Alexander Zelikovsky

2010-01-01

307

Regulation of Alternative Splicing by SRrp86 and Its Interacting Proteins  

Microsoft Academic Search

SRrp86 is a unique member of the SR protein superfamily containing one RNA recognition motif and two serine-arginine (SR)-rich domains separated by an unusual glutamic acid-lysine (EK)-rich region. Previously, we showed that SRrp86 could regulate alternative splicing by both positively and negatively modulating the activity of other SR proteins and that the unique EK domain could inhibit both constitutive and

Jun Li; Ian C. Hawkins; Christopher D. Harvey; Jennifer L. Jennings; Andrew J. Link; James G. Patton

2003-01-01

308

Alternative splicing of the FGF antisense gene: differential subcellular localization in human tissues and esophageal adenocarcinoma  

Microsoft Academic Search

Overexpression of FGF-2 is associated with tumor recurrence and reduced survival after surgical resection of esophageal cancer,\\u000a and these risks are reduced in tumors co-expressing the FGF antisense (FGF-AS) RNA. The aim of this study was to characterize\\u000a the expression of alternatively spliced FGF-AS transcripts and encoded nudix-motif proteins in normal human tissues and in\\u000a esophageal adenocarcinoma, and to correlate

Shuo Cheng Zhang; Christie Barclay; Leigh Ann Alexander; Laurette Geldenhuys; Geoffrey A. Porter; Alan G. Casson; Paul R. Murphy

2007-01-01

309

Alternative splicing and differential subcellular localization of the rat FGF antisense gene product  

Microsoft Academic Search

BACKGROUND: GFG\\/NUDT is a nudix hydrolase originally identified as the product of the fibroblast growth factor-2 antisense (FGF-AS) gene. While the FGF-AS RNA has been implicated as an antisense regulator of FGF-2 expression, the expression and function of the encoded GFG protein is largely unknown. Alternative splicing of the primary FGF-AS mRNA transcript predicts multiple GFG isoforms in many species

Shuo Cheng Zhang; Kimberley A MacDonald; Mark Baguma-Nibasheka; Laurette Geldenhuys; Alan G Casson; Paul R Murphy

2008-01-01

310

Alternative Splicing in the Differentiation of Human Embryonic Stem Cells into Cardiac Precursors  

Microsoft Academic Search

The role of alternative splicing in self-renewal, pluripotency and tissue lineage specification of human embryonic stem cells (hESCs) is largely unknown. To better define these regulatory cues, we modified the H9 hESC line to allow selection of pluripotent hESCs by neomycin resistance and cardiac progenitors by puromycin resistance. Exon-level microarray expression data from undifferentiated hESCs and cardiac and neural precursors

Nathan Salomonis; Brandon Nelson; Karen Vranizan; Alexander R. Pico; Kristina Hanspers; Allan Kuchinsky; Linda Ta; Mark Mercola; Bruce R. Conklin

2009-01-01

311

beta subunits modulate alternatively spliced, large conductance, calcium-activated potassium channels of avian hair cells.  

PubMed

Electrical tuning confers frequency selectivity onto sensory hair cells in the auditory periphery of frogs, turtles, and chicks. The resonant frequency is determined in large part by the number and kinetics of large conductance, calcium-activated potassium (BK) channels. BK channels in hair cells are encoded by the alternatively spliced slo gene and may include an accessory beta subunit. Here we examine the origins of kinetic variability among BK channels by heterologous expression of avian cochlear slo cDNAs. Four alternatively spliced forms of the slo-alpha gene from chick hair cells were co-expressed with accessory beta subunits (from quail cochlea) by transient transfection of human embryonic kidney 293 cells. Addition of the beta subunit increased steady-state calcium affinity, raised the Hill coefficient for calcium binding, and slowed channel deactivation rates, resulting in eight functionally distinct channels. For example, a naturally occurring splice variant containing three additional exons deactivated 20-fold more slowly when combined with beta. Deactivation kinetics were used to predict tuning frequencies and thus tonotopic location if hair cells were endowed with each of the expressed channels. All beta-containing channels were predicted to lie within the apical (low-frequency) 30% of the epithelium, consistent with previous in situ hybridization studies. Individual slo-alpha exons would be found anywhere within the apical 70%, depending on the presence of beta, and other alternative exons. Alternative splicing of the slo-alpha channel message provides intrinsic variability in gating kinetics that is expanded to a wider range of tuning by modulation with beta subunits. PMID:10684869

Ramanathan, K; Michael, T H; Fuchs, P A

2000-03-01

312

Tissue-specific expression of two alternatively spliced isoforms of the human insulin receptor protein  

Microsoft Academic Search

Two insulin receptor mRNA species are expressed in human tissues as a result of alternative splicing of exon 11. This event is regulated in a tissue-specific manner. To date, there is little information about the relative abundance of the two receptor protein isoforms on the cell surface. The aim of the present investigation was to assess whether the tissue-specific expression

G. Sesti; A. N. Tullio; R. D'Alfonso; M. L. Napolitano; M. A. Marini; P. Borboni; R. Longhi; L. Albonici; A. Fusco; A. M. Aglianò; V. Manzari; R. Lauro

1994-01-01

313

Alternate mRNA Splicing in Multiple Human Tryptase Genes Is Predicted to Regulate Tetramer Formation*  

PubMed Central

Tryptases are serine proteases that are thought to be uniquely and proteolytically active as tetramers. Crystallographic studies reveal that the active tetramer is a flat ring structure composed of four monomers, with their active sites arranged around a narrow central pore. This model explains why many of the preferred substrates of tryptase are short peptides; however, it does not explain how tryptase cleaves large protein substrates such as fibronectin, although a number of studies have reported in vitro mechanisms for generating active monomers that could digest larger substrates. Here we suggest that alternate mRNA splicing of human tryptase genes generates active tryptase monomers (or dimers). We have identified a conserved pattern of alternate splicing in four tryptase alleles (?II, ?I, ?III, and ?I), representing three distinct tryptase gene loci. When compared with their full-length counterparts, the splice variants use an alternate acceptor site within exon 4. This results in the deletion of 27 nucleotides within the central coding sequence and 9 amino acids from the translated protein product. Although modeling suggests that the deletion can be easily accommodated by the enzymes structurally, it is predicted to alter the specificity by enlarging the S1? or S2? binding pocket and results in the complete loss of the “47 loop,” reported to be critical for the formation of tetramers. Although active monomers can be generated in vitro using a range of artificial conditions, we suggest that alternate splicing is the in vivo mechanism used to generate active tryptase that can cleave large protein substrates.

Jackson, Nicole E.; Wang, Hong-Wei; Bryant, Katherine J.; McNeil, H. Patrick; Husain, Ahsan; Liu, Ke; Tedla, Nicodemus; Thomas, Paul S.; King, Garry C.; Hettiaratchi, Anusha; Cairns, Jennifer; Hunt, John E.

2008-01-01

314

Alternate mRNA splicing in multiple human tryptase genes is predicted to regulate tetramer formation.  

PubMed

Tryptases are serine proteases that are thought to be uniquely and proteolytically active as tetramers. Crystallographic studies reveal that the active tetramer is a flat ring structure composed of four monomers, with their active sites arranged around a narrow central pore. This model explains why many of the preferred substrates of tryptase are short peptides; however, it does not explain how tryptase cleaves large protein substrates such as fibronectin, although a number of studies have reported in vitro mechanisms for generating active monomers that could digest larger substrates. Here we suggest that alternate mRNA splicing of human tryptase genes generates active tryptase monomers (or dimers). We have identified a conserved pattern of alternate splicing in four tryptase alleles (alphaII, betaI, betaIII, and deltaI), representing three distinct tryptase gene loci. When compared with their full-length counterparts, the splice variants use an alternate acceptor site within exon 4. This results in the deletion of 27 nucleotides within the central coding sequence and 9 amino acids from the translated protein product. Although modeling suggests that the deletion can be easily accommodated by the enzymes structurally, it is predicted to alter the specificity by enlarging the S1' or S2' binding pocket and results in the complete loss of the "47 loop," reported to be critical for the formation of tetramers. Although active monomers can be generated in vitro using a range of artificial conditions, we suggest that alternate splicing is the in vivo mechanism used to generate active tryptase that can cleave large protein substrates. PMID:18854315

Jackson, Nicole E; Wang, Hong-Wei; Bryant, Katherine J; McNeil, H Patrick; Husain, Ahsan; Liu, Ke; Tedla, Nicodemus; Thomas, Paul S; King, Garry C; Hettiaratchi, Anusha; Cairns, Jennifer; Hunt, John E

2008-10-14

315

Alternative Splicing Regulates Prdm1/Blimp-1 DNA Binding Activities and Corepressor Interactions  

PubMed Central

Prdm1/Blimp-1 is a master regulator of gene expression in diverse tissues of the developing embryo and adult organism. Its C-terminal zinc finger domain mediates nuclear import, DNA binding, and recruitment of the corepressors G9a and HDAC1/2. Alternatively spliced transcripts lacking exon 7 sequences encode a structurally divergent isoform (Blimp-1?exon7) predicted to have distinct functions. Here we demonstrate that the short Blimp-1?exon7 isoform lacks DNA binding activity and fails to bind G9a or HDAC1/2 but retains the ability to interact with PRMT5. To investigate functional roles of alternative splicing in vivo, we engineered novel mouse strains via embryonic stem (ES) cell technology. Like null mutants, embryos carrying a targeted deletion of exon 7 and exclusively expressing Blimp-1?exon7 die at around embryonic day 10.5 (E10.5) due to placental defects. In heterozygous ?exon7 mice, there is no evidence of dominant-negative effects. Mice carrying a knock-in allele with an exon 6-exon 7 fusion express full-length Blimp-1 only, develop normally, are healthy and fertile as adults, and efficiently generate mature plasma cells. These findings strongly suggest that the short Blimp-1?exon7 isoform is dispensable. We propose that developmentally regulated alternative splicing is influenced by chromatin structure at the locus and fine-tunes Blimp-1's functional capabilities.

Morgan, Marc A. J.; Mould, Arne W.; Li, Li; Robertson, Elizabeth J.

2012-01-01

316

Identification of an Alternate Splice Form of Tapasin in Human Melanoma  

PubMed Central

Assembly of MHC class I molecules with peptide in the endoplasmic reticulum requires the assistance of tapasin. Alternative splicing, which is known to regulate many genes, has been reported for tapasin only in the context of mutations. Here, we report on an alternate splice form of tapasin (tpsn?Ex3) derived from a human melanoma cell line that does not appear to be due to mutations. Excision of exon 3 results in deletion of amino acids 70 to 156 within the beta barrel region, but the membrane proximal Ig domain, the transmembrane domain and cytoplasmic tail of tapasin are intact. Introduction of tpsn?Ex3 into a tapasin deficient cell line does not restore MHC class I expression at the cell surface. Similar to a previously described tapasin mutant (tpsn?N50), tpsn?Ex3 interacts with TAP. Therefore we utilized these altered forms of tapasin to test the importance of MHC class I interaction with TAP. In the presence of wild type tapasin, transfection of tpsn?N50, but not tpsn?Ex3, reduced MHC class I expression at the cell surface likely due its ability to compete MHC class I molecules from TAP. Together these findings suggest that tumor cells may contain alternate splice forms of tapasin which may regulate MHC class I antigen presentation.

Belicha-Villanueva, Alan; Golding, Michelle; McEvoy, Sarah; Sarvaiya, Nilofar; Cresswell, Peter; Gollnick, Sandra O.; Bangia, Naveen

2010-01-01

317

KISSPLICE: de-novo calling alternative splicing events from RNA-seq data  

PubMed Central

Background In this paper, we address the problem of identifying and quantifying polymorphisms in RNA-seq data when no reference genome is available, without assembling the full transcripts. Based on the fundamental idea that each polymorphism corresponds to a recognisable pattern in a De Bruijn graph constructed from the RNA-seq reads, we propose a general model for all polymorphisms in such graphs. We then introduce an exact algorithm, called KISSPLICE, to extract alternative splicing events. Results We show that KISSPLICE enables to identify more correct events than general purpose transcriptome assemblers. Additionally, on a 71 M reads dataset from human brain and liver tissues, KISSPLICE identified 3497 alternative splicing events, out of which 56% are not present in the annotations, which confirms recent estimates showing that the complexity of alternative splicing has been largely underestimated so far. Conclusions We propose new models and algorithms for the detection of polymorphism in RNA-seq data. This opens the way to a new kind of studies on large HTS RNA-seq datasets, where the focus is not the global reconstruction of full-length transcripts, but local assembly of polymorphic regions. KISSPLICE is available for download at http://alcovna.genouest.org/kissplice/.

2012-01-01

318

Alternative splicing regulates Prdm1/Blimp-1 DNA binding activities and corepressor interactions.  

PubMed

Prdm1/Blimp-1 is a master regulator of gene expression in diverse tissues of the developing embryo and adult organism. Its C-terminal zinc finger domain mediates nuclear import, DNA binding, and recruitment of the corepressors G9a and HDAC1/2. Alternatively spliced transcripts lacking exon 7 sequences encode a structurally divergent isoform (Blimp-1?exon7) predicted to have distinct functions. Here we demonstrate that the short Blimp-1?exon7 isoform lacks DNA binding activity and fails to bind G9a or HDAC1/2 but retains the ability to interact with PRMT5. To investigate functional roles of alternative splicing in vivo, we engineered novel mouse strains via embryonic stem (ES) cell technology. Like null mutants, embryos carrying a targeted deletion of exon 7 and exclusively expressing Blimp-1?exon7 die at around embryonic day 10.5 (E10.5) due to placental defects. In heterozygous ?exon7 mice, there is no evidence of dominant-negative effects. Mice carrying a knock-in allele with an exon 6-exon 7 fusion express full-length Blimp-1 only, develop normally, are healthy and fertile as adults, and efficiently generate mature plasma cells. These findings strongly suggest that the short Blimp-1?exon7 isoform is dispensable. We propose that developmentally regulated alternative splicing is influenced by chromatin structure at the locus and fine-tunes Blimp-1's functional capabilities. PMID:22733990

Morgan, Marc A J; Mould, Arne W; Li, Li; Robertson, Elizabeth J; Bikoff, Elizabeth K

2012-06-25

319

Eye development under the control of SRp55/B52-mediated alternative splicing of eyeless.  

PubMed

The genetic programs specifying eye development are highly conserved during evolution and involve the vertebrate Pax-6 gene and its Drosophila melanogaster homolog eyeless (ey). Here we report that the SR protein B52/SRp55 controls a novel developmentally regulated splicing event of eyeless that is crucial for eye growth and specification in Drosophila. B52/SRp55 generates two isoforms of eyeless differing by an alternative exon encoding a 60-amino-acid insert at the beginning of the paired domain. The long isoform has impaired ability to trigger formation of ectopic eyes and to bind efficiently Eyeless target DNA sequences in vitro. When over-produced in the eye imaginal disc, this isoform induces a small eye phenotype, whereas the isoform lacking the alternative exon triggers eye over-growth and strong disorganization. Our results suggest that B52/SRp55 splicing activity is used during normal eye development to control eye organogenesis and size through regulation of eyeless alternative splicing. PMID:17327915

Fic, Weronika; Juge, François; Soret, Johann; Tazi, Jamal

2007-02-28

320

Auto- and cross-regulation of the hnRNP L proteins by alternative splicing.  

PubMed

We recently characterized human hnRNP L as a global regulator of alternative splicing, binding to CA-repeat and CA-rich elements. Here we report that hnRNP L autoregulates its own expression on the level of alternative splicing. Intron 6 of the human hnRNP L gene contains a short exon that, if used, introduces a premature termination codon, resulting in nonsense-mediated decay (NMD). This "poison exon" is preceded by a highly conserved CA-rich cluster extending over 800 nucleotides that binds hnRNP L and functions as an unusually extended, intronic enhancer, promoting inclusion of the poison exon. As a result, excess hnRNP L activates NMD of its own mRNA, thereby creating a negative autoregulatory feedback loop and contributing to homeostasis of hnRNP L levels. We present experimental evidence for this mechanism, based on NMD inactivation, hnRNP L binding assays, and hnRNP L-dependent alternative splicing of heterologous constructs. In addition, we demonstrate that hnRNP L cross-regulates inclusion of an analogous poison exon in the hnRNP L-like pre-mRNA, which explains the reciprocal expression of the two closely related hnRNP L proteins. PMID:19124611

Rossbach, Oliver; Hung, Lee-Hsueh; Schreiner, Silke; Grishina, Inna; Heiner, Monika; Hui, Jingyi; Bindereif, Albrecht

2009-01-05

321

Auto- and Cross-Regulation of the hnRNP L Proteins by Alternative Splicing? ‡  

PubMed Central

We recently characterized human hnRNP L as a global regulator of alternative splicing, binding to CA-repeat and CA-rich elements. Here we report that hnRNP L autoregulates its own expression on the level of alternative splicing. Intron 6 of the human hnRNP L gene contains a short exon that, if used, introduces a premature termination codon, resulting in nonsense-mediated decay (NMD). This “poison exon” is preceded by a highly conserved CA-rich cluster extending over 800 nucleotides that binds hnRNP L and functions as an unusually extended, intronic enhancer, promoting inclusion of the poison exon. As a result, excess hnRNP L activates NMD of its own mRNA, thereby creating a negative autoregulatory feedback loop and contributing to homeostasis of hnRNP L levels. We present experimental evidence for this mechanism, based on NMD inactivation, hnRNP L binding assays, and hnRNP L-dependent alternative splicing of heterologous constructs. In addition, we demonstrate that hnRNP L cross-regulates inclusion of an analogous poison exon in the hnRNP L-like pre-mRNA, which explains the reciprocal expression of the two closely related hnRNP L proteins.

Rossbach, Oliver; Hung, Lee-Hsueh; Schreiner, Silke; Grishina, Inna; Heiner, Monika; Hui, Jingyi; Bindereif, Albrecht

2009-01-01

322

The Ewing sarcoma protein regulates DNA damage-induced alternative splicing.  

PubMed

The Ewing sarcoma (EWS) protein is a member of the TET (TLS/EWS/TAF15) family of RNA- and DNA-binding proteins whose expression is altered in cancer. We report that EWS depletion results in alternative splicing changes of genes involved in DNA repair and genotoxic stress signaling, including ABL1, CHEK2, and MAP4K2. Chromatin and RNA crosslinking immunoprecipitation results indicate that EWS cotranscriptionally binds to its target RNAs. This association is reduced upon irradiation of cells with ultraviolet light, concomitant with transient enrichment of EWS in nucleoli and with alternative splicing changes that parallel those induced by EWS depletion and that lead to reduced c-ABL protein expression. Consistent with the functional relevance of EWS-mediated alternative splicing regulation in DNA damage response, EWS depletion reduces cell viability and proliferation upon UV irradiation, effects that are attenuated by restoring c-ABL expression. These results provide insights into posttranscriptional mechanisms of DNA damage response by a TET protein. PMID:21816343

Paronetto, Maria Paola; Miñana, Belén; Valcárcel, Juan

2011-08-01

323

ASPicDB: a database of annotated transcript and protein variants generated by alternative splicing  

PubMed Central

Alternative splicing is emerging as a major mechanism for the expansion of the transcriptome and proteome diversity, particularly in human and other vertebrates. However, the proportion of alternative transcripts and proteins actually endowed with functional activity is currently highly debated. We present here a new release of ASPicDB which now provides a unique annotation resource of human protein variants generated by alternative splicing. A total of 256?939 protein variants from 17?191 multi-exon genes have been extensively annotated through state of the art machine learning tools providing information of the protein type (globular and transmembrane), localization, presence of PFAM domains, signal peptides, GPI-anchor propeptides, transmembrane and coiled-coil segments. Furthermore, full-length variants can be now specifically selected based on the annotation of CAGE-tags and polyA signal and/or polyA sites, marking transcription initiation and termination sites, respectively. The retrieval can be carried out at gene, transcript, exon, protein or splice site level allowing the selection of data sets fulfilling one or more features settled by the user. The retrieval interface also enables the selection of protein variants showing specific differences in the annotated features. ASPicDB is available at http://www.caspur.it/ASPicDB/.

Martelli, Pier L.; D'Antonio, Mattia; Bonizzoni, Paola; Castrignano, Tiziana; D'Erchia, Anna M.; D'Onorio De Meo, Paolo; Fariselli, Piero; Finelli, Michele; Licciulli, Flavio; Mangiulli, Marina; Mignone, Flavio; Pavesi, Giulio; Picardi, Ernesto; Rizzi, Raffaella; Rossi, Ivan; Valletti, Alessio; Zauli, Andrea; Zambelli, Federico; Casadio, Rita; Pesole, Graziano

2011-01-01

324

Alternative splicing of lola generates 19 transcription factors controlling axon guidance in Drosophila.  

PubMed

The Drosophila melanogaster transcription factor Lola (longitudinals lacking) is a pivotal regulator of neural wiring that sets the precise expression levels of proteins that execute specific axon guidance decisions. Lola has a zinc finger DNA binding domain and a BTB (for Broad-complex, Tramtrack and Bric a brac) dimerization motif. We now show that alternative splicing of the lola gene creates a family of 19 transcription factors. All lola isoforms share a common dimerization domain, but 17 have their own unique DNA-binding domains. Seven of these 17 isoforms are present in the distantly-related Dipteran Anopheles gambiae, suggesting that the properties of specific isoforms are likely to be critical to lola function. Analysis of the expression patterns of individual splice variants and of the phenotypes of mutants lacking single isoforms supports this idea and establishes that the alternative forms of lola are responsible for different functions of this gene. Thus, in this system, the alternative splicing of a key transcription factor helps to explain how a small genome encodes all the information that is necessary to specify the enormous diversity of axonal trajectories. PMID:12897787

Goeke, Scott; Greene, Elizabeth A; Grant, Paul K; Gates, Michael A; Crowner, Daniel; Aigaki, Toshiro; Giniger, Edward

2003-09-01

325

Alternative splicing of the mouse embryonic poly(A) binding protein (Epab) mRNA is regulated by an exonic splicing enhancer: a model for post-transcriptional control of gene expression in the oocyte  

Microsoft Academic Search

Embryonic poly(A) binding protein (EPAB), expressed in oocytes and early embryos, binds and stabilizes maternal mRNAs, and mediates initiation of their translation. We identified an alternatively spliced form of Epab lacking exon 10 (c.Ex10del) and inves- tigated the regulation of Epab mRNA alternative splicing as a model for alternative splicing in oocytes and early preimplantation embryos. Specifically, we evaluated the

Emre Seli; Aylin Yaba; Ozlem Guzeloglu-Kayisli; Maria D. Lalioti

2008-01-01

326

Review: Alternative Splicing (AS) of Genes As An Approach for Generating Protein Complexity  

PubMed Central

Prior to the completion of the human genome project, the human genome was thought to have a greater number of genes as it seemed structurally and functionally more complex than other simpler organisms. This along with the belief of “one gene, one protein”, were demonstrated to be incorrect. The inequality in the ratio of gene to protein formation gave rise to the theory of alternative splicing (AS). AS is a mechanism by which one gene gives rise to multiple protein products. Numerous databases and online bioinformatic tools are available for the detection and analysis of AS. Bioinformatics provides an important approach to study mRNA and protein diversity by various tools such as expressed sequence tag (EST) sequences obtained from completely processed mRNA. Microarrays and deep sequencing approaches also aid in the detection of splicing events. Initially it was postulated that AS occurred only in about 5% of all genes but was later found to be more abundant. Using bioinformatic approaches, the level of AS in human genes was found to be fairly high with 35-59% of genes having at least one AS form. Our ability to determine and predict AS is important as disorders in splicing patterns may lead to abnormal splice variants resulting in genetic diseases. In addition, the diversity of proteins produced by AS poses a challenge for successful drug discovery and therefore a greater understanding of AS would be beneficial.

Roy, Bishakha; Haupt, Larisa M; Griffiths, Lyn R

2013-01-01

327

Review: Alternative Splicing (AS) of Genes As An Approach for Generating Protein Complexity.  

PubMed

Prior to the completion of the human genome project, the human genome was thought to have a greater number of genes as it seemed structurally and functionally more complex than other simpler organisms. This along with the belief of "one gene, one protein", were demonstrated to be incorrect. The inequality in the ratio of gene to protein formation gave rise to the theory of alternative splicing (AS). AS is a mechanism by which one gene gives rise to multiple protein products. Numerous databases and online bioinformatic tools are available for the detection and analysis of AS. Bioinformatics provides an important approach to study mRNA and protein diversity by various tools such as expressed sequence tag (EST) sequences obtained from completely processed mRNA. Microarrays and deep sequencing approaches also aid in the detection of splicing events. Initially it was postulated that AS occurred only in about 5% of all genes but was later found to be more abundant. Using bioinformatic approaches, the level of AS in human genes was found to be fairly high with 35-59% of genes having at least one AS form. Our ability to determine and predict AS is important as disorders in splicing patterns may lead to abnormal splice variants resulting in genetic diseases. In addition, the diversity of proteins produced by AS poses a challenge for successful drug discovery and therefore a greater understanding of AS would be beneficial. PMID:24179441

Roy, Bishakha; Haupt, Larisa M; Griffiths, Lyn R

2013-05-01

328

An alternatively spliced cadherin-11 enhances human breast cancer cell invasion.  

PubMed

Although reduced levels of the epithelial cell adhesion molecule E-cadherin are often associated with poorly differentiated breast cancers, recent studies show that expression of other cadherins such as N-cadherin, P-cadherin, and the mesenchymal cadherin-11 is actually elevated in invasive breast cancers and cell lines. Cadherin-11 is unique among cadherins in that it exists as two alternatively spliced forms that are expressed together in the same cell. We now show that expression of wild-type cadherin-11, with or without coexpression of the COOH-terminal truncated splice variant, promotes epithelial differentiation of the cadherin-negative SKBR3 cell line. Exogenous wild-type cadherin-11 association with and membrane recruitment of beta-catenin and p120 are unaffected by coexpression of the truncated variant. Cadherin-11-expressing cells exhibit modest changes in cell proliferation and no change in anchorage-independent growth. However, coexpression of wild-type cadherin-11 and the splice variant promotes a dramatic increase in the ability of SKBR3 cells and E-cadherin-positive MCF7 cells to traverse Matrigel-coated filters. Biochemical studies indicate that the truncated variant may be secreted from the cell and/or enter a detergent-insoluble compartment. These data suggest that the presence of the cadherin-11 splice variant promotes invasion of cadherin-11-positive breast cancer cells. PMID:12438268

Feltes, Carolyn M; Kudo, Akira; Blaschuk, Orest; Byers, Stephen W

2002-11-15

329

Regulation of Neurexin 1[beta] Tertiary Structure and Ligand Binding through Alternative Splicing  

SciTech Connect

Neurexins and neuroligins play an essential role in synapse function, and their alterations are linked to autistic spectrum disorder. Interactions between neurexins and neuroligins regulate inhibitory and excitatory synaptogenesis in vitro through a splice-insert signaling code. In particular, neurexin 1{beta} carrying an alternative splice insert at site SS{number_sign}4 interacts with neuroligin 2 (found predominantly at inhibitory synapses) but much less so with other neuroligins (those carrying an insert at site B and prevalent at excitatory synapses). The structure of neurexin 1{beta}+SS{number_sign}4 reveals dramatic rearrangements to the 'hypervariable surface', the binding site for neuroligins. The splice insert protrudes as a long helix into space, triggers conversion of loop {beta}10-{beta}11 into a helix rearranging the binding site for neuroligins, and rearranges the Ca{sup 2+}-binding site required for ligand binding, increasing its affinity. Our structures reveal the mechanism by which neurexin 1{beta} isoforms acquire neuroligin splice isoform selectivity.

Shen, Kaiser C.; Kuczynska, Dorota A.; Wu, Irene J.; Murray, Beverly H.; Sheckler, Lauren R.; Rudenko, Gabby (Michigan)

2008-08-04

330

Extensive alternative splicing and dual promoter usage generates TCF-1 protein isoforms with differential transcription control properties  

Microsoft Academic Search

Previously, we reported the isolation of cDNA clones representing four alternative splice forms of TCF-1, a T-cell-specific transcription factor. In the present study, Western blotting (immunoblotting) yielded a multi- tude of TCF-1 proteins ranging from 25 to 55 kDa, a pattern not simply explained from the known splice alternatives. Subsequent cDNA cloning, PCR amplification, and analysis by rapid amplification of

M. van de Wetering; JAN CASTROP; VLADIMIR KORINEK; J. C. Clevers

1996-01-01

331

Computational analysis of candidate intron regulatory elements for tissue-specific alternative pre-mRNA splicing  

Microsoft Academic Search

Alternative pre-mRNA splicing is a major cellular process by which functionally diverse proteins can be generated from the primary transcript of a single gene, often in tissue-specific patterns. The current study investigates the hypothesis that splicing of tissue-specific alternative exons is regulated in part by control sequences in adjacent introns and that such elements may be recognized via computational analysis

Michael Brudno; Mikhail S. Gelfand; Sylvia Spengler; Manfred Zorn; Inna Dubchak; John G. Conboy

2001-01-01

332

Alternative Splicing Regulation of Cancer-Related Pathways in Caenorhabditis elegans: An In Vivo Model System with a Powerful Reverse Genetics Toolbox  

PubMed Central

Alternative splicing allows for the generation of protein diversity and fine-tunes gene expression. Several model systems have been used for the in vivo study of alternative splicing. Here we review the use of the nematode Caenorhabditis elegans to study splicing regulation in vivo. Recent studies have shown that close to 25% of genes in the worm genome undergo alternative splicing. A big proportion of these events are functional, conserved, and under strict regulation either across development or other conditions. Several techniques like genome-wide RNAi screens and bichromatic reporters are available for the study of alternative splicing in worms. In this review, we focus, first, on the main studies that have been performed to dissect alternative splicing in this system and later on examples from genes that have human homologs that are implicated in cancer. The significant advancement towards understanding the regulation of alternative splicing and cancer that the C. elegans system has offered is discussed.

Barberan-Soler, Sergio; Ragle, James Matthew

2013-01-01

333

RNA-Seq analysis in mutant zebrafish reveals role of U1C protein in alternative splicing regulation  

PubMed Central

Precise 5? splice-site recognition is essential for both constitutive and regulated pre-mRNA splicing. The U1 small nuclear ribonucleoprotein particle (snRNP)-specific protein U1C is involved in this first step of spliceosome assembly and important for stabilizing early splicing complexes. We used an embryonically lethal U1C mutant zebrafish, hi1371, to investigate the potential genomewide role of U1C for splicing regulation. U1C mutant embryos contain overall stable, but U1C-deficient U1 snRNPs. Surprisingly, genomewide RNA-Seq analysis of mutant versus wild-type embryos revealed a large set of specific target genes that changed their alternative splicing patterns in the absence of U1C. Injection of ZfU1C cRNA into mutant embryos and in vivo splicing experiments in HeLa cells after siRNA-mediated U1C knockdown confirmed the U1C dependency and specificity, as well as the functional conservation of the effects observed. In addition, sequence motif analysis of the U1C-dependent 5? splice sites uncovered an association with downstream intronic U-rich elements. In sum, our findings provide evidence for a new role of a general snRNP protein, U1C, as a mediator of alternative splicing regulation.

Rosel, Tanja Dorothe; Hung, Lee-Hsueh; Medenbach, Jan; Donde, Katrin; Starke, Stefan; Benes, Vladimir; Ratsch, Gunnar; Bindereif, Albrecht

2011-01-01

334

Phosphorylation-Dependent Regulation of PSF by GSK3 controls CD45 Alternative Splicing  

PubMed Central

Signal-induced alternative splicing of the CD45 gene in human T cells is essential for proper immune function. Skipping of the CD45 variable exons is controlled, in large part, by the recruitment of PSF to the pre-mRNA substrate upon T cell activation; however, the signaling cascade leading to exon exclusion has remained elusive. Here we demonstrate that in resting T cells PSF is directly phosphorylated by GSK3 thus promoting interaction of PSF with TRAP150 which prevents PSF from binding CD45 pre-mRNA. Upon T cell activation, reduced GSK3 activity leads to reduced PSF phosphorylation, releasing PSF from TRAP150 and allowing it to bind CD45 splicing regulatory elements and repress exon inclusion. Our data place two new players, GSK3 and TRAP150, in the complex network that regulates CD45 alternative splicing and demonstrate a new paradigm for signal transduction from the cell surface to the RNA processing machinery through the multi-functional protein PSF.

Heyd, Florian; Lynch, Kristen W.

2010-01-01

335

Oxidants induce alternative splicing of alpha-synuclein: Implications for Parkinson's disease.  

PubMed

alpha-Synuclein (alpha-syn) is a presynaptic protein that is widely implicated in the pathophysiology of Parkinson's disease (PD). Emerging evidence indicates a strong correlation between alpha-syn aggregation and proteasomal dysfunction as one of the major pathways responsible for destruction of the dopamine neurons. Using parkinsonism mimetics (MPP(+), rotenone) and related oxidants, we have identified an oxidant-induced alternative splicing of alpha-syn mRNA, generating a shorter isoform of alpha-syn with deleted exon-5 (112-syn). This spliced isoform has an altered localization and profoundly inhibits proteasomal function. The generation of 112-syn was suppressed by constitutively active MEK-1 and enhanced by inhibition of the Erk-MAP kinase pathway. Overexpression of 112-syn exacerbated cell death in a human dopaminergic cell line compared to full-length protein. Expression of 112-syn and proteasomal dysfunction were also evident in the substantia nigra and to a lesser extent in striatum, but not in the cortex of MPTP-treated mice. We conclude that oxidant-induced alternative splicing of alpha-syn plays a crucial role in the mechanism of dopamine neuron cell death and thus contributes to PD. PMID:19857570

Kalivendi, Shasi V; Yedlapudi, Deepthi; Hillard, Cecilia J; Kalyanaraman, B

2009-10-23

336

The distribution of phosphorylated SR proteins and alternative splicing are regulated by RANBP2  

PubMed Central

The mammalian cell nucleus is functionally compartmentalized into various substructures. Nuclear speckles, also known as interchromatin granule clusters, are enriched with SR splicing factors and are implicated in gene expression. Here we report that nuclear speckle formation is developmentally regulated; in certain cases phosphorylated SR proteins are absent from the nucleus and are instead localized at granular structures in the cytoplasm. To investigate how the nuclear architecture is formed, we performed a phenotypic screen of HeLa cells treated with a series of small interfering RNAs. Depletion of Ran-binding protein 2 induced cytoplasmic intermediates of nuclear speckles in G1 phase. Detailed analyses of these structures suggested that a late step in the sequential nuclear entry of mitotic interchromatin granule components was disrupted and that phosphorylated SR proteins were sequestered in an SR protein kinase–dependent manner. As a result, the cells had an imbalanced subcellular distribution of phosphorylated and hypophosphorylated SR proteins, which affected alternative splicing patterns. This study demonstrates that the speckled distribution of phosphorylated pre-mRNA processing factors is regulated by the nucleocytoplasmic transport system in mammalian cells and that it is important for alternative splicing.

Saitoh, Noriko; Sakamoto, Chiyomi; Hagiwara, Masatoshi; Agredano-Moreno, Lourdes T.; Jimenez-Garcia, Luis Felipe; Nakao, Mitsuyoshi

2012-01-01

337

Distinct types of disorder in the human proteome: functional implications for alternative splicing.  

PubMed

Intrinsically disordered regions have been associated with various cellular processes and are implicated in several human diseases, but their exact roles remain unclear. We previously defined two classes of conserved disordered regions in budding yeast, referred to as "flexible" and "constrained" conserved disorder. In flexible disorder, the property of disorder has been positionally conserved during evolution, whereas in constrained disorder, both the amino acid sequence and the property of disorder have been conserved. Here, we show that flexible and constrained disorder are widespread in the human proteome, and are particularly common in proteins with regulatory functions. Both classes of disordered sequences are highly enriched in regions of proteins that undergo tissue-specific (TS) alternative splicing (AS), but not in regions of proteins that undergo general (i.e., not tissue-regulated) AS. Flexible disorder is more highly enriched in TS alternative exons, whereas constrained disorder is more highly enriched in exons that flank TS alternative exons. These latter regions are also significantly more enriched in potential phosphosites and other short linear motifs associated with cell signaling. We further show that cancer driver mutations are significantly enriched in regions of proteins associated with TS and general AS. Collectively, our results point to distinct roles for TS alternative exons and flanking exons in the dynamic regulation of protein interaction networks in response to signaling activity, and they further suggest that alternatively spliced regions of proteins are often functionally altered by mutations responsible for cancer. PMID:23633940

Colak, Recep; Kim, TaeHyung; Michaut, Magali; Sun, Mark; Irimia, Manuel; Bellay, Jeremy; Myers, Chad L; Blencowe, Benjamin J; Kim, Philip M

2013-04-25

338

Whole genome exon arrays identify differential expression of alternatively spliced, cancer-related genes in lung cancer  

Microsoft Academic Search

Alternative processing of pre-mRNA transcripts is a major source of protein diversity in eukaryotes and has been implicated in several disease processes including cancer. In this study we have performed a genome wide analysis of alternative splicing events in lung adenocarcinoma. We found that 2369 of the 17800 core Refseq genes appear to have alternative transcripts that are differentially expressed

Liqiang Xi; Andrew Feber; Vanita Gupta; Maoxin Wu; Andrew D. Bergemann; Rodney J. Landreneau; Virginia R. Litle; Arjun Pennathur; James D. Luketich; Tony E. Godfrey

2008-01-01

339

Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts  

PubMed Central

Background Alternative splicing (AS) is a central mechanism in the generation of genomic complexity and is a major contributor to transcriptome and proteome diversity. Alterations of the splicing process can lead to deregulation of crucial cellular processes and have been associated with a large spectrum of human diseases. Cancer-associated transcripts are potential molecular markers and may contribute to the development of more accurate diagnostic and prognostic methods and also serve as therapeutic targets. Alternative splicing-enriched cDNA libraries have been used to explore the variability generated by alternative splicing. In this study, by combining the use of trapping heteroduplexes and RNA amplification, we developed a powerful approach that enables transcriptome-wide exploration of the AS repertoire for identifying AS variants associated with breast tumor cells modulated by ERBB2 (HER-2/neu) oncogene expression. Results The human breast cell line (C5.2) and a pool of 5 ERBB2 over-expressing breast tumor samples were used independently for the construction of two AS-enriched libraries. In total, 2,048 partial cDNA sequences were obtained, revealing 214 alternative splicing sequence-enriched tags (ASSETs). A subset with 79 multiple exon ASSETs was compared to public databases and reported 138 different AS events. A high success rate of RT-PCR validation (94.5%) was obtained, and 2 novel AS events were identified. The influence of ERBB2-mediated expression on AS regulation was evaluated by capillary electrophoresis and probe-ligation approaches in two mammary cell lines (Hb4a and C5.2) expressing different levels of ERBB2. The relative expression balance between AS variants from 3 genes was differentially modulated by ERBB2 in this model system. Conclusions In this study, we presented a method for exploring AS from any RNA source in a transcriptome-wide format, which can be directly easily adapted to next generation sequencers. We identified AS transcripts that were differently modulated by ERBB2-mediated expression and that can be tested as molecular markers for breast cancer. Such a methodology will be useful for completely deciphering the cancer cell transcriptome diversity resulting from AS and for finding more precise molecular markers.

2010-01-01

340

Complex changes in alternative pre-mRNA splicing play a central role in the Epithelial-Mesenchymal Transition (EMT)  

PubMed Central

The epithelial to mesenchymal transition (EMT) is an important developmental process that is also implicated in disease pathophysiology, such as cancer progression and metastasis. A wealth of literature in recent years has identified important transcriptional regulators and large-scale changes in gene expression programs that drive the phenotypic changes that occur during the EMT. However, in the past couple of years it has become apparent that extensive changes in alternative splicing also play a profound role in shaping the changes in cell behavior that characterize the EMT. While long known splicing switches in FGFR2 and p120-catenin provided hints of a larger program of EMT-associated alternative splicing, the recent identification of the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) began to reveal this genome-wide post-transcriptional network. Several studies have now demonstrated the truly vast extent of this alternative splicing program. The global switches in splicing associated with the EMT add an important additional layer of post-transcriptional control that works in harmony with transcriptional and epigenetic regulation to effect complex changes in cell shape, polarity, and behavior that mediate transitions between epithelial and mesenchymal cell states. Future challenges include the need to investigate the functional consequences of these splicing switches at both the individual gene as well as systems level.

Warzecha, Claude C.; Carstens, Russ P.

2012-01-01

341

Splicing Factor Tra2B1 Is Specifically Induced in Breast Cancer and Regulates Alternative Splicing of the CD44 Gene  

Microsoft Academic Search

The human CD44 gene undergoes extensive alternative splicing of multiple variable exons positioned in a cassette in the middle of the gene. Expression of alternative exons is often restricted to certain tissues and could be associated with tumor progression and metastasis of several human malignancies, including breast cancer. Exon v4 contains multiple copies of a C\\/A-rich exon enhancer sequence required

Dirk O. Watermann; Yesheng Tang; Markus Jager; Stefan Stamm; Elmar Stickeler

2006-01-01

342

Cell-type-specific expression of alternatively spliced human fibronectin IIICS mRNAs.  

PubMed Central

Fibronectin polypeptide diversity is generated to a large extent by alternative splicing of the fibronectin primary transcript at three sites: two extra domain exons encoding extra structural repeats and a region of nonhomologous sequence termed the type-III connecting segment (IIICS). A novel double primer extension assay was developed to identify and quantify simultaneously each of the five human IIICS mRNA splicing variants. Expression of the five IIICS variants was analyzed in a variety of human normal and tumor cell types as well as in human liver. Differences in IIICS expression patterns were observed among different cell types, among fibroblasts of different tissue origins, and between comparable normal and transformed cells. The most predominant cell-type-specific differences were in the abundance of the one IIICS- mRNA variant relative to the four IIICS+ variants. The percentage of O variant (IIICS-) mRNAs within the total fibronectin mRNA pool varied between 3 and 17% among tumor cells and between 7 and 46% among normal cells. The O variant composed 57% of the fibronectin mRNA in liver tissue, correlating with the previously described increased abundance of IIICS- polypeptide subunits in plasma fibronectin, compared with those in cellular fibronectins. Additional cell-type-specific changes among the expression levels of the four IIICS+ mRNA variants are consistent with a proposed model in which regulation of an alternative selection of a 3'splice site predominates over regulation of the selection of a 5' splice site in generating specific patterns of IIICS mRNA expression. Images

Hershberger, R P; Culp, L A

1990-01-01

343

PPS, a large multidomain protein, functions with sex-lethal to regulate alternative splicing in Drosophila.  

PubMed

Alternative splicing controls the expression of many genes, including the Drosophila sex determination gene Sex-lethal (Sxl). Sxl expression is controlled via a negative regulatory mechanism where inclusion of the translation-terminating male exon is blocked in females. Previous studies have shown that the mechanism leading to exon skipping is autoregulatory and requires the SXL protein to antagonize exon inclusion by interacting with core spliceosomal proteins, including the U1 snRNP protein Sans-fille (SNF). In studies begun by screening for proteins that interact with SNF, we identified PPS, a previously uncharacterized protein, as a novel component of the machinery required for Sxl male exon skipping. PPS encodes a large protein with four signature motifs, PHD, BRK, TFS2M, and SPOC, typically found in proteins involved in transcription. We demonstrate that PPS has a direct role in Sxl male exon skipping by showing first that loss of function mutations have phenotypes indicative of Sxl misregulation and second that the PPS protein forms a complex with SXL and the unspliced Sxl RNA. In addition, we mapped the recruitment of PPS, SXL, and SNF along the Sxl gene using chromatin immunoprecipitation (ChIP), which revealed that, like many other splicing factors, these proteins bind their RNA targets while in close proximity to the DNA. Interestingly, while SNF and SXL are specifically recruited to their predicted binding sites, PPS has a distinct pattern of accumulation along the Sxl gene, associating with a region that includes, but is not limited to, the SxlPm promoter. Together, these data indicate that PPS is different from other splicing factors involved in male-exon skipping and suggest, for the first time, a functional link between transcription and SXL-mediated alternative splicing. Loss of zygotic PPS function, however, is lethal to both sexes, indicating that its role may be of broad significance. PMID:20221253

Johnson, Matthew L; Nagengast, Alexis A; Salz, Helen K

2010-03-05

344

PPS, a Large Multidomain Protein, Functions with Sex-Lethal to Regulate Alternative Splicing in Drosophila  

PubMed Central

Alternative splicing controls the expression of many genes, including the Drosophila sex determination gene Sex-lethal (Sxl). Sxl expression is controlled via a negative regulatory mechanism where inclusion of the translation-terminating male exon is blocked in females. Previous studies have shown that the mechanism leading to exon skipping is autoregulatory and requires the SXL protein to antagonize exon inclusion by interacting with core spliceosomal proteins, including the U1 snRNP protein Sans-fille (SNF). In studies begun by screening for proteins that interact with SNF, we identified PPS, a previously uncharacterized protein, as a novel component of the machinery required for Sxl male exon skipping. PPS encodes a large protein with four signature motifs, PHD, BRK, TFS2M, and SPOC, typically found in proteins involved in transcription. We demonstrate that PPS has a direct role in Sxl male exon skipping by showing first that loss of function mutations have phenotypes indicative of Sxl misregulation and second that the PPS protein forms a complex with SXL and the unspliced Sxl RNA. In addition, we mapped the recruitment of PPS, SXL, and SNF along the Sxl gene using chromatin immunoprecipitation (ChIP), which revealed that, like many other splicing factors, these proteins bind their RNA targets while in close proximity to the DNA. Interestingly, while SNF and SXL are specifically recruited to their predicted binding sites, PPS has a distinct pattern of accumulation along the Sxl gene, associating with a region that includes, but is not limited to, the SxlPm promoter. Together, these data indicate that PPS is different from other splicing factors involved in male-exon skipping and suggest, for the first time, a functional link between transcription and SXL–mediated alternative splicing. Loss of zygotic PPS function, however, is lethal to both sexes, indicating that its role may be of broad significance.

Johnson, Matthew L.; Nagengast, Alexis A.; Salz, Helen K.

2010-01-01

345

CADM1 is expressed as multiple alternatively spliced functional and dysfunctional isoforms in human mast cells  

PubMed Central

Cell adhesion molecule 1 (CADM1) is implicated in the pathogenesis of several diseases and is responsible for adhesion and survival of mast cells (MCs). Differential expression of CADM1 isoforms was found in different species. We previously cloned SP4, SP1, SP6 and a dysfunctional isoform from human lung MCs (HLMCs) and the MC line HMC-1. The aim of this study was to identify all isoforms expressed in human MCs. The functional isoforms SP4, SP1, SP6 and SP3, with alternative splicing between exons 7/11, were detected in human MCs by RT-PCR. Two dysfunctional isoforms with alternative splicing of cryptic exons A and B between exons 1/2, leading to premature termination of translation, were found in ?40% of MC specimens. Sequencing of genomic DNA showed that splicing of cryptic exon B did not result from specific SNPs within this exon or its putative splice branch point. Highly glycosylated CADM1 (?105 kDa) was detected by western blotting, but an extracellular domain (?95 kDa) was found only in the culture medium from HLMCs, but not HMC-1 cells, indicating differential protein expression. Transfection of SP1 and SP6, but not SP4, reduced adhesion of HMC-1 cells to human lung fibroblasts but not airway smooth muscle cells. Hence, dysfunctional and functional CADM1 isoforms are found in human MCs. The longer SP1 and SP6 were most evident in differentiated HLMCs and displayed differential adhesion compared to SP4. These multiple isoforms are likely to contribute to MC function in both health and disease.

Moiseeva, Elena P.; Leyland, Mark L.; Bradding, Peter

2013-01-01

346

Prolyl 4-hydroxylase genes are subjected to alternative splicing in roots of maize seedlings under waterlogging  

PubMed Central

Background In animals, prolyl 4-hydroxylases (P4Hs) are regarded as oxygen sensors under hypoxia stress, but little is known about their role in the response to waterlogging in maize. Methods A comprehensive genome-wide analysis of P4H genes of maize (zmP4H genes) was carried out, including gene structures, phylogeny, protein motifs, chromosomal locations and expression patterns under waterlogging. Key Results Nine zmP4H genes were identified in maize, of which five were alternatively spliced into at least 19 transcripts. Different alternative splicing (AS) events were revealed in different inbred lines, even for the same gene, possibly because of organ and developmental specificities or different stresses. The signal strength of splice sites was strongly correlated with selection of donor and receptor sites, and ambiguous junction sites due to small direct repeats at the exon/intron junction frequently resulted in the selection of unconventional splicing sites. Eleven out of 14 transcripts resulting from AS harboured a premature termination codon, rendering them potential candidates for nonsense-mediated RNA degradation. Reverse transcription–PCR (RT–PCR) indicated that zmP4H genes displayed different expression patterns under waterlogging. The diverse transcripts generated from AS were expressed at different levels, suggesting that zmP4H genes were under specific control by post-transcriptional regulation under waterlogging stress in the line HZ32. Conclusions Our results provide a framework for future dissection of the function of the emerging zmP4H family and suggest that AS might have an important role in the regulation of the expression profile of this gene family under waterlogging stress.

Zou, Xiling; Jiang, Yuanyuan; Zheng, Yonglian; Zhang, Meidong; Zhang, Zuxin

2011-01-01

347

Poliovirus 2A Protease Triggers a Selective Nucleo-Cytoplasmic Redistribution of Splicing Factors to Regulate Alternative Pre-mRNA Splicing  

PubMed Central

Poliovirus protease 2A (2Apro) obstructs host gene expression by reprogramming transcriptional and post-transcriptional regulatory events during infection. Here we demonstrate that expression of 2Apro induces a selective nucleo-cytoplasm translocation of several important RNA binding proteins and splicing factors. Subcellular fractionation studies, together with immunofluorescence microscopy revealed an asymmetric distribution of HuR and TIA1/TIAR in 2Apro expressing cells, which modulates splicing of the human Fas exon 6. Consistent with this result, knockdown of HuR or overexpression of TIA1/TIAR, leads to Fas exon 6 inclusion in 2Apro-expressing cells. Therefore, poliovirus 2Apro can target alternative pre-mRNA splicing by regulating protein shuttling between the nucleus and the cytoplasm.

Alvarez, Enrique; Castello, Alfredo; Carrasco, Luis; Izquierdo, Jose M.

2013-01-01

348

Identification and characterization of a soluble cadherin-7 isoform produced by alternative splicing.  

PubMed

We identified an alternative mRNA encoding a novel cadherin-7 isoform by reverse transcriptase-PCR of RNA from day 12 chicken embryos. The alternative mRNA contains 49 bases of insertion in the premembrane region, leading to the substitution of 14 amino acids and the introduction of a premature stop codon. Identification of a 49-bp insertion sequence in the genomic DNA corresponding to the intron of the cadherin-7 gene suggests that alternative splicing is the cause of the alternative mRNA. Transient expression of the variant form in COS-7 or 293 cells produced a soluble protein. Aggregation assays and immunoprecipitation showed that the variant protein interacts with full-length cadherin-7 in vitro and in vivo and inhibits full-length cadherin-7-mediated cell adhesion. Immunohistochemistry revealed that the variant form was strongly expressed in dermomyotomes rather than in migrating neural crest cells, in contrast to the full-length cadherin-7, suggesting differential regulation of splicing and possible roles of variant cadherin-7 in the development of dermomyotomes and other tissues. PMID:12364338

Kawano, Rie; Matsuo, Noritaka; Tanaka, Hideaki; Nasu, Masaru; Yoshioka, Hidekatsu; Shirabe, Komei

2002-10-02

349

Functional characterization of alternative splicing in the C terminus of L-type CaV1.3 channels.  

PubMed

Ca(V)1.3 channels are unique among the high voltage-activated Ca(2+) channel family because they activate at the most negative potentials and display very rapid calcium-dependent inactivation. Both properties are of crucial importance in neurons of the suprachiasmatic nucleus and substantia nigra, where the influx of Ca(2+) ions at subthreshold membrane voltages supports pacemaking function. Previously, alternative splicing in the Ca(V)1.3 C terminus gives rise to a long (Ca(V)1.3(42)) and a short form (Ca(V)1.3(42A)), resulting in a pronounced activation at more negative voltages and faster inactivation in the latter. It was further shown that the C-terminal modulator in the Ca(V)1.3(42) isoforms modulates calmodulin binding to the IQ domain. Using splice variant-specific antibodies, we determined that protein localization of both splice variants in different brain regions were similar. Using the transcript-scanning method, we further identified alternative splicing at four loci in the C terminus of Ca(V)1.3 channels. Alternative splicing of exon 41 removes the IQ motif, resulting in a truncated Ca(V)1.3 protein with diminished inactivation. Splicing of exon 43 causes a frameshift and exhibits a robust inactivation of similar intensity to Ca(V)1.3(42A). Alternative splicing of exons 44 and 48 are in-frame, altering interaction of the distal modulator with the IQ domain and tapering inactivation slightly. Thus, alternative splicing in the C terminus of Ca(V)1.3 channels modulates its electrophysiological properties, which could in turn alter neuronal firing properties and functions. PMID:21998309

Tan, Bao Zhen; Jiang, Fengli; Tan, Ming Yeong; Yu, Dejie; Huang, Hua; Shen, Yiru; Soong, Tuck Wah

2011-10-13

350

Transcriptomic Analysis of PNN- and ESRP1-Regulated Alternative Pre-mRNA Splicing in Human Corneal Epithelial Cells  

PubMed Central

Purpose. We investigated the impact of PININ (PNN) and epithelial splicing regulatory protein 1 (ESRP1) on alternative pre-mRNA splicing in the corneal epithelial context. Methods. Isoform-specific RT-PCR assays were performed on wild-type and Pnn knockout mouse cornea. Protein interactions were examined by deconvolution microscopy and co-immunoprecipitation. For genome-wide alternative splicing study, immortalized human corneal epithelial cells (HCET) harboring doxycycline-inducible shRNA against PNN or ESRP1 were created. Total RNA was isolated from four biological replicates of control and knockdown HCET cells, and subjected to hGlue3_0 transcriptome array analysis. Results. Pnn depletion in developing mouse corneal epithelium led to disrupted alternative splicing of multiple ESRP-regulated epithelial-type exons. In HCET cells, ESRP1 and PNN displayed close localization in and around nuclear speckles, and their physical association in protein complexes was identified. Whole transcriptome array analysis on ESRP1 or PNN knockdown HCET cells revealed clear alterations in transcript profiles and splicing patterns of specific subsets of genes. Separate RT-PCR validation assays confirmed successfully specific changes in exon usage of several representative splice variants, including PAX6(5a), FOXJ3, ARHGEF11, and SLC37A2. Gene ontologic analyses on ESRP1- or PNN-regulated alternative exons suggested their roles in epithelial phenotypes, such as cell morphology and movement. Conclusions. Our data suggested that ESRP1 and PNN modulate alternative splicing of a specific subset of target genes, but not general splicing events, in HCET cells to maintain or enhance epithelial characteristics.

Joo, Jeong-Hoon; Correia, Greg P.; Li, Jian-Liang; Lopez, Maria-Cecilia; Baker, Henry V.; Sugrue, Stephen P.

2013-01-01

351

Alternative splicing and gene duplication in the evolution of the FoxP gene subfamily.  

PubMed

The FoxP gene subfamily of transcription factors is defined by its characteristic 110 amino acid long DNA-binding forkhead domain and plays essential roles in vertebrate biology. Its four members, FoxP1-P4, have been extensively characterized functionally. FoxP1, FoxP2, and FoxP4 are involved in lung, heart, gut, and central nervous system (CNS) development. FoxP3 is necessary and sufficient for the specification of regulatory T cells (Tregs) of the adaptive immune system. In Drosophila melanogaster, in silico predictions identify one unique FoxP subfamily gene member (CG16899) with no described function. We characterized this gene and established that it generates by alternative splicing two isoforms that differ in the forkhead DNA-binding domain. In D. melanogaster, both isoforms are expressed in the embryonic CNS, but in hemocytes, only isoform A is expressed, hinting to a putative modulation through alternative splicing of FoxP1 function in immunity and/or other hemocyte-dependent processes. Furthermore, we show that in vertebrates, this novel alternative splicing pattern is conserved for FoxP1. In mice, this new FoxP1 isoform is expressed in brain, liver, heart, testes, thymus, and macrophages (equivalent in function to hemocytes). This alternative splicing pattern has arisen at the base of the Bilateria, probably through exon tandem duplication. Moreover, our phylogenetic analysis suggests that in vertebrates, FoxP1 is more related to the FoxP gene ancestral form and the other three paralogues, originated through serial duplications, which only retained one of the alternative exons. Also, the newly described isoform differs from the other in amino acids critical for DNA-binding specificity. The integrity of its fold is maintained, but the molecule has lost the direct hydrogen bonding to DNA bases leading to a putatively lower specificity and possibly affinity toward DNA. With the present comparative study, through the integration of experimental and in silico studies of the FoxP gene subfamily across the animal kingdom, we establish a new model for the FoxP gene in invertebrates and for the vertebrate FoxP1 paralogue. Furthermore, we present a scenario for the structural evolution of this gene class and reveal new previously unsuspected levels of regulation for FoxP1 in the vertebrate system. PMID:20651048

Santos, M Emília; Athanasiadis, Alekos; Leitão, Alexandre B; DuPasquier, Louis; Sucena, Elio

2010-07-22

352

Alternative Splicing and Gene Duplication in the Evolution of the FoxP Gene Subfamily  

PubMed Central

The FoxP gene subfamily of transcription factors is defined by its characteristic 110 amino acid long DNA-binding forkhead domain and plays essential roles in vertebrate biology. Its four members, FoxP1–P4, have been extensively characterized functionally. FoxP1, FoxP2, and FoxP4 are involved in lung, heart, gut, and central nervous system (CNS) development. FoxP3 is necessary and sufficient for the specification of regulatory T cells (Tregs) of the adaptive immune system. In Drosophila melanogaster, in silico predictions identify one unique FoxP subfamily gene member (CG16899) with no described function. We characterized this gene and established that it generates by alternative splicing two isoforms that differ in the forkhead DNA-binding domain. In D. melanogaster, both isoforms are expressed in the embryonic CNS, but in hemocytes, only isoform A is expressed, hinting to a putative modulation through alternative splicing of FoxP1 function in immunity and/or other hemocyte-dependent processes. Furthermore, we show that in vertebrates, this novel alternative splicing pattern is conserved for FoxP1. In mice, this new FoxP1 isoform is expressed in brain, liver, heart, testes, thymus, and macrophages (equivalent in function to hemocytes). This alternative splicing pattern has arisen at the base of the Bilateria, probably through exon tandem duplication. Moreover, our phylogenetic analysis suggests that in vertebrates, FoxP1 is more related to the FoxP gene ancestral form and the other three paralogues, originated through serial duplications, which only retained one of the alternative exons. Also, the newly described isoform differs from the other in amino acids critical for DNA-binding specificity. The integrity of its fold is maintained, but the molecule has lost the direct hydrogen bonding to DNA bases leading to a putatively lower specificity and possibly affinity toward DNA. With the present comparative study, through the integration of experimental and in silico studies of the FoxP gene subfamily across the animal kingdom, we establish a new model for the FoxP gene in invertebrates and for the vertebrate FoxP1 paralogue. Furthermore, we present a scenario for the structural evolution of this gene class and reveal new previously unsuspected levels of regulation for FoxP1 in the vertebrate system.

Santos, M. Emilia; Athanasiadis, Alekos; Leitao, Alexandre B.; DuPasquier, Louis; Sucena, Elio

2011-01-01

353

ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43  

PubMed Central

Transactivating response region DNA binding protein (TDP-43) is the major protein component of ubiquitinated inclusions found in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitinated inclusions. Two ALS-causing mutants (TDP-43Q331K and TDP-43M337V), but not wild-type human TDP-43, are shown here to provoke age-dependent, mutant-dependent, progressive motor axon degeneration and motor neuron death when expressed in mice at levels and in a cell type-selective pattern similar to endogenous TDP-43. Mutant TDP-43-dependent degeneration of lower motor neurons occurs without: (i) loss of TDP-43 from the corresponding nuclei, (ii) accumulation of TDP-43 aggregates, and (iii) accumulation of insoluble TDP-43. Computational analysis using splicing-sensitive microarrays demonstrates alterations of endogenous TDP-43–dependent alternative splicing events conferred by both human wild-type and mutant TDP-43Q331K, but with high levels of mutant TDP-43 preferentially enhancing exon exclusion of some target pre-mRNAs affecting genes involved in neurological transmission and function. Comparison with splicing alterations following TDP-43 depletion demonstrates that TDP-43Q331K enhances normal TDP-43 splicing function for some RNA targets but loss-of-function for others. Thus, adult-onset motor neuron disease does not require aggregation or loss of nuclear TDP-43, with ALS-linked mutants producing loss and gain of splicing function of selected RNA targets at an early disease stage.

Arnold, Eveline S.; Ling, Shuo-Chien; Huelga, Stephanie C.; Lagier-Tourenne, Clotilde; Polymenidou, Magdalini; Ditsworth, Dara; Kordasiewicz, Holly B.; McAlonis-Downes, Melissa; Platoshyn, Oleksandr; Parone, Philippe A.; Da Cruz, Sandrine; Clutario, Kevin M.; Swing, Debbie; Tessarollo, Lino; Marsala, Martin; Shaw, Christopher E.; Yeo, Gene W.; Cleveland, Don W.

2013-01-01

354

Reduced fidelity of branch point recognition and alternative splicing induced by the anti-tumor drug spliceostatin A  

PubMed Central

Spliceostatin A (SSA) is a stabilized derivative of a Pseudomonas bacterial fermentation product that displays potent anti-proliferative and anti-tumor activities in cancer cells and animal models. The drug inhibits pre-mRNA splicing in vitro and in vivo and binds SF3b, a protein subcomplex of U2 small nuclear ribonucleoprotein (snRNP), which is essential for recognition of the pre-mRNA branch point. We report that SSA prevents interaction of an SF3b 155-kDa subunit with the pre-mRNA, concomitant with nonproductive recruitment of U2 snRNP to sequences 5? of the branch point. Differences in base-pairing potential with U2 snRNA in this region lead to different sensitivity of 3? splice sites to SSA, and to SSA-induced changes in alternative splicing. Indeed, rather than general splicing inhibition, splicing-sensitive microarray analyses reveal specific alternative splicing changes induced by the drug that significantly overlap with those induced by knockdown of SF3b 155. These changes lead to down-regulation of genes important for cell division, including cyclin A2 and Aurora A kinase, thus providing an explanation for the anti-proliferative effects of SSA. Our results reveal a mechanism that prevents nonproductive base-pairing interactions in the spliceosome, and highlight the regulatory and cancer therapeutic potential of perturbing the fidelity of splice site recognition.

Corrionero, Anna; Minana, Belen; Valcarcel, Juan

2011-01-01

355

Reduced fidelity of branch point recognition and alternative splicing induced by the anti-tumor drug spliceostatin A.  

PubMed

Spliceostatin A (SSA) is a stabilized derivative of a Pseudomonas bacterial fermentation product that displays potent anti-proliferative and anti-tumor activities in cancer cells and animal models. The drug inhibits pre-mRNA splicing in vitro and in vivo and binds SF3b, a protein subcomplex of U2 small nuclear ribonucleoprotein (snRNP), which is essential for recognition of the pre-mRNA branch point. We report that SSA prevents interaction of an SF3b 155-kDa subunit with the pre-mRNA, concomitant with nonproductive recruitment of U2 snRNP to sequences 5' of the branch point. Differences in base-pairing potential with U2 snRNA in this region lead to different sensitivity of 3' splice sites to SSA, and to SSA-induced changes in alternative splicing. Indeed, rather than general splicing inhibition, splicing-sensitive microarray analyses reveal specific alternative splicing changes induced by the drug that significantly overlap with those induced by knockdown of SF3b 155. These changes lead to down-regulation of genes important for cell division, including cyclin A2 and Aurora A kinase, thus providing an explanation for the anti-proliferative effects of SSA. Our results reveal a mechanism that prevents nonproductive base-pairing interactions in the spliceosome, and highlight the regulatory and cancer therapeutic potential of perturbing the fidelity of splice site recognition. PMID:21363963

Corrionero, Anna; Miñana, Belén; Valcárcel, Juan

2011-03-01

356

Polymer nanoparticle-mediated delivery of microRNA inhibition and alternative splicing.  

PubMed

The crux of current RNA-based therapeutics relies on association of synthetic nucleic acids with cellular RNA targets. Antisense oligonucleotide binding to mature microRNA and splicing junctions on pre-mRNA represent methods of gene therapy that respectively inhibit microRNA-mediated gene regulation and induce alternative splicing. We have developed biodegradable polymer nanoparticles, which are coated with cell-penetrating peptides, that can effectively deliver chemically modified oligonucleotide analogues to achieve these forms of gene regulation. We found that this nanoparticle system could block the activity of the oncogenic microRNA, miR-155, as well as modulate splicing to attenuate the expression of the proto-oncogene, Mcl-1. Regulation of these genes in human cancer cells reduced cell viability and produced pro-apoptotic effects. These findings establish polymer nanoparticles as delivery vectors for nonconventional forms of gene therapy activated by cellular delivery of RNA-targeted molecules, which have strong therapeutic implications. PMID:22482958

Cheng, Christopher J; Saltzman, W Mark

2012-04-18

357

HTLV-I antisense transcripts initiating in the 3'LTR are alternatively spliced and polyadenylated  

PubMed Central

Background Antisense transcription in retroviruses has been suggested for both HIV-1 and HTLV-I, although the existence and coding potential of these transcripts remain controversial. Thorough characterization is required to demonstrate the existence of these transcripts and gain insight into their role in retrovirus biology. Results This report provides the first complete characterization of an antisense retroviral transcript that encodes the previously described HTLV-I HBZ protein. In this study, we show that HBZ-encoding transcripts initiate in the 3' long terminal repeat (LTR) at several positions and consist of two alternatively spliced variants (SP1 and SP2). Expression of the most abundant HBZ spliced variant (SP1) could be detected in different HTLV-I-infected cell lines and importantly in cellular clones isolated from HTLV-I-infected patients. Polyadenylation of HBZ RNA occurred at a distance of 1450 nucleotides downstream of the HBZ stop codon in close proximity of a typical polyA signal. We have also determined that translation mostly initiates from the first exon located in the 3' LTR and that the HBZ isoform produced from the SP1 spliced variant demonstrated inhibition of Tax and c-Jun-dependent transcriptional activation. Conclusion These results conclusively demonstrate the existence of antisense transcription in retroviruses, which likely plays a role in HTLV-I-associated pathogenesis through HBZ protein synthesis.

Cavanagh, Marie-Helene; Landry, Sebastien; Audet, Brigitte; Arpin-Andre, Charlotte; Hivin, Patrick; Pare, Marie-Eve; Thete, Julien; Wattel, Eric; Marriott, Susan J; Mesnard, Jean-Michel; Barbeau, Benoit

2006-01-01

358

Novel alternative splice variants of chicken NPAS3 are expressed in the developing central nervous system.  

PubMed

We report isolation of novel splice variants of chicken Neuronal Per-Arnt-Sim domain protein 3 (cNPAS3) gene distinct from the previously predicted cNPAS3 at the 5' end. Newly identified cNPAS3 splice variants feature N-terminus coding sequences with high degrees of homology to human NPAS3 (hNAPS3). We also show that the alternative splicing pattern of NPAS3 is conserved between chicken and human. RNA in situ hybridization indicated that the expression of cNPAS3 in the developing central nervous system (CNS) is limited to the ventricular zone and only partially overlaps with that of chicken Reelin (cReelin), the only known regulatory target gene of NPAS3 in the adult brain. Overexpression of cNPAS3 by in ovo electroporation had little effect on the expression of Sox2, a marker for neural precursors, or of Isl1/2, a marker for early differentiating motor neurons. Taken together with the little effect of cNPAS3 overexpression on cReelin, it is noted that the function of NPAS3 in the developing CNS remains to be determined. Still, identification of proper cDNA sequences for cNPAS3 should represent a solid beginning of the understanding process. PMID:23962688

Shin, Jiheon; Kim, Jaesang

2013-08-18

359

Cross-regulation between an alternative splicing activator and a transcription repressor controls neurogenesis.  

PubMed

Neurogenesis requires the concerted action of numerous genes that are regulated at multiple levels. However, how different layers of gene regulation are coordinated to promote neurogenesis is not well understood. We show that the neural-specific Ser/Arg repeat-related protein of 100 kDa (nSR100/SRRM4) negatively regulates REST (NRSF), a transcriptional repressor of genes required for neurogenesis. nSR100 directly promotes alternative splicing of REST transcripts to produce a REST isoform (REST4) with greatly reduced repressive activity, thereby activating expression of REST targets in neural cells. Conversely, REST directly represses nSR100 in nonneural cells to prevent the activation of neural-specific splicing events. Consistent with a critical role for nSR100 in the inhibition of REST activity, blocking nSR100 expression in the developing mouse brain impairs neurogenesis. Our results thus reveal a fundamental role for direct regulatory interactions between a splicing activator and transcription repressor in the control of the multilayered regulatory programs required for neurogenesis. PMID:21884984

Raj, Bushra; O'Hanlon, Dave; Vessey, John P; Pan, Qun; Ray, Debashish; Buckley, Noel J; Miller, Freda D; Blencowe, Benjamin J

2011-09-01

360

ASPIC: a web resource for alternative splicing prediction and transcript isoforms characterization  

PubMed Central

Alternative splicing (AS) is now emerging as a major mechanism contributing to the expansion of the transcriptome and proteome complexity of multicellular organisms. The fact that a single gene locus may give rise to multiple mRNAs and protein isoforms, showing both major and subtle structural variations, is an exceptionally versatile tool in the optimization of the coding capacity of the eukaryotic genome. The huge and continuously increasing number of genome and transcript sequences provides an essential information source for the computational detection of genes AS pattern. However, much of this information is not optimally or comprehensively used in gene annotation by current genome annotation pipelines. We present here a web resource implementing the ASPIC algorithm which we developed previously for the investigation of AS of user submitted genes, based on comparative analysis of available transcript and genome data from a variety of species. The ASPIC web resource provides graphical and tabular views of the splicing patterns of all full-length mRNA isoforms compatible with the detected splice sites of genes under investigation as well as relevant structural and functional annotation. The ASPIC web resource—available at —is dynamically interconnected with the Ensembl and Unigene databases and also implements an upload facility.

Castrignano, Tiziana; Rizzi, Raffaella; Talamo, Ivano Giuseppe; De Meo, Paolo D'Onorio; Anselmo, Anna; Bonizzoni, Paola; Pesole, Graziano

2006-01-01

361

ASPIC: a web resource for alternative splicing prediction and transcript isoforms characterization.  

PubMed

Alternative splicing (AS) is now emerging as a major mechanism contributing to the expansion of the transcriptome and proteome complexity of multicellular organisms. The fact that a single gene locus may give rise to multiple mRNAs and protein isoforms, showing both major and subtle structural variations, is an exceptionally versatile tool in the optimization of the coding capacity of the eukaryotic genome. The huge and continuously increasing number of genome and transcript sequences provides an essential information source for the computational detection of genes AS pattern. However, much of this information is not optimally or comprehensively used in gene annotation by current genome annotation pipelines. We present here a web resource implementing the ASPIC algorithm which we developed previously for the investigation of AS of user submitted genes, based on comparative analysis of available transcript and genome data from a variety of species. The ASPIC web resource provides graphical and tabular views of the splicing patterns of all full-length mRNA isoforms compatible with the detected splice sites of genes under investigation as well as relevant structural and functional annotation. The ASPIC web resource-available at http://www.caspur.it/ASPIC/--is dynamically interconnected with the Ensembl and Unigene databases and also implements an upload facility. PMID:16845044

Castrignanò, Tiziana; Rizzi, Raffaella; Talamo, Ivano Giuseppe; De Meo, Paolo D'Onorio; Anselmo, Anna; Bonizzoni, Paola; Pesole, Graziano

2006-07-01

362

Alternative splicing due to an intronic SNP in HMSD generates a novel minor histocompatibility antigen.  

PubMed

Here we report the identification of a novel human leukocyte antigen (HLA)-B44-restricted minor histocompatibility antigen (mHA) with expression limited to hematopoietic cells. cDNA expression cloning studies demonstrated that the cytotoxic T lymphocyte (CTL) epitope of interest was encoded by a novel allelic splice variant of HMSD, hereafter designated as HMSD-v. The immunogenicity of the epitope was generated by differential protein expression due to alternative splicing, which was completely controlled by 1 intronic single-nucleotide polymorphism located in the consensus 5' splice site adjacent to an exon. Both HMSD-v and HMSD transcripts were selectively expressed at higher levels in mature dendritic cells and primary leukemia cells, especially those of myeloid lineage. Engraftment of mHA(+) myeloid leukemia stem cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID)/gammac(null) mice was completely inhibited by in vitro preincubation with the mHA-specific CTL clone, suggesting that this mHA is expressed on leukemic stem cells. The patient from whom the CTL clone was isolated demonstrated a significant increase of the mHA-specific T cells in posttransplantation peripheral blood, whereas mHA-specific T cells were undetectable in pretransplantation peripheral blood and in peripheral blood from his donor. These findings suggest that the HMSD-v-encoded mHA (designated ACC-6) could serve as a target antigen for immunotherapy against hematologic malignancies. PMID:17409267

Kawase, Takakazu; Akatsuka, Yoshiki; Torikai, Hiroki; Morishima, Satoko; Oka, Akira; Tsujimura, Akane; Miyazaki, Mikinori; Tsujimura, Kunio; Miyamura, Koichi; Ogawa, Seishi; Inoko, Hidetoshi; Morishima, Yasuo; Kodera, Yoshihisa; Kuzushima, Kiyotaka; Takahashi, Toshitada

2007-04-04

363

Differential regulation of alternative 3{prime} splicing of {epsilon} messenger RNA variants  

SciTech Connect

Alternative 3{prime} splicing of the one active human {epsilon} heavy chain gene results in variants of {epsilon} mRNA encoding distinct IgE proteins. The same relative amounts of these {epsilon} mRNA variants were produced by non-atopic donor B cells when driven in a variety of T-dependent or T-independent systems. The most abundant variants were those for classic secreted {epsilon} and a novel secreted form (CH4-M2{double_prime}). In contrast, cells from subjects with high levels of serum IgE secondary to parasitic infection or atopy spontaneously produced higher relative levels of the CH4-M2{prime} {epsilon} mRNA variant, lower relative amounts of both the membrane and CH4-M2{double_prime} secreted variants, and very low levels of the CH4{prime}-CH5 variant. The existence of and corresponding changes in levels of the CH4-M2{prime}-enclosed secreted protein were demonstrated. IL-10 induced this same differential expression of {epsilon} splice variants in vitro when used to costimulate IL-4 plus CD40-driven B cells and could differentially enhance the production of CH4-M2{prime} protein by established IgE-secreting cell lines. Inhibition of IgE by cross-linking the low affinity IgE receptor (CD23) decreased the levels of {epsilon} mRNA and resulted in a distinct pattern of {epsilon} mRNA characterized by a dramatic decrease in CH4-M2{prime} splice variant. IL-6, IL-2, or IFN-{gamma} did not change the {epsilon} mRNA pattern. Overall, the absolute and relative amounts of the different {epsilon} mRNA splice variants produced appear to be controlled in a differentiation-related fashion.

Diaz-Sanchez, D.; Zhang, K.; Saxon, A. [Univ. of California School of Medicine, Los Angeles, CA (United States)] [and others

1995-08-15

364

Disabled-1 Alternative Splicing in Human Fetal Retina and Neural Tumors  

PubMed Central

Background The Reelin-Dab1 signaling pathway plays a critical role in the positioning of migrating neurons, dendrite formation and lamination in the developing central nervous system. We have previously identified two alternatively spliced forms of Dab1 in the developing chick retina: an early form, Dab1-E, expressed in retinal progenitor cells, and a late form, Dab1 or Dab1-L, expressed in amacrine and ganglion cells. Compared to Dab1-L, Dab1-E lacks two exons that encode two Src family kinase (SFK) phosphorylation sites. Principal Findings Both Dab1-L and Dab1-E-like transcripts were identified in human fetal retina. Expression of human Dab1-L in primary chick retinal cultures resulted in Reelin-mediated induction of SFK phosphorylation and formation of neurite-like processes. In contrast, human Dab1-E-expressing cells retained an undifferentiated morphology. The human Dab1 gene is located within a common fragile site, and it has been postulated that it may function as a tumor suppressor. Analysis of Dab1 splice forms in retinoblastoma and neuroblastoma tumor cells revealed relative enrichment of Dab1-L-like (includes exons 7 and 8) and Dab1-E-like (excludes exons 7 and 8) transcripts in retinoblastoma and neuroblastoma, respectively. Treatment of retinoblastoma cell line RB522A with Reelin resulted in increased tyrosine phosphorylation of Dab1. As Nova2 has previously been implicated in the exclusion of exons 9B and 9C in Dab1, we examined the expression of this splicing factor in neuroblastoma and retinoblastoma cell lines. Nova2 was only detected in neuroblastoma cells, suggesting a correlation between Nova2 expression and increased levels of Dab1-E-like splice forms in neuroblastoma. Conclusions These results indicate that alternative splicing of Dab1 is conserved in avian and mammalian species, with Dab1-L driving SFK phosphorylation in both species. Dab1-E- and Dab-L-like isoforms are also expressed in childhood neural tumors, with preferential enrichment of Dab1-L-like and Dab1-E-like isoforms in retinoblastoma and neuroblastoma, respectively.

Katyal, Sachin; Glubrecht, Darryl D.; Li, Lei; Gao, Zhihua; Godbout, Roseline

2011-01-01

365

MADS: A new and improved method for analysis of differential alternative splicing by exon-tiling microarrays  

Microsoft Academic Search

We describe a method, microarray analysis of differential splicing (MADS), for discovery of differential alternative splicing from exon-tiling microarray data. MADS incorporates a series of low-level analysis algorithms motivated by the ''probe-rich'' design of exon arrays, including background correction, iterative probe selection, and removal of sequence-specific cross-hybridization to off-target transcripts. We used MADS to analyze Affymetrix Exon 1.0 array data

YI XING; PETER STOILOV; KAREN KAPUR; AREUM HAN; HUI JIANG; SHIHAO SHEN; DOUGLAS L. BLACK; WING HUNG WONG

2008-01-01

366

Structure and Expression of Phosphoenolpyruvate Carboxylase Kinase Genes in Solanaceae. A Novel Gene Exhibits Alternative Splicing1  

PubMed Central

Phosphorylation of phosphoenolpyruvate carboxylase (PEPc; EC 4.1.1.31) plays an important role in the control of central metabolism in higher plants. Two PPCK (PEPc kinase) genes have been identified in tomato (Lycopersicon esculentum cv Alicante), hereafter termed LePPCK1 and LePPCK2. The function of the gene products has been confirmed by transcription of full-length cDNAs, translation, and in vitro assay of kinase activity. Previously studied PPCK genes contain a single intron. LePPCK2 also contains a novel second intron that exhibits alternative splicing. The correctly spliced transcript encodes a functional PEPc kinase, whereas unspliced or incorrectly spliced transcripts encode a truncated, inactive protein. The relative abundance of the transcripts depends on tissue and conditions. Expression of LePPCK2 was markedly increased during fruit ripening. In ripe Alicante fruit, the locule and seeds contained only the correctly spliced LePPCK2 transcripts, whereas in ripe fruit of the tomato greenflesh mutant, they contained correctly and incorrectly spliced transcripts. Potato (Solanum tuberosum) contains genes that are very similar to LePPCK1, and LePPCK2; StPPCK2 exhibits alternative splicing. Aubergine (Solanum melongena) and tobacco (Nicotiana tabacum) also contain a PPCK2 gene; the sequence of the alternatively spliced intron is highly conserved between all four species. The data suggest that the two PPCK genes have different roles in tissue-specific regulation of PEPc and that the alternative splicing of PPCK2 transcripts is functionally significant.

Marsh, Justin T.; Sullivan, Stuart; Hartwell, James; Nimmo, Hugh G.

2003-01-01

367

Alternative splicing and nonsense-mediated decay regulate telomerase reverse transcriptase (TERT) expression during virus-induced lymphomagenesis in vivo  

Microsoft Academic Search

BACKGROUND: Telomerase activation, a critical step in cell immortalization and oncogenesis, is partly regulated by alternative splicing. In this study, we aimed to use the Marek's disease virus (MDV) T-cell lymphoma model to evaluate TERT regulation by splicing during lymphomagenesis in vivo, from the start point to tumor establishment. RESULTS: We first screened cDNA libraries from the chicken MDV lymphoma-derived

Souheila Amor; Sylvie Remy; Ginette Dambrine; Yves Le Vern; Denis Rasschaert; Sylvie Laurent

2010-01-01

368

Regulation of Multiple Core Spliceosomal Proteins by Alternative Splicing-Coupled Nonsense-Mediated mRNA Decay  

Microsoft Academic Search

Alternative splicing (AS) can regulate gene expression by introducing premature termination codons (PTCs) into spliced mRNA that subsequently elicit transcript degradation by the nonsense-mediated mRNA decay (NMD) pathway. However, the range of cellular functions controlled by this process and the factors required are poorly understood. By quantitative AS microarray profiling, we find that there are significant overlaps among the sets

Arneet L. Saltzman; Yoon Ki Kim; Qun Pan; Matthew M. Fagnani; Lynne E. Maquat; Benjamin J. Blencowe

2008-01-01

369

Support vector machines-based identification of alternative splicing in Arabidopsis thaliana from whole-genome tiling arrays  

Microsoft Academic Search

Background  Alternative splicing (AS) is a process which generates several distinct mRNA isoforms from the same gene by splicing different\\u000a portions out of the precursor transcript. Due to the (patho-)physiological importance of AS, a complete inventory of AS is\\u000a of great interest. While this is in reach for human and mammalian model organisms, our knowledge of AS in plants has remained

Johannes Eichner; Georg Zeller; Sascha Laubinger; Gunnar Rätsch

2011-01-01

370

A DNMT3B Alternatively Spliced Exon and Encoded Peptide Are Novel Biomarkers of Human Pluripotent Stem Cells  

Microsoft Academic Search

A major obstacle in human stem cell research is the limited number of reagents capable of distinguishing pluripotent stem cells from partially differentiated or incompletely reprogrammed derivatives. Although human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) express numerous alternatively spliced transcripts, little attention has been directed at developing splice variant-encoded protein isoforms as reagents for stem cell

Sailesh Gopalakrishna-Pillai; Linda E. Iverson

2011-01-01

371

Alternative splicing resulting in nonsense-mediated mRNA decay: what is the meaning of nonsense?  

Microsoft Academic Search

Alternative splicing (AS) strongly affects gene expres- sion by generating protein isoform diversity. However, up to one-third of human AS events create a premature termination codon that would cause the resulting mRNA to be degraded by nonsense-mediated mRNA decay (NMD). The extent to which such events represent func- tionally selected post-transcriptional gene control, as opposed to noise in the splicing

Nicholas J. McGlincy; Christopher W. J. Smith

2008-01-01

372

Molecular cloning, expression and subcellular distribution of an alternative splice variant of the porcine Sirt2 gene  

Microsoft Academic Search

Sirt2, a NAD+-dependent histone deacetylase, plays a critical role in regulating lifespan, metabolism, mitosis and adipocyte differentiation.\\u000a Here two bands of the porcine Sirt2 protein were found by western blotting, so we speculated existence of Sirt2 isoforms.\\u000a Next, we cloned the porcine Sirt2 gene, and also found its alternative splice variant and named the novel splicing variant\\u000a Sirt2T. The complete

Bingting Liu; Fei Liu; Liang Bai; Yucheng Li; Gongshe Yang

2010-01-01

373

Stress-induced alternative gene splicing in mind-body medicine.  

PubMed

Recent research documents how psychosocial stress can alter the expression of the acetylcholinesterase gene to generate at least 3 alternative proteins that are implicated in a wide variety of normal mind-body functions, as well as pathologies. These range from early embryological development, plasticity of the brain in adulthood, post-traumatic stress disorder (PTSD), and stress-associated dysfunctions of the central nervous, endocrine, and immune systems, to age-related neuropathologies. Such stress-induced alternative gene splicing is proposed here as a major mind-body pathway of psychosocial genomics-the modulation of gene expression by creative psychological, social, and cultural processes. We explore the types of research that are now needed to investigate how stress-induced alternative splicing of the acetylcholinesterase gene may play a pivotal role in the deep psychobiology of psychotherapy, meditation, spiritual rituals, and the experiencing of positive humanistic values that have been associated with mind-body medicine, such as compassion, beneficence, serenity, forgiveness, and gratitude. PMID:15356952

Rossi, Ernest Lawrence

2004-01-01

374

Alternative promoter usage and splicing of the human SCN5A gene contribute to transcript heterogeneity.  

PubMed

The sodium channel isoform Na(v)1.5 mediates sodium current, excitability, and electrical conduction in the human heart. Recent studies have indicated alternative splicing within the protein-coding portion of its gene, SCN5A, as a mechanism to generate diversity in Na(v)1.5 protein structure and function. In the present study we identified several novel SCN5A transcripts in human heart, displaying distinct 5?-untranslated regions but identical protein-coding sequences. These transcripts originated from the splicing of alternative exons 1 (designated 1A, 1B, 1C, and 1D) to the translational start codon-containing exon 2, and were preferentially expressed in the heart as compared to other tissues. Comparison of their expression level between adult and fetal heart demonstrated that exon 1C- and 1D-derived sequences were more prominent in adult than in fetal heart. Two new promoters (designated P2 and P3) for the SCN5A gene were identified and functionally characterized in myocardial- and nonmyocardial-derived cell lines. Translation of the transcript containing exon 1D-derived sequences proved to be significantly impaired in these cell lines, which could be restored by mutation of an upstream translational start codon. These results implicate the usage of alternative promoters and 5?-untranslated regions as new mechanisms in the regulation of human Na(v)1.5 expression. PMID:20618077

van Stuijvenberg, Leonie; Yildirim, Cansu; Kok, Bart G J M; van Veen, Toon A B; Varró, András; Winckels, Stephan K G; Vos, Marc A; Bierhuizen, Marti F A

2010-10-01

375

Polymorphism of alternative splicing of major histocompatibility complex transcripts in wild tiger salamanders.  

PubMed

Alternative splicing (AS) of mRNA transcripts is increasingly recognized as a source of transcriptome diversity. To date, most AS studies have focused either on comparisons across taxa or on intragenomic comparisons across gene families. We generated a novel data set that represents one of the first population genetic comparisons of AS across individuals. In ambystomatid salamanders, AS of the major histocompatibility complex (MHC) class IIbeta gene (Amti-DAB) produces two transcripts, one full-length and one truncated. The full-length transcript is functional, but the truncated transcript is missing the critical beta1 domain that forms half of the peptide binding region in the intact MHC class II molecule. We captured wild salamander larvae (Ambystoma tigrinum tigrinum) and genotyped them at Amti-DAB via DNA sequencing. From these same larvae, we extracted RNA from gill and spleen and evaluated the relative expression level of Amti-DAB in each tissue. Across individuals, 21% of the transcripts were truncated (alternatively spliced), and the absolute level of alternative transcript expression was higher in gill. The high level of nucleotide variation among seven Amti-DAB alleles provides the ability to detect substitutions (or linked DNA polymorphisms) that might have influenced AS. The data reveal no correlation between AS and haplotype, allele, or zygosity. However, indirect evidence (comparative expression patterns across 3 million years of evolution) suggests that the truncated Amti-DAB transcript may be functional and maintained by natural selection. PMID:18543016

Bulut, Zafer; McCormick, Cory R; Bos, David H; DeWoody, J Andrew

2008-06-10

376

Lysyl oxidase-like 4 is alternatively spliced in an anatomic site-specific manner in tumors involving the serosal cavities  

Microsoft Academic Search

Lysyl oxidase-like enzymes (LOXL) are expressed in various cancers. We analyzed the expression of LOXL2, LOXL3, and LOXL4\\u000a in cancers involving the serosal cavities—breast carcinoma, ovarian carcinoma, and malignant mesothelioma using reverse-transcriptase\\u000a polymerase chain reaction. We discovered two new alternative splice variants of LOXL4. The spliced segments were exon 9 (splice\\u000a variant 1) or both exons 8 and 9 (splice

Shulamit Sebban; Ben Davidson; Reuven Reich

2009-01-01

377

RNA-Seq of Arabidopsis Pollen Uncovers Novel Transcription and Alternative Splicing1[C][W][OA  

PubMed Central

Pollen grains of Arabidopsis (Arabidopsis thaliana) contain two haploid sperm cells enclosed in a haploid vegetative cell. Upon germination, the vegetative cell extrudes a pollen tube that carries the sperm to an ovule for fertilization. Knowing the identity, relative abundance, and splicing patterns of pollen transcripts will improve our understanding of pollen and allow investigation of tissue-specific splicing in plants. Most Arabidopsis pollen transcriptome studies have used the ATH1 microarray, which does not assay splice variants and lacks specific probe sets for many genes. To investigate the pollen transcriptome, we performed high-throughput sequencing (RNA-Seq) of Arabidopsis pollen and seedlings for comparison. Gene expression was more diverse in seedling, and genes involved in cell wall biogenesis were highly expressed in pollen. RNA-Seq detected at least 4,172 protein-coding genes expressed in pollen, including 289 assayed only by nonspecific probe sets. Additional exons and previously unannotated 5? and 3? untranslated regions for pollen-expressed genes were revealed. We detected regions in the genome not previously annotated as expressed; 14 were tested and 12 were confirmed by polymerase chain reaction. Gapped read alignments revealed 1,908 high-confidence new splicing events supported by 10 or more spliced read alignments. Alternative splicing patterns in pollen and seedling were highly correlated. For most alternatively spliced genes, the ratio of variants in pollen and seedling was similar, except for some encoding proteins involved in RNA splicing. This study highlights the robustness of splicing patterns in plants and the importance of ongoing annotation and visualization of RNA-Seq data using interactive tools such as Integrated Genome Browser.

Loraine, Ann E.; McCormick, Sheila; Estrada, April; Patel, Ketan; Qin, Peng

2013-01-01

378

Detecting tissue-specific alternative splicing and disease-associated aberrant splicing of the PTCH gene with exon junction microarrays  

Microsoft Academic Search

GenBank accession numbers{ Mutations in the human ortholog of Drosophila patched (PTCH) have been identified in patients with auto- somal dominant nevoid basal cell carcinoma syndrome (NBCCS), characterized by minor developmental anomalies and an increased incidence of cancers such as medulloblastoma and basal cell carcinoma. We identified many isoforms of PTCH mRNA involving exons 1-5, exon 10 and a novel

Kazuaki Nagao; Naoyuki Togawa; Katsunori Fujii; Hideki Uchikawa; Yoichi Kohno; Masao Yamada; Toshiyuki Miyashita

2005-01-01

379

Challenges in estimating percent inclusion of alternatively spliced junctions from RNA-seq data  

PubMed Central

Transcript quantification is a long-standing problem in genomics and estimating the relative abundance of alternatively-spliced isoforms from the same transcript is an important special case. Both problems have recently been illuminated by high-throughput RNA sequencing experiments which are quickly generating large amounts of data. However, much of the signal present in this data is corrupted or obscured by biases resulting in non-uniform and non-proportional representation of sequences from different transcripts. Many existing analyses attempt to deal with these and other biases with various task-specific approaches, which makes direct comparison between them difficult. However, two popular tools for isoform quantification, MISO and Cufflinks, have adopted a general probabilistic framework to model and mitigate these biases in a more general fashion. These advances motivate the need to investigate the effects of RNA-seq biases on the accuracy of different approaches for isoform quantification. We conduct the investigation by building models of increasing sophistication to account for noise introduced by the biases and compare their accuracy to the established approaches. We focus on methods that estimate the expression of alternatively-spliced isoforms with the percent-spliced-in (PSI) metric for each exon skipping event. To improve their estimates, many methods use evidence from RNA-seq reads that align to exon bodies. However, the methods we propose focus on reads that span only exon-exon junctions. As a result, our approaches are simpler and less sensitive to exon definitions than existing methods, which enables us to distinguish their strengths and weaknesses more easily. We present several probabilistic models of of position-specific read counts with increasing complexity and compare them to each other and to the current state-of-the-art methods in isoform quantification, MISO and Cufflinks. On a validation set with RT-PCR measurements for 26 cassette events, some of our methods are more accurate and some are significantly more consistent than these two popular tools. This comparison demonstrates the challenges in estimating the percent inclusion of alternatively spliced junctions and illuminates the tradeoffs between different approaches.

2012-01-01

380

Predicting the impact of alternative splicing on plant MADS domain protein function.  

PubMed

Several genome-wide studies demonstrated that alternative splicing (AS) significantly increases the transcriptome complexity in plants. However, the impact of AS on the functional diversity of proteins is difficult to assess using genome-wide approaches. The availability of detailed sequence annotations for specific genes and gene families allows for a more detailed assessment of the potential effect of AS on their function. One example is the plant MADS-domain transcription factor family, members of which interact to form protein complexes that function in transcription regulation. Here, we perform an in silico analysis of the potential impact of AS on the protein-protein interaction capabilities of MIKC-type MADS-domain proteins. We first confirmed the expression of transcript isoforms resulting from predicted AS events. Expressed transcript isoforms were considered functional if they were likely to be translated and if their corresponding AS events either had an effect on predicted dimerisation motifs or occurred in regions known to be involved in multimeric complex formation, or otherwise, if their effect was conserved in different species. Nine out of twelve MIKC MADS-box genes predicted to produce multiple protein isoforms harbored putative functional AS events according to those criteria. AS events with conserved effects were only found at the borders of or within the K-box domain. We illustrate how AS can contribute to the evolution of interaction networks through an example of selective inclusion of a recently evolved interaction motif in the MADS AFFECTING FLOWERING1-3 (MAF1-3) subclade. Furthermore, we demonstrate the potential effect of an AS event in SHORT VEGETATIVE PHASE (SVP), resulting in the deletion of a short sequence stretch including a predicted interaction motif, by overexpression of the fully spliced and the alternatively spliced SVP transcripts. For most of the AS events we were able to formulate hypotheses about the potential impact on the interaction capabilities of the encoded MIKC proteins. PMID:22295091

Severing, Edouard I; van Dijk, Aalt D J; Morabito, Giuseppa; Busscher-Lange, Jacqueline; Immink, Richard G H; van Ham, Roeland C H J

2012-01-25

381

Identification and characterization of the alternatively spliced nuclear receptor coactivator-6 isoforms.  

PubMed

The nuclear receptor coactivator-6 (NCOA6, AIB3, PRIP, ASC-2, TRBP, RAP250 or NRC) is a co-activator for nuclear hormone receptors and certain other transcription factors. NCOA6 plays an important role in embryonic development, adipocyte differentiation, metabolism and breast carcinogenesis. The human and mouse NCOA6 genes had 15 and 14 previously identified exons, respectively. This study further identified an alternatively spliced exon 11b (E11b) in human or E10b in mouse, which codes a short polypeptide and a Stop codon, resulting in splicing variants lacking the last four exon-coded polypeptide. Analyses of mouse testis NCOA6 mRNAs identified four alternatively spliced variants, NCOA6-? (without E10b), -? (without E10a and E10b), -? (with E10a and E10b) and -? (without E10a but with E10b). These isoforms were detected in multiple mouse tissues and in MDA-MB-435 human cells. NCOA6-? and -? are mainly located in the nucleus; NCOA6-? is located in both cytoplasm and nucleus; and NCOA6-? is mainly located in mitochondria. The C-terminus coded by the last four exons was responsible for locating NCOA6-? and -? into the nucleus. The human E11a or mouse E10a-coded region is responsible for distributing NCOA6-? in both cytoplasm and nucleus, while the region coded by E8-E9 in human or E7-E8 in mouse is responsible for directing NCOA6-? to mitochondria. Our assays also demonstrated that NCOA6-? and -? could significantly enhance estrogen receptor ?-mediated transcription, but NCOA6-? and -? were unable to do so. These results suggest that the diverse physiological function of NCOA6 may be mediated by multiple isoforms expressed in different tissues and localized in different subcellular compartments. PMID:21552418

Li, Qingtian; Xu, Jianming

2011-04-19

382

Quantification of type II procollagen splice forms using Alternative Transcript-qPCR (AT-qPCR)  

PubMed Central

During skeletal development, the onset of chondrogenic differentiation is marked by expression of the ?1(II) procollagen Col2a1) gene. Exon 2 of Col2a1 codes for a cysteine-rich von Willebrand factor C-like domain. Chondroprogenitors express the exon 2-containing IIA and IID splice forms by utilizing adjacent 5? splice sites separated by 3 base pairs. There is a shift to expression of the shorter, exon 2-lacking IIB splice form with further differentiation. Alternative splicing analysis of Col2a1 splice forms has often relied upon semi-quantitative PCR, using a single set of PCR primers to amplify multiple splice forms. We show that this widely used method is inaccurate due to mismatched amplification efficiency of different-sized PCR products. We have developed the TaqMan®-based AT-qPCR (Alternative Transcript-qPCR) assay to more accurately quantify alternatively spliced mRNA, and demonstrate the measurement of Col2a1 splice form expression in differentiating ATDC5 cells in vitro and in wild type mouse embryonic and postnatal cartilage in vivo. The AT-qPCR assay is based on the use of a multiple amplicon standard (MAS) plasmid, containing a chemically synthesized cluster of splice site-spanning PCR amplicons, to quantify alternative splice forms by standard curve-based qPCR. The MAS plasmid designed for Col2a1 also contained an 18S rRNA amplicon for sample normalization, and an amplicon corresponding to a region spanning exon 52-53 to measure total Col2a1 mRNA. In mouse E12.5 to P70 cartilage, we observed the expected switch between the IIA and IIB splice forms; no IID or IIC splice products were observed. However, in the ATDC5 cultures, predominant expression of the IIA and IID splice forms was found at all times in culture. Additionally, we observed that the sum of the IIA, IIB and IID splice forms comprises only a small fraction of Col2a1 transcripts containing the constitutive exon 52-53 junction. We conclude from our results that the majority of ATDC5 cells in the assay described in this study remained as chondroprogenitors during culture in standard differentiation conditions, and that additional Col2a1 transcripts may be present. The validity of this novel AT-qPCR assay was confirmed by demonstrating the expected Col2a1 isoform expression patterns in vivo in developing mouse cartilage. The ability to measure true levels of procollagen type II splice forms will provide better monitoring of chondrocyte differentiation in other model systems. In addition, the AT-qPCR assay described here could be applied to any gene of interest to detect and quantify known and predicted alternative splice forms and can be scaled up for high throughput assays.

McAlinden, Audrey; Shim, Kyu-Hwan; Wirthlin, Louisa; Ravindran, Soumya; Hering, Thomas M.

2012-01-01

383

Alternative splicing attenuates transgenic expression directed by the apolipoprotein E promoter-enhancer based expression vector pLIV11.  

PubMed

The plasmid vector pLIV11 is used commonly to achieve liver-specific expression of genes of interest in transgenic mice and rabbits. Expression is driven by the human apolipoprotein (apo)E 5' proximal promoter, which includes 5 kb of upstream sequence, exon 1, intron 1, and 5 bp of exon 2. A 3.8 kb 3' hepatic control region, derived from a region approximately 18 kb downstream of the apoE gene, enhances liver-specific expression. Here, we report that cDNA sequences inserted into the multiple cloning site (MCS) of pLIV11, which is positioned just downstream of truncated exon 2, can cause exon 2 skipping. Hence, splicing is displaced to downstream cryptic 3' splice acceptor sites causing deletion of cloned 5' untranslated mRNA sequences and, in some cases, deletion of the 5' end of an open reading frame. To prevent use of cryptic splice sites, the pLIV11 vector was modified with an engineered 3' splice acceptor site inserted immediately downstream of truncated apoE exon 2. Presence of this sequence fully shifted splicing of exon 1 from the native intron 1-exon 2 splice acceptor site to the engineered site. This finding confirmed that sequences inserted into the MCS of the vector pLIV11 can affect exon 2 recognition and provides a strategy to protect cloned sequences from alternative splicing and possible attenuation of transgenic expression. PMID:19965599

Cheng, Dongmei; MacArthur, Philip S; Rong, Shunxing; Parks, John S; Shelness, Gregory S

2009-10-27

384

Psip1\\/Ledgf p52 Binds Methylated Histone H3K36 and Splicing Factors and Contributes to the Regulation of Alternative Splicing  

Microsoft Academic Search

Increasing evidence suggests that chromatin modifications have important roles in modulating constitutive or alternative splicing. Here we demonstrate that the PWWP domain of the chromatin-associated protein Psip1\\/Ledgf can specifically recognize tri-methylated H3K36 and that, like this histone modification, the Psip1 short (p52) isoform is enriched at active genes. We show that the p52, but not the long (p75), isoform of

Madapura M. Pradeepa; Heidi G. Sutherland; Jernej Ule; Graeme R. Grimes; Wendy A. Bickmore

2012-01-01

385

DNA methylation dynamics, metabolic fluxes, gene splicing, and alternative phenotypes in honey bees  

PubMed Central

In honey bees (Apis mellifera), the development of a larva into either a queen or worker depends on differential feeding with royal jelly and involves epigenomic modifications by DNA methyltransferases. To understand the role of DNA methylation in this process we sequenced the larval methylomes in both queens and workers. We show that the number of differentially methylated genes (DMGs) in larval head is significantly increased relative to adult brain (2,399 vs. 560) with more than 80% of DMGs up-methylated in worker larvae. Several highly conserved metabolic and signaling pathways are enriched in methylated genes, underscoring the connection between dietary intake and metabolic flux. This includes genes related to juvenile hormone and insulin, two hormones shown previously to regulate caste determination. We also tie methylation data to expressional profiling and describe a distinct role for one of the DMGs encoding anaplastic lymphoma kinase (ALK), an important regulator of metabolism. We show that alk is not only differentially methylated and alternatively spliced in Apis, but also seems to be regulated by a cis-acting, anti-sense non–protein-coding transcript. The unusually complex regulation of ALK in Apis suggests that this protein could represent a previously unknown node in a process that activates downstream signaling according to a nutritional context. The correlation between methylation and alternative splicing of alk is consistent with the recently described mechanism involving RNA polymerase II pausing. Our study offers insights into diet-controlled development in Apis.

Foret, Sylvain; Kucharski, Robert; Pellegrini, Matteo; Feng, Suhua; Jacobsen, Steven E.; Robinson, Gene E.; Maleszka, Ryszard

2012-01-01

386

Alternative Cyclin D1 Splice Forms Differentially Regulate the DNA Damage Response  

PubMed Central

The DNA damage response (DDR) activates downstream pathways including cell cycle checkpoints. The cyclin D1 gene is overexpressed or amplified in many human cancers and is required for gastrointestinal, breast, and skin tumors in murine models. A common polymorphism in the human cyclin D1 gene is alternatively spliced, resulting in cyclin D1a and D1b proteins that differ in their carboxyl terminus. Cyclin D1 overexpression enhances DNA-damage induced apoptosis. The role of