Differential expression of conserved and novel microRNAs during tail regeneration in the lizard Anolis carolinensis.

PubMed

Hutchins, Elizabeth D; Eckalbar, Walter L; Wolter, Justin M; Mangone, Marco; Kusumi, Kenro

2016-05-05

Lizards are evolutionarily the most closely related vertebrates to humans that can lose and regrow an entire appendage. Regeneration in lizards involves differential expression of hundreds of genes that regulate wound healing, musculoskeletal development, hormonal response, and embryonic morphogenesis. While microRNAs are able to regulate large groups of genes, their role in lizard regeneration has not been investigated. MicroRNA sequencing of green anole lizard (Anolis carolinensis) regenerating tail and associated tissues revealed 350 putative novel and 196 known microRNA precursors. Eleven microRNAs were differentially expressed between the regenerating tail tip and base during maximum outgrowth (25 days post autotomy), including miR-133a, miR-133b, and miR-206, which have been reported to regulate regeneration and stem cell proliferation in other model systems. Three putative novel differentially expressed microRNAs were identified in the regenerating tail tip. Differentially expressed microRNAs were identified in the regenerating lizard tail, including known regulators of stem cell proliferation. The identification of 3 putative novel microRNAs suggests that regulatory networks, either conserved in vertebrates and previously uncharacterized or specific to lizards, are involved in regeneration. These findings suggest that differential regulation of microRNAs may play a role in coordinating the timing and expression of hundreds of genes involved in regeneration.

CID-miRNA: A web server for prediction of novel miRNA precursors in human genome

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

Tyagi, Sonika; Vaz, Candida; Gupta, Vipin

2008-08-08

microRNAs (miRNA) are a class of non-protein coding functional RNAs that are thought to regulate expression of target genes by direct interaction with mRNAs. miRNAs have been identified through both experimental and computational methods in a variety of eukaryotic organisms. Though these approaches have been partially successful, there is a need to develop more tools for detection of these RNAs as they are also thought to be present in abundance in many genomes. In this report we describe a tool and a web server, named CID-miRNA, for identification of miRNA precursors in a given DNA sequence, utilising secondary structure-based filteringmore » systems and an algorithm based on stochastic context free grammar trained on human miRNAs. CID-miRNA analyses a given sequence using a web interface, for presence of putative miRNA precursors and the generated output lists all the potential regions that can form miRNA-like structures. It can also scan large genomic sequences for the presence of potential miRNA precursors in its stand-alone form. The web server can be accessed at (http://mirna.jnu.ac.in/cidmirna/)« less

Undesired Small RNAs Originate from an Artificial microRNA Precursor in Transgenic Petunia (Petunia hybrida)

PubMed Central

Guo, Yulong; Han, Yao; Ma, Jing; Wang, Huiping; Sang, Xianchun; Li, Mingyang

2014-01-01

Although artificial microRNA (amiRNA) technology has been used frequently in gene silencing in plants, little research has been devoted to investigating the accuracy of amiRNA precursor processing. In this work, amiRNAchs1 (amiRchs1), based on the Arabidopsis miR319a precursor, was expressed in order to suppress the expression of CHS genes in petunia. The transgenic plants showed the CHS gene-silencing phenotype. A modified 5′ RACE technique was used to map small-RNA-directed cleavage sites and to detect processing intermediates of the amiRchs1 precursor. The results showed that the target CHS mRNAs were cut at the expected sites and that the amiRchs1 precursor was processed from loop to base. The accumulation of small RNAs in amiRchs1 transgenic petunia petals was analyzed using the deep-sequencing technique. The results showed that, alongside the accumulation of the desired artificial microRNAs, additional small RNAs that originated from other regions of the amiRNA precursor were also accumulated at high frequency. Some of these had previously been found to be accumulated at low frequency in the products of ath-miR319a precursor processing and some of them were accompanied by 3′-tailing variant. Potential targets of the undesired small RNAs were discovered in petunia and other Solanaceae plants. The findings draw attention to the potential occurrence of undesired target silencing induced by such additional small RNAs when amiRNA technology is used. No appreciable production of secondary small RNAs occurred, despite the fact that amiRchs1 was designed to have perfect complementarity to its CHS-J target. This confirmed that perfect pairing between an amiRNA and its targets is not the trigger for secondary small RNA production. In conjunction with the observation that amiRNAs with perfect complementarity to their target genes show high efficiency and specificity in gene silencing, this finding has an important bearing on future applications of amiRNAs in gene silencing in plants. PMID:24897430

Undesired small RNAs originate from an artificial microRNA precursor in transgenic petunia (Petunia hybrida).

PubMed

Guo, Yulong; Han, Yao; Ma, Jing; Wang, Huiping; Sang, Xianchun; Li, Mingyang

2014-01-01

Although artificial microRNA (amiRNA) technology has been used frequently in gene silencing in plants, little research has been devoted to investigating the accuracy of amiRNA precursor processing. In this work, amiRNAchs1 (amiRchs1), based on the Arabidopsis miR319a precursor, was expressed in order to suppress the expression of CHS genes in petunia. The transgenic plants showed the CHS gene-silencing phenotype. A modified 5' RACE technique was used to map small-RNA-directed cleavage sites and to detect processing intermediates of the amiRchs1 precursor. The results showed that the target CHS mRNAs were cut at the expected sites and that the amiRchs1 precursor was processed from loop to base. The accumulation of small RNAs in amiRchs1 transgenic petunia petals was analyzed using the deep-sequencing technique. The results showed that, alongside the accumulation of the desired artificial microRNAs, additional small RNAs that originated from other regions of the amiRNA precursor were also accumulated at high frequency. Some of these had previously been found to be accumulated at low frequency in the products of ath-miR319a precursor processing and some of them were accompanied by 3'-tailing variant. Potential targets of the undesired small RNAs were discovered in petunia and other Solanaceae plants. The findings draw attention to the potential occurrence of undesired target silencing induced by such additional small RNAs when amiRNA technology is used. No appreciable production of secondary small RNAs occurred, despite the fact that amiRchs1 was designed to have perfect complementarity to its CHS-J target. This confirmed that perfect pairing between an amiRNA and its targets is not the trigger for secondary small RNA production. In conjunction with the observation that amiRNAs with perfect complementarity to their target genes show high efficiency and specificity in gene silencing, this finding has an important bearing on future applications of amiRNAs in gene silencing in plants.

A high-content morphological screen identifies novel microRNAs that regulate neuroblastoma cell differentiation

PubMed Central

Zhao, Zhenze; Ma, Xiuye; Hsiao, Tzu-Hung; Lin, Gregory; Kosti, Adam; Yu, Xiaojie; Suresh, Uthra; Chen, Yidong; Tomlinson, Gail E.; Pertsemlidis, Alexander; Du, Liqin

2014-01-01

Neuroblastoma, the most common extracranial solid tumor of childhood, arises from neural crest cell precursors that fail to differentiate. Inducing cell differentiation is an important therapeutic strategy for neuroblastoma. We developed a direct functional high-content screen to identify differentiation-inducing microRNAs, in order to develop microRNA-based differentiation therapy for neuroblastoma. We discovered novel microRNAs, and more strikingly, three microRNA seed families that induce neuroblastoma cell differentiation. In addition, we showed that microRNA seed families were overrepresented in the identified group of fourteen differentiation-inducing microRNAs, suggesting that microRNA seed families are functionally more important in neuroblastoma differentiation than microRNAs with unique sequences. We further investigated the differentiation-inducing function of the microRNA-506-3p/microRNA-124-3p seed family, which was the most potent inducer of differentiation. We showed that the differentiation-inducing function of microRNA-506-3p/microRNA-124-3p is mediated, at least partially, by down-regulating expression of their targets CDK4 and STAT3. We further showed that expression of miR-506-3p, but not miR-124-3p, is dramatically upregulated in differentiated neuroblastoma cells, suggesting the important role of endogenous miR-506-3p in differentiation and tumorigenesis. Overall, our functional screen on microRNAs provided the first comprehensive analysis on the involvements of microRNA species in neuroblastoma cell differentiation and identified novel differentiation-inducing microRNAs. Further investigations are certainly warranted to fully characterize the function of the identified microRNAs in order to eventually benefit neuroblastoma therapy. PMID:24811707

Unravelling the complexity of microRNA-mediated gene regulation in black pepper (Piper nigrum L.) using high-throughput small RNA profiling.

PubMed

Asha, Srinivasan; Sreekumar, Sweda; Soniya, E V

2016-01-01

Analysis of high-throughput small RNA deep sequencing data, in combination with black pepper transcriptome sequences revealed microRNA-mediated gene regulation in black pepper ( Piper nigrum L.). Black pepper is an important spice crop and its berries are used worldwide as a natural food additive that contributes unique flavour to foods. In the present study to characterize microRNAs from black pepper, we generated a small RNA library from black pepper leaf and sequenced it by Illumina high-throughput sequencing technology. MicroRNAs belonging to a total of 303 conserved miRNA families were identified from the sRNAome data. Subsequent analysis from recently sequenced black pepper transcriptome confirmed precursor sequences of 50 conserved miRNAs and four potential novel miRNA candidates. Stem-loop qRT-PCR experiments demonstrated differential expression of eight conserved miRNAs in black pepper. Computational analysis of targets of the miRNAs showed 223 potential black pepper unigene targets that encode diverse transcription factors and enzymes involved in plant development, disease resistance, metabolic and signalling pathways. RLM-RACE experiments further mapped miRNA-mediated cleavage at five of the mRNA targets. In addition, miRNA isoforms corresponding to 18 miRNA families were also identified from black pepper. This study presents the first large-scale identification of microRNAs from black pepper and provides the foundation for the future studies of miRNA-mediated gene regulation of stress responses and diverse metabolic processes in black pepper.

Discovery of precursor and mature microRNAs and their putative gene targets using high-throughput sequencing in pineapple (Ananas comosus var. comosus).

PubMed

Yusuf, Noor Hydayaty Md; Ong, Wen Dee; Redwan, Raimi Mohamed; Latip, Mariam Abd; Kumar, S Vijay

2015-10-15

MicroRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that negatively regulate gene expression, resulting in the silencing of target mRNA transcripts through mRNA cleavage or translational inhibition. MiRNAs play significant roles in various biological and physiological processes in plants. However, the miRNA-mediated gene regulatory network in pineapple, the model tropical non-climacteric fruit, remains largely unexplored. Here, we report a complete list of pineapple mature miRNAs obtained from high-throughput small RNA sequencing and precursor miRNAs (pre-miRNAs) obtained from ESTs. Two small RNA libraries were constructed from pineapple fruits and leaves, respectively, using Illumina's Solexa technology. Sequence similarity analysis using miRBase revealed 579,179 reads homologous to 153 miRNAs from 41 miRNA families. In addition, a pineapple fruit transcriptome library consisting of approximately 30,000 EST contigs constructed using Solexa sequencing was used for the discovery of pre-miRNAs. In all, four pre-miRNAs were identified (MIR156, MIR399, MIR444 and MIR2673). Furthermore, the same pineapple transcriptome was used to dissect the function of the miRNAs in pineapple by predicting their putative targets in conjunction with their regulatory networks. In total, 23 metabolic pathways were found to be regulated by miRNAs in pineapple. The use of high-throughput sequencing in pineapples to unveil the presence of miRNAs and their regulatory pathways provides insight into the repertoire of miRNA regulation used exclusively in this non-climacteric model plant. Copyright © 2015 Elsevier B.V. All rights reserved.

Transcriptome-wide identification of Rauvolfia serpentina microRNAs and prediction of their potential targets.

PubMed

Prakash, Pravin; Rajakani, Raja; Gupta, Vikrant

2016-04-01

MicroRNAs (miRNAs) are small non-coding RNAs of ∼ 19-24 nucleotides (nt) in length and considered as potent regulators of gene expression at transcriptional and post-transcriptional levels. Here we report the identification and characterization of 15 conserved miRNAs belonging to 13 families from Rauvolfia serpentina through in silico analysis of available nucleotide dataset. The identified mature R. serpentina miRNAs (rse-miRNAs) ranged between 20 and 22nt in length, and the average minimal folding free energy index (MFEI) value of rse-miRNA precursor sequences was found to be -0.815 kcal/mol. Using the identified rse-miRNAs as query, their potential targets were predicted in R. serpentina and other plant species. Gene Ontology (GO) annotation showed that predicted targets of rse-miRNAs include transcription factors as well as genes involved in diverse biological processes such as primary and secondary metabolism, stress response, disease resistance, growth, and development. Few rse-miRNAs were predicted to target genes of pharmaceutically important secondary metabolic pathways such as alkaloids and anthocyanin biosynthesis. Phylogenetic analysis showed the evolutionary relationship of rse-miRNAs and their precursor sequences to homologous pre-miRNA sequences from other plant species. The findings under present study besides giving first hand information about R. serpentina miRNAs and their targets, also contributes towards the better understanding of miRNA-mediated gene regulatory processes in plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

Plant polycistronic precursors containing non-homologous microRNAs target transcripts encoding functionally related proteins

PubMed Central

2009-01-01

Background MicroRNAs (miRNAs) are endogenous single-stranded small RNAs that regulate the expression of specific mRNAs involved in diverse biological processes. In plants, miRNAs are generally encoded as a single species in independent transcriptional units, referred to as MIRNA genes, in contrast to animal miRNAs, which are frequently clustered. Results We performed a comparative genomic analysis in three model plants (rice, poplar and Arabidopsis) and characterized miRNA clusters containing two to eight miRNA species. These clusters usually encode miRNAs of the same family and certain share a common evolutionary origin across monocot and dicot lineages. In addition, we identified miRNA clusters harboring miRNAs with unrelated sequences that are usually not evolutionarily conserved. Strikingly, non-homologous miRNAs from the same cluster were predicted to target transcripts encoding related proteins. At least four Arabidopsis non-homologous clusters were expressed as single transcriptional units. Overexpression of one of these polycistronic precursors, producing Ath-miR859 and Ath-miR774, led to the DCL1-dependent accumulation of both miRNAs and down-regulation of their different mRNA targets encoding F-box proteins. Conclusions In addition to polycistronic precursors carrying related miRNAs, plants also contain precursors allowing coordinated expression of non-homologous miRNAs to co-regulate functionally related target transcripts. This mechanism paves the way for using polycistronic MIRNA precursors as a new molecular tool for plant biologists to simultaneously control the expression of different genes. PMID:19951405

miRBase: integrating microRNA annotation and deep-sequencing data.

PubMed

Kozomara, Ana; Griffiths-Jones, Sam

2011-01-01

miRBase is the primary online repository for all microRNA sequences and annotation. The current release (miRBase 16) contains over 15,000 microRNA gene loci in over 140 species, and over 17,000 distinct mature microRNA sequences. Deep-sequencing technologies have delivered a sharp rise in the rate of novel microRNA discovery. We have mapped reads from short RNA deep-sequencing experiments to microRNAs in miRBase and developed web interfaces to view these mappings. The user can view all read data associated with a given microRNA annotation, filter reads by experiment and count, and search for microRNAs by tissue- and stage-specific expression. These data can be used as a proxy for relative expression levels of microRNA sequences, provide detailed evidence for microRNA annotations and alternative isoforms of mature microRNAs, and allow us to revisit previous annotations. miRBase is available online at: http://www.mirbase.org/.

Characterization of basal and lipopolysaccharide-induced microRNA expression in equine peripheral blood mononuclear cells using Next-Generation Sequencing

PubMed Central

Buechner-Maxwell, Virginia A.; Witonsky, Sharon G.; Pleasant, R. Scott; Werre, Stephen R.; Ahmed, S. Ansar

2017-01-01

The innate immune response to lipopolysaccharide contributes substantially to the morbidity and mortality of gram-negative sepsis. Horses and humans share an exquisite sensitivity to lipopolysaccharide and thus the horse may provide valuable comparative insights into this aspect of the inflammatory response. MicroRNAs, small non-coding RNA molecules acting as post-transcriptional regulators of gene expression, have key roles in toll-like receptor signaling regulation but have not been studied in this context in horses. The central hypothesis of this study was that lipopolysaccharide induces differential microRNA expression in equine peripheral blood mononuclear cells in a manner comparable to humans. Illumina Next Generation Sequencing was used to characterize the basal microRNA transcriptome in isolated peripheral blood mononuclear cells from healthy adult horses, and to evaluate LPS-induced changes in microRNA expression in cells cultured for up to four hours. Selected expression changes were validated using quantitative reverse-transcriptase PCR. Only miR-155 was significantly upregulated by LPS, changing in parallel with supernatant tumor necrosis factor-α concentration. Eight additional microRNAs, including miR-146a and miR-146b, showed significant expression change with time in culture without a clear LPS effect. Target predictions indicated a number of potential immunity-associated targets for miR-155 in the horse, including SOCS1, TAB2 and elements of the PI3K signaling pathway, suggesting that it is likely to influence the acute inflammatory response to LPS. Gene alignment showed extensive conservation of the miR-155 precursor gene and associated promoter regions between horses and humans. The basal and LPS-stimulated microRNA expression pattern characterized here were similar to those described in human leukocytes. As well as providing a resource for further research into the roles of microRNAs in immune responses in horses, this will facilitate inter-species comparative study of the role of microRNAs in the inflammatory cascade during endotoxemia and sepsis. PMID:28552958

MicroRNA-like RNAs from the same miRNA precursors play a role in cassava chilling responses.

PubMed

Zeng, Changying; Xia, Jing; Chen, Xin; Zhou, Yufei; Peng, Ming; Zhang, Weixiong

2017-12-07

MicroRNAs (miRNAs) are known to play important roles in various cellular processes and stress responses. MiRNAs can be identified by analyzing reads from high-throughput deep sequencing. The reads realigned to miRNA precursors besides canonical miRNAs were initially considered as sequencing noise and ignored from further analysis. Here we reported a small-RNA species of phased and half-phased miRNA-like RNAs different from canonical miRNAs from cassava miRNA precursors detected under four distinct chilling conditions. They can form abundant multiple small RNAs arranged along precursors in a tandem and phased or half-phased fashion. Some of these miRNA-like RNAs were experimentally confirmed by re-amplification and re-sequencing, and have a similar qRT-PCR detection ratio as their cognate canonical miRNAs. The target genes of those phased and half-phased miRNA-like RNAs function in process of cell growth metabolism and play roles in protein kinase. Half-phased miR171d.3 was confirmed to have cleavage activities on its target gene P-glycoprotein 11, a broad substrate efflux pump across cellular membranes, which is thought to provide protection for tropical cassava during sharp temperature decease. Our results showed that the RNAs from miRNA precursors are miRNA-like small RNAs that are viable negative gene regulators and may have potential functions in cassava chilling responses.

High-Throughput Sequencing of Arabidopsis microRNAs: Evidence for Frequent Birth and Death of MIRNA Genes

PubMed Central

Fahlgren, Noah; Howell, Miya D.; Kasschau, Kristin D.; Chapman, Elisabeth J.; Sullivan, Christopher M.; Cumbie, Jason S.; Givan, Scott A.; Law, Theresa F.; Grant, Sarah R.; Dangl, Jeffery L.; Carrington, James C.

2007-01-01

In plants, microRNAs (miRNAs) comprise one of two classes of small RNAs that function primarily as negative regulators at the posttranscriptional level. Several MIRNA genes in the plant kingdom are ancient, with conservation extending between angiosperms and the mosses, whereas many others are more recently evolved. Here, we use deep sequencing and computational methods to identify, profile and analyze non-conserved MIRNA genes in Arabidopsis thaliana. 48 non-conserved MIRNA families, nearly all of which were represented by single genes, were identified. Sequence similarity analyses of miRNA precursor foldback arms revealed evidence for recent evolutionary origin of 16 MIRNA loci through inverted duplication events from protein-coding gene sequences. Interestingly, these recently evolved MIRNA genes have taken distinct paths. Whereas some non-conserved miRNAs interact with and regulate target transcripts from gene families that donated parental sequences, others have drifted to the point of non-interaction with parental gene family transcripts. Some young MIRNA loci clearly originated from one gene family but form miRNAs that target transcripts in another family. We suggest that MIRNA genes are undergoing relatively frequent birth and death, with only a subset being stabilized by integration into regulatory networks. PMID:17299599

A simple method for construction of artificial microRNA vector in plant.

PubMed

Li, Yang; Li, Yang; Zhao, Sunping; Zhong, Sheng; Wang, Zhaohai; Ding, Bo; Li, Yangsheng

2014-10-01

Artificial microRNA (amiRNA) is a powerful tool for silencing genes in many plant species. Here we provide an easy method to construct amiRNA vectors that reinvents the Golden Gate cloning approach and features a novel system called top speed amiRNA construction (TAC). This speedy approach accomplishes one restriction-ligation step in only 5 min, allowing easy and high-throughput vector construction. Three primers were annealed to be a specific adaptor, then digested and ligated on our novel vector pTAC. Importantly, this method allows the recombined amiRNA constructs to maintain the precursor of osa-miR528 with exception of the desired amiRNA/amiRNA* sequences. Using this method, our results showed the expected decrease of targeted genes in Nicotiana benthamiana and Oryza sativa.

High-Throughput Sequencing and Characterization of the Small RNA Transcriptome Reveal Features of Novel and Conserved MicroRNAs in Panax ginseng

PubMed Central

Ma, Yimian; Yuan, Lichai; Lu, Shanfa

2012-01-01

microRNAs (miRNAs) play vital regulatory roles in many organisms through direct cleavage of transcripts, translational repression, or chromatin modification. Identification of miRNAs has been carried out in various plant species. However, no information is available for miRNAs from Panax ginseng, an economically significant medicinal plant species. Using the next generation high-throughput sequencing technology, we obtained 13,326,328 small RNA reads from the roots, stems, leaves and flowers of P. ginseng. Analysis of these small RNAs revealed the existence of a large, diverse and highly complicated small RNA population in P. ginseng. We identified 73 conserved miRNAs, which could be grouped into 33 families, and 28 non-conserved ones belonging to 9 families. Characterization of P. ginseng miRNA precursors revealed many features, such as production of two miRNAs from distinct regions of a precursor, clusters of two precursors in a transcript, and generation of miRNAs from both sense and antisense transcripts. It suggests the complexity of miRNA production in P. gingseng. Using a computational approach, we predicted for the conserved and non-conserved miRNA families 99 and 31 target genes, respectively, of which eight were experimentally validated. Among all predicted targets, only about 20% are conserved among various plant species, whereas the others appear to be non-conserved, indicating the diversity of miRNA functions. Consistently, many miRNAs exhibited tissue-specific expression patterns. Moreover, we identified five dehydration- and ten heat-responsive miRNAs and found the existence of a crosstalk among some of the stress-responsive miRNAs. Our results provide the first clue to the elucidation of miRNA functions in P. ginseng. PMID:22962612

Identification of neglected cestode Taenia multiceps microRNAs by illumina sequencing and bioinformatic analysis

PubMed Central

2013-01-01

Background Worldwide, but especially in developing countries, coenurosis of sheep and other livestock is caused by Taenia multiceps larvae, and zoonotic infections occur in humans. Infections frequently lead to host death, resulting in huge socioeconomic losses. MicroRNAs (miRNAs) have important roles in the post-transcriptional regulation of a large number of animal genes by imperfectly binding target mRNAs. To date, there have been no reports of miRNAs in T. multiceps. Results In this study, we obtained 12.8 million high quality raw reads from adult T. multiceps small RNA library using Illumina sequencing technology. A total of 796 conserved miRNA families (containing 1,006 miRNAs) from 170,888 unique miRNAs were characterized using miRBase (Release 17.0). Here, we selected three conserved miRNA/miRNA* (antisense strand) duplexes at random and amplified their corresponding precursors using a PCR-based method. Furthermore, 20 candidate novel miRNA precursors were verified by genomic PCR. Among these, six corresponding T. multiceps miRNAs are considered specific for Taeniidae because no homologs were found in other species annotated in miRBase. In addition, 181,077 target sites within T. multiceps transcriptome were predicted for 20 candidate newly miRNAs. Conclusions Our large-scale investigation of miRNAs in adult T. multiceps provides a substantial platform for improving our understanding of the molecular regulation of T. multiceps and other cestodes development. PMID:23941076

MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression

PubMed Central

Grace, Christy R.; Ferreira, Antonio M.; Waddell, M. Brett; Ridout, Granger; Naeve, Deanna; Leuze, Michael; LoCascio, Philip F.; Panetta, John C.; Wilkinson, Mark R.; Pui, Ching-Hon; Naeve, Clayton W.; Uberbacher, Edward C.; Bonten, Erik J.; Evans, William E.

2016-01-01

MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA) and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR) that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 × 10−16) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription. PMID:26844769

RNAdualPF: software to compute the dual partition function with sample applications in molecular evolution theory.

PubMed

Garcia-Martin, Juan Antonio; Bayegan, Amir H; Dotu, Ivan; Clote, Peter

2016-10-19

RNA inverse folding is the problem of finding one or more sequences that fold into a user-specified target structure s 0 , i.e. whose minimum free energy secondary structure is identical to the target s 0 . Here we consider the ensemble of all RNA sequences that have low free energy with respect to a given target s 0 . We introduce the program RNAdualPF, which computes the dual partition function Z ∗ , defined as the sum of Boltzmann factors exp(-E(a,s 0 )/RT) of all RNA nucleotide sequences a compatible with target structure s 0 . Using RNAdualPF, we efficiently sample RNA sequences that approximately fold into s 0 , where additionally the user can specify IUPAC sequence constraints at certain positions, and whether to include dangles (energy terms for stacked, single-stranded nucleotides). Moreover, since we also compute the dual partition function Z ∗ (k) over all sequences having GC-content k, the user can require that all sampled sequences have a precise, specified GC-content. Using Z ∗ , we compute the dual expected energy 〈E ∗ 〉, and use it to show that natural RNAs from the Rfam 12.0 database have higher minimum free energy than expected, thus suggesting that functional RNAs are under evolutionary pressure to be only marginally thermodynamically stable. We show that C. elegans precursor microRNA (pre-miRNA) is significantly non-robust with respect to mutations, by comparing the robustness of each wild type pre-miRNA sequence with 2000 [resp. 500] sequences of the same GC-content generated by RNAdualPF, which approximately [resp. exactly] fold into the wild type target structure. We confirm and strengthen earlier findings that precursor microRNAs and bacterial small noncoding RNAs display plasticity, a measure of structural diversity. We describe RNAdualPF, which rapidly computes the dual partition function Z ∗ and samples sequences having low energy with respect to a target structure, allowing sequence constraints and specified GC-content. Using different inverse folding software, another group had earlier shown that pre-miRNA is mutationally robust, even controlling for compositional bias. Our opposite conclusion suggests a cautionary note that computationally based insights into molecular evolution may heavily depend on the software used. C/C++-software for RNAdualPF is available at http://bioinformatics.bc.edu/clotelab/RNAdualPF .

RISC RNA sequencing for context-specific identification of in vivo microRNA targets.

PubMed

Matkovich, Scot J; Van Booven, Derek J; Eschenbacher, William H; Dorn, Gerald W

2011-01-07

MicroRNAs (miRs) are expanding our understanding of cardiac disease and have the potential to transform cardiovascular therapeutics. One miR can target hundreds of individual mRNAs, but existing methodologies are not sufficient to accurately and comprehensively identify these mRNA targets in vivo. To develop methods permitting identification of in vivo miR targets in an unbiased manner, using massively parallel sequencing of mouse cardiac transcriptomes in combination with sequencing of mRNA associated with mouse cardiac RNA-induced silencing complexes (RISCs). We optimized techniques for expression profiling small amounts of RNA without introducing amplification bias and applied this to anti-Argonaute 2 immunoprecipitated RISCs (RISC-Seq) from mouse hearts. By comparing RNA-sequencing results of cardiac RISC and transcriptome from the same individual hearts, we defined 1645 mRNAs consistently targeted to mouse cardiac RISCs. We used this approach in hearts overexpressing miRs from Myh6 promoter-driven precursors (programmed RISC-Seq) to identify 209 in vivo targets of miR-133a and 81 in vivo targets of miR-499. Consistent with the fact that miR-133a and miR-499 have widely differing "seed" sequences and belong to different miR families, only 6 targets were common to miR-133a- and miR-499-programmed hearts. RISC-sequencing is a highly sensitive method for general RISC profiling and individual miR target identification in biological context and is applicable to any tissue and any disease state.

Identifying MicroRNAs and Transcript Targets in Jatropha Seeds

PubMed Central

Galli, Vanessa; Guzman, Frank; de Oliveira, Luiz F. V.; Loss-Morais, Guilherme; Körbes, Ana P.; Silva, Sérgio D. A.; Margis-Pinheiro, Márcia M. A. N.; Margis, Rogério

2014-01-01

MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play a key role in diverse plant biological processes. Jatropha curcas L. has received significant attention as a potential oilseed crop for the production of renewable oil. Here, a sRNA library of mature seeds and three mRNA libraries from three different seed development stages were generated by deep sequencing to identify and characterize the miRNAs and pre-miRNAs of J. curcas. Computational analysis was used for the identification of 180 conserved miRNAs and 41 precursors (pre-miRNAs) as well as 16 novel pre-miRNAs. The predicted miRNA target genes are involved in a broad range of physiological functions, including cellular structure, nuclear function, translation, transport, hormone synthesis, defense, and lipid metabolism. Some pre-miRNA and miRNA targets vary in abundance between the three stages of seed development. A search for sequences that produce siRNA was performed, and the results indicated that J. curcas siRNAs play a role in nuclear functions, transport, catalytic processes and disease resistance. This study presents the first large scale identification of J. curcas miRNAs and their targets in mature seeds based on deep sequencing, and it contributes to a functional understanding of these miRNAs. PMID:24551031

Genomic analysis suggests that mRNA destabilization by the microprocessor is specialized for the auto-regulation of Dgcr8.

PubMed

Shenoy, Archana; Blelloch, Robert

2009-09-11

The Microprocessor, containing the RNA binding protein Dgcr8 and RNase III enzyme Drosha, is responsible for processing primary microRNAs to precursor microRNAs. The Microprocessor regulates its own levels by cleaving hairpins in the 5'UTR and coding region of the Dgcr8 mRNA, thereby destabilizing the mature transcript. To determine whether the Microprocessor has a broader role in directly regulating other coding mRNA levels, we integrated results from expression profiling and ultra high-throughput deep sequencing of small RNAs. Expression analysis of mRNAs in wild-type, Dgcr8 knockout, and Dicer knockout mouse embryonic stem (ES) cells uncovered mRNAs that were specifically upregulated in the Dgcr8 null background. A number of these transcripts had evolutionarily conserved predicted hairpin targets for the Microprocessor. However, analysis of deep sequencing data of 18 to 200nt small RNAs in mouse ES, HeLa, and HepG2 indicates that exonic sequence reads that map in a pattern consistent with Microprocessor activity are unique to Dgcr8. We conclude that the Microprocessor's role in directly destabilizing coding mRNAs is likely specifically targeted to Dgcr8 itself, suggesting a specialized cellular mechanism for gene auto-regulation.

MicroRNAs form triplexes with double stranded DNA at sequence-specific binding sites; a eukaryotic mechanism via which microRNAs could directly alter gene expression

DOE PAGES

Paugh, Steven W.; Coss, David R.; Bao, Ju; ...

2016-02-04

MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA). Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence that microRNAs form triple-helical structures with duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show thatmore » several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 x 10 -16) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. As a result, this work has thus revealed a new mechanism by which microRNAs can interact with gene promoter regions to modify gene transcription.« less

MicroRNAs form triplexes with double stranded DNA at sequence-specific binding sites; a eukaryotic mechanism via which microRNAs could directly alter gene expression

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

Paugh, Steven W.; Coss, David R.; Bao, Ju

MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA). Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence that microRNAs form triple-helical structures with duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show thatmore » several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 x 10 -16) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. As a result, this work has thus revealed a new mechanism by which microRNAs can interact with gene promoter regions to modify gene transcription.« less

mESAdb: microRNA Expression and Sequence Analysis Database

PubMed Central

Kaya, Koray D.; Karakülah, Gökhan; Yakıcıer, Cengiz M.; Acar, Aybar C.; Konu, Özlen

2011-01-01

microRNA expression and sequence analysis database (http://konulab.fen.bilkent.edu.tr/mirna/) (mESAdb) is a regularly updated database for the multivariate analysis of sequences and expression of microRNAs from multiple taxa. mESAdb is modular and has a user interface implemented in PHP and JavaScript and coupled with statistical analysis and visualization packages written for the R language. The database primarily comprises mature microRNA sequences and their target data, along with selected human, mouse and zebrafish expression data sets. mESAdb analysis modules allow (i) mining of microRNA expression data sets for subsets of microRNAs selected manually or by motif; (ii) pair-wise multivariate analysis of expression data sets within and between taxa; and (iii) association of microRNA subsets with annotation databases, HUGE Navigator, KEGG and GO. The use of existing and customized R packages facilitates future addition of data sets and analysis tools. Furthermore, the ability to upload and analyze user-specified data sets makes mESAdb an interactive and expandable analysis tool for microRNA sequence and expression data. PMID:21177657

mESAdb: microRNA expression and sequence analysis database.

PubMed

Kaya, Koray D; Karakülah, Gökhan; Yakicier, Cengiz M; Acar, Aybar C; Konu, Ozlen

2011-01-01

microRNA expression and sequence analysis database (http://konulab.fen.bilkent.edu.tr/mirna/) (mESAdb) is a regularly updated database for the multivariate analysis of sequences and expression of microRNAs from multiple taxa. mESAdb is modular and has a user interface implemented in PHP and JavaScript and coupled with statistical analysis and visualization packages written for the R language. The database primarily comprises mature microRNA sequences and their target data, along with selected human, mouse and zebrafish expression data sets. mESAdb analysis modules allow (i) mining of microRNA expression data sets for subsets of microRNAs selected manually or by motif; (ii) pair-wise multivariate analysis of expression data sets within and between taxa; and (iii) association of microRNA subsets with annotation databases, HUGE Navigator, KEGG and GO. The use of existing and customized R packages facilitates future addition of data sets and analysis tools. Furthermore, the ability to upload and analyze user-specified data sets makes mESAdb an interactive and expandable analysis tool for microRNA sequence and expression data.

A transcriptome-wide, organ-specific regulatory map of Dendrobium officinale, an important traditional Chinese orchid herb

PubMed Central

Meng, Yijun; Yu, Dongliang; Xue, Jie; Lu, Jiangjie; Feng, Shangguo; Shen, Chenjia; Wang, Huizhong

2016-01-01

Dendrobium officinale is an important traditional Chinese herb. Here, we did a transcriptome-wide, organ-specific study on this valuable plant by combining RNA, small RNA (sRNA) and degradome sequencing. RNA sequencing of four organs (flower, root, leaf and stem) of Dendrobium officinale enabled us to obtain 536,558 assembled transcripts, from which 2,645, 256, 42 and 54 were identified to be highly expressed in the four organs respectively. Based on sRNA sequencing, 2,038, 2, 21 and 24 sRNAs were identified to be specifically accumulated in the four organs respectively. A total of 1,047 mature microRNA (miRNA) candidates were detected. Based on secondary structure predictions and sequencing, tens of potential miRNA precursors were identified from the assembled transcripts. Interestingly, phase-distributed sRNAs with degradome-based processing evidences were discovered on the long-stem structures of two precursors. Target identification was performed for the 1,047 miRNA candidates, resulting in the discovery of 1,257 miRNA--target pairs. Finally, some biological meaningful subnetworks involving hormone signaling, development, secondary metabolism and Argonaute 1-related regulation were established. All of the sequencing data sets are available at NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra/). Summarily, our study provides a valuable resource for the in-depth molecular and functional studies on this important Chinese orchid herb. PMID:26732614

Dual role for argonautes in microRNA processing and posttranscriptional regulation of microRNA expression.

PubMed

Diederichs, Sven; Haber, Daniel A

2007-12-14

MicroRNAs are small endogenous noncoding RNAs involved in posttranscriptional gene regulation. During microRNA biogenesis, Drosha and Dicer process the primary transcript (pri-miRNA) through a precursor hairpin (pre-miRNA) to the mature miRNA. The miRNA is incorporated into the RNA-Induced Silencing Complex (RISC) with Argonaute proteins, the effector molecules in RNA interference (RNAi). Here, we show that all Argonautes elevate mature miRNA expression posttranscriptionally, independent of RNase activity. Also, we identify a role for the RISC slicer Argonaute2 (Ago2) in cleaving the pre-miRNA to an additional processing intermediate, termed Ago2-cleaved precursor miRNA or ac-pre-miRNA. This endogenous, on-pathway intermediate results from cleavage of the pre-miRNA hairpin 12 nucleotides from its 3'-end. By analogy to siRNA processing, Ago2 cleavage may facilitate removal of the nicked passenger strand from RISC after maturation. The multiple roles of Argonautes in the RNAi effector phase and miRNA biogenesis and maturation suggest coordinate regulation of microRNA expression and function.

Transcriptome and Small RNA Deep Sequencing Reveals Deregulation of miRNA Biogenesis in Human Glioma

PubMed Central

Moore, Lynette M.; Kivinen, Virpi; Liu, Yuexin; Annala, Matti; Cogdell, David; Liu, Xiuping; Liu, Chang-Gong; Sawaya, Raymond; Yli-Harja, Olli; Shmulevich, Ilya; Fuller, Gregory N.; Zhang, Wei; Nykter, Matti

2013-01-01

Altered expression of oncogenic and tumor-suppressing microRNAs (miRNAs) is widely associated with tumorigenesis. However, the regulatory mechanisms underlying these alterations are poorly understood. We sought to shed light on the deregulation of miRNA biogenesis promoting the aberrant miRNA expression profiles identified in these tumors. Using sequencing technology to perform both whole-transcriptome and small RNA sequencing of glioma patient samples, we examined precursor and mature miRNAs to directly evaluate the miRNA maturation process, and interrogated expression profiles for genes involved in the major steps of miRNA biogenesis. We found that ratios of mature to precursor forms of a large number of miRNAs increased with the progression from normal brain to low-grade and then to high-grade gliomas. The expression levels of genes involved in each of the three major steps of miRNA biogenesis (nuclear processing, nucleo-cytoplasmic transport, and cytoplasmic processing) were systematically altered in glioma tissues. Survival analysis of an independent data set demonstrated that the alteration of genes involved in miRNA maturation correlates with survival in glioma patients. Direct quantification of miRNA maturation with deep sequencing demonstrated that deregulation of the miRNA biogenesis pathway is a hallmark for glioma genesis and progression. PMID:23007860

Identification and characterization of novel and differentially expressed microRNAs in peripheral blood from healthy and mastitis Holstein cattle by deep sequencing.

PubMed

Li, Zhixiong; Wang, Hongliang; Chen, Ling; Wang, Lijun; Liu, Xiaolin; Ru, Caixia; Song, Ailong

2014-02-01

MicroRNA (miRNA) mediates post-transcriptional gene regulation and plays an important role in regulating the development of immune cells and in modulating innate and adaptive immune responses in mammals, including cattle. In the present study, we identified novel and differentially expressed miRNAs in peripheral blood from healthy and mastitis Holstein cattle by Solexa sequencing and bioinformatics. In total, 608 precursor hairpins (pre-miRNAs) encoding for 753 mature miRNAs were detected. Statistically, 173 unique miRNAs (of 753, 22.98%) were identified that had significant differential expression between healthy and mastitis Holstein cattle (P < 0.001). Most differentially expressed miRNAs (118 of 173, 68.21%) belonged to the chemokine signaling pathway involved in the immune responses. This study expands the number of miRNAs known to be expressed in cattle. The patterns of miRNAs expression differed significantly between the peripheral blood from healthy and mastitis Holstein cattle, which provide important information on mastitis in miRNAs expression. Diverse miRNAs may play an important role in the treatment of mastitis in Holstein cattle. © 2013 Stichting International Foundation for Animal Genetics.

Computational Analysis of Mouse piRNA Sequence and Biogenesis

PubMed Central

Betel, Doron; Sheridan, Robert; Marks, Debora S; Sander, Chris

2007-01-01

The recent discovery of a new class of 30-nucleotide long RNAs in mammalian testes, called PIWI-interacting RNA (piRNA), with similarities to microRNAs and repeat-associated small interfering RNAs (rasiRNAs), has raised puzzling questions regarding their biogenesis and function. We report a comparative analysis of currently available piRNA sequence data from the pachytene stage of mouse spermatogenesis that sheds light on their sequence diversity and mechanism of biogenesis. We conclude that (i) there are at least four times as many piRNAs in mouse testes than currently known; (ii) piRNAs, which originate from long precursor transcripts, are generated by quasi-random enzymatic processing that is guided by a weak sequence signature at the piRNA 5′ends resulting in a large number of distinct sequences; and (iii) many of the piRNA clusters contain inverted repeats segments capable of forming double-strand RNA fold-back segments that may initiate piRNA processing analogous to transposon silencing. PMID:17997596

High-throughput amplification of mature microRNAs in uncharacterized animal models using polyadenylated RNA and stem-loop reverse transcription polymerase chain reaction.

PubMed

Biggar, Kyle K; Wu, Cheng-Wei; Storey, Kenneth B

2014-10-01

This study makes a significant advancement on a microRNA amplification technique previously used for expression analysis and sequencing in animal models without annotated mature microRNA sequences. As research progresses into the post-genomic era of microRNA prediction and analysis, the need for a rapid and cost-effective method for microRNA amplification is critical to facilitate wide-scale analysis of microRNA expression. To facilitate this requirement, we have reoptimized the design of amplification primers and introduced a polyadenylation step to allow amplification of all mature microRNAs from a single RNA sample. Importantly, this method retains the ability to sequence reverse transcription polymerase chain reaction (RT-PCR) products, validating microRNA-specific amplification. Copyright © 2014 Elsevier Inc. All rights reserved.

Revisiting Criteria for Plant MicroRNA Annotation in the Era of Big Data[OPEN

PubMed Central

2018-01-01

MicroRNAs (miRNAs) are ∼21-nucleotide-long regulatory RNAs that arise from endonucleolytic processing of hairpin precursors. Many function as essential posttranscriptional regulators of target mRNAs and long noncoding RNAs. Alongside miRNAs, plants also produce large numbers of short interfering RNAs (siRNAs), which are distinguished from miRNAs primarily by their biogenesis (typically processed from long double-stranded RNA instead of single-stranded hairpins) and functions (typically via roles in transcriptional regulation instead of posttranscriptional regulation). Next-generation DNA sequencing methods have yielded extensive data sets of plant small RNAs, resulting in many miRNA annotations. However, it has become clear that many miRNA annotations are questionable. The sheer number of endogenous siRNAs compared with miRNAs has been a major factor in the erroneous annotation of siRNAs as miRNAs. Here, we provide updated criteria for the confident annotation of plant miRNAs, suitable for the era of “big data” from DNA sequencing. The updated criteria emphasize replication and the minimization of false positives, and they require next-generation sequencing of small RNAs. We argue that improved annotation systems are needed for miRNAs and all other classes of plant small RNAs. Finally, to illustrate the complexities of miRNA and siRNA annotation, we review the evolution and functions of miRNAs and siRNAs in plants. PMID:29343505

Characterization of microRNAs Expressed during Secondary Wall Biosynthesis in Acacia mangium

PubMed Central

Ong, Seong Siang; Wickneswari, Ratnam

2012-01-01

MicroRNAs (miRNAs) play critical regulatory roles by acting as sequence specific guide during secondary wall formation in woody and non-woody species. Although thousands of plant miRNAs have been sequenced, there is no comprehensive view of miRNA mediated gene regulatory network to provide profound biological insights into the regulation of xylem development. Herein, we report the involvement of six highly conserved amg-miRNA families (amg-miR166, amg-miR172, amg-miR168, amg-miR159, amg-miR394, and amg-miR156) as the potential regulatory sequences of secondary cell wall biosynthesis. Within this highly conserved amg-miRNA family, only amg-miR166 exhibited strong differences in expression between phloem and xylem tissue. The functional characterization of amg-miR166 targets in various tissues revealed three groups of HD-ZIP III: ATHB8, ATHB15, and REVOLUTA which play pivotal roles in xylem development. Although these three groups vary in their functions, -psRNA target analysis indicated that miRNA target sequences of the nine different members of HD-ZIP III are always conserved. We found that precursor structures of amg-miR166 undergo exhaustive sequence variation even within members of the same family. Gene expression analysis showed three key lignin pathway genes: C4H, CAD, and CCoAOMT were upregulated in compression wood where a cascade of miRNAs was downregulated. This study offers a comprehensive analysis on the involvement of highly conserved miRNAs implicated in the secondary wall formation of woody plants. PMID:23251324

Genome-wide sequencing and quantification of circulating microRNAs for dogs with congestive heart failure secondary to myxomatous mitral valve degeneration.

PubMed

Jung, SeungWoo; Bohan, Amy

2018-02-01

OBJECTIVE To characterize expression profiles of circulating microRNAs via genome-wide sequencing for dogs with congestive heart failure (CHF) secondary to myxomatous mitral valve degeneration (MMVD). ANIMALS 9 healthy client-owned dogs and 8 age-matched client-owned dogs with CHF secondary to MMVD. PROCEDURES Blood samples were collected before administering cardiac medications for the management of CHF. Isolated microRNAs from plasma were classified into microRNA libraries and subjected to next-generation sequencing (NGS) for genome-wide sequencing analysis and quantification of circulating microRNAs. Quantitative reverse transcription PCR (qRT-PCR) assays were used to validate expression profiles of differentially expressed circulating microRNAs identified from NGS analysis of dogs with CHF. RESULTS 326 microRNAs were identified with NGS analysis. Hierarchical analysis revealed distinct expression patterns of circulating microRNAs between healthy dogs and dogs with CHF. Results of qRT-PCR assays confirmed upregulation of 4 microRNAs (miR-133, miR-1, miR-let-7e, and miR-125) and downregulation of 4 selected microRNAs (miR-30c, miR-128, miR-142, and miR-423). Results of qRT-PCR assays were highly correlated with NGS data and supported the specificity of circulating microRNA expression profiles in dogs with CHF secondary to MMVD. CONCLUSIONS AND CLINICAL RELEVANCE These results suggested that circulating microRNA expression patterns were unique and could serve as molecular biomarkers of CHF in dogs with MMVD.

De novo transcriptome sequencing reveals a considerable bias in the incidence of simple sequence repeats towards the downstream of 'Pre-miRNAs' of black pepper.

PubMed

Joy, Nisha; Asha, Srinivasan; Mallika, Vijayan; Soniya, Eppurathu Vasudevan

2013-01-01

Next generation sequencing has an advantageon transformational development of species with limited available sequence data as it helps to decode the genome and transcriptome. We carried out the de novo sequencing using illuminaHiSeq™ 2000 to generate the first leaf transcriptome of black pepper (Piper nigrum L.), an important spice variety native to South India and also grown in other tropical regions. Despite the economic and biochemical importance of pepper, a scientifically rigorous study at the molecular level is far from complete due to lack of sufficient sequence information and cytological complexity of its genome. The 55 million raw reads obtained, when assembled using Trinity program generated 2,23,386 contigs and 1,28,157 unigenes. Reports suggest that the repeat-rich genomic regions give rise to small non-coding functional RNAs. MicroRNAs (miRNAs) are the most abundant type of non-coding regulatory RNAs. In spite of the widespread research on miRNAs, little is known about the hair-pin precursors of miRNAs bearing Simple Sequence Repeats (SSRs). We used the array of transcripts generated, for the in silico prediction and detection of '43 pre-miRNA candidates bearing different types of SSR motifs'. The analysis identified 3913 different types of SSR motifs with an average of one SSR per 3.04 MB of thetranscriptome. About 0.033% of the transcriptome constituted 'pre-miRNA candidates bearing SSRs'. The abundance, type and distribution of SSR motifs studied across the hair-pin miRNA precursors, showed a significant bias in the position of SSRs towards the downstream of predicted 'pre-miRNA candidates'. The catalogue of transcripts identified, together with the demonstration of reliable existence of SSRs in the miRNA precursors, permits future opportunities for understanding the genetic mechanism of black pepper and likely functions of 'tandem repeats' in miRNAs.

De novo Transcriptome Sequencing Reveals a Considerable Bias in the Incidence of Simple Sequence Repeats towards the Downstream of ‘Pre-miRNAs’ of Black Pepper

PubMed Central

Joy, Nisha; Asha, Srinivasan; Mallika, Vijayan; Soniya, Eppurathu Vasudevan

2013-01-01

Next generation sequencing has an advantageon transformational development of species with limited available sequence data as it helps to decode the genome and transcriptome. We carried out the de novo sequencing using illuminaHiSeq™ 2000 to generate the first leaf transcriptome of black pepper (Piper nigrum L.), an important spice variety native to South India and also grown in other tropical regions. Despite the economic and biochemical importance of pepper, a scientifically rigorous study at the molecular level is far from complete due to lack of sufficient sequence information and cytological complexity of its genome. The 55 million raw reads obtained, when assembled using Trinity program generated 2,23,386 contigs and 1,28,157 unigenes. Reports suggest that the repeat-rich genomic regions give rise to small non-coding functional RNAs. MicroRNAs (miRNAs) are the most abundant type of non-coding regulatory RNAs. In spite of the widespread research on miRNAs, little is known about the hair-pin precursors of miRNAs bearing Simple Sequence Repeats (SSRs). We used the array of transcripts generated, for the in silico prediction and detection of ‘43 pre-miRNA candidates bearing different types of SSR motifs’. The analysis identified 3913 different types of SSR motifs with an average of one SSR per 3.04 MB of thetranscriptome. About 0.033% of the transcriptome constituted ‘pre-miRNA candidates bearing SSRs’. The abundance, type and distribution of SSR motifs studied across the hair-pin miRNA precursors, showed a significant bias in the position of SSRs towards the downstream of predicted ‘pre-miRNA candidates’. The catalogue of transcripts identified, together with the demonstration of reliable existence of SSRs in the miRNA precursors, permits future opportunities for understanding the genetic mechanism of black pepper and likely functions of ‘tandem repeats’ in miRNAs. PMID:23469176

MicroRNA drop in the bloodstream and microRNA boost in the tumour caused by treatment with ribonuclease A leads to an attenuation of tumour malignancy.

PubMed

Mironova, Nadezhda; Patutina, Olga; Brenner, Evgenyi; Kurilshikov, Alexander; Vlassov, Valentin; Zenkova, Marina

2013-01-01

Novel data showing an important role of microRNAs in mediating tumour progression opened a new field of possible molecular targets for cytotoxic ribonucleases. Recently, antitumour and antimetastatic activities of pancreatic ribonuclease A were demonstrated and here genome-wide profiles of microRNAs in the tumour and blood of mice bearing Lewis lung carcinoma after treatment with RNase A were analysed by high-throughput Sequencing by Oligonucleotide Ligation and Detection (SOLiD™) sequencing technology. Sequencing data showed that RNase A therapy resulted in the boost of 116 microRNAs in tumour tissue and a significant drop of 137 microRNAs in the bloodstream that were confirmed by qPCR. The microRNA boost in the tumour was accompanied by the overexpression of microRNA processing genes: RNASEN (Drosha), xpo5, dicer1, and eif2c2 (Ago2). Ribonuclease activity of RNase A was shown to be crucial for the activation of both microRNA synthesis and expression of the microRNA processing genes. In the tumour tissue, RNase A caused the upregulation of both oncomirs and tumour-suppressor microRNAs, including microRNAs of the let-7 family, known to negatively regulate tumour progression. Our results suggest that the alteration of microRNA signature caused by RNase A treatment leads to the attenuation of tumour malignancy.

miRanalyzer: a microRNA detection and analysis tool for next-generation sequencing experiments.

PubMed

Hackenberg, Michael; Sturm, Martin; Langenberger, David; Falcón-Pérez, Juan Manuel; Aransay, Ana M

2009-07-01

Next-generation sequencing allows now the sequencing of small RNA molecules and the estimation of their expression levels. Consequently, there will be a high demand of bioinformatics tools to cope with the several gigabytes of sequence data generated in each single deep-sequencing experiment. Given this scene, we developed miRanalyzer, a web server tool for the analysis of deep-sequencing experiments for small RNAs. The web server tool requires a simple input file containing a list of unique reads and its copy numbers (expression levels). Using these data, miRanalyzer (i) detects all known microRNA sequences annotated in miRBase, (ii) finds all perfect matches against other libraries of transcribed sequences and (iii) predicts new microRNAs. The prediction of new microRNAs is an especially important point as there are many species with very few known microRNAs. Therefore, we implemented a highly accurate machine learning algorithm for the prediction of new microRNAs that reaches AUC values of 97.9% and recall values of up to 75% on unseen data. The web tool summarizes all the described steps in a single output page, which provides a comprehensive overview of the analysis, adding links to more detailed output pages for each analysis module. miRanalyzer is available at http://web.bioinformatics.cicbiogune.es/microRNA/.

Deformability in the cleavage site of primary microRNA is not sensed by the double-stranded RNA binding domains in the microprocessor component DGCR8.

PubMed

Quarles, Kaycee A; Chadalavada, Durga; Showalter, Scott A

2015-06-01

The prevalence of double-stranded RNA (dsRNA) in eukaryotic cells has only recently been appreciated. Of interest here, RNA silencing begins with dsRNA substrates that are bound by the dsRNA-binding domains (dsRBDs) of their processing proteins. Specifically, processing of microRNA (miRNA) in the nucleus minimally requires the enzyme Drosha and its dsRBD-containing cofactor protein, DGCR8. The smallest recombinant construct of DGCR8 that is sufficient for in vitro dsRNA binding, referred to as DGCR8-Core, consists of its two dsRBDs and a C-terminal tail. As dsRBDs rarely recognize the nucleotide sequence of dsRNA, it is reasonable to hypothesize that DGCR8 function is dependent on the recognition of specific structural features in the miRNA precursor. Previously, we demonstrated that noncanonical structural elements that promote RNA flexibility within the stem of miRNA precursors are necessary for efficient in vitro cleavage by reconstituted Microprocessor complexes. Here, we combine gel shift assays with in vitro processing assays to demonstrate that neither the N-terminal dsRBD of DGCR8 in isolation nor the DGCR8-Core construct is sensitive to the presence of noncanonical structural elements within the stem of miRNA precursors, or to single-stranded segments flanking the stem. Extending DGCR8-Core to include an N-terminal heme-binding region does not change our conclusions. Thus, our data suggest that although the DGCR8-Core region is necessary for dsRNA binding and recruitment to the Microprocessor, it is not sufficient to establish the previously observed connection between RNA flexibility and processing efficiency. © 2015 Wiley Periodicals, Inc.

Functional relevance of “seed” and “non-seed” sequences in microRNA-mediated promotion of C. elegans developmental progression

PubMed Central

Zhang, Huibin; Artiles, Karen L.; Fire, Andrew Z.

2015-01-01

The founding heterochronic microRNAs, lin-4 and let-7, together with their validated targets and well-characterized phenotypes in C. elegans, offer an opportunity to test functionality of microRNAs in a developmental context. In this study, we defined sequence requirements at the microRNA level for these two microRNAs, evaluating lin-4 and let-7 mutant microRNAs for their ability to support temporal development under conditions where the wild-type lin-4 and let-7 gene products are absent. For lin-4, we found a strong requirement for seed sequences, with function drastically affected by several central mutations in the seed sequence, while rescue was retained by a set of mutations peripheral to the seed. let-7 rescuing activity was retained to a surprising degree by a variety of central seed mutations, while several non-seed mutant effects support potential noncanonical contributions to let-7 function. Taken together, this work illustrates both the functional partnership between seed and non-seed sequences in mediating C. elegans temporal development and a diversity among microRNA effectors in the contributions of seed and non-seed regions to activity. PMID:26385508

Deep sequencing of cardiac microRNA-mRNA interactomes in clinical and experimental cardiomyopathy

PubMed Central

Matkovich, Scot J.; Dorn, Gerald W.

2018-01-01

Summary MicroRNAs are a family of short (~21 nucleotide) noncoding RNAs that serve key roles in cellular growth and differentiation and the response of the heart to stress stimuli. As the sequence-specific recognition element of RNA-induced silencing complexes (RISCs), microRNAs bind mRNAs and prevent their translation via mechanisms that may include transcript degradation and/or prevention of ribosome binding. Short microRNA sequences and the ability of microRNAs to bind to mRNA sites having only partial/imperfect sequence complementarity complicates purely computational analyses of microRNA-mRNA interactomes. Furthermore, computational microRNA target prediction programs typically ignore biological context, and therefore the principal determinants of microRNA-mRNA binding: the presence and quantity of each. To address these deficiencies we describe an empirical method, developed via studies of stressed and failing hearts, to determine disease-induced changes in microRNAs, mRNAs, and the mRNAs targeted to the RISC, without cross-linking mRNAs to RISC proteins. Deep sequencing methods are used to determine RNA abundances, delivering unbiased, quantitative RNA data limited only by their annotation in the genome of interest. We describe the laboratory bench steps required to perform these experiments, experimental design strategies to achieve an appropriate number of sequencing reads per biological replicate, and computer-based processing tools and procedures to convert large raw sequencing data files into gene expression measures useful for differential expression analyses. PMID:25836573

Deep sequencing of cardiac microRNA-mRNA interactomes in clinical and experimental cardiomyopathy.

PubMed

Matkovich, Scot J; Dorn, Gerald W

2015-01-01

MicroRNAs are a family of short (~21 nucleotide) noncoding RNAs that serve key roles in cellular growth and differentiation and the response of the heart to stress stimuli. As the sequence-specific recognition element of RNA-induced silencing complexes (RISCs), microRNAs bind mRNAs and prevent their translation via mechanisms that may include transcript degradation and/or prevention of ribosome binding. Short microRNA sequences and the ability of microRNAs to bind to mRNA sites having only partial/imperfect sequence complementarity complicate purely computational analyses of microRNA-mRNA interactomes. Furthermore, computational microRNA target prediction programs typically ignore biological context, and therefore the principal determinants of microRNA-mRNA binding: the presence and quantity of each. To address these deficiencies we describe an empirical method, developed via studies of stressed and failing hearts, to determine disease-induced changes in microRNAs, mRNAs, and the mRNAs targeted to the RISC, without cross-linking mRNAs to RISC proteins. Deep sequencing methods are used to determine RNA abundances, delivering unbiased, quantitative RNA data limited only by their annotation in the genome of interest. We describe the laboratory bench steps required to perform these experiments, experimental design strategies to achieve an appropriate number of sequencing reads per biological replicate, and computer-based processing tools and procedures to convert large raw sequencing data files into gene expression measures useful for differential expression analyses.

Computational Characterization of Exogenous MicroRNAs that Can Be Transferred into Human Circulation

PubMed Central

Shu, Jiang; Chiang, Kevin; Zempleni, Janos; Cui, Juan

2015-01-01

MicroRNAs have been long considered synthesized endogenously until very recent discoveries showing that human can absorb dietary microRNAs from animal and plant origins while the mechanism remains unknown. Compelling evidences of microRNAs from rice, milk, and honeysuckle transported to human blood and tissues have created a high volume of interests in the fundamental questions that which and how exogenous microRNAs can be transferred into human circulation and possibly exert functions in humans. Here we present an integrated genomics and computational analysis to study the potential deciding features of transportable microRNAs. Specifically, we analyzed all publicly available microRNAs, a total of 34,612 from 194 species, with 1,102 features derived from the microRNA sequence and structure. Through in-depth bioinformatics analysis, 8 groups of discriminative features have been used to characterize human circulating microRNAs and infer the likelihood that a microRNA will get transferred into human circulation. For example, 345 dietary microRNAs have been predicted as highly transportable candidates where 117 of them have identical sequences with their homologs in human and 73 are known to be associated with exosomes. Through a milk feeding experiment, we have validated 9 cow-milk microRNAs in human plasma using microRNA-sequencing analysis, including the top ranked microRNAs such as bta-miR-487b, miR-181b, and miR-421. The implications in health-related processes have been illustrated in the functional analysis. This work demonstrates the data-driven computational analysis is highly promising to study novel molecular characteristics of transportable microRNAs while bypassing the complex mechanistic details. PMID:26528912

Computational Characterization of Exogenous MicroRNAs that Can Be Transferred into Human Circulation.

PubMed

Shu, Jiang; Chiang, Kevin; Zempleni, Janos; Cui, Juan

2015-01-01

MicroRNAs have been long considered synthesized endogenously until very recent discoveries showing that human can absorb dietary microRNAs from animal and plant origins while the mechanism remains unknown. Compelling evidences of microRNAs from rice, milk, and honeysuckle transported to human blood and tissues have created a high volume of interests in the fundamental questions that which and how exogenous microRNAs can be transferred into human circulation and possibly exert functions in humans. Here we present an integrated genomics and computational analysis to study the potential deciding features of transportable microRNAs. Specifically, we analyzed all publicly available microRNAs, a total of 34,612 from 194 species, with 1,102 features derived from the microRNA sequence and structure. Through in-depth bioinformatics analysis, 8 groups of discriminative features have been used to characterize human circulating microRNAs and infer the likelihood that a microRNA will get transferred into human circulation. For example, 345 dietary microRNAs have been predicted as highly transportable candidates where 117 of them have identical sequences with their homologs in human and 73 are known to be associated with exosomes. Through a milk feeding experiment, we have validated 9 cow-milk microRNAs in human plasma using microRNA-sequencing analysis, including the top ranked microRNAs such as bta-miR-487b, miR-181b, and miR-421. The implications in health-related processes have been illustrated in the functional analysis. This work demonstrates the data-driven computational analysis is highly promising to study novel molecular characteristics of transportable microRNAs while bypassing the complex mechanistic details.

High Class-Imbalance in pre-miRNA Prediction: A Novel Approach Based on deepSOM.

PubMed

Stegmayer, Georgina; Yones, Cristian; Kamenetzky, Laura; Milone, Diego H

2017-01-01

The computational prediction of novel microRNA within a full genome involves identifying sequences having the highest chance of being a miRNA precursor (pre-miRNA). These sequences are usually named candidates to miRNA. The well-known pre-miRNAs are usually only a few in comparison to the hundreds of thousands of potential candidates to miRNA that have to be analyzed, which makes this task a high class-imbalance classification problem. The classical way of approaching it has been training a binary classifier in a supervised manner, using well-known pre-miRNAs as positive class and artificially defining the negative class. However, although the selection of positive labeled examples is straightforward, it is very difficult to build a set of negative examples in order to obtain a good set of training samples for a supervised method. In this work, we propose a novel and effective way of approaching this problem using machine learning, without the definition of negative examples. The proposal is based on clustering unlabeled sequences of a genome together with well-known miRNA precursors for the organism under study, which allows for the quick identification of the best candidates to miRNA as those sequences clustered with known precursors. Furthermore, we propose a deep model to overcome the problem of having very few positive class labels. They are always maintained in the deep levels as positive class while less likely pre-miRNA sequences are filtered level after level. Our approach has been compared with other methods for pre-miRNAs prediction in several species, showing effective predictivity of novel miRNAs. Additionally, we will show that our approach has a lower training time and allows for a better graphical navegability and interpretation of the results. A web-demo interface to try deepSOM is available at http://fich.unl.edu.ar/sinc/web-demo/deepsom/.

Xenopus microRNA genes are predominantly located within introns and are differentially expressed in adult frog tissues via post-transcriptional regulation

PubMed Central

Tang, Guo-Qing; Maxwell, E. Stuart

2008-01-01

The amphibian Xenopus provides a model organism for investigating microRNA expression during vertebrate embryogenesis and development. Searching available Xenopus genome databases using known human pre-miRNAs as query sequences, more than 300 genes encoding 142 Xenopus tropicalis miRNAs were identified. Analysis of Xenopus tropicalis miRNA genes revealed a predominate positioning within introns of protein-coding and nonprotein-coding RNA Pol II-transcribed genes. MiRNA genes were also located in pre-mRNA exons and positioned intergenically between known protein-coding genes. Many miRNA species were found in multiple locations and in more than one genomic context. MiRNA genes were also clustered throughout the genome, indicating the potential for the cotranscription and coordinate expression of miRNAs located in a given cluster. Northern blot analysis confirmed the expression of many identified miRNAs in both X. tropicalis and X. laevis. Comparison of X. tropicalis and X. laevis blots revealed comparable expression profiles, although several miRNAs exhibited species-specific expression in different tissues. More detailed analysis revealed that for some miRNAs, the tissue-specific expression profile of the pri-miRNA precursor was distinctly different from that of the mature miRNA profile. Differential miRNA precursor processing in both the nucleus and cytoplasm was implicated in the observed tissue-specific differences. These observations indicated that post-transcriptional processing plays an important role in regulating miRNA expression in the amphibian Xenopus. PMID:18032731

Association of MicroRNAs with Antibody Response to Mycoplasma bovis in Beef Cattle

PubMed Central

Cai, Guohong; Kuehn, Larry A.; Register, Karen B.; McDaneld, Tara G.; Neill, John D.

2016-01-01

The objective of this study was to identify microRNAs associated with a serum antibody response to Mycoplasma bovis in beef cattle. Serum from sixteen beef calves was collected at three points: in summer after calves were born, in fall at weaning, and in the following spring. All sera collected in the summer were ELISA-negative for anti-M. bovis. By the fall, eight animals were seropositive for IgG (positive group), while eight remained negative (negative group). By spring, all animals in both groups were seropositive. MicroRNAs were extracted from sera and sequenced on the Illumina HiSeq next-generation sequencer. A total of 1,374,697 sequences mapped to microRNAs in the bovine genome. Of these, 82% of the sequences corresponded to 27 microRNAs, each represented by a minimum of 10,000 sequences. There was a statistically significant interaction between ELISA response and season for bta-miR-24-3p (P = 0.0268). All sera collected at the initial summer had a similar number of copies of this microRNA (P = 0.773). In the fall, the positive group had an increased number of copies when compared to the negative group (P = 0.021), and this grew more significant by the following spring (P = 0.0001). There were 21 microRNAs associated (P< 0.05) with season. These microRNAs could be evaluated further as candidates to potentially improve productivity in cattle. The microRNAs bta-let-7b, bta-miR- 24-3p, bta-miR- 92a, and bta-miR-423-5p, were significatly associated with ELISA status (P< 0.05). These microRNAs have been recognized as playing a role in the host defense against bacteria in humans, mice, and dairy cattle. Further studies are needed to establish if these microRNAs could be used as diagnostic marker or indicator of exposure, or whether intervention strategies could be developed as an alternative to antibiotics for controlling disease due to M. bovis. PMID:27537842

Kaposi's Sarcoma-Associated Herpesvirus MicroRNA Single-Nucleotide Polymorphisms Identified in Clinical Samples Can Affect MicroRNA Processing, Level of Expression, and Silencing Activity

PubMed Central

Han, Soo-Jin; Marshall, Vickie; Barsov, Eugene; Quiñones, Octavio; Ray, Alex; Labo, Nazzarena; Trivett, Matthew; Ott, David; Renne, Rolf

2013-01-01

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 pre-microRNAs that can produce 25 KSHV mature microRNAs. We previously reported single-nucleotide polymorphisms (SNPs) in KSHV-encoded pre-microRNA and mature microRNA sequences from clinical samples (V. Marshall et al., J. Infect. Dis., 195:645–659, 2007). To determine whether microRNA SNPs affect pre-microRNA processing and, ultimately, mature microRNA expression levels, we performed a detailed comparative analysis of (i) mature microRNA expression levels, (ii) in vitro Drosha/Dicer processing, and (iii) RNA-induced silencing complex-dependent targeting of wild-type (wt) and variant microRNA genes. Expression of pairs of wt and variant pre-microRNAs from retroviral vectors and measurement of KSHV mature microRNA expression by real-time reverse transcription-PCR (RT-PCR) revealed differential expression levels that correlated with the presence of specific sequence polymorphisms. Measurement of KSHV mature microRNA expression in a panel of primary effusion lymphoma cell lines by real-time RT-PCR recapitulated some observed expression differences but suggested a more complex relationship between sequence differences and expression of mature microRNA. Furthermore, in vitro maturation assays demonstrated significant SNP-associated changes in Drosha/DGCR8 and/or Dicer processing. These data demonstrate that SNPs within KSHV-encoded pre-microRNAs are associated with differential microRNA expression levels. Given the multiple reports on the involvement of microRNAs in cancer, the biological significance of these phenotypic and genotypic variants merits further studies in patients with KSHV-associated malignancies. PMID:24006441

smRNAome profiling to identify conserved and novel microRNAs in Stevia rebaudiana Bertoni

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) constitute a family of small RNA (sRNA) population that regulates the gene expression and plays an important role in plant development, metabolism, signal transduction and stress response. Extensive studies on miRNAs have been performed in different plants such as Arabidopsis thaliana, Oryza sativa etc. and volume of the miRNA database, mirBASE, has been increasing on day to day basis. Stevia rebaudiana Bertoni is an important perennial herb which accumulates high concentrations of diterpene steviol glycosides which contributes to its high indexed sweetening property with no calorific value. Several studies have been carried out for understanding molecular mechanism involved in biosynthesis of these glycosides, however, information about miRNAs has been lacking in S. rebaudiana. Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs irrespective of availability of genome sequence data. Results To identify miRNAs in S. rebaudiana, sRNA library was constructed and sequenced using Illumina genome analyzer II. A total of 30,472,534 reads representing 2,509,190 distinct sequences were obtained from sRNA library. Based on sequence similarity, we identified 100 miRNAs belonging to 34 highly conserved families. Also, we identified 12 novel miRNAs whose precursors were potentially generated from stevia EST and nucleotide sequences. All novel sequences have not been earlier described in other plant species. Putative target genes were predicted for most conserved and novel miRNAs. The predicted targets are mainly mRNA encoding enzymes regulating essential plant metabolic and signaling pathways. Conclusions This study led to the identification of 34 highly conserved miRNA families and 12 novel potential miRNAs indicating that specific miRNAs exist in stevia species. Our results provided information on stevia miRNAs and their targets building a foundation for future studies to understand their roles in key stevia traits. PMID:23116282

smRNAome profiling to identify conserved and novel microRNAs in Stevia rebaudiana Bertoni.

PubMed

Mandhan, Vibha; Kaur, Jagdeep; Singh, Kashmir

2012-11-01

MicroRNAs (miRNAs) constitute a family of small RNA (sRNA) population that regulates the gene expression and plays an important role in plant development, metabolism, signal transduction and stress response. Extensive studies on miRNAs have been performed in different plants such as Arabidopsis thaliana, Oryza sativa etc. and volume of the miRNA database, mirBASE, has been increasing on day to day basis. Stevia rebaudiana Bertoni is an important perennial herb which accumulates high concentrations of diterpene steviol glycosides which contributes to its high indexed sweetening property with no calorific value. Several studies have been carried out for understanding molecular mechanism involved in biosynthesis of these glycosides, however, information about miRNAs has been lacking in S. rebaudiana. Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs irrespective of availability of genome sequence data. To identify miRNAs in S. rebaudiana, sRNA library was constructed and sequenced using Illumina genome analyzer II. A total of 30,472,534 reads representing 2,509,190 distinct sequences were obtained from sRNA library. Based on sequence similarity, we identified 100 miRNAs belonging to 34 highly conserved families. Also, we identified 12 novel miRNAs whose precursors were potentially generated from stevia EST and nucleotide sequences. All novel sequences have not been earlier described in other plant species. Putative target genes were predicted for most conserved and novel miRNAs. The predicted targets are mainly mRNA encoding enzymes regulating essential plant metabolic and signaling pathways. This study led to the identification of 34 highly conserved miRNA families and 12 novel potential miRNAs indicating that specific miRNAs exist in stevia species. Our results provided information on stevia miRNAs and their targets building a foundation for future studies to understand their roles in key stevia traits.

Identification of microRNAs in Caragana intermedia by high-throughput sequencing and expression analysis of 12 microRNAs and their targets under salt stress.

PubMed

Zhu, Jianfeng; Li, Wanfeng; Yang, Wenhua; Qi, Liwang; Han, Suying

2013-09-01

142 miRNAs were identified and 38 miRNA targets were predicted, 4 of which were validated, in C. intermedia . The expression of 12 miRNAs in salt-stressed leaves was assessed by qRT-PCR. MicroRNAs (miRNAs) are endogenous small RNAs that play important roles in various biological and metabolic processes in plants. Caragana intermedia is an important ecological and economic tree species prominent in the desert environment of west and northwest China. To date, no investigation into C. intermedia miRNAs has been reported. In this study, high-throughput sequencing of small RNAs and analysis of transcriptome data were performed to identify both conserved and novel miRNAs, and also their target mRNA genes in C. intermedia. Based on sequence similarity and hairpin structure prediction, 132 putative conserved miRNAs (12 of which were confirmed to form hairpin precursors) belonging to 31 known miRNA families were identified. Ten novel miRNAs (including the miRNA* sequences of three novel miRNAs) were also discovered. Furthermore, 36 potential target genes of 17 known miRNA families and 2 potential target genes of 1 novel miRNA were predicted; 4 of these were validated by 5' RACE. The expression of 12 miRNAs was validated in different tissues, and these and five target mRNAs were assessed by qRT-PCR after salt treatment. The expression levels of seven miRNAs (cin-miR157a, cin-miR159a, cin-miR165a, cin-miR167b, cin-miR172b, cin-miR390a and cin-miR396a) were upregulated, while cin-miR398a expression was downregulated after salt treatment. The targets of cin-miR157a, cin-miR165a, cin-miR172b and cin-miR396a were downregulated and showed an approximately negative correlation with their corresponding miRNAs under salt treatment. These results would help further understanding of miRNA regulation in response to abiotic stress in C. intermedia.

MicroRNA biogenesis and function in plants.

PubMed

Chen, Xuemei

2005-10-31

A microRNA (miRNA) is a 21-24 nucleotide RNA product of a non-protein-coding gene. Plants, like animals, have a large number of miRNA-encoding genes in their genomes. The biogenesis of miRNAs in Arabidopsis is similar to that in animals in that miRNAs are processed from primary precursors by at least two steps mediated by RNAse III-like enzymes and that the miRNAs are incorporated into a protein complex named RISC. However, the biogenesis of plant miRNAs consists of an additional step, i.e., the miRNAs are methylated on the ribose of the last nucleotide by the miRNA methyltransferase HEN1. The high degree of sequence complementarity between plant miRNAs and their target mRNAs has facilitated the bioinformatic prediction of miRNA targets, many of which have been subsequently validated. Plant miRNAs have been predicted or confirmed to regulate a variety of processes, such as development, metabolism, and stress responses. A large category of miRNA targets consists of genes encoding transcription factors that play important roles in patterning the plant form.

In vitro quantification of specific microRNA using molecular beacons

PubMed Central

Baker, Meredith B.; Bao, Gang; Searles, Charles D.

2012-01-01

MicroRNAs (miRNAs), a class of non-coding RNAs, have become a major focus of molecular biology research because of their diverse genomic origin and ability to regulate an array of cellular processes. Although the biological functions of miRNA are yet to be fully understood, tissue levels of specific miRNAs have been shown to correlate with pathological development of disease. Here, we demonstrate that molecular beacons can readily distinguish mature- and pre-miRNAs, and reliably quantify miRNA expression. We found that molecular beacons with DNA, RNA and combined locked nucleic acid (LNA)–DNA backbones can all detect miRNAs of low (<1 nM) concentrations in vitro, with RNA beacons having the highest detection sensitivity. Furthermore, we found that molecular beacons have the potential to distinguish miRNAs that have slight variations in their nucleotide sequence. These results suggest that the molecular beacon-based approach to assess miRNA expression and distinguish mature and precursor miRNA species is quite robust, and has the promise for assessing miRNA levels in biological samples. PMID:22110035

Bleomycin Can Cleave an Oncogenic Noncoding RNA.

PubMed

Angelbello, Alicia J; Disney, Matthew D

2018-01-04

Noncoding RNAs are pervasive in cells and contribute to diseases such as cancer. A question in biomedical research is whether noncoding RNAs are targets of medicines. Bleomycin is a natural product that cleaves DNA; however, it is known to cleave RNA in vitro. Herein, an in-depth analysis of the RNA cleavage preferences of bleomycin A5 is presented. Bleomycin A5 prefers to cleave RNAs with stretches of AU base pairs. Based on these preferences and bioinformatic analysis, the microRNA-10b hairpin precursor was identified as a potential substrate for bleomycin A5. Both in vitro and cellular experiments demonstrated cleavage. Importantly, chemical cleavage by bleomycin A5 in the microRNA-10b hairpin precursors occurred near the Drosha and Dicer enzymatic processing sites and led to destruction of the microRNA. Evidently, oncogenic noncoding RNAs can be considered targets of cancer medicines and might elicit their pharmacological effects by targeting noncoding RNA. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparative analysis of the small RNA transcriptomes of Pinus contorta and Oryza sativa

PubMed Central

Morin, Ryan D.; Aksay, Gozde; Dolgosheina, Elena; Ebhardt, H. Alexander; Magrini, Vincent; Mardis, Elaine R.; Sahinalp, S. Cenk; Unrau, Peter J.

2008-01-01

The diversity of microRNAs and small-interfering RNAs has been extensively explored within angiosperms by focusing on a few key organisms such as Oryza sativa and Arabidopsis thaliana. A deeper division of the plants is defined by the radiation of the angiosperms and gymnosperms, with the latter comprising the commercially important conifers. The conifers are expected to provide important information regarding the evolution of highly conserved small regulatory RNAs. Deep sequencing provides the means to characterize and quantitatively profile small RNAs in understudied organisms such as these. Pyrosequencing of small RNAs from O. sativa revealed, as expected, ∼21- and ∼24-nt RNAs. The former contained known microRNAs, and the latter largely comprised intergenic-derived sequences likely representing heterochromatin siRNAs. In contrast, sequences from Pinus contorta were dominated by 21-nt small RNAs. Using a novel sequence-based clustering algorithm, we identified sequences belonging to 18 highly conserved microRNA families in P. contorta as well as numerous clusters of conserved small RNAs of unknown function. Using multiple methods, including expressed sequence folding and machine learning algorithms, we found a further 53 candidate novel microRNA families, 51 appearing specific to the P. contorta library. In addition, alignment of small RNA sequences to the O. sativa genome revealed six perfectly conserved classes of small RNA that included chloroplast transcripts and specific types of genomic repeats. The conservation of microRNAs and other small RNAs between the conifers and the angiosperms indicates that important RNA silencing processes were highly developed in the earliest spermatophytes. Genomic mapping of all sequences to the O. sativa genome can be viewed at http://microrna.bcgsc.ca/cgi-bin/gbrowse/rice_build_3/. PMID:18323537

Structural features of microRNA (miRNA) precursors and their relevance to miRNA biogenesis and small interfering RNA/short hairpin RNA design.

PubMed

Krol, Jacek; Sobczak, Krzysztof; Wilczynska, Urszula; Drath, Maria; Jasinska, Anna; Kaczynska, Danuta; Krzyzosiak, Wlodzimierz J

2004-10-01

We have established the structures of 10 human microRNA (miRNA) precursors using biochemical methods. Eight of these structures turned out to be different from those that were computer-predicted. The differences localized in the terminal loop region and at the opposite side of the precursor hairpin stem. We have analyzed the features of these structures from the perspectives of miRNA biogenesis and active strand selection. We demonstrated the different thermodynamic stability profiles for pre-miRNA hairpins harboring miRNAs at their 5'- and 3'-sides and discussed their functional implications. Our results showed that miRNA prediction based on predicted precursor structures may give ambiguous results, and the success rate is significantly higher for the experimentally determined structures. On the other hand, the differences between the predicted and experimentally determined structures did not affect the stability of termini produced through "conceptual dicing." This result confirms the value of thermodynamic analysis based on mfold as a predictor of strand section by RNAi-induced silencing complex (RISC).

Design of a small molecule against an oncogenic noncoding RNA.

PubMed

Velagapudi, Sai Pradeep; Cameron, Michael D; Haga, Christopher L; Rosenberg, Laura H; Lafitte, Marie; Duckett, Derek R; Phinney, Donald G; Disney, Matthew D

2016-05-24

The design of precision, preclinical therapeutics from sequence is difficult, but advances in this area, particularly those focused on rational design, could quickly transform the sequence of disease-causing gene products into lead modalities. Herein, we describe the use of Inforna, a computational approach that enables the rational design of small molecules targeting RNA to quickly provide a potent modulator of oncogenic microRNA-96 (miR-96). We mined the secondary structure of primary microRNA-96 (pri-miR-96) hairpin precursor against a database of RNA motif-small molecule interactions, which identified modules that bound RNA motifs nearby and in the Drosha processing site. Precise linking of these modules together provided Targaprimir-96 (3), which selectively modulates miR-96 production in cancer cells and triggers apoptosis. Importantly, the compound is ineffective on healthy breast cells, and exogenous overexpression of pri-miR-96 reduced compound potency in breast cancer cells. Chemical Cross-Linking and Isolation by Pull-Down (Chem-CLIP), a small-molecule RNA target validation approach, shows that 3 directly engages pri-miR-96 in breast cancer cells. In vivo, 3 has a favorable pharmacokinetic profile and decreases tumor burden in a mouse model of triple-negative breast cancer. Thus, rational design can quickly produce precision, in vivo bioactive lead small molecules against hard-to-treat cancers by targeting oncogenic noncoding RNAs, advancing a disease-to-gene-to-drug paradigm.

Isolation and identification of gene-specific microRNAs.

PubMed

Lin, Shi-Lung; Chang, Donald C; Ying, Shao-Yao

2006-01-01

Prediction of microRNA (miRNA) candidates using computer programming has identified hundreds and hundreds of genomic hairpin sequences, of which, the functions remain to be determined. Because direct transfection of hairpin-like miRNA precursors (pre)-miRNAs in mammalian cells is not always sufficient to trigger effective RNA-induced gene-silencing complex (RISC) assembly, a key step for RNA interference (RNAi)-related gene silencing, we developed an intronic miRNA-expressing system to overcome this problem, and successfully increased the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo. By insertion of a hairpin-like pre-miRNA structure into the intron region of a gene, this intronic miRNA biogenesis system has been found to depend on a coupled interaction of nascent precursor messenger RNA transcription and intron excision within a specific nuclear region proximal to genomic perichromatin fibrils. The intronic miRNA was transcribed by RNA type II polymerases, coexpressed with a primary gene transcript, and excised out of its encoding gene transcript by intracellular RNA splicing and processing mechanisms. Currently, some ribonuclease III endonucleases have been found to be involved in the processing of spliced introns and probably facilitating the intronic miRNA maturation. Using this miRNA-expressing system, we have shown for the first time that the intron-derived miRNAs were able to induce strong RNAi effects in not only human and mouse cells but also zebrafish, chicken embryos, and adult mice. Based on the strand complementarity between the designed miRNA and its target gene sequence, we have also developed a miRNA isolation protocol to purify and identify the mature miRNAs generated by the intronic miRNA-expressing system. Several intronic miRNA identities and structures are currently confirmed to be active in vitro and in vivo. According to this proof- of-principle method, we now have the knowledge to design pre-miRNA inserts that are more efficient and effective for the intronic miRNA-expressing system.

Isothermal circular-strand-displacement polymerization of DNA and microRNA in digital microfluidic devices.

PubMed

Giuffrida, Maria Chiara; Zanoli, Laura Maria; D'Agata, Roberta; Finotti, Alessia; Gambari, Roberto; Spoto, Giuseppe

2015-02-01

Nucleic-acid amplification is a crucial step in nucleic-acid-sequence-detection assays. The use of digital microfluidic devices to miniaturize amplification techniques reduces the required sample volume and the analysis time and offers new possibilities for process automation and integration in a single device. The recently introduced droplet polymerase-chain-reaction (PCR) amplification methods require repeated cycles of two or three temperature-dependent steps during the amplification of the nucleic-acid target sequence. In contrast, low-temperature isothermal-amplification methods have no need for thermal cycling, thus requiring simplified microfluidic-device features. Here, the combined use of digital microfluidics and molecular-beacon (MB)-assisted isothermal circular-strand-displacement polymerization (ICSDP) to detect microRNA-210 sequences is described. MicroRNA-210 has been described as the most consistently and predominantly upregulated hypoxia-inducible factor. The nmol L(-1)-pmol L(-1) detection capabilities of the method were first tested by targeting single-stranded DNA sequences from the genetically modified Roundup Ready soybean. The ability of the droplet-ICSDP method to discriminate between full-matched, single-mismatched, and unrelated sequences was also investigated. The detection of a range of nmol L(-1)-pmol L(-1) microRNA-210 solutions compartmentalized in nanoliter-sized droplets was performed, establishing the ability of the method to detect as little as 10(-18) mol of microRNA target sequences compartmentalized in 20 nL droplets. The suitability of the method for biological samples was tested by detecting microRNA-210 from transfected K562 cells.

Establishment of cells to monitor Microprocessor through fusion genes of microRNA and GFP

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

Tsutsui, Motomu; Hasegawa, Hitoki; Adachi, Koichi

Microprocessor, the complex of Drosha and DGCR8, promotes the processing of primary microRNA to precursor microRNA, which is a crucial step for microRNA maturation. So far, no convenient assay systems have been developed for observing this step in vivo. Here we report the establishment of highly sensitive cellular systems where we can visually monitor the function of Microprocessor. During a series of screening of transfectants with fusion genes of the EGFP cDNA and primary microRNA genes, we have obtained certain cell lines where introduction of siRNA against DGCR8 or Drosha strikingly augments GFP signals. In contrast, these cells have notmore » responded to Dicer siRNA; thus they have a unique character that GFP signals should be negatively and specifically correlated to the action of Microprocessor among biogenesis of microRNA. These cell lines can be useful tools for real-time analysis of Microprocessor action in vivo and identifying its novel modulators.« less

A-to-I editing of coding and non-coding RNAs by ADARs

PubMed Central

Nishikura, Kazuko

2016-01-01

Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA. This A-to-I editing occurs not only in protein-coding regions of mRNAs, but also frequently in non-coding regions that contain inverted Alu repeats. Editing of coding sequences can result in the expression of functionally altered proteins that are not encoded in the genome, whereas the significance of Alu editing remains largely unknown. Certain microRNA (miRNA) precursors are also edited, leading to reduced expression or altered function of mature miRNAs. Conversely, recent studies indicate that ADAR1 forms a complex with Dicer to promote miRNA processing, revealing a new function of ADAR1 in the regulation of RNA interference. PMID:26648264

In situ monitoring of cytoplasmic precursor and mature microRNA using gold nanoparticle and graphene oxide composite probes.

PubMed

Hong, Min; Sun, Hongxiao; Xu, Lidan; Yue, Qiaoli; Shen, Guodong; Li, Meifang; Tang, Bo; Li, Chen-Zhong

2018-08-27

This study strategically fabricates a nucleic acid functionalized gold nanoparticle and graphene oxide composite probe (AuNP/GO probe) to achieve both the recognition and in situ monitoring of cytoplasmic target precursor microRNAs (pre-miRNAs) and mature microRNAs (miRNAs) in living cells. The pre-miRNA-21 detection with AuNP probes has a good linear range of 0-300 nM and a limit of detection (LOD) of 4.5 nM, whereas the GO probe has a linear relationship with mature miRNA-21 from 0.1 to 10 nM with a LOD of 1.74 nM. This assay was utilized to directly visualize the relative expression levels of pre- and mature forms of miRNA-21 and let-7a. The results suggested that the expression levels of precursor miRNAs remain constant in cancer cells and normal cells. However, the expression levels of mature miRNAs vary widely, demonstrating the "up-regulation" of miRNA-21 and "down-regulation" of let-7a in cancer cells in contrast to that in normal cells. The practicality of this strategy was verified by in situ monitoring changes in cytoplasmic pre-miRNA-21 and mature miRNA-21 in response to small-molecule inhibitors of miRNA-21. Copyright © 2018 Elsevier B.V. All rights reserved.

Profiling of drought-responsive microRNA and mRNA in tomato using high-throughput sequencing.

PubMed

Liu, Minmin; Yu, Huiyang; Zhao, Gangjun; Huang, Qiufeng; Lu, Yongen; Ouyang, Bo

2017-06-26

Abiotic stresses cause severe loss of crop production. Among them, drought is one of the most frequent environmental stresses, which limits crop growth, development and productivity. Plant drought tolerance is fine-tuned by a complex gene regulatory network. Understanding the molecular regulation of this polygenic trait is crucial for the eventual success to improve plant yield and quality. Recent studies have demonstrated that microRNAs play critical roles in plant drought tolerance. However, little is known about the microRNA in drought response of the model plant tomato. Here, we described the profiling of drought-responsive microRNA and mRNA in tomato using high-throughput next-generation sequencing. Drought stress was applied on the seedlings of M82, a drought-sensitive cultivated tomato genotype, and IL9-1, a drought-tolerant introgression line derived from the stress-resistant wild species Solanum pennellii LA0716 and M82. Under drought, IL9-1 performed superior than M82 regarding survival rate, H 2 O 2 elimination and leaf turgor maintenance. A total of four small RNA and eight mRNA libraries were constructed and sequenced using Illumina sequencing technology. 105 conserved and 179 novel microRNAs were identified, among them, 54 and 98 were differentially expressed upon drought stress, respectively. The majority of the differentially-expressed conserved microRNAs was up-regulated in IL9-1 whereas down-regulated in M82. Under drought stress, 2714 and 1161 genes were found to be differentially expressed in M82 and IL9-1, respectively, and many of their homologues are involved in plant stress, such as genes encoding transcription factor and protein kinase. Various pathways involved in abiotic stress were revealed by Gene Ontology and pathway analysis. The mRNA sequencing results indicated that most of the target genes were regulated by their corresponding microRNAs, which suggested that microRNAs may play essential roles in the drought tolerance of tomato. In this study, numerous microRNAs and mRNAs involved in the drought response of tomato were identified using high-throughput sequencing, which will provide new insights into the complex regulatory network of plant adaption to drought stress. This work will also help to exploit new players functioning in plant drought-stress tolerance.

Defining RNA-Small Molecule Affinity Landscapes Enables Design of a Small Molecule Inhibitor of an Oncogenic Noncoding RNA.

PubMed

Velagapudi, Sai Pradeep; Luo, Yiling; Tran, Tuan; Haniff, Hafeez S; Nakai, Yoshio; Fallahi, Mohammad; Martinez, Gustavo J; Childs-Disney, Jessica L; Disney, Matthew D

2017-03-22

RNA drug targets are pervasive in cells, but methods to design small molecules that target them are sparse. Herein, we report a general approach to score the affinity and selectivity of RNA motif-small molecule interactions identified via selection. Named High Throughput Structure-Activity Relationships Through Sequencing (HiT-StARTS), HiT-StARTS is statistical in nature and compares input nucleic acid sequences to selected library members that bind a ligand via high throughput sequencing. The approach allowed facile definition of the fitness landscape of hundreds of thousands of RNA motif-small molecule binding partners. These results were mined against folded RNAs in the human transcriptome and identified an avid interaction between a small molecule and the Dicer nuclease-processing site in the oncogenic microRNA (miR)-18a hairpin precursor, which is a member of the miR-17-92 cluster. Application of the small molecule, Targapremir-18a, to prostate cancer cells inhibited production of miR-18a from the cluster, de-repressed serine/threonine protein kinase 4 protein (STK4), and triggered apoptosis. Profiling the cellular targets of Targapremir-18a via Chemical Cross-Linking and Isolation by Pull Down (Chem-CLIP), a covalent small molecule-RNA cellular profiling approach, and other studies showed specific binding of the compound to the miR-18a precursor, revealing broadly applicable factors that govern small molecule drugging of noncoding RNAs.

Defining RNA–Small Molecule Affinity Landscapes Enables Design of a Small Molecule Inhibitor of an Oncogenic Noncoding RNA

PubMed Central

2017-01-01

RNA drug targets are pervasive in cells, but methods to design small molecules that target them are sparse. Herein, we report a general approach to score the affinity and selectivity of RNA motif–small molecule interactions identified via selection. Named High Throughput Structure–Activity Relationships Through Sequencing (HiT-StARTS), HiT-StARTS is statistical in nature and compares input nucleic acid sequences to selected library members that bind a ligand via high throughput sequencing. The approach allowed facile definition of the fitness landscape of hundreds of thousands of RNA motif–small molecule binding partners. These results were mined against folded RNAs in the human transcriptome and identified an avid interaction between a small molecule and the Dicer nuclease-processing site in the oncogenic microRNA (miR)-18a hairpin precursor, which is a member of the miR-17-92 cluster. Application of the small molecule, Targapremir-18a, to prostate cancer cells inhibited production of miR-18a from the cluster, de-repressed serine/threonine protein kinase 4 protein (STK4), and triggered apoptosis. Profiling the cellular targets of Targapremir-18a via Chemical Cross-Linking and Isolation by Pull Down (Chem-CLIP), a covalent small molecule–RNA cellular profiling approach, and other studies showed specific binding of the compound to the miR-18a precursor, revealing broadly applicable factors that govern small molecule drugging of noncoding RNAs. PMID:28386598

OP17MICRORNA PROFILING USING SMALL RNA-SEQ IN PAEDIATRIC LOW GRADE GLIOMAS

PubMed Central

Jeyapalan, Jennie N.; Jones, Tania A.; Tatevossian, Ruth G.; Qaddoumi, Ibrahim; Ellison, David W.; Sheer, Denise

2014-01-01

INTRODUCTION: MicroRNAs regulate gene expression by targeting mRNAs for translational repression or degradation at the post-transcriptional level. In paediatric low-grade gliomas a few key genetic mutations have been identified, including BRAF fusions, FGFR1 duplications and MYB rearrangements. Our aim in the current study is to profile aberrant microRNA expression in paediatric low-grade gliomas and determine the role of epigenetic changes in the aetiology and behaviour of these tumours. METHOD: MicroRNA profiling of tumour samples (6 pilocytic, 2 diffuse, 2 pilomyxoid astrocytomas) and normal brain controls (4 adult normal brain samples and a primary glial progenitor cell-line) was performed using small RNA sequencing. Bioinformatic analysis included sequence alignment, analysis of the number of reads (CPM, counts per million) and differential expression. RESULTS: Sequence alignment identified 695 microRNAs, whose expression was compared in tumours v. normal brain. PCA and hierarchical clustering showed separate groups for tumours and normal brain. Computational analysis identified approximately 400 differentially expressed microRNAs in the tumours compared to matched location controls. Our findings will then be validated and integrated with extensive genetic and epigenetic information we have previously obtained for the full tumour cohort. CONCLUSION: We have identified microRNAs that are differentially expressed in paediatric low-grade gliomas. As microRNAs are known to target genes involved in the initiation and progression of cancer, they provide critical information on tumour pathogenesis and are an important class of biomarkers.

Small RNA deep sequencing identifies novel and salt-stress-regulated microRNAs from roots of Medicago sativa and Medicago truncatula.

PubMed

Long, Rui-Cai; Li, Ming-Na; Kang, Jun-Mei; Zhang, Tie-Jun; Sun, Yan; Yang, Qing-Chuan

2015-05-01

Small 21- to 24-nucleotide (nt) ribonucleic acids (RNAs), notably the microRNA (miRNA), are emerging as a posttranscriptional regulation mechanism. Salt stress is one of the primary abiotic stresses that cause the crop losses worldwide. In saline lands, root growth and function of plant are determined by the action of environmental salt stress through specific genes that adapt root development to the restrictive condition. To elucidate the role of miRNAs in salt stress regulation in Medicago, we used a high-throughput sequencing approach to analyze four small RNA libraries from roots of Zhongmu-1 (Medicago sativa) and Jemalong A17 (Medicago truncatula), which were treated with 300 mM NaCl for 0 and 8 h. Each library generated about 20 million short sequences and contained predominantly small RNAs of 24-nt length, followed by 21-nt and 22-nt small RNAs. Using sequence analysis, we identified 385 conserved miRNAs from 96 families, along with 68 novel candidate miRNAs. Of all the 68 predicted novel miRNAs, 15 miRNAs were identified to have miRNA*. Statistical analysis on abundance of sequencing read revealed specific miRNA showing contrasting expression patterns between M. sativa and M. truncatula roots, as well as between roots treated for 0 and 8 h. The expression of 10 conserved and novel miRNAs was also quantified by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The miRNA precursor and target genes were predicted by bioinformatics analysis. We concluded that the salt stress related conserved and novel miRNAs may have a large variety of target mRNAs, some of which might play key roles in salt stress regulation of Medicago. © 2014 Scandinavian Plant Physiology Society.

Deep sequencing and genome-wide analysis reveals the expansion of MicroRNA genes in the gall midge Mayetiola destructor

PubMed Central

2013-01-01

Background MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in regulating post transcriptional gene expression. Gall midges encompass a large group of insects that are of economic importance and also possess fascinating biological traits. The gall midge Mayetiola destructor, commonly known as the Hessian fly, is a destructive pest of wheat and model organism for studying gall midge biology and insect – host plant interactions. Results In this study, we systematically analyzed miRNAs from the Hessian fly. Deep-sequencing a Hessian fly larval transcriptome led to the identification of 89 miRNA species that are either identical or very similar to known miRNAs from other insects, and 184 novel miRNAs that have not been reported from other species. A genome-wide search through a draft Hessian fly genome sequence identified a total of 611 putative miRNA-encoding genes based on sequence similarity and the existence of a stem-loop structure for miRNA precursors. Analysis of the 611 putative genes revealed a striking feature: the dramatic expansion of several miRNA gene families. The largest family contained 91 genes that encoded 20 different miRNAs. Microarray analyses revealed the expression of miRNA genes was strictly regulated during Hessian fly larval development and abundance of many miRNA genes were affected by host genotypes. Conclusion The identification of a large number of miRNAs for the first time from a gall midge provides a foundation for further studies of miRNA functions in gall midge biology and behavior. The dramatic expansion of identical or similar miRNAs provides a unique system to study functional relations among miRNA iso-genes as well as changes in sequence specificity due to small changes in miRNAs and in their mRNA targets. These results may also facilitate the identification of miRNA genes for potential pest control through transgenic approaches. PMID:23496979

High-Throughput Sequencing Reveals Diverse Sets of Conserved, Nonconserved, and Species-Specific miRNAs in Jute

PubMed Central

Islam, Md. Tariqul; Ferdous, Ahlan Sabah; Najnin, Rifat Ara; Sarker, Suprovath Kumar; Khan, Haseena

2015-01-01

MicroRNAs play a pivotal role in regulating a broad range of biological processes, acting by cleaving mRNAs or by translational repression. A group of plant microRNAs are evolutionarily conserved; however, others are expressed in a species-specific manner. Jute is an agroeconomically important fibre crop; nonetheless, no practical information is available for microRNAs in jute to date. In this study, Illumina sequencing revealed a total of 227 known microRNAs and 17 potential novel microRNA candidates in jute, of which 164 belong to 23 conserved families and the remaining 63 belong to 58 nonconserved families. Among a total of 81 identified microRNA families, 116 potential target genes were predicted for 39 families and 11 targets were predicted for 4 among the 17 identified novel microRNAs. For understanding better the functions of microRNAs, target genes were analyzed by Gene Ontology and their pathways illustrated by KEGG pathway analyses. The presence of microRNAs identified in jute was validated by stem-loop RT-PCR followed by end point PCR and qPCR for randomly selected 20 known and novel microRNAs. This study exhaustively identifies microRNAs and their target genes in jute which will ultimately pave the way for understanding their role in this crop and other crops. PMID:25861616

Promoters, toll like receptors and microRNAs: a strange association.

PubMed

Korla, Kalyani; Arrigo, Patrizio; Mitra, Chanchal K

2013-06-01

Toll-like receptors (TLRs) are proteins that play key role in the innate immune system. In the present study, -1000 base pairs upstream are taken from the transcription start site of the various TLR genes (10 known) in human. About 40 microRNAs have been identified that share 12-19 nucleotide sequence similarity with the promoter regions of 10 TLRs. It is proposed that the microRNA performs potential role in identification of promoter sequence and initiation of transcription.

Next generation sequencing applications for microRNA biomarker discovery in toxicological studies

EPA Science Inventory

Next Generation Sequencing (NGS) technology will be reviewed for its base pair resolution, wide dynamic range, and insights into the genome and transcriptome, with special focus upon the biomarker potential of microRNAs (miRNAs). The first part of this presentation reviews commo...

Analysis of microRNA expression and function.

PubMed

Van Wynsberghe, Priscilla M; Chan, Shih-Peng; Slack, Frank J; Pasquinelli, Amy E

2011-01-01

Originally discovered in C. elegans, microRNAs (miRNAs) are small RNAs that regulate fundamental cellular processes in diverse organisms. MiRNAs are encoded within the genome and are initially transcribed as primary transcripts that can be several kilobases in length. Primary transcripts are successively cleaved by two RNase III enzymes, Drosha in the nucleus and Dicer in the cytoplasm, to produce ∼70 nucleotide (nt) long precursor miRNAs and 22 nt long mature miRNAs, respectively. Mature miRNAs regulate gene expression post-transcriptionally by imperfectly binding target mRNAs in association with the multiprotein RNA induced silencing complex (RISC). The conserved sequence, expression pattern, and function of some miRNAs across distinct species as well as the importance of specific miRNAs in many biological pathways have led to an explosion in the study of miRNA biogenesis, miRNA target identification, and miRNA target regulation. Many advances in our understanding of miRNA biology have come from studies in the powerful model organism C. elegans. This chapter reviews the current methods used in C. elegans to study miRNA biogenesis, small RNA populations, miRNA-protein complexes, and miRNA target regulation. Copyright © 2011 Elsevier Inc. All rights reserved.

The analysis of novel microRNA mimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detection.

PubMed

Winata, Patrick; Williams, Marissa; McGowan, Eileen; Nassif, Najah; van Zandwijk, Nico; Reid, Glen

2017-11-17

MicroRNAs are frequently downregulated in cancer, and restoring expression has tumour suppressive activity in tumour cells. Our recent phase I clinical trial investigated microRNA-based therapy in patients with malignant pleural mesothelioma. Treatment with TargomiRs, microRNA mimics with novel sequence packaged in EGFR antibody-targeted bacterial minicells, revealed clear signs of clinical activity. In order to detect delivery of microRNA mimics to tumour cells in future clinical trials, we tested hydrolysis probe-based assays specific for the sequence of the novel mimics in transfected mesothelioma cell lines using RT-qPCR. The custom assays efficiently and specifically amplified the consensus mimics. However, we found that these assays gave a signal when total RNA from untransfected and control mimic-transfected cells were used as templates. Further investigation revealed that the reverse transcription step using stem-loop primers appeared to introduce substantial non-specific amplification with either total RNA or synthetic RNA templates. This suggests that reverse transcription using stem-loop primers suffers from an intrinsic lack of specificity for the detection of highly similar microRNAs in the same family, especially when analysing total RNA. These results suggest that RT-qPCR is unlikely to be an effective means to detect delivery of microRNA mimic-based drugs to tumour cells in patients.

Design of a small molecule against an oncogenic noncoding RNA

PubMed Central

Velagapudi, Sai Pradeep; Cameron, Michael D.; Haga, Christopher L.; Rosenberg, Laura H.; Lafitte, Marie; Duckett, Derek R.; Phinney, Donald G.; Disney, Matthew D.

2016-01-01

The design of precision, preclinical therapeutics from sequence is difficult, but advances in this area, particularly those focused on rational design, could quickly transform the sequence of disease-causing gene products into lead modalities. Herein, we describe the use of Inforna, a computational approach that enables the rational design of small molecules targeting RNA to quickly provide a potent modulator of oncogenic microRNA-96 (miR-96). We mined the secondary structure of primary microRNA-96 (pri-miR-96) hairpin precursor against a database of RNA motif–small molecule interactions, which identified modules that bound RNA motifs nearby and in the Drosha processing site. Precise linking of these modules together provided Targaprimir-96 (3), which selectively modulates miR-96 production in cancer cells and triggers apoptosis. Importantly, the compound is ineffective on healthy breast cells, and exogenous overexpression of pri-miR-96 reduced compound potency in breast cancer cells. Chemical Cross-Linking and Isolation by Pull-Down (Chem-CLIP), a small-molecule RNA target validation approach, shows that 3 directly engages pri-miR-96 in breast cancer cells. In vivo, 3 has a favorable pharmacokinetic profile and decreases tumor burden in a mouse model of triple-negative breast cancer. Thus, rational design can quickly produce precision, in vivo bioactive lead small molecules against hard-to-treat cancers by targeting oncogenic noncoding RNAs, advancing a disease-to-gene-to-drug paradigm. PMID:27170187

Cloning and analysis of fetal ovary microRNAs in cattle.

PubMed

Tripurani, Swamy K; Xiao, Caide; Salem, Mohamed; Yao, Jianbo

2010-07-01

Ovarian folliculogenesis and early embryogenesis are complex processes, which require tightly regulated expression and interaction of a multitude of genes. Small endogenous RNA molecules, termed microRNAs (miRNAs), are involved in the regulation of gene expression during folliculogenesis and early embryonic development. To identify miRNAs in bovine oocytes/ovaries, a bovine fetal ovary miRNA library was constructed. Sequence analysis of random clones from the library identified 679 miRNA sequences, which represent 58 distinct bovine miRNAs. Of these distinct miRNAs, 42 are known bovine miRNAs present in the miRBase database and the remaining 16 miRNAs include 15 new bovine miRNAs that are homologous to miRNAs identified in other species, and one novel miRNA, which does not match any miRNAs in the database. The precursor sequences for 14 of the new 15 miRNAs as well as the novel miRNA were identified from the bovine genome database and their hairpin structures were predicted. Expression analysis of the 58 miRNAs in fetal ovaries in comparison to somatic tissue pools identified 8 miRNAs predominantly expressed in fetal ovaries. Further analysis of the eight miRNAs in germinal vesicle (GV) stage oocytes identified two miRNAs (bta-mir424 and bta-mir-10b), that are highly abundant in GV oocytes. Both miRNAs show similar expression patterns during oocyte maturation and preimplantation development of bovine embryos, being abundant in GV and MII stage oocytes, as well as in early stage embryos (until 16-cell stage). The amount of the novel miRNA is relatively small in oocytes and early cleavage embryos but greater in blastocysts, suggesting a role of this miRNA in blastocyst cell differentiation. Copyright 2010 Elsevier B.V. All rights reserved.

Identification and characterization of microRNAs in the pancreatic fluke Eurytrema pancreaticum.

PubMed

Xu, Min-Jun; Wang, Chun-Ren; Huang, Si-Yang; Fu, Jing-Hua; Zhou, Dong-Hui; Chang, Qiao-Cheng; Zheng, Xu; Zhu, Xing-Quan

2013-01-25

Eurytrema pancreaticum is one of the most common flukes, which mainly infects ruminants globally and infects human beings accidentally; causing eurytremiasis that has high veterinary and economic importance. MicroRNAs (miRNAs) are small non-coding RNAs and are now considered as a key mechanism of gene regulation at the post-transcription level. We investigated the global miRNA expression profile of E. pancreaticum adults using next-generation sequencing technology combined with real-time quantitative PCR. By using the genome of the closely-related species Schistosoma japonicum as reference, we obtained 27 miRNA candidates out of 16.45 million raw sequencing reads, with 13 of them found as known miRNAs in S. japonicum and/or S. mansoni, and the remaining 14 miRNAs were considered as novel. Five out of the 13 known miRNAs coming from one family named as sja-miR-2, including family members from miR-2a to miR-2e. Targets of 19 miRNAs were successfully predicated out of the 17401 mRNA and EST non-redundant sequences of S. japonicum. It was found that a significant high number of targets were related to "chch domain-containing protein mitochondrial precursor" (n = 29), "small subunit ribosomal protein s30e" (n = 21), and "insulin-induced gene 1 protein" (n = 9). Besides, "egg protein cp3842" (n = 2), "fumarate hydratase" (n = 2), "ubiquitin-conjugating enzyme" (n = 2), and "sperm-associated antigen 6" (n = 1) were also found as targets of the miRNAs of E. pancreaticum. The present study represents the first global characterization of E. pancreaticum miRNAs, which provides novel resources for a better understanding of the parasite, which, in turn, has implications for the effective control of the disease it causes.

miRNEST database: an integrative approach in microRNA search and annotation

PubMed Central

Szcześniak, Michał Wojciech; Deorowicz, Sebastian; Gapski, Jakub; Kaczyński, Łukasz; Makałowska, Izabela

2012-01-01

Despite accumulating data on animal and plant microRNAs and their functions, existing public miRNA resources usually collect miRNAs from a very limited number of species. A lot of microRNAs, including those from model organisms, remain undiscovered. As a result there is a continuous need to search for new microRNAs. We present miRNEST (http://mirnest.amu.edu.pl), a comprehensive database of animal, plant and virus microRNAs. The core part of the database is built from our miRNA predictions conducted on Expressed Sequence Tags of 225 animal and 202 plant species. The miRNA search was performed based on sequence similarity and as many as 10 004 miRNA candidates in 221 animal and 199 plant species were discovered. Out of them only 299 have already been deposited in miRBase. Additionally, miRNEST has been integrated with external miRNA data from literature and 13 databases, which includes miRNA sequences, small RNA sequencing data, expression, polymorphisms and targets data as well as links to external miRNA resources, whenever applicable. All this makes miRNEST a considerable miRNA resource in a sense of number of species (544) that integrates a scattered miRNA data into a uniform format with a user-friendly web interface. PMID:22135287

The use of high-throughput small RNA sequencing reveals differentially expressed microRNAs in response to aster yellows phytoplasma-infection in Vitis vinifera cv. ‘Chardonnay’

PubMed Central

Solofoharivelo, Marie-Chrystine; Souza-Richards, Rose; Stephan, Dirk; Murray, Shane; Burger, Johan T.

2017-01-01

Phytoplasmas are cell wall-less plant pathogenic bacteria responsible for major crop losses throughout the world. In grapevine they cause grapevine yellows, a detrimental disease associated with a variety of symptoms. The high economic impact of this disease has sparked considerable interest among researchers to understand molecular mechanisms related to pathogenesis. Increasing evidence exist that a class of small non-coding endogenous RNAs, known as microRNAs (miRNAs), play an important role in post-transcriptional gene regulation during plant development and responses to biotic and abiotic stresses. Thus, we aimed to dissect complex high-throughput small RNA sequencing data for the genome-wide identification of known and novel differentially expressed miRNAs, using read libraries constructed from healthy and phytoplasma-infected Chardonnay leaf material. Furthermore, we utilised computational resources to predict putative miRNA targets to explore the involvement of possible pathogen response pathways. We identified multiple known miRNA sequence variants (isomiRs), likely generated through post-transcriptional modifications. Sequences of 13 known, canonical miRNAs were shown to be differentially expressed. A total of 175 novel miRNA precursor sequences, each derived from a unique genomic location, were predicted, of which 23 were differentially expressed. A homology search revealed that some of these novel miRNAs shared high sequence similarity with conserved miRNAs from other plant species, as well as known grapevine miRNAs. The relative expression of randomly selected known and novel miRNAs was determined with real-time RT-qPCR analysis, thereby validating the trend of expression seen in the normalised small RNA sequencing read count data. Among the putative miRNA targets, we identified genes involved in plant morphology, hormone signalling, nutrient homeostasis, as well as plant stress. Our results may assist in understanding the role that miRNA pathways play during plant pathogenesis, and may be crucial in understanding disease symptom development in aster yellows phytoplasma-infected grapevines. PMID:28813447

MicroRNAs Promote Granule Cell Expansion in the Cerebellum Through Gli2.

PubMed

Constantin, Lena; Wainwright, Brandon J

2015-12-01

MicroRNAs (miRNAs) are important regulators of cerebellar function and homeostasis. Their deregulation results in cerebellar neuronal degeneration and spinocerebellar ataxia type 1 and contributes to medulloblastoma. Canonical miRNA processing involves Dicer, which cleaves precursor miRNAs into mature double-stranded RNA duplexes. In order to address the role of miRNAs in cerebellar granule cell precursor development, loxP-flanked exons of Dicer1 were conditionally inactivated using the granule cell precursor-specific Atoh1-Cre recombinase. A reduction of 87% in Dicer1 transcript was achieved in this conditional Dicer knockdown model. Although knockdown resulted in normal survival, mice had disruptions to the cortical layering of the anterior cerebellum, which resulted from the premature differentiation of granule cell precursors in this region during neonatal development. This defect manifested as a thinner external granular layer with ectopic mature granule cells, and a depleted internal granular layer. We found that expression of the activator components of the Hedgehog-Patched pathway, the Gli family of transcription factors, was perturbed in conditional Dicer knockdown mice. We propose that loss of Gli2 mRNA mediated the anterior-restricted defect in conditional Dicer knockdown mice and, as proof of principle, were able to show that miR-106b positively regulated Gli2 mRNA expression. These findings confirm the importance of miRNAs as positive mediators of Hedgehog-Patched signalling during granule cell precursor development.

Evolution of New miRNAs and Cerebro-Cortical Development.

PubMed

Kosik, Kenneth S; Nowakowski, Tomasz

2018-04-04

The noncoding portion of the genome, including microRNAs, has been fertile evolutionary soil for cortical development in primates. A major contribution to cortical expansion in primates is the generation of novel precursor cell populations. Because miRNA expression profiles track closely with cell identity, it is likely that numerous novel microRNAs have contributed to cellular diversity in the brain. The tools to determine the genomic context within which novel microRNAs emerge and how they become integrated into molecular circuitry are now in hand. Expected final online publication date for the Annual Review of Neuroscience Volume 41 is July 8, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

ADAR2 induces reproducible changes in sequence and abundance of mature microRNAs in the mouse brain

PubMed Central

Vesely, Cornelia; Tauber, Stefanie; Sedlazeck, Fritz J.; Tajaddod, Mansoureh; von Haeseler, Arndt; Jantsch, Michael F.

2014-01-01

Adenosine deaminases that act on RNA (ADARs) deaminate adenosines to inosines in double-stranded RNAs including miRNA precursors. A to I editing is widespread and required for normal life. By comparing deep sequencing data of brain miRNAs from wild-type and ADAR2 deficient mouse strains, we detect editing sites and altered miRNA processing at high sensitivity. We detect 48 novel editing events in miRNAs. Some editing events reach frequencies of up to 80%. About half of all editing events depend on ADAR2 while some miRNAs are preferentially edited by ADAR1. Sixty-four percent of all editing events are located within the seed region of mature miRNAs. For the highly edited miR-3099, we experimentally prove retargeting of the edited miRNA to novel 3′ UTRs. We show further that an abundant editing event in miR-497 promotes processing by Drosha of the corresponding pri-miRNA. We also detect reproducible changes in the abundance of specific miRNAs in ADAR2-deficient mice that occur independent of adjacent A to I editing events. This indicates that ADAR2 binding but not editing of miRNA precursors may influence their processing. Correlating with changes in miRNA abundance we find misregulation of putative targets of these miRNAs in the presence or absence of ADAR2. PMID:25260591

Droplet Microfluidic Device Fabrication and Use for Isothermal Amplification and Detection of MicroRNA.

PubMed

Giuffrida, Maria Chiara; D'Agata, Roberta; Spoto, Giuseppe

2017-01-01

Droplet microfluidics combined with the isothermal circular strand displacement polymerization (ICSDP) represents a powerful new technique to detect both single-stranded DNA and microRNA sequences. The method here described helps in overcoming some drawbacks of the lately introduced droplet polymerase chain reaction (PCR) amplification when implemented in microfluidic devices. The method also allows the detection of nanoliter droplets of nucleic acids sequences solutions, with a particular attention to microRNA sequences that are detected at the picomolar level. The integration of the ICSDP amplification protocol in droplet microfluidic devices reduces the time of analysis and the amount of sample required. In addition, there is also the possibility to design parallel analyses to be integrated in portable devices.

The expression of the Alzheimer’s Amyloid Precursor Protein-like gene is regulated by developmental timing microRNAs and their targets in Caenorhabditis elegans

PubMed Central

Niwa, Ryusuke; Zhou, Feng; Li, Chris; Slack, Frank J.

2008-01-01

Alzheimer’s Disease (AD) is a neurodegenerative disorder characterized by the accumulation of dense plaques in the brain, resulting in progressive dementia. A major plaque component is the β-amyloid peptide, which is a cleavage product of the amyloid precursor protein (APP). Studies of dominant inheritable familial AD support the hypothesis that APP is critical for AD development. On the other hand, the pathogenesis of amyloid plaque deposition in AD is thought to be the result of age-related changes with unknown mechanisms. Here we show that the Caenorhabditis elegans homolog of APP, APP-like-1 (apl-1), functions with and is under the control of molecules regulating developmental progression. In C. elegans, the timing of cell fate determination is controlled by the heterochronic genes, including let-7 microRNAs. C. elegans apl-1 shows significant genetic interactions with let-7 family microRNAs and let-7-targeted heterochronic genes, hbl-1, lin-41 and lin-42. apl-1 expression is upregulated during the last larval stage in hypodermal seam cells which is transcriptionally regulated by hbl-1, lin-41 and lin-42. Moreover, the levels of the apl-1 transcription are modulated by the activity of let-7 family microRNAs. Our works places apl-1 in a developmental timing pathway and may provide new insights into the time-dependent progression of AD. PMID:18262516

Epigenetic Deregulation of MicroRNAs in Rhabdomyosarcoma and Neuroblastoma and Translational Perspectives

PubMed Central

Romania, Paolo; Bertaina, Alice; Bracaglia, Giorgia; Locatelli, Franco; Fruci, Doriana; Rota, Rossella

2012-01-01

Gene expression control mediated by microRNAs and epigenetic remodeling of chromatin are interconnected processes often involved in feedback regulatory loops, which strictly guide proper tissue differentiation during embryonal development. Altered expression of microRNAs is one of the mechanisms leading to pathologic conditions, such as cancer. Several lines of evidence pointed to epigenetic alterations as responsible for aberrant microRNA expression in human cancers. Rhabdomyosarcoma and neuroblastoma are pediatric cancers derived from cells presenting features of skeletal muscle and neuronal precursors, respectively, blocked at different stages of differentiation. Consistently, tumor cells express tissue markers of origin but are unable to terminally differentiate. Several microRNAs playing a key role during tissue differentiation are often epigenetically downregulated in rhabdomyosarcoma and neuroblastoma and behave as tumor suppressors when re-expressed. Recently, inhibition of epigenetic modulators in adult tumors has provided encouraging results causing re-expression of anti-tumor master gene pathways. Thus, a similar approach could be used to correct the aberrant epigenetic regulation of microRNAs in rhabdomyosarcoma and neuroblastoma. The present review highlights the current insights on epigenetically deregulated microRNAs in rhabdomyosarcoma and neuroblastoma and their role in tumorigenesis and developmental pathways. The translational clinical implications and challenges regarding modulation of epigenetic chromatin remodeling/microRNAs interconnections are also discussed. PMID:23443118

MicroRNA profiling of antler stem cells in potentiated and dormant states and their potential roles in antler regeneration.

PubMed

Ba, Hengxing; Wang, Datao; Li, Chunyi

2016-04-01

MicroRNAs (miRNAs) can effectively regulate gene expression at the post-transcriptional level and play a critical role in tissue growth, development and regeneration. Our previous studies showed that antler regeneration is a stem cell-based process and antler stem cells reside in the periosteum of a pedicle, the permanent bony protuberance, from which antler regeneration takes place. Antlers are the only mammalian organ that can fully regenerate and hence provide a unique opportunity to identify miRNAs that are involved in organ regeneration. In the present study, we used next generation sequencing technology sequenced miRNAs of the stem cells derived from either the potentiated or the dormant pedicle periosteum. A population of both conserved and 20 deer-specific miRNAs was identified. These conserved miRNAs were derived from 453 homologous hairpin precursors across 88 animal species, and were further grouped into 167 miRNA families. Among them, the miR-296 is embryonic stem cell-specific. The potentiation process resulted in the significant regulation (>±2 Fold, q value <0.05) of conserved miRNAs; 8 miRNA transcripts were down- and 6 up-regulated. Several GO biology processes and the Wnt, MAPK and TGF-beta signaling pathways were found to be up-regulated as part of antlerogenic stem cell potentiation process. This research has identified miRNAs that are associated either with the dormant or the potentiated antler stem cells and identified some target miRNAs for further research into their role played in mammalian organ regeneration.

Micro-terminator: 'Hasta la vista, lncRNA!'.

PubMed

Diederichs, Sven

2015-04-01

Transcriptional termination is an important yet incompletely understood aspect of gene expression. Proudfoot, Jopling and colleagues now identify a new Microprocessor-mediated mechanism of transcriptional termination, which acts specifically on long noncoding transcripts that serve as microRNA precursors.

MicroRNAs as Key Effectors in the p53 Network.

PubMed

Goeman, Frauke; Strano, Sabrina; Blandino, Giovanni

2017-01-01

The guardian of the genome p53 is embedded in a fine-spun network of MicroRNAs. p53 is able to activate or repress directly the transcription of MicroRNAs that are participating in the tumor-suppressive mission of p53. On the other hand, the expression of p53 is under tight control of MicroRNAs that are either targeting directly p53 or factors that are modifying its protein level or activity. Although the most important function of p53 is suggested to be transcriptional regulation, there are several nontranscriptional functions described. One of those regards the modulation of MicroRNA biogenesis. Wild-type p53 is increasing the maturation of selected MicroRNAs from the primary transcript to the precursor MiRNA by interacting with the Microprocessor complex. Furthermore, p53 is modulating the mRNA accessibility for certain MicroRNAs by association with the RISC complex and transcriptional regulation of RNA-binding proteins. In this way p53 is able to remodel the MiRNA-mRNA interaction network. As wild-type p53 is employing MicroRNAs to suppress cancer development, gain-of-function mutant p53 proteins use MicroRNAs to confer oncogenic properties like chemoresistance and the ability to drive metastasis. Like its wild-type counterpart mutant p53 is able to regulate MicroRNAs transcriptionally and posttranscriptionally. Mutant p53 affects the MiRNA processing at two cleavage steps through interfering with the Microprocessor complex and by downregulating Dicer and KSRP, a modulator of MiRNA biogenesis. Thus, MicroRNAs are essential components in the p53 pathway, contributing substantially to combat or enhance tumor development depending on the wild-type or mutant p53 context. © 2017 Elsevier Inc. All rights reserved.

Isolation of Exosome-Like Nanoparticles and Analysis of MicroRNAs Derived from Coconut Water Based on Small RNA High-Throughput Sequencing.

PubMed

Zhao, Zhehao; Yu, Siran; Li, Min; Gui, Xin; Li, Ping

2018-03-21

In this study, the presence of microRNAs in coconut water was identified by real-time polymerase chain reaction (PCR) based on the results of high-throughput small RNA sequencing. In addition, the differences in microRNA content between immature and mature coconut water were compared. A total of 47 known microRNAs belonging to 25 families and 14 new microRNAs were identified in coconut endosperm. Through analysis using a target gene prediction software, potential microRNA target genes were identified in the human genome. Real-time PCR showed that the level of most microRNAs was higher in mature coconut water than in immature coconut water. Then, exosome-like nanoparticles were isolated from coconut water. After ultracentrifugation, some particle structures were seen in coconut water samples using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate fluorescence staining. Subsequent scanning electron microscopy observation and dynamic light scattering analysis also revealed some exosome-like nanoparticles in coconut water, and the mean diameters of the particles detected by the two methods were 13.16 and 59.72 nm, respectively. In conclusion, there are extracellular microRNAs in coconut water, and their levels are higher in mature coconut water than in immature coconut water. Some exosome-like nanoparticles were isolated from coconut water, and the diameter of these particles was smaller than that of animal-derived exosomes.

Identification and bioinformatics analysis of microRNAs from the sporophyte and gametophyte of Pyropia haitanensis

NASA Astrophysics Data System (ADS)

Huang, Aiyou; Wang, Guangce

2016-05-01

Pyropia haitanensis (T. J. Chang et B. F. Zheng) N. Kikuchi et M. Miyata ( Porphyra haitanensis) is an economically important genus that is cultured widely in China. P. haitanensis is cultured on a larger scale than Pyropia yezoensis, making up an important part of the total production of cultivated Pyropia in China. However, the majority of molecular mechanisms underlying the physiological processes of P. haitanensis remain unknown. P. haitanensis could utilize inorganic carbon and the sporophytes of P. haitanensis might possess a PCK-type C4-like carbon-fixation pathway. To identify microRNAs and their probable roles in sporophyte and gametophyte development, we constructed and sequenced small RNA libraries from sporophytes and gametophytes of P. haitanensis. Five microRNAs were identified that shared no sequence homology with known microRNAs. Our results indicated that P. haitanensis might posses a complex sRNA processing system in which the novel microRNAs act as important regulators of the development of different generations of P. haitanensis.

Identification of microRNAs and their targets in Finger millet by high throughput sequencing.

PubMed

Usha, S; Jyothi, M N; Sharadamma, N; Dixit, Rekha; Devaraj, V R; Nagesh Babu, R

2015-12-15

MicroRNAs are short non-coding RNAs which play an important role in regulating gene expression by mRNA cleavage or by translational repression. The majority of identified miRNAs were evolutionarily conserved; however, others expressed in a species-specific manner. Finger millet is an important cereal crop; nonetheless, no practical information is available on microRNAs to date. In this study, we have identified 95 conserved microRNAs belonging to 39 families and 3 novel microRNAs by high throughput sequencing. For the identified conserved and novel miRNAs a total of 507 targets were predicted. 11 miRNAs were validated and tissue specificity was determined by stem loop RT-qPCR, Northern blot. GO analyses revealed targets of miRNA were involved in wide range of regulatory functions. This study implies large number of known and novel miRNAs found in Finger millet which may play important role in growth and development. Copyright © 2015 Elsevier B.V. All rights reserved.

A database analysis method identifies an endogenous trans-acting short-interfering RNA that targets the Arabidopsis ARF2, ARF3, and ARF4 genes

PubMed Central

Williams, Leor; Carles, Cristel C.; Osmont, Karen S.; Fletcher, Jennifer C.

2005-01-01

Two classes of small RNAs, microRNAs and short-interfering RNA (siRNAs), have been extensively studied in plants and animals. In Arabidopsis, the capacity to uncover previously uncharacterized small RNAs by means of conventional strategies seems to be reaching its limits. To discover new plant small RNAs, we developed a protocol to mine an Arabidopsis nonannotated, noncoding EST database. Using this approach, we identified an endogenous small RNA, trans-acting short-interfering RNA–auxin response factor (tasiR-ARF), that shares a 21- and 22-nt region of sequence similarity with members of the ARF gene family. tasiR-ARF has characteristics of both short-interfering RNA and microRNA, recently defined as tasiRNA. Accumulation of trans-acting siRNA depends on DICER-LIKE1 and RNA-DEPENDENT RNA POLYMERASE6 but not RNA-DEPENDENT RNA POLYMERASE2. We demonstrate that tasiR-ARF targets three ARF genes, ARF2, ARF3/ETT, and ARF4, and that both the tasiR-ARF precursor and its target genes are evolutionarily conserved. The identification of tasiRNA-ARF as a low-abundance, previously uncharacterized small RNA species proves our method to be a useful tool to uncover additional small regulatory RNAs. PMID:15980147

PACCMIT/PACCMIT-CDS: identifying microRNA targets in 3′ UTRs and coding sequences

PubMed Central

Šulc, Miroslav; Marín, Ray M.; Robins, Harlan S.; Vaníček, Jiří

2015-01-01

The purpose of the proposed web server, publicly available at http://paccmit.epfl.ch, is to provide a user-friendly interface to two algorithms for predicting messenger RNA (mRNA) molecules regulated by microRNAs: (i) PACCMIT (Prediction of ACcessible and/or Conserved MIcroRNA Targets), which identifies primarily mRNA transcripts targeted in their 3′ untranslated regions (3′ UTRs), and (ii) PACCMIT-CDS, designed to find mRNAs targeted within their coding sequences (CDSs). While PACCMIT belongs among the accurate algorithms for predicting conserved microRNA targets in the 3′ UTRs, the main contribution of the web server is 2-fold: PACCMIT provides an accurate tool for predicting targets also of weakly conserved or non-conserved microRNAs, whereas PACCMIT-CDS addresses the lack of similar portals adapted specifically for targets in CDS. The web server asks the user for microRNAs and mRNAs to be analyzed, accesses the precomputed P-values for all microRNA–mRNA pairs from a database for all mRNAs and microRNAs in a given species, ranks the predicted microRNA–mRNA pairs, evaluates their significance according to the false discovery rate and finally displays the predictions in a tabular form. The results are also available for download in several standard formats. PMID:25948580

miRNAs involved in the development and differentiation of fertile and sterile flowers in Viburnum macrocephalum f. keteleeri.

PubMed

Li, Weixing; He, Zhichong; Zhang, Li; Lu, Zhaogeng; Xu, Jing; Cui, Jiawen; Wang, Li; Jin, Biao

2017-10-13

Sterile and fertile flowers are important evolutionary developmental phenotypes in angiosperm flowers. The development of floral organs, critical in angiosperm reproduction, is regulated by microRNAs (miRNAs). However, the mechanisms underpinning the miRNA regulation of the differentiation and development of sterile and fertile flowers remain unclear. Here, based on investigations of the morphological differences between fertile and sterile flowers, we used high-throughput sequencing to characterize the miRNAs in the differentiated floral organs of Viburnum macrocephalum f. keteleeri. We identified 49 known miRNAs and 67 novel miRNAs by small RNA (sRNA) sequencing and bioinformatics analysis, and 17 of these known and novel miRNA precursors were validated by polymerase chain reaction (PCR) and Sanger sequencing. Furthermore, by comparing the sequencing results of two sRNA libraries, we found that 30 known and 39 novel miRNA sequences were differentially expressed, and 35 were upregulated and 34 downregulated in sterile compared with fertile flowers. Combined with their predicted targets, the potential roles of miRNAs in V. macrocephalum f. keteleeri flowers include involvement in floral organogenesis, cell proliferation, hormonal pathways, and stress responses. miRNA precursors and targets were further validated by quantitative real-time PCR (qRT-PCR). Specifically, miR156a-5p, miR156g, and miR156j expression levels were significantly higher in fertile flowers than in sterile flowers, while SPL genes displayed the opposite expression pattern. Considering that the targets of miR156 are predicted to be SPL genes, we propose that miR156 may be involved in the regulation of stamen development in V. macrocephalum f. keteleeri. We identified miRNAs differentially expressed between fertile and sterile flowers in V. macrocephalum f. keteleeri and provided new insights into the important regulatory roles of miRNAs in the differentiation and development of fertile and sterile flowers.

Precursor microRNA Programmed Silencing Complex Assembly Pathways in Mammals

PubMed Central

Liu, Xuhang; Jin, Dong-Yan; McManus, Michael T.; Mourelatos, Zissimos

2012-01-01

Summary Assembly of microRNA Ribonucleoproteins (miRNPs) or RNA-Induced Silencing Complexes (RISCs) is essential for the function of miRNAs and initiates from processing of precursor miRNAs (pre-miRNAs) by Dicer or by Ago2. Here, we report an in-vitro miRNP/RISC assembly assay programmed by pre-miRNAs from mammalian cell lysates. Combining in-vivo studies in Dicer Knock-Out cells reconstituted with wild type or catalytically inactive Dicer, we find that the miRNA Loading Complex (miRLC) is the primary machinery linking pre-miRNA processing to miRNA loading. We show that a miRNA Precursor Deposit Complex (miPDC) plays a crucial role in Dicer-independent miRNA biogenesis and promotes miRNP assembly of certain Dicer-dependent miRNAs. Furthermore, we find that 5′-uridine, 3′-mid base pairing and 5′-mid mismatches within pre-miRNAs promote their assembly into miPDC. Our studies provide a comprehensive view of miRNP/RISC assembly pathways in mammals and our assay provides a versatile platform for further mechanistic dissection of such pathways in mammals. PMID:22503104

Microprocessor-dependent processing of Splice site Overlapping microRNA exons does not result in changes in alternative splicing.

PubMed

Pianigiani, Giulia; Licastro, Danilo; Fortugno, Paola; Castiglia, Daniele; Petrovic, Ivana; Pagani, Franco

2018-06-12

MicroRNAs are found throughout the genome and are processed by the microprocessor complex (MPC) from longer precursors. Some precursor miRNAs overlap intron:exon junctions. These Splice site Overlapping microRNAs (SO-miRNAs) are mostly located in coding genes. It has been intimated, in the rarer examples of SO-miRNAs in non-coding RNAs, that the competition between the spliceosome and the MPC modulates alternative splicing. However, the effect of this overlap on coding transcripts is unknown. Unexpectedly, we show that neither Drosha silencing nor SF3b1 silencing changed the inclusion ratio of SO-miRNA exons. Two SO-miRNAs, located in genes that code for basal membrane proteins, are known to inhibit proliferation in primary keratinocytes. These SO-miRNAs were upregulated during differentiation and the host mRNAs were downregulated, but again there was no change in inclusion ratio of the SO-miRNA exons. Interestingly, Drosha silencing increased nascent RNA density, on chromatin, downstream of SO-miRNA exons. Overall our data suggest a novel mechanism for regulating gene expression in which MPC-dependent cleavage of SO-miRNA exons could cause premature transcriptional termination of coding genes rather than affecting alternative splicing. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Increased precursor microRNA-21 following status epilepticus can compete with mature microRNA-21 to alter translation

PubMed Central

Chak, Kayam; Roy-Chaudhuri, Biswajoy; Kim, Hak Kyun; Kemp, Kayla C; Kay, Mark A

2016-01-01

MicroRNA-21 (miR-21) is consistently up-regulated in various neurological disorders, including epilepsy. Here, we show that the biogenesis of miR-21 is altered following pilocarpine status epilepticus (SE) with an increase in precursor miR-21 (pre-miR-21) in rats. We demonstrate that pre-miR-21 has an energetically favorable site overlapping with the miR-21 binding site and competes with mature miR-21 for binding in the 3′UTR of TGFBR2 mRNA, but not NT-3 mRNA in vitro. This binding competition influences miR-21-mediated repression in vitro and correlates with the increase in TGFBR2 and decrease in NT-3 following SE. Polysome profiling reveals co-localization of pre-miR-21 in the ribosome fraction with translating mRNAs in U-87 cells. The current work suggests that pre-miR-21 may post-transcriptionally counteract miR-21-mediated suppression following SE and could potentially lead to prolonged TGF-β receptor expression impacting epileptogenesis. The study further supports that the ratio of the pre to mature miRNA may be important in determining the regulatory effects of a miRNA gene. PMID:27725160

The expansion of the metazoan microRNA repertoire

PubMed Central

Hertel, Jana; Lindemeyer, Manuela; Missal, Kristin; Fried, Claudia; Tanzer, Andrea; Flamm, Christoph; Hofacker, Ivo L; Stadler, Peter F

2006-01-01

Background MicroRNAs have been identified as crucial regulators in both animals and plants. Here we report on a comprehensive comparative study of all known miRNA families in animals. We expand the MicroRNA Registry 6.0 by more than 1000 new homologs of miRNA precursors whose expression has been verified in at least one species. Using this uniform data basis we analyze their evolutionary history in terms of individual gene phylogenies and in terms of preservation of genomic nearness across species. This allows us to reliably identify microRNA clusters that are derived from a common transcript. Results We identify three episodes of microRNA innovation that correspond to major developmental innovations: A class of about 20 miRNAs is common to protostomes and deuterostomes and might be related to the advent of bilaterians. A second large wave of innovations maps to the branch leading to the vertebrates. The third significant outburst of miRNA innovation coincides with placental (eutherian) mammals. In addition, we observe the expected expansion of the microRNA inventory due to genome duplications in early vertebrates and in an ancestral teleost. The non-local duplications in the vertebrate ancestor are predated by local (tandem) duplications leading to the formation of about a dozen ancient microRNA clusters. Conclusion Our results suggest that microRNA innovation is an ongoing process. Major expansions of the metazoan miRNA repertoire coincide with the advent of bilaterians, vertebrates, and (placental) mammals. PMID:16480513

A Panel of MicroRNAs as Diagnostic Biomarkers for the Identification of Prostate Cancer.

PubMed

Daniel, Rhonda; Wu, Qianni; Williams, Vernell; Clark, Gene; Guruli, Georgi; Zehner, Zendra

2017-06-16

Prostate cancer is the most common non-cutaneous cancer among men; yet, current diagnostic methods are insufficient, and more reliable diagnostic markers need to be developed. One answer that can bridge this gap may lie in microRNAs. These small RNA molecules impact protein expression at the translational level, regulating important cellular pathways, the dysregulation of which can exert tumorigenic effects contributing to cancer. In this study, high throughput sequencing of small RNAs extracted from blood from 28 prostate cancer patients at initial stages of diagnosis and prior to treatment was used to identify microRNAs that could be utilized as diagnostic biomarkers for prostate cancer compared to 12 healthy controls. In addition, a group of four microRNAs (miR-1468-3p, miR-146a-5p, miR-1538 and miR-197-3p) was identified as normalization standards for subsequent qRT-PCR confirmation. qRT-PCR analysis corroborated microRNA sequencing results for the seven top dysregulated microRNAs. The abundance of four microRNAs (miR-127-3p, miR-204-5p, miR-329-3p and miR-487b-3p) was upregulated in blood, whereas the levels of three microRNAs (miR-32-5p, miR-20a-5p and miR-454-3p) were downregulated. Data analysis of the receiver operating curves for these selected microRNAs exhibited a better correlation with prostate cancer than PSA (prostate-specific antigen), the current gold standard for prostate cancer detection. In summary, a panel of seven microRNAs is proposed, many of which have prostate-specific targets, which may represent a significant improvement over current testing methods.

MicroRNA expression profile in bovine cumulus–oocyte complexes: Possible role of let-7 and miR-106a in the development of bovine oocytes

USDA-ARS?s Scientific Manuscript database

The expression of microRNAs (miRs) in bovine cumulus-oocyte complexes (COCs) during late oogenesis was profiled to determine the potential for regulation of maternal mRNAs by this class of small RNAs. A cDNA cloning and sequencing strategy resulted in 1812 putative miR sequences, representing 72 di...

CHO microRNA engineering is growing up: Recent successes and future challenges☆

PubMed Central

Jadhav, Vaibhav; Hackl, Matthias; Druz, Aliaksandr; Shridhar, Smriti; Chung, Cheng-Yu; Heffner, Kelley M.; Kreil, David P.; Betenbaugh, Mike; Shiloach, Joseph; Barron, Niall; Grillari, Johannes; Borth, Nicole

2013-01-01

microRNAs with their ability to regulate complex pathways that control cellular behavior and phenotype have been proposed as potential targets for cell engineering in the context of optimization of biopharmaceutical production cell lines, specifically of Chinese Hamster Ovary cells. However, until recently, research was limited by a lack of genomic sequence information on this industrially important cell line. With the publication of the genomic sequence and other relevant data sets for CHO cells since 2011, the doors have been opened for an improved understanding of CHO cell physiology and for the development of the necessary tools for novel engineering strategies. In the present review we discuss both knowledge on the regulatory mechanisms of microRNAs obtained from other biological models and proof of concepts already performed on CHO cells, thus providing an outlook of potential applications of microRNA engineering in production cell lines. PMID:23916872

PACCMIT/PACCMIT-CDS: identifying microRNA targets in 3' UTRs and coding sequences.

PubMed

Šulc, Miroslav; Marín, Ray M; Robins, Harlan S; Vaníček, Jiří

2015-07-01

The purpose of the proposed web server, publicly available at http://paccmit.epfl.ch, is to provide a user-friendly interface to two algorithms for predicting messenger RNA (mRNA) molecules regulated by microRNAs: (i) PACCMIT (Prediction of ACcessible and/or Conserved MIcroRNA Targets), which identifies primarily mRNA transcripts targeted in their 3' untranslated regions (3' UTRs), and (ii) PACCMIT-CDS, designed to find mRNAs targeted within their coding sequences (CDSs). While PACCMIT belongs among the accurate algorithms for predicting conserved microRNA targets in the 3' UTRs, the main contribution of the web server is 2-fold: PACCMIT provides an accurate tool for predicting targets also of weakly conserved or non-conserved microRNAs, whereas PACCMIT-CDS addresses the lack of similar portals adapted specifically for targets in CDS. The web server asks the user for microRNAs and mRNAs to be analyzed, accesses the precomputed P-values for all microRNA-mRNA pairs from a database for all mRNAs and microRNAs in a given species, ranks the predicted microRNA-mRNA pairs, evaluates their significance according to the false discovery rate and finally displays the predictions in a tabular form. The results are also available for download in several standard formats. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

miTRATA: a web-based tool for microRNA Truncation and Tailing Analysis.

PubMed

Patel, Parth; Ramachandruni, S Deepthi; Kakrana, Atul; Nakano, Mayumi; Meyers, Blake C

2016-02-01

We describe miTRATA, the first web-based tool for microRNA Truncation and Tailing Analysis--the analysis of 3' modifications of microRNAs including the loss or gain of nucleotides relative to the canonical sequence. miTRATA is implemented in Python (version 3) and employs parallel processing modules to enhance its scalability when analyzing multiple small RNA (sRNA) sequencing datasets. It utilizes miRBase, currently version 21, as a source of known microRNAs for analysis. miTRATA notifies user(s) via email to download as well as visualize the results online. miTRATA's strengths lie in (i) its biologist-focused web interface, (ii) improved scalability via parallel processing and (iii) its uniqueness as a webtool to perform microRNA truncation and tailing analysis. miTRATA is developed in Python and PHP. It is available as a web-based application from https://wasabi.dbi.udel.edu/∼apps/ta/. meyers@dbi.udel.edu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Small Molecule Targeting of a MicroRNA Associated with Hepatocellular Carcinoma.

PubMed

Childs-Disney, Jessica L; Disney, Matthew D

2016-02-19

Development of precision therapeutics is of immense interest, particularly as applied to the treatment of cancer. By analyzing the preferred cellular RNA targets of small molecules, we discovered that 5"-azido neomycin B binds the Drosha processing site in the microRNA (miR)-525 precursor. MiR-525 confers invasive properties to hepatocellular carcinoma (HCC) cells. Although HCC is one of the most common cancers, treatment options are limited, making the disease often fatal. Herein, we find that addition of 5"-azido neomycin B and its FDA-approved precursor, neomycin B, to an HCC cell line selectively inhibits production of the mature miRNA, boosts a downstream protein, and inhibits invasion. Interestingly, neomycin B is a second-line agent for hepatic encephalopathy (HE) and bacterial infections due to cirrhosis. Our results provocatively suggest that neomycin B, or second-generation derivatives, may be dual functioning molecules to treat both HE and HCC. Collectively, these studies show that rational design approaches can be tailored to disease-associated RNAs to afford potential lead therapeutics.

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

Jenny, Matthew J.; Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487; Aluru, Neelakanteswar

Although many drugs and environmental chemicals are teratogenic, the mechanisms by which most toxicants disrupt embryonic development are not well understood. MicroRNAs, single-stranded RNA molecules of ∼ 22 nt that regulate protein expression by inhibiting mRNA translation and promoting mRNA sequestration or degradation, are important regulators of a variety of cellular processes including embryonic development and cellular differentiation. Recent studies have demonstrated that exposure to xenobiotics can alter microRNA expression and contribute to the mechanisms by which environmental chemicals disrupt embryonic development. In this study we tested the hypothesis that developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a well-known teratogen, alters microRNAmore » expression during zebrafish development. We exposed zebrafish embryos to DMSO (0.1%) or TCDD (5 nM) for 1 h at 30 hours post fertilization (hpf) and measured microRNA expression using several methods at 36 and 60 hpf. TCDD caused strong induction of CYP1A at 36 hpf (62-fold) and 60 hpf (135-fold) as determined by real-time RT-PCR, verifying the effectiveness of the exposure. MicroRNA expression profiles were determined using microarrays (Agilent and Exiqon), next-generation sequencing (SOLiD), and real-time RT-PCR. The two microarray platforms yielded results that were similar but not identical; both showed significant changes in expression of miR-451, 23a, 23b, 24 and 27e at 60 hpf. Multiple analyses were performed on the SOLiD sequences yielding a total of 16 microRNAs as differentially expressed by TCDD in zebrafish embryos. However, miR-27e was the only microRNA to be identified as differentially expressed by all three methods (both microarrays, SOLiD sequencing, and real-time RT-PCR). These results suggest that TCDD exposure causes modest changes in expression of microRNAs, including some (miR-451, 23a, 23b, 24 and 27e) that are critical for hematopoiesis and cardiovascular development. -- Highlights: ► Zebrafish embryos were exposed to TCDD at two different developmental timepoints. ► Compared different methods in detecting global changes in microRNA expression. ► TCDD caused significant changes in microRNA expression in zebrafish embryos. ► Differentially expressed microRNAs have roles related to TCDD-induced phenotypes.« less

Cancer Exosomes Perform Cell-Independent MicroRNA Biogenesis and Promote Tumorigenesis

PubMed Central

Melo, Sonia A.; Sugimoto, Hikaru; O’Connell, Joyce T.; Kato, Noritoshi; Villanueva, Alberto; Vidal, August; Qiu, Le; Vitkin, Edward; Perelman, Lev T.; Melo, Carlos A.; Lucci, Anthony; Ivan, Cristina; Calin, George A.; Kalluri, Raghu

2014-01-01

SUMMARY Exosomes are secreted by all cell types and contain proteins and nucleic acids. Here, we report that breast cancer associated exosomes contain microRNAs (miRNAs) associated with the RISC Loading Complex (RLC) and display cell-independent capacity to process precursor microRNAs (pre-miRNAs) into mature miRNAs. Pre-miRNAs, along with Dicer, AGO2, and TRBP, are present in exosomes of cancer cells. CD43 mediates the accumulation of Dicer specifically in cancer exosomes. Cancer exosomes mediate an efficient and rapid silencing of mRNAs to reprogram the target cell transcriptome. Exosomes derived from cells and sera of patients with breast cancer instigate non-tumorigenic epithelial cells to form tumors in a Dicer-dependent manner. These findings offer opportunities for the development of exosomes based biomarkers and therapies. PMID:25446899

Epigenetics in myeloid derived suppressor cells: a sheathed sword towards cancer

PubMed Central

Zhang, Chao; Wang, Shuo; Liu, Yufeng; Yang, Cheng

2016-01-01

Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of cells composed of progenitors and precursors to myeloid cells, are deemed to participate in the development of tumor-favoring immunosuppressive microenvironment. Thus, the regulatory strategies targeting MDSCs' expansion, differentiation, accumulation and function could possibly be effective “weapons” in anti-tumor immunotherapies. Epigenetic mechanisms, which involve DNA modification, covalent histone modification and RNA interference, result in the heritable down-regulation or silencing of gene expression without a change in DNA sequences. Epigenetic modification of MDSC's functional plasticity leads to the remodeling of its characteristics, therefore reframing the microenvironment towards countering tumor growth and metastasis. This review summarized the pertinent findings on the DNA methylation, covalent histone modification, microRNAs and small interfering RNAs targeting MDSC in cancer genesis, progression and metastasis. The potentials as well as possible obstacles in translating into anti-cancer therapeutics were also discussed. PMID:27458169

GAMUT: GPU accelerated microRNA analysis to uncover target genes through CUDA-miRanda

PubMed Central

2014-01-01

Background Non-coding sequences such as microRNAs have important roles in disease processes. Computational microRNA target identification (CMTI) is becoming increasingly important since traditional experimental methods for target identification pose many difficulties. These methods are time-consuming, costly, and often need guidance from computational methods to narrow down candidate genes anyway. However, most CMTI methods are computationally demanding, since they need to handle not only several million query microRNA and reference RNA pairs, but also several million nucleotide comparisons within each given pair. Thus, the need to perform microRNA identification at such large scale has increased the demand for parallel computing. Methods Although most CMTI programs (e.g., the miRanda algorithm) are based on a modified Smith-Waterman (SW) algorithm, the existing parallel SW implementations (e.g., CUDASW++ 2.0/3.0, SWIPE) are unable to meet this demand in CMTI tasks. We present CUDA-miRanda, a fast microRNA target identification algorithm that takes advantage of massively parallel computing on Graphics Processing Units (GPU) using NVIDIA's Compute Unified Device Architecture (CUDA). CUDA-miRanda specifically focuses on the local alignment of short (i.e., ≤ 32 nucleotides) sequences against longer reference sequences (e.g., 20K nucleotides). Moreover, the proposed algorithm is able to report multiple alignments (up to 191 top scores) and the corresponding traceback sequences for any given (query sequence, reference sequence) pair. Results Speeds over 5.36 Giga Cell Updates Per Second (GCUPs) are achieved on a server with 4 NVIDIA Tesla M2090 GPUs. Compared to the original miRanda algorithm, which is evaluated on an Intel Xeon E5620@2.4 GHz CPU, the experimental results show up to 166 times performance gains in terms of execution time. In addition, we have verified that the exact same targets were predicted in both CUDA-miRanda and the original miRanda implementations through multiple test datasets. Conclusions We offer a GPU-based alternative to high performance compute (HPC) that can be developed locally at a relatively small cost. The community of GPU developers in the biomedical research community, particularly for genome analysis, is still growing. With increasing shared resources, this community will be able to advance CMTI in a very significant manner. Our source code is available at https://sourceforge.net/projects/cudamiranda/. PMID:25077821

Detection of microRNAs in color space.

PubMed

Marco, Antonio; Griffiths-Jones, Sam

2012-02-01

Deep sequencing provides inexpensive opportunities to characterize the transcriptional diversity of known genomes. The AB SOLiD technology generates millions of short sequencing reads in color-space; that is, the raw data is a sequence of colors, where each color represents 2 nt and each nucleotide is represented by two consecutive colors. This strategy is purported to have several advantages, including increased ability to distinguish sequencing errors from polymorphisms. Several programs have been developed to map short reads to genomes in color space. However, a number of previously unexplored technical issues arise when using SOLiD technology to characterize microRNAs. Here we explore these technical difficulties. First, since the sequenced reads are longer than the biological sequences, every read is expected to contain linker fragments. The color-calling error rate increases toward the 3(') end of the read such that recognizing the linker sequence for removal becomes problematic. Second, mapping in color space may lead to the loss of the first nucleotide of each read. We propose a sequential trimming and mapping approach to map small RNAs. Using our strategy, we reanalyze three published insect small RNA deep sequencing datasets and characterize 22 new microRNAs. A bash shell script to perform the sequential trimming and mapping procedure, called SeqTrimMap, is available at: http://www.mirbase.org/tools/seqtrimmap/ antonio.marco@manchester.ac.uk Supplementary data are available at Bioinformatics online.

Bio-barcode gel assay for microRNA

NASA Astrophysics Data System (ADS)

Lee, Hyojin; Park, Jeong-Eun; Nam, Jwa-Min

2014-02-01

MicroRNA has been identified as a potential biomarker because expression level of microRNA is correlated with various cancers. Its detection at low concentrations would be highly beneficial for cancer diagnosis. Here, we develop a new type of a DNA-modified gold nanoparticle-based bio-barcode assay that uses a conventional gel electrophoresis platform and potassium cyanide chemistry and show this assay can detect microRNA at aM levels without enzymatic amplification. It is also shown that single-base-mismatched microRNA can be differentiated from perfectly matched microRNA and the multiplexed detection of various combinations of microRNA sequences is possible with this approach. Finally, differently expressed microRNA levels are selectively detected from cancer cells using the bio-barcode gel assay, and the results are compared with conventional polymerase chain reaction-based results. The method and results shown herein pave the way for practical use of a conventional gel electrophoresis for detecting biomolecules of interest even at aM level without polymerase chain reaction amplification.

Adaptive evolution and functional innovation of Populus-specific recently evolved microRNAs.

PubMed

Xie, Jianbo; Yang, Xiaohui; Song, Yuepeng; Du, Qingzhang; Li, Ying; Chen, Jinhui; Zhang, Deqiang

2017-01-01

Lineage-specific microRNAs (miRNAs) undergo rapid turnover during evolution; however, their origin and functional importance have remained controversial. Here, we examine the origin, evolution, and potential roles in local adaptation of Populus-specific miRNAs, which originated after the recent salicoid-specific, whole-genome duplication. RNA sequencing was used to generate extensive, comparable miRNA and gene expression data for six tissues. A natural population of Populus trichocarpa and closely related species were used to study the divergence rates, evolution, and adaptive variation of miRNAs. MiRNAs that originated in 5' untranslated regions had higher expression levels and their expression showed high correlation with their host genes. Compared with conserved miRNAs, a significantly higher proportion of Populus-specific miRNAs appear to target genes that were duplicated in salicoids. Examination of single nucleotide polymorphisms in Populus-specific miRNA precursors showed high amounts of population differentiation. We also characterized the newly emerged MIR6445 family, which could trigger the production of phased small interfering RNAs from NAC mRNAs, which encode a transcription factor with primary roles in a variety of plant developmental processes. Together, these observations provide evolutionary insights into the birth and potential roles of Populus-specific miRNAs in genome maintenance, local adaptation, and functional innovation. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Identification and characterization of microRNAs in the screwworm flies Cochliomyia hominivorax and Cochliomyia macellaria (Diptera: Calliphoridae).

PubMed

Paulo, D F; Azeredo-Espin, A M L; Canesin, L E C; Vicentini, R; Junqueira, A C M

2017-02-01

MicroRNAs (miRNAs) are small noncoding RNAs that modulate gene expression through post-transcriptional regulation. Here, we report the identification and characterization of miRNAs in two closely related screwworm flies with different feeding habits: Cochliomyia hominivorax and Cochliomyia macellaria. The New World screwworm, C. hominivorax, is an obligatory parasite of warm-blooded vertebrates, whereas the secondary screwworm, C. macellaria, is a free-living organism that feeds on decaying organic matter. Here, the small RNA transcriptomes of adults and third-instar larvae of both species were sequenced. A total of 110 evolutionarily conserved miRNAs were identified, and 10 putative precursor miRNAs (pre-miRNAs) were predicted. The relative expression of six selected miRNAs was further investigated, including miRNAs that are related to reproduction and neural processes in other insects. Mature miRNAs were also characterized across an evolutionary time scale, suggesting that the majority of them have been conserved since the emergence of the Arthropoda [540 million years ago (Ma)], Hexapoda (488 Ma) and Brachycera (195 Ma) lineages. This study is the first report of miRNAs for screwworm flies. We also performed a comparative analysis with the hereby predicted miRNAs from the sheep blowfly, Lucilia cuprina. The results presented may advance our understanding of parasitic habits within Calliphoridae and assist further functional studies in blowflies. © 2016 The Royal Entomological Society.

Specific and Novel microRNAs Are Regulated as Response to Fungal Infection in Human Dendritic Cells.

PubMed

Dix, Andreas; Czakai, Kristin; Leonhardt, Ines; Schäferhoff, Karin; Bonin, Michael; Guthke, Reinhard; Einsele, Hermann; Kurzai, Oliver; Löffler, Jürgen; Linde, Jörg

2017-01-01

Within the last two decades, the incidence of invasive fungal infections has been significantly increased. They are characterized by high mortality rates and are often caused by Candida albicans and Aspergillus fumigatus . The increasing number of infections underlines the necessity for additional anti-fungal therapies, which require extended knowledge of gene regulations during fungal infection. MicroRNAs are regulators of important cellular processes, including the immune response. By analyzing their regulation and impact on target genes, novel therapeutic and diagnostic approaches may be developed. Here, we examine the role of microRNAs in human dendritic cells during fungal infection. Dendritic cells represent the bridge between the innate and the adaptive immune systems. Therefore, analysis of gene regulation of dendritic cells is of particular significance. By applying next-generation sequencing of small RNAs, we quantify microRNA expression in monocyte-derived dendritic cells after 6 and 12 h of infection with C. albicans and A. fumigatus as well as treatment with lipopolysaccharides (LPS). We identified 26 microRNAs that are differentially regulated after infection by the fungi or LPS. Three and five of them are specific for fungal infections after 6 and 12 h, respectively. We further validated interactions of miR-132-5p and miR-212-5p with immunological relevant target genes, such as FKBP1B, KLF4 , and SPN , on both RNA and protein level. Our results indicate that these microRNAs fine-tune the expression of immune-related target genes during fungal infection. Beyond that, we identified previously undiscovered microRNAs. We validated three novel microRNAs via qRT-PCR. A comparison with known microRNAs revealed possible relations with the miR-378 family and miR-1260a/b for two of them, while the third one features a unique sequence with no resemblance to known microRNAs. In summary, this study analyzes the effect of known microRNAs in dendritic cells during fungal infections and proposes novel microRNAs that could be experimentally verified.

A bioinformatics-based update on microRNAs and their targets in rainbow trout (Oncorhynchus mykiss).

PubMed

Yang, Liandong; He, Shunping

2014-01-01

MicroRNAs (miRNAs) participate in various vitally biological processes via controlling target genes activity and thousands of miRNAs have been identified in many species to date, including 18,698 known animal miRNA in miRBase. However, there are only limited studies reported in rainbow trout (Oncorhynchus mykiss) especially via the computational-based approaches. In present study, we systematically investigated the miRNAs in rainbow trout using a well-developed comparative genome-based homologue search. A total of 196 potential miRNAs, belonging to 124 miRNA families, were identified, most of which were firstly reported in rainbow trout. The length of miRNAs ranged from 17 to 24 nt with an average of 20 nt while the length of their precursors varied from 47 to 152 nt with an average of 85 nt. The identified miRNAs were not evenly distributed in each miRNA family, with only one member per family for a majority, and multiple members were also identified for several families. Nucleotide U was dominant in the pre-miRNAs with a percentage of 30.04%. The rainbow trout pre-miRNAs had relatively high negative minimal folding free energy (MFE) and adjusted MFE (AMFE). Not only the mature miRNAs but their precursor sequences are conserved among the living organisms. About 2466 O. mykiss genes were predicted as potential targets for 189 miRNAs. Gene Ontology (GO) analysis showed that nearly 2093, 2107, and 2081 target genes are involved in cellular component, molecular function, and biological processes respectively. KEGG pathway enrichment analysis illuminated that these miRNAs targets might regulate 105 metabolic pathways, including those of purine metabolism, nitrogen metabolism, and oxidative phosphorylation. This study has provided an update on rainbow trout miRNAs and their targets, which represents a foundation for future studies. © 2013.

microRNA expression profiling in fetal single ventricle malformation identified by deep sequencing.

PubMed

Yu, Zhang-Bin; Han, Shu-Ping; Bai, Yun-Fei; Zhu, Chun; Pan, Ya; Guo, Xi-Rong

2012-01-01

microRNAs (miRNAs) have emerged as key regulators in many biological processes, particularly cardiac growth and development, although the specific miRNA expression profile associated with this process remains to be elucidated. This study aimed to characterize the cellular microRNA profile involved in the development of congenital heart malformation, through the investigation of single ventricle (SV) defects. Comprehensive miRNA profiling in human fetal SV cardiac tissue was performed by deep sequencing. Differential expression of 48 miRNAs was revealed by sequencing by oligonucleotide ligation and detection (SOLiD) analysis. Of these, 38 were down-regulated and 10 were up-regulated in differentiated SV cardiac tissue, compared to control cardiac tissue. This was confirmed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Predicted target genes of the 48 differentially expressed miRNAs were analyzed by gene ontology and categorized according to cellular process, regulation of biological process and metabolic process. Pathway-Express analysis identified the WNT and mTOR signaling pathways as the most significant processes putatively affected by the differential expression of these miRNAs. The candidate genes involved in cardiac development were identified as potential targets for these differentially expressed microRNAs and the collaborative network of microRNAs and cardiac development related-mRNAs was constructed. These data provide the basis for future investigation of the mechanism of the occurrence and development of fetal SV malformations.

Small indels induced by CRISPR/Cas9 in the 5' region of microRNA lead to its depletion and Drosha processing retardance.

PubMed

Jiang, Qian; Meng, Xing; Meng, Lingwei; Chang, Nannan; Xiong, Jingwei; Cao, Huiqing; Liang, Zicai

2014-01-01

MicroRNA knockout by genome editing technologies is promising. In order to extend the application of the technology and to investigate the function of a specific miRNA, we used CRISPR/Cas9 to deplete human miR-93 from a cluster by targeting its 5' region in HeLa cells. Various small indels were induced in the targeted region containing the Drosha processing site and seed sequences. Interestingly, we found that even a single nucleotide deletion led to complete knockout of the target miRNA with high specificity. Functional knockout was confirmed by phenotype analysis. Furthermore, de novo microRNAs were not found by RNA-seq. Nevertheless, expression of the pri-microRNAs was increased. When combined with structural analysis, the data indicated that biogenesis was impaired. Altogether, we showed that small indels in the 5' region of a microRNA result in sequence depletion as well as Drosha processing retard.

Point-of-care diagnostic tools to detect circulating microRNAS as biomarkers of disease.

PubMed

Vaca, Luis

2014-05-22

MicroRNAs or miRNAs are a form of small non-coding RNAs (ncRNAs) of 19-22 nucleotides in length in their mature form. miRNAs are transcribed in the nucleus of all cells from large precursors, many of which have several kilobases in length. Originally identified as intracellular modulators of protein synthesis via posttranscriptional gene silencing, more recently it has been found that miRNAs can travel in extracellular human fluids inside specialized vesicles known as exosomes. We will be referring to this miRNAs as circulating microRNAs. More interestingly, the miRNA content inside exosomes changes during pathological events. In the present review we analyze the literature about circulating miRNAs and their possible use as biomarkers. Furthermore, we explore their future in point-of-care (POC) diagnostics and provide an example of a portable POC apparatus useful in the detection of circulating miRNAs.

Identification, characterization and expression analysis of pigeonpea miRNAs in response to Fusarium wilt.

PubMed

Hussain, Khalid; Mungikar, Kanak; Kulkarni, Abhijeet; Kamble, Avinash

2018-05-05

Upon confrontation with unfavourable conditions, plants invoke a very complex set of biochemical and physiological reactions and alter gene expression patterns to combat the situations. MicroRNAs (miRNAs), a class of small non-coding RNA, contribute extensively in regulation of gene expression through translation inhibition or degradation of their target mRNAs during such conditions. Therefore, identification of miRNAs and their targets holds importance in understanding the regulatory networks triggered during stress. Structure and sequence similarity based in silico prediction of miRNAs in Cajanus cajan L. (Pigeonpea) draft genome sequence has been carried out earlier. These annotations also appear in related GenBank genome sequence entries. However, there are no reports available on context dependent miRNA expression and their targets in pigeonpea. Therefore, in the present study we addressed these questions computationally, using pigeonpea EST sequence information. We identified five novel pigeonpea miRNA precursors, their mature forms and targets. Interestingly, only one of these miRNAs (miR169i-3p) was identified earlier in draft genome sequence. We then validated expression of these miRNAs, experimentally. It was also observed that these miRNAs show differential expression patterns in response to Fusarium inoculation indicating their biotic stress responsive nature. Overall these results will help towards better understanding the regulatory network of defense during pigeonpea -pathogen interactions and role of miRNAs in the process. Copyright © 2018 Elsevier B.V. All rights reserved.

miRNA Enriched in Human Neuroblast Nuclei Bind the MAZ Transcription Factor and Their Precursors Contain the MAZ Consensus Motif.

PubMed

Goldie, Belinda J; Fitzsimmons, Chantel; Weidenhofer, Judith; Atkins, Joshua R; Wang, Dan O; Cairns, Murray J

2017-01-01

While the cytoplasmic function of microRNA (miRNA) as post-transcriptional regulators of mRNA has been the subject of significant research effort, their activity in the nucleus is less well characterized. Here we use a human neuronal cell model to show that some mature miRNA are preferentially enriched in the nucleus. These molecules were predominantly primate-specific and contained a sequence motif with homology to the consensus MAZ transcription factor binding element. Precursor miRNA containing this motif were shown to have affinity for MAZ protein in nuclear extract. We then used Ago1/2 RIP-Seq to explore nuclear miRNA-associated mRNA targets. Interestingly, the genes for Ago2-associated transcripts were also significantly enriched with MAZ binding sites and neural function, whereas Ago1-transcripts were associated with general metabolic processes and localized with SC35 spliceosomes. These findings suggest the MAZ transcription factor is associated with miRNA in the nucleus and may influence the regulation of neuronal development through Ago2-associated miRNA induced silencing complexes. The MAZ transcription factor may therefore be important for organizing higher order integration of transcriptional and post-transcriptional processes in primate neurons.

Plasma exosome microRNAs are indicative of breast cancer.

PubMed

Hannafon, Bethany N; Trigoso, Yvonne D; Calloway, Cameron L; Zhao, Y Daniel; Lum, David H; Welm, Alana L; Zhao, Zhizhuang J; Blick, Kenneth E; Dooley, William C; Ding, W Q

2016-09-08

microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring. Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis. Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of breast cancer than their individual levels. Our results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of patients with breast cancer. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer.

Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC

PubMed Central

Castanotto, Daniela; Sakurai, Kumi; Lingeman, Robert; Li, Haitang; Shively, Louise; Aagaard, Lars; Soifer, Harris; Gatignol, Anne; Riggs, Arthur; Rossi, John J.

2007-01-01

Despite the great potential of RNAi, ectopic expression of shRNA or siRNAs holds the inherent risk of competition for critical RNAi components, thus altering the regulatory functions of some cellular microRNAs. In addition, specific siRNA sequences can potentially hinder incorporation of other siRNAs when used in a combinatorial approach. We show that both synthetic siRNAs and expressed shRNAs compete against each other and with the endogenous microRNAs for transport and for incorporation into the RNA induced silencing complex (RISC). The same siRNA sequences do not display competition when expressed from a microRNA backbone. We also show that TAR RNA binding protein (TRBP) is one of the sensors for selection and incorporation of the guide sequence of interfering RNAs. These findings reveal that combinatorial siRNA approaches can be problematic and have important implications for the methodology of expression and use of therapeutic interfering RNAs. PMID:17660190

Identification of miRNAs during mouse postnatal ovarian development and superovulation.

PubMed

Khan, Hamid Ali; Zhao, Yi; Wang, Li; Li, Qian; Du, Yu-Ai; Dan, Yi; Huo, Li-Jun

2015-07-08

MicroRNAs are small noncoding RNAs that play critical roles in regulation of gene expression in wide array of tissues including the ovary through sequence complementarity at post-transcriptional level. Tight regulation of multitude of genes involved in ovarian development and folliculogenesis could be regulated at transcription level by these miRNAs. Therefore, tissue specific miRNAs identification is considered a key step towards understanding the role of miRNAs in biological processes. To investigate the role of microRNAs during ovarian development and folliculogenesis we sequenced eight different libraries using Illumina deep sequencing technology. Different developmental stages were selected to explore miRNAs expression pattern at different stages of gonadal maturation with/without treatment of PMSG/hCG for superovulation. From massive sequencing reads, clean reads of 16-26 bp were selected for further analysis of differential expression analysis and novel microRNA annotation. Expression analysis of all miRNAs at different developmental stages showed that some miRNAs were present ubiquitously while others were differentially expressed at different stages. Among differentially expressed miRNAs we reported 61 miRNAs with a fold change of more than 2 at different developmental stages among all libraries. Among the up-regulated miRNAs, mmu-mir-1298 had the highest fold change with 4.025 while mmu-mir-150 was down-regulated more than 3 fold. Furthermore, we found 2659 target genes for 20 differentially expressed microRNAs using seven different target predictions programs (DIANA-mT, miRanda, miRDB, miRWalk, RNAhybrid, PICTAR5, TargetScan). Analysis of the predicted targets showed certain ovary specific genes targeted by single or multiple microRNAs. Furthermore, pathway annotation and Gene ontology showed involvement of these microRNAs in basic cellular process. These results suggest the presence of different miRNAs at different stages of ovarian development and superovulation. Potential role of these microRNAs was elucidated using bioinformatics tools in regulation of different pathways, biological functions and cellular components underlying ovarian development and superovulation. These results provide a framework for extended analysis of miRNAs and their roles during ovarian development and superovulation. Furthermore, this study provides a base for characterization of individual miRNAs to discover their role in ovarian development and female fertility.

MicroRNA Detection Using a Double Molecular Beacon Approach: Distinguishing Between miRNA and Pre-miRNA.

PubMed

James, Amanda Marie; Baker, Meredith B; Bao, Gang; Searles, Charles D

2017-01-01

MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression and are recognized for their roles both as modulators of disease progression and as biomarkers of disease activity, including neurological diseases, cancer, and cardiovascular disease (CVD). Commonly, miRNA abundance is assessed using quantitative real-time PCR (qRT-PCR), however, qRT-PCR for miRNA can be labor intensive, time consuming, and may lack specificity for detection of mature versus precursor forms of miRNA. Here, we describe a novel double molecular beacon approach to miRNA assessment that can distinguish and quantify mature versus precursor forms of miRNA in a single assay, an essential feature for use of miRNAs as biomarkers for disease. Using this approach, we found that molecular beacons with DNA or combined locked nucleic acid (LNA)-DNA backbones can detect mature and precursor miRNAs (pre-miRNAs) of low (< 1 nM) abundance in vitro . The double molecular beacon assay was accurate in assessing miRNA abundance in a sample containing a mixed population of mature and precursor miRNAs. In contrast, qRT-PCR and the single molecular beacon assay overestimated miRNA abundance. Additionally, the double molecular beacon assay was less labor intensive than traditional qRT-PCR and had 10-25% increased specificity. Our data suggest that the double molecular beacon-based approach is more precise and specific than previous methods, and has the promise of being the standard for assessing miRNA levels in biological samples.

MicroRNA Detection Using a Double Molecular Beacon Approach: Distinguishing Between miRNA and Pre-miRNA

PubMed Central

James, Amanda Marie; Baker, Meredith B.; Bao, Gang; Searles, Charles D.

2017-01-01

MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression and are recognized for their roles both as modulators of disease progression and as biomarkers of disease activity, including neurological diseases, cancer, and cardiovascular disease (CVD). Commonly, miRNA abundance is assessed using quantitative real-time PCR (qRT-PCR), however, qRT-PCR for miRNA can be labor intensive, time consuming, and may lack specificity for detection of mature versus precursor forms of miRNA. Here, we describe a novel double molecular beacon approach to miRNA assessment that can distinguish and quantify mature versus precursor forms of miRNA in a single assay, an essential feature for use of miRNAs as biomarkers for disease. Using this approach, we found that molecular beacons with DNA or combined locked nucleic acid (LNA)-DNA backbones can detect mature and precursor miRNAs (pre-miRNAs) of low (< 1 nM) abundance in vitro. The double molecular beacon assay was accurate in assessing miRNA abundance in a sample containing a mixed population of mature and precursor miRNAs. In contrast, qRT-PCR and the single molecular beacon assay overestimated miRNA abundance. Additionally, the double molecular beacon assay was less labor intensive than traditional qRT-PCR and had 10-25% increased specificity. Our data suggest that the double molecular beacon-based approach is more precise and specific than previous methods, and has the promise of being the standard for assessing miRNA levels in biological samples. PMID:28255356

Immunomodulation: A definitive role of microRNA-142.

PubMed

Sharma, Salil

2017-12-01

Majority of microRNAs are evolutionarily conserved in vertebrates. This is suggestive of their similar roles in regulation of gene networks. In addition to their conserved mature sequences and regulatory roles, a few microRNAs show very cell or tissue specific expression. These microRNAs are highly enriched in some cell types or organs. One such microRNA is microRNA-142 (miR-142). The classical stem-loop structure of miR142 encodes for two species of mature microRNAs; miR142-5p and miR142-3p. MiR-142 is abundant in cells of hematopoietic origin, and therefore, aptly plays a role in lineage differentiation of hematopoietic cells. Interestingly, over the years, miR-142 has gained considerable attention for its quintessential role in regulating immune response. This mini-review discusses the important functional roles of miR-142 in inflammatory and immune response in different physiological and disease setting. Copyright © 2017 Elsevier Ltd. All rights reserved.

A comparative analysis of high-throughput platforms for validation of a circulating microRNA signature in diabetic retinopathy.

PubMed

Farr, Ryan J; Januszewski, Andrzej S; Joglekar, Mugdha V; Liang, Helena; McAulley, Annie K; Hewitt, Alex W; Thomas, Helen E; Loudovaris, Tom; Kay, Thomas W H; Jenkins, Alicia; Hardikar, Anandwardhan A

2015-06-02

MicroRNAs are now increasingly recognized as biomarkers of disease progression. Several quantitative real-time PCR (qPCR) platforms have been developed to determine the relative levels of microRNAs in biological fluids. We systematically compared the detection of cellular and circulating microRNA using a standard 96-well platform, a high-content microfluidics platform and two ultra-high content platforms. We used extensive analytical tools to compute inter- and intra-run variability and concordance measured using fidelity scoring, coefficient of variation and cluster analysis. We carried out unprejudiced next generation sequencing to identify a microRNA signature for Diabetic Retinopathy (DR) and systematically assessed the validation of this signature on clinical samples using each of the above four qPCR platforms. The results indicate that sensitivity to measure low copy number microRNAs is inversely related to qPCR reaction volume and that the choice of platform for microRNA biomarker validation should be made based on the abundance of miRNAs of interest.

Rapid and sensitive microRNA detection with laminar flow-assisted dendritic amplification on power-free microfluidic chip.

PubMed

Arata, Hideyuki; Komatsu, Hiroshi; Hosokawa, Kazuo; Maeda, Mizuo

2012-01-01

Detection of microRNAs, small noncoding single-stranded RNAs, is one of the key topics in the new generation of cancer research because cancer in the human body can be detected or even classified by microRNA detection. This report shows rapid and sensitive microRNA detection using a power-free microfluidic device, which is driven by degassed poly(dimethylsiloxane), thus eliminating the need for an external power supply. MicroRNA is detected by sandwich hybridization, and the signal is amplified by laminar flow-assisted dendritic amplification. This method allows us to detect microRNA of specific sequences at a limit of detection of 0.5 pM from a 0.5 µL sample solution with a detection time of 20 min. Together with the advantages of self-reliance of this device, this method might contribute substantially to future point-of-care early-stage cancer diagnosis.

A Comprehensive Approach to Sequence-oriented IsomiR annotation (CASMIR): demonstration with IsomiR profiling in colorectal neoplasia.

PubMed

Wu, Chung Wah; Evans, Jared M; Huang, Shengbing; Mahoney, Douglas W; Dukek, Brian A; Taylor, William R; Yab, Tracy C; Smyrk, Thomas C; Jen, Jin; Kisiel, John B; Ahlquist, David A

2018-05-25

MicroRNA (miRNA) profiling is an important step in studying biological associations and identifying marker candidates. miRNA exists in isoforms, called isomiRs, which may exhibit distinct properties. With conventional profiling methods, limitations in assay and analysis platforms may compromise isomiR interrogation. We introduce a comprehensive approach to sequence-oriented isomiR annotation (CASMIR) to allow unbiased identification of global isomiRs from small RNA sequencing data. In this approach, small RNA reads are maintained as independent sequences instead of being summarized under miRNA names. IsomiR features are identified through step-wise local alignment against canonical forms and precursor sequences. Through customizing the reference database, CASMIR is applicable to isomiR annotation across species. To demonstrate its application, we investigated isomiR profiles in normal and neoplastic human colorectal epithelia. We also ran miRDeep2, a popular miRNA analysis algorithm to validate isomiRs annotated by CASMIR. With CASMIR, specific and biologically relevant isomiR patterns could be identified. We note that specific isomiRs are often more abundant than their canonical forms. We identify isomiRs that are commonly up-regulated in both colorectal cancer and advanced adenoma, and illustrate advantages in targeting isomiRs as potential biomarkers over canonical forms. Studying miRNAs at the isomiR level could reveal new insight into miRNA biology and inform assay design for specific isomiRs. CASMIR facilitates comprehensive annotation of isomiR features in small RNA sequencing data for isomiR profiling and differential expression analysis.

Despacito: the slow evolutionary changes in plant microRNAs.

PubMed

Baldrich, Patricia; Beric, Aleksandra; Meyers, Blake C

2018-02-12

MicroRNAs (miRNAs) are key regulators of gene expression. A handful of miRNAs are broadly conserved in land plants, while the majority are lineage specific; this review describes the processes by which new miRNAs are hypothesized to have emerged. Two major models describe miRNA origins, firstly, de novo emergence via inverted duplication of target gene fragments, and secondly, the expansion and neofunctionalization of existing miRNA families. The occasional acquisition of target sites by previously un-targeted genes adds further dynamism to the process by which miRNAs may shift roles during evolution. Additional factors guiding miRNA evolution include functional constraints on their length and the importance of precursor conservation that is observed in regions above or below the mature miRNA duplex; these regions represent recognition sites for components of biogenesis machinery and direct precursor processing. Insights into the mechanisms of miRNA emergence and divergence are important for understanding plant genome evolution and the impact of miRNA regulatory networks. Copyright © 2018 Elsevier Ltd. All rights reserved.

Sjögren Syndrome Antigen B (SSB)/La Promotes Global MicroRNA Expression by Binding MicroRNA Precursors through Stem-Loop Recognition*

PubMed Central

Liang, Chunyang; Xiong, Ke; Szulwach, Keith E.; Zhang, Yi; Wang, Zhaohui; Peng, Junmin; Fu, Mingui; Jin, Peng; Suzuki, Hiroshi I.; Liu, Qinghua

2013-01-01

MicroRNAs (miRNA) control numerous physiological and pathological processes. Typically, the primary miRNA (pri-miRNA) transcripts are processed by nuclear Drosha complex into ∼70-nucleotide stem-loop precursor miRNAs (pre-miRNA), which are further cleaved by cytoplasmic Dicer complex into ∼21-nucleotide mature miRNAs. However, it is unclear how nascent pre-miRNAs are protected from ribonucleases, such as MCPIP1, that degrade pre-miRNAs to abort miRNA production. Here, we identify Sjögren syndrome antigen B (SSB)/La as a pre-miRNA-binding protein that regulates miRNA processing in vitro. All three RNA-binding motifs (LAM, RRM1, and RRM2) of La/SSB are required for efficient pre-miRNA binding. Intriguingly, La/SSB recognizes the characteristic stem-loop structure of pre-miRNAs, of which the majority lack a 3′ UUU terminus. Moreover, La/SSB associates with endogenous pri-/pre-miRNAs and promotes miRNA biogenesis by stabilizing pre-miRNAs from nuclease (e.g. MCPIP1)-mediated decay in mammalian cells. Accordingly, we observed positive correlations between the expression status of La/SSB and Dicer in human cancer transcriptome and prognosis. These studies identify an important function of La/SSB as a global regulator of miRNA expression, and implicate stem-loop recognition as a major mechanism that mediates association between La/SSB and diverse RNA molecules. PMID:23129761

Sjogren syndrome antigen B (SSB)/La promotes global microRNA expression by binding microRNA precursors through stem-loop recognition.

PubMed

Liang, Chunyang; Xiong, Ke; Szulwach, Keith E; Zhang, Yi; Wang, Zhaohui; Peng, Junmin; Fu, Mingui; Jin, Peng; Suzuki, Hiroshi I; Liu, Qinghua

2013-01-04

MicroRNAs (miRNA) control numerous physiological and pathological processes. Typically, the primary miRNA (pri-miRNA) transcripts are processed by nuclear Drosha complex into ~70-nucleotide stem-loop precursor miRNAs (pre-miRNA), which are further cleaved by cytoplasmic Dicer complex into ~21-nucleotide mature miRNAs. However, it is unclear how nascent pre-miRNAs are protected from ribonucleases, such as MCPIP1, that degrade pre-miRNAs to abort miRNA production. Here, we identify Sjögren syndrome antigen B (SSB)/La as a pre-miRNA-binding protein that regulates miRNA processing in vitro. All three RNA-binding motifs (LAM, RRM1, and RRM2) of La/SSB are required for efficient pre-miRNA binding. Intriguingly, La/SSB recognizes the characteristic stem-loop structure of pre-miRNAs, of which the majority lack a 3' UUU terminus. Moreover, La/SSB associates with endogenous pri-/pre-miRNAs and promotes miRNA biogenesis by stabilizing pre-miRNAs from nuclease (e.g. MCPIP1)-mediated decay in mammalian cells. Accordingly, we observed positive correlations between the expression status of La/SSB and Dicer in human cancer transcriptome and prognosis. These studies identify an important function of La/SSB as a global regulator of miRNA expression, and implicate stem-loop recognition as a major mechanism that mediates association between La/SSB and diverse RNA molecules.

Analysis of secondary structural elements in human microRNA hairpin precursors.

PubMed

Liu, Biao; Childs-Disney, Jessica L; Znosko, Brent M; Wang, Dan; Fallahi, Mohammad; Gallo, Steven M; Disney, Matthew D

2016-03-01

MicroRNAs (miRNAs) regulate gene expression by targeting complementary mRNAs for destruction or translational repression. Aberrant expression of miRNAs has been associated with various diseases including cancer, thus making them interesting therapeutic targets. The composite of secondary structural elements that comprise miRNAs could aid the design of small molecules that modulate their function. We analyzed the secondary structural elements, or motifs, present in all human miRNA hairpin precursors and compared them to highly expressed human RNAs with known structures and other RNAs from various organisms. Amongst human miRNAs, there are 3808 are unique motifs, many residing in processing sites. Further, we identified motifs in miRNAs that are not present in other highly expressed human RNAs, desirable targets for small molecules. MiRNA motifs were incorporated into a searchable database that is freely available. We also analyzed the most frequently occurring bulges and internal loops for each RNA class and found that the smallest loops possible prevail. However, the distribution of loops and the preferred closing base pairs were unique to each class. Collectively, we have completed a broad survey of motifs found in human miRNA precursors, highly expressed human RNAs, and RNAs from other organisms. Interestingly, unique motifs were identified in human miRNA processing sites, binding to which could inhibit miRNA maturation and hence function.

MicroRNA-542-5p as a Novel Tumor Suppressor in Neuroblastoma

PubMed Central

Bray, Isabella; Tivnan, Amanda; Bryan, Kenneth; Foley, Niamh H; Watters, Karen M; Tracey, Lorraine; Davidoff, Andrew M; Stallings, Raymond L

2011-01-01

Several studies have implicated the dysregulation of microRNAs in neuroblastoma pathogenesis, an often fatal paediatric cancer arising from precursor cells of the sympathetic nervous system. Our group and others have demonstrated that lower expression of miR-542-5p is highly associated with poor patient survival, indicating a potential tumor suppressive function. Here, we demonstrate that ectopic over-expression of this miRNA decreases the invasive potential of neuroblastoma cell lines in vitro, along with primary tumor growth and metastases in an orthotopic mouse xenograft model, providing the first functional evidence for the involvement of miR-542-5p as a tumor suppressor in any type of cancer. PMID:21310526

Development of a graphical user interface for sgRNAcas9 and its application.

PubMed

Zhao, Chang-zhi; Zhang, Yi; Li, Guang-lei; Chen, Ji-liang; Li, Jing-Jin; Ren, Rui-min; Ni, Pan; Zhao, Shu-hong; Xie, Sheng-song

2015-10-01

The CRISPR/Cas9 genome editing technique is a powerful tool for researchers. However, off-target effects of the Cas9 nuclease activity is a recurrent concern of the CRISPR system. Thus, designing sgRNA (single guide RNA) with minimal off-target effects is very important. sgRNAcas9 is a software package, which can be used to design sgRNA and to evaluate potential off-target cleavage sites. In this study, a graphical user interface for sgRNAcas9 was developed using the Java programming language. In addition, off-target effect for sgRNAs was evaluated according to mismatched number and "seed sequence" specification. Moreover, sgRNAcas9 software was used to design 34 124 sgRNAs, which can target 4691 microRNA (miRNA) precursors from human, mouse, rat, pig, and chicken. In particular, the off-target effect of a sgRNA targeting to human miR-206 precursor was analyzed, and the on/off-target activity of this sgRNA was validated by T7E1 assay in vitro. Taken together, these data showed that the interface can simplify the usage of the sgRNAcas9 program, which can be used to design sgRNAs for the majority of miRNA precursors. We also found that the GC% of those sgRNAs ranged from 40% to 60%. In summary, the sgRNAcas9 software can be easily used to design sgRNA with minimal off-target effects for any species. The software can be downloaded from BiooTools website (http://www.biootools.com/).

Detection of human microRNAs across miRNA Array and Next Generation DNA Sequencing Platforms

EPA Science Inventory

microRNA (miRNAs) are non-coding RNA molecules between 19 and 30 nucleotides in length that are believed to regulate approximately 30 per cent of all human genes. They act as negative regulators of their gene targets in many biological processes. Recent developments in microar...

Genome-wide identification of microRNAs in pomegranate (Punica granatum L.) by high-throughput sequencing

USDA-ARS?s Scientific Manuscript database

Background: MicroRNAs (miRNAs), a class of small non-coding endogenous RNAs that regulate gene expression post-transcriptionally, play multiple key roles in plant growth and development and in biotic and abiotic stress response. Knowledge and roles of miRNAs in pomegranate fruit development have not...

TargetLink, a new method for identifying the endogenous target set of a specific microRNA in intact living cells.

PubMed

Xu, Yan; Chen, Yan; Li, Daliang; Liu, Qing; Xuan, Zhenyu; Li, Wen-Hong

2017-02-01

MicroRNAs are small non-coding RNAs acting as posttranscriptional repressors of gene expression. Identifying mRNA targets of a given miRNA remains an outstanding challenge in the field. We have developed a new experimental approach, TargetLink, that applied locked nucleic acid (LNA) as the affinity probe to enrich target genes of a specific microRNA in intact cells. TargetLink also consists a rigorous and systematic data analysis pipeline to identify target genes by comparing LNA-enriched sequences between experimental and control samples. Using miR-21 as a test microRNA, we identified 12 target genes of miR-21 in a human colorectal cancer cell by this approach. The majority of the identified targets interacted with miR-21 via imperfect seed pairing. Target validation confirmed that miR-21 repressed the expression of the identified targets. The cellular abundance of the identified miR-21 target transcripts varied over a wide range, with some targets expressed at a rather low level, confirming that both abundant and rare transcripts are susceptible to regulation by microRNAs, and that TargetLink is an efficient approach for identifying the target set of a specific microRNA in intact cells. C20orf111, one of the novel targets identified by TargetLink, was found to reside in the nuclear speckle and to be reliably repressed by miR-21 through the interaction at its coding sequence.

Toward the human cellular microRNAome.

PubMed

McCall, Matthew N; Kim, Min-Sik; Adil, Mohammed; Patil, Arun H; Lu, Yin; Mitchell, Christopher J; Leal-Rojas, Pamela; Xu, Jinchong; Kumar, Manoj; Dawson, Valina L; Dawson, Ted M; Baras, Alexander S; Rosenberg, Avi Z; Arking, Dan E; Burns, Kathleen H; Pandey, Akhilesh; Halushka, Marc K

2017-10-01

MicroRNAs are short RNAs that serve as regulators of gene expression and are essential components of normal development as well as modulators of disease. MicroRNAs generally act cell-autonomously, and thus their localization to specific cell types is needed to guide our understanding of microRNA activity. Current tissue-level data have caused considerable confusion, and comprehensive cell-level data do not yet exist. Here, we establish the landscape of human cell-specific microRNA expression. This project evaluated 8 billion small RNA-seq reads from 46 primary cell types, 42 cancer or immortalized cell lines, and 26 tissues. It identified both specific and ubiquitous patterns of expression that strongly correlate with adjacent superenhancer activity. Analysis of unaligned RNA reads uncovered 207 unknown minor strand (passenger) microRNAs of known microRNA loci and 495 novel putative microRNA loci. Although cancer cell lines generally recapitulated the expression patterns of matched primary cells, their isomiR sequence families exhibited increased disorder, suggesting DROSHA- and DICER1-dependent microRNA processing variability. Cell-specific patterns of microRNA expression were used to de-convolute variable cellular composition of colon and adipose tissue samples, highlighting one use of these cell-specific microRNA expression data. Characterization of cellular microRNA expression across a wide variety of cell types provides a new understanding of this critical regulatory RNA species. © 2017 McCall et al.; Published by Cold Spring Harbor Laboratory Press.

Differentially Expressed microRNAs and Target Genes Associated with Plastic Internode Elongation in Alternanthera philoxeroides in Contrasting Hydrological Habitats

PubMed Central

Li, Gengyun; Deng, Ying; Geng, Yupeng; Zhou, Chengchuan; Wang, Yuguo; Zhang, Wenju; Song, Zhiping; Gao, Lexuan; Yang, Ji

2017-01-01

Phenotypic plasticity is crucial for plants to survive in changing environments. Discovering microRNAs, identifying their targets and further inferring microRNA functions in mediating plastic developmental responses to environmental changes have been a critical strategy for understanding the underlying molecular mechanisms of phenotypic plasticity. In this study, the dynamic expression patterns of microRNAs under contrasting hydrological habitats in the amphibious species Alternanthera philoxeroides were identified by time course expression profiling using high-throughput sequencing technology. A total of 128 known and 18 novel microRNAs were found to be differentially expressed under contrasting hydrological habitats. The microRNA:mRNA pairs potentially associated with plastic internode elongation were identified by integrative analysis of microRNA and mRNA expression profiles, and were validated by qRT-PCR and 5′ RLM-RACE. The results showed that both the universal microRNAs conserved across different plants and the unique microRNAs novelly identified in A. philoxeroides were involved in the responses to varied water regimes. The results also showed that most of the differentially expressed microRNAs were transiently up-/down-regulated at certain time points during the treatments. The fine-scale temporal changes in microRNA expression highlighted the importance of time-series sampling in identifying stress-responsive microRNAs and analyzing their role in stress response/tolerance. PMID:29259617

Computational Prediction of miRNA Genes from Small RNA Sequencing Data

PubMed Central

Kang, Wenjing; Friedländer, Marc R.

2015-01-01

Next-generation sequencing now for the first time allows researchers to gage the depth and variation of entire transcriptomes. However, now as rare transcripts can be detected that are present in cells at single copies, more advanced computational tools are needed to accurately annotate and profile them. microRNAs (miRNAs) are 22 nucleotide small RNAs (sRNAs) that post-transcriptionally reduce the output of protein coding genes. They have established roles in numerous biological processes, including cancers and other diseases. During miRNA biogenesis, the sRNAs are sequentially cleaved from precursor molecules that have a characteristic hairpin RNA structure. The vast majority of new miRNA genes that are discovered are mined from small RNA sequencing (sRNA-seq), which can detect more than a billion RNAs in a single run. However, given that many of the detected RNAs are degradation products from all types of transcripts, the accurate identification of miRNAs remain a non-trivial computational problem. Here, we review the tools available to predict animal miRNAs from sRNA sequencing data. We present tools for generalist and specialist use cases, including prediction from massively pooled data or in species without reference genome. We also present wet-lab methods used to validate predicted miRNAs, and approaches to computationally benchmark prediction accuracy. For each tool, we reference validation experiments and benchmarking efforts. Last, we discuss the future of the field. PMID:25674563

Deep sequencing and proteomic analysis of the microRNA-induced silencing complex in human red blood cells.

PubMed

Azzouzi, Imane; Moest, Hansjoerg; Wollscheid, Bernd; Schmugge, Markus; Eekels, Julia J M; Speer, Oliver

2015-05-01

During maturation, erythropoietic cells extrude their nuclei but retain their ability to respond to oxidant stress by tightly regulating protein translation. Several studies have reported microRNA-mediated regulation of translation during terminal stages of erythropoiesis, even after enucleation. In the present study, we performed a detailed examination of the endogenous microRNA machinery in human red blood cells using a combination of deep sequencing analysis of microRNAs and proteomic analysis of the microRNA-induced silencing complex. Among the 197 different microRNAs detected, miR-451a was the most abundant, representing more than 60% of all read sequences. In addition, miR-451a and its known target, 14-3-3ζ mRNA, were bound to the microRNA-induced silencing complex, implying their direct interaction in red blood cells. The proteomic characterization of endogenous Argonaute 2-associated microRNA-induced silencing complex revealed 26 cofactor candidates. Among these cofactors, we identified several RNA-binding proteins, as well as motor proteins and vesicular trafficking proteins. Our results demonstrate that red blood cells contain complex microRNA machinery, which might enable immature red blood cells to control protein translation independent of de novo nuclei information. Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing.

PubMed

Chendrimada, Thimmaiah P; Gregory, Richard I; Kumaraswamy, Easwari; Norman, Jessica; Cooch, Neil; Nishikura, Kazuko; Shiekhattar, Ramin

2005-08-04

MicroRNAs (miRNAs) are generated by a two-step processing pathway to yield RNA molecules of approximately 22 nucleotides that negatively regulate target gene expression at the post-transcriptional level. Primary miRNAs are processed to precursor miRNAs (pre-miRNAs) by the Microprocessor complex. These pre-miRNAs are cleaved by the RNase III Dicer to generate mature miRNAs that direct the RNA-induced silencing complex (RISC) to messenger RNAs with complementary sequence. Here we show that TRBP (the human immunodeficiency virus transactivating response RNA-binding protein), which contains three double-stranded, RNA-binding domains, is an integral component of a Dicer-containing complex. Biochemical analysis of TRBP-containing complexes revealed the association of Dicer-TRBP with Argonaute 2 (Ago2), the catalytic engine of RISC. The physical association of Dicer-TRBP and Ago2 was confirmed after the isolation of the ternary complex using Flag-tagged Ago2 cell lines. In vitro reconstitution assays demonstrated that TRBP is required for the recruitment of Ago2 to the small interfering RNA (siRNA) bound by Dicer. Knockdown of TRBP results in destabilization of Dicer and a consequent loss of miRNA biogenesis. Finally, depletion of the Dicer-TRBP complex via exogenously introduced siRNAs diminished RISC-mediated reporter gene silencing. These results support a role of the Dicer-TRBP complex not only in miRNA processing but also as a platform for RISC assembly.

A deeper view into the significance of simple sequence repeats in pre-miRNAs provides clues for its possible roles in determining the function of microRNAs.

PubMed

Joy, Nisha; Maimoonath Beevi, Y P; Soniya, E V

2018-05-09

The central tenet of 'genome content' has been that the 'non-coding' parts are highly enriched with 'microsatellites' or 'Simple Sequence Repeats' (SSRs). We presume that the presence and change in number of repeat unit (n) of SSRs in different genomic locations may or may not become beneficial, depending on the position of SSRs in a gene. Very few studies have looked into the existence of SSRs in the hair-pin precursors of miRNAs (pre-miRNAs). The interplay between SSRs and miRNAs is not yet clearly understood. Considering the potential significance of SSRs in pre-miRNAs, we analysed the miRNA hair-pin precursors of 171 organisms, which revealed a noticeable (29.8%) existence of SSRs in their pre-miRNAs. The maintenance of SSRs in pre-miRNAs even in the complex, highly evolved phyla like Chordata and Magnoliophyta shed light upon its diverse functions. Putative effects of SSRs in either regulating the biogenesis or function of miRNAs were more underlined based on computational and experimental analysis. A preliminary computational analysis to explore the relevance of such SSRs maintained in pre-miRNA sequences led to the detection of splicing regulatory elements (SREs) either in or near to the SSRs. The absence of SSRs correspondingly decreased the detection of SREs. The present study is the first implication for the possible involvement of SSRs in shaping the SREs to undergo Alternative Splicing events to produce miRNA isoforms in accordance with different stress environments. This part of work well demonstrates the importance of studying such consistently maintained SSRs residing in pre-miRNAs and can enhance more and more research towards deciphering the exact function of SSRs in the near future.

IsomiR expression profiles in human lymphoblastoid cell lines exhibit population and gender dependencies

PubMed Central

Loher, Phillipe; Londin, Eric R.; Rigoutsos, Isidore

2014-01-01

For many years it was believed that each mature microRNA (miRNA) existed as a single entity with fixed endpoints and a ‘static’ and unchangeable primary sequence. However, recent evidence suggests that mature miRNAs are more ‘dynamic’ and that each miRNA precursor arm gives rise to multiple isoforms, the isomiRs. Here we report on our identification of numerous and abundant isomiRs in the lymphoblastoid cell lines (LCLs) of 452 men and women from five different population groups. Unexpectedly, we find that these isomiRs exhibit an expression profile that is population-dependent and gender-dependent. This is important as it indicates that the LCLs of each gender/population combination have their own unique collection of mature miRNA transcripts. Moreover, each identified isomiR has its own characteristic abundance that remains consistent across biological replicates indicating that these are not degradation products. The primary sequences of identified isomiRs differ from the known miRBase miRNA either at their 5´-endpoint (leads to a different ‘seed’ sequence and suggests a different targetome), their 3´-endpoint, or both simultaneously. Our analysis of Argonaute PAR-CLIP data from LCLs supports the association of many of these newly identified isomiRs with the Argonaute silencing complex and thus their functional roles through participation in the RNA interference pathway. PMID:25229428

IsomiR expression profiles in human lymphoblastoid cell lines exhibit population and gender dependencies.

PubMed

Loher, Phillipe; Londin, Eric R; Rigoutsos, Isidore

2014-09-30

For many years it was believed that each mature microRNA (miRNA) existed as a single entity with fixed endpoints and a 'static' and unchangeable primary sequence. However, recent evidence suggests that mature miRNAs are more 'dynamic' and that each miRNA precursor arm gives rise to multiple isoforms, the isomiRs. Here we report on our identification of numerous and abundant isomiRs in the lymphoblastoid cell lines (LCLs) of 452 men and women from five different population groups. Unexpectedly, we find that these isomiRs exhibit an expression profile that is population-dependent and gender-dependent. This is important as it indicates that the LCLs of each gender/population combination have their own unique collection of mature miRNA transcripts. Moreover, each identified isomiR has its own characteristic abundance that remains consistent across biological replicates indicating that these are not degradation products. The primary sequences of identified isomiRs differ from the known miRBase miRNA either at their 5´-endpoint (leads to a different 'seed' sequence and suggests a different targetome), their 3´-endpoint, or both simultaneously. Our analysis of Argonaute PAR-CLIP data from LCLs supports the association of many of these newly identified isomiRs with the Argonaute silencing complex and thus their functional roles through participation in the RNA interference pathway.

High-throughput and reliable protocols for animal microRNA library cloning.

PubMed

Xiao, Caide

2011-01-01

MicroRNAs are short single-stranded RNA molecules (18-25 nucleotides). Because of their ability to silence gene expressions, they can be used to diagnose and treat tumors. Experimental construction of microRNA libraries was the most important step to identify microRNAs from animal tissues. Although there are many commercial kits with special protocols to construct microRNA libraries, this chapter provides the most reliable, high-throughput, and affordable protocols for microRNA library construction. The high-throughput capability of our protocols came from a double concentration (3 and 15%, thickness 1.5 mm) polyacrylamide gel electrophoresis (PAGE), which could directly extract microRNA-size RNAs from up to 400 μg total RNA (enough for two microRNA libraries). The reliability of our protocols was assured by a third PAGE, which selected PCR products of microRNA-size RNAs ligated with 5' and 3' linkers by a miRCat™ kit. Also, a MathCAD program was provided to automatically search short RNAs inserted between 5' and 3' linkers from thousands of sequencing text files.

NAViGaTing the Micronome – Using Multiple MicroRNA Prediction Databases to Identify Signalling Pathway-Associated MicroRNAs

PubMed Central

Shirdel, Elize A.; Xie, Wing; Mak, Tak W.; Jurisica, Igor

2011-01-01

Background MicroRNAs are a class of small RNAs known to regulate gene expression at the transcript level, the protein level, or both. Since microRNA binding is sequence-based but possibly structure-specific, work in this area has resulted in multiple databases storing predicted microRNA:target relationships computed using diverse algorithms. We integrate prediction databases, compare predictions to in vitro data, and use cross-database predictions to model the microRNA:transcript interactome – referred to as the micronome – to study microRNA involvement in well-known signalling pathways as well as associations with disease. We make this data freely available with a flexible user interface as our microRNA Data Integration Portal — mirDIP (http://ophid.utoronto.ca/mirDIP). Results mirDIP integrates prediction databases to elucidate accurate microRNA:target relationships. Using NAViGaTOR to produce interaction networks implicating microRNAs in literature-based, KEGG-based and Reactome-based pathways, we find these signalling pathway networks have significantly more microRNA involvement compared to chance (p<0.05), suggesting microRNAs co-target many genes in a given pathway. Further examination of the micronome shows two distinct classes of microRNAs; universe microRNAs, which are involved in many signalling pathways; and intra-pathway microRNAs, which target multiple genes within one signalling pathway. We find universe microRNAs to have more targets (p<0.0001), to be more studied (p<0.0002), and to have higher degree in the KEGG cancer pathway (p<0.0001), compared to intra-pathway microRNAs. Conclusions Our pathway-based analysis of mirDIP data suggests microRNAs are involved in intra-pathway signalling. We identify two distinct classes of microRNAs, suggesting a hierarchical organization of microRNAs co-targeting genes both within and between pathways, and implying differential involvement of universe and intra-pathway microRNAs at the disease level. PMID:21364759

Polycistronic artificial miRNA-mediated resistance to Wheat dwarf virus in barley is highly efficient at low temperature.

PubMed

Kis, András; Tholt, Gergely; Ivanics, Milán; Várallyay, Éva; Jenes, Barnabás; Havelda, Zoltán

2016-04-01

Infection of Wheat dwarf virus (WDV) strains on barley results in dwarf disease, imposing severe economic losses on crop production. As the natural resistance resources against this virus are limited, it is imperative to elaborate a biotechnological approach that will provide effective and safe immunity to a wide range of WDV strains. Because vector insect-mediated WDV infection occurs during cool periods in nature, it is important to identify a technology which is effective at lower temperature. In this study, we designed artificial microRNAs (amiRNAs) using a barley miRNA precursor backbone, which target different conservative sequence elements of the WDV strains. Potential amiRNA sequences were selected to minimize the off-target effects and were tested in a transient sensor system in order to select the most effective constructs at low temperature. On the basis of the data obtained, a polycistronic amiRNA precursor construct (VirusBuster171) was built expressing three amiRNAs simultaneously. The construct was transformed into barley under the control of a constitutive promoter. The transgenic lines were kept at 12-15 °C to mimic autumn and spring conditions in which major WDV infection and accumulation take place. We were able to establish a stable barley transgenic line displaying resistance to insect-mediated WDV infection. Our study demonstrates that amiRNA technology can be an efficient tool for the introduction of highly efficient resistance in barley against a DNA virus belonging to the Geminiviridae family, and this resistance is effective at low temperature where the natural insect vector mediates the infection process. © 2015 BSPP and John Wiley & Sons Ltd.

Gene silencing efficiency and INF-β induction effects of splicing miRNA 155-based artificial miRNA with pre-miRNA stem-loop structures.

PubMed

Sin, Onsam; Mabiala, Prudence; Liu, Ye; Sun, Ying; Hu, Tao; Liu, Qingzhen; Guo, Deyin

2012-02-01

Artificial microRNA (miRNA) expression vectors have been developed and used for RNA interference. The secondary structure of artificial miRNA is important for RNA interference efficacy. We designed two groups of six artificial splicing miRNA 155-based miRNAs (SM155-based miRNAs) with the same target in the coding region or 3' UTR of a target gene and studied their RNA silencing efficiency and interferon β (IFN-β) induction effects. SM155-based miRNA with a mismatch at the +1 position and a bulge at the +11, +12 positions in a miRNA precursor stem-loop structure showed the highest gene silencing efficiency and lowest IFN-β induction effect (increased IFN-β mRNA level by 10% in both target cases), regardless of the specificity of the target sequence, suggesting that pSM155-based miRNA with this design could be a valuable miRNA expression vector.

Identification of novel microRNA genes in freshwater and marine ecotypes of the three-spined stickleback (Gasterosteus aculeatus).

PubMed

Rastorguev, S M; Nedoluzhko, A V; Sharko, F S; Boulygina, E S; Sokolov, A S; Gruzdeva, N M; Skryabin, K G; Prokhortchouk, E B

2016-11-01

The three-spined stickleback (Gasterosteus aculeatus L.) is an important model organism for studying the molecular mechanisms of speciation and adaptation to salinity. Despite increased interest to microRNA discovery and recent publication on microRNA prediction in the three-spined stickleback using bioinformatics approaches, there is still a lack of experimental support for these data. In this paper, high-throughput sequencing technology was applied to identify microRNA genes in gills of the three-spined stickleback. In total, 595 miRNA genes were discovered; half of them were predicted in previous computational studies and were confirmed here as microRNAs expressed in gill tissue. Moreover, 298 novel microRNA genes were identified. The presence of miRNA genes in selected 'divergence islands' was analysed and 10 miRNA genes were identified as not randomly located in 'divergence islands'. Regulatory regions of miRNA genes were found enriched with selective SNPs that may play a role in freshwater adaptation. © 2016 John Wiley & Sons Ltd.

RISC RNA sequencing for context-specific identification of in vivo miR targets

PubMed Central

Matkovich, Scot J; Van Booven, Derek J; Eschenbacher, William H; Dorn, Gerald W

2010-01-01

Rationale MicroRNAs (miRs) are expanding our understanding of cardiac disease and have the potential to transform cardiovascular therapeutics. One miR can target hundreds of individual mRNAs, but existing methodologies are not sufficient to accurately and comprehensively identify these mRNA targets in vivo. Objective To develop methods permitting identification of in vivo miR targets in an unbiased manner, using massively parallel sequencing of mouse cardiac transcriptomes in combination with sequencing of mRNA associated with mouse cardiac RNA-induced silencing complexes (RISCs). Methods and Results We optimized techniques for expression profiling small amounts of RNA without introducing amplification bias, and applied this to anti-Argonaute 2 immunoprecipitated RISCs (RISC-Seq) from mouse hearts. By comparing RNA-sequencing results of cardiac RISC and transcriptome from the same individual hearts, we defined 1,645 mRNAs consistently targeted to mouse cardiac RISCs. We employed this approach in hearts overexpressing miRs from Myh6 promoter-driven precursors (programmed RISC-Seq) to identify 209 in vivo targets of miR-133a and 81 in vivo targets of miR-499. Consistent with the fact that miR-133a and miR-499 have widely differing ‘seed’ sequences and belong to different miR families, only 6 targets were common to miR-133a- and miR-499-programmed hearts. Conclusions RISC-sequencing is a highly sensitive method for general RISC profiling and individual miR target identification in biological context, and is applicable to any tissue and any disease state. Summary MicroRNAs (miRs) are key regulators of mRNA translation in health and disease. While bioinformatic predictions suggest that a single miR may target hundreds of mRNAs, the number of experimentally verified targets of miRs is low. To enable comprehensive, unbiased examination of miR targets, we have performed deep RNA sequencing of cardiac transcriptomes in parallel with cardiac RNA-induced silencing complex (RISC)-associated RNAs (the RISCome), called RISC sequencing. We developed methods that did not require cross-linking of RNAs to RISCs or amplification of mRNA prior to sequencing, making it possible to rapidly perform RISC sequencing from intact tissue while avoiding amplification bias. Comparison of RISCome with transcriptome expression defined the degree of RISC enrichment for each mRNA. The majority of the mRNAs enriched in wild-type cardiac RISComes compared to transcriptomes were bioinformatically predicted to be targets of at least 1 of 139 cardiac-expressed miRs. Programming cardiomyocyte RISCs via transgenic overexpression in adult hearts of miR-133a or miR-499, two miRs that contain entirely different ‘seed’ sequences, elicited differing profiles of RISC-targeted mRNAs. Thus, RISC sequencing represents a highly sensitive method for general RISC profiling and individual miR target identification in biological context. PMID:21030712

Epitranscriptional orchestration of genetic reprogramming is an emergent property of stress-regulated cardiac microRNAs

PubMed Central

Hu, Yuanxin; Matkovich, Scot J.; Hecker, Peter A.; Zhang, Yan; Edwards, John R.; Dorn, Gerald W.

2012-01-01

Cardiac stress responses are driven by an evolutionarily conserved gene expression program comprising dozens of microRNAs and hundreds of mRNAs. Functionalities of different individual microRNAs are being studied, but the overall purpose of interactions between stress-regulated microRNAs and mRNAs and potentially distinct roles for microRNA-mediated epigenetic and conventional transcriptional genetic reprogramming of the stressed heart are unknown. Here we used deep sequencing to interrogate microRNA and mRNA regulation in pressure-overloaded mouse hearts, and performed a genome-wide examination of microRNA–mRNA interactions during early cardiac hypertrophy. Based on abundance and regulatory patterns, cardiac microRNAs were categorized as constitutively expressed housekeeping, regulated homeostatic, or dynamic early stress-responsive microRNAs. Regulation of 62 stress-responsive cardiac microRNAs directly affected levels of only 66 mRNAs, but the global impact of microRNA-mediated epigenetic regulation was amplified by preferential targeting of mRNAs encoding transcription factors, kinases, and phosphatases exerting amplified secondary effects. Thus, an emergent cooperative property of stress-regulated microRNAs is orchestration of transcriptional and posttranslational events that help determine the stress-reactive cardiac phenotype. This global functionality explains how large end-organ effects can be induced through modest individual changes in target mRNA and protein content by microRNAs that sense and respond dynamically to a changing physiological milieu. PMID:23150554

microRNAs Associated with Drought Response in the Bioenergy Crop Sugarcane (Saccharum spp.)

PubMed Central

Vilela, Romel Duarte; Costa, Gustavo Gilson Lacerda; Dias, Lara Isys; Endres, Laurício; Menossi, Marcelo

2012-01-01

Sugarcane (Saccharum spp.) is one of the most important crops in the world. Drought stress is a major abiotic stress factor that significantly reduces sugarcane yields. However the gene network that mediates plant responses to water stress remains largely unknown in several crop species. Although several microRNAs that mediate post-transcriptional regulation during water stress have been described in other species, the role of the sugarcane microRNAs during drought stress has not been studied. The objective of this work was to identify sugarcane miRNAs that are differentially expressed under drought stress and to correlate this expression with the behavior of two sugarcane cultivars with different drought tolerances. The sugarcane cultivars RB867515 (higher drought tolerance) and RB855536 (lower drought tolerance) were cultivated in a greenhouse for three months and then subjected to drought for 2, 4, 6 or 8 days. By deep sequencing of small RNAs, we were able to identify 18 miRNA families. Among all of the miRNAs thus identified, seven were differentially expressed during drought. Six of these miRNAs were differentially expressed at two days of stress, and five miRNAs were differentially expressed at four days. The expression levels of five miRNAs (ssp-miR164, ssp-miR394, ssp-miR397, ssp-miR399-seq 1 and miR528) were validated by RT-qPCR (quantitative reverse transcriptase PCR). Six precursors and the targets of the differentially expressed miRNA were predicted using an in silico approach and validated by RT-qPCR; many of these targets may play important roles in drought tolerance. These findings constitute a significant increase in the number of identified miRNAs in sugarcane and contribute to the elucidation of the complex regulatory network that is activated by drought stress. PMID:23071617

Small RNAs in plants: recent development and application for crop improvement

PubMed Central

Kamthan, Ayushi; Chaudhuri, Abira; Kamthan, Mohan; Datta, Asis

2015-01-01

The phenomenon of RNA interference (RNAi) which involves sequence-specific gene regulation by small non-coding RNAs, i.e., small interfering RNA (siRNA) and microRNA (miRNA) has emerged as one of most powerful approaches for crop improvement. RNAi based on siRNA is one of the widely used tools of reverse genetics which aid in revealing gene functions in many species. This technology has been extensively applied to alter the gene expression in plants with an aim to achieve desirable traits. RNAi has been used for enhancing the crop yield and productivity by manipulating the gene involved in biomass, grain yield and enhanced shelf life of fruits and vegetables. It has also been applied for developing resistance against various biotic (bacteria, fungi, viruses, nematodes, insects) and abiotic stresses (drought, salinity, cold, etc.). Nutritional improvements of crops have also been achieved by enriching the crops with essential amino acids, fatty acids, antioxidants and other nutrients beneficial for human health or by reducing allergens or anti-nutrients. microRNAs are key regulators of important plant processes like growth, development, and response to various stresses. In spite of similarity in size (20–24 nt), miRNA differ from siRNA in precursor structures, pathway of biogenesis, and modes of action. This review also highlights the miRNA based genetic modification technology where various miRNAs/artificial miRNAs and their targets can be utilized for improving several desirable plant traits. microRNA based strategies are much efficient than siRNA-based RNAi strategies due to its specificity and less undesirable off target effects. As per the FDA guidelines, small RNA (sRNA) based transgenics are much safer for consumption than those over-expressing proteins. This review thereby summarizes the emerging advances and achievement in the field of sRNAs and its application for crop improvement. PMID:25883599

Identification and characterization of microRNAs from Chinese pollination constant non-astringent persimmon using high-throughput sequencing.

PubMed

Luo, Yujie; Zhang, Xiaona; Luo, Zhengrong; Zhang, Qinglin; Liu, Jihong

2015-01-21

microRNAs (miRNAs) have been shown to play key roles in regulating gene expression at post-transcriptional level, but miRNAs associated with natural deastringency of Chinese pollination-constant nonastringent persimmon (CPCNA) have never been identified. In this study, two small RNA libraries established using 'Eshi No. 1' persimmon (Diospyros kaki Thunb.; CPCNA) fruits collected at 15 and 20 weeks after flowering (WAF) were sequenced through Solexa platform in order to identify miRNAs involved in deastringency of persimmon. A total of 6,258,487 and 7,634,169 reads were generated for the libraries at 15 and 20 WAF, respectively. Based on sequence similarity and hairpin structure prediction, 236 known miRNAs belonging to 65 miRNA families and 33 novel miRNAs were identified using persimmon transcriptome data. Sixty one of the characterized miRNAs exhibited pronounced difference in the expression levels between 15 and 20 WAF, 17 up-regulated and 44 down-regulated. Expression profiles of 12 conserved and 10 novel miRNAs were validated by stem loop qRT-PCR. A total of 198 target genes were predicted for the differentially expressed miRNAs, including several genes that have been reported to be implicated in proanthocyanidins (PAs, or called tannin) accumulation. In addition, two transcription factors, a GRF and a bHLH, were experimentally confirmed as the targets of dka-miR396 and dka-miR395, respectively. Taken together, the present data unraveled several important miRNAs in persimmon. Among them, miR395p-3p and miR858b may regulate bHLH and MYB, respectively, which are influenced by SPL under the control of miR156j-5p and in turn regulate the structural genes involved in PA biosynthesis. In addition, dka-miR396g and miR2911a may regulate their target genes associated with glucosylation and insolubilization of tannin precursors. All of these miRNAs might play key roles in the regulation of (de)astringency in persimmon fruits under normal development conditions.

Characterization of microRNAs in Mud Crab Scylla paramamosain under Vibrio parahaemolyticus Infection

PubMed Central

Li, Chuanbiao; Zhang, Zhao; Zhou, Lizhen; Wang, Shijia; Wang, Shuqi; Zhang, Yueling; Wen, Xiaobo

2013-01-01

Background Infection of bacterial Vibrio parahaemolyticus is common in mud crab farms. However, the mechanisms of the crab’s response to pathogenic V. parahaemolyticus infection are not fully understood. MicroRNAs (miRNAs) are a class of small noncoding RNAs that function as regulators of gene expression and play essential roles in various biological processes. To understand the underlying mechanisms of the molecular immune response of the crab to the pathogens, high-throughput Illumina/Solexa deep sequencing technology was used to investigate the expression profiles of miRNAs in S . paramamosain under V. parahaemolyticus infection. Methodology/Principal Findings Two mixed RNA pools of 7 tissues (intestine, heart, liver, gill, brain, muscle and blood) were obtained from V. parahaemolyticus infected crabs and the control groups, respectively. By aligning the sequencing data with known miRNAs, we characterized 421 miRNA families, and 133 conserved miRNA families in mud crab S . paramamosain were either identical or very similar to existing miRNAs in miRBase. Stem-loop qRT-PCRs were used to scan the expression levels of four randomly chosen differentially expressed miRNAs and tissue distribution. Eight novel potential miRNAs were confirmed by qRT-PCR analysis and the precursors of these novel miRNAs were verified by PCR amplification, cloning and sequencing in S . paramamosain . 161 miRNAs (106 of which up-regulated and 55 down-regulated) were significantly differentially expressed during the challenge and the potential targets of these differentially expressed miRNAs were predicted. Furthermore, we demonstrated evolutionary conservation of mud crab miRNAs in the animal evolution process. Conclusions/Significance In this study, a large number of miRNAs were identified in S . paramamosain when challenged with V. parahaemolyticus, some of which were differentially expressed. The results show that miRNAs might play some important roles in regulating gene expression in mud crab under V. parahaemolyticus infection, providing a basis for further investigation of miRNA-modulating networks in innate immunity of mud crab. PMID:24023678

Multifunctional silver nanocluster-hybrid oligonucleotide vehicle for cell imaging and microRNA-targeted gene silencing.

PubMed

Chen, Hau-Yun; Albert, Karunya; Wen, Cheng-Che; Hsieh, Pei-Ying; Chen, Sih-Yu; Huang, Nei-Chung; Lo, Shen-Chuan; Chen, Jen-Kun; Hsu, Hsin-Yun

2017-04-01

Novel therapeutics is urgently needed to prevent cancer-related deaths. MicroRNAs that act as tumor suppressors have been recognized as a next-generation tumor therapy, and the restoration of tumor-suppressive microRNAs using microRNA replacements or mimics may be a less toxic, more effective strategy due to fewer off-target effects. Here, we designed the novel multifunctional oligonucleotide nanocarrier complex composed of a tumor-targeting aptamer sequence specific to mucin 1 (MUC1), poly-cytosine region for fluorescent silver nanocluster (AgNC) synthesis, and complimentary sequence for microRNA miR-34a loading. MiR-34a was employed because of its therapeutic effect of inhibiting oncogene expression and inducing apoptosis in carcinomas. By monitoring the intrinsic fluorescence of AgNC, it was clearly shown that the constructed complex (MUC1-AgNC m -miR-34a) enters MCF-7 cells. To evaluate the efficacy of this nanocarrier for microRNA delivery, we investigated the gene and protein expression levels of downstream miR-34a targets (BCL-2, CDK6, and CCND1) by quantitative PCR and western blotting, respectively, and the results indicated their effective inhibition by miR-34a. This novel multifunctional AgNC-based nanocarrier can aid in improving the efficacy of breast cancer theranostics. Copyright © 2017 Elsevier B.V. All rights reserved.

Discovery and characterization of miRNA genes in atlantic salmon (Salmo salar) by use of a deep sequencing approach

PubMed Central

2013-01-01

Background MicroRNAs (miRNAs) are an abundant class of endogenous small RNA molecules that downregulate gene expression at the posttranscriptional level. They play important roles in multiple biological processes by regulating genes that control developmental timing, growth, stem cell division and apoptosis by binding to the mRNA of target genes. Despite the position Atlantic salmon (Salmo salar) has as an economically important domesticated animal, there has been little research on miRNAs in this species. Knowledge about miRNAs and their target genes may be used to control health and to improve performance of economically important traits. However, before their biological function can be unravelled they must be identified and annotated. The aims of this study were to identify and characterize miRNA genes in Atlantic salmon by deep sequencing analysis of small RNA libraries from nine different tissues. Results A total of 180 distinct mature miRNAs belonging to 106 families of evolutionary conserved miRNAs, and 13 distinct novel mature miRNAs were discovered and characterized. The mature miRNAs corresponded to 521 putative precursor sequences located at unique genome locations. About 40% of these precursors were part of gene clusters, and the majority of the Salmo salar gene clusters discovered were conserved across species. Comparison of expression levels in samples from different tissues applying DESeq indicated that there were tissue specific expression differences in three conserved and one novel miRNA. Ssa-miR 736 was detected in heart tissue only, while two other clustered miRNAs (ssa-miR 212 and132) seems to be at a higher expression level in brain tissue. These observations correlate well with their expected functions as regulators of signal pathways in cardiac and neuronal cells, respectively. Ssa-miR 8163 is one of the novel miRNAs discovered and its function remains unknown. However, differential expression analysis using DESeq suggests that this miRNA is enriched in liver tissue and the precursor was mapped to intron 7 of the transferrin gene. Conclusions The identification and annotation of evolutionary conserved and novel Salmo salar miRNAs as well as the characterization of miRNA gene clusters provide biological knowledge that will greatly facilitate further functional studies on miRNAs in this species. PMID:23865519

The MicroRNA-200 Family Is Upregulated in Endometrial Carcinoma

PubMed Central

Snowdon, Jaime; Zhang, Xiao; Childs, Tim; Tron, Victor A.; Feilotter, Harriet

2011-01-01

Background MicroRNAs (miRNAs, miRs) are small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. MicroRNAs are dysregulated in cancer and may play essential roles in tumorigenesis. Additionally, miRNAs have been shown to have prognostic and diagnostic value in certain types of cancer. The objective of this study was to identify dysregulated miRNAs in endometrioid endometrial adenocarcinoma (EEC) and the precursor lesion, complex atypical hyperplasia (CAH). Methodology We compared the expression profiles of 723 human miRNAs from 14 cases of EEC, 10 cases of CAH, and 10 normal proliferative endometria controls using Agilent Human miRNA arrays following RNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues. The expression of 4 dysregulated miRNAs was validated using real time reverse transcription-PCR. Results Forty-three miRNAs were dysregulated in EEC and CAH compared to normal controls (p<0.05). The entire miR-200 family (miR-200a/b/c, miR-141, and miR-429) was up-regulated in cases of EEC. Conclusions This information contributes to the candidate miRNA expression profile that has been generated for EEC and shows that certain miRNAs are dysregulated in the precursor lesion, CAH. These miRNAs in particular may play important roles in tumorigenesis. Examination of miRNAs that are consistently dysregulated in various studies of EEC, like the miR-200 family, will aid in the understanding of the role that miRNAs play in tumorigenesis in this tumour type. PMID:21897839

Targeted Deletion of miR-132/-212 Impairs Memory and Alters the Hippocampal Transcriptome

ERIC Educational Resources Information Center

Hansen, Katelin F.; Sakamoto, Kensuke; Aten, Sydney; Snider, Kaitlin H.; Loeser, Jacob; Hesse, Andrea M.; Page, Chloe E.; Pelz, Carl; Arthur, J. Simon C.; Impey, Soren; Obrietan, Karl

2016-01-01

miR-132 and miR-212 are structurally related microRNAs that have been found to exert powerful modulatory effects within the central nervous system (CNS). Notably, these microRNAs are tandomly processed from the same noncoding transcript, and share a common seed sequence: thus it has been difficult to assess the distinct contribution of each…

Isolation and Identification of Gene-Specific MicroRNAs.

PubMed

Lin, Shi-Lung; Chang, Donald C; Ying, Shao-Yao

2018-01-01

Computer programming has identified hundreds of genomic hairpin sequences, many with functions yet to be determined. Because transfection of hairpin-like microRNA precursors (pre-miRNAs) into mammalian cells is not always sufficient to trigger RNA-induced gene silencing complex (RISC) assembly, a key step for inducing RNA interference (RNAi)-related gene silencing, we have developed an intronic miRNA expression system to overcome this problem by inserting a hairpin-like pre-miRNA structure into the intron region of a gene, and hence successfully increase the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo. This intronic miRNA biogenesis mechanism has been found to depend on a coupled interaction of nascent messenger RNA transcription and intron excision within a specific nuclear region proximal to genomic perichromatin fibrils. The intronic miRNA so obtained is transcribed by type-II RNA polymerases, coexpressed within a primary gene transcript, and then excised out of the gene transcript by intracellular RNA splicing and processing machineries. After that, ribonuclease III (RNaseIII) endonucleases further process the spliced introns into mature miRNAs. Using this intronic miRNA expression system, we have shown for the first time that the intron-derived miRNAs are able to elicit strong RNAi effects in not only human and mouse cells in vitro but also in zebrafishes, chicken embryos, and adult mice in vivo. We have also developed a miRNA isolation protocol, based on the complementarity between the designed miRNA and its targeted gene sequence, to purify and identify the mature miRNAs generated. As a result, several intronic miRNA identities and structures have been confirmed. According to this proof-of-principle methodology, we now have full knowledge to design various intronic pre-miRNA inserts that are more efficient and effective for inducing specific gene silencing effects in vitro and in vivo.

MINTmap: fast and exhaustive profiling of nuclear and mitochondrial tRNA fragments from short RNA-seq data

PubMed Central

Loher, Phillipe; Telonis, Aristeidis G.; Rigoutsos, Isidore

2017-01-01

Transfer RNA fragments (tRFs) are an established class of constitutive regulatory molecules that arise from precursor and mature tRNAs. RNA deep sequencing (RNA-seq) has greatly facilitated the study of tRFs. However, the repeat nature of the tRNA templates and the idiosyncrasies of tRNA sequences necessitate the development and use of methodologies that differ markedly from those used to analyze RNA-seq data when studying microRNAs (miRNAs) or messenger RNAs (mRNAs). Here we present MINTmap (for MItochondrial and Nuclear TRF mapping), a method and a software package that was developed specifically for the quick, deterministic and exhaustive identification of tRFs in short RNA-seq datasets. In addition to identifying them, MINTmap is able to unambiguously calculate and report both raw and normalized abundances for the discovered tRFs. Furthermore, to ensure specificity, MINTmap identifies the subset of discovered tRFs that could be originating outside of tRNA space and flags them as candidate false positives. Our comparative analysis shows that MINTmap exhibits superior sensitivity and specificity to other available methods while also being exceptionally fast. The MINTmap codes are available through https://github.com/TJU-CMC-Org/MINTmap/ under an open source GNU GPL v3.0 license. PMID:28220888

Genome-wide identification of conserved microRNA and their response to drought stress in Dongxiang wild rice (Oryza rufipogon Griff.).

PubMed

Zhang, Fantao; Luo, Xiangdong; Zhou, Yi; Xie, Jiankun

2016-04-01

To identify drought stress-responsive conserved microRNA (miRNA) from Dongxiang wild rice (Oryza rufipogon Griff., DXWR) on a genome-wide scale, high-throughput sequencing technology was used to sequence libraries of DXWR samples, treated with and without drought stress. 505 conserved miRNAs corresponding to 215 families were identified. 17 were significantly down-regulated and 16 were up-regulated under drought stress. Stem-loop qRT-PCR revealed the same expression patterns as high-throughput sequencing, suggesting the accuracy of the sequencing result was high. Potential target genes of the drought-responsive miRNA were predicted to be involved in diverse biological processes. Furthermore, 16 miRNA families were first identified to be involved in drought stress response from plants. These results present a comprehensive view of the conserved miRNA and their expression patterns under drought stress for DXWR, which will provide valuable information and sequence resources for future basis studies.

DeepBase: annotation and discovery of microRNAs and other noncoding RNAs from deep-sequencing data.

PubMed

Yang, Jian-Hua; Qu, Liang-Hu

2012-01-01

Recent advances in high-throughput deep-sequencing technology have produced large numbers of short and long RNA sequences and enabled the detection and profiling of known and novel microRNAs (miRNAs) and other noncoding RNAs (ncRNAs) at unprecedented sensitivity and depth. In this chapter, we describe the use of deepBase, a database that we have developed to integrate all public deep-sequencing data and to facilitate the comprehensive annotation and discovery of miRNAs and other ncRNAs from these data. deepBase provides an integrative, interactive, and versatile web graphical interface to evaluate miRBase-annotated miRNA genes and other known ncRNAs, explores the expression patterns of miRNAs and other ncRNAs, and discovers novel miRNAs and other ncRNAs from deep-sequencing data. deepBase also provides a deepView genome browser to comparatively analyze these data at multiple levels. deepBase is available at http://deepbase.sysu.edu.cn/.

Biocomputational identification and validation of novel microRNAs predicted from bubaline whole genome shotgun sequences.

PubMed

Manku, H K; Dhanoa, J K; Kaur, S; Arora, J S; Mukhopadhyay, C S

2017-10-01

MicroRNAs (miRNAs) are small (19-25 base long), non-coding RNAs that regulate post-transcriptional gene expression by cleaving targeted mRNAs in several eukaryotes. The miRNAs play vital roles in multiple biological and metabolic processes, including developmental timing, signal transduction, cell maintenance and differentiation, diseases and cancers. Experimental identification of microRNAs is expensive and lab-intensive. Alternatively, computational approaches for predicting putative miRNAs from genomic or exomic sequences rely on features of miRNAs viz. secondary structures, sequence conservation, minimum free energy index (MFEI) etc. To date, not a single miRNA has been identified in bubaline (Bubalus bubalis), which is an economically important livestock. The present study aims at predicting the putative miRNAs of buffalo using comparative computational approach from buffalo whole genome shotgun sequencing data (INSDC: AWWX00000000.1). The sequences were blasted against the known mammalian miRNA. The obtained miRNAs were then passed through a series of filtration criteria to obtain the set of predicted (putative and novel) bubaline miRNA. Eight miRNAs were selected based on lowest E-value and validated by real time PCR (SYBR green chemistry) using RNU6 as endogenous control. The results from different trails of real time PCR shows that out of selected 8 miRNAs, only 2 (hsa-miR-1277-5p; bta-miR-2285b) are not expressed in bubaline PBMCs. The potential target genes based on their sequence complementarities were then predicted using miRanda. This work is the first report on prediction of bubaline miRNA from whole genome sequencing data followed by experimental validation. The finding could pave the way to future studies in economically important traits in buffalo. Copyright © 2017 Elsevier Ltd. All rights reserved.

A large microRNA cluster on chromosome 19 is a transcriptional hallmark of WHO type A and AB thymomas.

PubMed

Radovich, Milan; Solzak, Jeffrey P; Hancock, Bradley A; Conces, Madison L; Atale, Rutuja; Porter, Ryan F; Zhu, Jin; Glasscock, Jarret; Kesler, Kenneth A; Badve, Sunil S; Schneider, Bryan P; Loehrer, Patrick J

2016-02-16

Thymomas are one of the most rarely diagnosed malignancies. To better understand its biology and to identify therapeutic targets, we performed next-generation RNA sequencing. The RNA was sequenced from 13 thymic malignancies and 3 normal thymus glands. Validation of microRNA expression was performed on a separate set of 35 thymic malignancies. For cell-based studies, a thymoma cell line was used. Hierarchical clustering revealed 100% concordance between gene expression clusters and WHO subtype. A substantial differentiator was a large microRNA cluster on chr19q13.42 that was significantly overexpressed in all A and AB tumours and whose expression was virtually absent in the other thymomas and normal tissues. Overexpression of this microRNA cluster activates the PI3K/AKT/mTOR pathway. Treatment of a thymoma AB cell line with a panel of PI3K/AKT/mTOR inhibitors resulted in marked reduction of cell viability. A large microRNA cluster on chr19q13.42 is a transcriptional hallmark of type A and AB thymomas. Furthermore, this cluster activates the PI3K pathway, suggesting the possible exploration of PI3K inhibitors in patients with these subtypes of tumour. This work has led to the initiation of a phase II clinical trial of PI3K inhibition in relapsed or refractory thymomas (http://clinicaltrials.gov/ct2/show/NCT02220855).

A large microRNA cluster on chromosome 19 is a transcriptional hallmark of WHO type A and AB thymomas

PubMed Central

Radovich, Milan; Solzak, Jeffrey P; Hancock, Bradley A; Conces, Madison L; Atale, Rutuja; Porter, Ryan F; Zhu, Jin; Glasscock, Jarret; Kesler, Kenneth A; Badve, Sunil S; Schneider, Bryan P; Loehrer, Patrick J

2016-01-01

Background: Thymomas are one of the most rarely diagnosed malignancies. To better understand its biology and to identify therapeutic targets, we performed next-generation RNA sequencing. Methods: The RNA was sequenced from 13 thymic malignancies and 3 normal thymus glands. Validation of microRNA expression was performed on a separate set of 35 thymic malignancies. For cell-based studies, a thymoma cell line was used. Results: Hierarchical clustering revealed 100% concordance between gene expression clusters and WHO subtype. A substantial differentiator was a large microRNA cluster on chr19q13.42 that was significantly overexpressed in all A and AB tumours and whose expression was virtually absent in the other thymomas and normal tissues. Overexpression of this microRNA cluster activates the PI3K/AKT/mTOR pathway. Treatment of a thymoma AB cell line with a panel of PI3K/AKT/mTOR inhibitors resulted in marked reduction of cell viability. Conclusions: A large microRNA cluster on chr19q13.42 is a transcriptional hallmark of type A and AB thymomas. Furthermore, this cluster activates the PI3K pathway, suggesting the possible exploration of PI3K inhibitors in patients with these subtypes of tumour. This work has led to the initiation of a phase II clinical trial of PI3K inhibition in relapsed or refractory thymomas (http://clinicaltrials.gov/ct2/show/NCT02220855). PMID:26766736

MicroRNA-21 promotes proliferation of rat hepatocyte BRL-3A by targeting FASLG.

PubMed

Li, J J; Chan, W H; Leung, W Y; Wang, Y; Xu, C S

2015-04-27

Rat liver regeneration (RLR) induced by partial hepatectomy involves cell proliferation regulated by numerous factors, including microRNAs (miRNAs). miRNA high-throughput sequencing has been established and used to analyze miRNA expression profiles. This study showed that 39 miRNAs were related to RLR through the analysis of miRNA high-throughput sequencing. Their role toward rat normal hepatocyte line BRL-3A was studied by gain- and loss-of-function analyses, and one of them, microRNA-21 (miR-21), obviously upregulated and promoted BRL-3A cell proliferation. Using bioinformatics to search for miR-21 targets revealed that Fas ligand (FASLG) is one of miR-21's target genes. A dual-luciferase report assay and Western blot assay showed that miR-21 directly targeted the 3'-untranslated region of FASLG and inhibited the expression of FASLG, which suggests that miR-21 promoted BRL-3A cell proliferation by reducing FASLG expression.

RNAimmuno: A database of the nonspecific immunological effects of RNA interference and microRNA reagents

PubMed Central

Olejniczak, Marta; Galka-Marciniak, Paulina; Polak, Katarzyna; Fligier, Andrzej; Krzyzosiak, Wlodzimierz J.

2012-01-01

The RNAimmuno database was created to provide easy access to information regarding the nonspecific effects generated in cells by RNA interference triggers and microRNA regulators. Various RNAi and microRNA reagents, which differ in length and structure, often cause non-sequence-specific immune responses, in addition to triggering the intended sequence-specific effects. The activation of the cellular sensors of foreign RNA or DNA may lead to the induction of type I interferon and proinflammatory cytokine release. Subsequent changes in the cellular transcriptome and proteome may result in adverse effects, including cell death during therapeutic treatments or the misinterpretation of experimental results in research applications. The manually curated RNAimmuno database gathers the majority of the published data regarding the immunological side effects that are caused in investigated cell lines, tissues, and model organisms by different reagents. The database is accessible at http://rnaimmuno.ibch.poznan.pl and may be helpful in the further application and development of RNAi- and microRNA-based technologies. PMID:22411954

RNAimmuno: a database of the nonspecific immunological effects of RNA interference and microRNA reagents.

PubMed

Olejniczak, Marta; Galka-Marciniak, Paulina; Polak, Katarzyna; Fligier, Andrzej; Krzyzosiak, Wlodzimierz J

2012-05-01

The RNAimmuno database was created to provide easy access to information regarding the nonspecific effects generated in cells by RNA interference triggers and microRNA regulators. Various RNAi and microRNA reagents, which differ in length and structure, often cause non-sequence-specific immune responses, in addition to triggering the intended sequence-specific effects. The activation of the cellular sensors of foreign RNA or DNA may lead to the induction of type I interferon and proinflammatory cytokine release. Subsequent changes in the cellular transcriptome and proteome may result in adverse effects, including cell death during therapeutic treatments or the misinterpretation of experimental results in research applications. The manually curated RNAimmuno database gathers the majority of the published data regarding the immunological side effects that are caused in investigated cell lines, tissues, and model organisms by different reagents. The database is accessible at http://rnaimmuno.ibch.poznan.pl and may be helpful in the further application and development of RNAi- and microRNA-based technologies.

Identification and characterisation of a previously unknown drought tolerance-associated microRNA in barley.

PubMed

Zhou, Hui; Hussain, Syed Sarfraz; Hackenberg, Michael; Bazanova, Natalia; Eini, Omid; Li, Jie; Gustafson, Perry; Shi, Bujun

2018-04-22

Drought is the most serious abiotic stress, which causes crop losses on worldwide scale. The present study identified a previously unknown microRNA (designated as hvu-miRX) of 21 nucleotides (nt) in barley. Its precursor (designated pre-miRX) and primary transcript (designated pri-miRX) were also identified, with lengths of 73 nt and 559 nt, respectively. The identified upstream sequence of pri-miRX contains both the TATA box and the CAAT box, which are both required for transcription initiation. Transient promoter activation assays showed that the core promoter region of pri-miRX ranged 500 nt from the transcription start site. In transgenic barley over-expressing the wheat DREB3 transcription factor (TaDREB3) caused hvu-miRX to be highly expressed as compared to the same miRNA in non-transgenic barley. However, the high expression was not directly associated with TaDREB3. Genomic analysis revealed that the hvu-miRX gene was a single copy located on the short arm of chromosome 2 and appeared to be only conserved in Triticeae, but not in other plant species. Notably, transgenic barley overexpressing hvu-miRX showed drought tolerance. Degradome library analysis and other tests showed that hvu-miRX targeted various genes including transcription factors via the cleavage mode. Our data open an excellent opportunity to develop drought stress tolerant cereals with hvu-miRX. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

GSK3 inhibitors CHIR99021 and 6-bromoindirubin-3′-oxime inhibit microRNA maturation in mouse embryonic stem cells

PubMed Central

Wu, Yongyan; Liu, Fayang; Liu, Yingying; Liu, Xiaolei; Ai, Zhiying; Guo, Zekun; Zhang, Yong

2015-01-01

Wnt/β-catenin signalling plays a prominent role in maintaining self-renewal and pluripotency of mouse embryonic stem cells (mESCs). microRNAs (miRNAs) have critical roles in maintaining pluripotency and directing reprogramming. To investigate the effect of GSK3 inhibitors on miRNA expression, we analysed the miRNA expression profile of J1 mESCs in the absence or presence of CHIR99021 (CHIR) or 6-bromoindirubin-3′-oxime (BIO) by small RNA deep-sequencing. The results demonstrate that CHIR and BIO decrease mature miRNAs of most miRNA species, 90.4% and 98.1% of the differentially expressed miRNAs in BIO and CHIR treated cells were downregulated respectively. CHIR and BIO treatment leads to a slight upregulation of the primary transcripts of the miR-302–367 cluster and miR-181 family of miRNAs, these miRNAs are activated by Wnt/β-catenin signalling. However, the precursor and mature form of the miR-302–367 cluster and miR-181 family of miRNAs are downregulated by CHIR, suggesting CHIR inhibits maturation of primary miRNA. Western blot analysis shows that BIO and CHIR treatment leads to a reduction of the RNase III enzyme Drosha in the nucleus. These data suggest that BIO and CHIR inhibit miRNA maturation by disturbing nuclear localisation of Drosha. Results also show that BIO and CHIR induce miR-211 expression in J1 mESCs. PMID:25727520

Identification of microRNA precursor with the degenerate K-tuple or Kmer strategy.

PubMed

Liu, Bin; Fang, Longyun; Wang, Shanyi; Wang, Xiaolong; Li, Hongtao; Chou, Kuo-Chen

2015-11-21

The microRNA (miRNA), a small non-coding RNA molecule, plays an important role in transcriptional and post-transcriptional regulation of gene expression. Its abnormal expression, however, has been observed in many cancers and other disease states, implying that the miRNA molecules are also deeply involved in these diseases, particularly in carcinogenesis. Therefore, it is important for both basic research and miRNA-based therapy to discriminate the real pre-miRNAs from the false ones (such as hairpin sequences with similar stem-loops). Most existing methods in this regard were based on the strategy in which RNA samples were formulated by a vector formed by their Kmer components. But the length of Kmers must be very short; otherwise, the vector's dimension would be extremely large, leading to the "high-dimension disaster" or overfitting problem. Inspired by the concept of "degenerate energy levels" in quantum mechanics, we introduced the "degenerate Kmer" (deKmer) to represent RNA samples. By doing so, not only we can accommodate long-range coupling effects but also we can avoid the high-dimension problem. Rigorous jackknife tests and cross-species experiments indicated that our approach is very promising. It has not escaped our notice that the deKmer approach can also be applied to many other areas of computational biology. A user-friendly web-server for the new predictor has been established at http://bioinformatics.hitsz.edu.cn/miRNA-deKmer/, by which users can easily get their desired results. Copyright © 2015 Elsevier Ltd. All rights reserved.

Preliminary profiling of microRNA in the normal and regenerating liver of Chiloscyllium plagiosum.

PubMed

Cheng, Dandan; Chen, Yanna; Lu, Conger; Qian, Yuezhong; Lv, Zhengbing

2017-12-01

Liver is a vital organ present in animals for detoxification, protein synthesis, digestion and other functions and its powerful regenerative capacity is well known. C. plagiosum is an abundant fish that is representative of the cartilaginous class in the southeast coastal region of China and its liver accounts for >70% of the fish's visceral weight and contains many bioactive substances. MicroRNAs (microRNAs) play important roles in a wide range of biological processes in eukaryotes, including cell proliferation, differentiation, apoptosis. However, microRNAs in response to liver regeneration has not been well studied. This study aimed to identify the microRNAs that participate in liver regeneration and other liver-related diseases and to improve our understanding of the mechanisms of liver regeneration in sharks. To this end, normal and regenerating liver tissues from C. plagiosum were harvested 0, 3, 6, 12 and 24h after partial hepatectomy (pH) and were sequenced using the Illumina/Solexa platform. In total, 309 known microRNAs and 590 novel microRNAs were identified in C. plagiosum. There were many microRNAs differentially expressed in the normal and regenerating livers between time points. Using target prediction and GO analysis, most of the differentially expressed microRNAs were assigned to functional categories that may be involved in regulating liver regeneration, such as cell proliferation, differentiation and apoptosis. The microRNA expression profile of liver regeneration will pave the way for the development of effective strategies to fight against liver disease and other related disease. Copyright © 2017 Elsevier Inc. All rights reserved.

A novel method of predicting microRNA-disease associations based on microRNA, disease, gene and environment factor networks.

PubMed

Peng, Wei; Lan, Wei; Zhong, Jiancheng; Wang, Jianxin; Pan, Yi

2017-07-15

MicroRNAs have been reported to have close relationship with diseases due to their deregulation of the expression of target mRNAs. Detecting disease-related microRNAs is helpful for disease therapies. With the development of high throughput experimental techniques, a large number of microRNAs have been sequenced. However, it is still a big challenge to identify which microRNAs are related to diseases. Recently, researchers are interesting in combining multiple-biological information to identify the associations between microRNAs and diseases. In this work, we have proposed a novel method to predict the microRNA-disease associations based on four biological properties. They are microRNA, disease, gene and environment factor. Compared with previous methods, our method makes predictions not only by using the prior knowledge of associations among microRNAs, disease, environment factors and genes, but also by using the internal relationship among these biological properties. We constructed four biological networks based on the similarity of microRNAs, diseases, environment factors and genes, respectively. Then random walking was implemented on the four networks unequally. In the walking course, the associations can be inferred from the neighbors in the same networks. Meanwhile the association information can be transferred from one network to another. The results of experiment showed that our method achieved better prediction performance than other existing state-of-the-art methods. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification and Characterization of microRNA319a and Its Putative Target Gene, PvPCF5, in the Bioenergy Grass Switchgrass (Panicum virgatum).

PubMed

Xie, Qi; Liu, Xue; Zhang, Yinbing; Tang, Jinfu; Yin, Dedong; Fan, Bo; Zhu, Lihuang; Han, Liebao; Song, Guilong; Li, Dayong

2017-01-01

Due to its high biomass yield, low environmental impact, and widespread adaptability to poor soils and harsh conditions, switchgrass ( Panicum virgatum L.), a warm-region perennial herbaceous plant, has attracted much attention in recent years. However, little is known about microRNAs (miRNAs) and their functions in this bioenergy grass. Here, we identified and characterized a miRNA gene, Pvi-MIR319a , encoding microRNA319a in switchgrass. Transgenic rice lines generated by overexpressing the Pvi-MIR319a precursor gene exhibited broader leaves and delayed flowering compared with the control. Gene expression analysis indicated at least four putative target genes were downregulated. Additionally, we cloned a putative target gene ( PvPCF5 ) of Pvi-MIR319a from switchgrass. PvPCF5, a TCP transcription factor, is a nuclear-localized protein with transactivation activity and control the development of leaf. Our results suggest that Pvi-MIR319a and its target genes may be used as potential genetic regulators for future switchgrass genetic improvement.

MicroRNA-132 targets HB-EGF upon IgE-mediated activation in murine and human mast cells.

PubMed

Molnár, Viktor; Érsek, Barbara; Wiener, Zoltán; Tömböl, Zsófia; Szabó, Péter M; Igaz, Péter; Falus, András

2012-03-01

MicroRNAs provide an additional layer in the regulation of gene expression acting as repressors with several targets at the posttranscriptional level. This study describes microRNA expression patterns during differentiation and activation of mast cells. The expression levels of 567 different mouse miRNAs were compared by microarray between c-Kit+ committed progenitors, mucosal mast cells, resting and IgE-crosslinked BMMCs in vitro. The strongest upregulation of miR-132 upon IgE-mediated activation was validated in human cord blood-derived mast cells as well. HB-EGF growth factor also upregulated upon activation and was ranked high by more prediction algorithms. Co-transfection of miR-132 mimicking precursor and the 3'UTR of human Hbegf-containing luciferase vector proves that the predicted binding site is functional. In line with this, neutralization of miR-132 by anti-miR inhibitor leads to sustained production of HB-EGF protein in activated mast cells. Our data provide a novel example for negative regulation of a growth factor by an upregulated miRNA. © Springer Basel AG 2011

Target-induced Catalytic Assembly of Y-Shaped DNA and Its Application for In Situ Imaging of MicroRNAs.

PubMed

Xue, Chang; Zhang, Shu-Xin; Ouyang, Chang-He; Chang, Dingran; Salena, Bruno J; Li, Yingfu; Wu, Zai-Sheng

2018-06-14

DNA is a highly programmable material that can be configured into unique high-order structures, such as DNA branched junctions containing multiple helical arms converging at a center. Herein we show that DNA programmability can deliver in situ growth of a 3-way junction-based DNA structure (denoted Y-shaped DNA) with the use of three hairpin-shaped DNA molecules as precursors, a specific microRNA target as a recyclable trigger, and a DNA polymerase as a driver. We demonstrate that the Y-shaped configuration comes with the benefit of restricted freedom of movement in confined cellular environment, which makes the approach ideally suited for in situ imaging of small RNA targets, such as microRNAs. Comparative analysis illustrates that the proposed imaging technique is superior to both the classic fluorescence in situ hybridization (FISH) method and an analogous amplified imaging method via programmed growth of a double-stranded DNA (rather than Y-shaped DNA) product. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

MicroRNAs in the pathobiology of atherosclerosis

PubMed Central

Laffont, Benoit; Rayner, Katey J

2017-01-01

MicroRNAs are short non-coding RNAs, expressed in humans and involved in sequence-specific post-transcriptional regulation of gene expression. They have emerged as key players in a wide array of biological processes, and changes in their expression and/or function have been associated with plethora of human diseases. Atherosclerosis and its related clinical complications, such as myocardial infarction or stroke, represent the leading cause of death in the western world. Accumulating experimental evidence has revealed a key role for microRNAs in regulating cellular and molecular processes related to atherosclerosis development, ranging from risk factors, to plaque initiation and progression, up to atherosclerotic plaque rupture. In this review, we will focus on how microRNAs can influence atherosclerosis biology, as well as the potential clinical applications of microRNAs which are being developed as both targets and therapeutics for a growing industry hoping to harness the power of RNA-guided gene regulation to fight disease and infection. PMID:28232017

A systematic review of overlapping microRNA patterns in systemic sclerosis and idiopathic pulmonary fibrosis.

PubMed

Bagnato, Gianluca; Roberts, William Neal; Roman, Jesse; Gangemi, Sebastiano

2017-06-30

Lung fibrosis can be observed in systemic sclerosis and in idiopathic pulmonary fibrosis, two disorders where lung involvement carries a poor prognosis. Although much has been learned about the pathogenesis of these conditions, interventions capable of reversing or, at the very least, halting disease progression are not available. Recent studies point to the potential role of micro messenger RNAs (microRNAs) in cancer and tissue fibrogenesis. MicroRNAs are short non-coding RNA sequences (20-23 nucleotides) that are endogenous, evolutionarily conserved and encoded in the genome. By acting on several genes, microRNAs control protein expression. Considering the above, we engaged in a systematic review of the literature in search of overlapping observations implicating microRNAs in the pathogenesis of both idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc). Our objective was to uncover top microRNA candidates for further investigation based on their mechanisms of action and their potential for serving as targets for intervention against lung fibrosis. Our review points to microRNAs of the -29 family, -21-5p and -92a-3p, -26a-5p and let-7d-5p as having distinct and counter-balancing actions related to lung fibrosis. Based on this, we speculate that readjusting the disrupted balance between these microRNAs in lung fibrosis related to SSc and IPF may have therapeutic potential. Copyright ©ERS 2017.

THE INVOLVEMENT OF HUMAN MONOGENIC CARDIOMYOPATHY GENES IN EXPERIMENTAL POLYGENIC CARDIAC HYPERTROPHY.

PubMed

Prestes, Priscilla R; Marques, Francine Z; Lopez-Campos, Guillermo; Lewandowski, Paul; Delbridge, Lea M D; Charchar, Fadi J; Harrap, Stephen B

2018-05-18

Hypertrophic cardiomyopathy thickens heart muscles reducing functionality and increasing risk of cardiac disease and morbidity. Genetic factors are involved, but their contribution is poorly understood. We used the hypertrophic heart rat (HHR), a unique normotensive polygenic model of cardiac hypertrophy and heart failure to investigate the role of genes associated with monogenic human cardiomyopathy. We selected 42 genes involved in monogenic human cardiomyopathies to study: 1) DNA variants, by sequencing the whole-genome of 13-week old HHR and age-matched normal heart rat (NHR), its genetic control strain; 2) mRNA expression, by targeted RNA-sequencing in left ventricles of HHR and NHR at five ages (2-days old, 4-, 13-, 33- and 50-weeks old) compared to human idiopathic dilated data; and 3) microRNA expression, with rat microRNA microarrays in left ventricles of 2-days old HHR and age-matched NHR. We also investigated experimentally validated microRNA-mRNA interactions. Whole-genome sequencing revealed unique variants mostly located in non-coding regions of HHR and NHR. We found 29 genes differentially expressed in at least one age. Genes encoding desmoglein 2 (Dsg2) and transthyretin (Ttr) were significantly differentially expressed at all ages in the HHR, but only Ttr was also differentially expressed in human idiopathic cardiomyopathy. Lastly, only two microRNAs differentially expressed in the HHR were present in our comparison of validated microRNA-mRNA interactions. These two microRNAs interact with five of the genes studied. Our study shows that genes involved in monogenic forms of human cardiomyopathies may also influence polygenic forms of the disease.

Axonal localization and mitochondrial association of precursor microRNA 338

PubMed Central

Vargas, Jose Norberto S.; Kar, Amar N.; Kowalak, Jeffrey A.; Gale, Jenna R.; Aschrafi, Armaz; Chen, Cai-Yun; Gioio, Anthony E.; Kaplan, Barry B.

2016-01-01

microRNAs (miRNAs) selectively localize to subcompartments of the neuron, such as dendrites, axons and presynaptic terminals, where they regulate the local protein synthesis of their putative target genes. In addition to mature miRNAs, precursor miRNAs (pre-miRNAs) have also been shown to localize to somatodendritic and axonal compartments. miRNA-338 (miR-338) regulates the local expression of several nuclear-encoded mitochondrial mRNAs within axons of sympathetic neurons. Previous work has shown that precursor miR-338 (pre-miR-338) introduced into the axon can be locally processed into mature miR-338, where it can regulate local ATP synthesis. However, the mechanisms underlying the localization of pre-miRNAs to the axonal compartment remain unknown. In this study, we investigated the axonal localization of pre-miR-338. Using proteomic and biochemical approaches, we provide evidence for the localization of pre-miR-338 to distal neuronal compartments and identify several constituents of the pre-miR-338 ribonucleoprotein complex. Furthermore, we found that pre-miR-338 is associated with the mitochondria in axons of superior cervical ganglion (SCG) neurons. The maintenance of mitochondrial function within axons requires the precise spatio-temporal synthesis of nuclear-encoded mRNAs, some of which are regulated by miR-338. Therefore, the association of pre-miR-338 with axonal mitochondria could serve as a reservoir of mature, biologically active miRNAs, which could coordinate the intra-axonal expression of multiple nuclear-encoded mitochondrial mRNAs. PMID:27229124

Diverse correlation patterns between microRNAs and their targets during tomato fruit development indicates different modes of microRNA actions.

PubMed

Lopez-Gomollon, Sara; Mohorianu, Irina; Szittya, Gyorgy; Moulton, Vincent; Dalmay, Tamas

2012-12-01

MicroRNAs negatively regulate the accumulation of mRNAs therefore when they are expressed in the same cells their expression profiles show an inverse correlation. We previously described one positively correlated miRNA/target pair, but it is not known how widespread this phenomenon is. Here, we investigated the correlation between the expression profiles of differentially expressed miRNAs and their targets during tomato fruit development using deep sequencing, Northern blot and RT-qPCR. We found an equal number of positively and negatively correlated miRNA/target pairs indicating that positive correlation is more frequent than previously thought. We also found that the correlation between microRNA and target expression profiles can vary between mRNAs belonging to the same gene family and even for the same target mRNA at different developmental stages. Since microRNAs always negatively regulate their targets, the high number of positively correlated microRNA/target pairs suggests that mutual exclusion could be as widespread as temporal regulation. The change of correlation during development suggests that the type of regulatory circuit directed by a microRNA can change over time and can be different for individual gene family members. Our results also highlight potential problems for expression profiling-based microRNA target identification/validation.

miRDis: a Web tool for endogenous and exogenous microRNA discovery based on deep-sequencing data analysis.

PubMed

Zhang, Hanyuan; Vieira Resende E Silva, Bruno; Cui, Juan

2018-05-01

Small RNA sequencing is the most widely used tool for microRNA (miRNA) discovery, and shows great potential for the efficient study of miRNA cross-species transport, i.e., by detecting the presence of exogenous miRNA sequences in the host species. Because of the increased appreciation of dietary miRNAs and their far-reaching implication in human health, research interests are currently growing with regard to exogenous miRNAs bioavailability, mechanisms of cross-species transport and miRNA function in cellular biological processes. In this article, we present microRNA Discovery (miRDis), a new small RNA sequencing data analysis pipeline for both endogenous and exogenous miRNA detection. Specifically, we developed and deployed a Web service that supports the annotation and expression profiling data of known host miRNAs and the detection of novel miRNAs, other noncoding RNAs, and the exogenous miRNAs from dietary species. As a proof-of-concept, we analyzed a set of human plasma sequencing data from a milk-feeding study where 225 human miRNAs were detected in the plasma samples and 44 show elevated expression after milk intake. By examining the bovine-specific sequences, data indicate that three bovine miRNAs (bta-miR-378, -181* and -150) are present in human plasma possibly because of the dietary uptake. Further evaluation based on different sets of public data demonstrates that miRDis outperforms other state-of-the-art tools in both detection and quantification of miRNA from either animal or plant sources. The miRDis Web server is available at: http://sbbi.unl.edu/miRDis/index.php.

1994 Accident sequence precursor program results

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

Belles, R.J.; Cletcher, J.W.; Copinger, D.A.

1996-01-01

The Accident Sequence Precursor (ASP) Program involves the systematic review and evaluation of operational events that have occurred at light-water reactors to identify and categorize precursors to potential severe core damage accident sequences. The results of the ASP Program are published in an annual report. The most recent report, which contains the analyses of the precursors for 1994, is NUREG/CR-4674, Vols. 21 and 22, Precursors to Potential Severe Core Damage Accidents: 1994, A Status Report, published in December 1995. This article provides an overview of the ASP review and evaluation process and a summary of the results for 1994. 12more » refs., 2 figs., 4 tabs.« less

From CNS stem cells to neurons and glia: Sox for everyone.

PubMed

Reiprich, Simone; Wegner, Michael

2015-01-01

Neuroepithelial precursor cells of the vertebrate central nervous system either self-renew or differentiate into neurons, oligodendrocytes or astrocytes under the influence of a gene regulatory network that consists in transcription factors, epigenetic modifiers and microRNAs. Sox transcription factors are central to this regulatory network, especially members of the SoxB, SoxC, SoxD, SoxE and SoxF groups. These Sox proteins are widely expressed in neuroepithelial precursor cells and in newly specified, differentiating and mature neurons, oligodendrocytes and astrocytes and influence their identity, survival and development. They exert their effect predominantly at the transcriptional level but also have substantial impact on expression at the epigenetic and posttranscriptional levels with some Sox proteins acting as pioneer factors, recruiting chromatin-modifying and -remodelling complexes or influencing microRNA expression. They interact with a large variety of other transcription factors and influence the expression of regulatory molecules and effector genes in a cell-type-specific and temporally controlled manner. As versatile regulators with context-dependent functions, they are not only indispensable for central nervous system development but might also be instrumental for the development of reprogramming and cell conversion strategies for replacement therapies and for assisted regeneration after injury or degeneration-induced cell loss in the central nervous system.

microRNAs differentially modulated in response to heat and drought stress in durum wheat cultivars with contrasting water use efficiency.

PubMed

Giusti, Lorenzo; Mica, Erica; Bertolini, Edoardo; De Leonardis, Anna Maria; Faccioli, Primetta; Cattivelli, Luigi; Crosatti, Cristina

2017-05-01

Plant stress response is a complex molecular process based on transcriptional and posttranscriptional regulation of many stress-related genes. microRNAs are the best-studied class of small RNAs known to play key regulatory roles in plant response to stress, besides being involved in plant development and organogenesis. We analyzed the leaf miRNAome of two durum wheat cultivars (Cappelli and Ofanto) characterized by a contrasting water use efficiency, exposed to heat stress, and mild and severe drought stress. On the whole, we identified 98 miRNA highly similar to previously known miRNAs and grouped in 47 MIR families, as well as 85 novel candidate miRNA, putatively wheat specific. A total of 80 known and novel miRNA precursors were found differentially expressed between the two cultivars or modulated by stress and many of them showed a cultivar-specific expression profile. Interestingly, most in silico predicted targets of the miRNAs coming from the differentially expressed precursors have been experimentally linked in other species to mechanisms controlling stomatal movement, a finding in agreement with previous results showing that Cappelli has a lower stomatal conductance than Ofanto. Selected miRNAs were validated through a standardized and reliable stem-loop qRT-PCR procedure.

Dicer cleaves 5'-extended microRNA precursors originating from RNA polymerase II transcription start sites.

PubMed

Sheng, Peike; Fields, Christopher; Aadland, Kelsey; Wei, Tianqi; Kolaczkowski, Oralia; Gu, Tongjun; Kolaczkowski, Bryan; Xie, Mingyi

2018-05-09

MicroRNAs (miRNAs) are approximately 22 nucleotide (nt) long and play important roles in post-transcriptional regulation in both plants and animals. In animals, precursor (pre-) miRNAs are ∼70 nt hairpins produced by Drosha cleavage of long primary (pri-) miRNAs in the nucleus. Exportin-5 (XPO5) transports pre-miRNAs into the cytoplasm for Dicer processing. Alternatively, pre-miRNAs containing a 5' 7-methylguanine (m7G-) cap can be generated independently of Drosha and XPO5. Here we identify a class of m7G-capped pre-miRNAs with 5' extensions up to 39 nt long. The 5'-extended pre-miRNAs are transported by Exportin-1 (XPO1). Unexpectedly, a long 5' extension does not block Dicer processing. Rather, Dicer directly cleaves 5'-extended pre-miRNAs by recognizing its 3' end to produce mature 3p miRNA and extended 5p miRNA both in vivo and in vitro. The recognition of 5'-extended pre-miRNAs by the Dicer Platform-PAZ-Connector (PPC) domain can be traced back to ancestral animal Dicers, suggesting that this previously unrecognized Dicer reaction mode is evolutionarily conserved. Our work reveals additional genetic sources for small regulatory RNAs and substantiates Dicer's essential role in RNAi-based gene regulation.

Identification and characterization of the reptilian GnRH-II gene in the leopard gecko, Eublepharis macularius, and its evolutionary considerations.

PubMed

Ikemoto, Tadahiro; Park, Min Kyun

2003-10-16

To elucidate the molecular phylogeny and evolution of a particular peptide, one must analyze not the limited primary amino acid sequences of the low molecular weight mature polypeptide, but rather the sequences of the corresponding precursors from various species. Of all the structural variants of gonadotropin-releasing hormone (GnRH), GnRH-II (chicken GnRH-II, or cGnRH-II) is remarkably conserved without any sequence substitutions among vertebrates, but its precursor sequences vary considerably. We have identified and characterized the full-length complementary DNA (cDNA) encoding the GnRH-II precursor and determined its genomic structure, consisting of four exons and three introns, in a reptilian species, the leopard gecko Eublepharis macularius. This is the first report about the GnRH-II precursor cDNA/gene from reptiles. The deduced leopard gecko prepro-GnRH-II polypeptide had the highest identities with the corresponding polypeptides of amphibians. The GnRH-II precursor mRNA was detected in more than half of the tissues and organs examined. This widespread expression is consistent with the previous findings in several species, though the roles of GnRH outside the hypothalamus-pituitary-gonadal axis remain largely unknown. Molecular phylogenetic analysis combined with sequence comparison showed that the leopard gecko is more similar to fishes and amphibians than to eutherian mammals with respect to the GnRH-II precursor sequence. These results strongly suggest that the divergence of the GnRH-II precursor sequences seen in eutherian mammals may have occurred along with amniote evolution.

TWIST1-induced microRNA-424 reversibly drives mesenchymal programming while inhibiting tumor initiation

PubMed Central

Drasin, David J.; Guarnieri, Anna L.; Neelakantan, Deepika; Kim, Jihye; Cabrera, Joshua H.; Wang, Chu-An; Zaberezhnyy, Vadym; Gasparini, Pierluigi; Cascione, Luciano; Huebner, Kay; Tan, Aik-Choon; Ford, Heide L.

2015-01-01

Epithelial-to-mesenchymal transition (EMT) is a dynamic process that relies on cellular plasticity. Recently, the process of an oncogenic EMT, followed by a reverse mesenchymal-to-epithelial transition (MET), has been implicated as critical in the metastatic colonization of carcinomas. Unlike governance of epithelial programming, regulation of mesenchymal programming is not well understood in EMT. Here, we describe and characterize the first microRNA that enhances exclusively mesenchymal programming. We demonstrate that microRNA-424 is upregulated early during a TWIST1 or SNAI1-induced EMT, and that it causes cells to express mesenchymal genes without affecting epithelial genes, resulting in a mixed/intermediate EMT. Furthermore, microRNA-424 increases motility, decreases adhesion and induces a growth arrest, changes associated with a complete EMT, that can be reversed when microRNA-424 expression is lowered, concomitant with an MET-like process. Breast cancer patient microRNA-424 levels positively associate with TWIST1/2 and EMT-like gene signatures, and miR-424 is increased in primary tumors versus matched normal breast. However, microRNA-424 is downregulated in patient metastases versus matched primary tumors. Correspondingly, microRNA-424 decreases tumor initiation and is post-transcriptionally downregulated in macrometastases in mice, suggesting the need for biphasic expression of miR-424 to transit the EMT-MET axis. Next-generation RNA sequencing revealed microRNA-424 regulates numerous EMT and cancer stemness-associated genes, including TGFBR3, whose downregulation promotes mesenchymal phenotypes, but not tumor-initiating phenotypes. Instead, we demonstrate that increased MAPK/ERK signaling is critical for miR-424-mediated decreases in tumor-initiating phenotypes. These findings suggest microRNA-424 plays distinct roles in tumor progression, potentially facilitating earlier, but repressing later, stages of metastasis by regulating an EMT-MET axis. PMID:25716682

Dehydration triggers differential microRNA expression in Xenopus laevis brain.

PubMed

Luu, Bryan E; Storey, Kenneth B

2015-11-15

African clawed frogs, Xenopus laevis, although primarily aquatic, have a high tolerance for dehydration, being capable of withstanding the loss of up to 32-35% of total water body water. Recent studies have shown that microRNAs play a role in the response to dehydration by the liver, kidney and ventral skin of X. laevis. MicroRNAs act by modulating the expression of mRNA transcripts, thereby affecting diverse biochemical pathways. In this study, 43 microRNAs were assessed in frog brains comparing control and dehydrated (31.2±0.83% of total body water lost) conditions. MicroRNAs of interest were measured using a modified protocol which employs polyadenylation of microRNAs prior to reverse transcription and qPCR. Twelve microRNAs that showed a significant decrease in expression (to 41-77% of control levels) in brains from dehydrated frogs (xla-miR-15a, -150, -181a, -191, -211, -218, -219b, -30c, -30e, -31, -34a, and -34b) were identified. Genomic analysis showed that the sequences of these dehydration-responsive microRNAs were highly conserved as compared with the comparable microRNAs of mice (91-100%). Suppression of these microRNAs implies that translation of the mRNA transcripts under their control could be enhanced in response to dehydration. Bioinformatic analysis using the DIANA miRPath program (v.2.0) predicted the top two KEGG pathways that these microRNAs collectively regulate: 1. Axon guidance, and 2. Long-term potentiation. Previous studies indicated that suppression of these microRNAs promotes neuroprotective pathways by increasing the expression of brain-derived neurotrophic factor and activating anti-apoptotic pathways. This suggests that similar actions may be triggered in X. laevis brains as a protective response to dehydration. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

Sequence-specific inhibition of microRNA-130a gene by CRISPR/Cas9 system in breast cancer cell line

NASA Astrophysics Data System (ADS)

Ainina Abdollah, Nur; Das Kumitaa, Theva; Yusof Narazah, Mohd; Razak, Siti Razila Abdul

2017-05-01

MicroRNAs (miRNAs) are short stranded noncoding RNA that play important roles in apoptosis, cell survival, development and cell proliferation. However, gene expression control via small regulatory RNA, particularly miRNA in breast cancer is still less explored. Therefore, this project aims to develop an approach to target microRNA-130a using the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system in MCF7, breast cancer cell line. The 20 bp sequences target at stem loop, 3ʹ and 5ʹ end of miR130a were cloned into pSpCas9(BB)-2A-GFP (PX458) plasmid, and the positive clones were confirmed by sequencing. A total of 5 μg of PX458-miR130a was transfected to MCF7 using Lipofectamine® 3000 according to manufacturer’s protocol. The transfected cells were maintained in the incubator at 37 °C under humidified 5% CO2. After 48 hours, cells were harvested and total RNA was extracted using miRNeasy Mini Kit (Qiagen). cDNAs were synthesised specific to miR-130a using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). Then, qRT-PCR was carried out using TaqMan Universal Master Mix (Applied Biosystems) to quantify the knockdown level of mature miRNAs in the cells. Result showed that miR-130a-5p was significantly downregulated in MCF7 cell line. However, no significant changes were observed for sequences targeting miR-130a-3p and stem loop. Thus, this study showed that the expression of miR-130a-5p was successfully down-regulated using CRISPR silencing system. This technique may be useful to manipulate the level of miRNA in various cell types to answer clinical questions at the molecular level.

Small Molecule Chemical Probes of MicroRNA Function

PubMed Central

Velagapudi, Sai Pradeep; Vummidi, Balayeshwanth R.; Disney, Matthew D.

2015-01-01

MicroRNAs (miRNAs) are small, non-coding RNAs that control protein expression. Aberrant miRNA expression has been linked to various human diseases, and thus miRNAs have been explored as diagnostic markers and therapeutic targets. Although it is challenging to target RNA with small molecules in general, there have been successful campaigns that have identified small molecule modulators of miRNA function by targeting various pathways. For example, small molecules that modulate transcription and target nuclease processing sites in miRNA precursors have been identified. Herein, we describe challenges in developing chemical probes that target miRNAs and highlight aspects of miRNA cellular biology elucidated by using small molecule chemical probes. We expect that this area will expand dramatically in the near future as strides are made to understand small molecule recognition of RNA from a fundamental perspective. PMID:25500006

TmiRUSite and TmiROSite scripts: searching for mRNA fragments with miRNA binding sites with encoded amino acid residues.

PubMed

Berillo, Olga; Régnier, Mireille; Ivashchenko, Anatoly

2014-01-01

microRNAs are small RNA molecules that inhibit the translation of target genes. microRNA binding sites are located in the untranslated regions as well as in the coding domains. We describe TmiRUSite and TmiROSite scripts developed using python as tools for the extraction of nucleotide sequences for miRNA binding sites with their encoded amino acid residue sequences. The scripts allow for retrieving a set of additional sequences at left and at right from the binding site. The scripts presents all received data in table formats that are easy to analyse further. The predicted data finds utility in molecular and evolutionary biology studies. They find use in studying miRNA binding sites in animals and plants. TmiRUSite and TmiROSite scripts are available for free from authors upon request and at https: //sites.google.com/site/malaheenee/downloads for download.

Prediction of novel pre-microRNAs with high accuracy through boosting and SVM.

PubMed

Zhang, Yuanwei; Yang, Yifan; Zhang, Huan; Jiang, Xiaohua; Xu, Bo; Xue, Yu; Cao, Yunxia; Zhai, Qian; Zhai, Yong; Xu, Mingqing; Cooke, Howard J; Shi, Qinghua

2011-05-15

High-throughput deep-sequencing technology has generated an unprecedented number of expressed short sequence reads, presenting not only an opportunity but also a challenge for prediction of novel microRNAs. To verify the existence of candidate microRNAs, we have to show that these short sequences can be processed from candidate pre-microRNAs. However, it is laborious and time consuming to verify these using existing experimental techniques. Therefore, here, we describe a new method, miRD, which is constructed using two feature selection strategies based on support vector machines (SVMs) and boosting method. It is a high-efficiency tool for novel pre-microRNA prediction with accuracy up to 94.0% among different species. miRD is implemented in PHP/PERL+MySQL+R and can be freely accessed at http://mcg.ustc.edu.cn/rpg/mird/mird.php.

Predicting novel microRNA: a comprehensive comparison of machine learning approaches.

PubMed

Stegmayer, Georgina; Di Persia, Leandro E; Rubiolo, Mariano; Gerard, Matias; Pividori, Milton; Yones, Cristian; Bugnon, Leandro A; Rodriguez, Tadeo; Raad, Jonathan; Milone, Diego H

2018-05-23

The importance of microRNAs (miRNAs) is widely recognized in the community nowadays because these short segments of RNA can play several roles in almost all biological processes. The computational prediction of novel miRNAs involves training a classifier for identifying sequences having the highest chance of being precursors of miRNAs (pre-miRNAs). The big issue with this task is that well-known pre-miRNAs are usually few in comparison with the hundreds of thousands of candidate sequences in a genome, which results in high class imbalance. This imbalance has a strong influence on most standard classifiers, and if not properly addressed in the model and the experiments, not only performance reported can be completely unrealistic but also the classifier will not be able to work properly for pre-miRNA prediction. Besides, another important issue is that for most of the machine learning (ML) approaches already used (supervised methods), it is necessary to have both positive and negative examples. The selection of positive examples is straightforward (well-known pre-miRNAs). However, it is difficult to build a representative set of negative examples because they should be sequences with hairpin structure that do not contain a pre-miRNA. This review provides a comprehensive study and comparative assessment of methods from these two ML approaches for dealing with the prediction of novel pre-miRNAs: supervised and unsupervised training. We present and analyze the ML proposals that have appeared during the past 10 years in literature. They have been compared in several prediction tasks involving two model genomes and increasing imbalance levels. This work provides a review of existing ML approaches for pre-miRNA prediction and fair comparisons of the classifiers with same features and data sets, instead of just a revision of published software tools. The results and the discussion can help the community to select the most adequate bioinformatics approach according to the prediction task at hand. The comparative results obtained suggest that from low to mid-imbalance levels between classes, supervised methods can be the best. However, at very high imbalance levels, closer to real case scenarios, models including unsupervised and deep learning can provide better performance.

Hormonal activation of let-7-C microRNAs via EcR is required for adult Drosophila melanogaster morphology and function

PubMed Central

Chawla, Geetanjali; Sokol, Nicholas S.

2012-01-01

Steroid hormones and their nuclear receptors drive developmental transitions in diverse organisms, including mammals. In this study, we show that the Drosophila steroid hormone 20-hydroxyecdysone (20E) and its nuclear receptor directly activate transcription of the evolutionarily conserved let-7-complex (let-7-C) locus, which encodes the co-transcribed microRNAs miR-100, let-7 and miR-125. These small RNAs post-transcriptionally regulate the expression of target genes, and are required for the remodeling of the Drosophila neuromusculature during the larval-to-adult transition. Deletion of three 20E responsive elements located in the let-7-C locus results in reduced levels of let-7-C microRNAs, leading to neuromuscular and behavioral defects in adults. Given the evolutionary conservation of let-7-C microRNA sequences and temporal expression profiles, these findings indicate that steroid hormone-coupled control of let-7-C microRNAs is part of an ancestral pathway controlling the transition from larval-to-reproductive animal forms. PMID:22510985

Widespread long noncoding RNAs as endogenous target mimics for microRNAs in plants.

PubMed

Wu, Hua-Jun; Wang, Zhi-Min; Wang, Meng; Wang, Xiu-Jie

2013-04-01

Target mimicry is a recently identified regulatory mechanism for microRNA (miRNA) functions in plants in which the decoy RNAs bind to miRNAs via complementary sequences and therefore block the interaction between miRNAs and their authentic targets. Both endogenous decoy RNAs (miRNA target mimics) and engineered artificial RNAs can induce target mimicry effects. Yet until now, only the Induced by Phosphate Starvation1 RNA has been proven to be a functional endogenous microRNA target mimic (eTM). In this work, we developed a computational method and systematically identified intergenic or noncoding gene-originated eTMs for 20 conserved miRNAs in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). The predicted miRNA binding sites were well conserved among eTMs of the same miRNA, whereas sequences outside of the binding sites varied a lot. We proved that the eTMs of miR160 and miR166 are functional target mimics and identified their roles in the regulation of plant development. The effectiveness of eTMs for three other miRNAs was also confirmed by transient agroinfiltration assay.

Epigenetic regulation of oligodendrocyte identity

PubMed Central

Liu, Jia; Casaccia, Patrizia

2010-01-01

The interplay of transcription factors and epigenetic modifiers, including histone modifications, DNA methylation and microRNAs during development is essential for the acquisition of specific cell fates. Here we review the epigenetic “programming” of stem cells into oligodendrocytes, by analyzing three sequential stages of lineage progression. The first transition from pluripotent stem cell to neural precursor is characterized by repression of pluripotency genes and restriction of the lineage potential to the neural fate. The second transition from multipotential precursor to oligodendrocyte progenitor is associated with the progressive loss of plasticity and the repression of neuronal and astrocytic genes. The last step of differentiation of oligodendrocyte progenitors into myelin-forming cells is defined by a model of de-repression of myelin genes. PMID:20227775

A novel chlorophyll a/b binding (Cab) protein gene from petunia which encodes the lower molecular weight Cab precursor protein.

PubMed

Stayton, M M; Black, M; Bedbrook, J; Dunsmuir, P

1986-12-22

The 16 petunia Cab genes which have been characterized are all closely related at the nucleotide sequence level and they encode Cab precursor polypeptides which are similar in sequence and length. Here we describe a novel petunia Cab gene which encodes a unique Cab precursor protein. This protein is a member of the smallest class of Cab precursor proteins for which no gene has previously been assigned in petunia or any other species. The features of this Cab precursor protein are that it is shorter by 2-3 amino acids than the formerly characterized Cab precursors, its transit peptide sequence is unrelated, and the mature polypeptide is significantly diverged at the functionally important N terminus from other petunia Cab proteins. Gene structure also discriminates this gene which is the only intron containing Cab gene in petunia genomic DNA.

Comparative analysis of MicroRNA expression in dog lungs infected with the H3N2 and H5N1 canine influenza viruses.

PubMed

Zheng, Yun; Fu, Xinliang; Wang, Lifang; Zhang, Wenyan; Zhou, Pei; Zhang, Xin; Zeng, Weijie; Chen, Jidang; Cao, Zongxi; Jia, Kun; Li, Shoujun

2018-05-14

MicroRNAs, a class of noncoding RNAs 18 to 23 nucleotides (nt) in length, play critical roles in a wide variety of biological processes. The objective of this study was to examine differences in microRNA expression profiles derived from the lungs of beagle dogs infected with the avian-origin H3N2 canine influenza virus (CIV) or the highly pathogenic avian influenza (HPAI) H5N1 virus (canine-origin isolation strain). After dogs were infected with H3N2 or H5N1, microRNA expression in the lungs was assessed using a deep-sequencing approach. To identify the roles of microRNAs in viral pathogenicity and the host immune response, microRNA target genes were predicted, and their functions were analyzed using bioinformatics software. A total of 229 microRNAs were upregulated in the H5N1 infection group compared with those in the H3N2 infection group, and 166 microRNAs were downregulated. MicroRNA target genes in the H5N1 group were more significantly involved in metabolic pathways, such as glycerolipid metabolism and glycerophospholipid metabolism, than those in the H3N2 group. The inhibition of metabolic pathways may lead to appetite loss, weight loss and weakened immunity. Moreover, miR-485, miR-144, miR-133b, miR-4859-5p, miR-6902-3p, miR-7638, miR-1307-3p and miR-1346 were significantly altered microRNAs that potentially led to the inhibition of innate immune pathways and the heightened pathogenicity of H5N1 compared with that of H3N2 in dogs. This study deepens our understanding of the complex relationships among microRNAs, the influenza virus-mediated immune response and immune injury in dogs. Copyright © 2018 Elsevier Ltd. All rights reserved.

Isolation and identification of gene-specific microRNAs.

PubMed

Lin, Shi-Lung; Chang, Donald C; Ying, Shao-Yao

2013-01-01

Computer programming has identified hundreds of genomic hairpin sequences, many with functions remain to be determined. Because direct transfection of hairpin-like miRNA precursors (pre)-miRNAs in mammalian cells is not always sufficient to trigger effective RNA-induced gene silencing complex (RISC) assembly, a key step for RNA interference (RNAi)-related gene silencing, we developed an intronic miRNA-expressing system to overcome this problem by inserting a hairpin-like pre-miRNA structure into the intron region of a gene and successfully increased the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo. This intronic miRNA biogenesis has been found to depend on a coupled interaction of nascent precursor messenger RNA transcription and intron excision within a specific nuclear region proximal to genomic perichromatin fibrils. The intronic miRNA was transcribed by RNA type II polymerases, coexpressed with a primary gene transcript, and excised out of its encoding gene transcript by intracellular RNA splicing and processing mechanisms. Currently, some ribonuclease III endonucleases have been found to be involved in the processing of spliced introns and probably facilitating the intronic miRNA maturation. Using this miRNA generation system, we have shown for the first time that the intron-derived miRNAs were able to induce strong RNAi effects in not only human and mouse cells but also zebrafishes, chicken embryos, and adult mice. We have also developed an miRNA isolation protocol, based on the complementarity between the designed miRNA and its target gene sequence, to purify and identify the mature miRNAs generated by the intronic miRNA-expressing system. Several intronic miRNA identities and structures are currently confirmed to be active in vitro and in vivo. According to this proven-of-principle method, we now have full knowledge to design pre-miRNA inserts that are more efficient and effective for the intronic miRNA-expressing systems.

Identification of novel microRNAs in Hevea brasiliensis and computational prediction of their targets

PubMed Central

2012-01-01

Background Plants respond to external stimuli through fine regulation of gene expression partially ensured by small RNAs. Of these, microRNAs (miRNAs) play a crucial role. They negatively regulate gene expression by targeting the cleavage or translational inhibition of target messenger RNAs (mRNAs). In Hevea brasiliensis, environmental and harvesting stresses are known to affect natural rubber production. This study set out to identify abiotic stress-related miRNAs in Hevea using next-generation sequencing and bioinformatic analysis. Results Deep sequencing of small RNAs was carried out on plantlets subjected to severe abiotic stress using the Solexa technique. By combining the LeARN pipeline, data from the Plant microRNA database (PMRD) and Hevea EST sequences, we identified 48 conserved miRNA families already characterized in other plant species, and 10 putatively novel miRNA families. The results showed the most abundant size for miRNAs to be 24 nucleotides, except for seven families. Several MIR genes produced both 20-22 nucleotides and 23-27 nucleotides. The two miRNA class sizes were detected for both conserved and putative novel miRNA families, suggesting their functional duality. The EST databases were scanned with conserved and novel miRNA sequences. MiRNA targets were computationally predicted and analysed. The predicted targets involved in "responses to stimuli" and to "antioxidant" and "transcription activities" are presented. Conclusions Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs when the complete genome is not yet available. Our study provided additional information for evolutionary studies and revealed potentially specific regulation of the control of redox status in Hevea. PMID:22330773

In-vivo quantification of primary microRNA processing by Drosha with a luciferase based system

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

Allegra, Danilo; Cooperation Unit 'Mechanisms of Leukemogenesis', B061, DKFZ, Im Neuenheimer Feld 280, 69120 Heidelberg; Mertens, Daniel, E-mail: daniel.mertens@uniklinik-ulm.de

2011-03-25

Research highlights: {yields} Posttranscriptional regulation of miRNA processing is difficult to quantify. {yields} Our in-vivo processing assay can quantify Drosha cleavage in live cells. {yields} It is based on luciferase reporters fused with pri-miRNAs. {yields} The assay validates the processing defect caused by a mutation in pri-16-1. {yields} It is a sensitive method to quantify pri-miRNA cleavage by Drosha in live cells. -- Abstract: The RNAse III Drosha is responsible for the first step of microRNA maturation, the cleavage of primary miRNA to produce the precursor miRNA. Processing by Drosha is finely regulated and influences the amount of mature microRNAmore » in a cell. We describe in the present work a method to quantify Drosha processing activity in-vivo, which is applicable to any microRNA. With respect to other methods for measuring Drosha activity, our system is faster and scalable, can be used with any cellular system and does not require cell sorting or use of radioactive isotopes. This system is useful to study regulation of Drosha activity in physiological and pathological conditions.« less

Identification of a precursor genomic segment that provided a sequence unique to glycophorin B and E genes

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

Onda, M.; Kudo, S.; Fukuda, M.

Human glycophorin A, B, and E (GPA, GPB, and GPE) genes belong to a gene family located at the long arm of chromosome 4. These three genes are homologous from the 5'-flanking sequence to the Alu sequence, which is 1 kb downstream from the exon encoding the transmembrane domain. Analysis of the Alu sequence and flanking direct repeat sequences suggested that the GPA gene most closely resembles the ancestral gene, whereas the GPB and GPE gene arose by homologous recombination within the Alu sequence, acquiring 3' sequences from an unrelated precursor genomic segment. Here the authors describe the identification ofmore » this putative precursor genomic segment. A human genomic library was screened by using the sequence of the 3' region of the GPB gene as a probe. The genomic clones isolated were found to contain an Alu sequence that appeared to be involved in the recombination. Downstream from the Alu sequence, the nucleotide sequence of the precursor genomic segment is almost identical to that of the GPB or GPE gene. In contrast, the upstream sequence of the genomic segment differs entirely from that of the GPA, GPB, and GPE genes. Conservation of the direct repeats flanking the Alu sequence of the genomic segment strongly suggests that the sequence of this genomic segment has been maintained during evolution. This identified genomic segment was found to reside downstream from the GPA gene by both gene mapping and in situ chromosomal localization. The precursor genomic segment was also identified in the orangutan genome, which is known to lack GPB and GPE genes. These results indicate that one of the duplicated ancestral glycophorin genes acquired a unique 3' sequence by unequal crossing-over through its Alu sequence and the further downstream Alu sequence present in the duplicated gene. Further duplication and divergence of this gene yielded the GPB and GPE genes. 37 refs., 5 figs.« less

Analysis of host microRNA function uncovers a role for miR-29b-2-5p in Shigella capture by filopodia

PubMed Central

Silva, Ricardo Jorge; Cruz, Ana Rita; Mano, Miguel

2017-01-01

MicroRNAs play an important role in the interplay between bacterial pathogens and host cells, participating as host defense mechanisms, as well as exploited by bacteria to subvert host cellular functions. Here, we show that microRNAs modulate infection by Shigella flexneri, a major causative agent of bacillary dysentery in humans. Specifically, we characterize the dual regulatory role of miR-29b-2-5p during infection, showing that this microRNA strongly favors Shigella infection by promoting both bacterial binding to host cells and intracellular replication. Using a combination of transcriptome analysis and targeted high-content RNAi screening, we identify UNC5C as a direct target of miR-29b-2-5p and show its pivotal role in the modulation of Shigella binding to host cells. MiR-29b-2-5p, through repression of UNC5C, strongly enhances filopodia formation thus increasing Shigella capture and promoting bacterial invasion. The increase of filopodia formation mediated by miR-29b-2-5p is dependent on RhoF and Cdc42 Rho-GTPases. Interestingly, the levels of miR-29b-2-5p, but not of other mature microRNAs from the same precursor, are decreased upon Shigella replication at late times post-infection, through degradation of the mature microRNA by the exonuclease PNPT1. While the relatively high basal levels of miR-29b-2-5p at the start of infection ensure efficient Shigella capture by host cell filopodia, dampening of miR-29b-2-5p levels later during infection may constitute a bacterial strategy to favor a balanced intracellular replication to avoid premature cell death and favor dissemination to neighboring cells, or alternatively, part of the host response to counteract Shigella infection. Overall, these findings reveal a previously unappreciated role of microRNAs, and in particular miR-29b-2-5p, in the interaction of Shigella with host cells. PMID:28394930

Analysis of host microRNA function uncovers a role for miR-29b-2-5p in Shigella capture by filopodia.

PubMed

Sunkavalli, Ushasree; Aguilar, Carmen; Silva, Ricardo Jorge; Sharan, Malvika; Cruz, Ana Rita; Tawk, Caroline; Maudet, Claire; Mano, Miguel; Eulalio, Ana

2017-04-01

MicroRNAs play an important role in the interplay between bacterial pathogens and host cells, participating as host defense mechanisms, as well as exploited by bacteria to subvert host cellular functions. Here, we show that microRNAs modulate infection by Shigella flexneri, a major causative agent of bacillary dysentery in humans. Specifically, we characterize the dual regulatory role of miR-29b-2-5p during infection, showing that this microRNA strongly favors Shigella infection by promoting both bacterial binding to host cells and intracellular replication. Using a combination of transcriptome analysis and targeted high-content RNAi screening, we identify UNC5C as a direct target of miR-29b-2-5p and show its pivotal role in the modulation of Shigella binding to host cells. MiR-29b-2-5p, through repression of UNC5C, strongly enhances filopodia formation thus increasing Shigella capture and promoting bacterial invasion. The increase of filopodia formation mediated by miR-29b-2-5p is dependent on RhoF and Cdc42 Rho-GTPases. Interestingly, the levels of miR-29b-2-5p, but not of other mature microRNAs from the same precursor, are decreased upon Shigella replication at late times post-infection, through degradation of the mature microRNA by the exonuclease PNPT1. While the relatively high basal levels of miR-29b-2-5p at the start of infection ensure efficient Shigella capture by host cell filopodia, dampening of miR-29b-2-5p levels later during infection may constitute a bacterial strategy to favor a balanced intracellular replication to avoid premature cell death and favor dissemination to neighboring cells, or alternatively, part of the host response to counteract Shigella infection. Overall, these findings reveal a previously unappreciated role of microRNAs, and in particular miR-29b-2-5p, in the interaction of Shigella with host cells.

Genome-Wide Posttranscriptional Dysregulation by MicroRNAs in Human Asthma as Revealed by Frac-seq.

PubMed

Martinez-Nunez, Rocio T; Rupani, Hitasha; Platé, Manuela; Niranjan, Mahesan; Chambers, Rachel C; Howarth, Peter H; Sanchez-Elsner, Tilman

2018-05-16

MicroRNAs are small noncoding RNAs that inhibit gene expression posttranscriptionally, implicated in virtually all biological processes. Although the effect of individual microRNAs is generally studied, the genome-wide role of multiple microRNAs is less investigated. We assessed paired genome-wide expression of microRNAs with total (cytoplasmic) and translational (polyribosome-bound) mRNA levels employing subcellular fractionation and RNA sequencing (Frac-seq) in human primary bronchoepithelium from healthy controls and severe asthmatics. Severe asthma is a chronic inflammatory disease of the airways characterized by poor response to therapy. We found genes (i.e., isoforms of a gene) and mRNA isoforms differentially expressed in asthma, with novel inflammatory and structural pathophysiological mechanisms related to bronchoepithelium disclosed solely by polyribosome-bound mRNAs (e.g., IL1A and LTB genes or ITGA6 and ITGA2 alternatively spliced isoforms). Gene expression (i.e., isoforms of a gene) and mRNA expression analysis revealed different molecular candidates and biological pathways, with differentially expressed polyribosome-bound and total mRNAs also showing little overlap. We reveal a hub of six dysregulated microRNAs accounting for ∼90% of all microRNA targeting, displaying preference for polyribosome-bound mRNAs. Transfection of this hub in bronchial epithelial cells from healthy donors mimicked asthma characteristics. Our work demonstrates extensive posttranscriptional gene dysregulation in human asthma, in which microRNAs play a central role, illustrating the feasibility and importance of assessing posttranscriptional gene expression when investigating human disease. Copyright © 2018 by The American Association of Immunologists, Inc.

Immunological and biochemical characterization of processing products from the neurotensin/neuromedin N precursor in the rat medullary thyroid carcinoma 6-23 cell line.

PubMed Central

Bidard, J N; de Nadai, F; Rovere, C; Moinier, D; Laur, J; Martinez, J; Cuber, J C; Kitabgi, P

1993-01-01

Neurotensin (NT) and neuromedin N (NN) are two related biologically active peptides that are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide starting with an N-terminal signal peptide and containing in its C-terminal region one copy each of NT and NN. NN precedes NT and is separated from it by a Lys-Arg sequence. Two other Lys-Arg sequences flank the N-terminus of NN and the C-terminus of NT. A fourth Lys-Arg sequence occurs near the middle of the precursor and is followed by an NN-like sequence. Finally, an Arg-Arg pair is present within the NT moiety. The four Lys-Arg doublets represent putative processing sites in the precursor molecule. The present study was designed to investigate the post-translational processing of the NT/NN precursor in the rat medullary thyroid carcinoma (rMTC) 6-23 cell line, which synthesizes large amounts of NT upon dexamethasone treatment. Five region-specific antisera recognizing the free N- or C-termini of sequences adjacent to the basic doublets were produced, characterized and used for immunoblotting and radioimmunoassay studies in combination with gel filtration, reverse-phase h.p.l.c. and trypsin digestion of rMTC 6-23 cell extracts. Because two of the antigenic sequences, i.e. NN and the NN-like sequence, start with a lysine residue that is essential for recognition by their respective antisera, a micromethod by which trypsin specifically cleaves at arginine residues was developed. The results show that dexamethasone-treated rMTC 6-23 cells produced comparable amounts of NT, NN and a peptide corresponding to a large N-terminal precursor fragment lacking the NN and NT moieties. This large fragment was purified. N-Terminal sequencing revealed that it started at residue Ser23 of the prepro-NT/NN sequence, and thus established the Cys22-Ser23 bond as the cleavage site of the signal peptide. Two other large N-terminal fragments bearing respectively the NN and NT sequences at their C-termini were present in lower amounts. The NN-like sequence was internal to all the large fragments. There was no evidence for the presence of peptides with the NN-like sequence at their N-termini. This shows that, in rMTC 6-23 cells, the precursor is readily processed at the three Lys-Arg doublets that flank and separate the NT and NN sequences. In contrast, the Lys-Arg doublet that precedes the NN-like sequence is not processed in this system.(ABSTRACT TRUNCATED AT 400 WORDS) Images Figure 3 PMID:8471039

A Common Genetic Variant in the 3′-UTR of Vacuolar H+-ATPase ATP6V0A1 Creates a Micro-RNA Motif to Alter Chromogranin A (CHGA) Processing and Hypertension Risk

PubMed Central

Wei, Zhiyun; Biswas, Nilima; Wang, Lei; Courel, Maite; Zhang, Kuixing; Soler-Jover, Alex; Taupenot, Laurent; O’Connor, Daniel T.

2012-01-01

Background The catecholamine release-inhibitor catestatin and its precursor chromogranin A (CHGA) may constitute “intermediate phenotypes” in analysis of genetic risk for cardiovascular disease such as hypertension. Previously, the vacuolar H+-ATPase subunit gene ATP6V0A1 was found within the confidence interval for linkage with catestatin secretion in a genome-wide study, and its 3′-UTR polymorphism T+3246C (rs938671) was associated with both catestatin processing from CHGA, as well as population blood pressure (BP). Here we explored the molecular mechanism of this effect by experiments with transfected chimeric photoproteins in chromaffin cells. Methods and Results Placing the ATP6V0A1 3′-UTR downstream of a luciferase reporter, we found that the C (variant) allele decreased overall gene expression. The 3′-UTR effect was verified by coupled in vitro transcription/translation of the entire/intact human ATP6V0A1 mRNA. Chromaffin granule pH, monitored by fluorescence a CHGA/EGFP chimera during vesicular H+-ATPase inhibition by bafilomycin A1, was more easily perturbed during co-expression of the ATP6V0A1 3′-UTR C-allele than the T-allele. After bafilomycin A1 treatment, the ratio of CHGA precursor to its catestatin fragments in PC12 cells was substantially diminished, though the qualitative composition of such fragments was not affected (on immunoblot or MALDI mass spectrometry). Bafilomycin A1 treatment also decreased exocytotic secretion from the regulated pathway, monitored by a CHGA chimera tagged with embryonic alkaline phosphatase (EAP). 3′-UTR T+3246C created a binding motif for micro-RNA hsa-miR-637; co-transfection of hsa-miR-637 precursor or antagomir/inhibitor oligonucleotides yielded the predicted changes in expression of luciferase reporter/ATP6V0A1-3′-UTR plasmids varying at T+3246C. Conclusions The results suggest a series of events whereby ATP6V0A1 3′-UTR variant T+3246C functioned: ATP6V0A1 expression was affected likely through differential micro-RNA effects, altering vacuolar pH and consequently CHGA processing and exocytotic secretion. PMID:21558123

Ribosome-targeting antibiotics as inhibitors of oncogenic microRNAs biogenesis: Old scaffolds for new perspectives in RNA targeting.

PubMed

Tran, Thi Phuong Anh; Vo, Duc Duy; Di Giorgio, Audrey; Duca, Maria

2015-09-01

MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression at the post-transcriptional level. It is now well established that the overexpression of some miRNAs (oncogenic miRNAs) is responsible for initiation and progression of human cancers and the discovery of new molecules able to interfere with their production and/or function represents one of the most important challenges of current medicinal chemistry of RNA ligands. In this work, we studied the ability of 18 different antibiotics, known as prokaryotic ribosomal RNA, to bind to oncogenic miRNA precursors (stem-loop structured pre-miRNAs) in order to inhibit miRNAs production. In vitro inhibition, binding constants, thermodynamic parameters and binding sites were investigated and highlighted that aminoglycosides and tetracyclines represent interesting pre-miRNA ligands with the ability to inhibit Dicer processing. Copyright © 2015 Elsevier Ltd. All rights reserved.

Small molecule chemical probes of microRNA function.

PubMed

Velagapudi, Sai Pradeep; Vummidi, Balayeshwanth R; Disney, Matthew D

2015-02-01

MicroRNAs (miRNAs) are small, non-coding RNAs that control protein expression. Aberrant miRNA expression has been linked to various human diseases, and thus miRNAs have been explored as diagnostic markers and therapeutic targets. Although it is challenging to target RNA with small molecules in general, there have been successful campaigns that have identified small molecule modulators of miRNA function by targeting various pathways. For example, small molecules that modulate transcription and target nuclease processing sites in miRNA precursors have been identified. Herein, we describe challenges in developing chemical probes that target miRNAs and highlight aspects of miRNA cellular biology elucidated by using small molecule chemical probes. We expect that this area will expand dramatically in the near future as progress is made in understanding small molecule recognition of RNA. Copyright © 2014. Published by Elsevier Ltd.

Human cytomegalovirus microRNA miR-US4-1 inhibits CD8+ T cell response by targeting the aminopeptidase ERAP1

PubMed Central

Kim, Sungchul; Lee, Sanghyun; Shin, Jinwook; Kim, Youngkyun; Evnouchidou, Irini; Kim, Donghyun; Kim, Young-Kook; Kim, Young-Eui; Ahn, Jin-Hyun; Riddell, Stanley R.; Stratikos, Efstratios; Kim, V. Narry; Ahn, Kwangseog

2012-01-01

The major histocompatibility complex (MHC) class I molecules present peptides on the cell surface by CD8+ T cells, which is critical for killing of virally infected or transformed cells. Precursors of MHC class I-presented peptides are trimmed to mature epitopes by endoplasmic reticulum aminopeptidase 1 (ERAP1). The US2-US11 genomic region of human cytomegalovirus (HCMV) is dispensable for viral replication and harbors 3 microRNAs (miRNAs). We show here the HCMV miR-US4-1 specifically down-regulates ERAP1 expression during viral infection. Accordingly, the trimming of HCMV-derived peptides is inhibited, leading to reduced susceptibility of infected cells to HCMV-specific cytotoxic T lymphocytes (CTLs). Our findings reveal a novel viral miRNA-based CTL evasion mechanism that targets a key step in the MHC class I antigen-processing pathway. PMID:21892175

Characterization of skin ulceration syndrome associated microRNAs in sea cucumber Apostichopus japonicus by deep sequencing.

PubMed

Li, Chenghua; Feng, Weida; Qiu, Lihua; Xia, Changge; Su, Xiurong; Jin, Chunhua; Zhou, Tingting; Zeng, Yuan; Li, Taiwu

2012-08-01

MicroRNAs (miRNAs) constitute a family of small RNA species which have been demonstrated to be one of key effectors in mediating host-pathogen interaction. In this study, two haemocytes miRNA libraries were constructed with deep sequenced by illumina Hiseq2000 from healthy (L1) and skin ulceration syndrome Apostichopus japonicus (L2). The high throughput solexa sequencing resulted in 9,579,038 and 7,742,558 clean data from L1 and L2, respectively. Sequences analysis revealed that 40 conserved miRNAs were found in both libraries, in which let-7 and mir-125 were speculated to be clustered together and expressed accordingly. Eighty-six miRNA candidates were also identified by reference genome search and stem-loop structure prediction. Importantly, mir-31 and mir-2008 displayed significant differential expression between the two libraries according to FPKM model, which might be considered as promising targets for elucidating the intrinsic mechanism of skin ulceration syndrome outbreak in the species. Copyright © 2012 Elsevier Ltd. All rights reserved.

The RNA-binding region of human TRBP interacts with microRNA precursors through two independent domains

PubMed Central

Benoit, Matthieu P. M. H.; Imbert, Lionel; Palencia, Andrés; Pérard, Julien; Ebel, Christine; Boisbouvier, Jérôme; Plevin, Michael J.

2013-01-01

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through RNA interference. Human miRNAs are generated through a series of enzymatic processing steps. The precursor miRNA (pre-miRNA) is recognized and cleaved by a complex containing Dicer and several non-catalytic accessory proteins. HIV TAR element binding protein (TRBP) is a constituent of the Dicer complex, which augments complex stability and potentially functions in substrate recognition and product transfer to the RNA-induced silencing complex. Here we have analysed the interaction between the RNA-binding region of TRBP and an oncogenic human miRNA, miR-155, at different stages in the biogenesis pathway. We show that the region of TRBP that binds immature miRNAs comprises two independent double-stranded RNA-binding domains connected by a 60-residue flexible linker. No evidence of contact between the two double-stranded RNA-binding domains was observed either in the apo- or RNA-bound state. We establish that the RNA-binding region of TRBP interacts with both pre-miR-155 and the miR-155/miR-155* duplex through the same binding surfaces and with similar affinities, and that two protein molecules can simultaneously interact with each immature miRNA. These data suggest that TRBP could play a role before and after processing of pre-miRNAs by Dicer. PMID:23435228

The construction and partial characterization of plasmids containing complementary DNA sequences to human calcitonin precursor polyprotein.

PubMed Central

Allison, J; Hall, L; MacIntyre, I; Craig, R K

1981-01-01

(1) Total poly(A)-containing RNA isolated from human thyroid medullary carcinoma tissue was shown to direct the synthesis in the wheat germ cell-free system of a major (Mr 21000) and several minor forms of human calcitonin precursor polyproteins. Evidence for processing of these precursor(s) by the wheat germ cell-free system is also presented. (2) A small complementary DNA (cDNA) plasmid library has been constructed in the PstI site of the plasmid pAT153, using total human thyroid medullary carcinoma poly(A)-containing RNA as the starting material. (3) Plasmids containing abundant cDNA sequences were selected by hybridization in situ, and two of these (ph T-B3 and phT-B6) were characterized by hybridization--translation and restriction analysis. Each was shown to contain human calcitonin precursor polyprotein cDNA sequences. (4) RNA blotting techniques demonstrate that the human calcitonin precursor polyprotein is encoded within a mRNA containing 1000 bases. (5) The results demonstrate that human calcitonin is synthesized as a precursor polyprotein. Images Fig. 1. Fig. 2. Fig. 3. PMID:6896146

Viability, Longevity, and Egg Production of Drosophila melanogaster Are Regulated by the miR-282 microRNA

PubMed Central

Vilmos, Péter; Bujna, Ágnes; Szuperák, Milán; Havelda, Zoltán; Várallyay, Éva; Szabad, János; Kucerova, Lucie; Somogyi, Kálmán; Kristó, Ildikó; Lukácsovich, Tamás; Jankovics, Ferenc; Henn, László; Erdélyi, Miklós

2013-01-01

The first microRNAs were discovered some 20 years ago, but only a small fraction of the microRNA-encoding genes have been described in detail yet. Here we report the molecular analysis of a computationally predicted Drosophila melanogaster microRNA gene, mir-282. We show that the mir-282 gene is the source of a 4.9-kb-long primary transcript with a 5′ cap and a 3′-poly(A) sequence and a mature microRNA of ∼25 bp. Our data strongly suggest the existence of an independent mir-282 gene conserved in holometabolic insects. We give evidence that the mir-282 locus encodes a functional transcript that influences viability, longevity, and egg production in Drosophila. We identify the nervous system-specific adenylate cyclase (rutabaga) as a target of miR-282 and assume that one of the main functions of mir-282 is the regulation of adenylate cyclase activity in the nervous system during metamorphosis. PMID:23852386

Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

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

Chattopadhyay, Saket; Ely, Abdullah; Bloom, Kristie

2009-11-20

RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR)more » shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.« less

Coordination of Myeloid Differentiation with Reduced Cell Cycle Progression by PU.1 Induction of MicroRNAs Targeting Cell Cycle Regulators and Lipid Anabolism.

PubMed

Solomon, Lauren A; Podder, Shreya; He, Jessica; Jackson-Chornenki, Nicholas L; Gibson, Kristen; Ziliotto, Rachel G; Rhee, Jess; DeKoter, Rodney P

2017-05-15

During macrophage development, myeloid progenitor cells undergo terminal differentiation coordinated with reduced cell cycle progression. Differentiation of macrophages from myeloid progenitors is accompanied by increased expression of the E26 transformation-specific transcription factor PU.1. Reduced PU.1 expression leads to increased proliferation and impaired differentiation of myeloid progenitor cells. It is not understood how PU.1 coordinates macrophage differentiation with reduced cell cycle progression. In this study, we utilized cultured PU.1-inducible myeloid cells to perform genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis coupled with gene expression analysis to determine targets of PU.1 that may be involved in regulating cell cycle progression. We found that genes encoding cell cycle regulators and enzymes involved in lipid anabolism were directly and inducibly bound by PU.1 although their steady-state mRNA transcript levels were reduced. Inhibition of lipid anabolism was sufficient to reduce cell cycle progression in these cells. Induction of PU.1 reduced expression of E2f1 , an important activator of genes involved in cell cycle and lipid anabolism, indirectly through microRNA 223. Next-generation sequencing identified microRNAs validated as targeting cell cycle and lipid anabolism for downregulation. These results suggest that PU.1 coordinates cell cycle progression with differentiation through induction of microRNAs targeting cell cycle regulators and lipid anabolism. Copyright © 2017 American Society for Microbiology.

MicroRNA miR-27b rescues bone marrow-derived angiogenic cell function and accelerates wound healing in type 2 diabetes mellitus.

PubMed

Wang, Jie-Mei; Tao, Jun; Chen, Dan-Dan; Cai, Jing-Jing; Irani, Kaikobad; Wang, Qinde; Yuan, Hong; Chen, Alex F

2014-01-01

Vascular precursor cells with angiogenic potentials are important for tissue repair, which is impaired in diabetes mellitus. MicroRNAs are recently discovered key regulators of gene expression, but their role in vascular precursor cell-mediated angiogenesis in diabetes mellitus is unknown. We tested the hypothesis that the microRNA miR-27b rescues impaired bone marrow-derived angiogenic cell (BMAC) function in vitro and in vivo in type 2 diabetic mice. BMACs from adult male type 2 diabetic db/db and from normal littermate db/+ mice were used. miR-27b expression was decreased in db/db BMACs. miR-27b mimic improved db/db BMAC function, including proliferation, adhesion, tube formation, and delayed apoptosis, but it did not affect migration. Elevated thrombospondin-1 (TSP-1) protein in db/db BMACs was suppressed on miR-27b mimic transfection. Inhibition of miR-27b in db/+ BMACs reduced angiogenesis, which was reversed by TSP-1 small interfering RNA (siRNA). miR-27b suppressed the pro-oxidant protein p66(shc) and mitochondrial oxidative stress, contributing to its protection of BMAC function. miR-27b also suppressed semaphorin 6A to improve BMAC function in diabetes mellitus. Luciferase binding assay suggested that miR-27b directly targeted TSP-1, TSP-2, p66(shc), and semaphorin 6A. miR-27b improved topical cell therapy of diabetic BMACs on diabetic skin wound closure, with a concomitant augmentation of wound perfusion and capillary formation. Normal BMAC therapy with miR-27b inhibition demonstrated reduced efficacy in wound closure, perfusion, and capillary formation. Local miR-27b delivery partly improved wound healing in diabetic mice. miR-27b rescues impaired BMAC angiogenesis via TSP-1 suppression, semaphorin 6A expression, and p66shc-dependent mitochondrial oxidative stress and improves BMAC therapy in wound healing in type 2 diabetic mice.

Cocaine Modulates the Expression of Opioid Receptors and miR-let-7d in Zebrafish Embryos

PubMed Central

López-Bellido, Roger; Barreto-Valer, Katherine; Sánchez-Simón, Fátima Macho; Rodríguez, Raquel E.

2012-01-01

Prenatal exposure to cocaine, in mammals, has been shown to interfere with the expression of opioid receptors, which can have repercussions in its activity. Likewise, microRNAs, such as let-7, have been shown to regulate the expression of opioid receptors and hence their functions in mammals and in vitro experiments. In light of this, using the zebrafish embryos as a model our aim here was to evaluate the actions of cocaine in the expression of opioid receptors and let-7d miRNA during embryogenesis. In order to determine the effects produced by cocaine on the opioid receptors (zfmor, zfdor1 and zfdor2) and let-7d miRNA (dre-let-7d) and its precursors (dre-let-7d-1 and dre-let-7d-2), embryos were exposed to 1.5 µM cocaine hydrochloride (HCl). Our results revealed that cocaine upregulated dre-let-7d and its precursors, and also increased the expression of zfmor, zfdor1 and zfdor2 during early developmental stages and decreased them in late embryonic stages. The changes observed in the expression of opioid receptors might occur through dre-let-7d, since DNA sequences and the morpholinos of opioid receptors microinjections altered the expression of dre-let-7d and its precursors. Likewise, opioid receptors and dre-let-7d showed similar distributions in the central nervous system (CNS) and at the periphery, pointing to a possible interrelationship between them. In conclusion, the silencing and overexpression of opioid receptors altered the expression of dre-let-7d, which points to the notion that cocaine via dre-let-7 can modulate the expression of opioid receptors. Our study provides new insights into the actions of cocaine during zebrafish embryogenesis, indicating a role of miRNAs, let-7d, in development and its relationship with gene expression of opioid receptors, related to pain and addiction process. PMID:23226419

Evaluation of microRNA alignment techniques

PubMed Central

Kaspi, Antony; El-Osta, Assam

2016-01-01

Genomic alignment of small RNA (smRNA) sequences such as microRNAs poses considerable challenges due to their short length (∼21 nucleotides [nt]) as well as the large size and complexity of plant and animal genomes. While several tools have been developed for high-throughput mapping of longer mRNA-seq reads (>30 nt), there are few that are specifically designed for mapping of smRNA reads including microRNAs. The accuracy of these mappers has not been systematically determined in the case of smRNA-seq. In addition, it is unknown whether these aligners accurately map smRNA reads containing sequence errors and polymorphisms. By using simulated read sets, we determine the alignment sensitivity and accuracy of 16 short-read mappers and quantify their robustness to mismatches, indels, and nontemplated nucleotide additions. These were explored in the context of a plant genome (Oryza sativa, ∼500 Mbp) and a mammalian genome (Homo sapiens, ∼3.1 Gbp). Analysis of simulated and real smRNA-seq data demonstrates that mapper selection impacts differential expression results and interpretation. These results will inform on best practice for smRNA mapping and enable more accurate smRNA detection and quantification of expression and RNA editing. PMID:27284164

Widespread Long Noncoding RNAs as Endogenous Target Mimics for MicroRNAs in Plants1[W

PubMed Central

Wu, Hua-Jun; Wang, Zhi-Min; Wang, Meng; Wang, Xiu-Jie

2013-01-01

Target mimicry is a recently identified regulatory mechanism for microRNA (miRNA) functions in plants in which the decoy RNAs bind to miRNAs via complementary sequences and therefore block the interaction between miRNAs and their authentic targets. Both endogenous decoy RNAs (miRNA target mimics) and engineered artificial RNAs can induce target mimicry effects. Yet until now, only the Induced by Phosphate Starvation1 RNA has been proven to be a functional endogenous microRNA target mimic (eTM). In this work, we developed a computational method and systematically identified intergenic or noncoding gene-originated eTMs for 20 conserved miRNAs in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). The predicted miRNA binding sites were well conserved among eTMs of the same miRNA, whereas sequences outside of the binding sites varied a lot. We proved that the eTMs of miR160 and miR166 are functional target mimics and identified their roles in the regulation of plant development. The effectiveness of eTMs for three other miRNAs was also confirmed by transient agroinfiltration assay. PMID:23429259

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

Muhlheim, M.D.; Belles, R.J.; Cletcher, J.W.

The Accident Sequence Precursor (ASP) Program involves the systematic review and evaluation of operational events that have occurred at light-water reactors to identify and categorize precursors to potential severe core damage accident sequences. The results of the ASP Program are published in an annual report. The most recent report, which contains the precursors for 1995, is NUREG/CR-4674, Volume 23, Precursors to Potential Severe Core Damage Accidents: 1995, A Status Report, published in April 1997. This article provides an overview of the ASP review and evaluation process and a summary of the results for 1995.

MicroRNA Targets of Human Androgen Receptor

DTIC Science & Technology

2013-05-01

Clin 62, 10- 29. Siegel R., Naishadham D. & Jemal A. (2013) Cancer statistics, 2013. CA Cancer J Clin 63, 11- 30. Sikand K., Barik S. & Shukla G.C...Wongvipat J ., Arora V.K., Watson P.A., Chen Y., Greene G.L., Shen Y. & Sawyers C.L. (2013) Overcoming mutation-based resistance to antiandrogens...Marzo A.M., Meeker A.K., Zha S., Luo J ., Nakayama M., Platz E.A., Isaacs W.B. & Nelson W.G. (2003) Human prostate cancer precursors and pathobiology

Phylogenetic Relationship of Necoclí Virus to Other South American Hantaviruses (Bunyaviridae: Hantavirus).

PubMed

Montoya-Ruiz, Carolina; Cajimat, Maria N B; Milazzo, Mary Louise; Diaz, Francisco J; Rodas, Juan David; Valbuena, Gustavo; Fulhorst, Charles F

2015-07-01

The results of a previous study suggested that Cherrie's cane rat (Zygodontomys cherriei) is the principal host of Necoclí virus (family Bunyaviridae, genus Hantavirus) in Colombia. Bayesian analyses of complete nucleocapsid protein gene sequences and complete glycoprotein precursor gene sequences in this study confirmed that Necoclí virus is phylogenetically closely related to Maporal virus, which is principally associated with the delicate pygmy rice rat (Oligoryzomys delicatus) in western Venezuela. In pairwise comparisons, nonidentities between the complete amino acid sequence of the nucleocapsid protein of Necoclí virus and the complete amino acid sequences of the nucleocapsid proteins of other hantaviruses were ≥8.7%. Likewise, nonidentities between the complete amino acid sequence of the glycoprotein precursor of Necoclí virus and the complete amino acid sequences of the glycoprotein precursors of other hantaviruses were ≥11.7%. Collectively, the unique association of Necoclí virus with Z. cherriei in Colombia, results of the Bayesian analyses of complete nucleocapsid protein gene sequences and complete glycoprotein precursor gene sequences, and results of the pairwise comparisons of amino acid sequences strongly support the notion that Necoclí virus represents a novel species in the genus Hantavirus. Further work is needed to determine whether Calabazo virus (a hantavirus associated with Z. brevicauda cherriei in Panama) and Necoclí virus are conspecific.

Comprehensive modeling of microRNA targets predicts functional non-conserved and non-canonical sites.

PubMed

Betel, Doron; Koppal, Anjali; Agius, Phaedra; Sander, Chris; Leslie, Christina

2010-01-01

mirSVR is a new machine learning method for ranking microRNA target sites by a down-regulation score. The algorithm trains a regression model on sequence and contextual features extracted from miRanda-predicted target sites. In a large-scale evaluation, miRanda-mirSVR is competitive with other target prediction methods in identifying target genes and predicting the extent of their downregulation at the mRNA or protein levels. Importantly, the method identifies a significant number of experimentally determined non-canonical and non-conserved sites.

Normalization for Relative Quantification of mRNA and microRNA in Soybean Exposed to Various Abiotic Stresses

PubMed Central

Zhou, Yonggang; Chen, Huan; Dong, Yuanyuan; Wang, Nan; Li, Xiaowei; Jameel, Aysha; Yang, He; Zhang, Min; Chen, Kai; Wang, Fawei; Li, Haiyan

2016-01-01

Plant microRNAs are small non-coding, endogenic RNA molecule (containing 20–24 nucleotides) produced from miRNA precursors (pri-miRNA and pre-miRNA). Evidence suggests that up and down regulation of the miRNA targets the mRNA genes involved in resistance against biotic and abiotic stresses. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful technique to analyze variations in mRNA levels. Normalizing the data using reference genes is essential for the analysis of reliable RT-qPCR data. In this study, two groups of candidate reference mRNAs and miRNAs in soybean leaves and roots treated with various abiotic stresses (PEG-simulated drought, salinity, alkalinity, salinity+alkalinity, and abscisic acid) were analyzed by RT-qPCR. We analyzed the most appropriate reference mRNA/miRNAs using the geNorm, NormFinder, and BestKeeper algorithms. According to the results, Act and EF1b were the most suitable reference mRNAs in leaf and root samples, for mRNA and miRNA precursor data normalization. The most suitable reference miRNAs found in leaf and root samples were 166a and 167a for mature miRNA data normalization. Hence the best combinations of reference mRNAs for mRNA and miRNA precursor data normalization were EF1a + Act or EF1b + Act in leaf samples, and EF1a + EF1b or 60s + EF1b in root samples. For mature miRNA data normalization, the most suitable combinations of reference miRNAs were 166a + 167d in leaf samples, and 171a + 156a or 167a + 171a in root samples. We identified potential reference mRNA/miRNAs for accurate RT-qPCR data normalization for mature miRNA, miRNA precursors, and their targeted mRNAs. Our results promote miRNA-based studies on soybean plants exposed to abiotic stress conditions. PMID:27176476

Processing of the precursor of protamine P2 in mouse. Peptide mapping and N-terminal sequence analysis of intermediates.

PubMed Central

Carré-Eusèbe, D; Lederer, F; Lê, K H; Elsevier, S M

1991-01-01

Protamine P2, the major basic chromosomal protein of mouse spermatozoa, is synthesized as a precursor almost twice as long as the mature protein, its extra length arising from an N-terminal extension of 44 amino acid residues. This precursor is integrated into chromatin of spermatids, and the extension is processed during chromatin condensation in the haploid cells. We have studied processing in the mouse and have identified two intermediates generated by proteolytic cleavage of the precursor. H.p.l.c. separated protamine P2 from four other spermatid proteins, including the precursor and three proteins known to possess physiological characteristics expected of processing intermediates. Peptide mapping indicated that all of these proteins were structurally similar. Two major proteins were further purified by PAGE, transferred to poly(vinylidene difluoride) membranes and submitted to automated N-terminal sequence analysis. Both sequences were found within the deduced sequence of the precursor extension. The N-terminus of the larger intermediate, PP2C, was Gly-12, whereas the N-terminus of the smaller, PP2D, was His-21. Both processing sites involved a peptide bond in which the carbonyl function was contributed by an acidic amino acid. Images Fig. 1. Fig. 3. Fig. 4. PMID:1854346

TargetSpy: a supervised machine learning approach for microRNA target prediction.

PubMed

Sturm, Martin; Hackenberg, Michael; Langenberger, David; Frishman, Dmitrij

2010-05-28

Virtually all currently available microRNA target site prediction algorithms require the presence of a (conserved) seed match to the 5' end of the microRNA. Recently however, it has been shown that this requirement might be too stringent, leading to a substantial number of missed target sites. We developed TargetSpy, a novel computational approach for predicting target sites regardless of the presence of a seed match. It is based on machine learning and automatic feature selection using a wide spectrum of compositional, structural, and base pairing features covering current biological knowledge. Our model does not rely on evolutionary conservation, which allows the detection of species-specific interactions and makes TargetSpy suitable for analyzing unconserved genomic sequences.In order to allow for an unbiased comparison of TargetSpy to other methods, we classified all algorithms into three groups: I) no seed match requirement, II) seed match requirement, and III) conserved seed match requirement. TargetSpy predictions for classes II and III are generated by appropriate postfiltering. On a human dataset revealing fold-change in protein production for five selected microRNAs our method shows superior performance in all classes. In Drosophila melanogaster not only our class II and III predictions are on par with other algorithms, but notably the class I (no-seed) predictions are just marginally less accurate. We estimate that TargetSpy predicts between 26 and 112 functional target sites without a seed match per microRNA that are missed by all other currently available algorithms. Only a few algorithms can predict target sites without demanding a seed match and TargetSpy demonstrates a substantial improvement in prediction accuracy in that class. Furthermore, when conservation and the presence of a seed match are required, the performance is comparable with state-of-the-art algorithms. TargetSpy was trained on mouse and performs well in human and drosophila, suggesting that it may be applicable to a broad range of species. Moreover, we have demonstrated that the application of machine learning techniques in combination with upcoming deep sequencing data results in a powerful microRNA target site prediction tool http://www.targetspy.org.

TargetSpy: a supervised machine learning approach for microRNA target prediction

PubMed Central

2010-01-01

Background Virtually all currently available microRNA target site prediction algorithms require the presence of a (conserved) seed match to the 5' end of the microRNA. Recently however, it has been shown that this requirement might be too stringent, leading to a substantial number of missed target sites. Results We developed TargetSpy, a novel computational approach for predicting target sites regardless of the presence of a seed match. It is based on machine learning and automatic feature selection using a wide spectrum of compositional, structural, and base pairing features covering current biological knowledge. Our model does not rely on evolutionary conservation, which allows the detection of species-specific interactions and makes TargetSpy suitable for analyzing unconserved genomic sequences. In order to allow for an unbiased comparison of TargetSpy to other methods, we classified all algorithms into three groups: I) no seed match requirement, II) seed match requirement, and III) conserved seed match requirement. TargetSpy predictions for classes II and III are generated by appropriate postfiltering. On a human dataset revealing fold-change in protein production for five selected microRNAs our method shows superior performance in all classes. In Drosophila melanogaster not only our class II and III predictions are on par with other algorithms, but notably the class I (no-seed) predictions are just marginally less accurate. We estimate that TargetSpy predicts between 26 and 112 functional target sites without a seed match per microRNA that are missed by all other currently available algorithms. Conclusion Only a few algorithms can predict target sites without demanding a seed match and TargetSpy demonstrates a substantial improvement in prediction accuracy in that class. Furthermore, when conservation and the presence of a seed match are required, the performance is comparable with state-of-the-art algorithms. TargetSpy was trained on mouse and performs well in human and drosophila, suggesting that it may be applicable to a broad range of species. Moreover, we have demonstrated that the application of machine learning techniques in combination with upcoming deep sequencing data results in a powerful microRNA target site prediction tool http://www.targetspy.org. PMID:20509939

Passenger strand loading in overexpression experiments using microRNA mimics.

PubMed

Søkilde, Rolf; Newie, Inga; Persson, Helena; Borg, Åke; Rovira, Carlos

2015-01-01

MicroRNAs (miRNAs) are important regulators of gene function and manipulation of miRNAs is a central component of basic research. Modulation of gene expression by miRNA gain-of-function can be based on different approaches including transfection with miRNA mimics; artificial, chemically modified miRNA-like small RNAs. These molecules are intended to mimic the function of a miRNA guide strand while bypassing the maturation steps of endogenous miRNAs. Due to easy accessibility through commercial providers this approach has gained popularity, and accuracy is often assumed without prior independent testing. Our in silico analysis of over-represented sequence motifs in microarray expression data and sequencing of AGO-associated small RNAs indicate, however, that miRNA mimics may be associated with considerable side-effects due to the unwanted activity of the miRNA mimic complementary strand.

The proteolytic processing site of the precursor of lysyl oxidase.

PubMed Central

Cronshaw, A D; Fothergill-Gilmore, L A; Hulmes, D J

1995-01-01

The precise cleavage site of the N-terminal propeptide region of the precursor of lysyl oxidase has not yet been established, due to N-terminal blocking of the mature protein. Using a combination of peptide fragmentation, amino acid sequencing, time-of-flight m.s. and partial chemical unblocking procedures, it is shown that the mature form of lysyl oxidase begins at residue Asp-169 of the precursor protein (numbered according to the human sequence). The cleavage site is 28 residues to the C-terminal side of the site previously suggested on the basis of apparant molecular mass by SDS/PAGE, with the consequence that the two putative, N-linked glycosylation sites and the position of the Arg/Gln sequence polymorphism are now all in the precursor region. PMID:7864821

Coevolution Pattern and Functional Conservation or Divergence of miR167s and their targets across Diverse Plant Species

PubMed Central

Barik, Suvakanta; Kumar, Ashutosh; Sarkar Das, Shabari; Yadav, Sandeep; Gautam, Vibhav; Singh, Archita; Singh, Sharmila; Sarkar, Ananda K.

2015-01-01

microRNAs (miRNAs), a class of endogenously produced small non-coding RNAs of 20–21 nt length, processed from precursor miRNAs, regulate many developmental processes by negatively regulating the target genes in both animals and plants. The coevolutionary pattern of a miRNA family and their targets underscores its functional conservation or diversification. The miR167 regulates various aspects of plant development in Arabidopsis by targeting ARF6 and ARF8. The evolutionary conservation or divergence of miR167s and their target genes are poorly understood till now. Here we show the evolutionary relationship among 153 MIR167 genes obtained from 33 diverse plant species. We found that out of the 153 of miR167 sequences retrieved from the “miRBase”, 27 have been annotated to be processed from the 3′ end, and have diverged distinctively from the other miR167s produced from 5′ end. Our analysis reveals that gma-miR167h/i and mdm-miR167a are processed from 3′ end and have evolved separately, diverged most resulting in novel targets other than their known ones, and thus led to functional diversification, especially in apple and soybean. We also show that mostly conserved miR167 sequences and their target AUXIN RESPONSE FACTORS (ARFs) have gone through parallel evolution leading to functional diversification among diverse plant species. PMID:26459056

Coevolution Pattern and Functional Conservation or Divergence of miR167s and their targets across Diverse Plant Species.

PubMed

Barik, Suvakanta; Kumar, Ashutosh; Sarkar Das, Shabari; Yadav, Sandeep; Gautam, Vibhav; Singh, Archita; Singh, Sharmila; Sarkar, Ananda K

2015-10-13

microRNAs (miRNAs), a class of endogenously produced small non-coding RNAs of 20-21 nt length, processed from precursor miRNAs, regulate many developmental processes by negatively regulating the target genes in both animals and plants. The coevolutionary pattern of a miRNA family and their targets underscores its functional conservation or diversification. The miR167 regulates various aspects of plant development in Arabidopsis by targeting ARF6 and ARF8. The evolutionary conservation or divergence of miR167s and their target genes are poorly understood till now. Here we show the evolutionary relationship among 153 MIR167 genes obtained from 33 diverse plant species. We found that out of the 153 of miR167 sequences retrieved from the "miRBase", 27 have been annotated to be processed from the 3' end, and have diverged distinctively from the other miR167s produced from 5' end. Our analysis reveals that gma-miR167h/i and mdm-miR167a are processed from 3' end and have evolved separately, diverged most resulting in novel targets other than their known ones, and thus led to functional diversification, especially in apple and soybean. We also show that mostly conserved miR167 sequences and their target AUXIN RESPONSE FACTORS (ARFs) have gone through parallel evolution leading to functional diversification among diverse plant species.

Deep Sequence Analysis of AgoshRNA Processing Reveals 3' A Addition and Trimming.

PubMed

Harwig, Alex; Herrera-Carrillo, Elena; Jongejan, Aldo; van Kampen, Antonius Hubertus; Berkhout, Ben

2015-07-14

The RNA interference (RNAi) pathway, in which microprocessor and Dicer collaborate to process microRNAs (miRNA), was recently expanded by the description of alternative processing routes. In one of these noncanonical pathways, Dicer action is replaced by the Argonaute2 (Ago2) slicer function. It was recently shown that the stem-length of precursor-miRNA or short hairpin RNA (shRNA) molecules is a major determinant for Dicer versus Ago2 processing. Here we present the results of a deep sequence study on the processing of shRNAs with different stem length and a top G·U wobble base pair (bp). This analysis revealed some unexpected properties of these so-called AgoshRNA molecules that are processed by Ago2 instead of Dicer. First, we confirmed the gradual shift from Dicer to Ago2 processing upon shortening of the hairpin length. Second, hairpins with a stem larger than 19 base pair are inefficiently cleaved by Ago2 and we noticed a shift in the cleavage site. Third, the introduction of a top G·U bp in a regular shRNA can promote Ago2-cleavage, which coincides with a loss of Ago2-loading of the Dicer-cleaved 3' strand. Fourth, the Ago2-processed AgoshRNAs acquire a short 3' tail of 1-3 A-nucleotides (nt) and we present evidence that this product is subsequently trimmed by the poly(A)-specific ribonuclease (PARN).

Deep Sequence Analysis of AgoshRNA Processing Reveals 3' A Addition and Trimming

PubMed Central

Harwig, Alex; Herrera-Carrillo, Elena; Jongejan, Aldo; van Kampen, Antonius Hubertus; Berkhout, Ben

2015-01-01

The RNA interference (RNAi) pathway, in which microprocessor and Dicer collaborate to process microRNAs (miRNA), was recently expanded by the description of alternative processing routes. In one of these noncanonical pathways, Dicer action is replaced by the Argonaute2 (Ago2) slicer function. It was recently shown that the stem-length of precursor-miRNA or short hairpin RNA (shRNA) molecules is a major determinant for Dicer versus Ago2 processing. Here we present the results of a deep sequence study on the processing of shRNAs with different stem length and a top G·U wobble base pair (bp). This analysis revealed some unexpected properties of these so-called AgoshRNA molecules that are processed by Ago2 instead of Dicer. First, we confirmed the gradual shift from Dicer to Ago2 processing upon shortening of the hairpin length. Second, hairpins with a stem larger than 19 base pair are inefficiently cleaved by Ago2 and we noticed a shift in the cleavage site. Third, the introduction of a top G·U bp in a regular shRNA can promote Ago2-cleavage, which coincides with a loss of Ago2-loading of the Dicer-cleaved 3' strand. Fourth, the Ago2-processed AgoshRNAs acquire a short 3' tail of 1–3 A-nucleotides (nt) and we present evidence that this product is subsequently trimmed by the poly(A)-specific ribonuclease (PARN). PMID:26172504

Exploring the Genes of Yerba Mate (Ilex paraguariensis A. St.-Hil.) by NGS and De Novo Transcriptome Assembly

PubMed Central

Aguilera, Patricia M.; Bubillo, Rosana E.; Otegui, Mónica B.; Ducasse, Daniel A.; Zapata, Pedro D.; Marti, Dardo A.

2014-01-01

Yerba mate (Ilex paraguariensis A. St.-Hil.) is an important subtropical tree crop cultivated on 326,000 ha in Argentina, Brazil and Paraguay, with a total yield production of more than 1,000,000 t. Yerba mate presents a strong limitation regarding sequence information. The NCBI GenBank lacks an EST database of yerba mate and depicts only 80 DNA sequences, mostly uncharacterized. In this scenario, in order to elucidate the yerba mate gene landscape by means of NGS, we explored and discovered a vast collection of I. paraguariensis transcripts. Total RNA from I. paraguariensis was sequenced by Illumina HiSeq-2000 obtaining 72,031,388 pair-end 100 bp sequences. High quality reads were de novo assembled into 44,907 transcripts encompassing 40 million bases with an estimated coverage of 180X. Multiple sequence analysis allowed us to predict that yerba mate contains ∼32,355 genes and 12,551 gene variants or isoforms. We identified and categorized members of more than 100 metabolic pathways. Overall, we have identified ∼1,000 putative transcription factors, genes involved in heat and oxidative stress, pathogen response, as well as disease resistance and hormone response. We have also identified, based in sequence homology searches, novel transcripts related to osmotic, drought, salinity and cold stress, senescence and early flowering. We have also pinpointed several members of the gene silencing pathway, and characterized the silencing effector Argonaute1. We predicted a diverse supply of putative microRNA precursors involved in developmental processes. We present here the first draft of the transcribed genomes of the yerba mate chloroplast and mitochondrion. The putative sequence and predicted structure of the caffeine synthase of yerba mate is presented. Moreover, we provide a collection of over 10,800 SSR accessible to the scientific community interested in yerba mate genetic improvement. This contribution broadly expands the limited knowledge of yerba mate genes, and is presented as the first genomic resource of this important crop. PMID:25330175

microRNA therapies in cancer.

PubMed

Rothschild, Sacha I

2014-01-01

MicroRNAs (miRNAs or miRs) are a family of small non-coding RNA species that have been implicated in the control of many fundamental cellular and physiological processes such as cellular differentiation, proliferation, apoptosis and stem cell maintenance. miRNAs regulate gene expression by the sequence-selective targeting of mRNAs, leading to translational repression or mRNA degradation. Some microRNAs have been categorized as "oncomiRs" as opposed to "tumor suppressor miRs" Modulating the miRNA activities may provide exciting opportunities for cancer therapy. This review highlights the latest discovery of miRNAs involved in carcinogenesis as well as the potential applications of miRNA regulations in cancer treatment. Several studies have demonstrated the feasibility of restoring tumor suppressive miRNAs and targeting oncogenic miRNAs for cancer therapy using in vivo model systems.

Evaluation of normalization methods in mammalian microRNA-Seq data

PubMed Central

Garmire, Lana Xia; Subramaniam, Shankar

2012-01-01

Simple total tag count normalization is inadequate for microRNA sequencing data generated from the next generation sequencing technology. However, so far systematic evaluation of normalization methods on microRNA sequencing data is lacking. We comprehensively evaluate seven commonly used normalization methods including global normalization, Lowess normalization, Trimmed Mean Method (TMM), quantile normalization, scaling normalization, variance stabilization, and invariant method. We assess these methods on two individual experimental data sets with the empirical statistical metrics of mean square error (MSE) and Kolmogorov-Smirnov (K-S) statistic. Additionally, we evaluate the methods with results from quantitative PCR validation. Our results consistently show that Lowess normalization and quantile normalization perform the best, whereas TMM, a method applied to the RNA-Sequencing normalization, performs the worst. The poor performance of TMM normalization is further evidenced by abnormal results from the test of differential expression (DE) of microRNA-Seq data. Comparing with the models used for DE, the choice of normalization method is the primary factor that affects the results of DE. In summary, Lowess normalization and quantile normalization are recommended for normalizing microRNA-Seq data, whereas the TMM method should be used with caution. PMID:22532701

Identification of microRNAs differentially expressed involved in male flower development.

PubMed

Wang, Zhengjia; Huang, Jianqin; Sun, Zhichao; Zheng, Bingsong

2015-03-01

Hickory (Carya cathayensis Sarg.) is one of the most economically important woody trees in eastern China, but its long flowering phase delays yield. Our understanding of the regulatory roles of microRNAs (miRNAs) in male flower development in hickory remains poor. Using high-throughput sequencing technology, we have pyrosequenced two small RNA libraries from two male flower differentiation stages in hickory. Analysis of the sequencing data identified 114 conserved miRNAs that belonged to 23 miRNA families, five novel miRNAs including their corresponding miRNA*s, and 22 plausible miRNA candidates. Differential expression analysis revealed 12 miRNA sequences that were upregulated in the later (reproductive) stage of male flower development. Quantitative real-time PCR showed similar expression trends as that of the deep sequencing. Novel miRNAs and plausible miRNA candidates were predicted using bioinformatic analysis methods. The miRNAs newly identified in this study have increased the number of known miRNAs in hickory, and the identification of differentially expressed miRNAs will provide new avenues for studies into miRNAs involved in the process of male flower development in hickory and other related trees.

Inaugural Genomics Automation Congress and the coming deluge of sequencing data.

PubMed

Creighton, Chad J

2010-10-01

Presentations at Select Biosciences's first 'Genomics Automation Congress' (Boston, MA, USA) in 2010 focused on next-generation sequencing and the platforms and methodology around them. The meeting provided an overview of sequencing technologies, both new and emerging. Speakers shared their recent work on applying sequencing to profile cells for various levels of biomolecular complexity, including DNA sequences, DNA copy, DNA methylation, mRNA and microRNA. With sequencing time and costs continuing to drop dramatically, a virtual explosion of very large sequencing datasets is at hand, which will probably present challenges and opportunities for high-level data analysis and interpretation, as well as for information technology infrastructure.

Characterization and differential expression of microRNAs elicited by sulfur deprivation in Chlamydomonas reinhardtii

PubMed Central

2012-01-01

Background microRNAs (miRNAs) have been found to play an essential role in the modulation of numerous biological processes in eukaryotes. Chlamydomonas reinhardtii is an ideal model organism for the study of many metabolic processes including responses to sulfur-deprivation. We used a deep sequencing platform to extensively profile and identify changes in the miRNAs expression that occurred under sulfur-replete and sulfur-deprived conditions. The aim of our research was to characterize the differential expression of Chlamydomonas miRNAs under sulfur-deprived conditions, and subsequently, the target genes of miRNA involved in sulfur-deprivation were further predicted and analyzed. Results By using high-throughput sequencing, we characterized the microRNA transcriptomes under sulphur-replete and sulfur-deprived conditions in Chlamydomonas reinhardtii. We predicted a total of 310 miRNAs which included 85 known miRNAs and 225 novel miRNAs. 13 miRNAs were the specific to the sulfur-deprived conditions. 47 miRNAs showed significantly differential expressions responding to sulfur-deprivation, and most were up-regulated in the small RNA libraries with sulfur-deprivation. Using a web-based integrated system (Web MicroRNAs Designer 3) and combing the former information from a transcriptome of Chlamydomonas reinhardtii, 22 miRNAs and their targets involved in metabolism regulation with sulfur-deprivation were verified. Conclusions Our results indicate that sulfur-deprivation may have a significant influence on small RNA expression patterns, and the differential expressions of miRNAs and interactions between miRNA and its targets might further reveal the molecular mechanism responding to sulfur-deprivation in Chlamydomonas reinhardtii. PMID:22439676

Characterization and Comparative Profiling of MiRNA Transcriptomes in Bighead Carp and Silver Carp

PubMed Central

Chi, Wei; Tong, Chaobo; Gan, Xiaoni; He, Shunping

2011-01-01

MicroRNAs (miRNAs) are small non-coding RNA molecules that are processed from large ‘hairpin’ precursors and function as post-transcriptional regulators of target genes. Although many individual miRNAs have recently been extensively studied, there has been very little research on miRNA transcriptomes in teleost fishes. By using high throughput sequencing technology, we have identified 167 and 166 conserved miRNAs (belonging to 108 families) in bighead carp (Hypophthalmichthys nobilis) and silver carp (Hypophthalmichthys molitrix), respectively. We compared the expression patterns of conserved miRNAs by means of hierarchical clustering analysis and log2 ratio. Results indicated that there is not a strong correlation between sequence conservation and expression conservation, most of these miRNAs have similar expression patterns. However, high expression differences were also identified for several individual miRNAs. Several miRNA* sequences were also found in our dataset and some of them may have regulatory functions. Two computational strategies were used to identify novel miRNAs from un-annotated data in the two carps. A first strategy based on zebrafish genome, identified 8 and 22 novel miRNAs in bighead carp and silver carp, respectively. We postulate that these miRNAs should also exist in the zebrafish, but the methodologies used have not allowed for their detection. In the second strategy we obtained several carp-specific miRNAs, 31 in bighead carp and 32 in silver carp, which showed low expression. Gain and loss of family members were observed in several miRNA families, which suggests that duplication of animal miRNA genes may occur through evolutionary processes which are similar to the protein-coding genes. PMID:21858165

Molecular cloning and functional characterization of an antifungal PR-5 protein from Ocimum basilicum.

PubMed

Rather, Irshad Ahmad; Awasthi, Praveen; Mahajan, Vidushi; Bedi, Yashbir S; Vishwakarma, Ram A; Gandhi, Sumit G

2015-03-01

Pathogenesis-related (PR) proteins are involved in biotic and abiotic stress responses of plants and are grouped into 17 families (PR-1 to PR-17). PR-5 family includes proteins related to thaumatin and osmotin, with several members possessing antimicrobial properties. In this study, a PR-5 gene showing a high degree of homology with osmotin-like protein was isolated from sweet basil (Ocimum basilicum L.). A complete open reading frame consisting of 675 nucleotides, coding for a precursor protein, was obtained by PCR amplification. Based on sequence comparisons with tobacco osmotin and other osmotin-like proteins (OLPs), this protein was named ObOLP. The predicted mature protein is 225 amino acids in length and contains 16 cysteine residues that may potentially form eight disulfide bonds, a signature common to most PR-5 proteins. Among the various abiotic stress treatments tested, including high salt, mechanical wounding and exogenous phytohormone/elicitor treatments; methyl jasmonate (MeJA) and mechanical wounding significantly induced the expression of ObOLP gene. The coding sequence of ObOLP was cloned and expressed in a bacterial host resulting in a 25kDa recombinant-HIS tagged protein, displaying antifungal activity. The ObOLP protein sequence appears to contain an N-terminal signal peptide with signatures of secretory pathway. Further, our experimental data shows that ObOLP expression is regulated transcriptionally and in silico analysis suggests that it may be post-transcriptionally and post-translationally regulated through microRNAs and post-translational protein modifications, respectively. This study appears to be the first report of isolation and characterization of osmotin-like protein gene from O. basilicum. Copyright © 2014 Elsevier B.V. All rights reserved.

Accident sequence precursor events with age-related contributors

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

Murphy, G.A.; Kohn, W.E.

1995-12-31

The Accident Sequence Precursor (ASP) Program at ORNL analyzed about 14.000 Licensee Event Reports (LERs) filed by US nuclear power plants 1987--1993. There were 193 events identified as precursors to potential severe core accident sequences. These are reported in G/CR-4674. Volumes 7 through 20. Under the NRC Nuclear Plant Aging Research program, the authors evaluated these events to determine the extent to which component aging played a role. Events were selected that involved age-related equipment degradation that initiated an event or contributed to an event sequence. For the 7-year period, ORNL identified 36 events that involved aging degradation as amore » contributor to an ASP event. Except for 1992, the percentage of age-related events within the total number of ASP events over the 7-year period ({approximately}19%) appears fairly consistent up to 1991. No correlation between plant ape and number of precursor events was found. A summary list of the age-related events is presented in the report.« less

Next-Generation Sequence Analysis of the Genome of RFHVMn, the Macaque Homolog of Kaposi's Sarcoma (KS)-Associated Herpesvirus, from a KS-Like Tumor of a Pig-Tailed Macaque

PubMed Central

Bruce, A. Gregory; Ryan, Jonathan T.; Thomas, Mathew J.; Peng, Xinxia; Grundhoff, Adam; Tsai, Che-Chung

2013-01-01

The complete sequence of retroperitoneal fibromatosis-associated herpesvirus Macaca nemestrina (RFHVMn), the pig-tailed macaque homolog of Kaposi's sarcoma-associated herpesvirus (KSHV), was determined by next-generation sequence analysis of a Kaposi's sarcoma (KS)-like macaque tumor. Colinearity of genes was observed with the KSHV genome, and the core herpesvirus genes had strong sequence homology to the corresponding KSHV genes. RFHVMn lacked homologs of open reading frame 11 (ORF11) and KSHV ORFs K5 and K6, which appear to have been generated by duplication of ORFs K3 and K4 after the divergence of KSHV and RFHV. RFHVMn contained positional homologs of all other unique KSHV genes, although some showed limited sequence similarity. RFHVMn contained a number of candidate microRNA genes. Although there was little sequence similarity with KSHV microRNAs, one candidate contained the same seed sequence as the positional homolog, kshv-miR-K12-10a, suggesting functional overlap. RNA transcript splicing was highly conserved between RFHVMn and KSHV, and strong sequence conservation was noted in specific promoters and putative origins of replication, predicting important functional similarities. Sequence comparisons indicated that RFHVMn and KSHV developed in long-term synchrony with the evolution of their hosts, and both viruses phylogenetically group within the RV1 lineage of Old World primate rhadinoviruses. RFHVMn is the closest homolog of KSHV to be completely sequenced and the first sequenced RV1 rhadinovirus homolog of KSHV from a nonhuman Old World primate. The strong genetic and sequence similarity between RFHVMn and KSHV, coupled with similarities in biology and pathology, demonstrate that RFHVMn infection in macaques offers an important and relevant model for the study of KSHV in humans. PMID:24109218

Discovery and profiling of novel and conserved microRNAs during flower development in Carya cathayensis via deep sequencing.

PubMed

Wang, Zheng Jia; Huang, Jian Qin; Huang, You Jun; Li, Zheng; Zheng, Bing Song

2012-08-01

Hickory (Carya cathayensis Sarg.) is an economically important woody plant in China, but its long juvenile phase delays yield. MicroRNAs (miRNAs) are critical regulators of genes and important for normal plant development and physiology, including flower development. We used Solexa technology to sequence two small RNA libraries from two floral differentiation stages in hickory to identify miRNAs related to flower development. We identified 39 conserved miRNA sequences from 114 loci belonging to 23 families as well as two novel and ten potential novel miRNAs belonging to nine families. Moreover, 35 conserved miRNA*s and two novel miRNA*s were detected. Twenty miRNA sequences from 49 loci belonging to 11 families were differentially expressed; all were up-regulated at the later stage of flower development in hickory. Quantitative real-time PCR of 12 conserved miRNA sequences, five novel miRNA families, and two novel miRNA*s validated that all were expressed during hickory flower development, and the expression patterns were similar to those detected with Solexa sequencing. Finally, a total of 146 targets of the novel and conserved miRNAs were predicted. This study identified a diverse set of miRNAs that were closely related to hickory flower development and that could help in plant floral induction.

Elevated microRNA miR-21 Levels in Pancreatic Cyst Fluid Are Predictive of Mucinous Precursor Lesions of Ductal Adenocarcinoma

PubMed Central

Ryu, Ji Kon; Matthaei, Hanno; dal Molin, Marco; Hong, Seung-Mo; Canto, Marcia I.; Schulick, Richard D.; Wolfgang, Christopher; Goggins, Michael G.; Hruban, Ralph H.; Cope, Leslie; Maitra, Anirban

2011-01-01

Background Biomarkers for the diagnostic classification of pancreatic cysts are urgently needed. Deregulated microRNA (miRNAs) expression is widespread in pancreatic cancer. We assessed whether aberrant miRNAs in pancreatic cyst fluid could be used as potential biomarkers for cystic precursor lesions of pancreatic cancer. Methods Cyst fluid specimens were prospectively collected from 40 surgically resected pancreatic cysts, and small RNAs were extracted. The ‘mucinous’ cohort included 14 intraductal papillary mucinous neoplasms (including 3 with an associated adenocarcinoma) and 10 mucinous cystic neoplasms; the ‘nonmucinous’ cohort included 11 serous cystadenomas and 5 other benign cysts. Quantitative reverse transcription PCR was performed for five miRNAs (miR-21, miR-155, miR-221, miR-17-3p, miR-191), which were previously reported as overexpressed in pancreatic adenocarcinomas. Results Significantly higher expression of miR-21, miR-221, and miR-17-3p was observed in the mucinous versus nonmucinous cysts (p < 0.01), with the mean relative fold differences being 7.0-, 7.9-, and 5.4-fold, respectively. Receiver operating characteristic curves demonstrated the highest median area under the curve for miR-21, with a median specificity of 76%, at a sensitivity of 80%. Conclusion This pilot study demonstrates that profiling miRNAs in pancreatic cyst fluid samples is feasible and can yield potential biomarkers for the classification of cystic lesions of the pancreas. PMID:21757972

MicroRNA let-7d regulates the TLX/microRNA-9 cascade to control neural cell fate and neurogenesis

PubMed Central

Zhao, Chunnian; Sun, GuoQiang; Ye, Peng; Li, Shengxiu; Shi, Yanhong

2013-01-01

MicroRNAs have important functions in the nervous system through post-transcriptional regulation of neurogenesis genes. Here we show that microRNA let-7d, which has been implicated in cocaine addiction and other neurological disorders, targets the neural stem cell regulator TLX. Overexpression of let-7d in vivo reduced neural stem cell proliferation and promoted premature neuronal differentiation and migration, a phenotype similar to those induced by TLX knockdown or overexpression of its negatively-regulated target, microRNA-9. We found a let-7d binding sequence in the tlx 3′ UTR and demonstrated that let-7d reduced TLX expression levels in neural stem cells, which in turn, up-regulated miR-9 expression. Moreover, co-expression of let-7d and TLX lacking its 3′ UTR in vivo restored neural stem cell proliferation and reversed the premature neuronal differentiation and migration. Therefore, manipulating let-7d and its downstream targets could be a novel strategy to unravel neurogenic signaling pathways and identify potential interventions for relevant neurological disorders. PMID:23435502

MicroRNA let-7d regulates the TLX/microRNA-9 cascade to control neural cell fate and neurogenesis.

PubMed

Zhao, Chunnian; Sun, GuoQiang; Ye, Peng; Li, Shengxiu; Shi, Yanhong

2013-01-01

MicroRNAs have important functions in the nervous system through post-transcriptional regulation of neurogenesis genes. Here we show that microRNA let-7d, which has been implicated in cocaine addiction and other neurological disorders, targets the neural stem cell regulator TLX. Overexpression of let-7d in vivo reduced neural stem cell proliferation and promoted premature neuronal differentiation and migration, a phenotype similar to those induced by TLX knockdown or overexpression of its negatively-regulated target, microRNA-9. We found a let-7d binding sequence in the tlx 3' UTR and demonstrated that let-7d reduced TLX expression levels in neural stem cells, which in turn, up-regulated miR-9 expression. Moreover, co-expression of let-7d and TLX lacking its 3' UTR in vivo restored neural stem cell proliferation and reversed the premature neuronal differentiation and migration. Therefore, manipulating let-7d and its downstream targets could be a novel strategy to unravel neurogenic signaling pathways and identify potential interventions for relevant neurological disorders.

Rapid Communication: MiR-92a as a housekeeping gene for analysis of bovine mastitis-related microRNA in milk.

PubMed

Lai, Y C; Fujikawa, T; Ando, T; Kitahara, G; Koiwa, M; Kubota, C; Miura, N

2017-06-01

Our aim was to identify a suitable microRNA housekeeping gene for real-time PCR analysis of bovine mastitis-related microRNA in milk. We identified , , and as housekeeping gene candidates on the basis of previous Solexa sequencing results. Threshold cycle (CT) values for , , and did not differ between milk from control cows and milk from mastitis-affected cows. NormFinder software identified as the most stable single housekeeping gene. We evaluated the suitability of the housekeeping gene candidates by using them to assess expression levels of the inflammation-related gene . Regardless of the housekeeping gene candidates used for normalization, relative expression levels of were significantly higher in mastitis-affected samples than in control samples. However, of all the housekeeping genes and gene combinations investigated, normalization with alone generated the difference in relative expression between mastitis-affected and control samples with the highest significance. These results suggest that is suitable for use as a housekeeping gene for analysis of bovine mastitis-related microRNA in milk.

[Genetic variants in miRNAs and its association with breast cancer].

PubMed

Méndez-Gómez, Susana; Ruiz Esparza-Garrido, Ruth; Velázquez-Flores, Miguel; Dolores-Vergara, Maria; Salamanca-Gómez, Fabio; Arenas-Aranda, Diego Julio

2014-01-01

In Mexico, breast cancer represents the first cause of cancer death in females. At the molecular level, non-coding RNAs and especially microRNAs have played an important role in the origin and development of this neoplasm In the Anglo-Saxon population, diverse genetic variants in microRNA genes and in their targets are associated with the development of this disease. In the Mexican population it is not known if these or other variants exist. Identification of these or new variants in our population is fundamental in order to have a better understanding of cancer development and to help establish a better diagnostic strategy. DNA was isolated from mammary tumors, adjacent tissue and peripheral blood of Mexican females with or without cancer. From DNA, five microRNA genes and three of their targets were amplified and sequenced. Genetic variants associated with breast cancer in an Anglo- Saxon population have been previously identified in these sequences. In the samples studied we identified seven single nucleotide polymorphisms (SNPs). Two had not been previously described and were identified only in women with cancer. The new variants may be genetic predisposition factors for the development of breast cancer in our population. Further experiments are needed to determine the involvement of these variants in the development, establishment and progression of breast cancer.

Heart structure-specific transcriptomic atlas reveals conserved microRNA-mRNA interactions.

PubMed

Vacchi-Suzzi, Caterina; Hahne, Florian; Scheubel, Philippe; Marcellin, Magali; Dubost, Valerie; Westphal, Magdalena; Boeglen, Catherine; Büchmann-Møller, Stine; Cheung, Ming Sin; Cordier, André; De Benedetto, Christopher; Deurinck, Mark; Frei, Moritz; Moulin, Pierre; Oakeley, Edward; Grenet, Olivier; Grevot, Armelle; Stull, Robert; Theil, Diethilde; Moggs, Jonathan G; Marrer, Estelle; Couttet, Philippe

2013-01-01

MicroRNAs are short non-coding RNAs that regulate gene expression at the post-transcriptional level and play key roles in heart development and cardiovascular diseases. Here, we have characterized the expression and distribution of microRNAs across eight cardiac structures (left and right ventricles, apex, papillary muscle, septum, left and right atrium and valves) in rat, Beagle dog and cynomolgus monkey using microRNA sequencing. Conserved microRNA signatures enriched in specific heart structures across these species were identified for cardiac valve (miR-let-7c, miR-125b, miR-127, miR-199a-3p, miR-204, miR-320, miR-99b, miR-328 and miR-744) and myocardium (miR-1, miR-133b, miR-133a, miR-208b, miR-30e, miR-499-5p, miR-30e*). The relative abundance of myocardium-enriched (miR-1) and valve-enriched (miR-125b-5p and miR-204) microRNAs was confirmed using in situ hybridization. MicroRNA-mRNA interactions potentially relevant for cardiac functions were explored using anti-correlation expression analysis and microRNA target prediction algorithms. Interactions between miR-1/Timp3, miR-125b/Rbm24, miR-204/Tgfbr2 and miR-208b/Csnk2a2 were identified and experimentally investigated in human pulmonary smooth muscle cells and luciferase reporter assays. In conclusion, we have generated a high-resolution heart structure-specific mRNA/microRNA expression atlas for three mammalian species that provides a novel resource for investigating novel microRNA regulatory circuits involved in cardiac molecular physiopathology.

Serum microRNAs in patients with genetic amyotrophic lateral sclerosis and pre-manifest mutation carriers.

PubMed

Freischmidt, Axel; Müller, Kathrin; Zondler, Lisa; Weydt, Patrick; Volk, Alexander E; Božič, Anže Lošdorfer; Walter, Michael; Bonin, Michael; Mayer, Benjamin; von Arnim, Christine A F; Otto, Markus; Dieterich, Christoph; Holzmann, Karlheinz; Andersen, Peter M; Ludolph, Albert C; Danzer, Karin M; Weishaupt, Jochen H

2014-11-01

Knowledge about the nature of pathomolecular alterations preceding onset of symptoms in amyotrophic lateral sclerosis is largely lacking. It could not only pave the way for the discovery of valuable therapeutic targets but might also govern future concepts of pre-manifest disease modifying treatments. MicroRNAs are central regulators of transcriptome plasticity and participate in pathogenic cascades and/or mirror cellular adaptation to insults. We obtained comprehensive expression profiles of microRNAs in the serum of patients with familial amyotrophic lateral sclerosis, asymptomatic mutation carriers and healthy control subjects. We observed a strikingly homogenous microRNA profile in patients with familial amyotrophic lateral sclerosis that was largely independent from the underlying disease gene. Moreover, we identified 24 significantly downregulated microRNAs in pre-manifest amyotrophic lateral sclerosis mutation carriers up to two decades or more before the estimated time window of disease onset; 91.7% of the downregulated microRNAs in mutation carriers overlapped with the patients with familial amyotrophic lateral sclerosis. Bioinformatic analysis revealed a consensus sequence motif present in the vast majority of downregulated microRNAs identified in this study. Our data thus suggest specific common denominators regarding molecular pathogenesis of different amyotrophic lateral sclerosis genes. We describe the earliest pathomolecular alterations in amyotrophic lateral sclerosis mutation carriers known to date, which provide a basis for the discovery of novel therapeutic targets and strongly argue for studies evaluating presymptomatic disease-modifying treatment in amyotrophic lateral sclerosis. © The Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Existence of a microRNA pathway in anucleate platelets

PubMed Central

Landry, Patricia; Plante, Isabelle; Ouellet, Dominique L; Perron, Marjorie P; Rousseau, Guy; Provost, Patrick

2010-01-01

Platelets play a critical role in the maintenance of hemostasis as well as in thrombosis and vessel occlusion that underlie stroke and acute coronary syndromes. Anucleate platelets contain messenger RNAs (mRNAs) and are capable of protein synthesis, raising the issue of how these mRNAs are regulated. Here we show that human platelets harbor an abundant and diverse array of microRNAs (miRNAs), which are known as key regulators of mRNA translation. Further analyses revealed that platelets contain Dicer and Argonaute 2 (Ago2) complexes functional in exogenously supplied miRNA precursor (pre-miRNA) processing and the control of specific reporter transcripts, respectively. Detection of the receptor P2Y12 mRNA in Ago2 immunoprecipitates suggests that P2Y12 expression may be subjected to miRNA control in human platelets. Our study lends an additional level of complexity to the control of gene expression in these anucleate elements of the cardiovascular system. PMID:19668211

Small RNA Deep Sequencing and the Effects of microRNA408 on Root Gravitropic Bending in Arabidopsis

NASA Astrophysics Data System (ADS)

Li, Huasheng; Lu, Jinying; Sun, Qiao; Chen, Yu; He, Dacheng; Liu, Min

2015-11-01

MicroRNA (miRNA) is a non-coding small RNA composed of 20 to 24 nucleotides that influences plant root development. This study analyzed the miRNA expression in Arabidopsis root tip cells using Illumina sequencing and real-time PCR before (sample 0) and 15 min after (sample 15) a 3-D clinostat rotational treatment was administered. After stimulation was performed, the expression levels of seven miRNA genes, including Arabidopsis miR160, miR161, miR394, miR402, miR403, miR408, and miR823, were significantly upregulated. Illumina sequencing results also revealed two novel miRNAsthat have not been previously reported, The target genes of these miRNAs included pentatricopeptide repeat-containing protein and diadenosine tetraphosphate hydrolase. An overexpression vector of Arabidopsis miR408 was constructed and transferred to Arabidopsis plant. The roots of plants over expressing miR408 exhibited a slower reorientation upon gravistimulation in comparison with those of wild-type. This result indicate that miR408 could play a role in root gravitropic response.

Characterization of gonadotrophin-releasing hormone precursor cDNA in the Old World mole-rat Cryptomys hottentotus pretoriae: high degree of identity with the New World guinea pig sequence.

PubMed

Kalamatianos, T; du Toit, L; Hrabovszky, E; Kalló, I; Marsh, P J; Bennett, N C; Coen, C W

2005-05-01

Regulation of pituitary gonadotrophins by the decapeptide gonadotrophin-releasing hormone 1 (GnRH1) is crucial for the development and maintenance of reproductive functions. A common amino acid sequence for this decapeptide, designated as 'mammalian' GnRH, has been identified in all mammals thus far investigated with the exception of the guinea pig, in which there are two amino acid substitutions. Among hystricognath rodents, the members of the family Bathyergidae regulate reproduction in response to diverse cues. Thus, highveld mole-rats (Cryptomys hottentotus pretoriae) are social bathyergids in which breeding is restricted to a particular season in the dominant female, but continuously suppressed in subordinate colony members. Elucidation of reproductive control in these animals will be facilitated by characterization of their GnRH1 gene. A partial sequence of GnRH1 precursor cDNA was isolated and characterized. Comparative analysis revealed the highest degree of identity (86%) to guinea pig GnRH1 precursor mRNA. Nevertheless, the deduced amino acid sequence of the mole-rat decapeptide is identical to the 'mammalian' sequence rather than that of guinea pigs. Successful detection of GnRH1-synthesizing neurones using either a guinea pig GnRH1 riboprobe or an antibody against the 'mammalian' decapeptide is consistent with the guinea pig-like sequence for the precursor and the classic 'mammalian' form for the decapeptide. The high degree of identity in the GnRH1 precursor sequence between this Old World mole-rat and the New World guinea pig is consistent with the theory that caviomorphs and phiomorphs originated from a common ancestral line in the Palaeocene to mid Eocene, some 63-45 million years ago.

High-Throughput Sequencing of microRNAs in Glucocorticoid Sensitive Paediatric Inflammatory Bowel Disease Patients.

PubMed

De Iudicibus, Sara; Lucafò, Marianna; Vitulo, Nicola; Martelossi, Stefano; Zimbello, Rosanna; De Pascale, Fabio; Forcato, Claudio; Naviglio, Samuele; Di Silvestre, Alessia; Gerdol, Marco; Stocco, Gabriele; Valle, Giorgio; Ventura, Alessandro; Bramuzzo, Matteo; Decorti, Giuliana

2018-05-08

The aim of this research was the identification of novel pharmacogenomic biomarkers for better understanding the complex gene regulation mechanisms underpinning glucocorticoid (GC) action in paediatric inflammatory bowel disease (IBD). This goal was achieved by evaluating high-throughput microRNA (miRNA) profiles during GC treatment, integrated with the assessment of expression changes in GC receptor (GR) heterocomplex genes. Furthermore, we tested the hypothesis that differentially expressed miRNAs could be directly regulated by GCs through investigating the presence of GC responsive elements (GREs) in their gene promoters. Ten IBD paediatric patients responding to GCs were enrolled. Peripheral blood was obtained at diagnosis (T0) and after four weeks of steroid treatment (T4). MicroRNA profiles were analyzed using next generation sequencing, and selected significantly differentially expressed miRNAs were validated by quantitative reverse transcription-polymerase chain reaction. In detail, 18 miRNAs were differentially expressed from T0 to T4, 16 of which were upregulated and 2 of which were downregulated. Out of these, three miRNAs (miR-144, miR-142, and miR-96) could putatively recognize the 3’UTR of the GR gene and three miRNAs (miR-363, miR-96, miR-142) contained GREs sequences, thereby potentially enabling direct regulation by the GR. In conclusion, we identified miRNAs differently expressed during GC treatment and miRNAs which could be directly regulated by GCs in blood cells of young IBD patients. These results could represent a first step towards their translation as pharmacogenomic biomarkers.

Deep sequencing leads to the identification of eukaryotic translation initiation factor 5A as a key element in Rsv1-mediated lethal systemic hypersensitive response to Soybean mosaic virus infection in soybean.

PubMed

Chen, Hui; Adam Arsovski, Andrej; Yu, Kangfu; Wang, Aiming

2017-04-01

Rsv1, a single dominant resistance locus in soybean, confers extreme resistance to the majority of Soybean mosaic virus (SMV) strains, but is susceptible to the G7 strain. In Rsv1-genotype soybean, G7 infection provokes a lethal systemic hypersensitive response (LSHR), a delayed host defence response. The Rsv1-mediated LSHR signalling pathway remains largely unknown. In this study, we employed a genome-wide investigation to gain an insight into the molecular interplay between SMV G7 and Rsv1-genotype soybean. Small RNA (sRNA), degradome and transcriptome sequencing analyses were used to identify differentially expressed genes (DEGs) and microRNAs (DEMs) in response to G7 infection. A number of DEGs, DEMs and microRNA targets, and the interaction network of DEMs and their target mRNAs responsive to G7 infection, were identified. Knock-down of one of the identified DEGs, the eukaryotic translation initiation factor 5A (eIF5A), diminished the LSHR and enhanced viral accumulation, suggesting the essential role of eIF5A in the G7-induced, Rsv1-mediated LSHR signalling pathway. This work provides an in-depth genome-wide analysis of high-throughput sequencing data, and identifies multiple genes and microRNA signatures that are associated with the Rsv1-mediated LSHR. © 2016 HER MAJESTY THE QUEEN IN RIGHT OF CANADA MOLECULAR PLANT PATHOLOGY © 2016 BSPP AND JOHN WILEY & SONS LTD.

Carica papaya microRNAs are responsive to Papaya meleira virus infection.

PubMed

Abreu, Paolla M V; Gaspar, Clicia G; Buss, David S; Ventura, José A; Ferreira, Paulo C G; Fernandes, Patricia M B

2014-01-01

MicroRNAs are implicated in the response to biotic stresses. Papaya meleira virus (PMeV) is the causal agent of sticky disease, a commercially important pathology in papaya for which there are currently no resistant varieties. PMeV has a number of unusual features, such as residence in the laticifers of infected plants, and the response of the papaya to PMeV infection is not well understood. The protein levels of 20S proteasome subunits increase during PMeV infection, suggesting that proteolysis could be an important aspect of the plant defense response mechanism. To date, 10,598 plant microRNAs have been identified in the Plant miRNAs Database, but only two, miR162 and miR403, are from papaya. In this study, known plant microRNA sequences were used to search for potential microRNAs in the papaya genome. A total of 462 microRNAs, representing 72 microRNA families, were identified. The expression of 11 microRNAs, whose targets are involved in 20S and 26S proteasomal degradation and in other stress response pathways, was compared by real-time PCR in healthy and infected papaya leaf tissue. We found that the expression of miRNAs involved in proteasomal degradation increased in response to very low levels of PMeV titre and decreased as the viral titre increased. In contrast, miRNAs implicated in the plant response to biotic stress decreased their expression at very low level of PMeV and increased at high PMeV levels. Corroborating with this results, analysed target genes for this miRNAs had their expression modulated in a dependent manner. This study represents a comprehensive identification of conserved miRNAs inpapaya. The data presented here might help to complement the available molecular and genomic tools for the study of papaya. The differential expression of some miRNAs and identifying their target genes will be helpful for understanding the regulation and interaction of PMeV and papaya.

Carica papaya MicroRNAs Are Responsive to Papaya meleira virus Infection

PubMed Central

Abreu, Paolla M. V.; Gaspar, Clicia G.; Buss, David S.; Ventura, José A.; Ferreira, Paulo C. G.; Fernandes, Patricia M. B.

2014-01-01

MicroRNAs are implicated in the response to biotic stresses. Papaya meleira virus (PMeV) is the causal agent of sticky disease, a commercially important pathology in papaya for which there are currently no resistant varieties. PMeV has a number of unusual features, such as residence in the laticifers of infected plants, and the response of the papaya to PMeV infection is not well understood. The protein levels of 20S proteasome subunits increase during PMeV infection, suggesting that proteolysis could be an important aspect of the plant defense response mechanism. To date, 10,598 plant microRNAs have been identified in the Plant miRNAs Database, but only two, miR162 and miR403, are from papaya. In this study, known plant microRNA sequences were used to search for potential microRNAs in the papaya genome. A total of 462 microRNAs, representing 72 microRNA families, were identified. The expression of 11 microRNAs, whose targets are involved in 20S and 26S proteasomal degradation and in other stress response pathways, was compared by real-time PCR in healthy and infected papaya leaf tissue. We found that the expression of miRNAs involved in proteasomal degradation increased in response to very low levels of PMeV titre and decreased as the viral titre increased. In contrast, miRNAs implicated in the plant response to biotic stress decreased their expression at very low level of PMeV and increased at high PMeV levels. Corroborating with this results, analysed target genes for this miRNAs had their expression modulated in a dependent manner. This study represents a comprehensive identification of conserved miRNAs inpapaya. The data presented here might help to complement the available molecular and genomic tools for the study of papaya. The differential expression of some miRNAs and identifying their target genes will be helpful for understanding the regulation and interaction of PMeV and papaya. PMID:25072834

Genetic and mechanistic diversity of piRNA 3' end formation

PubMed Central

Handler, Dominik; Mohn, Fabio; Ameres, Stefan L.; Brennecke, Julius

2016-01-01

Small regulatory RNAs guide Argonaute (Ago) proteins in a sequence-specific manner to their targets and thereby play important roles in eukaryotic gene silencing1. Of the three small RNA classes, microRNAs and siRNAs are processed from double-stranded precursors into defined 21- to 23-mers by Dicer, an endoribonuclease with intrinsic ruler function. piRNAs—the 22-30 nt long guides for PIWI-clade Ago proteins that silence transposons in animal gonads—are generated Dicer-independently from single-stranded precursors2,3. piRNA 5' ends are defined either by Zucchini, a mitochondria-anchored endonuclease4,5, or by piRNA-guided target cleavage6,7. Formation of piRNA 3' ends is poorly understood. Here, we find that two genetically and mechanistically distinct pathways generate piRNA 3' ends in Drosophila. The initiating nucleases are either Zucchini or the PIWI-clade proteins Aubergine (Aub)/Ago3. While Zucchini-mediated cleavages directly define mature piRNA 3' ends8,9, Aub/Ago3-mediated cleavages liberate pre-piRNAs that require extensive resection by the 3'-to-5' exoribonuclease Nibbler/Mut-710–13. The relative activity of these two pathways dictates the extent to which piRNAs are fueled into cytoplasmic or nuclear PIWI-clade proteins and thereby sets the balance between post-transcriptional and transcriptional silencing. Strikingly, loss of both Zucchini and Nibbler reveals a minimal, Argonaute-driven small RNA biogenesis pathway where piRNA 5' and 3' ends are directly produced by closely spaced Aub/Ago3-mediated cleavage events. Our data establish a coherent blueprint for piRNA biogenesis, and set the stage for the mechanistic dissection of the processes that govern piRNA 3' end formation. PMID:27851737

Characterization of a molt-inhibiting hormone (MIH) of the crayfish, Orconectes limosus, by cDNA cloning and mass spectrometric analysis.

PubMed

Bulau, Patrick; Okuno, Atsuro; Thome, Elke; Schmitz, Tina; Peter-Katalinic, Jasna; Keller, Rainer

2005-11-01

The structure of the precursor of a molt-inhibiting hormone (MIH) of the American crayfish, Orconectes limosus was determined by cloning of a cDNA based on RNA from the neurosecretory perikarya of the X-organ in the eyestalk ganglia. The open reading frame includes the complete precursor sequence, consisting of a signal peptide of 29, and the MIH sequence of 77 amino acids. In addition, the mature peptide was isolated by HPLC from the neurohemal sinus gland and analyzed by ESI-MS and MALDI-TOF-MS peptide mapping. This showed that the mature peptide (Mass 8664.29 Da) consists of only 75 amino acids, having Ala75-NH2 as C-terminus. Thus, C-terminal Arg77 of the precursor is removed during processing, and Gly76 serves as an amide donor. Sequence comparison confirms this peptide as a novel member of the large family, which includes crustacean hyperglycaemic hormone (CHH), MIH and gonad (vitellogenesis)-inhibiting hormone (GIH/VIH). The lack of a CPRP (CHH-precursor related peptide) in the hormone precursor, the size and specific sequence characteristics show that Orl MIH belongs to the MIH/GIH(VIH) subgroup of this larger family. Comparison with the MIH of Procambarus clarkii, the only other MIH that has thus far been identified in freshwater crayfish, shows extremely high sequence conservation. Both MIHs differ in only one amino acid residue ( approximately 99% identity), whereas the sequence identity to several other known MIHs is between 40 and 46%.

Simultaneous and Sequential MS/MS Scan Combinations and Permutations in a Linear Quadrupole Ion Trap.

PubMed

Snyder, Dalton T; Szalwinski, Lucas J; Cooks, R Graham

2017-10-17

Methods of performing precursor ion scans as well as neutral loss scans in a single linear quadrupole ion trap have recently been described. In this paper we report methodology for performing permutations of MS/MS scan modes, that is, ordered combinations of precursor, product, and neutral loss scans following a single ion injection event. Only particular permutations are allowed; the sequences demonstrated here are (1) multiple precursor ion scans, (2) precursor ion scans followed by a single neutral loss scan, (3) precursor ion scans followed by product ion scans, and (4) segmented neutral loss scans. (5) The common product ion scan can be performed earlier in these sequences, under certain conditions. Simultaneous scans can also be performed. These include multiple precursor ion scans, precursor ion scans with an accompanying neutral loss scan, and multiple neutral loss scans. We argue that the new capability to perform complex simultaneous and sequential MS n operations on single ion populations represents a significant step in increasing the selectivity of mass spectrometry.

Identification and Characterization of Liver MicroRNAs of the Chinese Tree Shrew via Deep Sequencing.

PubMed

Feng, Yue; Feng, Yue-Mei; Feng, Yang; Lu, Caixia; Liu, Li; Sun, Xiaomei; Dai, Jiejie; Xia, Xueshan

2015-10-01

Chinese tree shrew (Tupaia belangeri chinensis) is a small animal that possess many features, which are valuable in biomedical research, as experimental models. Currently, there are numerous attempts to utilize tree shrews as models for hepatitis C virus (HCV) infection. This study aimed to construct a liver microRNA (miRNA) data of the tree shrew. Three second filial generation tree shrews were used in this study. Total RNA was extracted from each liver of the tree shrew and equal quality mixed, then reverse-transcribed to complementary DNA (cDNA). The cDNAs were amplified by polymerase chain reaction and subjected to high-throughput sequencing. A total of 2060 conserved miRNAs were identified through alignment with the mature miRNAs in miRBase 20.0 database. The gene ontology and Kyoto encyclopedia of genes and genomes analyses of the target genes of the miRNAs revealed several candidate miRNAs, genes and pathways that may involve in the process of HCV infection. The abundance of miR-122 and Let-7 families and their other characteristics provided us more evidences for the utilization of this animal, as a potential model for HCV infection and other related biomedical research. Moreover, 80 novel microRNAs were predicted using the software Mireap. The top 3 abundant miRNAs were validated in other tree samples, based on stem-loop quantitative reverse transcription-polymerase chain reaction. According to the liver microRNA data of Chinese tree shrew, characteristics of the miR-122 and Let-7 families further highlight the suitability of tree shrew as the animal model in HCV research.

Identification of MicroRNAs in the Coral Stylophora pistillata

PubMed Central

Liew, Yi Jin; Aranda, Manuel; Carr, Adrian; Baumgarten, Sebastian; Zoccola, Didier; Tambutté, Sylvie; Allemand, Denis; Micklem, Gos; Voolstra, Christian R.

2014-01-01

Coral reefs are major contributors to marine biodiversity. However, they are in rapid decline due to global environmental changes such as rising sea surface temperatures, ocean acidification, and pollution. Genomic and transcriptomic analyses have broadened our understanding of coral biology, but a study of the microRNA (miRNA) repertoire of corals is missing. miRNAs constitute a class of small non-coding RNAs of ∼22 nt in size that play crucial roles in development, metabolism, and stress response in plants and animals alike. In this study, we examined the coral Stylophora pistillata for the presence of miRNAs and the corresponding core protein machinery required for their processing and function. Based on small RNA sequencing, we present evidence for 31 bona fide microRNAs, 5 of which (miR-100, miR-2022, miR-2023, miR-2030, and miR-2036) are conserved in other metazoans. Homologues of Argonaute, Piwi, Dicer, Drosha, Pasha, and HEN1 were identified in the transcriptome of S. pistillata based on strong sequence conservation with known RNAi proteins, with additional support derived from phylogenetic trees. Examination of putative miRNA gene targets indicates potential roles in development, metabolism, immunity, and biomineralisation for several of the microRNAs. Here, we present first evidence of a functional RNAi machinery and five conserved miRNAs in S. pistillata, implying that miRNAs play a role in organismal biology of scleractinian corals. Analysis of predicted miRNA target genes in S. pistillata suggests potential roles of miRNAs in symbiosis and coral calcification. Given the importance of miRNAs in regulating gene expression in other metazoans, further expression analyses of small non-coding RNAs in transcriptional studies of corals should be informative about miRNA-affected processes and pathways. PMID:24658574

Environmental contaminants and microRNA regulation: Transcription factors as regulators of toxicant-altered microRNA expression

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

Sollome, James; Martin, Elizabeth

MicroRNAs (miRNAs) regulate gene expression by binding mRNA and inhibiting translation and/or inducing degradation of the associated transcripts. Expression levels of miRNAs have been shown to be altered in response to environmental toxicants, thus impacting cellular function and influencing disease risk. Transcription factors (TFs) are known to be altered in response to environmental toxicants and play a critical role in the regulation of miRNA expression. To date, environmentally-responsive TFs that are important for regulating miRNAs remain understudied. In a state-of-the-art analysis, we utilized an in silico bioinformatic approach to characterize potential transcriptional regulators of environmentally-responsive miRNAs. Using the miRStart database,more » genomic sequences of promoter regions for all available human miRNAs (n = 847) were identified and promoter regions were defined as − 1000/+500 base pairs from the transcription start site. Subsequently, the promoter region sequences of environmentally-responsive miRNAs (n = 128) were analyzed using enrichment analysis to determine overrepresented TF binding sites (TFBS). While most (56/73) TFs differed across environmental contaminants, a set of 17 TFs was enriched for promoter binding among miRNAs responsive to numerous environmental contaminants. Of these, one TF was common to miRNAs altered by the majority of environmental contaminants, namely SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 3 (SMARCA3). These identified TFs represent candidate common transcriptional regulators of miRNAs perturbed by environmental toxicants. - Highlights: • Transcription factors that regulate environmentally-modulated miRNA expression are understudied • Transcription factor binding sites (TFBS) located within DNA promoter regions of miRNAs were identified. • Specific transcription factors may serve as master regulators of environmentally-mediated microRNA expression.« less

Profilings of MicroRNAs in the Liver of Common Carp (Cyprinus carpio) Infected with Flavobacterium columnare

PubMed Central

Zhao, Lijuan; Lu, Hong; Meng, Qinglei; Wang, Jinfu; Wang, Weimin; Yang, Ling; Lin, Li

2016-01-01

MicroRNAs (miRNAs) play important roles in regulation of many biological processes in eukaryotes, including pathogen infection and host interactions. Flavobacterium columnare (FC) infection can cause great economic loss of common carp (Cyprinus carpio) which is one of the most important cultured fish in the world. However, miRNAs in response to FC infection in common carp has not been characterized. To identify specific miRNAs involved in common carp infected with FC, we performed microRNA sequencing using livers of common carp infected with and without FC. A total of 698 miRNAs were identified, including 142 which were identified and deposited in the miRbase database (Available online: http://www.mirbase.org/) and 556 had only predicted miRNAs. Among the deposited miRNAs, eight miRNAs were first identified in common carp. Thirty of the 698 miRNAs were differentially expressed miRNAs (DIE-miRNAs) between the FC infected and control samples. From the DIE-miRNAs, seven were selected randomly and their expression profiles were confirmed to be consistent with the microRNA sequencing results using RT-PCR and qRT-PCR. In addition, a total of 27,363 target genes of the 30 DIE-miRNAs were predicted. The target genes were enriched in five Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including focal adhesion, extracellular matrix (ECM)-receptor interaction, erythroblastic leukemia viral oncogene homolog (ErbB) signaling pathway, regulation of actin cytoskeleton, and adherent junction. The miRNA expression profile of the liver of common carp infected with FC will pave the way for the development of effective strategies to fight against FC infection. PMID:27092486

Expression levels of the microRNA maturing microprocessor complex component DGCR8 and the RNA-induced silencing complex (RISC) components argonaute-1, argonaute-2, PACT, TARBP1, and TARBP2 in epithelial skin cancer.

PubMed

Sand, Michael; Skrygan, Marina; Georgas, Dimitrios; Arenz, Christoph; Gambichler, Thilo; Sand, Daniel; Altmeyer, Peter; Bechara, Falk G

2012-11-01

The microprocessor complex mediates intranuclear biogenesis of precursor microRNAs from the primary microRNA transcript. Extranuclear, mature microRNAs are incorporated into the RNA-induced silencing complex (RISC) before interaction with complementary target mRNA leads to transcriptional repression or cleavage. In this study, we investigated the expression profiles of the microprocessor complex subunit DiGeorge syndrome critical region gene 8 (DGCR8) and the RISC components argonaute-1 (AGO1), argonaute-2 (AGO2), as well as double-stranded RNA-binding proteins PACT, TARBP1, and TARBP2 in epithelial skin cancer and its premalignant stage. Patients with premalignant actinic keratoses (AK, n = 6), basal cell carcinomas (BCC, n = 15), and squamous cell carcinomas (SCC, n = 7) were included in the study. Punch biopsies were harvested from the center of the tumors (lesional), from healthy skin sites (intraindividual controls), and from healthy skin sites in a healthy control group (n = 16; interindividual control). The DGCR8, AGO1, AGO2, PACT, TARBP1, and TARBP2 mRNA expression levels were detected by quantitative real-time reverse transcriptase polymerase chain reaction. The DGCR8, AGO1, AGO2, PACT, and TARBP1 expression levels were significantly higher in the AK, BCC, and SCC groups than the healthy controls (P < 0.05). There was no significant difference in the TARBP2 expression levels between groups (P > 0.05). This study indicates that major components of the miRNA pathway, such as the microprocessor complex and RISC, are dysregulated in epithelial skin cancer. Copyright © 2011 Wiley Periodicals, Inc.

Expression profiling of microRNAs in human bone tissue from postmenopausal women.

PubMed

De-Ugarte, Laura; Serra-Vinardell, Jenny; Nonell, Lara; Balcells, Susana; Arnal, Magdalena; Nogues, Xavier; Mellibovsky, Leonardo; Grinberg, Daniel; Diez-Perez, Adolfo; Garcia-Giralt, Natalia

2018-01-01

Bone tissue is composed of several cell types, which express their own microRNAs (miRNAs) that will play a role in cell function. The set of total miRNAs expressed in all cell types configures the specific signature of the bone tissue in one physiological condition. The aim of this study was to explore the miRNA expression profile of bone tissue from postmenopausal women. Tissue was obtained from trabecular bone and was analyzed in fresh conditions (n = 6). Primary osteoblasts were also obtained from trabecular bone (n = 4) and human osteoclasts were obtained from monocyte precursors after in vitro differentiation (n = 5). MicroRNA expression profiling was obtained for each sample by microarray and a global miRNA analysis was performed combining the data acquired in all the microarray experiments. From the 641 miRNAs detected in bone tissue samples, 346 (54%) were present in osteoblasts and/or osteoclasts. The other 46% were not identified in any of the bone cells analyzed. Intersection of osteoblast and osteoclast arrays identified 101 miRNAs shared by both cell types, which accounts for 30-40% of miRNAs detected in these cells. In osteoblasts, 266 miRNAs were detected, of which 243 (91%) were also present in the total bone array, representing 38% of all bone miRNAs. In osteoclasts, 340 miRNAs were detected, of which 196 (58%) were also present in the bone tissue array, representing 31% of all miRNAs detected in total bone. These analyses provide an overview of miRNAs expressed in bone tissue, broadening our knowledge in the microRNA field.

The contrasting microRNA content of a drought tolerant and a drought susceptible wheat cultivar.

PubMed

Bakhshi, Behnam; Fard, Ehsan Mohseni; Gharechahi, Javad; Safarzadeh, Mahdieh; Nikpay, Nava; Fotovat, Reza; Azimi, Mohammad Reza; Salekdeh, Ghasem Hosseini

2017-09-01

Drought stress represents one of the most common stresses affecting the productivity of crop plants. A rather recently discovered component of the plant response to drought is the cellular population of microRNAs. Here, the microRNA content was revealed of two bread wheat cultivars contrasting strongly with respect to the ability to withstand drought stress. A total of 1813 miRNAs was identified, grouped into 106 families. Some 104 of these miRNAs were predicted to match 212 novel miRNA precursors. In the drought tolerant cultivar (SM), 105 (33 known and 72 novel) miRNAs were altered in abundance by the imposition of drought stress, while the equivalent number in the more sensitive cultivar (SW) was 51 (20 and 31). An in silico analysis predicted that these miRNAs target at least 1959 genes in SM and 1111 in SW, suggesting their broad contribution to the drought stress response. Among the target genes were several known stress-related genes, encoding, for example, superoxide dismutase, various MYB transcription factors, various ABA signaling proteins and various MADS-box transcription factors. In many cases, the more susceptible cultivar SW behaved in a contrasting manner. The suggestion is that miRNAs represent an important aspect of the drought stress response, post-transcriptionally regulating a range of stress-related genes. Copyright © 2017 Elsevier GmbH. All rights reserved.

MicroRNA-211 Regulates Oxidative Phosphorylation and Energy Metabolism in Human Vitiligo.

PubMed

Sahoo, Anupama; Lee, Bongyong; Boniface, Katia; Seneschal, Julien; Sahoo, Sanjaya K; Seki, Tatsuya; Wang, Chunyan; Das, Soumen; Han, Xianlin; Steppie, Michael; Seal, Sudipta; Taieb, Alain; Perera, Ranjan J

2017-09-01

Vitiligo is a common chronic skin disorder characterized by loss of epidermal melanocytes and progressive depigmentation. Vitiligo has complex immune, genetic, environmental, and biochemical causes, but the exact molecular mechanisms of vitiligo development and progression, particularly those related to metabolic control, are poorly understood. In this study we characterized the human vitiligo cell line PIG3V and the normal human melanocyte line HEM-l by RNA sequencing, targeted metabolomics, and shotgun lipidomics. Melanocyte-enriched microRNA-211, a known metabolic switch in nonpigmented melanoma cells, was severely down-regulated in vitiligo cell line PIG3V and skin biopsy samples from vitiligo patients, whereas its predicted targets PPARGC1A, RRM2, and TAOK1 were reciprocally up-regulated. microRNA-211 binds to PGC1-α 3' untranslated region locus and represses it. Although mitochondrial numbers were constant, mitochondrial complexes I, II, and IV and respiratory responses were defective in vitiligo cells. Nanoparticle-coated microRNA-211 partially augmented the oxygen consumption rate in PIG3V cells. The lower oxygen consumption rate, changes in lipid and metabolite profiles, and increased reactive oxygen species production observed in vitiligo cells appear to be partly due to abnormal regulation of microRNA-211 and its target genes. These genes represent potential biomarkers and therapeutic targets in human vitiligo. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Next-generation small RNA sequencing for microRNAs profiling in the honey bee Apis mellifera.

PubMed

Chen, X; Yu, X; Cai, Y; Zheng, H; Yu, D; Liu, G; Zhou, Q; Hu, S; Hu, F

2010-12-01

MicroRNAs (miRNAs) are key regulators in various physiological and pathological processes via post-transcriptional regulation of gene expression. The honey bee (Apis mellifera) is a key model for highly social species, and its complex social behaviour can be interpreted theoretically as changes in gene regulation, in which miRNAs are thought to be involved. We used the SOLiD sequencing system to identify the repertoire of miRNAs in the honey bee by sequencing a mixed small RNA library from different developmental stages. We obtained a total of 36,796,459 raw sequences; of which 5,491,100 short sequences were fragments of mRNA and other noncoding RNAs (ncRNA), and 1,759,346 reads mapped to the known miRNAs. We predicted 267 novel honey bee miRNAs representing 380,182 short reads, including eight miRNAs of other insects in 14,107,583 genome-mapped sequences. We verified 50 of them using stem-loop reverse-transcription PCR (RT-PCR), in which 35 yielded PCR products. Cross-species analyses showed 81 novel miRNAs with homologues in other insects, suggesting that they were authentic miRNAs and have similar functions. The results of this study provide a basis for studies of the miRNA-modulating networks in development and some intriguing phenomena such as caste differentiation in A. mellifera. © 2010 The Authors. Insect Molecular Biology © 2010 The Royal Entomological Society.

Characterization of microRNAs from goat (Capra hircus) by Solexa deep-sequencing technology.

PubMed

Ling, Y H; Ding, J P; Zhang, X D; Wang, L J; Zhang, Y H; Li, Y S; Zhang, Z J; Zhang, X R

2013-06-13

MicroRNAs (miRNAs) are an important class of small noncoding RNAs that are highly conserved in plants and animals. Many miRNAs are known to mediate a myriad of cell processes, including proliferation and differentiation, via the regulation of some transcription and signaling factors, which are closely related to muscle development and disease. In this study, small RNA cDNA libraries of Boer goats were constructed. In addition, we obtained the goat muscle miRNAs by using Solexa deep-sequencing technology and analyzed these miRNA characteristics by combining it with the bioinformatics technology. Based on Solexa sequencing and bioinformatics analysis, 562 species-conserved and 5 goat genome-specific miRNAs were identified, 322 of which exceeded 100 in the expression levels. The results of real-time quantitative polymerase chain reaction from 8 randomly selected miRNAs showed that the 8 miRNAs were expressed in goat muscle, and the expression patterns were consistent with the Solexa sequencing results. The identification and characterization of miRNAs in goat muscle provide important information on the role of miRNA regulation in muscle growth and development. These data will help to facilitate studies on the regulatory roles played by miRNAs during goat growth and development.

MicroRNA repertoire for functional genome research in tilapia identified by deep sequencing.

PubMed

Yan, Biao; Wang, Zhen-Hua; Zhu, Chang-Dong; Guo, Jin-Tao; Zhao, Jin-Liang

2014-08-01

The Nile tilapia (Oreochromis niloticus; Cichlidae) is an economically important species in aquaculture and occupies a prominent position in the aquaculture industry. MicroRNAs (miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression involved in diverse biological and metabolic processes. To increase the repertoire of miRNAs characterized in tilapia, we used the Illumina/Solexa sequencing technology to sequence a small RNA library using pooled RNA sample isolated from the different developmental stages of tilapia. Bioinformatic analyses suggest that 197 conserved and 27 novel miRNAs are expressed in tilapia. Sequence alignments indicate that all tested miRNAs and miRNAs* are highly conserved across many species. In addition, we characterized the tissue expression patterns of five miRNAs using real-time quantitative PCR. We found that miR-1/206, miR-7/9, and miR-122 is abundantly expressed in muscle, brain, and liver, respectively, implying a potential role in the regulation of tissue differentiation or the maintenance of tissue identity. Overall, our results expand the number of tilapia miRNAs, and the discovery of miRNAs in tilapia genome contributes to a better understanding the role of miRNAs in regulating diverse biological processes.

Simultaneous visualization of the subfemtomolar expression of microRNA and microRNA target gene using HILO microscopy.

PubMed

Lin, Yi-Zhen; Ou, Da-Liang; Chang, Hsin-Yuan; Lin, Wei-Yu; Hsu, Chiun; Chang, Po-Ling

2017-09-01

The family of microRNAs (miRNAs) not only plays an important role in gene regulation but is also useful for the diagnosis of diseases. A reliable method with high sensitivity may allow researchers to detect slight fluctuations in ultra-trace amounts of miRNA. In this study, we propose a sensitive imaging method for the direct probing of miR-10b (miR-10b-3p, also called miR-10b*) and its target ( HOXD10 mRNA) in fixed cells based on the specific recognition of molecular beacons combined with highly inclined and laminated optical sheet (HILO) fluorescence microscopy. The designed dye-quencher-labelled molecular beacons offer excellent efficiencies of fluorescence resonance energy transfer that allow us to detect miRNA and the target mRNA simultaneously in hepatocellular carcinoma cells using HILO fluorescence microscopy. Not only can the basal trace amount of miRNA be observed in each individual cell, but the obtained images also indicate that this method is useful for monitoring the fluctuations in ultra-trace amounts of miRNA when the cells are transfected with a miRNA precursor or a miRNA inhibitor (anti-miR). Furthermore, a reasonable causal relation between the miR-10b and HOXD10 expression levels was observed in miR-10b* precursor-transfected cells and miR-10b* inhibitor-transfected cells. The trends of the miRNA alterations obtained using HILO microscopy completely matched the RT-qPCR data and showed remarkable reproducibility (the coefficient of variation [CV] = 0.86%) and sensitivity (<1.0 fM). This proposed imaging method appears to be useful for the simultaneous visualisation of ultra-trace amounts of miRNA and target mRNA and excludes the procedures for RNA extraction and amplification. Therefore, the visualisation of miRNA and the target mRNA should facilitate the exploration of the functions of ultra-trace amounts of miRNA in fixed cells in biological studies and may serve as a powerful tool for diagnoses based on circulating cancer cells.

Inflammatory gene networks in term human decidual cells define a potential signature for cytokine-mediated parturition.

PubMed

Ibrahim, Sherrine A; Ackerman, William E; Summerfield, Taryn L; Lockwood, Charles J; Schatz, Frederick; Kniss, Douglas A

2016-02-01

Inflammation is a proximate mediator of preterm birth and fetal injury. During inflammation several microRNAs (22 nucleotide noncoding ribonucleic acid (RNA) molecules) are up-regulated in response to cytokines such as interleukin-1β. MicroRNAs, in most cases, fine-tune gene expression, including both up-regulation and down-regulation of their target genes. However, the role of pro- and antiinflammatory microRNAs in this process is poorly understood. The principal goal of the work was to examine the inflammatory genomic profile of human decidual cells challenged with a proinflammatory cytokine known to be present in the setting of preterm parturition. We determined the coding (messenger RNA) and noncoding (microRNA) sequences to construct a network of interacting genes during inflammation using an in vitro model of decidual stromal cells. The effects of interleukin-1β exposure on mature microRNA expression were tested in human decidual cell cultures using the multiplexed NanoString platform, whereas the global inflammatory transcriptional response was measured using oligonucleotide microarrays. Differential expression of select transcripts was confirmed by quantitative real time-polymerase chain reaction. Bioinformatics tools were used to infer transcription factor activation and regulatory interactions. Interleukin-1β elicited up- and down-regulation of 350 and 78 nonredundant transcripts (false discovery rate < 0.1), respectively, including induction of numerous cytokines, chemokines, and other inflammatory mediators. Whereas this transcriptional response included marked changes in several microRNA gene loci, the pool of fully processed, mature microRNA was comparatively stable following a cytokine challenge. Of a total of 6 mature microRNAs identified as being differentially expressed by NanoString profiling, 2 (miR-146a and miR-155) were validated by quantitative real time-polymerase chain reaction. Using complementary bioinformatics approaches, activation of several inflammatory transcription factors could be inferred downstream of interleukin-1β based on the overall transcriptional response. Further analysis revealed that miR-146a and miR-155 both target genes involved in inflammatory signaling, including Toll-like receptor and mitogen-activated protein kinase pathways. Stimulation of decidual cells with interleukin-1β alters the expression of microRNAs that function to temper proinflammatory signaling. In this setting, some microRNAs may be involved in tissue-level inflammation during the bulk of gestation and assist in pregnancy maintenance. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification and Characterization of Cyprinid Herpesvirus-3 (CyHV-3) Encoded MicroRNAs

PubMed Central

Donohoe, Owen H.; Henshilwood, Kathy; Way, Keith; Hakimjavadi, Roya; Stone, David M.; Walls, Dermot

2015-01-01

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in post-transcriptional gene regulation. Some viruses encode their own miRNAs and these are increasingly being recognized as important modulators of viral and host gene expression. Cyprinid herpesvirus 3 (CyHV-3) is a highly pathogenic agent that causes acute mass mortalities in carp (Cyprinus carpio carpio) and koi (Cyprinus carpio koi) worldwide. Here, bioinformatic analyses of the CyHV-3 genome suggested the presence of non-conserved precursor miRNA (pre-miRNA) genes. Deep sequencing of small RNA fractions prepared from in vitro CyHV-3 infections led to the identification of potential miRNAs and miRNA–offset RNAs (moRNAs) derived from some bioinformatically predicted pre-miRNAs. DNA microarray hybridization analysis, Northern blotting and stem-loop RT-qPCR were then used to definitively confirm that CyHV-3 expresses two pre-miRNAs during infection in vitro. The evidence also suggested the presence of an additional four high-probability and two putative viral pre-miRNAs. MiRNAs from the two confirmed pre-miRNAs were also detected in gill tissue from CyHV-3-infected carp. We also present evidence that one confirmed miRNA can regulate the expression of a putative CyHV-3-encoded dUTPase. Candidate homologues of some CyHV-3 pre-miRNAs were identified in CyHV-1 and CyHV-2. This is the first report of miRNA and moRNA genes encoded by members of the Alloherpesviridae family, a group distantly related to the Herpesviridae family. The discovery of these novel CyHV-3 genes may help further our understanding of the biology of this economically important virus and their encoded miRNAs may have potential as biomarkers for the diagnosis of latent CyHV-3. PMID:25928140

Mining, identification and function analysis of microRNAs and target genes in peanut (Arachis hypogaea L.).

PubMed

Zhang, Tingting; Hu, Shuhao; Yan, Caixia; Li, Chunjuan; Zhao, Xiaobo; Wan, Shubo; Shan, Shihua

2017-02-01

In the present investigation, a total of 60 conserved peanut (Arachis hypogaea L.) microRNA (miRNA) sequences, belonging to 16 families, were identified using bioinformatics methods. There were 392 target gene sequences, identified from 58 miRNAs with Target-align software and BLASTx analyses. Gene Ontology (GO) functional analysis suggested that these target genes were involved in mediating peanut growth and development, signal transduction and stress resistance. There were 55 miRNA sequences, verified employing a poly (A) tailing test, with a success rate of up to 91.67%. Twenty peanut target gene sequences were randomly selected, and the 5' rapid amplification of the cDNA ends (5'-RACE) method were used to validate the cleavage sites of these target genes. Of these, 14 (70%) peanut miRNA targets were verified by means of gel electrophoresis, cloning and sequencing. Furthermore, functional analysis and homologous sequence retrieval were conducted for target gene sequences, and 26 target genes were chosen as the objects for stress resistance experimental study. Real-time fluorescence quantitative PCR (qRT-PCR) technology was applied to measure the expression level of resistance-associated miRNAs and their target genes in peanut exposed to Aspergillus flavus (A. flavus) infection and drought stress, respectively. In consequence, 5 groups of miRNAs & targets were found accorded with the mode of miRNA negatively controlling the expression of target genes. This study, preliminarily determined the biological functions of some resistance-associated miRNAs and their target genes in peanut. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Molecular characterization of an ependymin precursor from goldfish brain.

PubMed

Königstorfer, A; Sterrer, S; Eckerskorn, C; Lottspeich, F; Schmidt, R; Hoffmann, W

1989-01-01

Ependymins are thought to be implicated in fundamental processes involved in plasticity of the goldfish CNS. Gas-phase sequencing of purified ependymins beta and gamma revealed that they share the same N-terminal sequence. Each sequence displays microheterogeneities at several positions. Based on the protein sequences obtained, we constructed synthetic oligonucleotides and used them as hybridization probes for screening cDNA libraries of goldfish brain. In this article we describe the full-length sequence of a mRNA encoding a precursor of ependymins. A cleavable signal sequence characteristic of secretory proteins is located at the N-terminal end, followed directly by the ependymin sequence. Also, two potential N-glycosylation sites were detected. A computer search revealed that ependymins form a novel family of unique proteins.

Discovery of novel representatives of bilaterian neuropeptide families and reconstruction of neuropeptide precursor evolution in ophiuroid echinoderms

PubMed Central

Abylkassimova, Nikara; Hugall, Andrew F.; O'Hara, Timothy D.; Elphick, Maurice R.

2017-01-01

Neuropeptides are a diverse class of intercellular signalling molecules that mediate neuronal regulation of many physiological and behavioural processes. Recent advances in genome/transcriptome sequencing are enabling identification of neuropeptide precursor proteins in species from a growing variety of animal taxa, providing new insights into the evolution of neuropeptide signalling. Here, detailed analysis of transcriptome sequence data from three brittle star species, Ophionotus victoriae, Amphiura filiformis and Ophiopsila aranea, has enabled the first comprehensive identification of neuropeptide precursors in the class Ophiuroidea of the phylum Echinodermata. Representatives of over 30 bilaterian neuropeptide precursor families were identified, some of which occur as paralogues. Furthermore, homologues of endothelin/CCHamide, eclosion hormone, neuropeptide-F/Y and nucleobinin/nesfatin were discovered here in a deuterostome/echinoderm for the first time. The majority of ophiuroid neuropeptide precursors contain a single copy of a neuropeptide, but several precursors comprise multiple copies of identical or non-identical, but structurally related, neuropeptides. Here, we performed an unprecedented investigation of the evolution of neuropeptide copy number over a period of approximately 270 Myr by analysing sequence data from over 50 ophiuroid species, with reference to a robust phylogeny. Our analysis indicates that the composition of neuropeptide ‘cocktails’ is functionally important, but with plasticity over long evolutionary time scales. PMID:28878039

Conductance of single microRNAs chains related to the autism spectrum disorder

NASA Astrophysics Data System (ADS)

Oliveira, J. I. N.; Albuquerque, E. L.; Fulco, U. L.; Mauriz, P. W.; Sarmento, R. G.; Caetano, E. W. S.; Freire, V. N.

2014-09-01

The charge transport properties of single-stranded microRNAs (miRNAs) chains associated to autism disorder were investigated. The computations were performed within a tight-binding model, together with a transfer matrix technique, with ionization energies and hopping parameters obtained by quantum chemistry method. Current-voltage (I× V) curves of twelve miRNA chains related to the autism spectrum disorders were calculated and analysed. We have obtained both semiconductor and insulator behavior, and a relationship between the current intensity and the autism-related miRNA bases sequencies, suggesting that a kind of electronic biosensor can be developed to distinguish different profiles of autism disorders.

Multilevel regulation of gene expression by microRNAs.

PubMed

Makeyev, Eugene V; Maniatis, Tom

2008-03-28

MicroRNAs (miRNAs) are approximately 22-nucleotide-long noncoding RNAs that normally function by suppressing translation and destabilizing messenger RNAs bearing complementary target sequences. Some miRNAs are expressed in a cell- or tissue-specific manner and may contribute to the establishment and/or maintenance of cellular identity. Recent studies indicate that tissue-specific miRNAs may function at multiple hierarchical levels of gene regulatory networks, from targeting hundreds of effector genes incompatible with the differentiated state to controlling the levels of global regulators of transcription and alternative pre-mRNA splicing. This multilevel regulation may allow individual miRNAs to profoundly affect the gene expression program of differentiated cells.

Direct Downregulation of B-Cell Translocation Gene 3 by microRNA-93 Is Required for Desensitizing Esophageal Cancer to Radiotherapy.

PubMed

Cui, Hujun; Zhang, Shengqiang; Zhou, Hongbo; Guo, Ling

2017-08-01

Esophageal squamous carcinoma (ESC) is one of the most fatal malignancies worldwide with increasing occurrences yet poor outcome. MicroRNAs were reported to play roles in ESC. We aimed to understand how miRNAs affect the radiotherapy resistance of ESC. MicroRNA assays, real-time PCR, and Western blot were performed for expression analysis of miR-93 and BTG3. Luciferase activity assay was conducted with mutated B-cell translocation gene 3 (BTG3) 3'-UTR sequence in the 3' end of luciferase sequence with miR-93 inhibitor. ESC cells were treated with irradiation (IR) and clonogenic assay was utilized to detect the cell viability. Human ESC xenograft mouse model was established and subjected to target IR treatment followed by tumor size analysis. MiR-93 was decreased and BTG3 was increased in ESC cells, with negative correlation of their expression in ESC tissues. MiR-93 directly targeted BTG3 3'-UTR by luciferase activity assay. Either miR-93 inhibition or BTG3 overexpression decreased radiation resistance. Furthermore, miR-93 inhibition suppressed radiation resistance through BTG3. Direct downregulation of BTG3 by miR-93 is able to render ESC resistant to radiotherapy, and both BTG3 and miR-93 may potentially serve as clinical markers for ESC and contribute to the treatment of ESC.

Low-Temperature Molecular Layer Deposition Using Monofunctional Aromatic Precursors and Ozone-Based Ring-Opening Reactions.

PubMed

Svärd, Laura; Putkonen, Matti; Kenttä, Eija; Sajavaara, Timo; Krahl, Fabian; Karppinen, Maarit; Van de Kerckhove, Kevin; Detavernier, Christophe; Simell, Pekka

2017-09-26

Molecular layer deposition (MLD) is an increasingly used deposition technique for producing thin coatings consisting of purely organic or hybrid inorganic-organic materials. When organic materials are prepared, low deposition temperatures are often required to avoid decomposition, thus causing problems with low vapor pressure precursors. Monofunctional compounds have higher vapor pressures than traditional bi- or trifunctional MLD precursors, but do not offer the required functional groups for continuing the MLD growth in subsequent deposition cycles. In this study, we have used high vapor pressure monofunctional aromatic precursors in combination with ozone-triggered ring-opening reactions to achieve sustained sequential growth. MLD depositions were carried out by using three different aromatic precursors in an ABC sequence, namely with TMA + phenol + O 3 , TMA + 3-(trifluoromethyl)phenol + O 3 , and TMA + 2-fluoro-4-(trifluoromethyl)benzaldehyde + O 3 . Furthermore, the effect of hydrogen peroxide as a fourth step was evaluated for all studied processes resulting in a four-precursor ABCD sequence. According to the characterization results by ellipsometry, infrared spectroscopy, and X-ray reflectivity, self-limiting MLD processes could be obtained between 75 and 150 °C with each of the three aromatic precursors. In all cases, the GPC (growth per cycle) decreased with increasing temperature. In situ infrared spectroscopy indicated that ring-opening reactions occurred in each ABC sequence. Compositional analysis using time-of-flight elastic recoil detection indicated that fluorine could be incorporated into the film when 3-(trifluoromethyl)phenol and 2-fluoro-4-(trifluoromethyl)benzaldehyde were used as precursors.

Identification of new stress-induced microRNA and their targets in wheat using computational approach.

PubMed

Pandey, Bharati; Gupta, Om Prakash; Pandey, Dev Mani; Sharma, Indu; Sharma, Pradeep

2013-05-01

MicroRNAs (miRNAs) are a class of short endogenous non-coding small RNA molecules of about 18-22 nucleotides in length. Their main function is to downregulate gene expression in different manners like translational repression, mRNA cleavage and epigenetic modification. Computational predictions have raised the number of miRNAs in wheat significantly using an EST based approach. Hence, a combinatorial approach which is amalgamation of bioinformatics software and perl script was used to identify new miRNA to add to the growing database of wheat miRNA. Identification of miRNAs was initiated by mining the EST (Expressed Sequence Tags) database available at National Center for Biotechnology Information. In this investigation, 4677 mature microRNA sequences belonging to 50 miRNA families from different plant species were used to predict miRNA in wheat. A total of five abiotic stress-responsive new miRNAs were predicted and named Ta-miR5653, Ta-miR855, Ta-miR819k, Ta-miR3708 and Ta-miR5156. In addition, four previously identified miRNA, i.e., Ta-miR1122, miR1117, Ta-miR1134 and Ta-miR1133 were predicted in newly identified EST sequence and 14 potential target genes were subsequently predicted, most of which seems to encode ubiquitin carrier protein, serine/threonine protein kinase, 40S ribosomal protein, F-box/kelch-repeat protein, BTB/POZ domain-containing protein, transcription factors which are involved in growth, development, metabolism and stress response. Our result has increased the number of miRNAs in wheat, which should be useful for further investigation into the biological functions and evolution of miRNAs in wheat and other plant species.

Small RNA sequencing and functional characterization reveals microRNA-143 tumor suppressor activity in liposarcoma

PubMed Central

Ugras, Stacy; Brill, Elliott; Jacobsen, Anders; Hafner, Markus; Socci, Nicholas D.; DeCarolis, Penelope L.; Khanin, Raya; O'Connor, Rachael; Mihailovic, Aleksandra; Taylor, Barry S.; Sheridan, Robert; Gimble, Jeffrey M.; Viale, Agnes; Crago, Aimee; Antonescu, Cristina R.; Sander, Chris; Tuschl, Thomas; Singer, Samuel

2011-01-01

Liposarcoma remains the most common mesenchymal cancer, with a mortality rate of 60% among patients with this disease. To address the present lack of therapeutic options, we embarked upon a study of microRNA (miRNA) expression alterations associated with liposarcomagenesis with the goal of exploiting differentially expressed miRNAs and the gene products they regulate as potential therapeutic targets. MicroRNA expression was profiled in samples of normal adipose tissue, well-differentiated liposarcoma, and dedifferentiated liposarcoma by both deep sequencing of small RNA libraries and hybridization-based Agilent microarrays. The expression profiles discriminated liposarcoma from normal adipose tissue and well-differentiated from dedifferentiated disease. We defined over 40 miRNAs that were dysregulated in dedifferentiated liposarcomas in both the sequencing and the microarray analysis. The upregulated miRNAs included two cancer-associated species (miR-21, miR-26a), and the downregulated miRNAs included two species that were highly abundant in adipose tissue (miR-143, miR-145). Restoring miR-143 expression in dedifferentiated liposarcoma cells inhibited proliferation, induced apoptosis, and decreased expression of BCL2, TOP2A, PRC1, and PLK1. The downregulation of PRC1 and its docking partner PLK1 suggests that miR-143 inhibits cytokinesis in these cells. In support of this idea, treatment with a PLK1 inhibitor potently induced G2/M growth arrest and apoptosis in liposarcoma cells. Taken together, our findings suggest that miR-143 re-expression vectors or selective agents directed at miR-143 or its targets may have therapeutic value in dedifferentiated liposarcoma. PMID:21693658

Sequence of a cDNA encoding pancreatic preprosomatostatin-22.

PubMed Central

Magazin, M; Minth, C D; Funckes, C L; Deschenes, R; Tavianini, M A; Dixon, J E

1982-01-01

We report the nucleotide sequence of a precursor to somatostatin that upon proteolytic processing may give rise to a hormone of 22 amino acids. The nucleotide sequence of a cDNA from the channel catfish (Ictalurus punctatus) encodes a precursor to somatostatin that is 105 amino acids (Mr, 11,500). The cDNA coding for somatostatin-22 consists of 36 nucleotides in the 5' untranslated region, 315 nucleotides that code for the precursor to somatostatin-22, 269 nucleotides at the 3' untranslated region, and a variable length of poly(A). The putative preprohormone contains a sequence of hydrophobic amino acids at the amino terminus that has the properties of a "signal" peptide. A connecting sequence of approximately 57 amino acids is followed by a single Arg-Arg sequence, which immediately precedes the hormone. Somatostatin-22 is homologous to somatostatin-14 in 7 of the 14 amino acids, including the Phe-Trp-Lys sequence. Hybridization selection of mRNA, followed by its translation in a wheat germ cell-free system, resulted in the synthesis of a single polypeptide having a molecular weight of approximately 10,000 as estimated on Na-DodSO4/polyacrylamide gels. Images PMID:6127673

Serum miRNAs Signature Plays an Important Role in Keloid Disease.

PubMed

Luan, Y; Liu, Y; Liu, C; Lin, Q; He, F; Dong, X; Xiao, Z

2016-01-01

The molecular mechanism underlying the pathogenesis of keloid is largely unknown. MicroRNA (miRNA) is a class of small regulatory RNA that has emerged as a group of posttranscriptional gene repressors, participating in diverse pathophysiological processes of skin diseases. We investigated the expression profiles of miRNAs in the sera of patients to decipher the complicated factors involved in the development of keloid disease. MiRNA expression profiling in the sera from 9 keloid patients and 7 normal controls were characterized using a miRNA microarray containing established human mature and precursor miRNA sequences. Quantitative real-time PCR was performed to confirm the expression of miRNAs. The putative targets of differentially expressed miRNAs were functionally annotated by bioinformatics. MiRNA microarray analysis identified 37 differentially expressed miRNAs (17 upregulated and 20 downregulated) in keloid patients, compared to the healthy controls. Functional annotations revealed that the targets of those differentially expressed miRNAs were enriched in signaling pathways essential for scar formation and wound healing. The expression profiling of miRNAs is altered in the keloid, providing a clue for the molecular mechanisms underlying its initiation and progression. MiRNAs may partly contribute to the etiology of keloids by affecting the critical signaling pathways relevant to keloid pathogenesis.

The small RNA profile in latex from Hevea brasiliensis trees is affected by tapping panel dryness.

PubMed

Gébelin, Virginie; Leclercq, Julie; Kuswanhadi; Argout, Xavier; Chaidamsari, Tetty; Hu, Songnian; Tang, Chaorong; Sarah, Gautier; Yang, Meng; Montoro, Pascal

2013-10-01

Natural rubber is harvested by tapping Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg. Harvesting stress can lead to tapping panel dryness (TPD). MicroRNAs (miRNAs) are induced by abiotic stress and regulate gene expression by targeting the cleavage or translational inhibition of target messenger RNAs. This study set out to sequence miRNAs expressed in latex cells and to identify TPD-related putative targets. Deep sequencing of small RNAs was carried out on latex from trees affected by TPD using Solexa technology. The most abundant small RNA class size was 21 nucleotides for TPD trees compared with 24 nucleotides in healthy trees. By combining the LeARN pipeline, data from the Plant MicroRNA database and Hevea EST sequences, we identified 19 additional conserved and four putative species-specific miRNA families not found in previous studies on rubber. The relative transcript abundance of the Hbpre-MIR159b gene increased with TPD. This study revealed a small RNA-specific signature of TPD-affected trees. Both RNA degradation and a shift in miRNA biogenesis are suggested to explain the general decline in small RNAs and, particularly, in miRNAs.

Identification and characterization of novel serum microRNA candidates from deep sequencing in cervical cancer patients.

PubMed

Juan, Li; Tong, Hong-li; Zhang, Pengjun; Guo, Guanghong; Wang, Zi; Wen, Xinyu; Dong, Zhennan; Tian, Ya-ping

2014-09-03

Small non-coding microRNAs (miRNAs) are involved in cancer development and progression, and serum profiles of cervical cancer patients may be useful for identifying novel miRNAs. We performed deep sequencing on serum pools of cervical cancer patients and healthy controls with 3 replicates and constructed a small RNA library. We used MIREAP to predict novel miRNAs and identified 2 putative novel miRNAs between serum pools of cervical cancer patients and healthy controls after filtering out pseudo-pre-miRNAs using Triplet-SVM analysis. The 2 putative novel miRNAs were validated by real time PCR and were significantly decreased in cervical cancer patients compared with healthy controls. One novel miRNA had an area under curve (AUC) of 0.921 (95% CI: 0.883, 0.959) with a sensitivity of 85.7% and a specificity of 88.2% when discriminating between cervical cancer patients and healthy controls. Our results suggest that characterizing serum profiles of cervical cancers by Solexa sequencing may be a good method for identifying novel miRNAs and that the validated novel miRNAs described here may be cervical cancer-associated biomarkers.

Apple ring rot-responsive putative microRNAs revealed by high-throughput sequencing in Malus × domestica Borkh.

PubMed

Yu, Xin-Yi; Du, Bei-Bei; Gao, Zhi-Hong; Zhang, Shi-Jie; Tu, Xu-Tong; Chen, Xiao-Yun; Zhang, Zhen; Qu, Shen-Chun

2014-08-01

MicroRNAs (miRNAs) are small non-coding RNAs, which silence target mRNA via cleavage or translational inhibition to function in regulating gene expression. MiRNAs act as important regulators of plant development and stress response. For understanding the role of miRNAs responsive to apple ring rot stress, we identified disease-responsive miRNAs using high-throughput sequencing in Malus × domestica Borkh.. Four small RNA libraries were constructed from two control strains in M. domestica, crabapple (CKHu) and Fuji Naga-fu No. 6 (CKFu), and two disease stress strains, crabapple (DSHu) and Fuji Naga-fu No. 6 (DSFu). A total of 59 miRNA families were identified and five miRNAs might be responsive to apple ring rot infection and validated via qRT-PCR. Furthermore, we predicted 76 target genes which were regulated by conserved miRNAs potentially. Our study demonstrated that miRNAs was responsive to apple ring rot infection and may have important implications on apple disease resistance.

Identification of MicroRNAs and transcript targets in Camelina sativa by deep sequencing and computational methods

DOE PAGES

Poudel, Saroj; Aryal, Niranjan; Lu, Chaofu; ...

2015-03-31

Camelina sativa is an annual oilseed crop that is under intensive development for renewable resources of biofuels and industrial oils. MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play key roles in diverse plant biological processes. Here, we conducted deep sequencing on small RNA libraries prepared from camelina leaves, flower buds and two stages of developing seeds corresponding to initial and peak storage products accumulation. Computational analyses identified 207 known miRNAs belonging to 63 families, as well as 5 novel miRNAs. These miRNAs, especially members of the miRNA families, varied greatly in different tissues and developmental stages. The predictedmore » miRNA target genes are involved in a broad range of physiological functions including lipid metabolism. This report is the first step toward elucidating roles of miRNAs in C. sativa and will provide additional tools to improve this oilseed crop for biofuels and biomaterials.« less

A discovery of novel microRNAs in the silkworm (Bombyx mori) genome.

PubMed

Yu, Xiaomin; Zhou, Qing; Cai, Yimei; Luo, Qibin; Lin, Hongbin; Hu, Songnian; Yu, Jun

2009-12-01

MicroRNAs (miRNAs) are pivotal regulators involved in various physiological and pathological processes via their post-transcriptional regulation of gene expressions. We sequenced 14 libraries of small RNAs constructed from samples spanning the life cycle of silkworms, and discovered 50 novel miRNAs previously not known in animals and verified 43 of them using stem-loop RT-PCR. Our genome-wide analyses of 27 species-specific miRNAs suggest they arise from transposable elements, protein-coding genes duplication/transposition and random foldback sequences; which is consistent with the idea that novel animal miRNAs may evolve from incomplete self-complementary transcripts and become fixed in the process of co-adaptation with their targets. Computational prediction suggests that the silkworm-specific miRNAs may have a preference of regulating genes that are related to life-cycle-associated traits, and these genes can serve as potential targets for subsequent studies of the modulating networks in the development of Bombyx mori.

MicroRNAs play critical roles during plant development and in response to abiotic stresses.

PubMed

de Lima, Júlio César; Loss-Morais, Guilherme; Margis, Rogerio

2012-12-01

MicroRNAs (miRNAs) have been identified as key molecules in regulatory networks. The fine-tuning role of miRNAs in addition to the regulatory role of transcription factors has shown that molecular events during development are tightly regulated. In addition, several miRNAs play crucial roles in the response to abiotic stress induced by drought, salinity, low temperatures, and metals such as aluminium. Interestingly, several miRNAs have overlapping roles with regard to development, stress responses, and nutrient homeostasis. Moreover, in response to the same abiotic stresses, different expression patterns for some conserved miRNA families among different plant species revealed different metabolic adjustments. The use of deep sequencing technologies for the characterisation of miRNA frequency and the identification of new miRNAs adds complexity to regulatory networks in plants. In this review, we consider the regulatory role of miRNAs in plant development and abiotic stresses, as well as the impact of deep sequencing technologies on the generation of miRNA data.

miR-137 Targets p160 Steroid Receptor Coactivators SRC1, SRC2, and SRC3 and Inhibits Cell Proliferation

PubMed Central

Eedunuri, Vijay Kumar; Rajapakshe, Kimal; Fiskus, Warren; Geng, Chuandong; Chew, Sue Anne; Foley, Christopher; Shah, Shrijal S.; Shou, John; Mohamed, Junaith S.; O'Malley, Bert W.

2015-01-01

The p160 family of steroid receptor coactivators (SRCs) are pleiotropic transcription factor coactivators and “master regulators” of gene expression that promote cancer cell proliferation, survival, metabolism, migration, invasion, and metastasis. Cancers with high p160 SRC expression exhibit poor clinical outcomes and resistance to therapy, highlighting the SRCs as critical oncogenic drivers and, thus, therapeutic targets. microRNAs are important epigenetic regulators of protein expression. To examine the regulation of p160 SRCs by microRNAs, we used and combined 4 prediction algorithms to identify microRNAs that could target SRC1, SRC2, and SRC3 expression. For validation of these predictions, we assessed p160 SRC protein expression and cell viability after transfection of corresponding microRNA mimetics in breast cancer, uveal melanoma, and prostate cancer (PC) cell lines. Transfection of selected microRNA mimetics into breast cancer, uveal melanoma, and PC cells depleted SRC protein expression levels and exerted potent antiproliferative activity in these cell types. In particular, microRNA-137 (miR-137) depleted expression of SRC1, SRC2, and very potently, SRC3. The latter effect can be attributed to the presence of 3 miR-137 recognition sequences within the SRC3 3′-untranslated region. Using reverse phase protein array analysis, we identified a network of proteins, in addition to SRC3, that were modulated by miR-137 in PC cells. We also found that miR-137 and its host gene are epigenetically silenced in human cancer specimens and cell lines. These results support the development and testing of microRNA-based therapies (in particular based on restoring miR-137 levels) for targeting the oncogenic family of p160 SRCs in cancer. PMID:26066330

Precursor microRNA-programmed silencing complex assembly pathways in mammals.

PubMed

Liu, Xuhang; Jin, Dong-Yan; McManus, Michael T; Mourelatos, Zissimos

2012-05-25

Assembly of microRNA ribonucleoproteins (miRNPs) or RNA-induced silencing complexes (RISCs) is essential for the function of miRNAs and initiates from processing of precursor miRNAs (pre-miRNAs) by Dicer or by Ago2. Here, we report an in vitro miRNP/RISC assembly assay programmed by pre-miRNAs from mammalian cell lysates. Combining in vivo studies in Dicer Knockout cells reconstituted with wild-type or catalytically inactive Dicer, we find that the miRNA loading complex (miRLC) is the primary machinery linking pre-miRNA processing to miRNA loading. We show that a miRNA precursor deposit complex (miPDC) plays a crucial role in Dicer-independent miRNA biogenesis and promotes miRNP assembly of certain Dicer-dependent miRNAs. Furthermore, we find that 5'-uridine, 3'-mid base pairing, and 5'-mid mismatches within pre-miRNAs promote their assembly into miPDC. Our studies provide a comprehensive view of miRNP/RISC assembly pathways in mammals, and our assay provides a versatile platform for further mechanistic dissection of such pathways in mammals. Copyright © 2012 Elsevier Inc. All rights reserved.

Native gel analysis for RISC assembly.

PubMed

Kawamata, Tomoko; Tomari, Yukihide

2011-01-01

Small-interfering RNAs (siRNAs) and microRNAs (miRNAs) regulate expression of their target mRNAs via the RNA-induced silencing complex (RISC). A core component of RISC is the Argonaute (Ago) protein, which dictates the RISC function. In Drosophila, miRNAs and siRNAs are generally loaded into Ago1-containing RISC (Ago1-RISC) and Ago2-containing RISC (Ago2-RISC), respectively. We developed a native agarose gel system to directly detect Ago1-RISC, Ago2-RISC, and their precursor complexes. Methods presented here will provide powerful tools to biochemically dissect the RISC assembly pathways.

Differential expression analysis of Paralichthys olivaceus microRNAs in adult ovary and testis by deep sequencing.

PubMed

Gu, Yifeng; Zhang, Lei; Chen, Xiaowu

2014-08-01

MicroRNAs (miRNAs) play an important role in gonadal development and differentiation in fish. However, understanding of the mechanism of this process is hindered by our poor knowledge of miRNA expression patterns in fish gonads. In this study, miRNA libraries derived from adult gonads of Paralichthys olivaceus were generated by using next-generation sequencing (NGS) technology. Bioinformatics analysis was performed to distinguish mature miRNA sequences from two classes of small RNAs represented in the sequencing data. A total of 141 mature miRNAs were identified, in which 21 miRNAs were found in P. olivaceus for the first time. Variance and preference of miRNAs expression were concluded from the deep sequencing reads. Some miRNAs, such as pol-miR-143, pol-miR-26a and pol-let-7a were found with quite high expression levels in both gonads, while some exhibited a clear sex-biased expression in different gonad. Approximate 20.0% and 13.1% of the isolated miRNAs were preferentially expressed in the testis (FC<0.5) or ovary (FC>2), respectively. The identification and the preliminary analysis of the sex-biased expression of miRNAs in P. olivaceus gonads in our work by using NGS will provide us a basic catalog of miRNAs to facilitate future improvement and exploitation of sexual regulatory mechanisms in P. olivaceus. Copyright © 2014. Published by Elsevier Inc.

MicroRNA categorization using sequence motifs and k-mers.

PubMed

Yousef, Malik; Khalifa, Waleed; Acar, İlhan Erkin; Allmer, Jens

2017-03-14

Post-transcriptional gene dysregulation can be a hallmark of diseases like cancer and microRNAs (miRNAs) play a key role in the modulation of translation efficiency. Known pre-miRNAs are listed in miRBase, and they have been discovered in a variety of organisms ranging from viruses and microbes to eukaryotic organisms. The computational detection of pre-miRNAs is of great interest, and such approaches usually employ machine learning to discriminate between miRNAs and other sequences. Many features have been proposed describing pre-miRNAs, and we have previously introduced the use of sequence motifs and k-mers as useful ones. There have been reports of xeno-miRNAs detected via next generation sequencing. However, they may be contaminations and to aid that important decision-making process, we aimed to establish a means to differentiate pre-miRNAs from different species. To achieve distinction into species, we used one species' pre-miRNAs as the positive and another species' pre-miRNAs as the negative training and test data for the establishment of machine learned models based on sequence motifs and k-mers as features. This approach resulted in higher accuracy values between distantly related species while species with closer relation produced lower accuracy values. We were able to differentiate among species with increasing success when the evolutionary distance increases. This conclusion is supported by previous reports of fast evolutionary changes in miRNAs since even in relatively closely related species a fairly good discrimination was possible.

Insilico profiling of microRNAs in Korean ginseng (Panax ginseng Meyer)

PubMed Central

Mathiyalagan, Ramya; Subramaniyam, Sathiyamoorthy; Natarajan, Sathishkumar; Kim, Yeon Ju; Sun, Myung Suk; Kim, Se Young; Kim, Yu-Jin; Yang, Deok Chun

2013-01-01

MicroRNAs (miRNAs) are a class of recently discovered non-coding small RNA molecules, on average approximately 21 nucleotides in length, which underlie numerous important biological roles in gene regulation in various organisms. The miRNA database (release 18) has 18,226 miRNAs, which have been deposited from different species. Although miRNAs have been identified and validated in many plant species, no studies have been reported on discovering miRNAs in Panax ginseng Meyer, which is a traditionally known medicinal plant in oriental medicine, also known as Korean ginseng. It has triterpene ginseng saponins called ginsenosides, which are responsible for its various pharmacological activities. Predicting conserved miRNAs by homology-based analysis with available expressed sequence tag (EST) sequences can be powerful, if the species lacks whole genome sequence information. In this study by using the EST based computational approach, 69 conserved miRNAs belonging to 44 miRNA families were identified in Korean ginseng. The digital gene expression patterns of predicted conserved miRNAs were analyzed by deep sequencing using small RNA sequences of flower buds, leaves, and lateral roots. We have found that many of the identified miRNAs showed tissue specific expressions. Using the insilico method, 346 potential targets were identified for the predicted 69 conserved miRNAs by searching the ginseng EST database, and the predicted targets were mainly involved in secondary metabolic processes, responses to biotic and abiotic stress, and transcription regulator activities, as well as a variety of other metabolic processes. PMID:23717176

Isolation and Characterization of a microRNA-size Secretable Small RNA in Streptococcus sanguinis.

PubMed

Choi, Ji-Woong; Kwon, Tae-Yub; Hong, Su-Hyung; Lee, Heon-Jin

2018-06-01

MicroRNAs in eukaryotic cells are thought to control highly complex signal transduction and other biological processes by regulating coding transcripts, accounting for their important role in cellular events in eukaryotes. Recently, a novel class of bacterial RNAs similar in size [18-22 nucleotides (nt)] to microRNAs has been reported. Herein, we describe microRNAs, small RNAs from the oral pathogen Streptococcus sanguinis. The bacteria are normally present in the oral cavities and cause endocarditis by contaminating bloodstreams. Small RNAs were analyzed by deep sequencing. Selected highly expressed small RNAs were further validated by real-time polymerase chain reaction and northern blot analyses. We found that skim milk supplement changed the expression of small RNAs S.S-1964 in tandem with the nearby SSA_0513 gene involved in vitamin B 12 conversion. We furthermore observed small RNAs secreted via bacterial membrane vesicles. Although their precise function remains unclear, secretable small RNAs may represent an entirely new area of study in bacterial genetics.

Lateral flow nucleic acid biosensor for sensitive detection of microRNAs based on the dual amplification strategy of duplex-specific nuclease and hybridization chain reaction.

PubMed

Ying, Na; Ju, Chuanjing; Sun, Xiuwei; Li, Letian; Chang, Hongbiao; Song, Guangping; Li, Zhongyi; Wan, Jiayu; Dai, Enyong

2017-01-01

MicroRNAs (miRNAs) constitute novel biomarkers for various diseases. Accurate and quantitative analysis of miRNA expression is critical for biomedical research and clinical theranostics. In this study, a method was developed for sensitive and specific detection of miRNAs via dual signal amplification based on duplex specific nuclease (DSN) and hybridization chain reaction (HCR). A reporter probe (RP), comprising recognition sequence (3' end modified with biotin) for a target miRNA of miR-21 and capture sequence (5' end modified with Fam) for HCR product, was designed and synthesized. HCR was initiated by partial sequence of initiator probe (IP), the other part of which can hybridize with capture sequence of RP, and was assembled by hairpin probes modified with biotin (H1-bio and H2-bio). A miR-21 triggered cyclical DSN cleavage of RP, which was immobilized to a streptavidin (SA) coated magnetic bead (MB). The released Fam labeled capture sequence then hybridized with the HCR product to generate a detectable dsDNA. This polymer was then dropped on lateral flow strip and positive result was observed. The proposed method allowed quantitative sequence-specific detection of miR-21 (with a detection limit of 2.1 fM, S/N = 3) in a dynamic range from 100 fM to 100 pM, with an excellent ability to discriminate differences in miRNAs. The method showed acceptable testing recoveries for the determination of miRNAs in serum.

Identification of microRNAs from Amur grape (Vitis amurensis Rupr.) by deep sequencing and analysis of microRNA variations with bioinformatics.

PubMed

Wang, Chen; Han, Jian; Liu, Chonghuai; Kibet, Korir Nicholas; Kayesh, Emrul; Shangguan, Lingfei; Li, Xiaoying; Fang, Jinggui

2012-03-29

MicroRNA (miRNA) is a class of functional non-coding small RNA with 19-25 nucleotides in length while Amur grape (Vitis amurensis Rupr.) is an important wild fruit crop with the strongest cold resistance among the Vitis species, is used as an excellent breeding parent for grapevine, and has elicited growing interest in wine production. To date, there is a relatively large number of grapevine miRNAs (vv-miRNAs) from cultivated grapevine varieties such as Vitis vinifera L. and hybrids of V. vinifera and V. labrusca, but there is no report on miRNAs from Vitis amurensis Rupr, a wild grapevine species. A small RNA library from Amur grape was constructed and Solexa technology used to perform deep sequencing of the library followed by subsequent bioinformatics analysis to identify new miRNAs. In total, 126 conserved miRNAs belonging to 27 miRNA families were identified, and 34 known but non-conserved miRNAs were also found. Significantly, 72 new potential Amur grape-specific miRNAs were discovered. The sequences of these new potential va-miRNAs were further validated through miR-RACE, and accumulation of 18 new va-miRNAs in seven tissues of grapevines confirmed by real time RT-PCR (qRT-PCR) analysis. The expression levels of va-miRNAs in flowers and berries were found to be basically consistent in identity to those from deep sequenced sRNAs libraries of combined corresponding tissues. We also describe the conservation and variation of va-miRNAs using miR-SNPs and miR-LDs during plant evolution based on comparison of orthologous sequences, and further reveal that the number and sites of miR-SNP in diverse miRNA families exhibit distinct divergence. Finally, 346 target genes for the new miRNAs were predicted and they include a number of Amur grape stress tolerance genes and many genes regulating anthocyanin synthesis and sugar metabolism. Deep sequencing of short RNAs from Amur grape flowers and berries identified 72 new potential miRNAs and 34 known but non-conserved miRNAs, indicating that specific miRNAs exist in Amur grape. These results show that a number of regulatory miRNAs exist in Amur grape and play an important role in Amur grape growth, development, and response to abiotic or biotic stress.

Unique preservation of neural cells in Hutchinson- Gilford progeria syndrome is due to the expression of the neural-specific miR-9 microRNA.

PubMed

Nissan, Xavier; Blondel, Sophie; Navarro, Claire; Maury, Yves; Denis, Cécile; Girard, Mathilde; Martinat, Cécile; De Sandre-Giovannoli, Annachiara; Levy, Nicolas; Peschanski, Marc

2012-07-26

One puzzling observation in patients affected with Hutchinson-Gilford progeria syndrome (HGPS), who overall exhibit systemic and dramatic premature aging, is the absence of any conspicuous cognitive impairment. Recent studies based on induced pluripotent stem cells derived from HGPS patient cells have revealed a lack of expression in neural derivatives of lamin A, a major isoform of LMNA that is initially produced as a precursor called prelamin A. In HGPS, defective maturation of a mutated prelamin A induces the accumulation of toxic progerin in patient cells. Here, we show that a microRNA, miR-9, negatively controls lamin A and progerin expression in neural cells. This may bear major functional correlates, as alleviation of nuclear blebbing is observed in nonneural cells after miR-9 overexpression. Our results support the hypothesis, recently proposed from analyses in mice, that protection of neural cells from progerin accumulation in HGPS is due to the physiologically restricted expression of miR-9 to that cell lineage. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

Regulation of FLOWERING LOCUS T by a MicroRNA in Brachypodium distachyon[C][W

PubMed Central

Wu, Liang; Liu, Dongfeng; Wu, Jiajie; Zhang, Rongzhi; Qin, Zhengrui; Liu, Danmei; Li, Aili; Fu, Daolin; Zhai, Wenxue; Mao, Long

2013-01-01

The highly conserved florigen gene FLOWERING LOCUS T (FT) functions at the core of the flowering pathways. Extensive studies have examined the transcriptional regulation of FT; however, other layers of FT regulation remain unclear. Here, we identified miR5200 a Pooideae-specific microRNA that is expressed in leaves and targets Brachypodium distachyon FT orthologs for mRNA cleavage. miR5200 was abundantly expressed in plants grown under short-day (SD) conditions but was dramatically repressed in plants transferred to long-day (LD) conditions. We also found that the epigenetic chromatin status, specifically the levels of histone methylation marks, at miR5200 precursor loci changed in response to daylength. Moreover, artificial interruption of miR5200 activity by target mimicry in B. distachyon altered flowering time in SD but not in LD conditions, suggesting that miR5200 functions in photoperiod-mediated flowering time regulation. Together, these findings illustrate a posttranscriptional regulation mechanism of FT and provide insights into understanding of the multiple concerted pathways for flowering time control in plants. PMID:24285787

Genome-wide identification and characterization of putative lncRNAs in the diamondback moth, Plutella xylostella (L.).

PubMed

Wang, Yue; Xu, Tingting; He, Weiyi; Shen, Xiujing; Zhao, Qian; Bai, Jianlin; You, Minsheng

2018-01-01

Long non-coding RNAs (lncRNAs) are of particular interest because of their contributions to many biological processes. Here, we present the genome-wide identification and characterization of putative lncRNAs in a global insect pest, Plutella xylostella. A total of 8096 lncRNAs were identified and classified into three groups. The average length of exons in lncRNAs was longer than that in coding genes and the GC content was lower than that in mRNAs. Most lncRNAs were flanked by canonical splice sites, similar to mRNAs. Expression profiling identified 114 differentially expressed lncRNAs during the DBM development and found that majority were temporally specific. While the biological functions of lncRNAs remain uncharacterized, many are microRNA precursors or competing endogenous RNAs involved in micro-RNA regulatory pathways. This work provides a valuable resource for further studies on molecular bases for development of DBM and lay the foundation for discovery of lncRNA functions in P. xylostella. Copyright © 2017 Elsevier Inc. All rights reserved.

Precursors of vertebrate peptide antibiotics dermaseptin b and adenoregulin have extensive sequence identities with precursors of opioid peptides dermorphin, dermenkephalin, and deltorphins.

PubMed

Amiche, M; Ducancel, F; Mor, A; Boulain, J C; Menez, A; Nicolas, P

1994-07-08

The dermaseptins are a family of broad spectrum antimicrobial peptides, 27-34 amino acids long, involved in the defense of the naked skin of frogs against microbial invasion. They are the first vertebrate peptides to show lethal effects against the filamentous fungi responsible for severe opportunistic infections accompanying immunodeficiency syndrome and the use of immunosuppressive agents. A cDNA library was constructed from skin poly(A+) RNA of the arboreal frog Phyllomedusa bicolor and screened with an oligonucleotide probe complementary to the COOH terminus of dermaseptin b. Several clones contained a full-length DNA copy of a 443-nucleotide mRNA that encoded a 78-residue dermaseptin b precursor protein. The deduced precursor contained a putative signal sequence at the NH2 terminus, a 20-residue spacer sequence extremely rich (60%) in glutamic and aspartic acids, and a single copy of a dermaseptin b progenitor sequence at the COOH terminus. One clone contained a complete copy of adenoregulin, a 33-residue peptide reported to enhance the binding of agonists to the A1 adenosine receptor. The mRNAs encoding adenoregulin and dermaseptin b were very similar: 70 and 75% nucleotide identities between the 5'- and 3'-untranslated regions, respectively; 91% amino acid identity between the signal peptides; 82% identity between the acidic spacer sequences; and 38% identity between adenoregulin and dermaseptin b. Because adenoregulin and dermaseptin b have similar precursor designs and antimicrobial spectra, adenoregulin should be considered as a new member of the dermaseptin family and alternatively named dermaseptin b II. Preprodermaseptin b and preproadenoregulin have considerable sequence identities to the precursors encoding the opioid heptapeptides dermorphin, dermenkephalin, and deltorphins. This similarity extended into the 5'-untranslated regions of the mRNAs. These findings suggest that the genes encoding the four preproproteins are all members of the same family despite the fact that they encode end products having very different biological activities. These genes might contain a homologous export exon comprising the 5'-untranslated region, the 22-residue signal peptide, the 20-24-residue acidic spacer, and the basic pair Lys-Arg.

Label-free technology for the amplified detection of microRNA based on the allosteric hairpin DNA switch and hybridization chain reaction.

PubMed

Cai, Sheng; Cao, Zhijuan; Lau, Choiwan; Lu, Jianzhong

2014-11-21

By using the allosteric hairpin DNA switch, a novel assay for the detection of microRNA (miRNA) let-7a via a hybridization chain reaction (HCR) was introduced. Briefly, the hairpin DNA switch probe is a single-stranded DNA consisting of a streptavidin (SA) aptamer sequence, a target binding sequence and a certain sequence that acts as a trigger of the HCR. In the presence of target let-7a, the hairpin DNA switch would open and expose the stem region sequences, where a part of this sequence acts as initiator sequence strands for the HCR and triggers a cascade of hybridization events that yields nicked double helices analogous to alternating copolymers, another part is the SA aptamer sequence which activates its binding affinity to SA on SA-coated magnetic particles. The hybridization event could be sensitively detected via an instantaneous derivatization reaction between a special chemiluminescence (CL) reagent, 3,4,5-trimethoxylphenylglyoxal (TMPG) and the guanine nucleotides within the target, the hairpin DNA switch probe, and HCR helices to form an unstable CL intermediate for the generation of light. Our results show that the coupling of the hairpin DNA switch probe and the HCR for the amplified detection of let-7a achieves a better performance (e.g. wide linear response range: 0.1-1000 fmol, low detection limit: 0.1 fmol, and high specificity). Furthermore, this approach could be easily applied to the detection of let-7a in human lung cells, and extended to detect other types of miRNA and proteins such as PDGF based on aptamers. We believe such advancements will represent a significant step towards improved diagnostics and more personalized medical treatment.

Investigation of phase evolution of CaCu{sub 3}Ti{sub 4}O{sub 12} (CCTO) by in situ synchrotron high-temperature powder diffraction

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

Ouyang, Xin; Huang, Saifang; School of Materials Science and Technology, China University of Geosciences

2014-03-15

In situ synchrotron X-ray powder diffraction was used to study the high-temperature phase evolution of CaCu{sub 3}Ti{sub 4}O{sub 12} (CCTO) precursors prepared via solid-state and sol–gel methods. After the precursors are heated to 1225 °C, the CCTO phase is the main phase observed in the calcined powder, with the presence of some minor impurities. Comparing the two precursors, we found that the onset temperature for the CCTO phase formation is 800 °C in the sol–gel precursor, lower than that in the solid-state precursor (875 °C). Intermediate phases were only observed in the sol–gel precursor. Both precursors are able to bemore » calcined to sub-micrometric sized powders. Based on the synchrotron data along with differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA), the phase formation sequence and mechanism during calcination are proposed in this study. -- Graphical abstract: The in situ synchrotron HT-XRD patterns of CCTO sol–gel and solid-state precursor. Highlights: • Phase formation sequence/mechanism in two CCTO precursors has been established. • Formation temperature of CCTO via sol–gel method is lower than solid-state method. • Intermediate phases are only observed in the sol–gel precursor. • Both precursors are able to be calcined into sub-micrometric sized powders.« less

YM500: a small RNA sequencing (smRNA-seq) database for microRNA research

PubMed Central

Cheng, Wei-Chung; Chung, I-Fang; Huang, Tse-Shun; Chang, Shih-Ting; Sun, Hsing-Jen; Tsai, Cheng-Fong; Liang, Muh-Lii; Wong, Tai-Tong; Wang, Hsei-Wei

2013-01-01

MicroRNAs (miRNAs) are small RNAs ∼22 nt in length that are involved in the regulation of a variety of physiological and pathological processes. Advances in high-throughput small RNA sequencing (smRNA-seq), one of the next-generation sequencing applications, have reshaped the miRNA research landscape. In this study, we established an integrative database, the YM500 (http://ngs.ym.edu.tw/ym500/), containing analysis pipelines and analysis results for 609 human and mice smRNA-seq results, including public data from the Gene Expression Omnibus (GEO) and some private sources. YM500 collects analysis results for miRNA quantification, for isomiR identification (incl. RNA editing), for arm switching discovery, and, more importantly, for novel miRNA predictions. Wetlab validation on >100 miRNAs confirmed high correlation between miRNA profiling and RT-qPCR results (R = 0.84). This database allows researchers to search these four different types of analysis results via our interactive web interface. YM500 allows researchers to define the criteria of isomiRs, and also integrates the information of dbSNP to help researchers distinguish isomiRs from SNPs. A user-friendly interface is provided to integrate miRNA-related information and existing evidence from hundreds of sequencing datasets. The identified novel miRNAs and isomiRs hold the potential for both basic research and biotech applications. PMID:23203880

A MicroRNA Superfamily Regulates Nucleotide Binding Site–Leucine-Rich Repeats and Other mRNAs[W][OA

PubMed Central

Shivaprasad, Padubidri V.; Chen, Ho-Ming; Patel, Kanu; Bond, Donna M.; Santos, Bruno A.C.M.; Baulcombe, David C.

2012-01-01

Analysis of tomato (Solanum lycopersicum) small RNA data sets revealed the presence of a regulatory cascade affecting disease resistance. The initiators of the cascade are microRNA members of an unusually diverse superfamily in which miR482 and miR2118 are prominent members. Members of this superfamily are variable in sequence and abundance in different species, but all variants target the coding sequence for the P-loop motif in the mRNA sequences for disease resistance proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) motifs. We confirm, using transient expression in Nicotiana benthamiana, that miR482 targets mRNAs for NBS-LRR disease resistance proteins with coiled-coil domains at their N terminus. The targeting causes mRNA decay and production of secondary siRNAs in a manner that depends on RNA-dependent RNA polymerase 6. At least one of these secondary siRNAs targets other mRNAs of a defense-related protein. The miR482-mediated silencing cascade is suppressed in plants infected with viruses or bacteria so that expression of mRNAs with miR482 or secondary siRNA target sequences is increased. We propose that this process allows pathogen-inducible expression of NBS-LRR proteins and that it contributes to a novel layer of defense against pathogen attack. PMID:22408077

Characterization of Urtica dioica agglutinin isolectins and the encoding gene family.

PubMed

Does, M P; Ng, D K; Dekker, H L; Peumans, W J; Houterman, P M; Van Damme, E J; Cornelissen, B J

1999-01-01

Urtica dioica agglutinin (UDA) has previously been found in roots and rhizomes of stinging nettles as a mixture of UDA-isolectins. Protein and cDNA sequencing have shown that mature UDA is composed of two hevein domains and is processed from a precursor protein. The precursor contains a signal peptide, two in-tandem hevein domains, a hinge region and a carboxyl-terminal chitinase domain. Genomic fragments encoding precursors for UDA-isolectins have been amplified by five independent polymerase chain reactions on genomic DNA from stinging nettle ecotype Weerselo. One amplified gene was completely sequenced. As compared to the published cDNA sequence, the genomic sequence contains, besides two basepair substitutions, two introns located at the same positions as in other plant chitinases. By partial sequence analysis of 40 amplified genes, 16 different genes were identified which encode seven putative UDA-isolectins. The deduced amino acid sequences share 78.9-98.9% identity. In extracts of roots and rhizomes of stinging nettle ecotype Weerselo six out of these seven isolectins were detected by mass spectrometry. One of them is an acidic form, which has not been identified before. Our results demonstrate that UDA is encoded by a large gene family.

Suppression of miR-19b enhanced the cytotoxic effects of mTOR inhibitors in human neuroblastoma cells.

PubMed

Chen, Yun; Tsai, Ya-Hui; Tseng, Bor-Jiun; Pan, Hsin-Yen; Tseng, Sheng-Hong

2016-11-01

Mammalian target of rapamycin (mTOR) inhibitors exert significant antitumor effects on several cancer cell types. In this study, we investigated the effects of mTOR inhibitors, in particular the regulation of the microRNA, in neuroblastoma cells. AZD8055 (a new mTOR inhibitor)- or rapamycin-induced cytotoxic effects on neuroblastoma cells were studied. Western blotting was used to investigate the expression of various proteins in the mTOR pathway. MicroRNA precursors and antagomirs were transfected into cells to manipulate the expression of target microRNA. AZD8055 exerted stronger cytotoxic effects than rapamycin in neuroblastoma cells (p<0.03). In addition, AZD8055 suppressed the mTOR pathway and increased the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in the neuroblastoma cells. AZD8055 significantly decreased miR-19b expression (p<0.005); in contrast, rapamycin increased miR-19b expression (p<0.05). Transfection of miR-19b antagomir into the neuroblastoma cells mimicked the effects of AZD8055 treatment, whereas miR-19b overexpression reversed the effects of AZD8055. Combination of miR-19b knockdown and rapamycin treatment significantly improved the sensitivity of neuroblastoma cells to rapamycin (p<0.02). Suppression of miR-19b may enhance the cytotoxic effects of mTOR inhibitors in neuroblastoma cells. Copyright © 2016 Elsevier Inc. All rights reserved.

A microRNA detection system based on padlock probes and rolling circle amplification

PubMed Central

Jonstrup, Søren Peter; Koch, Jørn; Kjems, Jørgen

2006-01-01

The differential expression and the regulatory roles of microRNAs (miRNAs) are being studied intensively these years. Their minute size of only 19–24 nucleotides and strong sequence similarity among related species call for enhanced methods for reliable detection and quantification. Moreover, miRNA expression is generally restricted to a limited number of specific cells within an organism and therefore requires highly sensitive detection methods. Here we present a simple and reliable miRNA detection protocol based on padlock probes and rolling circle amplification. It can be performed without specialized equipment and is capable of measuring the content of specific miRNAs in a few nanograms of total RNA. PMID:16888321

A microRNA detection system based on padlock probes and rolling circle amplification.

PubMed

Jonstrup, Søren Peter; Koch, Jørn; Kjems, Jørgen

2006-09-01

The differential expression and the regulatory roles of microRNAs (miRNAs) are being studied intensively these years. Their minute size of only 19-24 nucleotides and strong sequence similarity among related species call for enhanced methods for reliable detection and quantification. Moreover, miRNA expression is generally restricted to a limited number of specific cells within an organism and therefore requires highly sensitive detection methods. Here we present a simple and reliable miRNA detection protocol based on padlock probes and rolling circle amplification. It can be performed without specialized equipment and is capable of measuring the content of specific miRNAs in a few nanograms of total RNA.

Utility of MicroRNAs and siRNAs in Cervical Carcinogenesis

PubMed Central

Díaz-González, Sacnite del Mar; Benítez-Boijseauneau, Odelia; Gómez-Cerón, Claudia; Bermúdez-Morales, Victor Hugo; Rodríguez-Dorantes, Mauricio; Pérez-Plasencia, Carlos; Peralta-Zaragoza, Oscar

2015-01-01

MicroRNAs and siRNAs belong to a family of small noncoding RNAs which bind through partial sequence complementarity to 3′-UTR regions of mRNA from target genes, resulting in the regulation of gene expression. MicroRNAs have become an attractive target for genetic and pharmacological modulation due to the critical function of their target proteins in several signaling pathways, and their expression profiles have been found to be altered in various cancers. A promising technology platform for selective silencing of cell and/or viral gene expression using siRNAs is currently in development. Cervical cancer is the most common cancer in women in the developing world and sexually transmitted infection with HPV is the cause of this malignancy. Therefore, a cascade of abnormal events is induced during cervical carcinogenesis, including the induction of genomic instability, reprogramming of cellular metabolic pathways, deregulation of cell proliferation, inhibition of apoptotic mechanisms, disruption of cell cycle control mechanisms, and alteration of gene expression. Thus, in the present review article, we highlight new research on microRNA expression profiles which may be utilized as biomarkers for cervical cancer. Furthermore, we discuss selective silencing of HPV E6 and E7 with siRNAs which represents a potential gene therapy strategy against cervical cancer. PMID:25874209

Utility of microRNAs and siRNAs in cervical carcinogenesis.

PubMed

Díaz-González, Sacnite del Mar; Deas, Jessica; Benítez-Boijseauneau, Odelia; Gómez-Cerón, Claudia; Bermúdez-Morales, Victor Hugo; Rodríguez-Dorantes, Mauricio; Pérez-Plasencia, Carlos; Peralta-Zaragoza, Oscar

2015-01-01

MicroRNAs and siRNAs belong to a family of small noncoding RNAs which bind through partial sequence complementarity to 3'-UTR regions of mRNA from target genes, resulting in the regulation of gene expression. MicroRNAs have become an attractive target for genetic and pharmacological modulation due to the critical function of their target proteins in several signaling pathways, and their expression profiles have been found to be altered in various cancers. A promising technology platform for selective silencing of cell and/or viral gene expression using siRNAs is currently in development. Cervical cancer is the most common cancer in women in the developing world and sexually transmitted infection with HPV is the cause of this malignancy. Therefore, a cascade of abnormal events is induced during cervical carcinogenesis, including the induction of genomic instability, reprogramming of cellular metabolic pathways, deregulation of cell proliferation, inhibition of apoptotic mechanisms, disruption of cell cycle control mechanisms, and alteration of gene expression. Thus, in the present review article, we highlight new research on microRNA expression profiles which may be utilized as biomarkers for cervical cancer. Furthermore, we discuss selective silencing of HPV E6 and E7 with siRNAs which represents a potential gene therapy strategy against cervical cancer.

An Internal Signal Sequence Directs Intramembrane Proteolysis of a Cellular Immunoglobulin Domain Protein*S⃞

PubMed Central

Robakis, Thalia; Bak, Beata; Lin, Shu-huei; Bernard, Daniel J.; Scheiffele, Peter

2008-01-01

Precursor proteolysis is a crucial mechanism for regulating protein structure and function. Signal peptidase (SP) is an enzyme with a well defined role in cleaving N-terminal signal sequences but no demonstrated function in the proteolysis of cellular precursor proteins. We provide evidence that SP mediates intraprotein cleavage of IgSF1, a large cellular Ig domain protein that is processed into two separate Ig domain proteins. In addition, our results suggest the involvement of signal peptide peptidase (SPP), an intramembrane protease, which acts on substrates that have been previously cleaved by SP. We show that IgSF1 is processed through sequential proteolysis by SP and SPP. Cleavage is directed by an internal signal sequence and generates two separate Ig domain proteins from a polytopic precursor. Our findings suggest that SP and SPP function are not restricted to N-terminal signal sequence cleavage but also contribute to the processing of cellular transmembrane proteins. PMID:18981173

Human adenovirus serotype 12 virion precursors pMu and pVI are cleaved at amino-terminal and carboxy-terminal sites that conform to the adenovirus 2 endoproteinase cleavage consensus sequence.

PubMed

Freimuth, P; Anderson, C W

1993-03-01

The sequence of a 1158-base pair fragment of the human adenovirus serotype 12 (Ad12) genome was determined. This segment encodes the precursors for virion components Mu and VI. Both Ad12 precursors contain two sequences that conform to a consensus sequence motif for cleavage by the endoproteinase of adenovirus 2 (Ad2). Analysis of the amino terminus of VI and of the peptide fragments found in Ad12 virions demonstrated that these sites are cleaved during Ad12 maturation. This observation suggests that the recognition motif for adenovirus endoproteinases is highly conserved among human serotypes. The adenovirus 2 endoproteinase polypeptide requires additional co-factors for activity (C. W. Anderson, Protein Expression Purif., 1993, 4, 8-15). Synthetic Ad12 or Ad2 pVI carboxy-terminal peptides each permitted efficient cleavage of an artificial endoproteinase substrate by recombinant Ad2 endoproteinase polypeptide.

MicroRNA-133a Regulates Insulin-like Growth Factor-1 Receptor Expression and Vascular Smooth Muscle Cell Proliferation in Murine Atherosclerosis

PubMed Central

Gao, Song; Wassler, Michael; Zhang, Lulu; Li, Yangxin; Wang, Jun; Zhang, Yi; Shelat, Harnath; Williams, Jason; Geng, Yong-Jian

2014-01-01

Objective MicroRNA-133a (miR-133a) and insulin-like growth factor-1 (IGF-1) are two different molecules known to regulate cardiovascular cell proliferation. This study tested whether miR-133a affects expression of IGF-1 receptor (IGF-1R) and proliferation of IGF-1-stimulated vascular smooth muscle cells (VSMC) in a murine model of atherosclerosis. Methods and Results Expression of IGF-1R was analyzed by immuno-fluorescence and immuno-blotting, and miR-133a by qRT-PCR in the aortas of wild-type C57BL/6J (WT) and apolipoprotein-E deficient (ApoE−/−) mice. Compared to those in WT aortas, the IGF-1R and miR-133a levels were lower in ApoE−/− aortas. ApoE−/− VSMC grew slower than WT cells in the cultures with IGF-1-containing medium. MiR-133a-specific inhibitor decreased miR-133a, IGF-1R expression, IGF-1-stimulated VSMC growth in lipoprotein-deficient media. By contrast, miR-133a precursor increased IGF-1R levels and promoted IGF-1-induced VSMC proliferation. In the luciferase-IGF-1R 3’UTR reporter system, the reporter luciferase activity was not inhibited in VSMC with miR-133a overexpression. IGF-1R mRNA half-life in ApoE−/− VSMC was shorter than that in WT VSMC. MiR-133a inhibitor reduced but precursor increased the mRNA half-life, although the effects appeared less striking in ApoE−/− VSMC than in WT cells. Conclusion MiR-133a serves as a stimulatory factor for IGF-1R expression through prolonging IGF-1R mRNA half-life. In atherosclerosis induced by ApoE deficiency, reduced miR-133a expression is associated with lower IGF-1R levels and suppressive VSMC growth. Administration of miR-133a precursor may potentiate IGF-1 stimulated VSMC survival and growth. PMID:24401233

Distinct MicroRNA Expression Profile and Targeted Biological Pathways in Functional Myeloid-derived Suppressor Cells Induced by Δ9-Tetrahydrocannabinol in Vivo

PubMed Central

Hegde, Venkatesh L.; Tomar, Sunil; Jackson, Austin; Rao, Roshni; Yang, Xiaoming; Singh, Udai P.; Singh, Narendra P.; Nagarkatti, Prakash S.; Nagarkatti, Mitzi

2013-01-01

Δ9-Tetrahydrocannabinol (THC), the major bioactive component of marijuana, has been shown to induce functional myeloid-derived suppressor cells (MDSCs) in vivo. Here, we studied the involvement of microRNA (miRNA) in this process. CD11b+Gr-1+ MDSCs were purified from peritoneal exudates of mice administered with THC and used for genome-wide miRNA profiling. Expression of CD31 and Ki-67 confirmed that the THC-MDSCs were immature and proliferating. THC-induced MDSCs exhibited distinct miRNA expression signature relative to various myeloid cells and BM precursors. We identified 13 differentially expressed (>2-fold) miRNA in THC-MDSCs relative to control BM precursors. In silico target prediction for these miRNA and pathway analysis using multiple bioinformatics tools revealed significant overrepresentation of Gene Ontology clusters within hematopoiesis, myeloid cell differentiation, and regulation categories. Insulin-like growth factor 1 signaling involved in cell growth and proliferation, and myeloid differentiation pathways were among the most significantly enriched canonical pathways. Among the differentially expressed, miRNA-690 was highly overexpressed in THC-MDSCs (∼16-fold). Transcription factor CCAAT/enhancer-binding protein α (C/EBPα) was identified as a potential functional target of miR-690. Supporting this, C/EBPα expression was attenuated in THC-MDSCs as compared with BM precursors and exhibited an inverse relation with miR-690. miR-690 knockdown using peptide nucleic acid-antagomiR was able to unblock and significantly increase C/EBPα expression establishing the functional link. Further, CD11b+Ly6G+Ly6C+ and CD11b+Ly6G−Ly6C+ purified subtypes showed high levels of miR-690 with attenuated C/EBPα expression. Moreover, EL-4 tumor-elicited MDSCs showed increased miR-690 expression. In conclusion, miRNA are significantly altered during the generation of functional MDSC from BM. Select miRNA such as miR-690 targeting genes involved in myeloid expansion and differentiation likely play crucial roles in this process and therefore in cannabinoid-induced immunosuppression. PMID:24202177

A common anchor facilitated GO-DNA nano-system for multiplex microRNA analysis in live cells.

PubMed

Yu, Jiantao; He, Sihui; Shao, Chen; Zhao, Haoran; Li, Jing; Tian, Leilei

2018-04-19

The design of a nano-system for the detection of intracellular microRNAs is challenging as it must fulfill complex requirements, i.e., it must have a high sensitivity to determine the dynamic expression level, a good reliability for multiplex and simultaneous detection, and a satisfactory biostability to work in biological environments. Instead of employing a commonly used physisorption or a full-conjugation strategy, here, a GO-DNA nano-system was developed under graft/base-pairing construction. The common anchor sequence was chemically grafted to GO to base-pair with various microRNA probes; and the hybridization with miRNAs drives the dyes on the probes to leave away from GO, resulting in "turned-on" fluorescence. This strategy not only simplifies the synthesis but also efficiently balances the loading yields of different probes. Moreover, the conjugation yield of GO with a base-paired hybrid has been improved by more than two-fold compared to that of the conjugation with a single strand. We demonstrated that base-paired DNA probes could be efficiently delivered into cells along with GO and are properly stabilized by the conjugated anchor sequence. The resultant GO-DNA nano-system exhibited high stability in a complex biological environment and good resistance to nucleases, and was able to accurately discriminate various miRNAs without cross-reaction. With all of these positive features, the GO-DNA nano-system can simultaneously detect three miRNAs and monitor their dynamic expression levels.

Profile of microRNA in Giant Panda Blood: A Resource for Immune-Related and Novel microRNAs

PubMed Central

Yang, Mingyu; Du, Lianming; Li, Wujiao; Shen, Fujun; Fan, Zhenxin; Jian, Zuoyi; Hou, Rong; Shen, Yongmei; Yue, Bisong; Zhang, Xiuyue

2015-01-01

The giant panda (Ailuropoda melanoleuca) is one of the world’s most beloved endangered mammals. Although the draft genome of this species had been assembled, little was known about the composition of its microRNAs (miRNAs) or their functional profiles. Recent studies demonstrated that changes in the expression of miRNAs are associated with immunity. In this study, miRNAs were extracted from the blood of four healthy giant pandas and sequenced by Illumina next generation sequencing technology. As determined by miRNA screening, a total of 276 conserved miRNAs and 51 novel putative miRNAs candidates were detected. After differential expression analysis, we noticed that the expressions of 7 miRNAs were significantly up-regulated in young giant pandas compared with that of adults. Moreover, 2 miRNAs were up-regulated in female giant pandas and 1 in the male individuals. Target gene prediction suggested that the miRNAs of giant panda might be relevant to the expressions of 4,602 downstream genes. Subseuqently, the predicted target genes were conducted to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis and we found that these genes were mainly involved in host immunity, including the Ras signaling pathway, the PI3K-Akt signaling pathway, and the MAPK signaling pathway. In conclusion, our results provide the first miRNA profiles of giant panda blood, and the predicted functional analyses may open an avenue for further study of giant panda immunity. PMID:26599861

Profile of microRNA in Giant Panda Blood: A Resource for Immune-Related and Novel microRNAs.

PubMed

Yang, Mingyu; Du, Lianming; Li, Wujiao; Shen, Fujun; Fan, Zhenxin; Jian, Zuoyi; Hou, Rong; Shen, Yongmei; Yue, Bisong; Zhang, Xiuyue

2015-01-01

The giant panda (Ailuropoda melanoleuca) is one of the world's most beloved endangered mammals. Although the draft genome of this species had been assembled, little was known about the composition of its microRNAs (miRNAs) or their functional profiles. Recent studies demonstrated that changes in the expression of miRNAs are associated with immunity. In this study, miRNAs were extracted from the blood of four healthy giant pandas and sequenced by Illumina next generation sequencing technology. As determined by miRNA screening, a total of 276 conserved miRNAs and 51 novel putative miRNAs candidates were detected. After differential expression analysis, we noticed that the expressions of 7 miRNAs were significantly up-regulated in young giant pandas compared with that of adults. Moreover, 2 miRNAs were up-regulated in female giant pandas and 1 in the male individuals. Target gene prediction suggested that the miRNAs of giant panda might be relevant to the expressions of 4,602 downstream genes. Subseuqently, the predicted target genes were conducted to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis and we found that these genes were mainly involved in host immunity, including the Ras signaling pathway, the PI3K-Akt signaling pathway, and the MAPK signaling pathway. In conclusion, our results provide the first miRNA profiles of giant panda blood, and the predicted functional analyses may open an avenue for further study of giant panda immunity.

TAM: a method for enrichment and depletion analysis of a microRNA category in a list of microRNAs.

PubMed

Lu, Ming; Shi, Bing; Wang, Juan; Cao, Qun; Cui, Qinghua

2010-08-09

MicroRNAs (miRNAs) are a class of important gene regulators. The number of identified miRNAs has been increasing dramatically in recent years. An emerging major challenge is the interpretation of the genome-scale miRNA datasets, including those derived from microarray and deep-sequencing. It is interesting and important to know the common rules or patterns behind a list of miRNAs, (i.e. the deregulated miRNAs resulted from an experiment of miRNA microarray or deep-sequencing). For the above purpose, this study presents a method and develops a tool (TAM) for annotations of meaningful human miRNAs categories. We first integrated miRNAs into various meaningful categories according to prior knowledge, such as miRNA family, miRNA cluster, miRNA function, miRNA associated diseases, and tissue specificity. Using TAM, given lists of miRNAs can be rapidly annotated and summarized according to the integrated miRNA categorical data. Moreover, given a list of miRNAs, TAM can be used to predict novel related miRNAs. Finally, we confirmed the usefulness and reliability of TAM by applying it to deregulated miRNAs in acute myocardial infarction (AMI) from two independent experiments. TAM can efficiently identify meaningful categories for given miRNAs. In addition, TAM can be used to identify novel miRNA biomarkers. TAM tool, source codes, and miRNA category data are freely available at http://cmbi.bjmu.edu.cn/tam.

SERS-based inverse molecular sentinel (iMS) nanoprobes for multiplexed detection of microRNA cancer biomarkers in biological samples

NASA Astrophysics Data System (ADS)

Crawford, Bridget M.; Wang, Hsin-Neng; Fales, Andrew M.; Bowie, Michelle L.; Seewaldt, Victoria L.; Vo-Dinh, Tuan

2017-02-01

The development of sensitive and selective biosensing techniques is of great interest for clinical diagnostics. Here, we describe the development and application of a surface enhanced Raman scattering (SERS) sensing technology, referred to as "inverse Molecular Sentinel (iMS)" nanoprobes, for the detection of nucleic acid biomarkers in biological samples. This iMS nanoprobe involves the use of plasmonic-active nanostars as the sensing platform for a homogenous assay for multiplexed detection of nucleic acid biomarkers, including DNA, RNA and microRNA (miRNA). The "OFF-to-ON" signal switch is based on a non-enzymatic strand-displacement process and the conformational change of stem-loop (hairpin) oligonucleotide probes upon target binding. Here, we demonstrate the development of iMS nanoprobes for the detection of DNA sequences as well as a modified design of the nanoprobe for the detection of short (22-nt) microRNA sequences. The application of iMS nanoprobes to detect miRNAs in real biological samples was performed with total small RNA extracted from breast cancer cell lines. The multiplex capability of the iMS technique was demonstrated using a mixture of the two differently labeled nanoprobes to detect miR-21 and miR-34a miRNA biomarkers for breast cancer. The results of this study demonstrate the feasibility of applying the iMS technique for multiplexed detection of nucleic acid biomarkers, including short miRNAs molecules.

Diversity and functional convergence of small noncoding RNAs in male germ cell differentiation and fertilization

PubMed Central

García-López, Jesús; Alonso, Lola; Cárdenas, David B.; Artaza-Alvarez, Haydeé; Hourcade, Juan de Dios; Martínez, Sergio; Brieño-Enríquez, Miguel A.; del Mazo, Jesús

2015-01-01

The small noncoding RNAs (sncRNAs) are considered as post-transcriptional key regulators of male germ cell development. In addition to microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), other sncRNAs generated from small nucleolar RNAs (snoRNAs), tRNAs, or rRNAs processing may also play important regulatory roles in spermatogenesis. By next-generation sequencing (NGS), we characterized the sncRNA populations detected at three milestone stages in male germ differentiation: primordial germ cells (PGCs), pubertal spermatogonia cells, and mature spermatozoa. To assess their potential transmission through the spermatozoa during fertilization, the sncRNAs of mouse oocytes and zygotes were also analyzed. Both, microRNAs and snoRNA-derived small RNAs are abundantly expressed in PGCs but transiently replaced by piRNAs in spermatozoa and endo-siRNAs in oocytes and zygotes. Exhaustive analysis of miRNA sequence variants also shows an increment of noncanonical microRNA forms along male germ cell differentiation. RNAs-derived from tRNAs and rRNAs interacting with PIWI proteins are not generated by the ping-pong pathway and could be a source of primary piRNAs. Moreover, our results strongly suggest that the small RNAs-derived from tRNAs and rRNAs are interacting with PIWI proteins, and specifically with MILI. Finally, computational analysis revealed their potential involvement in post-transcriptional regulation of mRNA transcripts suggesting functional convergence among different small RNA classes in germ cells and zygotes. PMID:25805854

RACK-1 regulates let-7 microRNA expression and terminal cell differentiation in Caenorhabditis elegans

PubMed Central

Chu, Yu-De; Wang, Wei-Chieh; Chen, Shi-An A; Hsu, Yen-Ting; Yeh, Meng-Wei; Slack, Frank J; Chan, Shih-Peng

2014-01-01

The let-7 microRNA (miRNA) regulates cell cycle exit and terminal differentiation in the C. elegans heterochronic gene pathway. Low expression of let-7 results in retarded vulva and hypodermal cell development in C. elegans and has been associated with several human cancers. Previously, the versatile scaffold protein receptor for activated C kinase 1 (RACK1) was proposed to facilitate recruitment of the miRNA-induced silencing complex (miRISC) to the polysome and to be required for miRNA function in C. elegans and humans. Here, we show that depletion of C. elegans RACK-1 by RNAi increases let-7 miRNA levels and suppresses the retarded terminal differentiation of lateral hypodermal seam cells in mutants carrying the hypomorphic let-7(n2853) allele or lacking the let-7 family miRNA genes mir-48 and mir-241. Depletion of RACK-1 also increases the levels of precursor let-7 miRNA. When Dicer is knocked down and pre-miRNA processing is inhibited, depletion of RACK-1 still leads to increased levels of pre-let-7, suggesting that RACK-1 affects a biogenesis mechanism upstream of Dicer. No changes in the activity of the let-7 promoter or the levels of primary let-7 miRNA are associated with depletion of RACK-1, suggesting that RACK-1 affects let-7 miRNA biogenesis at the post-transcriptional level. Interestingly, rack-1 knockdown also increases the levels of a few other precursor miRNAs. Our results reveal that RACK-1 controls the biogenesis of a subset of miRNAs, including let-7, and in this way plays a role in the heterochronic gene pathway during C. elegans development. PMID:24776851

Small Molecule Inhibition of microRNA-210 Reprograms an Oncogenic Hypoxic Circuit.

PubMed

Costales, Matthew G; Haga, Christopher L; Velagapudi, Sai Pradeep; Childs-Disney, Jessica L; Phinney, Donald G; Disney, Matthew D

2017-03-08

A hypoxic state is critical to the metastatic and invasive characteristics of cancer. Numerous pathways play critical roles in cancer maintenance, many of which include noncoding RNAs such as microRNA (miR)-210 that regulates hypoxia inducible factors (HIFs). Herein, we describe the identification of a small molecule named Targapremir-210 that binds to the Dicer site of the miR-210 hairpin precursor. This interaction inhibits production of the mature miRNA, derepresses glycerol-3-phosphate dehydrogenase 1-like enzyme (GPD1L), a hypoxia-associated protein negatively regulated by miR-210, decreases HIF-1α, and triggers apoptosis of triple negative breast cancer cells only under hypoxic conditions. Further, Targapremir-210 inhibits tumorigenesis in a mouse xenograft model of hypoxic triple negative breast cancer. Many factors govern molecular recognition of biological targets by small molecules. For protein, chemoproteomics and activity-based protein profiling are invaluable tools to study small molecule target engagement and selectivity in cells. Such approaches are lacking for RNA, leaving a void in the understanding of its druggability. We applied Chemical Cross-Linking and Isolation by Pull Down (Chem-CLIP) to study the cellular selectivity and the on- and off-targets of Targapremir-210. Targapremir-210 selectively recognizes the miR-210 precursor and can differentially recognize RNAs in cells that have the same target motif but have different expression levels, revealing this important feature for selectively drugging RNAs for the first time. These studies show that small molecules can be rapidly designed to selectively target RNAs and affect cellular responses to environmental conditions, resulting in favorable benefits against cancer. Further, they help define rules for identifying druggable targets in the transcriptome.

MicroRNA-153 Physiologically Inhibits Expression of Amyloid-β Precursor Protein in Cultured Human Fetal Brain Cells and Is Dysregulated in a Subset of Alzheimer Disease Patients*

PubMed Central

Long, Justin M.; Ray, Balmiki; Lahiri, Debomoy K.

2012-01-01

Regulation of amyloid-β (Aβ) precursor protein (APP) expression is complex. MicroRNAs (miRNAs) are expected to participate in the molecular network that controls this process. The composition of this network is, however, still undefined. Elucidating the complement of miRNAs that regulate APP expression should reveal novel drug targets capable of modulating Aβ production in AD. Here, we investigated the contribution of miR-153 to this regulatory network. A miR-153 target site within the APP 3′-untranslated region (3′-UTR) was predicted by several bioinformatic algorithms. We found that miR-153 significantly reduced reporter expression when co-transfected with an APP 3′-UTR reporter construct. Mutation of the predicted miR-153 target site eliminated this reporter response. miR-153 delivery in both HeLa cells and primary human fetal brain cultures significantly reduced APP expression. Delivery of a miR-153 antisense inhibitor to human fetal brain cultures significantly elevated APP expression. miR-153 delivery also reduced expression of the APP paralog APLP2. High functional redundancy between APP and APLP2 suggests that miR-153 may target biological pathways in which they both function. Interestingly, in a subset of human AD brain specimens with moderate AD pathology, miR-153 levels were reduced. This same subset also exhibited elevated APP levels relative to control specimens. Therefore, endogenous miR-153 inhibits expression of APP in human neurons by specifically interacting with the APP 3′-UTR. This regulatory interaction may have relevance to AD etiology, where low miR-153 levels may drive increased APP expression in a subset of AD patients. PMID:22733824

MicroRNA160 Modulates Plant Development and Heat Shock Protein Gene Expression to Mediate Heat Tolerance in Arabidopsis

PubMed Central

Lin, Jeng-Shane; Kuo, Chia-Chia; Yang, I-Chu; Tsai, Wei-An; Shen, Yu-Hsing; Lin, Chih-Ching; Liang, Yi-Chen; Li, Yu-Chi; Kuo, Yun-Wei; King, Yu-Chi; Lai, Hsi-Mei; Jeng, Shih-Tong

2018-01-01

Global warming is causing a negative impact on plant growth and adversely impacts on crop yield. MicroRNAs (miRNAs) are critical in regulating the expression of genes involved in plant development as well as defense responses. The effects of miRNAs on heat-stressed Arabidopsis warrants further investigation. Heat stress increased the expression of miR160 and its precursors but considerably reduced that of its targets, ARF10, ARF16, and ARF17. To study the roles of miR160 during heat stress, transgenic Arabidopsis plants overexpressing miR160 precursor a (160OE) and artificial miR160 (MIM160), which mimics an inhibitor of miR160, were created. T-DNA insertion mutants of miR160 targets were also used to examine their tolerances to heat stress. Results presented that overexpressing miR160 improved seed germination and seedling survival under heat stress. The lengths of hypocotyl elongation and rachis were also longer in 160OE than the wild-type (WT) plants under heat stress. Interestingly, MIM160 plants showed worse adaption to heat. In addition, arf10, arf16, and arf17 mutants presented similar phenotypes to 160OE under heat stress to advance abilities of thermotolerance. Moreover, transcriptome and qRT-PCR analyses revealed that HSP17.6A, HSP17.6II, HSP21, and HSP70B expression levels were regulated by heat in 160OE, MIM160, arf10, arf16, and arf17 plants. Hence, miR160 altered the expression of the heat shock proteins and plant development to allow plants to survive heat stress. PMID:29449855

MiR-422a as a Potential Cellular MicroRNA Biomarker for Postmenopausal Osteoporosis

PubMed Central

Cao, Zheng; Moore, Benjamin T.; Wang, Yang; Peng, Xian-Hao; Lappe, Joan M.; Recker, Robert R.; Xiao, Peng

2014-01-01

Background MicroRNAs (miRNAs) are a class of short non-coding RNA molecules that regulate gene expression by targeting mRNAs. Recently, miRNAs have been shown to play important roles in the etiology of various diseases. However, little is known about their roles in the development of osteoporosis. Circulating monocytes are osteoclast precursors that also produce various factors important for osteoclastogenesis. Previously, we have identified a potential biomarker miR-133a in circulating monocytes for postmenopausal osteoporosis. In this study, we aimed to further identify significant miRNA biomarkers in human circulating monocytes underlying postmenopausal osteoporosis. Methodology/Principal Findings We used ABI TaqMan miRNA array followed by qRT-PCR validation in human circulating monocytes from 10 high BMD and 10 low BMD postmenopausal Caucasian women to identify miRNA biomarkers. MiR-422a was up-regulated with marginal significance (P = 0.065) in the low compared with the high BMD group in the array analysis. However, a significant up-regulation of miR-422a was identified in the low BMD group by qRT-PCR analysis (P = 0.029). We also performed bioinformatic target gene analysis and found several potential target genes of miR-422a which are involved in osteoclastogenesis. Further qRT-PCR analyses of the target genes in the same study subjects showed that the expression of five of these genes (CBL, CD226, IGF1, PAG1, and TOB2) correlated negatively with miR-422a expression. Conclusions/Significance Our study suggests that miR-422a in human circulating monocytes (osteoclast precursors) is a potential miRNA biomarker underlying postmenopausal osteoporosis. PMID:24820117

MiR-422a as a potential cellular microRNA biomarker for postmenopausal osteoporosis.

PubMed

Cao, Zheng; Moore, Benjamin T; Wang, Yang; Peng, Xian-Hao; Lappe, Joan M; Recker, Robert R; Xiao, Peng

2014-01-01

MicroRNAs (miRNAs) are a class of short non-coding RNA molecules that regulate gene expression by targeting mRNAs. Recently, miRNAs have been shown to play important roles in the etiology of various diseases. However, little is known about their roles in the development of osteoporosis. Circulating monocytes are osteoclast precursors that also produce various factors important for osteoclastogenesis. Previously, we have identified a potential biomarker miR-133a in circulating monocytes for postmenopausal osteoporosis. In this study, we aimed to further identify significant miRNA biomarkers in human circulating monocytes underlying postmenopausal osteoporosis. We used ABI TaqMan miRNA array followed by qRT-PCR validation in human circulating monocytes from 10 high BMD and 10 low BMD postmenopausal Caucasian women to identify miRNA biomarkers. MiR-422a was up-regulated with marginal significance (P = 0.065) in the low compared with the high BMD group in the array analysis. However, a significant up-regulation of miR-422a was identified in the low BMD group by qRT-PCR analysis (P = 0.029). We also performed bioinformatic target gene analysis and found several potential target genes of miR-422a which are involved in osteoclastogenesis. Further qRT-PCR analyses of the target genes in the same study subjects showed that the expression of five of these genes (CBL, CD226, IGF1, PAG1, and TOB2) correlated negatively with miR-422a expression. Our study suggests that miR-422a in human circulating monocytes (osteoclast precursors) is a potential miRNA biomarker underlying postmenopausal osteoporosis.

Molecular cloning of a cDNA encoding the precursor of adenoregulin from frog skin. Relationships with the vertebrate defensive peptides, dermaseptins.

PubMed

Amiche, M; Ducancel, F; Lajeunesse, E; Boulain, J C; Ménez, A; Nicolas, P

1993-03-31

Adenoregulin has recently been isolated from Phyllomedusa skin as a 33 amino acid residues peptide which enhanced binding of agonists to the A1 adenosine receptor. In order to study the structure of the precursor of adenoregulin we constructed a cDNA library from mRNAs extracted from the skin of Phyllomedusa bicolor. We detected the complete nucleotide sequence of a cDNA encoding the adenoregulin biosynthetic precursor. The deduced sequence of the precursor is 81 amino acids long, exhibits a putative signal sequence at the NH2 terminus and contains a single copy of the biologically active peptide at the COOH terminus. Structural and conformational homologies that are observed between adenoregulin and the dermaseptins, antimicrobial peptides exhibiting strong membranolytic activities against various pathogenic agents, suggest that adenoregulin is an additional member of the growing family of cytotropic antimicrobial peptides that allow vertebrate animals to defend themselves against microorganisms. As such, the adenosine receptor regulating activity of adenoregulin could be due to its ability to interact with and disrupt membranes lipid bilayers.

Amyloid-like aggregation of provasopressin in diabetes insipidus and secretory granule sorting.

PubMed

Beuret, Nicole; Hasler, Franziska; Prescianotto-Baschong, Cristina; Birk, Julia; Rutishauser, Jonas; Spiess, Martin

2017-01-26

Aggregation of peptide hormone precursors in the trans-Golgi network is an essential process in the biogenesis of secretory granules in endocrine cells. It has recently been proposed that this aggregation corresponds to the formation of functional amyloids. Our previous finding that dominant mutations in provasopressin, which cause cell degeneration and diabetes insipidus, prevent native folding and produce fibrillar aggregates in the endoplasmic reticulum (ER) might thus reflect mislocalized amyloid formation by sequences that evolved to mediate granule sorting. Here we identified two sequences responsible for fibrillar aggregation of mutant precursors in the ER: the N-terminal vasopressin nonapeptide and the C-terminal glycopeptide. To test their role in granule sorting, the glycopeptide was deleted and/or vasopressin mutated to inactivate ER aggregation while still permitting precursor folding and ER exit. These mutations strongly reduced sorting into granules and regulated secretion in endocrine AtT20 cells. The same sequences - vasopressin and the glycopeptide - mediate physiological aggregation of the wild-type hormone precursor into secretory granules and the pathological fibrillar aggregation of disease mutants in the ER. These findings support the amyloid hypothesis for secretory granule biogenesis.

Targeted deletion of miR-132/-212 impairs memory and alters the hippocampal transcriptome.

PubMed

Hansen, Katelin F; Sakamoto, Kensuke; Aten, Sydney; Snider, Kaitlin H; Loeser, Jacob; Hesse, Andrea M; Page, Chloe E; Pelz, Carl; Arthur, J Simon C; Impey, Soren; Obrietan, Karl

2016-02-01

miR-132 and miR-212 are structurally related microRNAs that have been found to exert powerful modulatory effects within the central nervous system (CNS). Notably, these microRNAs are tandomly processed from the same noncoding transcript, and share a common seed sequence: thus it has been difficult to assess the distinct contribution of each microRNA to gene expression within the CNS. Here, we employed a combination of conditional knockout and transgenic mouse models to examine the contribution of the miR-132/-212 gene locus to learning and memory, and then to assess the distinct effects that each microRNA has on hippocampal gene expression. Using a conditional deletion approach, we show that miR-132/-212 double-knockout mice exhibit significant cognitive deficits in spatial memory, recognition memory, and in tests of novel object recognition. Next, we utilized transgenic miR-132 and miR-212 overexpression mouse lines and the miR-132/-212 double-knockout line to explore the distinct effects of these two miRNAs on the transcriptional profile of the hippocampus. Illumina sequencing revealed that miR-132/-212 deletion increased the expression of 1138 genes; Venn analysis showed that 96 of these genes were also downregulated in mice overexpressing miR-132. Of the 58 genes that were decreased in animals overexpressing miR-212, only four of them were also increased in the knockout line. Functional gene ontology analysis of downregulated genes revealed significant enrichment of genes related to synaptic transmission, neuronal proliferation, and morphogenesis, processes known for their roles in learning, and memory formation. These data, coupled with previous studies, firmly establish a role for the miR-132/-212 gene locus as a key regulator of cognitive capacity. Further, although miR-132 and miR-212 share a seed sequence, these data indicate that these miRNAs do not exhibit strongly overlapping mRNA targeting profiles, thus indicating that these two genes may function in a complex, nonredundant manner to shape the transcriptional profile of the CNS. The dysregulation of miR-132/-212 expression could contribute to signaling mechanisms that are involved in an array of cognitive disorders. © 2016 Hansen et al.; Published by Cold Spring Harbor Laboratory Press.

Targeted deletion of miR-132/-212 impairs memory and alters the hippocampal transcriptome

PubMed Central

Hansen, Katelin F.; Sakamoto, Kensuke; Aten, Sydney; Snider, Kaitlin H.; Loeser, Jacob; Hesse, Andrea M.; Page, Chloe E.; Pelz, Carl; Arthur, J. Simon C.; Impey, Soren

2016-01-01

miR-132 and miR-212 are structurally related microRNAs that have been found to exert powerful modulatory effects within the central nervous system (CNS). Notably, these microRNAs are tandomly processed from the same noncoding transcript, and share a common seed sequence: thus it has been difficult to assess the distinct contribution of each microRNA to gene expression within the CNS. Here, we employed a combination of conditional knockout and transgenic mouse models to examine the contribution of the miR-132/-212 gene locus to learning and memory, and then to assess the distinct effects that each microRNA has on hippocampal gene expression. Using a conditional deletion approach, we show that miR-132/-212 double-knockout mice exhibit significant cognitive deficits in spatial memory, recognition memory, and in tests of novel object recognition. Next, we utilized transgenic miR-132 and miR-212 overexpression mouse lines and the miR-132/-212 double-knockout line to explore the distinct effects of these two miRNAs on the transcriptional profile of the hippocampus. Illumina sequencing revealed that miR-132/-212 deletion increased the expression of 1138 genes; Venn analysis showed that 96 of these genes were also downregulated in mice overexpressing miR-132. Of the 58 genes that were decreased in animals overexpressing miR-212, only four of them were also increased in the knockout line. Functional gene ontology analysis of downregulated genes revealed significant enrichment of genes related to synaptic transmission, neuronal proliferation, and morphogenesis, processes known for their roles in learning, and memory formation. These data, coupled with previous studies, firmly establish a role for the miR-132/-212 gene locus as a key regulator of cognitive capacity. Further, although miR-132 and miR-212 share a seed sequence, these data indicate that these miRNAs do not exhibit strongly overlapping mRNA targeting profiles, thus indicating that these two genes may function in a complex, nonredundant manner to shape the transcriptional profile of the CNS. The dysregulation of miR-132/-212 expression could contribute to signaling mechanisms that are involved in an array of cognitive disorders. PMID:26773099

Identification and Characterization of miRNA Transcriptome in Asiatic Cotton (Gossypium arboreum) Using High Throughput Sequencing

PubMed Central

Farooq, Muhammad; Mansoor, Shahid; Guo, Hui; Amin, Imran; Chee, Peng W.; Azim, M. Kamran; Paterson, Andrew H.

2017-01-01

MicroRNAs (miRNAs) are small 20–24nt molecules that have been well studied over the past decade due to their important regulatory roles in different cellular processes. The mature sequences are more conserved across vast phylogenetic scales than their precursors and some are conserved within entire kingdoms, hence, their loci and function can be predicted by homology searches. Different studies have been performed to elucidate miRNAs using de novo prediction methods but due to complex regulatory mechanisms or false positive in silico predictions, not all of them express in reality and sometimes computationally predicted mature transcripts differ from the actual expressed ones. With the availability of a complete genome sequence of Gossypium arboreum, it is important to annotate the genome for both coding and non-coding regions using high confidence transcript evidence, for this cotton species that is highly resistant to various biotic and abiotic stresses. Here we have analyzed the small RNA transcriptome of G. arboreum leaves and provided genome annotation of miRNAs with evidence from miRNA/miRNA∗ transcripts. A total of 446 miRNAs clustered into 224 miRNA families were found, among which 48 families are conserved in other plants and 176 are novel. Four short RNA libraries were used to shortlist best predictions based on high reads per million. The size, origin, copy numbers and transcript depth of all miRNAs along with their isoforms and targets has been reported. The highest gene copy number was observed for gar-miR7504 followed by gar-miR166, gar-miR8771, gar-miR156, and gar-miR7484. Altogether, 1274 target genes were found in G. arboreum that are enriched for 216 KEGG pathways. The resultant genomic annotations are provided in UCSC, BED format. PMID:28663752

Human L-DOPA decarboxylase mRNA is a target of miR-145: A prediction to validation workflow.

PubMed

Papadopoulos, Emmanuel I; Fragoulis, Emmanuel G; Scorilas, Andreas

2015-01-10

l-DOPA decarboxylase (DDC) is a multiply-regulated gene which encodes the enzyme that catalyzes the biosynthesis of dopamine in humans. MicroRNAs comprise a novel class of endogenously transcribed small RNAs that can post-transcriptionally regulate the expression of various genes. Given that the mechanism of microRNA target recognition remains elusive, several genes, including DDC, have not yet been identified as microRNA targets. Nevertheless, a number of specifically designed bioinformatic algorithms provide candidate miRNAs for almost every gene, but still their results exhibit moderate accuracy and should be experimentally validated. Motivated by the above, we herein sought to discover a microRNA that regulates DDC expression. By using the current algorithms according to bibliographic recommendations we found that miR-145 could be predicted with high specificity as a candidate regulatory microRNA for DDC expression. Thus, a validation experiment followed by firstly transfecting an appropriate cell culture system with a synthetic miR-145 sequence and sequentially assessing the mRNA and protein levels of DDC via real-time PCR and Western blotting, respectively. Our analysis revealed that miR-145 had no significant impact on protein levels of DDC but managed to dramatically downregulate its mRNA expression. Overall, the experimental and bioinformatic analysis conducted herein indicate that miR-145 has the ability to regulate DDC mRNA expression and potentially this occurs by recognizing its mRNA as a target. Copyright © 2014. Published by Elsevier B.V.

Identification of evolutionarily conserved Momordica charantia microRNAs using computational approach and its utility in phylogeny analysis.

PubMed

Thirugnanasambantham, Krishnaraj; Saravanan, Subramanian; Karikalan, Kulandaivelu; Bharanidharan, Rajaraman; Lalitha, Perumal; Ilango, S; HairulIslam, Villianur Ibrahim

2015-10-01

Momordica charantia (bitter gourd, bitter melon) is a monoecious Cucurbitaceae with anti-oxidant, anti-microbial, anti-viral and anti-diabetic potential. Molecular studies on this economically valuable plant are very essential to understand its phylogeny and evolution. MicroRNAs (miRNAs) are conserved, small, non-coding RNA with ability to regulate gene expression by bind the 3' UTR region of target mRNA and are evolved at different rates in different plant species. In this study we have utilized homology based computational approach and identified 27 mature miRNAs for the first time from this bio-medically important plant. The phylogenetic tree developed from binary data derived from the data on presence/absence of the identified miRNAs were noticed to be uncertain and biased. Most of the identified miRNAs were highly conserved among the plant species and sequence based phylogeny analysis of miRNAs resolved the above difficulties in phylogeny approach using miRNA. Predicted gene targets of the identified miRNAs revealed their importance in regulation of plant developmental process. Reported miRNAs held sequence conservation in mature miRNAs and the detailed phylogeny analysis of pre-miRNA sequences revealed genus specific segregation of clusters. Copyright © 2015 Elsevier Ltd. All rights reserved.

Target mimicry provides a new mechanism for regulation of microRNA activity.

PubMed

Franco-Zorrilla, José Manuel; Valli, Adrián; Todesco, Marco; Mateos, Isabel; Puga, María Isabel; Rubio-Somoza, Ignacio; Leyva, Antonio; Weigel, Detlef; García, Juan Antonio; Paz-Ares, Javier

2007-08-01

MicroRNAs (miRNA) regulate key aspects of development and physiology in animals and plants. These regulatory RNAs act as guides of effector complexes to recognize specific mRNA sequences based on sequence complementarity, resulting in translational repression or site-specific cleavage. In plants, most miRNA targets are cleaved and show almost perfect complementarity with the miRNAs around the cleavage site. Here, we examined the non-protein coding gene IPS1 (INDUCED BY PHOSPHATE STARVATION 1) from Arabidopsis thaliana. IPS1 contains a motif with sequence complementarity to the phosphate (Pi) starvation-induced miRNA miR-399, but the pairing is interrupted by a mismatched loop at the expected miRNA cleavage site. We show that IPS1 RNA is not cleaved but instead sequesters miR-399. Thus, IPS1 overexpression results in increased accumulation of the miR-399 target PHO2 mRNA and, concomitantly, in reduced shoot Pi content. Engineering of IPS1 to be cleavable abolishes its inhibitory activity on miR-399. We coin the term 'target mimicry' to define this mechanism of inhibition of miRNA activity. Target mimicry can be generalized beyond the control of Pi homeostasis, as demonstrated using artificial target mimics.

Regulation of Plant Microprocessor Function in Shaping microRNA Landscape.

PubMed

Dolata, Jakub; Taube, Michał; Bajczyk, Mateusz; Jarmolowski, Artur; Szweykowska-Kulinska, Zofia; Bielewicz, Dawid

2018-01-01

MicroRNAs are small molecules (∼21 nucleotides long) that are key regulators of gene expression. They originate from long stem-loop RNAs as a product of cleavage by a protein complex called Microprocessor. The core components of the plant Microprocessor are the RNase type III enzyme Dicer-Like 1 (DCL1), the zinc finger protein Serrate (SE), and the double-stranded RNA binding protein Hyponastic Leaves 1 (HYL1). Microprocessor assembly and its processing of microRNA precursors have been reported to occur in discrete nuclear bodies called Dicing bodies. The accessibility of and modifications to Microprocessor components affect microRNA levels and may have dramatic consequences in plant development. Currently, numerous lines of evidence indicate that plant Microprocessor activity is tightly regulated. The cellular localization of HYL1 is dependent on a specific KETCH1 importin, and the E3 ubiquitin ligase COP1 indirectly protects HYL1 from degradation in a light-dependent manner. Furthermore, proper localization of HYL1 in Dicing bodies is regulated by MOS2. On the other hand, the Dicing body localization of DCL1 is regulated by NOT2b, which also interacts with SE in the nucleus. Post-translational modifications are substantial factors that contribute to protein functional diversity and provide a fine-tuning system for the regulation of protein activity. The phosphorylation status of HYL1 is crucial for its activity/stability and is a result of the interplay between kinases (MPK3 and SnRK2) and phosphatases (CPL1 and PP4). Additionally, MPK3 and SnRK2 are known to phosphorylate SE. Several other proteins (e.g., TGH, CDF2, SIC, and RCF3) that interact with Microprocessor have been found to influence its RNA-binding and processing activities. In this minireview, recent findings on the various modes of Microprocessor activity regulation are discussed.

Regulation of Plant Microprocessor Function in Shaping microRNA Landscape

PubMed Central

Dolata, Jakub; Taube, Michał; Bajczyk, Mateusz; Jarmolowski, Artur; Szweykowska-Kulinska, Zofia; Bielewicz, Dawid

2018-01-01

MicroRNAs are small molecules (∼21 nucleotides long) that are key regulators of gene expression. They originate from long stem–loop RNAs as a product of cleavage by a protein complex called Microprocessor. The core components of the plant Microprocessor are the RNase type III enzyme Dicer-Like 1 (DCL1), the zinc finger protein Serrate (SE), and the double-stranded RNA binding protein Hyponastic Leaves 1 (HYL1). Microprocessor assembly and its processing of microRNA precursors have been reported to occur in discrete nuclear bodies called Dicing bodies. The accessibility of and modifications to Microprocessor components affect microRNA levels and may have dramatic consequences in plant development. Currently, numerous lines of evidence indicate that plant Microprocessor activity is tightly regulated. The cellular localization of HYL1 is dependent on a specific KETCH1 importin, and the E3 ubiquitin ligase COP1 indirectly protects HYL1 from degradation in a light-dependent manner. Furthermore, proper localization of HYL1 in Dicing bodies is regulated by MOS2. On the other hand, the Dicing body localization of DCL1 is regulated by NOT2b, which also interacts with SE in the nucleus. Post-translational modifications are substantial factors that contribute to protein functional diversity and provide a fine-tuning system for the regulation of protein activity. The phosphorylation status of HYL1 is crucial for its activity/stability and is a result of the interplay between kinases (MPK3 and SnRK2) and phosphatases (CPL1 and PP4). Additionally, MPK3 and SnRK2 are known to phosphorylate SE. Several other proteins (e.g., TGH, CDF2, SIC, and RCF3) that interact with Microprocessor have been found to influence its RNA-binding and processing activities. In this minireview, recent findings on the various modes of Microprocessor activity regulation are discussed. PMID:29922322

Partial structure of the phylloxin gene from the giant monkey frog, Phyllomedusa bicolor: parallel cloning of precursor cDNA and genomic DNA from lyophilized skin secretion.

PubMed

Chen, Tianbao; Gagliardo, Ron; Walker, Brian; Zhou, Mei; Shaw, Chris

2005-12-01

Phylloxin is a novel prototype antimicrobial peptide from the skin of Phyllomedusa bicolor. Here, we describe parallel identification and sequencing of phylloxin precursor transcript (mRNA) and partial gene structure (genomic DNA) from the same sample of lyophilized skin secretion using our recently-described cloning technique. The open-reading frame of the phylloxin precursor was identical in nucleotide sequence to that previously reported and alignment with the nucleotide sequence derived from genomic DNA indicated the presence of a 175 bp intron located in a near identical position to that found in the dermaseptins. The highly-conserved structural organization of skin secretion peptide genes in P. bicolor can thus be extended to include that encoding phylloxin (plx). These data further reinforce our assertion that application of the described methodology can provide robust genomic/transcriptomic/peptidomic data without the need for specimen sacrifice.

Genome-wide identification and functional annotation of miRNAs in anti-inflammatory plant and their cross-kingdom regulation in Homo sapiens.

PubMed

Sharma, Ankita; Sahu, Sarika; Kumari, Pooja; Gopi, Soundhara Rajan; Malhotra, Rajesh; Biswas, Sagarika

2017-05-01

MicroRNAs (miRNAs) are newly discovered non-coding small (~17-24 nucleotide) RNAs that regulate gene expression of its target mRNA at the post-transcriptional levels. In this study, total 12,593 ESTs of Curcuma longa were taken from database of expressed sequence tags (dbEST) and clustered into 2821 contigs using EGassembler web server. Precursor miRNAs (pre-miRNAs) were predicted from these contigs that folded into stem-loop structure using MFold server. Thirty-four mature C. longa miRNAs (clo-miRNAs) were identified from pre-miRNAs having targets involved in various important functions of plant such as self-defence, growth and development, alkaloid metabolic pathway and ethylene signalling process. Sequence analysis of identified clo-miRNAs indicated that 56% miRNAs belong to ORF and 44% belong to non-ORF region. clo-mir-5 and clo-mir-6 were established as the conserved miRNAs, whereas clo-mir-20 was predicted to be the most stable miRNA. Phylogenetic analysis carried out by molecular evolutionary genetics analysis (MEGA) software indicated close evolutionary relationship of clo-mir-5075 with osa-MIR5075. Further, identified clo-miRNAs were checked for their cross-kingdom regulatory potential. clo-mir-14 was found to regulate various gene transcripts in humans that has been further investigated for its biostability in foetal bovine serum (FBS). The results indicated higher degree of stability of clo-mir-14 (48 h) in FBS. Thus, contribution of this miRNA to the cellular immune response during the inflamed condition of rheumatoid arthritis and adequate stability may make it a good choice for the therapeutic agent in near future.

Circulating MicroRNAs in Serum from Cattle Challenged with Bovine Viral Diarrhea Virus.

PubMed

Taxis, Tasia M; Bauermann, Fernando V; Ridpath, Julia F; Casas, Eduardo

2017-01-01

Bovine viral diarrhea virus (BVDV) is an RNA virus that is often associated with respiratory disease in cattle. MicroRNAs have been proposed as indicators of exposure to respiratory pathogens. The objective of this study was to identify microRNAs in cattle that had been challenged with a non-cytopathic field strain of BVDV. Five colostrum deprived neonate Holstein calves were inoculated with BVDV (challenged) and 4 were mock challenged (control). Serum from all calves was collected at four different times: prior to challenge (day 0) and at 4, 9, and 16 days post-challenge. RNA was extracted from sera, and expression, via read counts, of small non-coding RNAs were obtained using next-generation sequencing. A total of 905,861 sequences identified 427 microRNAs. Sixty-two microRNAs had >1,000 total reads across all samples. Bta-miR-339a, bta-miR-185, bta-miR-486, Bta-miR-92a, bta-miR-30e-5p, bta-let-7c, and bta-miR-2284x were significantly different ( P < 0.05) across time regardless of challenge status. Bta-miR-423-5p ( P = 0.008) and bta-miR-151-3p ( P = 0.005) were significantly different between challenged and control animals across time. In challenged animals, bta-miR-423-5p peaked in number of reads by day 4 and steadily declined from day 4 to day 16. In control animals, bta-miR-423-5p declined from day 0 to day 9 and increased in number by day 16. By day 16, both challenged and control animals had similar levels of bta-miR-423-5p, and these levels were similar to day 0 levels. Bta-miR-151-3p peaked at day 9 in challenged animals, while control animals decreased across time. By day 16, the number of reads of bta-miR-151-3p were similar between challenged and control animals. The level in challenged animals had returned to day 0 levels by day 16, whereas the levels for control animals was significantly lower ( P = 0.006) than day 0. Further studies are needed to establish if bta-miR-423-5p or bta-miR-151-3p could be used as a biomarker for exposure to BVDV.

Circulating MicroRNAs in Serum from Cattle Challenged with Bovine Viral Diarrhea Virus‡

PubMed Central

Taxis, Tasia M.; Bauermann, Fernando V.; Ridpath, Julia F.; Casas, Eduardo

2017-01-01

Bovine viral diarrhea virus (BVDV) is an RNA virus that is often associated with respiratory disease in cattle. MicroRNAs have been proposed as indicators of exposure to respiratory pathogens. The objective of this study was to identify microRNAs in cattle that had been challenged with a non-cytopathic field strain of BVDV. Five colostrum deprived neonate Holstein calves were inoculated with BVDV (challenged) and 4 were mock challenged (control). Serum from all calves was collected at four different times: prior to challenge (day 0) and at 4, 9, and 16 days post-challenge. RNA was extracted from sera, and expression, via read counts, of small non-coding RNAs were obtained using next-generation sequencing. A total of 905,861 sequences identified 427 microRNAs. Sixty-two microRNAs had >1,000 total reads across all samples. Bta-miR-339a, bta-miR-185, bta-miR-486, Bta-miR-92a, bta-miR-30e-5p, bta-let-7c, and bta-miR-2284x were significantly different (P < 0.05) across time regardless of challenge status. Bta-miR-423-5p (P = 0.008) and bta-miR-151-3p (P = 0.005) were significantly different between challenged and control animals across time. In challenged animals, bta-miR-423-5p peaked in number of reads by day 4 and steadily declined from day 4 to day 16. In control animals, bta-miR-423-5p declined from day 0 to day 9 and increased in number by day 16. By day 16, both challenged and control animals had similar levels of bta-miR-423-5p, and these levels were similar to day 0 levels. Bta-miR-151-3p peaked at day 9 in challenged animals, while control animals decreased across time. By day 16, the number of reads of bta-miR-151-3p were similar between challenged and control animals. The level in challenged animals had returned to day 0 levels by day 16, whereas the levels for control animals was significantly lower (P = 0.006) than day 0. Further studies are needed to establish if bta-miR-423-5p or bta-miR-151-3p could be used as a biomarker for exposure to BVDV. PMID:28702050

Delivery and detection of dietary plant-based miRNAs in animal tissues

USDA-ARS?s Scientific Manuscript database

It has been proposed that genetic material, namely microRNAs (miRNAs), consumed in plant-based diets can affect animal gene expression. Though deep sequencing reveals the low-level presence of plant miRNAs in animal tissues, many groups have been thus far unable to replicate the finding that a rice ...

DNA strand-displacement-induced fluorescence enhancement for highly sensitive and selective assay of multiple microRNA in cancer cells.

PubMed

Wu, Ping; Tu, Yunqiu; Qian, Yingdan; Zhang, Hui; Cai, Chenxin

2014-01-28

We report a new strategy for evaluating multiple miRNA expressions in cancer cells based on DNA strand-displacement-induced fluorescence enhancement. This assay has the ability to discriminate the target from even single-base mismatched sequences or other miRNAs.

Differentially expressed JAK-STAT signaling pathway genes and target microRNAs in the spleen of necrotic enteritis-afflicted chicken lines

USDA-ARS?s Scientific Manuscript database

The JAK signal transducer and STAT signaling pathway is an important regulator of cell proliferation, differentiation, survival, motility, apoptosis, immune response, and development. In this study, we used RNA-Sequencing, qRT-PCR, and bioinformatics tools to investigate the differential expression ...

Identification of Differentially Expressed Micrornas Associate with Glucose Metabolism in Different Organs of Blunt Snout Bream (Megalobrama amblycephala)

PubMed Central

Miao, Ling-Hong; Lin, Yan; Pan, Wen-Jing; Huang, Xin; Ge, Xian-Ping; Ren, Ming-Chun; Zhou, Qun-Lan; Liu, Bo

2017-01-01

Blunt snout bream (Megalobrama amblycephala) is a widely favored herbivorous fish species and is a frequentlyused fish model for studying the metabolism physiology. This study aimed to provide a comprehensive illustration of the mechanisms of a high-starch diet (HSD) induced lipid metabolic disorder by identifying microRNAs (miRNAs) controlled pathways in glucose and lipid metabolism in fish using high-throughput sequencing technologies. Small RNA libraries derived from intestines, livers, and brains of HSD and normal-starch diet (NSD) treated M. amblycephala were sequenced and 79, 124 and 77 differentially expressed miRNAs (DEMs) in intestines, livers, and brains of HSD treated fish were identified, respectively. Bioinformatics analyses showed that these DEMs targeted hundreds of predicted genes were enriched into metabolic pathways and biosynthetic processes, including peroxisome proliferator-activated receptor (PPAR), glycolysis/gluconeogenesis, and insulin signaling pathway. These analyses confirmed that miRNAs play crucial roles in glucose and lipid metabolism related to high wheat starch treatment. These results provide information on further investigation of a DEM-related mechanism dysregulated by a high carbohydrate diet. PMID:28561770

Mycobacterium tuberculosis Exploits a Molecular Off Switch of the Immune System for Intracellular Survival.

PubMed

von Both, Ulrich; Berk, Maurice; Agapow, Paul-Michael; Wright, Joseph D; Git, Anna; Hamilton, Melissa Shea; Goldgof, Greg; Siddiqui, Nazneen; Bellos, Evangelos; Wright, Victoria J; Coin, Lachlan J; Newton, Sandra M; Levin, Michael

2018-01-12

Mycobacterium tuberculosis (M. tuberculosis) survives and multiplies inside human macrophages by subversion of immune mechanisms. Although these immune evasion strategies are well characterised functionally, the underlying molecular mechanisms are poorly understood. Here we show that during infection of human whole blood with M. tuberculosis, host gene transcriptional suppression, rather than activation, is the predominant response. Spatial, temporal and functional characterisation of repressed genes revealed their involvement in pathogen sensing and phagocytosis, degradation within the phagolysosome and antigen processing and presentation. To identify mechanisms underlying suppression of multiple immune genes we undertook epigenetic analyses. We identified significantly differentially expressed microRNAs with known targets in suppressed genes. In addition, after searching regions upstream of the start of transcription of suppressed genes for common sequence motifs, we discovered novel enriched composite sequence patterns, which corresponded to Alu repeat elements, transposable elements known to have wide ranging influences on gene expression. Our findings suggest that to survive within infected cells, mycobacteria exploit a complex immune "molecular off switch" controlled by both microRNAs and Alu regulatory elements.

The Complex Exogenous RNA Spectra in Human Plasma: An Interface with Human Gut Biota?

PubMed Central

Wang, Kai; Li, Hong; Yuan, Yue; Etheridge, Alton; Zhou, Yong; Huang, David; Wilmes, Paul; Galas, David

2012-01-01

Human plasma has long been a rich source for biomarker discovery. It has recently become clear that plasma RNA molecules, such as microRNA, in addition to proteins are common and can serve as biomarkers. Surveying human plasma for microRNA biomarkers using next generation sequencing technology, we observed that a significant fraction of the circulating RNA appear to originate from exogenous species. With careful analysis of sequence error statistics and other controls, we demonstrated that there is a wide range of RNA from many different organisms, including bacteria and fungi as well as from other species. These RNAs may be associated with protein, lipid or other molecules protecting them from RNase activity in plasma. Some of these RNAs are detected in intracellular complexes and may be able to influence cellular activities under in vitro conditions. These findings raise the possibility that plasma RNAs of exogenous origin may serve as signaling molecules mediating for example the human-microbiome interaction and may affect and/or indicate the state of human health. PMID:23251414

RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA

PubMed Central

Cui, Yalei; Huang, Tianzhi; Zhang, Xiaobo

2015-01-01

MicroRNAs (miRNAs) integrate with Argonaut (Ago) to create the RNA-induced silencing complex, and regulate gene expression by silencing target mRNAs. RNA editing of miRNA may affect miRNA processing, assembly of the Ago complex and target mRNA binding. However, the function of edited miRNA, assembled within the Ago complex, has not been extensively investigated. In this study, sequence analysis of the Ago complex of Marsupenaeus japonicus shrimp infected with white spot syndrome virus (WSSV) revealed that host ADAR (adenosine deaminase acting on RNA) catalysed A-to-I RNA editing of a viral miRNA (WSSV-miR-N12) at the +16 site. This editing of the non-seed sequence did not affect association of the edited miRNA with the Ago protein, but inhibited interaction between the miRNA and its target gene (wsv399). The WSSV early gene wsv399 inhibited WSSV infection. As a result, the RNA editing of miRNA caused virus latency. Our results highlight a novel example of miRNA editing in the miRNA-induced silencing complex. PMID:26674414

Identification and profiling of conserved and novel microRNAs involved in oil and oleic acid production during embryogenesis in Carya cathayensis Sarg.

PubMed

Wang, Zhengjia; Huang, Ruiming; Sun, Zhichao; Zhang, Tong; Huang, Jianqin

2017-05-01

MicroRNAs (miRNAs) are important regulators of plant development and fruit formation. Mature embryos of hickory (Carya cathayensis Sarg.) nuts contain more than 70% oil (comprising 90% unsaturated fatty acids), along with a substantial amount of oleic acid. To understand the roles of miRNAs involved in oil and oleic acid production during hickory embryogenesis, three small RNA libraries from different stages of embryogenesis were constructed. Deep sequencing of these three libraries identified 95 conserved miRNAs with 19 miRNA*s, 7 novel miRNAs (as well as their corresponding miRNA*s), and 26 potentially novel miRNAs. The analysis identified 15 miRNAs involved in oil and oleic acid production that are differentially expressed during embryogenesis in hickory. Among them, nine miRNA sequences, including eight conserved and one novel, were confirmed by qRT-PCR. In addition, 145 target genes of the novel miRNAs were predicted using a bioinformatic approach. Our results provide a framework for better understanding the roles of miRNAs during embryogenesis in hickory.

High-Throughput Sequencing of Plasma MicroRNA in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis

PubMed Central

Brenu, Ekua W.; Ashton, Kevin J.; Batovska, Jana; Staines, Donald R.; Marshall-Gradisnik, Sonya M.

2014-01-01

Background MicroRNAs (miRNAs) are known to regulate many biological processes and their dysregulation has been associated with a variety of diseases including Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME). The recent discovery of stable and reproducible miRNA in plasma has raised the possibility that circulating miRNAs may serve as novel diagnostic markers. The objective of this study was to determine the role of plasma miRNA in CFS/ME. Results Using Illumina high-throughput sequencing we identified 19 miRNAs that were differentially expressed in the plasma of CFS/ME patients in comparison to non-fatigued controls. Following RT-qPCR analysis, we were able to confirm the significant up-regulation of three miRNAs (hsa-miR-127-3p, hsa-miR-142-5p and hsa-miR-143-3p) in the CFS/ME patients. Conclusion Our study is the first to identify circulating miRNAs from CFS/ME patients and also to confirm three differentially expressed circulating miRNAs in CFS/ME patients, providing a basis for further study to find useful CFS/ME biomarkers. PMID:25238588

Revised annotation of Plutella xylostella microRNAs and their genome-wide target identification.

PubMed

Etebari, K; Asgari, S

2016-12-01

The diamondback moth, Plutella xylostella, is the most devastating pest of brassica crops worldwide. Although 128 mature microRNAs (miRNAs) have been annotated from this species in miRBase, there is a need to extend and correct the current P. xylostella miRNA repertoire as a result of its recently improved genome assembly and more available small RNA sequence data. We used our new ultra-deep sequence data and bioinformatics to re-annotate the P. xylostella genome for high confidence miRNAs with the correct 5p and 3p arm features. Furthermore, all the P. xylostella annotated genes were also screened to identify potential miRNA binding sites using three target-predicting algorithms. In total, 203 mature miRNAs were annotated, including 33 novel miRNAs. We identified 7691 highly confident binding sites for 160 pxy-miRNAs. The data provided here will facilitate future studies involving functional analyses of P. xylostella miRNAs as a platform to introduce novel approaches for sustainable management of this destructive pest. © 2016 The Royal Entomological Society.

miR-328 Functions as an RNA Decoy to Modulate hnRNP E2 Regulation of mRNA Translation in Leukemic Blasts

PubMed Central

Eiring, Anna M.; Harb, Jason G.; Neviani, Paolo; Garton, Christopher; Oaks, Joshua J.; Spizzo, Riccardo; Liu, Shujun; Schwind, Sebastian; Santhanam, Ramasamy; Hickey, Christopher J.; Becker, Heiko; Chandler, Jason C.; Andino, Raul; Cortes, Jorge; Hokland, Peter; Huettner, Claudia S.; Bhatia, Ravi; Roy, Denis C.; Liebhaber, Stephen A.; Caligiuri, Michael A.; Marcucci, Guido; Garzon, Ramiro; Croce, Carlo M.; Calin, George A.; Perrotti, Danilo

2010-01-01

SUMMARY MicroRNAs and heterogeneous ribonucleoproteins (hnRNPs) are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner. Here, we report that loss of miR-328 occurs in blast crisis chronic myelogenous leukemia (CML-BC) in a BCR/ABL dose- and kinase-dependent manner through the MAPK-hnRNP E2 pathway. Restoration of miR-328 expression rescues differentiation and impairs survival of leukemic blasts by simultaneously interacting with the translational regulator poly(rC)-binding protein hnRNP E2 and with the mRNA encoding the survival factor PIM1, respectively. The interaction with hnRNP E2 is independent of the microRNA’s seed sequence and it leads to release of CEBPA mRNA from hnRNP E2-mediated translational inhibition. Altogether, these data reveal the dual ability of a microRNA to control cell fate both through base pairing with mRNA targets and through a decoy activity that interferes with the function of regulatory proteins. PMID:20211135

Angiostrongylus cantonensis: identification and characterization of microRNAs in male and female adults.

PubMed

Chen, Mu-Xin; Ai, Lin; Xu, Min-Jun; Zhang, Ren-Li; Chen, Shao-Hong; Zhang, Yong-Nian; Guo, Jian; Cai, Yu-Chun; Tian, Li-Guang; Zhang, Ling-Ling; Zhu, Xing-Quan; Chen, Jia-Xu

2011-06-01

Angiostrongylus cantonensis causes eosinophilic meningitis and eosinophilic pleocytosis in humans and is of significant socio-economic importance globally. microRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in gene expression regulation, cellular function and defense, homeostasis and pathogenesis. They have been identified in a diverse range of organisms. The objective of this study was to determine and characterize miRNAs of female and male adults of A. cantonensis by Solexa deep sequencing. A total of 8,861,260 and 10,957,957 high quality reads with 20 and 23 conserved miRNAs were obtained in females and males, respectively. No new miRNA sequence was found. Nucleotide bias analysis showed that uracil was the prominent nucleotide, particularly at positions of 1, 10, 14, 17 and 22, approximately at the beginning, middle and the end of the conserved miRNAs. To our knowledge, this is the first report of miRNA profiles in A. cantonensis, which may represent a new platform for studying regulation of genes and their networks in A. cantonensis. Copyright © 2011 Elsevier Inc. All rights reserved.

The Mechanism of Synchronous Precise Regulation of Two Shrimp White Spot Syndrome Virus Targets by a Viral MicroRNA

PubMed Central

He, Yaodong; Ma, Tiantian; Zhang, Xiaobo

2017-01-01

MicroRNAs (miRNAs), important factors in animal innate immunity, suppress the expressions of their target genes by binding to target mRNA’s 3′ untranslated regions (3′UTRs). However, the mechanism of synchronous regulation of multiple targets by a single miRNA remains unclear. In this study, the interaction between a white spot syndrome virus (WSSV) miRNA (WSSV-miR-N32) and its two viral targets (wsv459 and wsv322) was characterized in WSSV-infected shrimp. The outcomes indicated that WSSV-encoded miRNA (WSSV-miR-N32) significantly inhibited virus infection by simultaneously targeting wsv459 and wsv322. The silencing of wsv459 or wsv322 by siRNA led to significant decrease of WSSV copies in shrimp, showing that the two viral genes were required for WSSV infection. WSSV-miR-N32 could mediate 5′–3′ exonucleolytic digestion of its target mRNAs, which stopped at the sites of target mRNA 3′UTRs close to the sequence complementary to the miRNA seed sequence. The complementary bases (to the target mRNA sequence) of a miRNA 9th–18th non-seed sequence were essential for the miRNA targeting. Therefore, our findings presented novel insights into the mechanism of miRNA-mediated suppression of target gene expressions, which would be helpful for understanding the roles of miRNAs in innate immunity of invertebrate. PMID:29230209

MicroRNA in Glioblastoma: An Overview

PubMed Central

Banelli, Barbara; Forlani, Alessandra; Allemanni, Giorgio; Morabito, Anna; Pistillo, Maria Pia

2017-01-01

Glioblastoma is the most aggressive brain tumor and, even with the current multimodal therapy, is an invariably lethal cancer with a life expectancy that depends on the tumor subtype but, even in the most favorable cases, rarely exceeds 2 years. Epigenetic factors play an important role in gliomagenesis, are strong predictors of outcome, and are important determinants for the resistance to radio- and chemotherapy. The latest addition to the epigenetic machinery is the noncoding RNA (ncRNA), that is, RNA molecules that are not translated into a protein and that exert their function by base pairing with other nucleic acids in a reversible and nonmutational mode. MicroRNAs (miRNA) are a class of ncRNA of about 22 bp that regulate gene expression by binding to complementary sequences in the mRNA and silence its translation into proteins. MicroRNAs reversibly regulate transcription through nonmutational mechanisms; accordingly, they can be considered as epigenetic effectors. In this review, we will discuss the role of miRNA in glioma focusing on their role in drug resistance and on their potential applications in the therapy of this tumor. PMID:29234674

mirEX: a platform for comparative exploration of plant pri-miRNA expression data.

PubMed

Bielewicz, Dawid; Dolata, Jakub; Zielezinski, Andrzej; Alaba, Sylwia; Szarzynska, Bogna; Szczesniak, Michal W; Jarmolowski, Artur; Szweykowska-Kulinska, Zofia; Karlowski, Wojciech M

2012-01-01

mirEX is a comprehensive platform for comparative analysis of primary microRNA expression data. RT-qPCR-based gene expression profiles are stored in a universal and expandable database scheme and wrapped by an intuitive user-friendly interface. A new way of accessing gene expression data in mirEX includes a simple mouse operated querying system and dynamic graphs for data mining analyses. In contrast to other publicly available databases, the mirEX interface allows a simultaneous comparison of expression levels between various microRNA genes in diverse organs and developmental stages. Currently, mirEX integrates information about the expression profile of 190 Arabidopsis thaliana pri-miRNAs in seven different developmental stages: seeds, seedlings and various organs of mature plants. Additionally, by providing RNA structural models, publicly available deep sequencing results, experimental procedure details and careful selection of auxiliary data in the form of web links, mirEX can function as a one-stop solution for Arabidopsis microRNA information. A web-based mirEX interface can be accessed at http://bioinfo.amu.edu.pl/mirex.

MicroRNAs Change the Landscape of Cancer Resistance.

PubMed

Zhu, Jun; Zhu, Wei; Wu, Wei

2018-01-01

One of the major challenges in the cancer treatment is the development of drug resistance. It represents a major obstacle to curing cancer with constrained efficacy of both conventional chemotherapy and targeted therapies, even recent immune checkpoint blockade therapy. Deciphering the mechanisms of resistance is critical to further understanding the multifactorial pathways involved, and developing more specific targeted treatments. To date, numerous studies have reported the potential role of microRNAs (miRNAs) in the resistance to various cancer treatments. MicroRNAs are a family of small noncoding RNAs that regulate gene expression by sequence-specific targeting of mRNAs causing translational repression or mRNA degradation. More than 1200 validated human miRNAs have been identified in human genome. While one miRNA can regulate hundreds of targets, a single target can also be affected by multiple miRNAs. Evidence suggests that dysregulation of specific miRNAs may be involved in the acquisition of resistance, thereby modulating the sensitivity of cancer cells to treatment. Therefore, manipulation of miRNAs may be an attractive strategy for more effective individualized therapies through reprograming resistant network in cancer cells.

RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.).

PubMed

Patade, Vikas Yadav; Khatri, Deepti; Kumar, Kamal; Grover, Atul; Kumari, Maya; Gupta, Sanjay Mohan; Kumar, Devender; Nasim, Mohammed

2014-07-01

Curcin, a type I ribosomal inhibiting protein-RIP, encoded by curcin precursor gene, is a phytotoxin present in Jatropha (Jatropha curcas L.). Here, we report designing of RNAi construct for the curcin precursor gene and further its genetic transformation of Jatropha to reduce its transcript expression. Curcin precursor gene was first cloned from Jatropha strain DARL-2 and part of the gene sequence was cloned in sense and antisense orientation separated by an intron sequence in plant expression binary vector pRI101 AN. The construction of the RNAi vector was confirmed by double digestion and nucleotide sequencing. The vector was then mobilized into Agrobacterium tumefaciens strain GV 3101 and used for tissue culture independent in planta transformation protocol optimized for Jatropha. Germinating seeds were injured with a needle before infection with Agrobacterium and then transferred to sterilized sand medium. The seedlings were grown for 90 days and genomic DNA was isolated from leaves for transgenic confirmation based on real time PCR with NPT II specific dual labeled probe. Result of the transgenic confirmation analysis revealed presence of the gene silencing construct in ten out of 30 tested seedlings. Further, quantitative transcript expression analysis of the curcin precursor gene revealed reduction in the transcript abundance by more than 98% to undetectable level. The transgenic plants are being grown in containment for further studies on reduction in curcin protein content in Jatropha seeds.

Genetic variants in microRNA and microRNA biogenesis pathway genes and breast cancer risk among women of African ancestry

PubMed Central

Qian, Frank; Feng, Ye; Zheng, Yonglan; Ogundiran, Temidayo O.; Ojengbede, Oladosu; Zheng, Wei; Blot, William; Ambrosone, Christine B.; John, Esther M.; Bernstein, Leslie; Hu, Jennifer J.; Ziegler, Regina G.; Nyante, Sarah; Bandera, Elisa V.; Ingles, Sue A.; Press, Michael F.; Nathanson, Katherine L.; Hennis, Anselm; Nemesure, Barbara; Ambs, Stefan; Kolonel, Laurence N.; Olopade, Olufunmilayo I.; Haiman, Christopher A.; Huo, Dezheng

2016-01-01

Background MicroRNAs (miRNA) regulate breast biology by binding to specific RNA sequences, leading to RNA degradation and inhibition of translation of their target genes. While germline genetic variations may disrupt some of these interactions between miRNAs and their targets, studies assessing the relationship between genetic variations in the miRNA network and breast cancer risk are still limited, particularly among women of African ancestry. Methods We systematically put together a list of 822 and 10,468 genetic variants among primary miRNA sequences and 38 genes in the miRNA biogenesis pathway, respectively; and examined their association with breast cancer risk in the ROOT consortium which includes women of African ancestry. Findings were replicated in an independent consortium. Logistic regression was used to estimate the odds ratio (OR) and 95% confidence intervals (CI). Results For overall breast cancer risk, three single nucleotide polymorphisms (SNPs) in miRNA biogenesis genes DROSHA rs78393591 (OR=0.69, 95% CI: 0.55–0.88, P=0.003), ESR1 rs523736 (OR=0.88, 95% CI: 0.82–0.95, P=3.99×10−4), and ZCCHC11 rs114101502 (OR=1.33, 95% CI: 1.11–1.59, P=0.002) and one SNP in primary miRNA sequence (rs116159732 in miR-6826, OR=0.74, 95% CI: 0.63–0.89, P=0.001) were found to have significant associations in both discovery and validation phases. In a subgroup analysis, two SNPs were associated with risk of estrogen receptor (ER)-negative breast cancer and three SNPs were associated with risk of ER-positive breast cancer. Conclusion Several variants in miRNA and miRNA biogenesis pathway genes were associated with breast cancer risk. Risk associations varied by ER status, suggesting potential new mechanisms in etiology. PMID:27380242

Genetic variants in microRNA and microRNA biogenesis pathway genes and breast cancer risk among women of African ancestry.

PubMed

Qian, Frank; Feng, Ye; Zheng, Yonglan; Ogundiran, Temidayo O; Ojengbede, Oladosu; Zheng, Wei; Blot, William; Ambrosone, Christine B; John, Esther M; Bernstein, Leslie; Hu, Jennifer J; Ziegler, Regina G; Nyante, Sarah; Bandera, Elisa V; Ingles, Sue A; Press, Michael F; Nathanson, Katherine L; Hennis, Anselm; Nemesure, Barbara; Ambs, Stefan; Kolonel, Laurence N; Olopade, Olufunmilayo I; Haiman, Christopher A; Huo, Dezheng

2016-10-01

MicroRNAs (miRNA) regulate breast biology by binding to specific RNA sequences, leading to RNA degradation and inhibition of translation of their target genes. While germline genetic variations may disrupt some of these interactions between miRNAs and their targets, studies assessing the relationship between genetic variations in the miRNA network and breast cancer risk are still limited, particularly among women of African ancestry. We systematically put together a list of 822 and 10,468 genetic variants among primary miRNA sequences and 38 genes in the miRNA biogenesis pathway, respectively; and examined their association with breast cancer risk in the ROOT consortium which includes women of African ancestry. Findings were replicated in an independent consortium. Logistic regression was used to estimate the odds ratio (OR) and 95 % confidence intervals (CI). For overall breast cancer risk, three single-nucleotide polymorphisms (SNPs) in miRNA biogenesis genes DROSHA rs78393591 (OR = 0.69, 95 % CI: 0.55-0.88, P = 0.003), ESR1 rs523736 (OR = 0.88, 95 % CI: 0.82-0.95, P = 3.99 × 10(-4)), and ZCCHC11 rs114101502 (OR = 1.33, 95 % CI: 1.11-1.59, P = 0.002), and one SNP in primary miRNA sequence (rs116159732 in miR-6826, OR = 0.74, 95 % CI: 0.63-0.89, P = 0.001) were found to have significant associations in both discovery and validation phases. In a subgroup analysis, two SNPs were associated with risk of estrogen receptor (ER)-negative breast cancer, and three SNPs were associated with risk of ER-positive breast cancer. Several variants in miRNA and miRNA biogenesis pathway genes were associated with breast cancer risk. Risk associations varied by ER status, suggesting potential new mechanisms in etiology.

Identification of microRNAs from Amur grape (vitis amurensis Rupr.) by deep sequencing and analysis of microRNA variations with bioinformatics

PubMed Central

2012-01-01

Background MicroRNA (miRNA) is a class of functional non-coding small RNA with 19-25 nucleotides in length while Amur grape (Vitis amurensis Rupr.) is an important wild fruit crop with the strongest cold resistance among the Vitis species, is used as an excellent breeding parent for grapevine, and has elicited growing interest in wine production. To date, there is a relatively large number of grapevine miRNAs (vv-miRNAs) from cultivated grapevine varieties such as Vitis vinifera L. and hybrids of V. vinifera and V. labrusca, but there is no report on miRNAs from Vitis amurensis Rupr, a wild grapevine species. Results A small RNA library from Amur grape was constructed and Solexa technology used to perform deep sequencing of the library followed by subsequent bioinformatics analysis to identify new miRNAs. In total, 126 conserved miRNAs belonging to 27 miRNA families were identified, and 34 known but non-conserved miRNAs were also found. Significantly, 72 new potential Amur grape-specific miRNAs were discovered. The sequences of these new potential va-miRNAs were further validated through miR-RACE, and accumulation of 18 new va-miRNAs in seven tissues of grapevines confirmed by real time RT-PCR (qRT-PCR) analysis. The expression levels of va-miRNAs in flowers and berries were found to be basically consistent in identity to those from deep sequenced sRNAs libraries of combined corresponding tissues. We also describe the conservation and variation of va-miRNAs using miR-SNPs and miR-LDs during plant evolution based on comparison of orthologous sequences, and further reveal that the number and sites of miR-SNP in diverse miRNA families exhibit distinct divergence. Finally, 346 target genes for the new miRNAs were predicted and they include a number of Amur grape stress tolerance genes and many genes regulating anthocyanin synthesis and sugar metabolism. Conclusions Deep sequencing of short RNAs from Amur grape flowers and berries identified 72 new potential miRNAs and 34 known but non-conserved miRNAs, indicating that specific miRNAs exist in Amur grape. These results show that a number of regulatory miRNAs exist in Amur grape and play an important role in Amur grape growth, development, and response to abiotic or biotic stress. PMID:22455456

A systemic identification approach for primary transcription start site of Arabidopsis miRNAs from multidimensional omics data.

PubMed

You, Qi; Yan, Hengyu; Liu, Yue; Yi, Xin; Zhang, Kang; Xu, Wenying; Su, Zhen

2017-05-01

The 22-nucleotide non-coding microRNAs (miRNAs) are mostly transcribed by RNA polymerase II and are similar to protein-coding genes. Unlike the clear process from stem-loop precursors to mature miRNAs, the primary transcriptional regulation of miRNA, especially in plants, still needs to be further clarified, including the original transcription start site, functional cis-elements and primary transcript structures. Due to several well-characterized transcription signals in the promoter region, we proposed a systemic approach integrating multidimensional "omics" (including genomics, transcriptomics, and epigenomics) data to improve the genome-wide identification of primary miRNA transcripts. Here, we used the model plant Arabidopsis thaliana to improve the ability to identify candidate promoter locations in intergenic miRNAs and to determine rules for identifying primary transcription start sites of miRNAs by integrating high-throughput omics data, such as the DNase I hypersensitive sites, chromatin immunoprecipitation-sequencing of polymerase II and H3K4me3, as well as high throughput transcriptomic data. As a result, 93% of refined primary transcripts could be confirmed by the primer pairs from a previous study. Cis-element and secondary structure analyses also supported the feasibility of our results. This work will contribute to the primary transcriptional regulatory analysis of miRNAs, and the conserved regulatory pattern may be a suitable miRNA characteristic in other plant species.

Loop nucleotides control primary and mature miRNA function in target recognition and repression

PubMed Central

Yue, Si-Biao; Deis Trujillo, Robin; Tang, Yujie; O'Gorman, William E

2011-01-01

MicroRNA (miRNA) genes produce three major RNA products; primary (pri-), precursor (pre-), and mature miRNAs. Each product includes sequences complementary to cognate targets, thus they all can in principle interact with the targets. In a recent study we showed that pri-miRNAs play a direct role in target recognition and repression in the absence of functional mature miRNAs. Here we examined the functional contribution of pri-miRNAs in target regulation when full-length functional miRNAs are present. We found that pri-let-7 loop nucleotides control the production of the 5′ end of mature miRNAs and modulate the activity of the miRNA gene. This insight enabled us to modulate biogenesis of functional mature miRNAs and dissect the causal relationships between mature miRNA biogenesis and target repression. We demonstrate that both pri- and mature miRNAs can contribute to target repression and that their contributions can be distinguished by the differences between the pri- and mature miRNAs' sensitivity to bind to the first seed nucleotide. Our results demonstrate that the regulatory information encoded in the pri-/pre-miRNA loop nucleotides controls the activities of pri-miRNAs and mature let-7 by influencing pri-miRNA and target complex formation and the fidelity of mature miRNA seed generation. PMID:22142974

An alternative approach in regulation of expression of a transgene by endogenous miR-145 in carcinoma and normal breast cell lines.

PubMed

Ghanbari Safari, Maryam; Baesi, Kazem; Hosseinkhani, Saman

2017-03-01

MicroRNAs are small noncoding RNAs that regulate gene expression by repressing translation of target cellular transcripts. Increasing evidences indicate that miRNAs have different expression profiles and play crucial roles in numerous cellular processes. Delivery and expression of transgenes for cancer therapy must be specific for tumors to avoid killing of healthy tissues. Many investigators have shown that transgene expression can be suppressed in normal cells using vectors that are responsive to microRNA regulation. To overcome this problem, miR-145 that exhibits downregulation in many types of cancer cells was chosen for posttranscriptional regulatory systems mediated by microRNAs. In this study, a psiCHECK-145T vector carrying four tandem copies of target sequences of miR-145 into 3'-UTR of the Renilla luciferase gene was constructed. Renilla luciferase activity from the psiCHECK-145T vector was 57% lower in MCF10A cells with high miR-145 expression as compared to a control condition. Additionally, overexpression of miR-145 in MCF-7 cells with low expression level of miR-145 showed more than 76% reduction in the Renilla luciferase activity from the psiCHECK-145T vector. Inclusion of miR-145 target sequences into the 3'-UTR of the Renilla luciferase gene is a feasible strategy for restricting transgene expression in a breast cancer cell line while sparing a breast normal cell line. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

Solexa sequencing and custom microRNA chip reveal repertoire of microRNAs in mammary gland of bovine suffering from natural infectious mastitis.

PubMed

Ju, Zhihua; Jiang, Qiang; Liu, Gang; Wang, Xiuge; Luo, Guojing; Zhang, Yan; Zhang, Jibin; Zhong, Jifeng; Huang, Jinming

2018-02-01

Identification of microRNAs (miRNAs), target genes and regulatory networks associated with innate immune and inflammatory responses and tissue damage is essential to elucidate the molecular and genetic mechanisms for resistance to mastitis. In this study, a combination of Solexa sequencing and custom miRNA chip approaches was used to profile the expression of miRNAs in bovine mammary gland at the late stage of natural infection with Staphylococcus aureus, a widespread mastitis pathogen. We found 383 loci corresponding to 277 known and 49 putative novel miRNAs, two potential mitrons and 266 differentially expressed miRNAs in the healthy and mastitic cows' mammary glands. Several interaction networks and regulators involved in mastitis susceptibility, such as ALCAM, COL1A1, APOP4, ITIH4, CRP and fibrinogen alpha (FGA), were highlighted. Significant down-regulation and location of bta-miR-26a, which targets FGA in the mastitic mammary glands, were validated using quantitative real-time PCR, in situ hybridization and dual-luciferase reporter assays. We propose that the observed miRNA variations in mammary glands of mastitic cows are related to the maintenance of immune and defense responses, cell proliferation and apoptosis, and tissue injury and healing during the late stage of infection. Furthermore, the effect of bta-miR-26a in mastitis, mediated at least in part by enhancing FGA expression, involves host defense, inflammation and tissue damage. © 2018 Stichting International Foundation for Animal Genetics.

MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias

PubMed Central

Calin, George Adrian; Liu, Chang-Gong; Sevignani, Cinzia; Ferracin, Manuela; Felli, Nadia; Dumitru, Calin Dan; Shimizu, Masayoshi; Cimmino, Amelia; Zupo, Simona; Dono, Mariella; Dell'Aquila, Marie L.; Alder, Hansjuerg; Rassenti, Laura; Kipps, Thomas J.; Bullrich, Florencia; Negrini, Massimo; Croce, Carlo M.

2004-01-01

Little is known about the expression levels or function of micro-RNAs (miRNAs) in normal and neoplastic cells, although it is becoming clear that miRNAs play important roles in the regulation of gene expression during development [Ambros, V. (2003) Cell 113, 673–676; McManus, M. T. (2003) Semin. Cancer Biol. 13, 253–258]. We now report the genomewide expression profiling of miRNAs in human B cell chronic lymphocytic leukemia (CLL) by using a microarray containing hundreds of human precursor and mature miRNA oligonucleotide probes. This approach allowed us to identify significant differences in miRNome expression between CLL samples and normal CD5+ B cells; data were confirmed by Northern blot analyses and real-time RT-PCR. At least two distinct clusters of CLL samples can be identified that were associated with the presence or absence of Zap-70 expression, a predictor of early disease progression. Two miRNA signatures were associated with the presence or absence of mutations in the expressed Ig variableregion genes or with deletions at 13q14, respectively. These data suggest that miRNA expression patterns have relevance to the biological and clinical behavior of this leukemia. PMID:15284443

Using Markov chains of nucleotide sequences as a possible precursor to predict functional roles of human genome: a case study on inactive chromatin regions.

PubMed

Lee, K-E; Lee, E-J; Park, H-S

2016-08-30

Recent advances in computational epigenetics have provided new opportunities to evaluate n-gram probabilistic language models. In this paper, we describe a systematic genome-wide approach for predicting functional roles in inactive chromatin regions by using a sequence-based Markovian chromatin map of the human genome. We demonstrate that Markov chains of sequences can be used as a precursor to predict functional roles in heterochromatin regions and provide an example comparing two publicly available chromatin annotations of large-scale epigenomics projects: ENCODE project consortium and Roadmap Epigenomics consortium.

MicroRNA-31 is a positive modulator of endothelial-mesenchymal transition and associated secretory phenotype induced by TGF-β.

PubMed

Katsura, Akihiro; Suzuki, Hiroshi I; Ueno, Toshihide; Mihira, Hajime; Yamazaki, Tomoko; Yasuda, Takahiko; Watabe, Tetsuro; Mano, Hiroyuki; Yamada, Yoshitsugu; Miyazono, Kohei

2016-01-01

Transforming growth factor-β (TGF-β) plays central roles in endothelial-mesenchymal transition (EndMT) involved in development and pathogenesis. Although EndMT and epithelial-mesenchymal transition are similar processes, roles of microRNAs in EndMT are largely unknown. Here, we report that constitutively active microRNA-31 (miR-31) is a positive regulator of TGF-β-induced EndMT. Although the expression is not induced by TGF-β, miR-31 is required for induction of mesenchymal genes including α-SMA, actin reorganization and MRTF-A activation during EndMT. We identified VAV3, a regulator of actin remodeling and MRTF-A activity, as a miR-31 target. Global transcriptome analysis further showed that miR-31 positively regulates EndMT-associated unique secretory phenotype (EndMT-SP) characterized by induction of multiple inflammatory chemokines and cytokines including CCL17, CX3CL1, CXCL16, IL-6 and Angptl2. As a mechanism for this phenomenon, TGF-β and miR-31 suppress Stk40, a negative regulator of NF-κB pathway. Interestingly, TGF-β induces alternative polyadenylation (APA)-coupled miR-31-dependent Stk40 suppression without concomitant miR-31 induction, and APA-mediated exclusion of internal poly(A) sequence in Stk40 3'UTR enhances target efficiency of Stk40. Finally, miR-31 functions as a molecular hub to integrate TGF-β and TNF-α signaling to enhance EndMT. These data confirm that constitutively active microRNAs, as well as inducible microRNAs, serve as phenotypic modifiers interconnected with transcriptome dynamics during EndMT. © 2015 The Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

Genetic rearrangements, hotspot mutations, and microRNA expression in the progression of metastatic adenoid cystic carcinoma of the salivary gland

PubMed Central

Andreasen, Simon; Agander, Tina Klitmøller; Bjørndal, Kristine; Erentaite, Daiva; Heegaard, Steffen; Larsen, Stine R.; Melchior, Linea Cecilie; Tan, Qihua; Ulhøi, Benedicte Parm; Wessel, Irene; Homøe, Preben

2018-01-01

Adenoid cystic carcinoma (ACC) is among the most common salivary gland malignancies, and is notorious for its unpredictable clinical course with frequent local recurrences and metastatic spread. However, the molecular mechanisms for metastatic spread are poorly understood. This malignancy is known to frequently harbor gene fusions involving MYB, MYBL1, and NFIB, and to have a low mutational burden. Most studies have focused on primary tumors to understand the biology of ACC, but this has not revealed a genetic cause for metastatic dissemination in the majority of cases. Hence, other molecular mechanisms are likely to be involved. Here, we characterize the genetic and microRNA expressional landscape of primary ACC and corresponding metastatic lesions from 11 patients. FISH demonstrated preservation of MYB aberrations between primary tumors and metastases, and targeted next-generation sequencing identified mutations exclusive for the metastatic lesions in 3/11 cases (27.3%). Global microRNA profiling identified several differentially expressed miRNAs between primary ACC and metastases as compared to normal salivary gland tissue. Interestingly, individual tumor pairs differed in miRNA profile, but there was no general difference between primary ACCs and metastases. Collectively, we show that MYB and NFIB aberrations are consistently preserved in ACC metastatic lesions, and that additional mutations included in the 50-gene hotspot panel used are infrequently acquired by the metastatic lesions. In contrast, tumor pairs differ in microRNA expression and our data suggest that they are heterogeneous according to their microRNA profile. This adds an additional layer to the complex process of ACC metastatic spread. PMID:29731974

Circulating Plasma microRNAs can differentiate Human Sepsis and Systemic Inflammatory Response Syndrome (SIRS).

PubMed

Caserta, Stefano; Kern, Florian; Cohen, Jonathan; Drage, Stephen; Newbury, Sarah F; Llewelyn, Martin J

2016-06-20

Systemic inflammation in humans may be triggered by infection, termed sepsis, or non-infective processes, termed non-infective systemic inflammatory response syndrome (SIRS). MicroRNAs regulate cellular processes including inflammation and may be detected in blood. We aimed to establish definitive proof-of-principle that circulating microRNAs are differentially affected during sepsis and non-infective SIRS. Critically ill patients with severe (n = 21) or non-severe (n = 8) intra-abdominal sepsis; severe (n = 23) or non-severe (n = 21) non-infective SIRS; or no SIRS (n = 16) were studied. Next-generation sequencing and qRT-PCR were used to measure plasma microRNAs. Detectable blood miRNAs (n = 116) were generally up-regulated in SIRS compared to no-SIRS patients. Levels of these 'circulating inflammation-related microRNAs' (CIR-miRNAs) were 2.64 (IQR: 2.10-3.29) and 1.52 (IQR: 1.15-1.92) fold higher for non-infective SIRS and sepsis respectively (p < 0.0001), hence CIR-miRNAs appeared less abundant in sepsis than in SIRS. Six CIR-miRNAs (miR-30d-5p, miR-30a-5p, miR-192-5p, miR-26a-5p, miR-23a-5p, miR-191-5p) provided good-to-excellent discrimination of severe sepsis from severe SIRS (0.742-0.917 AUC of ROC curves). CIR-miRNA levels inversely correlated with pro-inflammatory cytokines (IL-1, IL-6 and others). Thus, among critically ill patients, sepsis and non-infective SIRS are associated with substantial, differential changes in CIR-miRNAs. CIR-miRNAs may be regulators of inflammation and warrant thorough evaluation as diagnostic and therapeutic targets.

Uncoupling GP1 and GP2 Expression in the Lassa Virus Glycoprotein Complex: Implications for GP1 Ectodomain Shedding

DTIC Science & Technology

2008-12-23

glycoprotein precursor (GPC) signal peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain... peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain was deleted, the...terminal signal peptide (SP), which directs the precursor to the endoplasmic retic- ulum (ER) for further processing [11]. The SP, which has been

Center for Regenerative Biology and Medicine at Mount Desert Island Biological Laboratory

DTIC Science & Technology

2012-06-01

Code Axolotl microRNAs Zebrafish Polypterus 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF...controlled in both Polypterus and axolotl samples. These comparisons revealed a total of 2779 shared genes that are significantly upregulated during...UPREGULATED DOWNREGULATED Figure 1: Venn diagram of UniProt protein sequence IDs among Axolotl and Polypterus contigs that were up-regulated

microRNA profiling in the zoonotic parasite Echinococcus canadensis using a high-throughput approach.

PubMed

Macchiaroli, Natalia; Cucher, Marcela; Zarowiecki, Magdalena; Maldonado, Lucas; Kamenetzky, Laura; Rosenzvit, Mara Cecilia

2015-02-06

microRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression at post-transcriptional level and play essential roles in fundamental biological processes such as development and metabolism. The particular developmental and metabolic characteristics of cestode parasites highlight the importance of studying miRNA gene regulation in these organisms. Here, we perform a comprehensive analysis of miRNAs in the parasitic cestode Echinococcus canadensis G7, one of the causative agents of the neglected zoonotic disease cystic echinococcosis. Small RNA libraries from protoscoleces and cyst walls of E. canadensis G7 and protoscoleces of E. granulosus sensu stricto G1 were sequenced using Illumina technology. For miRNA prediction, miRDeep2 core algorithm was used. The output list of candidate precursors was manually curated to generate a high confidence set of miRNAs. Differential expression analysis of miRNAs between stages or species was estimated with DESeq. Expression levels of selected miRNAs were validated using poly-A RT-qPCR. In this study we used a high-throughput approach and found transcriptional evidence of 37 miRNAs thus expanding the miRNA repertoire of E. canadensis G7. Differential expression analysis showed highly regulated miRNAs between life cycle stages, suggesting a role in maintaining the features of each developmental stage or in the regulation of developmental timing. In this work we characterize conserved and novel Echinococcus miRNAs which represent 30 unique miRNA families. Here we confirmed the remarkable loss of conserved miRNA families in E. canadensis, reflecting their low morphological complexity and high adaptation to parasitism. We performed the first in-depth study profiling of small RNAs in the zoonotic parasite E. canadensis G7. We found that miRNAs are the preponderant small RNA silencing molecules, suggesting that these small RNAs could be an essential mechanism of gene regulation in this species. We also identified both parasite specific and divergent miRNAs which are potential biomarkers of infection. This study will provide valuable information for better understanding of the complex biology of this parasite and could help to find new potential targets for therapy and/or diagnosis.

Investigation of relationship between precursor of miRNA-944 and its mature form in lung squamous-cell carcinoma - the diagnostic value.

PubMed

Powrózek, Tomasz; Mlak, Radosław; Dziedzic, Marcin; Małecka-Massalska, Teresa; Sagan, Dariusz

2018-03-01

MicroRNA (miRNA) are attractive markers of lung cancer, due to their regulatory role in cell cycle. However, we know more about function of miRNA in cancer development, there is still little known about role of their precursors (primary miRNA; pri-miRNA) in tumorgenesis. In present study we investigated potential role of miRNA-944 and its precursor pri-miRNA-944 in development of squamous-cell lung cancer (SCC) and explored interdependence between miRNA precursor and its mature form. This is a first available literature report analyzing pri-miRNA as a cancer diagnostic marker. Expression of miRNA-944 and its precursor was analyzed in 58 fresh-frozen tissues of non-small cell lung cancer and corresponding adjacent non-cancerous tissues using qRT-PCR. Expression of pri-miRNA-944 was correlated with TP63 and miRNA-944. Using ROC analysis diagnostic accuracy of studied markers was evaluated. miRNA-944 and its precursor were significantly overexspressed in SCC compared to adenocarcinoma (AC) and non-cancerous tissue. pri-miRNA-944 strongly and positively correlated with TP63 (r = 0.739, p < 0.001) and with mature miRNA-944 expression (r = 0.691, p < 0.001). Also, TP63 expression significantly correlated with mature miRNA (r = 0.785, p < 0.001). Combined analysis of pri-miRNA-944 and mature miRNA-944 allowed to distinguish SCC tissue form AC with sensitivity of 93.3% and specificity of 100% (AUC = 0.978), and SCC from non-cancerous tissue with 92.9% sensitivity and 100% specificity (AUC = 0.992). We assumed that pri-miRNA-944 and miRNA-944 may be involved in early squamous-type differentiation of lung tumors. Moreover, analysis of both markers provided high diagnostic accuracy for SCC detection. Copyright © 2018 Elsevier GmbH. All rights reserved.

Genome-wide discovery and differential regulation of conserved and novel microRNAs in chickpea via deep sequencing.

PubMed

Jain, Mukesh; Chevala, V V S Narayana; Garg, Rohini

2014-11-01

MicroRNAs (miRNAs) are essential components of complex gene regulatory networks that orchestrate plant development. Although several genomic resources have been developed for the legume crop chickpea, miRNAs have not been discovered until now. For genome-wide discovery of miRNAs in chickpea (Cicer arietinum), we sequenced the small RNA content from seven major tissues/organs employing Illumina technology. About 154 million reads were generated, which represented more than 20 million distinct small RNA sequences. We identified a total of 440 conserved miRNAs in chickpea based on sequence similarity with known miRNAs in other plants. In addition, 178 novel miRNAs were identified using a miRDeep pipeline with plant-specific scoring. Some of the conserved and novel miRNAs with significant sequence similarity were grouped into families. The chickpea miRNAs targeted a wide range of mRNAs involved in diverse cellular processes, including transcriptional regulation (transcription factors), protein modification and turnover, signal transduction, and metabolism. Our analysis revealed several miRNAs with differential spatial expression. Many of the chickpea miRNAs were expressed in a tissue-specific manner. The conserved and differential expression of members of the same miRNA family in different tissues was also observed. Some of the same family members were predicted to target different chickpea mRNAs, which suggested the specificity and complexity of miRNA-mediated developmental regulation. This study, for the first time, reveals a comprehensive set of conserved and novel miRNAs along with their expression patterns and putative targets in chickpea, and provides a framework for understanding regulation of developmental processes in legumes. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Small RNA profiling of low biomass samples: identification and removal of contaminants.

PubMed

Heintz-Buschart, Anna; Yusuf, Dilmurat; Kaysen, Anne; Etheridge, Alton; Fritz, Joëlle V; May, Patrick; de Beaufort, Carine; Upadhyaya, Bimal B; Ghosal, Anubrata; Galas, David J; Wilmes, Paul

2018-05-14

Sequencing-based analyses of low-biomass samples are known to be prone to misinterpretation due to the potential presence of contaminating molecules derived from laboratory reagents and environments. DNA contamination has been previously reported, yet contamination with RNA is usually considered to be very unlikely due to its inherent instability. Small RNAs (sRNAs) identified in tissues and bodily fluids, such as blood plasma, have implications for physiology and pathology, and therefore the potential to act as disease biomarkers. Thus, the possibility for RNA contaminants demands careful evaluation. Herein, we report on the presence of small RNA (sRNA) contaminants in widely used microRNA extraction kits and propose an approach for their depletion. We sequenced sRNAs extracted from human plasma samples and detected important levels of non-human (exogenous) sequences whose source could be traced to the microRNA extraction columns through a careful qPCR-based analysis of several laboratory reagents. Furthermore, we also detected the presence of artefactual sequences related to these contaminants in a range of published datasets, thereby arguing in particular for a re-evaluation of reports suggesting the presence of exogenous RNAs of microbial and dietary origin in blood plasma. To avoid artefacts in future experiments, we also devise several protocols for the removal of contaminant RNAs, define minimal amounts of starting material for artefact-free analyses, and confirm the reduction of contaminant levels for identification of bona fide sequences using 'ultra-clean' extraction kits. This is the first report on the presence of RNA molecules as contaminants in RNA extraction kits. The described protocols should be applied in the future to avoid confounding sRNA studies.

Genome-wide discovery of novel and conserved microRNAs in white shrimp (Litopenaeus vannamei).

PubMed

Xi, Qian-Yun; Xiong, Yuan-Yan; Wang, Yuan-Mei; Cheng, Xiao; Qi, Qi-En; Shu, Gang; Wang, Song-Bo; Wang, Li-Na; Gao, Ping; Zhu, Xiao-Tong; Jiang, Qing-Yan; Zhang, Yong-Liang; Liu, Li

2015-01-01

Of late years, a large amount of conserved and species-specific microRNAs (miRNAs) have been performed on identification from species which are economically important but lack a full genome sequence. In this study, Solexa deep sequencing and cross-species miRNA microarray were used to detect miRNAs in white shrimp. We identified 239 conserved miRNAs, 14 miRNA* sequences and 20 novel miRNAs by bioinformatics analysis from 7,561,406 high-quality reads representing 325,370 distinct sequences. The all 20 novel miRNAs were species-specific in white shrimp and not homologous in other species. Using the conserved miRNAs from the miRBase database as a query set to search for homologs from shrimp expressed sequence tags (ESTs), 32 conserved computationally predicted miRNAs were discovered in shrimp. In addition, using microarray analysis in the shrimp fed with Panax ginseng polysaccharide complex, 151 conserved miRNAs were identified, 18 of which were significant up-expression, while 49 miRNAs were significant down-expression. In particular, qRT-PCR analysis was also performed for nine miRNAs in three shrimp tissues such as muscle, gill and hepatopancreas. Results showed that these miRNAs expression are tissue specific. Combining results of the three methods, we detected 20 novel and 394 conserved miRNAs. Verification with quantitative reverse transcription (qRT-PCR) and Northern blot showed a high confidentiality of data. The study provides the first comprehensive specific miRNA profile of white shrimp, which includes useful information for future investigations into the function of miRNAs in regulation of shrimp development and immunology.

Identification of MicroRNAs in Helicoverpa armigera and Spodoptera litura Based on Deep Sequencing and Homology Analysis

PubMed Central

Ge, Xie; Zhang, Yong; Jiang, Jianhao; Zhong, Yi; Yang, Xiaonan; Li, Zhiqian; Huang, Yongping; Tan, Anjiang

2013-01-01

The current identification of microRNAs (miRNAs) in insects is largely dependent on genome sequences. However, the lack of available genome sequences inhibits the identification of miRNAs in various insect species. In this study, we used a miRNA database of the silkworm Bombyx mori as a reference to identify miRNAs in Helicoverpa armigera and Spodoptera litura using deep sequencing and homology analysis. Because all three species belong to the Lepidoptera, the experiment produced reliable results. Our study identified 97 and 91 conserved miRNAs in H. armigera and S. litura, respectively. Using the genome of B. mori and BAC sequences of H. armigera as references, 1 novel miRNA and 8 novel miRNA candidates were identified in H. armigera, and 4 novel miRNA candidates were identified in S. litura. An evolutionary analysis revealed that most of the identified miRNAs were insect-specific, and more than 20 miRNAs were Lepidoptera-specific. The investigation of the expression patterns of miR-2a, miR-34, miR-2796-3p and miR-11 revealed their potential roles in insect development. miRNA target prediction revealed that conserved miRNA target sites exist in various genes in the 3 species. Conserved miRNA target sites for the Hsp90 gene among the 3 species were validated in the mammalian 293T cell line using a dual-luciferase reporter assay. Our study provides a new approach with which to identify miRNAs in insects lacking genome information and contributes to the functional analysis of insect miRNAs. PMID:23289012

Identifying functional cancer-specific miRNA-mRNA interactions in testicular germ cell tumor.

PubMed

Sedaghat, Nafiseh; Fathy, Mahmood; Modarressi, Mohammad Hossein; Shojaie, Ali

2016-09-07

Testicular cancer is the most common cancer in men aged between 15 and 35 and more than 90% of testicular neoplasms are originated at germ cells. Recent research has shown the impact of microRNAs (miRNAs) in different types of cancer, including testicular germ cell tumor (TGCT). MicroRNAs are small non-coding RNAs which affect the development and progression of cancer cells by binding to mRNAs and regulating their expressions. The identification of functional miRNA-mRNA interactions in cancers, i.e. those that alter the expression of genes in cancer cells, can help delineate post-regulatory mechanisms and may lead to new treatments to control the progression of cancer. A number of sequence-based methods have been developed to predict miRNA-mRNA interactions based on the complementarity of sequences. While necessary, sequence complementarity is, however, not sufficient for presence of functional interactions. Alternative methods have thus been developed to refine the sequence-based interactions using concurrent expression profiles of miRNAs and mRNAs. This study aims to find functional cancer-specific miRNA-mRNA interactions in TGCT. To this end, the sequence-based predicted interactions are first refined using an ensemble learning method, based on two well-known methods of learning miRNA-mRNA interactions, namely, TaLasso and GenMiR++. Additional functional analyses were then used to identify a subset of interactions to be most likely functional and specific to TGCT. The final list of 13 miRNA-mRNA interactions can be potential targets for identifying TGCT-specific interactions and future laboratory experiments to develop new therapies. Copyright © 2016 Elsevier Ltd. All rights reserved.

miR-ID: A novel, circularization-based platform for detection of microRNAs

PubMed Central

Kumar, Pavan; Johnston, Brian H.; Kazakov, Sergei A.

2011-01-01

MicroRNAs (miRNAs) are important regulators of gene expression and have great potential as biomarkers, prognostic indicators, and therapeutic targets. Determining the expression patterns of these molecules is essential for elucidating their biogenesis, regulation, relation to disease, and response to therapy. Although PCR-based assays are commonly used for expression profiling of miRNAs, the small size, sequence heterogeneity, and (in some cases) end modifications of miRNAs constrain the performance of existing PCR methods. Here we introduce miR-ID, a novel method that avoids these constraints while providing superior sensitivity and sequence specificity at a lower cost. It also has the unique ability to differentiate unmodified small RNAs from those carrying 2′-OMe groups at their 3′-ends while detecting both forms. miR-ID is comprised of the following steps: (1) circularization of the miRNA by a ligase; (2) reverse transcription of the circularized miRNA (RTC), producing tandem repeats of a DNA sequence complementary to the miRNA; and (3) qPCR amplification of segments of this multimeric cDNA using 5′-overlapping primers and a nonspecific dye such as SYBR Green. No chemically modified probes (e.g., TaqMan) or primers (e.g., LNA) are required. The circular RNA and multimeric cDNA templates provide unmatched flexibility in the positioning of primers, which may include straddling the boundaries between these repetitive miRNA sequences. miR-ID is based on new findings that are themselves of general interest, including reverse transcription of small RNA circles and the use of 5′-overlapping primers for detection of repetitive sequences by qPCR. PMID:21169480

Sequencing and Characterisation of an Extensive Atlantic Salmon (Salmo salar L.) MicroRNA Repertoire

PubMed Central

Bekaert, Michaël; Lowe, Natalie R.; Bishop, Stephen C.; Bron, James E.; Taggart, John B.; Houston, Ross D.

2013-01-01

Atlantic salmon (Salmo salar L.), a member of the family Salmonidae, is a totemic species of ecological and cultural significance that is also economically important in terms of both sports fisheries and aquaculture. These factors have promoted the continuous development of genomic resources for this species, furthering both fundamental and applied research. MicroRNAs (miRNA) are small endogenous non-coding RNA molecules that control spatial and temporal expression of targeted genes through post-transcriptional regulation. While miRNA have been characterised in detail for many other species, this is not yet the case for Atlantic salmon. To identify miRNAs from Atlantic salmon, we constructed whole fish miRNA libraries for 18 individual juveniles (fry, four months post hatch) and characterised them by Illumina high-throughput sequencing (total of 354,505,167 paired-ended reads). We report an extensive and partly novel repertoire of miRNA sequences, comprising 888 miRNA genes (547 unique mature miRNA sequences), quantify their expression levels in basal conditions, examine their homology to miRNAs from other species and identify their predicted target genes. We also identify the location and putative copy number of the miRNA genes in the draft Atlantic salmon reference genome sequence. The Atlantic salmon miRNAs experimentally identified in this study provide a robust large-scale resource for functional genome research in salmonids. There is an opportunity to explore the evolution of salmonid miRNAs following the relatively recent whole genome duplication event in salmonid species and to investigate the role of miRNAs in the regulation of gene expression in particular their contribution to variation in economically and ecologically important traits. PMID:23922936

miRCat2: accurate prediction of plant and animal microRNAs from next-generation sequencing datasets

PubMed Central

Paicu, Claudia; Mohorianu, Irina; Stocks, Matthew; Xu, Ping; Coince, Aurore; Billmeier, Martina; Dalmay, Tamas; Moulton, Vincent; Moxon, Simon

2017-01-01

Abstract Motivation MicroRNAs are a class of ∼21–22 nt small RNAs which are excised from a stable hairpin-like secondary structure. They have important gene regulatory functions and are involved in many pathways including developmental timing, organogenesis and development in eukaryotes. There are several computational tools for miRNA detection from next-generation sequencing datasets. However, many of these tools suffer from high false positive and false negative rates. Here we present a novel miRNA prediction algorithm, miRCat2. miRCat2 incorporates a new entropy-based approach to detect miRNA loci, which is designed to cope with the high sequencing depth of current next-generation sequencing datasets. It has a user-friendly interface and produces graphical representations of the hairpin structure and plots depicting the alignment of sequences on the secondary structure. Results We test miRCat2 on a number of animal and plant datasets and present a comparative analysis with miRCat, miRDeep2, miRPlant and miReap. We also use mutants in the miRNA biogenesis pathway to evaluate the predictions of these tools. Results indicate that miRCat2 has an improved accuracy compared with other methods tested. Moreover, miRCat2 predicts several new miRNAs that are differentially expressed in wild-type versus mutants in the miRNA biogenesis pathway. Availability and Implementation miRCat2 is part of the UEA small RNA Workbench and is freely available from http://srna-workbench.cmp.uea.ac.uk/. Contact v.moulton@uea.ac.uk or s.moxon@uea.ac.uk Supplementary information Supplementary data are available at Bioinformatics online. PMID:28407097

The evolution of microRNAs in plants

PubMed Central

Cui, Jie; You, Chenjiang; Chen, Xuemei

2016-01-01

MicroRNAs (miRNAs) are a central player in post-transcriptional regulation of gene expression and are involved in numerous biological processes in eukaryotes. Knowledge of the origins and divergence of miRNAs paves the way for a better understanding of the complexity of the regulatory networks that they participate in. The biogenesis, degradation, and regulatory activities of miRNAs are relatively better understood, but the evolutionary history of miRNAs still needs more exploration. Inverted duplication of target genes, random hairpin sequences and small transposable elements constitute three main models that explain the origination of miRNA genes (MIR). Both inter- and intra-species divergence of miRNAs exhibits functional adaptation and adaptation to changing environments in evolution. Here we summarize recent progress in studies on the evolution of MIR and related genes. PMID:27886593

Fluorescence-based high-throughput screening of dicer cleavage activity.

PubMed

Podolska, Katerina; Sedlak, David; Bartunek, Petr; Svoboda, Petr

2014-03-01

Production of small RNAs by ribonuclease III Dicer is a key step in microRNA and RNA interference pathways, which employ Dicer-produced small RNAs as sequence-specific silencing guides. Further studies and manipulations of microRNA and RNA interference pathways would benefit from identification of small-molecule modulators. Here, we report a study of a fluorescence-based in vitro Dicer cleavage assay, which was adapted for high-throughput screening. The kinetic assay can be performed under single-turnover conditions (35 nM substrate and 70 nM Dicer) in a small volume (5 µL), which makes it suitable for high-throughput screening in a 1536-well format. As a proof of principle, a small library of bioactive compounds was analyzed, demonstrating potential of the assay.

MicroRNAs Expression Profiles in Cardiovascular Diseases

PubMed Central

Bronze-da-Rocha, Elsa

2014-01-01

The current search for new markers of cardiovascular diseases (CVDs) is explained by the high morbidity and mortality still observed in developed and developing countries due to cardiovascular events. Recently, microRNAs (miRNAs or miRs) have emerged as potential new biomarkers and are small sequences of RNAs that regulate gene expression at posttranscriptional level by inhibiting translation or inducing degradation of the target mRNAs. Circulating miRNAs are involved in the regulation of signaling pathways associated to aging and can be used as novel diagnostic markers for acute and chronic diseases such as cardiovascular pathologies. This review summarizes the biogenesis, maturation, and stability of miRNAs and their use as potential biomarkers for coronary artery disease (CAD), myocardial infarction (MI), and heart failure (HF). PMID:25013816

Proteolytic processing of the vitellogenin precursor in the boll weevil, Anthonomus grandis.

PubMed

Heilmann, L J; Trewitt, P M; Kumaran, A K

1993-01-01

The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M(r)s of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M(r) vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M(r) honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M(r) boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10-15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein.

Whole-Genome Sequencing for Optimized Patient Management

PubMed Central

Bainbridge, Matthew N.; Wiszniewski, Wojciech; Murdock, David R.; Friedman, Jennifer; Gonzaga-Jauregui, Claudia; Newsham, Irene; Reid, Jeffrey G.; Fink, John K.; Morgan, Margaret B.; Gingras, Marie-Claude; Muzny, Donna M.; Hoang, Linh D.; Yousaf, Shahed; Lupski, James R.; Gibbs, Richard A.

2012-01-01

Whole-genome sequencing of patient DNA can facilitate diagnosis of a disease, but its potential for guiding treatment has been under-realized. We interrogated the complete genome sequences of a 14-year-old fraternal twin pair diagnosed with dopa (3,4-dihydroxyphenylalanine)–responsive dystonia (DRD; Mendelian Inheritance in Man #128230). DRD is a genetically heterogeneous and clinically complex movement disorder that is usually treated with l-dopa, a precursor of the neurotransmitter dopamine. Whole-genome sequencing identified compound heterozygous mutations in the SPR gene encoding sepiapterin reductase. Disruption of SPR causes a decrease in tetrahydrobiopterin, a cofactor required for the hydroxylase enzymes that synthesize the neurotransmitters dopamine and serotonin. Supplementation of l-dopa therapy with 5-hydroxytryptophan, a serotonin precursor, resulted in clinical improvements in both twins. PMID:21677200

Massive Analysis of Rice Small RNAs: Mechanistic Implications of Regulated MicroRNAs and Variants for Differential Target RNA Cleavage[W][OA

PubMed Central

Jeong, Dong-Hoon; Park, Sunhee; Zhai, Jixian; Gurazada, Sai Guna Ranjan; De Paoli, Emanuele; Meyers, Blake C.; Green, Pamela J.

2011-01-01

Small RNAs have a variety of important roles in plant development, stress responses, and other processes. They exert their influence by guiding mRNA cleavage, translational repression, and chromatin modification. To identify previously unknown rice (Oryza sativa) microRNAs (miRNAs) and those regulated by environmental stress, 62 small RNA libraries were constructed from rice plants and used for deep sequencing with Illumina technology. The libraries represent several tissues from control plants and plants subjected to different environmental stress treatments. More than 94 million genome-matched reads were obtained, resulting in more than 16 million distinct small RNA sequences. This allowed an evaluation of ~400 annotated miRNAs with current criteria and the finding that among these, ~150 had small interfering RNA–like characteristics. Seventy-six new miRNAs were found, and miRNAs regulated in response to water stress, nutrient stress, or temperature stress were identified. Among the new examples of miRNA regulation were members of the same miRNA family that were differentially regulated in different organs and had distinct sequences Some of these distinct family members result in differential target cleavage and provide new insight about how an agriculturally important rice phenotype could be regulated in the panicle. This high-resolution analysis of rice miRNAs should be relevant to plant miRNAs in general, particularly in the Poaceae. PMID:22158467

miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades.

PubMed

Friedländer, Marc R; Mackowiak, Sebastian D; Li, Na; Chen, Wei; Rajewsky, Nikolaus

2012-01-01

microRNAs (miRNAs) are a large class of small non-coding RNAs which post-transcriptionally regulate the expression of a large fraction of all animal genes and are important in a wide range of biological processes. Recent advances in high-throughput sequencing allow miRNA detection at unprecedented sensitivity, but the computational task of accurately identifying the miRNAs in the background of sequenced RNAs remains challenging. For this purpose, we have designed miRDeep2, a substantially improved algorithm which identifies canonical and non-canonical miRNAs such as those derived from transposable elements and informs on high-confidence candidates that are detected in multiple independent samples. Analyzing data from seven animal species representing the major animal clades, miRDeep2 identified miRNAs with an accuracy of 98.6-99.9% and reported hundreds of novel miRNAs. To test the accuracy of miRDeep2, we knocked down the miRNA biogenesis pathway in a human cell line and sequenced small RNAs before and after. The vast majority of the >100 novel miRNAs expressed in this cell line were indeed specifically downregulated, validating most miRDeep2 predictions. Last, a new miRNA expression profiling routine, low time and memory usage and user-friendly interactive graphic output can make miRDeep2 useful to a wide range of researchers.

ISRNA: an integrative online toolkit for short reads from high-throughput sequencing data.

PubMed

Luo, Guan-Zheng; Yang, Wei; Ma, Ying-Ke; Wang, Xiu-Jie

2014-02-01

Integrative Short Reads NAvigator (ISRNA) is an online toolkit for analyzing high-throughput small RNA sequencing data. Besides the high-speed genome mapping function, ISRNA provides statistics for genomic location, length distribution and nucleotide composition bias analysis of sequence reads. Number of reads mapped to known microRNAs and other classes of short non-coding RNAs, coverage of short reads on genes, expression abundance of sequence reads as well as some other analysis functions are also supported. The versatile search functions enable users to select sequence reads according to their sub-sequences, expression abundance, genomic location, relationship to genes, etc. A specialized genome browser is integrated to visualize the genomic distribution of short reads. ISRNA also supports management and comparison among multiple datasets. ISRNA is implemented in Java/C++/Perl/MySQL and can be freely accessed at http://omicslab.genetics.ac.cn/ISRNA/.

A novel peptide from the ACEI/BPP-CNP precursor in the venom of Crotalus durissus collilineatus.

PubMed

Higuchi, Shigesada; Murayama, Nobuhiro; Saguchi, Ken-ichi; Ohi, Hiroaki; Fujita, Yoshiaki; da Silva, Nelson Jorge; de Siqueira, Rodrigo José Bezerra; Lahlou, Saad; Aird, Steven D

2006-10-01

In crotaline venoms, angiotensin-converting enzyme inhibitors [ACEIs, also known as bradykinin potentiating peptides (BPPs)], are products of a gene coding for an ACEI/BPP-C-type natriuretic peptide (CNP) precursor. In the genes from Bothrops jararaca and Gloydius blomhoffii, ACEI/BPP sequences are repeated. Sequencing of a cDNA clone from venom glands of Crotalus durissus collilineatus showed that two ACEIs/BPPs are located together at the N-terminus, but without repeats. An additional sequence for CNP was unexpectedly found at the C-terminus. Homologous genes for the ACEI/BPP-CNP precursor suggest that most crotaline venoms contain both ACEIs/BPPs and CNP. The sequence of ACEIs/BPPs is separated from the CNP sequence by a long spacer sequence. Previously, there was no evidence that this spacer actually coded any expressed peptides. Aird and Kaiser (1986, unpublished) previously isolated and sequenced a peptide of 11 residues (TPPAGPDVGPR) from Crotalus viridis viridis venom. In the present study, analysis of the cDNA clone from C. d. collilineatus revealed a nearly identical sequence in the ACEI/BPP-CNP spacer. Fractionation of the crude venom by reverse phase HPLC (C(18)), and analysis of the fractions by mass spectrometry (MS) indicated a component of 1020.5 Da. Amino acid sequencing by MS/MS confirmed that C. d. collilineatus venom contains the peptide TPPAGPDGGPR. Its high proline content and paired proline residues are typical of venom hypotensive peptides, although it lacks the usual N-terminal pyroglutamate. It has no demonstrable hypotensive activity when injected intravenously in rats; however, its occurrence in the venoms of dissimilar species suggests that its presence is not accidental. Evidence suggests that these novel toxins probably activate anaphylatoxin C3a receptors.

Adenosine-to-Inosine Editing of MicroRNA-487b Alters Target Gene Selection After Ischemia and Promotes Neovascularization.

PubMed

van der Kwast, Reginald V C T; van Ingen, Eva; Parma, Laura; Peters, Hendrika A B; Quax, Paul H A; Nossent, A Yaël

2018-02-02

Adenosine-to-inosine editing of microRNAs has the potential to cause a shift in target site selection. 2'-O-ribose-methylation of adenosine residues, however, has been shown to inhibit adenosine-to-inosine editing. To investigate whether angiomiR miR487b is subject to adenosine-to-inosine editing or 2'-O-ribose-methylation during neovascularization. Complementary DNA was prepared from C57BL/6-mice subjected to hindlimb ischemia. Using Sanger sequencing and endonuclease digestion, we identified and validated adenosine-to-inosine editing of the miR487b seed sequence. In the gastrocnemius muscle, pri-miR487b editing increased from 6.7±0.4% before to 11.7±1.6% ( P =0.02) 1 day after ischemia. Edited pri-miR487b is processed into a novel microRNA, edited miR487b, which is also upregulated after ischemia. We confirmed editing of miR487b in multiple human primary vascular cell types. Short interfering RNA-mediated knockdown demonstrated that editing is adenosine deaminase acting on RNA 1 and 2 dependent. Using reverse-transcription at low dNTP concentrations followed by quantitative-PCR, we found that the same adenosine residue is methylated in mice and human primary cells. In the murine gastrocnemius, the estimated methylation fraction increased from 32.8±14% before to 53.6±12% 1 day after ischemia. Short interfering RNA knockdown confirmed that methylation is fibrillarin dependent. Although we could not confirm that methylation directly inhibits editing, we do show that adenosine deaminase acting on RNA 1 and 2 and fibrillarin negatively influence each other's expression. Using multiple luciferase reporter gene assays, we could demonstrate that editing results in a complete switch of target site selection. In human primary cells, we confirmed the shift in miR487b targeting after editing, resulting in a edited miR487b targetome that is enriched for multiple proangiogenic pathways. Furthermore, overexpression of edited miR487b, but not wild-type miR487b, stimulates angiogenesis in both in vitro and ex vivo assays. MiR487b is edited in the seed sequence in mice and humans, resulting in a novel, proangiogenic microRNA with a unique targetome. The rate of miR487b editing, as well as 2'-O-ribose-methylation, is increased in murine muscle tissue during postischemic neovascularization. Our findings suggest miR487b editing plays an intricate role in postischemic neovascularization. © 2017 American Heart Association, Inc.

Sox2: A multitasking networker

PubMed Central

Reiprich, Simone; Wegner, Michael

2014-01-01

The transcription factor Sox2 is best known as a pluripotency factor in stem and precursor cells and its expression generally correlates with an undifferentiated state. Proposed modes of action include those as classical transcription factor and pre-patterning factor with influence on histone modifications and chromatin structure. Recently, we provided the first detailed analysis of Sox2 expression and function during development of oligodendrocytes, the myelin-forming cells of the CNS. Surprisingly, we found evidence for a role of Sox2 as differentiation factor and found it to act through modulation of microRNA levels. Thus, we add new facets to the functional repertoire of Sox2 and throw light on the networking activity of this multitasking developmental regulator. PMID:27502481

Memetic algorithms for de novo motif-finding in biomedical sequences.

PubMed

Bi, Chengpeng

2012-09-01

The objectives of this study are to design and implement a new memetic algorithm for de novo motif discovery, which is then applied to detect important signals hidden in various biomedical molecular sequences. In this paper, memetic algorithms are developed and tested in de novo motif-finding problems. Several strategies in the algorithm design are employed that are to not only efficiently explore the multiple sequence local alignment space, but also effectively uncover the molecular signals. As a result, there are a number of key features in the implementation of the memetic motif-finding algorithm (MaMotif), including a chromosome replacement operator, a chromosome alteration-aware local search operator, a truncated local search strategy, and a stochastic operation of local search imposed on individual learning. To test the new algorithm, we compare MaMotif with a few of other similar algorithms using simulated and experimental data including genomic DNA, primary microRNA sequences (let-7 family), and transmembrane protein sequences. The new memetic motif-finding algorithm is successfully implemented in C++, and exhaustively tested with various simulated and real biological sequences. In the simulation, it shows that MaMotif is the most time-efficient algorithm compared with others, that is, it runs 2 times faster than the expectation maximization (EM) method and 16 times faster than the genetic algorithm-based EM hybrid. In both simulated and experimental testing, results show that the new algorithm is compared favorably or superior to other algorithms. Notably, MaMotif is able to successfully discover the transcription factors' binding sites in the chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) data, correctly uncover the RNA splicing signals in gene expression, and precisely find the highly conserved helix motif in the transmembrane protein sequences, as well as rightly detect the palindromic segments in the primary microRNA sequences. The memetic motif-finding algorithm is effectively designed and implemented, and its applications demonstrate it is not only time-efficient, but also exhibits excellent performance while compared with other popular algorithms. Copyright © 2012 Elsevier B.V. All rights reserved.

Adaptive evolution of newly emerged micro-RNA genes in Drosophila.

PubMed

Lu, Jian; Fu, Yonggui; Kumar, Supriya; Shen, Yang; Zeng, Kai; Xu, Anlong; Carthew, Richard; Wu, Chung-I

2008-05-01

How often micro-RNA (miRNA) genes emerged and how fast they evolved soon after their emergence are some of the central questions in the evolution of miRNAs. Because most known miRNA genes are ancient and highly conserved, these questions can be best answered by identifying newly emerged miRNA genes. Among the 78 miRNA genes in Drosophila reported before 2007, only 5 are confirmed to be newly emerged in the genus (although many more can be found in the newly reported data set; e.g., Ruby et al. 2007; Stark et al. 2007; Lu et al. 2008). These new miRNA genes have undergone numerous changes, even in the normally invariant mature sequences. Four of them (the miR-310/311/312/313 cluster, denoted miR-310s) were duplicated from other conserved miRNA genes. The fifth one (miR-303) appears to be a very young gene, originating de novo from a non-miRNA sequence recently. We sequenced these 5 miRNA genes and their neighboring regions from a worldwide collection of Drosophila melanogaster lines. The levels of divergence and polymorphism in these miRNA genes, vis-à-vis those of the neighboring DNA sequences, suggest that these 5 genes are evolving adaptively. Furthermore, the polymorphism pattern of miR-310s in D. melanogaster is indicative of hitchhiking under positive selection. Thus, a large number of adaptive changes over a long period of time may be essential for the evolution of newly emerged miRNA genes.

Sequencing of small RNAs of the fern Pleopeltis minima (Polypodiaceae) offers insight into the evolution of the microrna repertoire in land plants

PubMed Central

Berruezo, Florencia; de Souza, Flávio S. J.; Picca, Pablo I.; Nemirovsky, Sergio I.; Martínez Tosar, Leandro; Rivero, Mercedes; Mentaberry, Alejandro N.

2017-01-01

MicroRNAs (miRNAs) are short, single stranded RNA molecules that regulate the stability and translation of messenger RNAs in diverse eukaryotic groups. Several miRNA genes are of ancient origin and have been maintained in the genomes of animal and plant taxa for hundreds of millions of years, playing key roles in development and physiology. In the last decade, genome and small RNA (sRNA) sequencing of several plant species have helped unveil the evolutionary history of land plants. Among these, the fern group (monilophytes) occupies a key phylogenetic position, as it represents the closest extant cousin taxon of seed plants, i.e. gymno- and angiosperms. However, in spite of their evolutionary, economic and ecological importance, no fern genome has been sequenced yet and few genomic resources are available for this group. Here, we sequenced the small RNA fraction of an epiphytic South American fern, Pleopeltis minima (Polypodiaceae), and compared it to plant miRNA databases, allowing for the identification of miRNA families that are shared by all land plants, shared by all vascular plants (tracheophytes) or shared by euphyllophytes (ferns and seed plants) only. Using the recently described transcriptome of another fern, Lygodium japonicum, we also estimated the degree of conservation of fern miRNA targets in relation to other plant groups. Our results pinpoint the origin of several miRNA families in the land plant evolutionary tree with more precision and are a resource for future genomic and functional studies of fern miRNAs. PMID:28494025

Identification and characterization of lipid metabolism-related microRNAs in the liver of genetically improved farmed tilapia (GIFT, Oreochromis niloticus) by deep sequencing.

PubMed

Tao, Yi-Fan; Qiang, Jun; Yin, Guo-Jun; Xu, Pao; Shi, Qiong; Bao, Jing-Wen

2017-10-01

MicroRNAs (miRNAs) play vital roles in modulating diverse metabolic processes in the liver, including lipid metabolism. Genetically improved farmed tilapia (GIFT, Oreochromis niloticus), an important aquaculture species in China, is susceptible to hepatic steatosis when reared in intensive culture systems. To investigate the miRNAs involved in GIFT lipid metabolism, two hepatic small RNA libraries from high-fat diet-fed and normal-fat diet-fed GIFT were constructed and sequenced using high-throughput sequencing technology. A total of 204 known and 56 novel miRNAs were identified by aligning the sequencing data with known Danio rerio miRNAs listed in miRBase 21.0. Six known miRNAs (miR-30a-5p, miR-34a, miR-145-5p, miR-29a, miR-205-5p, and miR-23a-3p) that were differentially expressed between the high-fat diet and normal-fat diet groups were validated by quantitative real-time PCR. Bioinformatics tools were used to predict the potential target genes of these differentially expressed miRNAs, and Gene Ontology enrichment analysis indicated that these miRNAs may play important roles in diet-induced hepatic steatosis in GIFT. Our results provide a foundation for further studies of the role of miRNAs in tilapia lipid homeostasis regulation, and may help to identify novel targets for therapeutic interventions to reduce the occurrence of fatty liver disease in farmed tilapia. Copyright © 2017. Published by Elsevier Ltd.

Highly-sensitive microRNA detection based on bio-bar-code assay and catalytic hairpin assembly two-stage amplification.

PubMed

Tang, Songsong; Gu, Yuan; Lu, Huiting; Dong, Haifeng; Zhang, Kai; Dai, Wenhao; Meng, Xiangdan; Yang, Fan; Zhang, Xueji

2018-04-03

Herein, a highly-sensitive microRNA (miRNA) detection strategy was developed by combining bio-bar-code assay (BBA) with catalytic hairpin assembly (CHA). In the proposed system, two nanoprobes of magnetic nanoparticles functionalized with DNA probes (MNPs-DNA) and gold nanoparticles with numerous barcode DNA (AuNPs-DNA) were designed. In the presence of target miRNA, the MNP-DNA and AuNP-DNA hybridized with target miRNA to form a "sandwich" structure. After "sandwich" structures were separated from the solution by the magnetic field and dehybridized by high temperature, the barcode DNA sequences were released by dissolving AuNPs. The released barcode DNA sequences triggered the toehold strand displacement assembly of two hairpin probes, leading to recycle of barcode DNA sequences and producing numerous fluorescent CHA products for miRNA detection. Under the optimal experimental conditions, the proposed two-stage amplification system could sensitively detect target miRNA ranging from 10 pM to 10 aM with a limit of detection (LOD) down to 97.9 zM. It displayed good capability to discriminate single base and three bases mismatch due to the unique sandwich structure. Notably, it presented good feasibility for selective multiplexed detection of various combinations of synthetic miRNA sequences and miRNAs extracted from different cell lysates, which were in agreement with the traditional polymerase chain reaction analysis. The two-stage amplification strategy may be significant implication in the biological detection and clinical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Sequencing of small RNAs of the fern Pleopeltis minima (Polypodiaceae) offers insight into the evolution of the microrna repertoire in land plants.

PubMed

Berruezo, Florencia; de Souza, Flávio S J; Picca, Pablo I; Nemirovsky, Sergio I; Martínez Tosar, Leandro; Rivero, Mercedes; Mentaberry, Alejandro N; Zelada, Alicia M

2017-01-01

MicroRNAs (miRNAs) are short, single stranded RNA molecules that regulate the stability and translation of messenger RNAs in diverse eukaryotic groups. Several miRNA genes are of ancient origin and have been maintained in the genomes of animal and plant taxa for hundreds of millions of years, playing key roles in development and physiology. In the last decade, genome and small RNA (sRNA) sequencing of several plant species have helped unveil the evolutionary history of land plants. Among these, the fern group (monilophytes) occupies a key phylogenetic position, as it represents the closest extant cousin taxon of seed plants, i.e. gymno- and angiosperms. However, in spite of their evolutionary, economic and ecological importance, no fern genome has been sequenced yet and few genomic resources are available for this group. Here, we sequenced the small RNA fraction of an epiphytic South American fern, Pleopeltis minima (Polypodiaceae), and compared it to plant miRNA databases, allowing for the identification of miRNA families that are shared by all land plants, shared by all vascular plants (tracheophytes) or shared by euphyllophytes (ferns and seed plants) only. Using the recently described transcriptome of another fern, Lygodium japonicum, we also estimated the degree of conservation of fern miRNA targets in relation to other plant groups. Our results pinpoint the origin of several miRNA families in the land plant evolutionary tree with more precision and are a resource for future genomic and functional studies of fern miRNAs.

Accelerated Evolution of the Pituitary Adenylate Cyclase-Activating Polypeptide Precursor Gene During Human Origin

PubMed Central

Wang, Yin-qiu; Qian, Ya-ping; Yang, Su; Shi, Hong; Liao, Cheng-hong; Zheng, Hong-Kun; Wang, Jun; Lin, Alice A.; Cavalli-Sforza, L. Luca; Underhill, Peter A.; Chakraborty, Ranajit; Jin, Li; Su, Bing

2005-01-01

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide abundantly expressed in the central nervous system and involved in regulating neurogenesis and neuronal signal transduction. The amino acid sequence of PACAP is extremely conserved across vertebrate species, indicating a strong functional constraint during the course of evolution. However, through comparative sequence analysis, we demonstrated that the PACAP precursor gene underwent an accelerated evolution in the human lineage since the divergence from chimpanzees, and the amino acid substitution rate in humans is at least seven times faster than that in other mammal species resulting from strong Darwinian positive selection. Eleven human-specific amino acid changes were identified in the PACAP precursors, which are conserved from murine to African apes. Protein structural analysis suggested that a putative novel neuropeptide might have originated during human evolution and functioned in the human brain. Our data suggested that the PACAP precursor gene underwent adaptive changes during human origin and may have contributed to the formation of human cognition. PMID:15834139

The Plant Peptidome: An Expanding Repertoire of Structural Features and Biological Functions[OPEN

PubMed Central

Tavormina, Patrizia; De Coninck, Barbara; Nikonorova, Natalia; De Smet, Ive; Cammue, Bruno P.A.

2015-01-01

Peptides fulfill a plethora of functions in plant growth, development, and stress responses. They act as key components of cell-to-cell communication, interfere with signaling and response pathways, or display antimicrobial activity. Strikingly, both the diversity and amount of plant peptides have been largely underestimated. Most characterized plant peptides to date acting as small signaling peptides or antimicrobial peptides are derived from nonfunctional precursor proteins. However, evidence is emerging on peptides derived from a functional protein, directly translated from small open reading frames (without the involvement of a precursor) or even encoded by primary transcripts of microRNAs. These novel types of peptides further add to the complexity of the plant peptidome, even though their number is still limited and functional characterization as well as translational evidence are often controversial. Here, we provide a comprehensive overview of the reported types of plant peptides, including their described functional and structural properties. We propose a novel, unifying peptide classification system to emphasize the enormous diversity in peptide synthesis and consequent complexity of the still expanding knowledge on the plant peptidome. PMID:26276833

TGFβ1-induced down-regulation of microRNA-138 contributes to epithelial-mesenchymal transition in primary lung cancer cells.

PubMed

Zhang, Fang; Li, Tiepeng; Han, Lu; Qin, Peng; Wu, Zhao; Xu, Benling; Gao, Quanli; Song, Yongping

2018-02-19

The existence of cancer stem cells within the tumor could lead to cancer therapy resistance. TGFβ1 is considered as one of the most powerful players in the generation of CSCs through induction of epithelial-mesenchymal transition in different types of cancer including lung cancer, however, the detailed mechanisms by which TGFβ1 contribute to EMT induction and CSC maintenance remains unclear. Here, we showed primary lung cancer cells treated by TGFβ1 exhibit mesenchymal features, including morphology and expression of mesenchymal marker in a time-dependent manner. We also observed long-term TGFβ1 exposure leads to an enrichment of a sub-population of CD44 + CD90 + cells which represent CSCs in lung cancer cells. Moreover, the differential expression microRNAs between CSCs and non-CSCs were identified using next-generation sequencing to screen key miRNAs which might contribute to TGFβ1-induced EMT and CSCs generation. Among those differentially expressed miRNAs, the expression of microRNA-138 was time-dependently down-regulated by TGFβ1 treatment. We further demonstrated primary lung cancer cells, in which we knockdown the expression of miR-138, exhibit mesenchymal phenotypes and stem cell properties. Taken together, these findings indicate TGFβ1-induced down-regulation of microRNA-138 contributes to EMT in primary lung cancer cells, and suggest that miR-138 might serve as a potential therapeutic target. Copyright © 2018 Elsevier Inc. All rights reserved.

Single-Molecule Electrical Random Resequencing of DNA and RNA

NASA Astrophysics Data System (ADS)

Ohshiro, Takahito; Matsubara, Kazuki; Tsutsui, Makusu; Furuhashi, Masayuki; Taniguchi, Masateru; Kawai, Tomoji

2012-07-01

Two paradigm shifts in DNA sequencing technologies--from bulk to single molecules and from optical to electrical detection--are expected to realize label-free, low-cost DNA sequencing that does not require PCR amplification. It will lead to development of high-throughput third-generation sequencing technologies for personalized medicine. Although nanopore devices have been proposed as third-generation DNA-sequencing devices, a significant milestone in these technologies has been attained by demonstrating a novel technique for resequencing DNA using electrical signals. Here we report single-molecule electrical resequencing of DNA and RNA using a hybrid method of identifying single-base molecules via tunneling currents and random sequencing. Our method reads sequences of nine types of DNA oligomers. The complete sequence of 5'-UGAGGUA-3' from the let-7 microRNA family was also identified by creating a composite of overlapping fragment sequences, which was randomly determined using tunneling current conducted by single-base molecules as they passed between a pair of nanoelectrodes.

Differential expression profiles of miRNAs induced by vaccination followed by Marek’s disease virus challenge at cytolytic stage in chickens resistant or susceptible to Marek’s disease

USDA-ARS?s Scientific Manuscript database

Mounting evidence shows microRNAs (miRNAs) directly regulate gene expression post-transcriptionally through base-pairing with regions in the 3’-untranslated sequences of target gene mRNAs, which results in dysregulation of gene expression/translation and subsequently modulates cellular processes. We...

Transcriptome profiling of microRNA by next-gen deep sequencing reveals known and novel miRNA species in the lipid fraction of human breast milk

USDA-ARS?s Scientific Manuscript database

While breast milk has unique health advantages for infants, the mechanisms by which it regulates the physiology of newborns are incompletely understood. miRNAs have been described as functioning transcellularly, and have been previously isolated in cell-free and exosomal form from bodily liquids (se...

Discovery and Analysis of MicroRNAs in Leymus chinensis under Saline-Alkali and Drought Stress Using High-Throughput Sequencing

PubMed Central

Wang, Qi; Wang, Nan; Wang, Fawei; Liu, Weican; Li, Xiaowei; Chen, Huan; Yao, Na; Guan, Lili; Chen, Kai; Cui, Xiyan; Yang, Meiying; Li, Haiyan

2014-01-01

Leymus chinensis (Trin.) Tzvel. is a perennial rhizome grass of the Poaceae (also called Gramineae) family, which adapts well to drought, saline and alkaline conditions. However, little is known about the stress tolerance of L. chinensis at the molecular level. microRNAs (miRNAs) are known to play critical roles in nutrient homeostasis, developmental processes, pathogen responses, and abiotic stress in plants. In this study, we used Solexa sequencing technology to generate high-quality small RNA data from three L. chinensis groups: a control group, a saline-alkaline stress group (100 mM NaCl and 200 mM NaHCO3), and a drought stress group (20% polyethylene glycol 2000). From these data we identified 132 known miRNAs and 16 novel miRNAs candidates. For these miRNAs we also identified target genes that encode a broad range of proteins that may be correlated with abiotic stress regulation. This is the first study to demonstrate differentially expressed miRNAs in L. chinensis under saline-alkali and drought stress. These findings may help explain the saline-alkaline and drought stress responses in L. chinensis. PMID:25369004

Discovery and analysis of microRNAs in Leymus chinensis under saline-alkali and drought stress using high-throughput sequencing.

PubMed

Zhai, Junfeng; Dong, Yuanyuan; Sun, Yepeng; Wang, Qi; Wang, Nan; Wang, Fawei; Liu, Weican; Li, Xiaowei; Chen, Huan; Yao, Na; Guan, Lili; Chen, Kai; Cui, Xiyan; Yang, Meiying; Li, Haiyan

2014-01-01

Leymus chinensis (Trin.) Tzvel. is a perennial rhizome grass of the Poaceae (also called Gramineae) family, which adapts well to drought, saline and alkaline conditions. However, little is known about the stress tolerance of L. chinensis at the molecular level. microRNAs (miRNAs) are known to play critical roles in nutrient homeostasis, developmental processes, pathogen responses, and abiotic stress in plants. In this study, we used Solexa sequencing technology to generate high-quality small RNA data from three L. chinensis groups: a control group, a saline-alkaline stress group (100 mM NaCl and 200 mM NaHCO3), and a drought stress group (20% polyethylene glycol 2000). From these data we identified 132 known miRNAs and 16 novel miRNAs candidates. For these miRNAs we also identified target genes that encode a broad range of proteins that may be correlated with abiotic stress regulation. This is the first study to demonstrate differentially expressed miRNAs in L. chinensis under saline-alkali and drought stress. These findings may help explain the saline-alkaline and drought stress responses in L. chinensis.

miPrimer: an empirical-based qPCR primer design method for small noncoding microRNA

PubMed Central

Kang, Shih-Ting; Hsieh, Yi-Shan; Feng, Chi-Ting; Chen, Yu-Ting; Yang, Pok Eric; Chen, Wei-Ming

2018-01-01

MicroRNAs (miRNAs) are 18–25 nucleotides (nt) of highly conserved, noncoding RNAs involved in gene regulation. Because of miRNAs’ short length, the design of miRNA primers for PCR amplification remains a significant challenge. Adding to the challenge are miRNAs similar in sequence and miRNA family members that often only differ in sequences by 1 nt. Here, we describe a novel empirical-based method, miPrimer, which greatly reduces primer dimerization and increases primer specificity by factoring various intrinsic primer properties and employing four primer design strategies. The resulting primer pairs displayed an acceptable qPCR efficiency of between 90% and 110%. When tested on miRNA families, miPrimer-designed primers are capable of discriminating among members of miRNA families, as validated by qPCR assays using Quark Biosciences’ platform. Of the 120 miRNA primer pairs tested, 95.6% and 93.3% were successful in amplifying specifically non-family and family miRNA members, respectively, after only one design trial. In summary, miPrimer provides a cost-effective and valuable tool for designing miRNA primers. PMID:29208706

RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA.

PubMed

Cui, Yalei; Huang, Tianzhi; Zhang, Xiaobo

2015-12-01

MicroRNAs (miRNAs) integrate with Argonaut (Ago) to create the RNA-induced silencing complex, and regulate gene expression by silencing target mRNAs. RNA editing of miRNA may affect miRNA processing, assembly of the Ago complex and target mRNA binding. However, the function of edited miRNA, assembled within the Ago complex, has not been extensively investigated. In this study, sequence analysis of the Ago complex of Marsupenaeus japonicus shrimp infected with white spot syndrome virus (WSSV) revealed that host ADAR (adenosine deaminase acting on RNA) catalysed A-to-I RNA editing of a viral miRNA (WSSV-miR-N12) at the +16 site. This editing of the non-seed sequence did not affect association of the edited miRNA with the Ago protein, but inhibited interaction between the miRNA and its target gene (wsv399). The WSSV early gene wsv399 inhibited WSSV infection. As a result, the RNA editing of miRNA caused virus latency. Our results highlight a novel example of miRNA editing in the miRNA-induced silencing complex. © 2015 The Authors.

Identification and characterization of microRNAs and their target genes from Nile tilapia (Oreochromis niloticus).

PubMed

Huang, Yong; Ma, Xiu Ying; Yang, You Bing; Ren, Hong Tao; Sun, Xi Hong; Wang, Li Rui

MicroRNAs (miRNAs) are a class of small single-stranded, endogenous 21-22 nt non-coding RNAs that regulate their target mRNA levels by causing either inactivation or degradation of the mRNAs. In recent years, miRNA genes have been identified from mammals, insects, worms, plants, and viruses. In this research, bioinformatics approaches were used to predict potential miRNAs and their targets in Nile tilapia from the expressed sequence tag (EST) and genomic survey sequence (GSS) database, respectively, based on the conservation of miRNAs in many animal species. A total of 19 potential miRNAs were detected following a range of strict filtering criteria. To test the validity of the bioinformatics method, seven predicted Nile tilapia miRNA genes were selected for further biological validation, and their mature miRNA transcripts were successfully detected by stem-loop RT-PCR experiments. Using these potential miRNAs, we found 56 potential targets in this species. Most of the target mRNAs appear to be involved in development, metabolism, signal transduction, transcription regulation and stress responses. Overall, our findings will provide an important foundation for further research on miRNAs function in the Nile tilapia.

Genome-wide computational identification of microRNAs and their targets in the deep-branching eukaryote Giardia lamblia.

PubMed

Zhang, Yan-Qiong; Chen, Dong-Liang; Tian, Hai-Feng; Zhang, Bao-Hong; Wen, Jian-Fan

2009-10-01

Using a combined computational program, we identified 50 potential microRNAs (miRNAs) in Giardia lamblia, one of the most primitive unicellular eukaryotes. These miRNAs are unique to G. lamblia and no homologues have been found in other organisms; miRNAs, currently known in other species, were not found in G. lamblia. This suggests that miRNA biogenesis and miRNA-mediated gene regulation pathway may evolve independently, especially in evolutionarily distant lineages. A majority (43) of the predicted miRNAs are located at one single locus; however, some miRNAs have two or more copies in the genome. Among the 58 miRNA genes, 28 are located in the intergenic regions whereas 30 are present in the anti-sense strands of the protein-coding sequences. Five predicted miRNAs are expressed in G. lamblia trophozoite cells evidenced by expressed sequence tags or RT-PCR. Thirty-seven identified miRNAs may target 50 protein-coding genes, including seven variant-specific surface proteins (VSPs). Our findings provide a clue that miRNA-mediated gene regulation may exist in the early stage of eukaryotic evolution, suggesting that it is an important regulation system ubiquitous in eukaryotes.

A New Module in Neural Differentiation Control: Two MicroRNAs Upregulated by Retinoic Acid, miR-9 and -103, Target the Differentiation Inhibitor ID2

PubMed Central

Savino, Mauro; Laneve, Pietro; Caffarelli, Elisa; Nasi, Sergio

2012-01-01

The transcription factor ID2 is an important repressor of neural differentiation strongly implicated in nervous system cancers. MicroRNAs (miRNAs) are increasingly involved in differentiation control and cancer development. Here we show that two miRNAs upregulated on differentiation of neuroblastoma cells – miR-9 and miR-103 – restrain ID2 expression by directly targeting the coding sequence and 3′ untranslated region of the ID2 encoding messenger RNA, respectively. Notably, the two miRNAs show an inverse correlation with ID2 during neuroblastoma cell differentiation induced by retinoic acid. Overexpression of miR-9 and miR-103 in neuroblastoma cells reduces proliferation and promotes differentiation, as it was shown to occur upon ID2 inhibition. Conversely, an ID2 mutant that cannot be targeted by either miRNA prevents retinoic acid-induced differentiation more efficient than wild-type ID2. These findings reveal a new regulatory module involving two microRNAs upregulated during neural differentiation that directly target expression of the key differentiation inhibitor ID2, suggesting that its alteration may be involved in neural cancer development. PMID:22848373

A new module in neural differentiation control: two microRNAs upregulated by retinoic acid, miR-9 and -103, target the differentiation inhibitor ID2.

PubMed

Annibali, Daniela; Gioia, Ubaldo; Savino, Mauro; Laneve, Pietro; Caffarelli, Elisa; Nasi, Sergio

2012-01-01

The transcription factor ID2 is an important repressor of neural differentiation strongly implicated in nervous system cancers. MicroRNAs (miRNAs) are increasingly involved in differentiation control and cancer development. Here we show that two miRNAs upregulated on differentiation of neuroblastoma cells--miR-9 and miR-103--restrain ID2 expression by directly targeting the coding sequence and 3' untranslated region of the ID2 encoding messenger RNA, respectively. Notably, the two miRNAs show an inverse correlation with ID2 during neuroblastoma cell differentiation induced by retinoic acid. Overexpression of miR-9 and miR-103 in neuroblastoma cells reduces proliferation and promotes differentiation, as it was shown to occur upon ID2 inhibition. Conversely, an ID2 mutant that cannot be targeted by either miRNA prevents retinoic acid-induced differentiation more efficient than wild-type ID2. These findings reveal a new regulatory module involving two microRNAs upregulated during neural differentiation that directly target expression of the key differentiation inhibitor ID2, suggesting that its alteration may be involved in neural cancer development.

Biomarkers in Immunoglobulin Light Chain Amyloidosis.

PubMed

Kufová, Z; Sevcikova, T; Growkova, K; Vojta, P; Filipová, J; Adam, Z; Pour, L; Penka, M; Rysava, R; Němec, P; Brozova, L; Vychytilova, P; Jurczyszyn, A; Grosicki, S; Barchnicka, A; Hajdúch, M; Simicek, M; Hájek, R

2017-01-01

Immunoglobulin light chain amyloidosis (AL amyloidosis - ALA) is a monoclonal gammopathy characterized by presence of aberrant plasma cells producing amyloidogenic immunoglobulin light chains. This leads to formation of amyloid fibrils in various organs and tissues, mainly in heart and kidney, and causes their dysfunction. As amyloid depositing in target organs is irreversible, there is a big effort to identify biomarker that could help to distinguish ALA from other monoclonal gammopathies in the early stages of disease, when amyloid deposits are not fatal yet. High throughput technologies bring new opportunities to modern cancer research as they enable to study disease within its complexity. Sophisticated methods such as next generation sequencing, gene expression profiling and circulating microRNA profiling are new approaches to study aberrant plasma cells from patients with light chain amyloidosis and related diseases. While generally known mutation in multiple myeloma patients (KRAS, NRAS, MYC, TP53) were not found in ALA, number of mutated genes is comparable. Transcriptome of ALA patients proves to be more similar to monoclonal gammopathy of undetermined significance patients, moreover level of circulating microRNA, that are known to correlate with heart damage, is increased in ALA patients, where heart damage in ALA typical symptom.Key words: amyloidosis - plasma cell - genome - transcriptome - microRNA.

MicroRNA-944 Affects Cell Growth by Targeting EPHA7 in Non-Small Cell Lung Cancer.

PubMed

Liu, Minxia; Zhou, Kecheng; Cao, Yi

2016-09-26

MicroRNAs (miRNAs) have critical roles in lung tumorigenesis and development. To determine aberrantly expressed miRNAs involved in non-small cell lung cancer (NSCLC) and investigate pathophysiological functions and mechanisms, we firstly carried out small RNA deep sequencing in NSCLC cell lines (EPLC-32M1, A549 and 801D) and a human immortalized cell line 16HBE, we then studied miRNA function by cell proliferation and apoptosis. cDNA microarray, luciferase reporter assay and miRNA transfection were used to investigate interaction between the miRNA and target gene. miR-944 was significantly down-regulated in NSCLC and had many putative targets. Moreover, the forced expression of miR-944 significantly inhibited the proliferation of NSCLC cells in vitro. By integrating mRNA expression data and miR-944-target prediction, we disclosed that EPHA7 was a potential target of miR-944, which was further verified by luciferase reporter assay and microRNA transfection. Our data indicated that miR-944 targets EPHA7 in NSCLC and regulates NSCLC cell proliferation, which may offer a new mechanism underlying the development and progression of NSCLC.

SC1 Promotes MiR124-3p Expression to Maintain the Self-Renewal of Mouse Embryonic Stem Cells by Inhibiting the MEK/ERK Pathway.

PubMed

Wei, Qing; Liu, Hongliang; Ai, Zhiying; Wu, Yongyan; Liu, Yingxiang; Shi, Zhaopeng; Ren, Xuexue; Guo, Zekun

2017-01-01

Self-renewal is one of the most important features of embryonic stem (ES) cells. SC1 is a small molecule modulator that effectively maintains the self-renewal of mouse ES cells in the absence of leukemia inhibitory factor (LIF), serum and feeder cells. However, the mechanism by which SC1 maintains the undifferentiated state of mouse ES cells remains unclear. In this study, microarray and small RNA deep-sequencing experiments were performed on mouse ES cells treated with or without SC1 to identify the key genes and microRNAs that contributed to self-renewal. SC1 regulates the expressions of pluripotency and differentiation factors, and antagonizes the retinoic acid (RA)-induced differentiation in the presence or absence of LIF. SC1 inhibits the MEK/ERK pathway through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and pathway reporting experiments. Small RNA deep-sequencing revealed that SC1 significantly modulates the expression of multiple microRNAs with crucial functions in ES cells. The expression of miR124-3p is upregulated in SC1-treated ES cells, which significantly inhibits the MEK/ERK pathway by targeting Grb2, Sos2 and Egr1. SC1 enhances the self-renewal capacity of mouse ES cells by modulating the expression of key regulatory genes and pluripotency-associated microRNAs. SC1 significantly upregulates miR124-3p expression to further inhibit the MEK/ ERK pathway by targeting Grb2, Sos2 and Egr1. © 2017 The Author(s). Published by S. Karger AG, Basel.

De novo characterization of microRNAs in oriental fruit moth Grapholita molesta and selection of reference genes for normalization of microRNA expression

PubMed Central

Zhang, Jing; Zhang, Qingwen; Liu, Xiaoxia; Li, Zhen

2017-01-01

MicroRNAs (miRNAs) are a group of endogenous non-coding small RNAs that have critical regulatory functions in almost all known biological processes at the post-transcriptional level in a variety of organisms. The oriental fruit moth Grapholita molesta is one of the most serious pests in orchards worldwide and threatens the production of Rosacea fruits. In this study, a de novo small RNA library constructed from mixed stages of G. molesta was sequenced through Illumina sequencing platform and a total of 536 mature miRNAs consisting of 291 conserved and 245 novel miRNAs were identified. Most of the conserved and novel miRNAs were detected with moderate abundance. The miRNAs in the same cluster normally showed correlated expressional profiles. A comparative analysis of the 79 conserved miRNA families within 31 arthropod species indicated that these miRNA families were more conserved among insects and within orders of closer phylogenetic relationships. The KEGG pathway analysis and network prediction of target genes indicated that the complex composed of miRNAs, clock genes and developmental regulation genes may play vital roles to regulate the developmental circadian rhythm of G. molesta. Furthermore, based on the sRNA library of G. molesta, suitable reference genes were selected and validated for study of miRNA transcriptional profile in G. molesta under two biotic and six abiotic experimental conditions. This study systematically documented the miRNA profile in G. molesta, which could lay a foundation for further understanding of the regulatory roles of miRNAs in the development and metabolism in this pest and might also suggest clues to the development of genetic-based techniques for agricultural pest control. PMID:28158242

Evolutionary relationships between miRNA genes and their activity.

PubMed

Zhu, Yan; Skogerbø, Geir; Ning, Qianqian; Wang, Zhen; Li, Biqing; Yang, Shuang; Sun, Hong; Li, Yixue

2012-12-22

The emergence of vertebrates is characterized by a strong increase in miRNA families. MicroRNAs interact broadly with many transcripts, and the evolution of such a system is intriguing. However, evolutionary questions concerning the origin of miRNA genes and their subsequent evolution remain unexplained. In order to systematically understand the evolutionary relationship between miRNAs gene and their function, we classified human known miRNAs into eight groups based on their evolutionary ages estimated by maximum parsimony method. New miRNA genes with new functional sequences accumulated more dynamically in vertebrates than that observed in Drosophila. Different levels of evolutionary selection were observed over miRNA gene sequences with different time of origin. Most genic miRNAs differ from their host genes in time of origin, there is no particular relationship between the age of a miRNA and the age of its host genes, genic miRNAs are mostly younger than the corresponding host genes. MicroRNAs originated over different time-scales are often predicted/verified to target the same or overlapping sets of genes, opening the possibility of substantial functional redundancy among miRNAs of different ages. Higher degree of tissue specificity and lower expression level was found in young miRNAs. Our data showed that compared with protein coding genes, miRNA genes are more dynamic in terms of emergence and decay. Evolution patterns are quite different between miRNAs of different ages. MicroRNAs activity is under tight control with well-regulated expression increased and targeting decreased over time. Our work calls attention to the study of miRNA activity with a consideration of their origin time.

Beta.-glucosidase coding sequences and protein from orpinomyces PC-2

DOEpatents

Li, Xin-Liang; Ljungdahl, Lars G.; Chen, Huizhong; Ximenes, Eduardo A.

2001-02-06

Provided is a novel .beta.-glucosidase from Orpinomyces sp. PC2, nucleotide sequences encoding the mature protein and the precursor protein, and methods for recombinant production of this .beta.-glucosidase.

Individual microRNAs (miRNAs) display distinct mRNA targeting "rules".

PubMed

Wang, Wang-Xia; Wilfred, Bernard R; Xie, Kevin; Jennings, Mary H; Hu, Yanling Hu; Stromberg, Arnold J; Nelson, Peter T

2010-01-01

MicroRNAs (miRNAs) guide Argonaute (AGO)-containing microribonucleoprotein (miRNP) complexes to target mRNAs.It has been assumed that miRNAs behave similarly to each other with regard to mRNA target recognition. The usual assumptions, which are based on prior studies, are that miRNAs target preferentially sequences in the 3'UTR of mRNAs,guided by the 5' "seed" portion of the miRNAs. Here we isolated AGO- and miRNA-containing miRNPs from human H4 tumor cells by co-immunoprecipitation (co-IP) with anti-AGO antibody. Cells were transfected with miR-107, miR-124,miR-128, miR-320, or a negative control miRNA. Co-IPed RNAs were subjected to downstream high-density Affymetrix Human Gene 1.0 ST microarray analyses using an assay we validated previously-a "RIP-Chip" experimental design. RIP-Chip data provided a list of mRNAs recruited into the AGO-miRNP in correlation to each miRNA. These experimentally identified miRNA targets were analyzed for complementary six nucleotide "seed" sequences within the transfected miRNAs. We found that miR-124 targets tended to have sequences in the 3'UTR that would be recognized by the 5' seed of miR-124, as described in previous studies. By contrast, miR-107 targets tended to have 'seed' sequences in the mRNA open reading frame, but not the 3' UTR. Further, mRNA targets of miR-128 and miR-320 are less enriched for 6-mer seed sequences in comparison to miR-107 and miR-124. In sum, our data support the importance of the 5' seed in determining binding characteristics for some miRNAs; however, the "binding rules" are complex, and individual miRNAs can have distinct sequence determinants that lead to mRNA targeting.

Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus.

PubMed Central

Laprevotte, I; Hampe, A; Sherr, C J; Galibert, F

1984-01-01

The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing. PMID:6328019

Conservation of small RNA pathways in platypus

PubMed Central

Murchison, Elizabeth P.; Kheradpour, Pouya; Sachidanandam, Ravi; Smith, Carly; Hodges, Emily; Xuan, Zhenyu; Kellis, Manolis; Grützner, Frank; Stark, Alexander; Hannon, Gregory J.

2008-01-01

Small RNA pathways play evolutionarily conserved roles in gene regulation and defense from parasitic nucleic acids. The character and expression patterns of small RNAs show conservation throughout animal lineages, but specific animal clades also show variations on these recurring themes, including species-specific small RNAs. The monotremes, with only platypus and four species of echidna as extant members, represent the basal branch of the mammalian lineage. Here, we examine the small RNA pathways of monotremes by deep sequencing of six platypus and echidna tissues. We find that highly conserved microRNA species display their signature tissue-specific expression patterns. In addition, we find a large rapidly evolving cluster of microRNAs on platypus chromosome X1, which is unique to monotremes. Platypus and echidna testes contain a robust Piwi-interacting (piRNA) system, which appears to be participating in ongoing transposon defense. PMID:18463306

Computational Identification of MicroRNAs and Their Targets from Finger Millet (Eleusine coracana).

PubMed

Usha, S; Jyothi, M N; Suchithra, B; Dixit, Rekha; Rai, D V; Nagesh Babu, R

2017-03-01

MicroRNAs are endogenous small RNAs regulating intrinsic normal growth and development of plant. Discovering miRNAs, their targets and further inferring their functions had become routine process to comprehend the normal biological processes of miRNAs and their roles in plant development. In this study, we used homology-based analysis with available expressed sequence tag of finger millet (Eleusine coracana) to predict conserved miRNAs. Three potent miRNAs targeting 88 genes were identified. The newly identified miRNAs were found to be homologous with miR166 and miR1310. The targets recognized were transcription factors and enzymes, and GO analysis showed these miRNAs played varied roles in gene regulation. The identification of miRNAs and their targets is anticipated to hasten the pace of key epigenetic regulators in plant development.

Nonlinear Optical Properties of ZnO for BioimagingCell and Cell Destruction

NASA Astrophysics Data System (ADS)

Urban, Ben; Chakki, Samudyatha; Senthilkumar, Os; Senthilkumar, Kasilingam; Fujita, Yasuhisa; Neogi, Arup

2011-03-01

As of recent years nanotechnology has been at the forefront of scientific research. It promises to have a broad range of applications from turning unhealthy foods into health foods, making computers faster and curing cancer. We present results on using nonlinear optical processes of ZnO nano-crystals to detect, track and destroy cells. By incorporating ZnO into a hydrophobic nano-hydrogel matrix with trace amounts of H2 O2 , we can attach antibodies or microRNA for specific cell targeting and, using the heat generating properties of the third order nonlinear process, release H2 O2 in the cell causing instant cell death. Theoretically, with the appropriate sequence for microRNA or the appropriate antibodies, we could target cancer cells in the body and destroy them. This presentation gives our results until now.

Conservation of small RNA pathways in platypus.

PubMed

Murchison, Elizabeth P; Kheradpour, Pouya; Sachidanandam, Ravi; Smith, Carly; Hodges, Emily; Xuan, Zhenyu; Kellis, Manolis; Grützner, Frank; Stark, Alexander; Hannon, Gregory J

2008-06-01

Small RNA pathways play evolutionarily conserved roles in gene regulation and defense from parasitic nucleic acids. The character and expression patterns of small RNAs show conservation throughout animal lineages, but specific animal clades also show variations on these recurring themes, including species-specific small RNAs. The monotremes, with only platypus and four species of echidna as extant members, represent the basal branch of the mammalian lineage. Here, we examine the small RNA pathways of monotremes by deep sequencing of six platypus and echidna tissues. We find that highly conserved microRNA species display their signature tissue-specific expression patterns. In addition, we find a large rapidly evolving cluster of microRNAs on platypus chromosome X1, which is unique to monotremes. Platypus and echidna testes contain a robust Piwi-interacting (piRNA) system, which appears to be participating in ongoing transposon defense.

The interplay of microRNA and neuronal activity in health and disease

PubMed Central

Eacker, Stephen M.; Dawson, Ted M.; Dawson, Valina L.

2013-01-01

MicroRNAs (miRNAs) are small 19–23 nucleotide regulatory RNAs that function by modulating mRNA translation and/or turnover in a sequence-specific fashion. In the nervous system, miRNAs regulate the production of numerous proteins involved in synaptic transmission. In turn, neuronal activity can regulate the production and turnover of miRNA through a variety of mechanisms. In this way, miRNAs and neuronal activity are in a reciprocal homeostatic relationship that balances neuronal function. The miRNA function is critical in pathological states related to overexcitation such as epilepsy and stroke, suggesting miRNA’s potential as a therapeutic target. We review the current literature relating the interplay of miRNA and neuronal activity and provide future directions for defining miRNA’s role in disease. PMID:23986658

Identification of MicroRNA Targets of Capsicum spp. Using MiRTrans—a Trans-Omics Approach

PubMed Central

Zhang, Lu; Qin, Cheng; Mei, Junpu; Chen, Xiaocui; Wu, Zhiming; Luo, Xirong; Cheng, Jiaowen; Tang, Xiangqun; Hu, Kailin; Li, Shuai C.

2017-01-01

The microRNA (miRNA) can regulate the transcripts that are involved in eukaryotic cell proliferation, differentiation, and metabolism. Especially for plants, our understanding of miRNA targets, is still limited. Early attempts of prediction on sequence alignments have been plagued by enormous false positives. It is helpful to improve target prediction specificity by incorporating the other data sources such as the dependency between miRNA and transcript expression or even cleaved transcripts by miRNA regulations, which are referred to as trans-omics data. In this paper, we developed MiRTrans (Prediction of MiRNA targets by Trans-omics data) to explore miRNA targets by incorporating miRNA sequencing, transcriptome sequencing, and degradome sequencing. MiRTrans consisted of three major steps. First, the target transcripts of miRNAs were predicted by scrutinizing their sequence characteristics and collected as an initial potential targets pool. Second, false positive targets were eliminated if the expression of miRNA and its targets were weakly correlated by lasso regression. Third, degradome sequencing was utilized to capture the miRNA targets by examining the cleaved transcripts that regulated by miRNAs. Finally, the predicted targets from the second and third step were combined by Fisher's combination test. MiRTrans was applied to identify the miRNA targets for Capsicum spp. (i.e., pepper). It can generate more functional miRNA targets than sequence-based predictions by evaluating functional enrichment. MiRTrans identified 58 miRNA-transcript pairs with high confidence from 18 miRNA families conserved in eudicots. Most of these targets were transcription factors; this lent support to the role of miRNA as key regulator in pepper. To our best knowledge, this work is the first attempt to investigate the miRNA targets of pepper, as well as their regulatory networks. Surprisingly, only a small proportion of miRNA-transcript pairs were shared between degradome sequencing and expression dependency predictions, suggesting that miRNA targets predicted by a single technology alone may be prone to report false negatives. PMID:28443105

MicroRNA-339-5p down-regulates protein expression of β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) in human primary brain cultures and is reduced in brain tissue specimens of Alzheimer disease subjects.

PubMed

Long, Justin M; Ray, Balmiki; Lahiri, Debomoy K

2014-02-21

Alzheimer disease (AD) results, in part, from the excess accumulation of the amyloid-β (Aβ) peptide as neuritic plaques in the brain. The short Aβ peptide is derived from the large transmembrane Aβ precursor protein (APP). The rate-limiting step in the production of Aβ from APP is mediated by the β-site APP-cleaving enzyme 1 (BACE1). Dysregulation of BACE1 levels leading to excess Aβ deposition is implicated in sporadic AD. Thus, elucidating the full complement of regulatory pathways that control BACE1 expression is key to identifying novel drug targets central to the Aβ-generating process. MicroRNAs (miRNAs) are expected to participate in this molecular network. Here, we identified a known miRNA, miR-339-5p, as a key contributor to this regulatory network. Two distinct miR-339-5p target sites were predicted in the BACE1 3'-UTR by in silico analyses. Co-transfection of miR-339-5p with a BACE1 3'-UTR reporter construct resulted in significant reduction in reporter expression. Mutation of both target sites eliminated this effect. Delivery of the miR-339-5p mimic also significantly inhibited expression of BACE1 protein in human glioblastoma cells and human primary brain cultures. Delivery of target protectors designed against the miR-339-5p BACE1 3'-UTR target sites in primary human brain cultures significantly elevated BACE1 expression. Finally, miR-339-5p levels were found to be significantly reduced in brain specimens isolated from AD patients as compared with age-matched controls. Therefore, miR-339-5p regulates BACE1 expression in human brain cells and is most likely dysregulated in at least a subset of AD patients making this miRNA a novel drug target.

MicroRNA-339-5p Down-regulates Protein Expression of β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 (BACE1) in Human Primary Brain Cultures and Is Reduced in Brain Tissue Specimens of Alzheimer Disease Subjects*

PubMed Central

Long, Justin M.; Ray, Balmiki; Lahiri, Debomoy K.

2014-01-01

Alzheimer disease (AD) results, in part, from the excess accumulation of the amyloid-β (Aβ) peptide as neuritic plaques in the brain. The short Aβ peptide is derived from the large transmembrane Aβ precursor protein (APP). The rate-limiting step in the production of Aβ from APP is mediated by the β-site APP-cleaving enzyme 1 (BACE1). Dysregulation of BACE1 levels leading to excess Aβ deposition is implicated in sporadic AD. Thus, elucidating the full complement of regulatory pathways that control BACE1 expression is key to identifying novel drug targets central to the Aβ-generating process. MicroRNAs (miRNAs) are expected to participate in this molecular network. Here, we identified a known miRNA, miR-339-5p, as a key contributor to this regulatory network. Two distinct miR-339-5p target sites were predicted in the BACE1 3′-UTR by in silico analyses. Co-transfection of miR-339-5p with a BACE1 3′-UTR reporter construct resulted in significant reduction in reporter expression. Mutation of both target sites eliminated this effect. Delivery of the miR-339-5p mimic also significantly inhibited expression of BACE1 protein in human glioblastoma cells and human primary brain cultures. Delivery of target protectors designed against the miR-339-5p BACE1 3′-UTR target sites in primary human brain cultures significantly elevated BACE1 expression. Finally, miR-339-5p levels were found to be significantly reduced in brain specimens isolated from AD patients as compared with age-matched controls. Therefore, miR-339-5p regulates BACE1 expression in human brain cells and is most likely dysregulated in at least a subset of AD patients making this miRNA a novel drug target. PMID:24352696

Sample sequencing of vascular plants demonstrates widespread conservation and divergence of microRNAs.

PubMed

Chávez Montes, Ricardo A; de Fátima Rosas-Cárdenas, Flor; De Paoli, Emanuele; Accerbi, Monica; Rymarquis, Linda A; Mahalingam, Gayathri; Marsch-Martínez, Nayelli; Meyers, Blake C; Green, Pamela J; de Folter, Stefan

2014-04-23

Small RNAs are pivotal regulators of gene expression that guide transcriptional and post-transcriptional silencing mechanisms in eukaryotes, including plants. Here we report a comprehensive atlas of sRNA and miRNA from 3 species of algae and 31 representative species across vascular plants, including non-model plants. We sequence and quantify sRNAs from 99 different tissues or treatments across species, resulting in a data set of over 132 million distinct sequences. Using miRBase mature sequences as a reference, we identify the miRNA sequences present in these libraries. We apply diverse profiling methods to examine critical sRNA and miRNA features, such as size distribution, tissue-specific regulation and sequence conservation between species, as well as to predict putative new miRNA sequences. We also develop database resources, computational analysis tools and a dedicated website, http://smallrna.udel.edu/. This study provides new insights on plant sRNAs and miRNAs, and a foundation for future studies.

Processing of intervening sequences: a new yeast mutant which fails to excise intervening sequences from precursor tRNAs.

PubMed

Hopper, A K; Schultz, L D; Shapiro, R A

1980-03-01

By using conditional loss of suppression an an assay, we have been successful in screening for a yeast mutant which is defective in tRNA processing. The los1-1 mutation causes an accumulation of a subset of precursor tRNAs at the nonpermissive temperature. These pre-tRNAs are like those which accumulate in the yeast mutant ts 136 (rna1) in that they have transcribed intervening sequences. The mutations at los1-1 and rna1 complement and segregate independently of each other. The los1-1 mutation affects the expression of all 8 tyrosine-inserting suppressor loci, but does not seem to affect rRNA or mRNA synthesis.

Characterizing the Specificity and Co-operation of Aminopeptidases in the Cytosol and ER During MHC Class I antigen Presentation1

PubMed Central

Hearn, Arron; York, Ian A.; Bishop, Courtney; Rock, Kenneth L.

2010-01-01

Many MHC class I binding peptides are generated as N-extended precursors during protein degradation by the proteasome. These peptides can be subsequently trimmed by aminopeptidases in the cytosol and/or the ER to produce mature epitope. However, the contribution and specificity of each of these subcellular compartments in removing N-terminal amino acids for antigen presentation is not well defined. Here we investigate this issue for antigenic precursors that are expressed in the cytosol. By systematically varying the N-terminal flanking sequences of peptides we show that the amino acids upstream of an epitope precursor are a major determinant of the amount of antigen presentation. In many cases MHC class I binding peptides are produced through sequential trimming in both the cytosol and ER. Trimming of flanking residues in the cytosol contributes most to sequences that are poorly trimmed in the ER. Since N-terminal trimming has different specificity in the cytosol and ER, the cleavage of peptides in both of these compartments serves to broaden the repertoire of sequences that are presented. PMID:20351195

miR-150 Regulates Memory CD8 T Cell Differentiation via c-Myb.

PubMed

Chen, Zeyu; Stelekati, Erietta; Kurachi, Makoto; Yu, Sixiang; Cai, Zhangying; Manne, Sasikanth; Khan, Omar; Yang, Xiaolu; Wherry, E John

2017-09-12

MicroRNAs play an important role in T cell responses. However, how microRNAs regulate CD8 T cell memory remains poorly defined. Here, we found that miR-150 negatively regulates CD8 T cell memory in vivo. Genetic deletion of miR-150 disrupted the balance between memory precursor and terminal effector CD8 T cells following acute viral infection. Moreover, miR-150-deficient memory CD8 T cells were more protective upon rechallenge. A key circuit whereby miR-150 repressed memory CD8 T cell development through the transcription factor c-Myb was identified. Without miR-150, c-Myb was upregulated and anti-apoptotic targets of c-Myb, such as Bcl-2 and Bcl-xL, were also increased, suggesting a miR-150-c-Myb survival circuit during memory CD8 T cell development. Indeed, overexpression of non-repressible c-Myb rescued the memory CD8 T cell defects caused by overexpression of miR-150. Overall, these results identify a key role for miR-150 in memory CD8 T cells through a c-Myb-controlled enhanced survival circuit. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Neurotrophin-3 mRNA a putative target of miR21 following status epilepticus.

PubMed

Risbud, Rashmi M; Lee, Carolyn; Porter, Brenda E

2011-11-18

Status epilepticus induces a cascade of protein expression changes contributing to the subsequent development of epilepsy. By identifying the cascade of molecular changes that contribute to the development of epilepsy we hope to be able to design therapeutics for preventing epilepsy. MicroRNAs influence gene expression by altering mRNA stability and/or translation and have been implicated in the pathology of multiple diseases. MiR21 and its co-transcript miR21, microRNAs produced from either the 5' or 3' ends of the same precursor RNA strand, are increased in the hippocampus following status epilepticus. We have identified a miR21 binding site, in the 3' UTR of neurotrophin-3 that inhibits translation. Neurotrophin-3 mRNA levels decrease in the hippocampus following SE concurrent with the increase in miR21. MiR21 levels in cultured hippocampal neurons inversely correlate with neurotrophin-3 mRNA levels. Treatment of hippocampal neuronal cultures with excess K(+)Cl(-), a depolarizing agent mimicking the episode of status epilepticus, also results in an increase in miR21 and a decrease in neurotrophin-3 mRNA. MiR21 is a candidate for regulating neurotrophin-3 signaling in the hippocampus following status epilepticus. Copyright © 2011 Elsevier B.V. All rights reserved.

MicroRNA Functions in Osteogenesis and Dysfunctions in Osteoporosis

PubMed Central

van Wijnen, Andre J.; van de Peppel, Jeroen; van Leeuwen, Johannes P.; Lian, Jane B.; Stein, Gary S.; Westendorf, Jennifer J.; Oursler, Merry-Jo; Sampen, Hee-Jeong Im; Taipaleenmaki, Hanna; Hesse, Eric; Riester, Scott; Kakar, Sanjeev

2013-01-01

MicroRNAs (miRNAs) are critical post-transcriptional regulators of gene expression that control osteoblast mediated bone formation and osteoclast-related bone remodelling. Deregulation of miRNA mediated mechanisms is emerging as an important pathological factor in bone degeneration (e.g., osteoporosis) and other bone-related diseases. MiRNAs are intriguing regulatory molecules that are networked with cell signaling pathways and intricate transcriptional programs through ingenuous circuits with remarkably simple logic. This overview examines key principles by which miRNAs control differentiation of osteoblasts as they evolve from mesenchymal stromal cells during osteogenesis, or of osteoclasts as they originate from monocytic precursors in the hematopoietic lineage during osteoclastogenesis. Of particular note are miRNAs that are temporally up-regulated during osteoblastogenesis (e.g., miR-218) or osteoclastogenesis (e.g., miR-148a). Each miRNA stimulates differentiation by suppressing inhibitory signalling pathways (‘double-negative’ regulation). The excitement surrounding miRNAs in bone biology stems from the prominent effects that individual miRNAs can have on biological transitions during differentiation of skeletal cells and correlations of miRNA dysfunction with bone diseases. MiRNAs have significant clinical potential which is reflected by their versatility as disease-specific biomarkers and their promise as therapeutic agents to ameliorate or reverse bone tissue degeneration. PMID:23605904

The nucleotide sequence of the intergenic region between the 5.8S and 26S rRNA genes of the yeast ribosomal RNA operon. Possible implications for the interaction between 5.8S and 26S rRNA and the processing of the primary transcript.

PubMed Central

Veldman, G M; Klootwijk, J; van Heerikhuizen, H; Planta, R J

1981-01-01

We have determined the nucleotide sequence of part of a cloned yeast ribosomal RNA operon extending from the 5.8S RNA gene downstream into the 5' -terminal region of the 26S RNA gene. We mapped the pertinent processing sites, viz. the 5' end of 26S rRNA and the 3'ends of 5.8S rRNA and its immediate precursor, 7S RNA. At the 3' end of 7S RNA we find the sequence UCGUUU which is very similar to the type I consensus sequence UCAUUA/U present at the 3' ends of 17S, 5.8S and 26S rRNA as well as 18S precursor rRNA in yeast. At the 5' end of the 26S RNA gene we find a sequence of thirteen nucleotides which is homologous to the type II sequence present at the 5' termini of both the 17S and the 5.8S RNA gene. These findings further support the suggestion put forward earlier (G.M. Veldman et al. (1980) Nucl. Acids Res. 8, 2907-2920) that both consensus sequences are involved in the recognition of precursor rRNA by the processing nuclease(s). We discuss a model for the processing of yeast rRNA in which a processing enzyme sequentially recognizes several combinations of a type I and a type II consensus sequence. We also describe the existence of a significant base complementarity between sequences in the 5' -terminal region of 26S rRNA and the 3' -terminal region of 5.8S rRNA. We suggest that base pairing between these sequences contributes to the binding between 5.8S and 26S rRNA. Images PMID:7312619

The Center for Regenerative Biology and Medicine at Mount Desert Island Biological Laboratory

DTIC Science & Technology

2015-08-01

TERMS limb regeneration Positional Memory Code Axolotl microRNAs Zebrafish Polypterus 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18...injury induced regeneration of limbs/appendage tissues in Ambystoma ( axolotl ) and Polypterus animals. The defining feature of limb/appendage...downregulated in UPREGULATED DOWNREGULATED Figure 1: Venn diagram of UniProt protein sequence IDs among Axolotl and Polypterus contigs that

Changes in miRNAs Signal High-Risk HPV Infections | Center for Cancer Research

Cancer.gov

microRNAs (miRNAs) are approximately 21 nucleotide long, non-coding RNAs that regulate the expression of certain proteins. As part of the RNA-induced silencing complex or RISC, miRNAs bind to complementary sequences in the 3’ untranslated regions of target messenger RNAs, blocking protein synthesis and sometimes leading to the destruction of the target RNA. Numerous studies

Transposable element-associated microRNA hairpins produce 21-nt sRNAs integrated into typical microRNA pathways in rice

PubMed Central

Ou-Yang, Fangqian; Luo, Qing-Jun; Zhang, Yue; Richardson, Casey R.; Jiang, Yingwen; Rock, Christopher D.

2013-01-01

microRNAs (miRNAs) are a class of small RNAs (sRNAs) of ~21 nucleotides (nt) in length processed from foldback hairpins by DICER-LIKE1 (DCL1) or DCL4. They regulate the expression of target mRNAs by base pairing through RNA-Induced Silencing Complex (RISC). In the RISC, ARGONAUTE1 (AGO1) is the key protein that cleaves miRNA targets at position ten of a miRNA:target duplex. The authenticity of many annotated rice miRNA hairpins is under debate because of their homology to repeat sequences. Some of them, like miR1884b, have been removed from the current release of miRBase based on incomplete information. In this study, we investigated the association of transposable element (TE)-derived miRNAs with typical miRNA pathways (DCL1/4- and AGO1-dependent) using publicly available deep sequencing datasets. Seven miRNA hairpins with 13 unique sRNAs were specifically enriched in AGO1 immunoprecipitation samples and relatively reduced in DCL1/4 knockdown genotypes. Interestingly, these species are ~21-nt long, instead of 24-nt as annotated in miRBase and the literature. Their expression profiles meet current criteria for functional annotation of miRNAs. In addition, diagnostic cleavage tags were found in degradome datasets for predicted target mRNAs. Most of these miRNA hairpins share significant homology with miniature inverted-repeat transposable elements (MITEs), one type of abundant DNA transposons in rice. Finally, the root-specific production of a 24 nt miRNA-like sRNA was confirmed by RNA blot for a novel EST that maps to the 3'-UTR of a candidate pseudogene showing extensive sequence homology to miR1884b hairpin. Our data are consistent with the hypothesis that TEs can serve as a driving force for the evolution of some MIRNAs, where co-opting of DICER-LIKE1/4 processing and integration into AGO1 could exapt transcribed TE-associated hairpins into typical miRNA pathways. PMID:23420033

High-resolution identification and abundance profiling of cassava (Manihot esculenta Crantz) microRNAs.

PubMed

Khatabi, Behnam; Arikit, Siwaret; Xia, Rui; Winter, Stephan; Oumar, Doungous; Mongomake, Kone; Meyers, Blake C; Fondong, Vincent N

2016-01-28

Small RNAs (sRNAs) are endogenous sRNAs that play regulatory roles in plant growth, development, and biotic and abiotic stress responses. In plants, one subset of sRNAs, microRNAs (miRNAs) exhibit tissue-differential expression and regulate gene expression mainly through direct cleavage of mRNA or indirectly via production of secondary phased siRNAs (phasiRNAs) that silence cognate target transcripts in trans. Here, we have identified cassava (Manihot esculenta Crantz) miRNAs using high resolution sequencing of sRNA libraries from leaf, stem, callus, male and female flower tissues. To analyze the data, we built a cassava genome database and, via sequence analysis and secondary structure prediction, 38 miRNAs not previously reported in cassava were identified. These new cassava miRNAs included two miRNAs not previously been reported in any plant species. The miRNAs exhibited tissue-differential accumulation as confirmed by quantitative RT-PCR and Northern blot analysis, largely reflecting levels observed in sequencing data. Some of the miRNAs identified were predicted to trigger production of secondary phased siRNAs (phasiRNAs) from 80 PHAS loci. Cassava is a woody perennial shrub, grown principally for its starch-rich storage roots, which are rich in calories. In this study, new miRNAs were identified and their expression was validated using qRT-PCR of RNA from five different tissues. The data obtained expand the list of annotated miRNAs and provide additional new resources for cassava improvement research.

Characterization of cold-associated microRNAs in the freeze-tolerant gall fly Eurosta solidaginis using high-throughput sequencing.

PubMed

Lyons, Pierre J; Govaere, Louise; Crapoulet, Nicolas; Storey, Kenneth B; Morin, Pier Jr

2016-12-01

Significant physiological and biochemical changes are observed in freeze-tolerant insects when confronted with cold temperatures. These insects have adapted to winter by retreating into a hypometabolic state of diapause and implementing cryoprotective mechanisms that allow them to survive whole body freezing. MicroRNAs (miRNAs), a family of short ribonucleic acids, are emerging as likely molecular players underlying the process of cold adaptation. Unfortunately, the data is sparse concerning the signature of miRNAs that are modulated following cold exposure in the freeze-tolerant goldenrod gall fly Eurosta solidaginis. Leveraging for the first time a next-generation sequencing approach, differentially expressed miRNAs were evaluated in 5°C and -15°C-exposed E. solidaginis larvae. Next-generation sequencing expression data was subsequently validated by qRT-PCR for selected miRNA targets. Results demonstrate 24 differentially expressed freeze-responsive miRNAs. Notable, miR-1-3p, a miRNA modulated at low temperature in another cold-hardy insect, and miR-14-3p, a miRNA associated with stress response in the fruit fly, were shown to be significantly up-regulated in -15°C-exposed larvae. Overall, this work identifies, for the first time in a high-throughput manner, differentially expressed miRNAs in cold-exposed E. solidaginis larvae and further clarifies an emerging signature of miRNAs modulated at low temperatures in cold-hardy insects. Copyright © 2016 Elsevier Inc. All rights reserved.

The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system.

PubMed

Vonk, Freek J; Casewell, Nicholas R; Henkel, Christiaan V; Heimberg, Alysha M; Jansen, Hans J; McCleary, Ryan J R; Kerkkamp, Harald M E; Vos, Rutger A; Guerreiro, Isabel; Calvete, Juan J; Wüster, Wolfgang; Woods, Anthony E; Logan, Jessica M; Harrison, Robert A; Castoe, Todd A; de Koning, A P Jason; Pollock, David D; Yandell, Mark; Calderon, Diego; Renjifo, Camila; Currier, Rachel B; Salgado, David; Pla, Davinia; Sanz, Libia; Hyder, Asad S; Ribeiro, José M C; Arntzen, Jan W; van den Thillart, Guido E E J M; Boetzer, Marten; Pirovano, Walter; Dirks, Ron P; Spaink, Herman P; Duboule, Denis; McGlinn, Edwina; Kini, R Manjunatha; Richardson, Michael K

2013-12-17

Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection.

Crypteins derived from the mouse neuropeptide FF (NPFF)A precursor display NPFF-like effects in nociceptive tests in mice.

PubMed

Kotlinska, Jolanta H; Gibula-Bruzda, Ewa; Suder, Piotr; Wasielak, Magdalena; Bray, Lauriane; Raoof, Hana; Bodzon-Kulakowska, Anna; Silberring, Jerzy

2012-07-01

NPFF precursor, pro-NPFF(A) contains three known bioactive sequences: NPFF (FLFQPQRF-NH(2)), neuropeptide AF (NPAF; AGEGLSSPFWSLAAPQRF-NH(2)) and neuropeptide SF (NPSF; SLAAPQRF-NH(2)). The key-feature of these fragments is their common PQRF-amidated sequence at their C termini. Here, we evaluated the biological activity of two other sequences derived from the mouse NPFF(A) precursor, that does not have PQRF-amidated C-terminus. One peptide was residing between positions 85 and 99 in the mice pro-NPFF(A). This peptide was referred to as neuropeptide SA (NPSA; SAWGSWSKEQLNPQA), assigned due to its flanking amino acids. Another sequence used in the experiments was N-terminal fragment of NPSA, here referred to as neuropeptide SS (NPSS; SAWGSWS). These two peptides, classified as crypteins, were synthesized and tested in the hot-plate and tail immersion tests in mice for their pharmacological activity in morphine-induced antinociception. The effects of both crypteins were compared to NPFF. Our experiments indicated that both crypteins inhibited morphine antinociception and their effects were reversed by RF9, an antagonist of NPFF receptors. These data show that NPSA and NPSS possess NPFF-like anti-opioid activity in these behavioral tests. Copyright © 2012 Elsevier Inc. All rights reserved.

Ginsenoside 20(S)-Rg3 Inhibits the Warburg Effect Via Modulating DNMT3A/ MiR-532-3p/HK2 Pathway in Ovarian Cancer Cells.

PubMed

Zhou, Yuanyuan; Zheng, Xia; Lu, Jiaojiao; Chen, Wei; Li, Xu; Zhao, Le

2018-01-01

The Warburg effect is one of the main energy metabolism features supporting cancer cell growth. 20(S)-Rg3 exerts anti-tumor effect on ovarian cancer partly by inhibiting the Warburg effect. microRNAs are important regulators of the Warburg effect. However, the microRNA regulatory network mediating the anti-Warburg effect of 20(S)-Rg3 was largely unknown. microRNA deep sequencing was performed to identify the 20(S)-Rg3-influenced microRNAs in SKOV3 ovarian cancer cells. miR-532-3p was overexpressed by mimic532-3p transfection in SKOV3 and A2780 cells or inhibited by inhibitor532-3p transfection in 20(S)-Rg3-treated cells to examine the changes in HK2 and PKM2 expression, glucose consumption, lactate production and cell growth. Dual-luciferase reporter assay was conducted to verify the direct binding of miR-532-3p to HK2. The methylation status in the promoter region of pre-miR-532-3p gene was examined by methylation-specific PCR. Expression changes of key molecules controlling DNA methylation including DNMT1, DNMT3A, DNMT3B, and TET1-3 were examined in 20(S)-Rg3-treated cells. DNMT3A was overexpressed in 20(S)-Rg3-treated cells to examine its influence on miR-532-3p level, HK2 and PKM2 expression, glucose consumption and lactate production. Deep sequencing results showed that 11 microRNAs were increased and 9 microRNAs were decreased by 20(S)-Rg3 in SKOV3 cells, which were verified by qPCR. More than 2-fold increase of miR-532-3p was found in 20(S)-Rg3-treated SKOV3 cells. Forced expression of miR-532-3p reduced HK2 and PKM2 expression, glucose consumption and lactate production in SKOV3 and A2780 ovarian cancer cells. Inhibition of miR-532-3p antagonized the suppressive effect of 20(S)-Rg3 on HK2 and PKM2 expression, glucose consumption and lactate production in ovarian cancer cells. Dual-luciferase reporter assay showed that miR-532-3p directly suppressed HK2 rather than PKM2. miR-532-3p level was controlled by the methylation in the promoter region of its host gene. 20(S)-Rg3 inhibited DNMT3A expression while exerted insignificant effect on DNMT1, DNMT3B and TET1-3. 20(S)-Rg3 reversed DNMT3A-mediated methylation in the promoter of the host gene of miR-532-3p, and thus elevated miR-532-3p level followed by suppression of HK2 and PKM2 expression, glucose consumption and lactate production. 20(S)-Rg3 modulated microRNAs to exert the anti-tumor effect in ovarian cancer. 20(S)-Rg3 lessened the DNMT3A-mediated methylation and promoted the suppression of miR-532-3p on HK2 to antagonize the Warburg effect of ovarian cancer cells. © 2018 The Author(s). Published by S. Karger AG, Basel.

The Draft Genome and Transcriptome of Panagrellus redivivus Are Shaped by the Harsh Demands of a Free-Living Lifestyle

PubMed Central

Srinivasan, Jagan; Dillman, Adler R.; Macchietto, Marissa G.; Heikkinen, Liisa; Lakso, Merja; Fracchia, Kelley M.; Antoshechkin, Igor; Mortazavi, Ali; Wong, Garry; Sternberg, Paul W.

2013-01-01

Nematodes compose an abundant and diverse invertebrate phylum with members inhabiting nearly every ecological niche. Panagrellus redivivus (the “microworm”) is a free-living nematode frequently used to understand the evolution of developmental and behavioral processes given its phylogenetic distance to Caenorhabditis elegans. Here we report the de novo sequencing of the genome, transcriptome, and small RNAs of P. redivivus. Using a combination of automated gene finders and RNA-seq data, we predict 24,249 genes and 32,676 transcripts. Small RNA analysis revealed 248 microRNA (miRNA) hairpins, of which 63 had orthologs in other species. Fourteen miRNA clusters containing 42 miRNA precursors were found. The RNA interference, dauer development, and programmed cell death pathways are largely conserved. Analysis of protein family domain abundance revealed that P. redivivus has experienced a striking expansion of BTB domain-containing proteins and an unprecedented expansion of the cullin scaffold family of proteins involved in multi-subunit ubiquitin ligases, suggesting proteolytic plasticity and/or tighter regulation of protein turnover. The eukaryotic release factor protein family has also been dramatically expanded and suggests an ongoing evolutionary arms race with viruses and transposons. The P. redivivus genome provides a resource to advance our understanding of nematode evolution and biology and to further elucidate the genomic architecture leading to free-living lineages, taking advantage of the many fascinating features of this worm revealed by comparative studies. PMID:23410827

Identification and characterization of wheat long non-protein coding RNAs responsive to powdery mildew infection and heat stress by using microarray analysis and SBS sequencing

PubMed Central

2011-01-01

Background Biotic and abiotic stresses, such as powdery mildew infection and high temperature, are important limiting factors for yield and grain quality in wheat production. Emerging evidences suggest that long non-protein coding RNAs (npcRNAs) are developmentally regulated and play roles in development and stress responses of plants. However, identification of long npcRNAs is limited to a few plant species, such as Arabidopsis, rice and maize, no systematic identification of long npcRNAs and their responses to abiotic and biotic stresses is reported in wheat. Results In this study, by using computational analysis and experimental approach we identified 125 putative wheat stress responsive long npcRNAs, which are not conserved among plant species. Among them, some were precursors of small RNAs such as microRNAs and siRNAs, two long npcRNAs were identified as signal recognition particle (SRP) 7S RNA variants, and three were characterized as U3 snoRNAs. We found that wheat long npcRNAs showed tissue dependent expression patterns and were responsive to powdery mildew infection and heat stress. Conclusion Our results indicated that diverse sets of wheat long npcRNAs were responsive to powdery mildew infection and heat stress, and could function in wheat responses to both biotic and abiotic stresses, which provided a starting point to understand their functions and regulatory mechanisms in the future. PMID:21473757

Next-generation sequencing identifies the natural killer cell microRNA transcriptome

PubMed Central

Fehniger, Todd A.; Wylie, Todd; Germino, Elizabeth; Leong, Jeffrey W.; Magrini, Vincent J.; Koul, Sunita; Keppel, Catherine R.; Schneider, Stephanie E.; Koboldt, Daniel C.; Sullivan, Ryan P.; Heinz, Michael E.; Crosby, Seth D.; Nagarajan, Rakesh; Ramsingh, Giridharan; Link, Daniel C.; Ley, Timothy J.; Mardis, Elaine R.

2010-01-01

Natural killer (NK) cells are innate lymphocytes important for early host defense against infectious pathogens and surveillance against malignant transformation. Resting murine NK cells regulate the translation of effector molecule mRNAs (e.g., granzyme B, GzmB) through unclear molecular mechanisms. MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate the translation of their mRNA targets, and are therefore candidates for mediating this control process. While the expression and importance of miRNAs in T and B lymphocytes have been established, little is known about miRNAs in NK cells. Here, we used two next-generation sequencing (NGS) platforms to define the miRNA transcriptomes of resting and cytokine-activated primary murine NK cells, with confirmation by quantitative real-time PCR (qRT-PCR) and microarrays. We delineate a bioinformatics analysis pipeline that identified 302 known and 21 novel mature miRNAs from sequences obtained from NK cell small RNA libraries. These miRNAs are expressed over a broad range and exhibit isomiR complexity, and a subset is differentially expressed following cytokine activation. Using these miRNA NGS data, miR-223 was identified as a mature miRNA present in resting NK cells with decreased expression following cytokine activation. Furthermore, we demonstrate that miR-223 specifically targets the 3′ untranslated region of murine GzmB in vitro, indicating that this miRNA may contribute to control of GzmB translation in resting NK cells. Thus, the sequenced NK cell miRNA transcriptome provides a valuable framework for further elucidation of miRNA expression and function in NK cell biology. PMID:20935160

microRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3' untranslated region: function in replication and influence of RNA secondary structure.

PubMed

Gerresheim, Gesche K; Dünnes, Nadia; Nieder-Röhrmann, Anika; Shalamova, Lyudmila A; Fricke, Markus; Hofacker, Ivo; Höner Zu Siederdissen, Christian; Marz, Manja; Niepmann, Michael

2017-02-01

We have analyzed the binding of the liver-specific microRNA-122 (miR-122) to three conserved target sites of hepatitis C virus (HCV) RNA, two in the non-structural protein 5B (NS5B) coding region and one in the 3' untranslated region (3'UTR). miR-122 binding efficiency strongly depends on target site accessibility under conditions when the range of flanking sequences available for the formation of local RNA secondary structures changes. Our results indicate that the particular sequence feature that contributes most to the correlation between target site accessibility and binding strength varies between different target sites. This suggests that the dynamics of miRNA/Ago2 binding not only depends on the target site itself but also on flanking sequence context to a considerable extent, in particular in a small viral genome in which strong selection constraints act on coding sequence and overlapping cis-signals and model the accessibility of cis-signals. In full-length genomes, single and combination mutations in the miR-122 target sites reveal that site 5B.2 is positively involved in regulating overall genome replication efficiency, whereas mutation of site 5B.3 showed a weaker effect. Mutation of the 3'UTR site and double or triple mutants showed no significant overall effect on genome replication, whereas in a translation reporter RNA, the 3'UTR target site inhibits translation directed by the HCV 5'UTR. Thus, the miR-122 target sites in the 3'-region of the HCV genome are involved in a complex interplay in regulating different steps of the HCV replication cycle.

Characterization and Evolution of Conserved MicroRNA through Duplication Events in Date Palm (Phoenix dactylifera)

PubMed Central

Yang, Yaodong; Mason, Annaliese S.; Lei, Xintao; Ma, Zilong

2013-01-01

MicroRNAs (miRNAs) are important regulators of gene expression at the post-transcriptional level in a wide range of species. Highly conserved miRNAs regulate ancestral transcription factors common to all plants, and control important basic processes such as cell division and meristem function. We selected 21 conserved miRNA families to analyze the distribution and maintenance of miRNAs. Recently, the first genome sequence in Palmaceae was released: date palm (Phoenix dactylifera). We conducted a systematic miRNA analysis in date palm, computationally identifying and characterizing the distribution and duplication of conserved miRNAs in this species compared to other published plant genomes. A total of 81 miRNAs belonging to 18 miRNA families were identified in date palm. The majority of miRNAs in date palm and seven other well-studied plant species were located in intergenic regions and located 4 to 5 kb away from the nearest protein-coding genes. Sequence comparison showed that 67% of date palm miRNA members were present in duplicated segments, and that 135 pairs of miRNA-containing segments were duplicated in Arabidopsis, tomato, orange, rice, apple, poplar and soybean with a high similarity of non coding sequences between duplicated segments, indicating genomic duplication was a major force for expansion of conserved miRNAs. Duplicated miRNA pairs in date palm showed divergence in pre-miRNA sequence and in number of promoters, implying that these duplicated pairs may have undergone divergent evolution. Comparisons between date palm and the seven other plant species for the gain/loss of miR167 loci in an ancient segment shared between monocots and dicots suggested that these conserved miRNAs were highly influenced by and diverged as a result of genomic duplication events. PMID:23951162

Characterization and evolution of conserved MicroRNA through duplication events in date palm (Phoenix dactylifera).

PubMed

Xiao, Yong; Xia, Wei; Yang, Yaodong; Mason, Annaliese S; Lei, Xintao; Ma, Zilong

2013-01-01

MicroRNAs (miRNAs) are important regulators of gene expression at the post-transcriptional level in a wide range of species. Highly conserved miRNAs regulate ancestral transcription factors common to all plants, and control important basic processes such as cell division and meristem function. We selected 21 conserved miRNA families to analyze the distribution and maintenance of miRNAs. Recently, the first genome sequence in Palmaceae was released: date palm (Phoenix dactylifera). We conducted a systematic miRNA analysis in date palm, computationally identifying and characterizing the distribution and duplication of conserved miRNAs in this species compared to other published plant genomes. A total of 81 miRNAs belonging to 18 miRNA families were identified in date palm. The majority of miRNAs in date palm and seven other well-studied plant species were located in intergenic regions and located 4 to 5 kb away from the nearest protein-coding genes. Sequence comparison showed that 67% of date palm miRNA members were present in duplicated segments, and that 135 pairs of miRNA-containing segments were duplicated in Arabidopsis, tomato, orange, rice, apple, poplar and soybean with a high similarity of non coding sequences between duplicated segments, indicating genomic duplication was a major force for expansion of conserved miRNAs. Duplicated miRNA pairs in date palm showed divergence in pre-miRNA sequence and in number of promoters, implying that these duplicated pairs may have undergone divergent evolution. Comparisons between date palm and the seven other plant species for the gain/loss of miR167 loci in an ancient segment shared between monocots and dicots suggested that these conserved miRNAs were highly influenced by and diverged as a result of genomic duplication events.

High-Throughput Sequencing of microRNAs in Peripheral Blood Mononuclear Cells: Identification of Potential Weight Loss Biomarkers

PubMed Central

Milagro, Fermín I.; Miranda, Jonatan; Portillo, María P.; Fernandez-Quintela, Alfredo; Campión, Javier; Martínez, J. Alfredo

2013-01-01

Introduction MicroRNAs (miRNAs) are being increasingly studied in relation to energy metabolism and body composition homeostasis. Indeed, the quantitative analysis of miRNAs expression in different adiposity conditions may contribute to understand the intimate mechanisms participating in body weight control and to find new biomarkers with diagnostic or prognostic value in obesity management. Objective The aim of this study was the search for miRNAs in blood cells whose expression could be used as prognostic biomarkers of weight loss. Methods Ten Caucasian obese women were selected among the participants in a weight-loss trial that consisted in following an energy-restricted treatment. Weight loss was considered unsuccessful when <5% of initial body weight (non-responders) and successful when >5% (responders). At baseline, total miRNA isolated from peripheral blood mononuclear cells (PBMC) was sequenced with SOLiD v4. The miRNA sequencing data were validated by RT-PCR. Results Differential baseline expression of several miRNAs was found between responders and non-responders. Two miRNAs were up-regulated in the non-responder group (mir-935 and mir-4772) and three others were down-regulated (mir-223, mir-224 and mir-376b). Both mir-935 and mir-4772 showed relevant associations with the magnitude of weight loss, although the expression of other transcripts (mir-874, mir-199b, mir-766, mir-589 and mir-148b) also correlated with weight loss. Conclusions This research addresses the use of high-throughput sequencing technologies in the search for miRNA expression biomarkers in obesity, by determining the miRNA transcriptome of PBMC. Basal expression of different miRNAs, particularly mir-935 and mir-4772, could be prognostic biomarkers and may forecast the response to a hypocaloric diet. PMID:23335998

Decoy Oligonucleotide Rescues IGF1R Expression from MicroRNA-223 Suppression

PubMed Central

Wang, Rong; He, Bao Mei; Qi, Bing; Xu, Chang Jun; Wu, Xing Zhong

2013-01-01

A mature miRNA generally suppresses hundreds of mRNA targets. To evaluate the selective effect of synthetic oligonucleotide decoys on hsa-miR-223 activity, reporters containing 3’ untranslated regions (UTR) of IGF1R, FOXO1, POLR3G, FOXO3, CDC27, FBXW7 and PAXIP1 mRNAs were constructed for the luciferase assay. The oligonucleotide decoys were designed and synthesized according to mature miR-223 sequence and its target mRNA sequence. Quantitative RT-PCR & western analysis were used to measure miR-223-targeted mRNA expression, Interestingly, apart from the antisense oligonucleotide, decoy nucleotides which were complementary to the 5’, central or 3’ region of mature miR-223 suppressed miR-223 targeting the 3’UTR of IGF1R, FOXO1, FOXO3, CDC27, POLR3G, and FBXW7 mRNAs and rescued the expression of these genes to varying degrees from miR-223 suppression at both mRNA and protein levels. All decoys had no effect on PAXIP1 which was not targeted by miR-223. The decoy 1 that was based on the sequence of IGF1R 3’UTR rescued the expression of IGF1R more significantly than other decoy nucleotides except the antisense decoy 4. Decoy 1 also rescued the expression of FOXO3 and POLR3G of which their 3’UTRs have similar binding sites for miR-223 with IGF1R 3’UTR. However decoy 1 failed to recover Sp1, CDC27 and FBXW7 expression. These data support that the sequence-specific decoy oligonucleotides might represent exogenous competing RNA which selectively inhibits microRNA targeting. PMID:24324762

Decoy oligonucleotide rescues IGF1R expression from MicroRNA-223 suppression.

PubMed

Wu, Li Hui; Cai, Qian Qian; Dong, Yi Wei; Wang, Rong; He, Bao Mei; Qi, Bing; Xu, Chang Jun; Wu, Xing Zhong

2013-01-01

A mature miRNA generally suppresses hundreds of mRNA targets. To evaluate the selective effect of synthetic oligonucleotide decoys on hsa-miR-223 activity, reporters containing 3' untranslated regions (UTR) of IGF1R, FOXO1, POLR3G, FOXO3, CDC27, FBXW7 and PAXIP1 mRNAs were constructed for the luciferase assay. The oligonucleotide decoys were designed and synthesized according to mature miR-223 sequence and its target mRNA sequence. Quantitative RT-PCR & western analysis were used to measure miR-223-targeted mRNA expression, Interestingly, apart from the antisense oligonucleotide, decoy nucleotides which were complementary to the 5', central or 3' region of mature miR-223 suppressed miR-223 targeting the 3'UTR of IGF1R, FOXO1, FOXO3, CDC27, POLR3G, and FBXW7 mRNAs and rescued the expression of these genes to varying degrees from miR-223 suppression at both mRNA and protein levels. All decoys had no effect on PAXIP1 which was not targeted by miR-223. The decoy 1 that was based on the sequence of IGF1R 3'UTR rescued the expression of IGF1R more significantly than other decoy nucleotides except the antisense decoy 4. Decoy 1 also rescued the expression of FOXO3 and POLR3G of which their 3'UTRs have similar binding sites for miR-223 with IGF1R 3'UTR. However decoy 1 failed to recover Sp1, CDC27 and FBXW7 expression. These data support that the sequence-specific decoy oligonucleotides might represent exogenous competing RNA which selectively inhibits microRNA targeting.

Comparison analysis of microRNAs in response to dengue virus type 2 infection between the Vero cell-adapted strain and its source, the clinical C6/36 isolated strain.

PubMed

Yang, Jiajia; Lin, Yao; Jiang, Liming; Xi, Juemin; Wang, Xiaodan; Guan, Jiaoqiong; Chen, Junying; Pan, Yue; Luo, Jia; Ye, Chao; Sun, Qiangming

2018-05-02

To elucidate the differences in microRNAs during dengue virus infection between Vero cell-adapted strain (DENV-2-Vero) and its source, the clinical C6/36 isolated strain (DENV-2-C6/36), a comparison analysis was performed in Vero cells by high throughput sequencing. The results showed that the expression of 16 known and 3 novel miRNAs exhibited marked differences. 5 known miRNAs were up-regulated in DENV-2-C6/36 group, while 11 known microRNAs were down-regulated in DENV-2-Vero group. The GO enrichment and KEGG pathway analysis showed that there was a distinct difference in regulating viral replication between two strains. In DENV-2-Vero infection group, significantly enriched GO terms included virion attachment to host cells, viral structural protein/genome processing and packaging. Meanwhile, the regulation of cell death and apoptosis between two groups were different in the early stage of infection. KEGG enrichment analysis showed that DENV-2-C6/36 infection induced more intense regulation of immune-related pathways, including Fc gamma R-mediated phagocytosis, etc. DENV-2-Vero infection could partially alleviate the immune defense of Vero cells compared with DENV-2-C6/36. The results indicated that the distinct microRNA changes induced by two DENV-2 strains may be partly related to their infective abilities. Our data provide useful insights that help elucidate the host-pathogen interactions following DENV infection. Copyright © 2018 Elsevier B.V. All rights reserved.

Molecular aspects of diabetes mellitus: Resistin, microRNA, and exosome.

PubMed

Saeedi Borujeni, Mohammad Javad; Esfandiary, Ebrahim; Taheripak, Gholamreza; Codoñer-Franch, Pilar; Alonso-Iglesias, Eulalia; Mirzaei, Hamed

2018-02-01

Diabetes mellitus (DM) is known as one of important common endocrine disorders which could due to deregulation of a variety of cellular and molecular pathways. A large numbers studies indicated that various pathogenesis events including mutation, serin phosphorylation, and increasing/decreasing expression of many genes could contribute to initiation and progression of DM. Insulin resistance is one of important factors which could play critical roles in DM pathogenesis. It has been showed that insulin resistance via targeting a sequence of cellular and molecular pathways (eg, PI3 kinases, PPARγ co-activator-1, microRNAs, serine/threonine kinase Akt, and serin phosphorylation) could induce DM. Among of various factors involved in DM pathogenesis, microRNAs, and exosomes have been emerged as effective factors in initiation and progression of DM. A variety of studies indicated that deregulation of these molecules could change behavior of various types of cells and contribute to progression of DM. Resistin is other main factor which is known as signal molecule involved in insulin resistance. Multiple lines evidence indicated that resistin exerts its effects via affecting on glucose metabolism, inhibition of fatty acid uptake and metabolism with affecting on a variety of targets such as CD36, fatty acid transport protein 1, Acetyl-CoA carboxylase, and AMP-activated protein kinase. Here, we summarized various molecular aspects are associated with DM particularly the molecular pathways involved in insulin resistance and resistin in DM. Moreover, we highlighted exosomes and microRNAs as effective players in initiation and progression of DM. © 2017 Wiley Periodicals, Inc.

Genome-wide profiling of microRNAs reveals novel insights into the interactions between H9N2 avian influenza virus and avian dendritic cells.

PubMed

Lin, Jian; Xia, Jing; Zhang, Tian; Zhang, Keyun; Yang, Qian

2018-05-10

The antigen-presenting ability of dendritic cells (DCs) plays an important and irreplaceable role in recognising and clearing viruses. Antiviral responses must rapidly defend against infection while minimising inflammatory damage, but the mechanisms that regulate the magnitude of response within an infected cell are not well understood. MicroRNAs (microRNAs), small non-coding RNAs, can regulate mouse or avian DCs to inhibit the infection and replication of avian influenza virus (AIV). Here, we performed a global analysis to understand how avian DCs respond to H9N2 AIV and provide a potential mechanism to explain how avian microRNAs can defend against H9N2 AIV replication. First, we found that both active and inactive H9N2 AIV enhanced the ability of DCs to present antigens and activate T lymphocytes. Next, total microarray analyses suggested that H9N2 AIV stimulation involved protein localisation, nucleotide binding, leucocyte transendothelial migration and MAPK signalling. Moreover, we constructed 551 transcription factor (TF)-miRNA-mRNA loops based on the above analyses. Furthermore, we found that the haemagglutinin (HA) fragment, neither H5N1-HA or H9N2-HA, could not activate DCs, while truncated HA greatly increased the immune function of DCs by activating ERK and STAT3 signalling pathways. Lastly, our results not only suggested that gga-miR1644 targets muscleblind-like protein 2 (MBNL2) to enhance the ability of avian DCs to inhibit virus replication, but also suggested that gga-miR6675 targets the nuclear localisation sequence of polymerase basic protein 1 (PB1) to trigger the silencing of PB1 genes, resulting in the inhibition of H9N2 AIV replication. Altogether, our innovative study will shed new light on the role of avian microRNAs in evoking avian DCs and inhibiting virus replication.

Let-7 miRNA Precursors Co-express with LIN28B in Cervical Cells.

PubMed

Zamora-Contreras, Aida Margarita; Alvarez-Salas, Luis Marat

2018-01-01

The let-7 microRNAs (miRNAs) are frequently dysregulated in carcinogenic processes, including cervical cancer. LIN28 proteins regulate let-7 biogenesis by binding to conserved sequences within the pre-miRNA structure. Nevertheless, recent research has shown that some let-7 miRNAs may escape LIN28 regulation. Correlate pre-let-7 miRNAs and LIN28B levels in cervical cell lines with different malignancy and HPV content. Pre-let-7 levels were determined by RTqPCR. LIN28B and other let-7 targets were analyzed by immunoblot. In silico tools were used to correlate let-7 and LIN28B expression and to analyze prelet- 7 sequences and structures. Lin28B protein was detected in all tested cell lines although it was more expressed in tumor cell lines. High levels of pre-let-7c/f-1 and pre-miR-98 were present in almost all cell lines regardless malignancy and LIN28B expression. Pre-let-7g/i were mainly expressed in tumor cell lines, pre-let-7e and pre-let-7-a3 were absent in all cell lines and pre-let-7a-2 showed indistinct expression. LIN28B showed positive correlation with pre-let-7i/g/f-1 and pre-miR-98 in tumor cell lines, suggesting escape from regulation. Sequence alignment and analysis of pre-let-7 miRNAs showed distinctive structural features within the preE region that may influence the ideal pre-let-7 structuring for LIN28B interaction. Short preE-stems were present in pre-let-7 that may escape LIN28B regulation, but long preEstems were mostly associated with high-level pre-let-7 miRNAs. The observed differences of pre-let-7 levels in cervical cell lines may be the result of alternative preE structuring affecting interaction with LIN28B thus resulting in differential let-7 regulation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Evolution of Fish Let-7 MicroRNAs and Their Expression Correlated to Growth Development in Blunt Snout Bream

PubMed Central

Zhao, Bo-Wen; Zhou, Lai-Fang; Liu, Yu-Long; Wan, Shi-Ming; Gao, Ze-Xia

2017-01-01

The lethal-7 (let-7) miRNA, known as one of the first founding miRNAs, is present in multiple copies in a genome and has diverse functions in animals. In this study, comparative genomic analysis of let-7 miRNAs members in fish species indicated that let-7 miRNA is a sequence conserved family in fish, while different species have the variable gene copy numbers. Among the ten members including let-7a/b/c/d/e/f/g/h/i/j, the let-7a precursor sequence was more similar to ancestral sequences, whereas other let-7 miRNA members were separate from the late differentiation of let-7a. The mostly predicted target genes of let-7 miRNAs are involved in biological process, especially developmental process and growth through Gene Ontology (GO) enrichment analysis. In order to identify the possible different functions of these ten miRNAs in fish growth development, their expression levels were quantified in adult males and females of Megalobrama amblycephala, as well as in 3-, 6-, and 12-months-old individuals with relatively slow- and fast-growth rates. These ten miRNAs had similar tissue expression patterns between males and females, with higher expression levels in the brain and pituitary than that in other tissues (p < 0.05). Among these miRNAs, the relative expression level of let-7a was the highest among almost all the tested tissues, followed by let-7b, let-7d and let-7c/e/f/g/h/i/j. As to the groups with different growth rates, the expression levels of let-7 miRNAs in pituitary and brain from the slow-growth group were always significantly higher than that in the fast-growth group (p < 0.05). These results suggest that let-7 miRNA members could play an important role in the regulation of growth development in M. amblycephala through negatively regulating expression of their target genes. PMID:28300776

BIOPEP database and other programs for processing bioactive peptide sequences.

PubMed

Minkiewicz, Piotr; Dziuba, Jerzy; Iwaniak, Anna; Dziuba, Marta; Darewicz, Małgorzata

2008-01-01

This review presents the potential for application of computational tools in peptide science based on a sample BIOPEP database and program as well as other programs and databases available via the World Wide Web. The BIOPEP application contains a database of biologically active peptide sequences and a program enabling construction of profiles of the potential biological activity of protein fragments, calculation of quantitative descriptors as measures of the value of proteins as potential precursors of bioactive peptides, and prediction of bonds susceptible to hydrolysis by endopeptidases in a protein chain. Other bioactive and allergenic peptide sequence databases are also presented. Programs enabling the construction of binary and multiple alignments between peptide sequences, the construction of sequence motifs attributed to a given type of bioactivity, searching for potential precursors of bioactive peptides, and the prediction of sites susceptible to proteolytic cleavage in protein chains are available via the Internet as are other approaches concerning secondary structure prediction and calculation of physicochemical features based on amino acid sequence. Programs for prediction of allergenic and toxic properties have also been developed. This review explores the possibilities of cooperation between various programs.

The involvement of InMIR167 in the regulation of expression of its target gene InARF8, and their participation in the vegetative and generative development of Ipomoea nil plants.

PubMed

Glazińska, Paulina; Wojciechowski, Waldemar; Wilmowicz, Emilia; Zienkiewicz, Agnieszka; Frankowski, Kamil; Kopcewicz, Jan

2014-02-15

The plant hormone auxin plays a critical role in regulating plant growth and development. Recent advances have been made that having improved our understanding of auxin response pathways, primarily by characterizing the genes encoding auxin response factors (ARFs) in Arabidopsis. In addition, the expression of some ARFs is regulated by microRNAs (miRNAs). In Arabidopsis thaliana, ARF6 and ARF8 are targeted by miR167, whereas ARF10, ARF16 and ARF17 are targeted by miR160. Nevertheless, little is known about any possible interactions between miRNAs and the auxin signaling pathway during plant development. In this study, we isolated the miR167 target gene InARF8 cDNA from the cotyledons of the short day plant (SDP) Ipomoea nil (named also Pharbitis nil). Additionally, the In-miR167 precursor was identified from the I. nil EST database and analyses of InARF8 mRNA, In-pre-miR167 and mature miR167 accumulation in the plant's vegetative and generative organs were performed. The identified cDNA of InARF8 contains a miR167 complementary sequence and shows significant similarity to ARF8 cDNAs of other plant species. The predicted amino acid sequence of InARF8 includes all of the characteristic domains for ARF family transcription factors (B3 DNA-binding domain, AUX/IAA-CTD and a glutamine-rich region). Quantitative RT-PCR reactions and in situ hybridization indicated that InARF8 was expressed primarily in the shoot apices, leaf primordia and hypocotyls of I. nil seedlings, as well as in flower pistils and petals. The InARF8 transcript level increased consistently during the entire period of pistil development, whereas in the stamens, the greatest transcriptional activity occurred only during the intensive elongation phase. Additionally, an expression analysis of both the precursor In-pre-miR167 molecules identified and mature miRNA was performed. We observed that, in most of the organs examined, the InARF8 expression pattern was opposite to that of MIR167, indicating that the gene's activity was regulated by mRNA cleavage. Our findings suggested that InARF8 and InMIR167 participated in the development of young tissues, especially the shoot apices and flower elements. The main function of MIR167 appears to be to regulate InARF8 organ localization. Copyright © 2013 Elsevier GmbH. All rights reserved.

[The role of microRNAs in molecular pathology of esophageal cancer and their potential usage in clinical oncology].

PubMed

Kovaříková, A; Héžová, R; Srovnal, J; Rédová-Lojová, M; Slabý, O

2014-01-01

MicroRNAs are an abundant class of noncoding RNAs (approx. 18- 25 nucleotides in length) that suppress translation through binding to their target mRNAs, eventually leading to mRNAs degradation. Sequences of these endogenous RNA molecules are highly conserved, even among unrelated species, indicating their involvement in basic bio-logical processes, such as development, differentiation, proliferation or apoptosis. MiRNAs also participate on regulation of cancer stem cell functioning, immune system and malignant transformation. This review provides a comprehensive overview of miRNAs functions in esophageal cancer, their roles in key pathogenetic pathways and disease development, as well as their potential usage in clinical routine as bio-markers improving dia-gnosis, prognosis and prediction of therapeutic response. Through regulation of signaling pathways important in malignant transformation, miRNAs present also promising therapeutic targets.

MicroRNAs and atherosclerosis: new actors for an old movie.

PubMed

Santovito, D; Mezzetti, A; Cipollone, F

2012-11-01

To date, cardiovascular diseases (CVDs) are the leading causes of morbidity and mortality worldwide. MicroRNAs (miRNAs) are endogenous, short, non-coding RNA sequences able to regulate gene expression principally at the post-transcriptional level. Initially, they were thought to be involved only in developmental timing of worms. Their involvement in human biology was recently discovered and many studies have been performed to demonstrate the role of miRNA in human cancer. Since the first observation in 2005 of their implication in cardiac biology, many studies have demonstrated their role in the genetic modulation of cardiovascular development and in cardiovascular diseases such as cardial remodeling and heart failure, cardiac arrhythmias, cardiac ischaemia, cardiac fibrosis, atherosclerosis and stroke. Thus, the aim of this review is to describe the role of miRNA in atherosclerosis development and evolution and to individuate their role as potential therapeutic target. Copyright © 2012 Elsevier B.V. All rights reserved.

Orthologous plant microRNAs: microregulators with great potential for improving stress tolerance in plants.

PubMed

Rajwanshi, Ravi; Chakraborty, Sreejita; Jayanandi, Karam; Deb, Bibhas; Lightfoot, David A

2014-12-01

Small RNAs that are highly conserved across many plant species are involved in stress responses. Plants are exposed to many types of unfavorable conditions during their life cycle that result in some degree of stress. Recent studies on microRNAs (miRNAs) have highlighted their great potential as regulators of stress tolerance in plants. One of the possible ways in which plants counter environmental stresses is by altering their gene expression by the action of miRNAs. miRNAs regulate the expression of target genes by hybridizing to their nascent reverse complementary sequences marking them for cleavage in the nucleus or translational repression in the cytoplasm. Some miRNAs have been reported to be key regulators in biotic as well as abiotic stress responses across many species. The present review highlights some of the regulatory roles of orthologous plant miRNAs in response to various types of stress conditions.

Functional characterization of an alpha-factor-like Sordaria macrospora peptide pheromone and analysis of its interaction with its cognate receptor in Saccharomyces cerevisiae.

PubMed

Mayrhofer, Severine; Pöggeler, Stefanie

2005-04-01

The homothallic filamentous ascomycete Sordaria macrospora possesses genes which are thought to encode two pheromone precursors and two seven-transmembrane pheromone receptors. The pheromone precursor genes are termed ppg1 and ppg2. The putative products derived from the gene sequence show structural similarity to the alpha-factor precursors and a-factor precursors of the yeast Saccharomyces cerevisiae. Likewise, sequence similarity has been found between the putative products of the pheromone receptor genes pre2 and pre1 and the S. cerevisiae Ste2p alpha-factor receptor and Ste3p a-factor receptor, respectively. To investigate whether the alpha-factor-like pheromone-receptor pair of S. macrospora is functional, a heterologous yeast assay was used. Our results show that the S. macrospora alpha-factor-like pheromone precursor PPG1 is processed into an active pheromone by yeast MATalpha cells. The S. macrospora PRE2 protein was demonstrated to be a peptide pheromone receptor. In yeast MATa cells lacking the endogenous Ste2p receptor, the S. macrospora PRE2 receptor facilitated all aspects of the pheromone response. Using a synthetic peptide, we can now predict the sequence of one active form of the S. macrospora peptide pheromone. We proved that S. macrospora wild-type strains secrete an active pheromone into the culture medium and that disruption of the ppg1 gene in S. macrospora prevents pheromone production. However, loss of the ppg1 gene does not affect vegetative growth or fertility. Finally, we established the yeast assay as an easy and useful system for analyzing pheromone production in developmental mutants of S. macrospora.

Highly sensitive dual mode electrochemical platform for microRNA detection

NASA Astrophysics Data System (ADS)

Jolly, Pawan; Batistuti, Marina R.; Miodek, Anna; Zhurauski, Pavel; Mulato, Marcelo; Lindsay, Mark A.; Estrela, Pedro

2016-11-01

MicroRNAs (miRNAs) play crucial regulatory roles in various human diseases including cancer, making them promising biomarkers. However, given the low levels of miRNAs present in blood, their use as cancer biomarkers requires the development of simple and effective analytical methods. Herein, we report the development of a highly sensitive dual mode electrochemical platform for the detection of microRNAs. The platform was developed using peptide nucleic acids as probes on gold electrode surfaces to capture target miRNAs. A simple amplification strategy using gold nanoparticles has been employed exploiting the inherent charges of the nucleic acids. Electrochemical impedance spectroscopy was used to monitor the changes in capacitance upon any binding event, without the need for any redox markers. By using thiolated ferrocene, a complementary detection mode on the same sensor was developed where the increasing peaks of ferrocene were recorded using square wave voltammetry with increasing miRNA concentration. This dual-mode approach allows detection of miRNA with a limit of detection of 0.37 fM and a wide dynamic range from 1 fM to 100 nM along with clear distinction from mismatched target miRNA sequences. The electrochemical platform developed can be easily expanded to other miRNA/DNA detection along with the development of microarray platforms.

Different processing of CAPA and pyrokinin precursors in the giant mealworm beetle Zophobas atratus (Tenebrionidae) and the boll weevil Anthonomus grandis grandis (Curculionidae).

PubMed

Neupert, Susanne; Marciniak, Pawel; Köhler, Rene; Nachman, Ronald J; Suh, Charles P-C; Predel, Reinhard

2018-03-01

Capa and pyrokinin (pk) genes in hexapods share a common evolutionary origin. Using transcriptomics and peptidomics, we analyzed products of these genes in two beetles, the giant mealworm beetle (Zophobas atratus; Tenebrionidae) and the boll weevil (Anthonomus grandis grandis; Curculionidae). Our data revealed that even within Coleoptera, which represents a very well-defined group of insects, highly different evolutionary developments occurred in the neuropeptidergic system. These differences, however, primarily affect the general structure of the precursors and differential processing of mature peptides and, to a lesser degree, the sequences of the active core motifs. With the differential processing of the CAPA-precursor in Z. atratus we found a perfect example of completely different products cleaved from a single neuropeptide precursor in different cells. The CAPA precursor in abdominal ganglia of this species yields primarily periviscerokinins (PVKs) whereas processing of the same precursor in neurosecretory cells of the subesophageal ganglion results in CAPA-tryptoPK and a novel CAPA-PK. Particularly important was the detection of that CAPA-PK which has never been observed in the CNS of insects before. The three different types of CAPA peptides (CAPA-tryptoPK, CAPA-PK, PVK) each represent potential ligands which activate different receptors. In contrast to the processing of the CAPA precursor from Z. atratus, no indications of a differential processing of the CAPA precursor were found in A. g. grandis. These data suggest that rapid evolutionary changes regarding the processing of CAPA precursors were still going on when the different beetle lineages diverged. The sequence of the single known PVK of A. g. grandis occupies a special position within the known PVKs of insects and might serve asa basis to develop lineage-specific peptidomimetics capable of disrupting physiological processes regulated by PVKs. Copyright © 2017 Elsevier Inc. All rights reserved.

MicroRNA-281 regulates the expression of ecdysone receptor (EcR) isoform B in the silkworm, Bombyx mori

USDA-ARS?s Scientific Manuscript database

Hundreds of Bombyx mori miRNAs had been identified in recent years, but their function in vivo remains poorly understood. The silkworm EcR gene (BmEcR) has three transcriptional isoforms, A, B1 and B2. Isoform sequences are different in the 3’UTR region of the gene, which is the case only in insects...

Novel determinants of mammalian primary microRNA processing revealed by systematic evaluation of hairpin-containing transcripts and human genetic variation

PubMed Central

Roden, Christine; Gaillard, Jonathan; Kanoria, Shaveta; Rennie, William; Barish, Syndi; Cheng, Jijun; Pan, Wen; Liu, Jun; Cotsapas, Chris; Ding, Ye; Lu, Jun

2017-01-01

Mature microRNAs (miRNAs) are processed from hairpin-containing primary miRNAs (pri-miRNAs). However, rules that distinguish pri-miRNAs from other hairpin-containing transcripts in the genome are incompletely understood. By developing a computational pipeline to systematically evaluate 30 structural and sequence features of mammalian RNA hairpins, we report several new rules that are preferentially utilized in miRNA hairpins and govern efficient pri-miRNA processing. We propose that a hairpin stem length of 36 ± 3 nt is optimal for pri-miRNA processing. We identify two bulge-depleted regions on the miRNA stem, located ∼16–21 nt and ∼28–32 nt from the base of the stem, that are less tolerant of unpaired bases. We further show that the CNNC primary sequence motif selectively enhances the processing of optimal-length hairpins. We predict that a small but significant fraction of human single-nucleotide polymorphisms (SNPs) alter pri-miRNA processing, and confirm several predictions experimentally including a disease-causing mutation. Our study enhances the rules governing mammalian pri-miRNA processing and suggests a diverse impact of human genetic variation on miRNA biogenesis. PMID:28087842

Small RNA Profiling of Two Important Cultivars of Banana and Overexpression of miRNA156 in Transgenic Banana Plants

PubMed Central

Ganapathi, Thumballi R.

2015-01-01

Micro RNAs (miRNAs) are a class of non-coding, short RNAs having important roles in regulation of gene expression. Although plant miRNAs have been studied in detail in some model plants, less is known about these miRNAs in important fruit plants like banana. miRNAs have pivotal roles in plant growth and development, and in responses to diverse biotic and abiotic stress stimuli. Here, we have analyzed the small RNA expression profiles of two different economically significant banana cultivars by using high-throughput sequencing technology. We identified a total of 170 and 244 miRNAs in the two libraries respectively derived from cv. Grand Naine and cv. Rasthali leaves. In addition, several cultivar specific microRNAs along with their putative target transcripts were also detected in our studies. To validate our findings regarding the small RNA profiles, we also undertook overexpression of a common microRNA, MusamiRNA156 in transgenic banana plants. The transgenic plants overexpressing the stem-loop sequence derived from MusamiRNA156 gene were stunted in their growth together with peculiar changes in leaf anatomy. These results provide a foundation for further investigations into important physiological and metabolic pathways operational in banana in general and cultivar specific traits in particular. PMID:25962076

Role of bioinformatics in establishing microRNAs as modulators of abiotic stress responses: the new revolution

PubMed Central

Tripathi, Anita; Goswami, Kavita; Sanan-Mishra, Neeti

2015-01-01

microRNAs (miRs) are a class of 21–24 nucleotide long non-coding RNAs responsible for regulating the expression of associated genes mainly by cleavage or translational inhibition of the target transcripts. With this characteristic of silencing, miRs act as an important component in regulation of plant responses in various stress conditions. In recent years, with drastic change in environmental and soil conditions different type of stresses have emerged as a major challenge for plants growth and productivity. The identification and profiling of miRs has itself been a challenge for research workers given their small size and large number of many probable sequences in the genome. Application of computational approaches has expedited the process of identification of miRs and their expression profiling in different conditions. The development of High-Throughput Sequencing (HTS) techniques has facilitated to gain access to the global profiles of the miRs for understanding their mode of action in plants. Introduction of various bioinformatics databases and tools have revolutionized the study of miRs and other small RNAs. This review focuses the role of bioinformatics approaches in the identification and study of the regulatory roles of plant miRs in the adaptive response to stresses. PMID:26578966

Deep sequencing of the small RNA transcriptome of normal and malignant human B cells identifies hundreds of novel microRNAs

PubMed Central

Jima, Dereje D.; Zhang, Jenny; Jacobs, Cassandra; Richards, Kristy L.; Dunphy, Cherie H.; Choi, William W. L.; Yan Au, Wing; Srivastava, Gopesh; Czader, Magdalena B.; Rizzieri, David A.; Lagoo, Anand S.; Lugar, Patricia L.; Mann, Karen P.; Flowers, Christopher R.; Bernal-Mizrachi, Leon; Naresh, Kikkeri N.; Evens, Andrew M.; Gordon, Leo I.; Luftig, Micah; Friedman, Daphne R.; Weinberg, J. Brice; Thompson, Michael A.; Gill, Javed I.; Liu, Qingquan; How, Tam; Grubor, Vladimir; Gao, Yuan; Patel, Amee; Wu, Han; Zhu, Jun; Blobe, Gerard C.; Lipsky, Peter E.; Chadburn, Amy

2010-01-01

A role for microRNA (miRNA) has been recognized in nearly every biologic system examined thus far. A complete delineation of their role must be preceded by the identification of all miRNAs present in any system. We elucidated the complete small RNA transcriptome of normal and malignant B cells through deep sequencing of 31 normal and malignant human B-cell samples that comprise the spectrum of B-cell differentiation and common malignant phenotypes. We identified the expression of 333 known miRNAs, which is more than twice the number previously recognized in any tissue type. We further identified the expression of 286 candidate novel miRNAs in normal and malignant B cells. These miRNAs were validated at a high rate (92%) using quantitative polymerase chain reaction, and we demonstrated their application in the distinction of clinically relevant subgroups of lymphoma. We further demonstrated that a novel miRNA cluster, previously annotated as a hypothetical gene LOC100130622, contains 6 novel miRNAs that regulate the transforming growth factor-β pathway. Thus, our work suggests that more than a third of the miRNAs present in most cellular types are currently unknown and that these miRNAs may regulate important cellular functions. PMID:20733160

Bioinformatic Identification of Potential MicroRNAs and Their Targets in the Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Higher Basidiomycetes).

PubMed

Mu, Da-Shuai; Li, Chenyang; Shi, Liang; Zhang, Xuchen; Ren, Ang; Zhao, Ming-Wen

2015-01-01

MicroRNAs (miRNAs) are a class of small, endogenous, noncoding RNA molecules that negatively regulate gene expression at the transcriptional or the post-transcriptional level. Although a large number of miRNAs have been identified in many species, especially model plants and animals, miRNAs in fungi remain largely unknown. In this study, based on a database of expressed sequence tags in Ganoderma lucidum, 89 potential miRNAs were identified using computational methods. Real-time polymerase chain reaction analysis of miRNA-like samples prepared from G. lucidum at different development stages revealed that miRNA-like RNAs were differentially expressed in different stages. Furthermore, a total of 28 potential targets were found based on near-perfect or perfect complementarity between the randomly selected 9 miRNA-like RNAs and the target sequences, and potential targets for G. lucidum miRNA-like RNAs were predicted. Finally, we studied the expression pattern of 4 target genes in 3 different development stages of G. lucidum to further understand the mechanism of interaction between miRNA-like RNAs and their target genes. Our analysis paves the way toward identifying fungal miRNA-like RNAs that might be involved in various physiological and cellular differentiation processes.

Characterization of microRNAs of Beta macrocarpa and their responses to Beet necrotic yellow vein virus infection.

PubMed

Liu, Jun-Ying; Fan, Hui-Yan; Wang, Ying; Zhang, Yong-Liang; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

2017-01-01

Plant microRNAs (miRNAs) are a class of non-coding RNAs that play important roles in plant development, defense, and symptom development. Here, 547 known miRNAs representing 129 miRNA families, and 282 potential novel miRNAs were identified in Beta macrocarpa using small RNA deep sequencing. A phylogenetic analysis was performed, and 8 Beta lineage-specific miRNAs were identified. Through a differential expression analysis, miRNAs associated with Beet necrotic yellow vein virus (BNYVV) infection were identified and confirmed using a microarray analysis and stem-loop RT-qPCR. In total, 103 known miRNAs representing 38 miRNA families, and 45 potential novel miRNAs were differentially regulated, with at least a two-fold change, in BNYVV-infected plants compared with that of the mock-inoculated control. Targets of these differentially expressed miRNAs were also predicted by degradome sequencing. These differentially expressed miRNAs were involved in hormone biosynthesis and signal transduction pathways, and enhanced axillary bud development and plant defenses. This work is the first to describe miRNAs of the plant genus Beta and may offer a reference for miRNA research in other species in the genus. It provides valuable information on the pathogenicity mechanisms of BNYVV.