Comparative RNA-Seq analysis reveals pervasive tissue-specific alternative polyadenylation in Caenorhabditis elegans intestine and muscles

Blazie, Stephen M; Babb, Cody S.; Wilky, Henry; Rawls, Alan; Park, Jin G.; Mangone, Marco

doi:10.1186/s12915-015-0116-6

Cited by 75 publications

(163 citation statements)

References 82 publications

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“…( C ) The expression levels of the muscle, intestine and hypodermis marker genes detected in various muscle transcriptomes. modENCODE: mean level of whole body expression across 19 stages and conditions; mixed tissues: SRT-based expression profile derived from PU2-3::tag::SL1(G16C) transgenic worms; PAB-RNA-IP: RNA-seq data derived from PAB-mediated RNA-IP (44). According to WormBase, some intestine or hypodermis marker genes were also described as muscle, expressed by fluorescent reporter (**) or other muscle-specific high-throughput profile (*) (13).…”

Section: Resultsmentioning

confidence: 99%

Analysis ofC. elegansmuscle transcriptome using trans-splicing-based RNA tagging (SRT)

Zhan

Sleumer

et al. 2016

Nucleic Acids Res

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Current approaches to profiling tissue-specific gene expression in C. elegans require delicate manipulation and are difficult under certain conditions, e.g. from dauer or aging worms. We have developed an easy and robust method for tissue-specific RNA-seq by taking advantage of the endogenous trans-splicing process. In this method, transgenic worms are generated in which a spliced leader (SL) RNA gene is fused with a sequence tag and driven by a tissue-specific promoter. Only in the tissue of interest, the tagged SL RNA gene is transcribed and then trans-spliced onto mRNAs. The tag allows enrichment and sequencing of mRNAs from that tissue only. As a proof of principle, we profiled the muscle transcriptome, which showed high coverage and efficient enrichment of muscle specific genes, with low background noise. To demonstrate the robustness of our method, we profiled muscle gene expression in dauer larvae and aging worms, revealing gene expression changes consistent with the physiology of these stages. The resulting muscle transcriptome also revealed 461 novel RNA transcripts, likely muscle-expressed long non-coding RNAs. In summary, the splicing-based RNA tagging (SRT) method provides a convenient and robust tool to profile trans-spliced genes and identify novel transcripts in a tissue-specific manner, with a low false positive rate.

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Section: Resultsmentioning

confidence: 99%

Analysis ofC. elegansmuscle transcriptome using trans-splicing-based RNA tagging (SRT)

Zhan

Sleumer

et al. 2016

Nucleic Acids Res

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“…This indicates, surprisingly, a lower translation efficiency in mutants with little to no miR-58 family activity. As distinct tissues and cellular contexts can employ different mechanisms for miRNA silencing (Subtelny et al 2014) or use different 3 ′ UTR isoforms (Blazie et al 2015), we analyzed whether miR-58 targets expressed in the germline or in the soma might be regulated differently. Even though we observed similar additive effects on protein and target mRNA regulation, we found that the observed reduced translational efficiency in the miR-58 family mutants stems from the germline-expressed targets (Supplemental Fig.…”

Section: Reduced Translational Efficiency Of Derepressed Targetsmentioning

confidence: 99%

Cooperative target mRNA destabilization and translation inhibition by miR-58 microRNA family in C. elegans

Subasic

Brümmer

et al. 2015

Genome Res.

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In animals, microRNAs frequently form families with related sequences. The functional relevance of miRNA families and the relative contribution of family members to target repression have remained, however, largely unexplored. Here, we used the Caenorhabditis elegans miR-58 miRNA family, composed primarily of the four highly abundant members miR-58.1, miR-80, miR-81, and miR-82, as a model to investigate the redundancy of miRNA family members and their impact on target expression in an in vivo setting. We found that miR-58 family members repress largely overlapping sets of targets in a predominantly additive fashion. Progressive deletions of miR-58 family members lead to cumulative up-regulation of target protein and RNA levels. Phenotypic defects could only be observed in the family quadruple mutant, which also showed the strongest change in target protein levels. Interestingly, although the seed sequences of miR-80 and miR-58.1 differ in a single nucleotide, predicted canonical miR-80 targets were efficiently up-regulated in the mir-58.1 single mutant, indicating functional redundancy of distinct members of this miRNA family. At the aggregate level, target binding leads mainly to mRNA degradation, although we also observed some degree of translational inhibition, particularly in the single miR-58 family mutants. These results provide a framework for understanding how miRNA family members interact to regulate target mRNAs.

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“…The 3 0 UTR sequences were taken from the worm UTRome (http://tomato.biodesign.asu.edu/cgibin/UTRome/utrome.cgi). 61 The pADH1::GFP:unc-54:MCS:Ribozyme plasmid expression vector was generated using sequential PCR stitching and gap repair of DNA constructs 62 into the pDest22 backbone (Thermo Fisher Scientific). The S65T GFP sequence was amplified from pFA6:GFP (kindly provided by Paul Kaufman).…”

Section: Cloning Of Rna Elements and Rbpsmentioning

confidence: 99%

PRIMA: a gene-centered, RNA-to-protein method for mapping RNA-protein interactions

et al. 2017

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Interactions between RNA binding proteins (RBPs) and mRNAs are critical to post-transcriptional gene regulation. Eukaryotic genomes encode thousands of mRNAs and hundreds of RBPs. However, in contrast to interactions between transcription factors (TFs) and DNA, the interactome between RBPs and RNA has been explored for only a small number of proteins and RNAs. This is largely because the focus has been on using 'protein-centered' (RBP-to-RNA) interaction mapping methods that identify the RNAs with which an individual RBP interacts. While powerful, these methods cannot as of yet be applied to the entire RBPome. Moreover, it may be desirable for a researcher to identify the repertoire of RBPs that can interact with an mRNA of interest-in a 'gene-centered' manner-yet few such techniques are available. Here, we present Protein-RNA Interaction Mapping Assay (PRIMA) with which an RNA 'bait' can be tested versus multiple RBP 'preys' in a single experiment. PRIMA is a translation-based assay that examines interactions in the yeast cytoplasm, the cellular location of mRNA translation. We show that PRIMA can be used with small RNA elements, as well as with full-length Caenorhabditis elegans 3 0 UTRs. PRIMA faithfully recapitulated numerous well-characterized RNA-RBP interactions and also identified novel interactions, some of which were confirmed in vivo. We envision that PRIMA will provide a complementary tool to expand the depth and scale with which the RNA-RBP interactome can be explored.

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Comparative RNA-Seq analysis reveals pervasive tissue-specific alternative polyadenylation in Caenorhabditis elegans intestine and muscles

Cited by 75 publications

References 82 publications

Analysis ofC. elegansmuscle transcriptome using trans-splicing-based RNA tagging (SRT)

Analysis ofC. elegansmuscle transcriptome using trans-splicing-based RNA tagging (SRT)

Cooperative target mRNA destabilization and translation inhibition by miR-58 microRNA family in C. elegans

PRIMA: a gene-centered, RNA-to-protein method for mapping RNA-protein interactions

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