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dc.contributor.authorKovalak, Carrie
dc.contributor.authorMetkar, Mihir
dc.contributor.authorMoore, Melissa J.
dc.date2022-08-11T08:08:24.000
dc.date.accessioned2022-08-23T15:53:45Z
dc.date.available2022-08-23T15:53:45Z
dc.date.issued2019-11-19
dc.date.submitted2019-11-27
dc.identifier.citation<p>bioRxiv 847004; doi: https://doi.org/10.1101/847004. <a href="https://doi.org/10.1101/847004" target="_blank">Link to preprint on bioRxiv service.</a></p>
dc.identifier.doi10.1101/847004
dc.identifier.urihttp://hdl.handle.net/20.500.14038/29417
dc.description.abstractBackground The ability to generate multiple mRNA isoforms from a single gene by alternative splicing (AS) is crucial for the regulation of eukaryotic gene expression. Because different mRNA isoforms can have widely differing decay rates, however, the flux through competing AS pathways cannot be determined by traditional RNA-Seq data alone. Further, some mRNA isoforms with extremely short half-lives, such as those subject to translation-dependent nonsense-mediated decay (AS-NMD), may be completely overlooked in even the most extensive RNA-Seq analyses. Results RNA immunoprecipitation in tandem (RIPiT) of exon junction complex (EJC) components allows for the purification of post-splicing mRNA-protein particles (mRNPs) not yet subject to translation (pre-translational mRNPs) and translation-dependent mRNA decay. Here we compared EJC RIPiT-Seq to whole cell and cytoplasmic RNA-Seq data from HEK293 cells. Consistent with expectations, we found that the flux through known AS-NMD pathways is substantially higher than what is captured by RNA-Seq. We also identified thousands of previously unannotated splicing events; while many can be attributed to “splicing noise”, others are evolutionarily-conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis. Conclusions Deep sequencing of RNAs in post-splicing, pre-translational mRNPs provides a means to identify and quantify splicing events without the confounding influence of differential mRNA decay. For many known AS-NMD targets, the NMD-linked AS pathway dominates. EJC RIPiT-Seq also enabled identification of numerous conserved but previously unknown AS-NMD events.
dc.language.isoen_US
dc.rightsThe copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subjectBioinformatics
dc.subjectmRNA
dc.subjectDeep sequencing
dc.subjectAS-NMD
dc.subjectExon junctions
dc.subjectmRNA isoforms
dc.subjectAS-NMD
dc.subjectRIPiT-Seq
dc.subjectpre-translational mRNPs
dc.subjectsplicing noise
dc.subjectAmino Acids, Peptides, and Proteins
dc.subjectBioinformatics
dc.subjectGenetic Phenomena
dc.subjectGenetics and Genomics
dc.subjectNucleic Acids, Nucleotides, and Nucleosides
dc.titleDeep sequencing of pre-translational mRNPs reveals hidden flux through evolutionarily conserved AS-NMD pathways [preprint]
dc.typePreprint
dc.source.journaltitlebioRxiv
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=2661&amp;context=faculty_pubs&amp;unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/faculty_pubs/1645
dc.identifier.contextkey15882329
refterms.dateFOA2022-08-23T15:53:46Z
html.description.abstract<p><p id="x-x-x-x-p-3"><strong>Background</strong> The ability to generate multiple mRNA isoforms from a single gene by alternative splicing (AS) is crucial for the regulation of eukaryotic gene expression. Because different mRNA isoforms can have widely differing decay rates, however, the flux through competing AS pathways cannot be determined by traditional RNA-Seq data alone. Further, some mRNA isoforms with extremely short half-lives, such as those subject to translation-dependent nonsense-mediated decay (AS-NMD), may be completely overlooked in even the most extensive RNA-Seq analyses. <p id="x-x-x-x-p-4"><strong>Results</strong> RNA immunoprecipitation in tandem (RIPiT) of exon junction complex (EJC) components allows for the purification of post-splicing mRNA-protein particles (mRNPs) not yet subject to translation (pre-translational mRNPs) and translation-dependent mRNA decay. Here we compared EJC RIPiT-Seq to whole cell and cytoplasmic RNA-Seq data from HEK293 cells. Consistent with expectations, we found that the flux through known AS-NMD pathways is substantially higher than what is captured by RNA-Seq. We also identified thousands of previously unannotated splicing events; while many can be attributed to “splicing noise”, others are evolutionarily-conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis. <p id="x-x-x-x-p-5"><strong>Conclusions</strong> Deep sequencing of RNAs in post-splicing, pre-translational mRNPs provides a means to identify and quantify splicing events without the confounding influence of differential mRNA decay. For many known AS-NMD targets, the NMD-linked AS pathway dominates. EJC RIPiT-Seq also enabled identification of numerous conserved but previously unknown AS-NMD events.</p>
dc.identifier.submissionpathfaculty_pubs/1645
dc.contributor.departmentGraduate School of Biomedical Sciences
dc.contributor.departmentDepartment of Biochemistry and Molecular Pharmacology
dc.contributor.departmentRNA Therapeutics Institute


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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
Except where otherwise noted, this item's license is described as The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.