Deep sequencing of pre-translational mRNPs reveals hidden flux through evolutionarily conserved AS-NMD pathways [preprint]
UMass Chan Affiliations
Graduate School of Biomedical SciencesDepartment of Biochemistry and Molecular Pharmacology
RNA Therapeutics Institute
Document Type
PreprintPublication Date
2019-11-19Keywords
BioinformaticsmRNA
Deep sequencing
AS-NMD
Exon junctions
mRNA isoforms
AS-NMD
RIPiT-Seq
pre-translational mRNPs
splicing noise
Amino Acids, Peptides, and Proteins
Bioinformatics
Genetic Phenomena
Genetics and Genomics
Nucleic Acids, Nucleotides, and Nucleosides
Metadata
Show full item recordAbstract
Background 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.Source
bioRxiv 847004; doi: https://doi.org/10.1101/847004. Link to preprint on bioRxiv service.
DOI
10.1101/847004Permanent Link to this Item
http://hdl.handle.net/20.500.14038/29417Rights
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.Distribution License
http://creativecommons.org/licenses/by-nc-nd/4.0/ae974a485f413a2113503eed53cd6c53
10.1101/847004
Scopus Count
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.