Deep sequencing of pre-translational mRNPs reveals hidden flux through evolutionarily conserved AS-NMD pathways

Abstract

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.

Deep sequencing of pre-translational mRNPs reveals hidden flux through evolutionarily conserved alternative splicing nonsense-mediated decay pathways

“…Paired-end deep sequencing of these EJC RIPiT-Seq libraries resulted in 19-25 million mate pairs each (Additional file 1: Table S1). To enable comparison to RNA-Seq for the current study, we created and sequenced rRNA-depleted whole cell RNA-Seq libraries (84-93 million mate pairs each) [15] wherein the captured fragments were of similar length (220-500 nts) to our previously published EJC RIPiT-Seq libraries. The new RNA-Seq libraries were generated from cultures (three biological replicates each) that were (+) or were not (−) subjected to a 1 h pre-treatment with harringtonine.…”

“…Paired-end sequencing (150 nt reads) on the Illumina NextSeq platform resulted in 18-24 million mate pairs per replicate [13,14]. For RNA-Seq libraries [15], FLAG-Magoh HEK293 (validated in [9,13]) cells were grown as in [13] and subjected (+) or not (−) to a 1 h treatment with harringtonine (2 ng/mL) prior to cell harvest. RNA was isolated in TRIzol using the Direct-zol RNA Kit (Zymo Research, R2062).…”

Nanopore sequencing reveals endogenous NMD-targeted isoforms in human cells

“…As an alternative approach to assess the transcriptome of spliced mRNAs prior to translation, Kovalak and colleagues captured newly synthesized mRNAs by tandem immunoprecipitation of epitope-tagged and untagged EJC components, a technique known as RNA/protein immunoprecipitation in tandem (RIPiT) (79). This analysis revealed that EJC-bound RNAs are highly enriched for spliced transcripts that are subject to rapid clearance by translation-dependent decay (80). Our analysis points to the same direction, because after inhibiting NMD, we enriched for mRNAs with exon junctions in the 3΄UTR that likely still contain EJCs.…”