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

BACKGROUND: Alternative splicing, which generates multiple mRNA isoforms from single genes, is crucial for the regulation of eukaryotic gene expression. The flux through competing splicing pathways cannot be determined by traditional RNA-Seq, however, because different mRNA isoforms can have widely differing decay rates. Indeed, 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 components allows for purification of post-splicing mRNA-protein particles (mRNPs) not yet subject to translation (pre-translational mRNPs) and, therefore, translation-dependent mRNA decay. Here we compare exon junction complex RIPiT-Seq to whole cell RNA-Seq data from HEK293 cells. Consistent with expectation, the flux through known AS-NMD pathways is substantially higher than that captured by RNA-Seq. Our RIPiT-Seq also definitively demonstrates that the splicing machinery itself has no ability to detect reading frame. We identify 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 nonsense-mediated decay-linked alternative splicing pathway predominates. Exon junction complex RIPiT-Seq also revealed numerous conserved but previously unannotated AS-NMD events.

The DEAD-box protein Dhh1 promotes decapping by slowing ribosome movement.

Translational control and messenger RNA (mRNA) decay represent important control points in the regulation of gene expression. In yeast, the major pathway for mRNA decay is initiated by deadenylation followed by decapping and 5'-3' exonucleolytic digestion of the mRNA. Proteins that activate decapping, such as the DEAD-box RNA helicase Dhh1, have been postulated to function by limiting translation initiation, thereby promoting a ribosome-free mRNA that is targeted for decapping. In contrast to this model, we show here that Dhh1 represses translation in vivo at a step subsequent to initiation. First, we establish that Dhh1 represses translation independent of initiation factors eIF4E and eIF3b. Second, we show association of Dhh1 on an mRNA leads to the accumulation of ribosomes on the transcript. Third, we demonstrate that endogenous Dhh1 accompanies slowly translocating polyribosomes. Lastly, Dhh1 activates decapping in response to impaired ribosome elongation. Together, these findings suggest that changes in ribosome transit rate represent a key event in the decapping and turnover of mRNA.