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
