Splicing fidelity infuences the fate of mRNA molecules

RNA splicing has historically been thought to be highly efficient and accurate, with little opportunity for deviation from regulated alternative splicing decisions. This dogma has been challenged by recent observations that suggest that biological noise may contribute substantially to transcriptome diversity. However, quantitative understanding of stochastic variations in splicing is challenging because these transcripts are likely subject to rapid degradation. Here, we use ultra-deep sequencing across RNA compartments to track splicing intermediates in human cells and see abundant cryptic splicing associated with genomic features that promote splicing noise. We observe pervasive usage of low-fidelity splice sites, likely due to stochasticity in recruitment or binding of the spliceosome. These sites are most likely degraded in the nucleus rather than targeted by translation-dependent degradation processes, suggesting widespread surveillance and quality control of non-productive RNA transcripts. Our findings provide unprecedented insights into the propensity for error in RNA processing mechanisms to buffer mechanistic noise. Overall design: RNAseq from nascent, nuclear and polyA enriched fractions from K562, KNS60, AM38, SY5Y and HepG2 cell lines. K562 cells were treated with cycloheximide, NMDi14, uridine, 4SU and DMSO, followed by polyA enriched RNAseq. K562 cells were treated with cycloheximide and NMDi14 followed by polyA enriched RNAseq. K562 cells were treated with IdU, with or without 4sU labelling, followed by nuclear isolation, without or without 4sU pulldown and RNAseq