RNA-Seq reveals to what extent splice-variant transcripts are translated into protein products

High-throughput RNA sequencing (RNA-seq) has revealed an enormous complexity of alternative splicing (AS) across diverse cell and tissue types. However, it is currently unknown to what extent repertoires of splice-variant transcripts are translated into protein products.

Here, University of Toronto researchers surveyed AS events engaged by the ribosome. Notably, at least 75% of human exon-skipping events detected in transcripts with medium-to-high abundance in RNA-seq data were also detected in ribosome profiling data. Furthermore, relatively small subsets of functionally related splice variants are engaged by ribosomes at levels that do not reflect their absolute abundance, thus indicating a role for AS in modulating translational output. This mode of regulation is associated with control of the mammalian cell cycle.

Most cassette AS events in transcripts with medium-to-high abundance are engaged by the ribosome

(a) Box plots showing AS frequency (scored as the fraction of annotated exons in canonical transcripts showing skipping) for genes with different levels of RNA-seq or ribosome profiling read coverage in human cells. (b) Stacked bar plot comparing percentages of constitutive versus alternatively spliced exons for all genes with at least three exons detected. (c) Bar plot comparing fractions of total AS events identified in RNA-seq data that were also identified as alternatively spliced in ribosome profiling (RP) data, at different expression levels. (d) Bar plots showing fractions of coding AS events detected in matched ribosome profiling (n = 2,431 events) and RNA-seq (n = 8,797 events) data sets comprising exons divisible by 3 and not encoding an in-frame premature stop codon, or not divisible by 3 but partially overlapping a UTR sequence. (e) Bar plot showing fractions of coding AS events not divisible by 3 within CDS that display changes in PSI between ribosomal profiling and matched RNA-seq data (n = 1,226 events).

These results thus suggest that a major fraction of splice variants is translated and that specific cellular functions including cell-cycle control are subject to AS-dependent modulation of translation output.