Frac-seq reveals isoform-specific recruitment to polyribosomes

Abstract

Pre-mRNA splicing is required for the accurate expression of virtually all human protein coding genes. However, splicing also plays important roles in coordinating subsequent steps of pre-mRNA processing such as polyadenylation and mRNA export. Here, we test the hypothesis that nuclear pre-mRNA processing influences the polyribosome association of alternative mRNA isoforms. By comparing isoform ratios in cytoplasmic and polyribosomal extracts, we determined that the alternative products of ∼30% (597/1954) of mRNA processing events are differentially partitioned between these subcellular fractions. Many of the events exhibiting isoform-specific polyribosome association are highly conserved across mammalian genomes, underscoring their possible biological importance. We find that differences in polyribosome association may be explained, at least in part by the observation that alternative splicing alters the cis-regulatory landscape of mRNAs isoforms. For example, inclusion or exclusion of upstream open reading frames (uORFs) in the 5′UTR as well as Alu-elements and microRNA target sites in the 3′UTR have a strong influence on polyribosome association of alternative mRNA isoforms. Taken together, our data demonstrate for the first time the potential link between alternative splicing and translational control of the resultant mRNA isoforms.

The landscape of human mutually exclusive splicing

“…However, ribosome profiling data showed high frequencies of ribosome engagement of cassette exons indicating that these isoforms are likely translated (Weatheritt et al , 2016). Similar results have been obtained through polyribosome profiling (Sterne‐Weiler et al , 2013; Floor & Doudna, 2016). These observations suggest that most of the MXEs evaluated at the transcript level will also be found in the proteome.…”

Widespread Translational Remodeling during Human Neuronal Differentiation

“…Even if the coding sequence is identical between two alternatively processed transcripts of a gene, the level of protein output can differ due to changes in the regulatory composition of untranslated regions at the 5′ and 3′ ends (Arribere and Gilbert, 2013; Floor and Doudna, 2016; Hinnebusch et al, 2016; Mayr, 2016; Mayr and Bartel, 2009; Sandberg et al, 2008; Sterne-Weiler et al, 2013). We therefore compared transcript isoform properties in polysome-low versus polysome-high clusters that might influence translation, such as the density of various regulatory elements in the 3′ UTR or codon usage (Figure S4A).…”

The Clothes Make the mRNA: Past and Present Trends in mRNP Fashion

“…In a recent study, mRNA isoforms generated by ~30% of AS events were found to impact regulatory cis elements in either 5′ UTRs (short upstream ORFs, internal ribosome entry sites) or 3′ UTRs (miRNA-binding sites; Alu elements), leading to differential isoform association with polyribosomes (73). Finally, by introducing an upstream ORF into the 5′ UTR or a premature translation termination codon into the main ORF, many AS events simply redefine the 3′-UTR boundary without significantly altering overall mRNP composition.…”