mTOR-driven widespread exon skipping renders multifaceted gene regulation and proteome complexity

Abstract Mammalian target of rapamycin (mTOR) pathway is crucial in cell proliferation. Many have associated its dysregulation with numerous human pathogenic processes. Previously, we reported 3’-untranslated region (UTR) length dynamics by alternative polyadenylation in the mTOR-activated transcriptome and its impact on the proteome. Here, we further explored the mTOR-activated transcriptome with regard to alternative splicing (AS) events and their functional relevance. We employed cellular models with genetic or pharmacological manipulation of mTOR activity and investigated the changes of transcriptome profile using RNA-Seq experiments and a custom-developed AS-Quant pipeline. Strikingly, we found that hyperactivation of mTOR in cells promotes transcriptome-wide exon skipping/exclusion. These AS events multifariously regulate the proteome: exon-skipping in the coding regions widely affects functional domains in the proteome; exon-skipping in the 5’-UTR can control translation efficiency. Moreover, a number of these exon-skipping events affects potential ubiquitination and phosphorylation sites and produces protein isoforms with varying stabilities. Furthermore, some of these exon-skipping events can switch transcripts from being coding to non-coding or vice versa, suggesting AS as a built-in post-transcriptional molecular switch for gene expression and/or functional regulation. Notably, we found that mTOR-activated widespread exon skipping is in part facilitated by splicing factor Srsf3. Our study reveals previously unappreciated mTOR-coordinated post-transcriptional pathways that regulate cellular proteome. These findings also highlight multifaceted mechanisms of AS in controlling functional proteome.