Mechanisms and impact of Post-transcriptional Exon Shuffling (PTES)

Abstract

Most eukaryotic genes undergo splicing to remove introns and join exons sequentially to produce protein-coding or non-coding transcripts. Post-transcriptional Exon Shuffling (PTES) describes a new class of RNA molecules, characterized by exon order different from the underlying genomic context. PTES can result in linear and circular RNA (circRNA) molecules and enhance the complexity of transcriptomes. Prior to my studies, I developed PTESFinder, a computational tool for PTES identification from high-throughput RNAseq data. As various sources of artefacts (including pseudogenes, template-switching and others) can confound PTES identification, I first assessed the effectiveness of filters within PTESFinder devised to systematically exclude artefacts. When compared to 4 published methods, PTESFinder achieves the highest specificity (~0.99) and comparable sensitivity (~0.85). To define sub-cellular distribution of PTES, I performed in silico analyses of data from various cellular compartments and revealed diverse populations of PTES in nuclei and enrichment in cytosol of various cell lines. Identification of PTES from chromatin-associated RNAseq data and an assessment of co-transcriptional splicing, established that PTES may occur during transcription. To assess if PTES contribute to the proteome, I analyzed sucrose-gradient fractionated data from HEK293, treated with arsenite to induce translational arrest and dislodge ribosomes. My results showed no effect of arsenite treatment on ribosome occupancy within PTES transcripts, indicating that these transcripts are not generally bound by polysomes and do not contribute to the proteome. To investigate the impact of differential degradation on expression levels of linear and circRNAs, I analyzed the PTES population within RNAseq data of anucleate cells and established that most PTES transcripts are circular and are enriched in platelets 17-to-188-fold relative to nucleated tissues. For some genes, only reads from circRNA exons were detectable, suggesting that platelets have lost >90% of their progenitor mRNAs, consistent with timedependent degradation of platelets transcriptomes. However, some circRNAs exhibit read density patterns suggestive of miRNA induced degradation. Finally, a linear PTES from RMST locus has been implicated in pluripotency maintenance using limited RNAseq data from human embryonic stem cells (hESC). To identify other PTES transcripts with similar expression patterns, I analyzed RNAseq data from H9 ESC differentiation series. Statistical analyses of PTES transcripts identified during cellular differentiation established that PTES expression changes track with that of cognate linear transcripts and accumulate upon differentiation. Contrary to previous reports, the dominant transcript from RMST is circular and increases in abundance during differentiation. Functional Abstract iii analyses demonstrating the role of RMST in pluripotency maintenance had targeted exons within the predicted circRNA, suggesting previously unreported functional relevance for circRNAs.