Nuclear Surveillance Pathways Play a Key Role in Regulating the Transcription Landscape of Eukaryotic Genomes

The bulk of the eukaryotic genome is pervasively transcribed, the largest proportion of which represents a diverse collection of non-coding RNA transcripts. Nuclear surveillance pathways play an integral role in regulating the expression of pervasive transcripts and protect the integrity of the transcriptome by engaging the activity of several nuclear exoribonucleases. In human nuclei, Xrn2 degrades RNA with 5’→3’ directionality, whereas the exosome complex contains two catalytic subunits: Exosc10 and Dis3 capable of 3’→5’ RNA decay. Functional studies of nuclear surveillance pathways in the past used RNA interference (RNAi) mediated protein depletion. Although informative, RNAi requires prolonged periods of gene downregulation which can often be incomplete and introduces indirect effects, further obfuscating the immediate function of nuclear exoribonucleases. The rise in popularity and efficiency of CRISPR/Cas9 mediated gene editing have stimulated a renaissance in the field of functional genomics for those wishing to apply a more direct approach within human models. Combining CRISPR/Cas9 with a post-translational degron depletion system, human cell lines can be engineered to undergo rapid, conditional and reversible downregulation of gene expression. As such, three auxin-inducible degron HCT116 cells lines were generated in this study with the aim to dissect the three major exonucleases: Xrn2, Dis3 and Exosc10. High-throughput RNA sequencing of nuclear transcriptomes in each scenario have identified distinct substrates for each exoribonuclease and new layers of gene regulation. The data presented hereafter highlights the extent of pervasive transcription and the separate nuclear surveillance pathways available within human nuclei.