Human DIS3 shapes the RNA polymerase II transcriptome degrading variety of unwanted transcripts.. Homo sapiens

Human DIS3 is a nuclear, catalytic subunit of the exosome complex containing exonucleolytic and endonucleolytic active domains. To identify DIS3 targets genome-wide we conducted comprehensive transcriptomic analysis of HEK293 cells producing mutated DIS3 versions and Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) experiments. Pervasive transcription products like Promoter Upstream Transcripts (PROMPTs) accumulated robustly in catalytic DIS3 mutants, representing ~8% of PAR-CLIP reads. Importantly, RNAs originating from unannotated genomic regions increased ~2.5 times in double DIS3 mutants, covering ~70% of genome and allowing for discovery of thousands of novel transcripts. The first intron of many pre-mRNAs accumulated in DIS3 mutants indicating a widespread premature RNA polymerase II termination. The short form of NEAT1 lincRNA was overexpressed in DIS3 mutants, leading to increased number of paraspeckles. Moreover, there was a global deregulation of mRNAs in DIS3 double mutant. Finally, snoRNA precursors accumulated, which correlated with a strong PAR-CLIP signal indicating that DIS3 but not RRP6 is a main snoRNA processing enzyme. In aggregate, we demonstrate that DIS3 is a major nucleoplasmic activity responsible for shaping the human transcriptome. Overall design: RNA-seq experiments were performed in triplicates for DIS3 wild type (control), DIS3 PIN, DIS3 RNB domain mutants and DIS3 PIN RNB double mutant. RNA-seq samples from DIS3 wild type and DIS3 double mutant were additionally sequenced in deeper resolution, also in triplicates. DIS3 PAR-CLIP experiment was performed in duplicate. Pol II ChIP-seq experiment in WT and DIS3 PIN RNB double-mutants cells was performed in triplicates.