RESUMEN
Cell-based studies of human ribonucleases traditionally rely on methods that deplete proteins slowly. We engineered cells in which the 3'â5' exoribonucleases of the exosome complex, DIS3 and EXOSC10, can be rapidly eliminated to assess their immediate roles in nuclear RNA biology. The loss of DIS3 has the greatest impact, causing the substantial accumulation of thousands of transcripts within 60 min. These transcripts include enhancer RNAs, promoter upstream transcripts (PROMPTs), and products of premature cleavage and polyadenylation (PCPA). These transcripts are unaffected by the rapid loss of EXOSC10, suggesting that they are rarely targeted to it. More direct detection of EXOSC10-bound transcripts revealed its substrates to prominently include short 3' extended ribosomal and small nucleolar RNAs. Finally, the 5'â3' exoribonuclease, XRN2, has little activity on exosome substrates, but its elimination uncovers different mechanisms for the early termination of transcription from protein-coding gene promoters.
Asunto(s)
Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , ARN Nuclear/metabolismo , ARN/metabolismo , Exorribonucleasas/deficiencia , Exorribonucleasas/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/deficiencia , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Regulación de la Expresión Génica , Células HCT116 , Células HEK293 , Humanos , ARN/genética , ARN Nuclear/genética , Transcripción GenéticaRESUMEN
The vast majority of the mammalian genome has the potential to express noncoding RNA (ncRNA). The 11-subunit RNA exosome complex is the main source of cellular 3'-5' exoribonucleolytic activity and potentially regulates the mammalian noncoding transcriptome. Here we generated a mouse model in which the essential subunit Exosc3 of the RNA exosome complex can be conditionally deleted. Exosc3-deficient B cells lack the ability to undergo normal levels of class switch recombination and somatic hypermutation, two mutagenic DNA processes used to generate antibody diversity via the B-cell mutator protein activation-induced cytidine deaminase (AID). The transcriptome of Exosc3-deficient B cells has revealed the presence of many novel RNA exosome substrate ncRNAs. RNA exosome substrate RNAs include xTSS-RNAs, transcription start site (TSS)-associated antisense transcripts that can exceed 500 base pairs in length and are transcribed divergently from cognate coding gene transcripts. xTSS-RNAs are most strongly expressed at genes that accumulate AID-mediated somatic mutations and/or are frequent translocation partners of DNA double-strand breaks generated at Igh in B cells. Strikingly, translocations near TSSs or within gene bodies occur over regions of RNA exosome substrate ncRNA expression. These RNA exosome-regulated, antisense-transcribed regions of the B-cell genome recruit AID and accumulate single-strand DNA structures containing RNA-DNA hybrids. We propose that RNA exosome regulation of ncRNA recruits AID to single-strand DNA-forming sites of antisense and divergent transcription in the B-cell genome, thereby creating a link between ncRNA transcription and overall maintenance of B-cell genomic integrity.