Rapid Depletion of DIS3, EXOSC10, or XRN2 Reveals the Immediate Impact of Exoribonucleolysis on Nuclear RNA Metabolism and Transcriptional Control

Abstract

Summary Cell-based studies of human ribonucleases traditionally rely on methods that deplete proteins slowly. We engineered cells in which the 3′→5′ 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′→3′ 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.

High-throughput siRNA screening reveals functional interactions and redundancies of human processive exoribonucleases

“…The GIs that were mapped in the present study support the unique functions of each of the examined ribonucleases, which is consistent with distinct substrate specificity (Szczepińska et al, 2015;Tomecki et al, 2010;Staals et al, 2010;Ustianenko et al, 2013;Davidson et al, 2012Davidson et al, , 2019Lubas et al, 2013), which can be explained by their different modes of substrate selection, catalytic activity, and cellular compartmentalization in mammalian cells.…”

Identification and dynamic quantification of regulatory elements using total RNA

“…The transcriptome encodes an abundance of short-lived transcripts that are rapidly degraded by the DIS3 exoribonuclease component of the exosome (Szczepinśka et al 2015;Davidson et al 2019). Sensitive identification of these transcripts and their TSSs usually requires live cells to isolate nuclear RNA or nuclear runon products (Core et al 2008(Core et al , 2014Nechaev et al 2010;Lam et al 2013).…”

“…Sensitive identification of these transcripts and their TSSs usually requires live cells to isolate nuclear RNA or nuclear runon products (Core et al 2008(Core et al , 2014Nechaev et al 2010;Lam et al 2013). To test whether these transcripts can be readily detected from only total RNA, we performed csRNA-seq in HCT116 cells, for which RNA-seq data for both DIS3 exoribonuclease degradation (Davidson et al 2019) and nascent transcription (PRO-seq) (Rao et al 2017) are available for comparison. TSSs were called and stability of the associated transcripts was inferred by integrating csRNA-seq and total RNA-seq data.…”

“…In contrast, methods that capture nascent RNA detect transcripts and their TSSs independent of their stability. These methods include using nuclear or chromatin run-on reactions with modified nucleotides to isolate nascent transcripts (GRO-seq [Core et al 2008], PRO-seq [Kwak et al 2013], ChRO-seq [Chu et al 2018]), sequencing RNA polymerase-associated RNAs (NET-seq; Churchman and Weissman 2011), in vivo labeling RNA and enrichment of newly synthesized RNA (Salic and Mitchison 2008;Duffy et al 2015;Schwalb et al 2016), or depletion of cellular components to deter the degradation of unstable RNAs (Preker et al 2008;Davidson et al 2019). These methods faithfully map transcribed regulatory elements and reveal the transcriptome at an unprecedented scale.…”

A unified allosteric/torpedo mechanism for transcriptional termination on human protein-coding genes

“…S2B). We performed this gene editing in our recently described DIS3-AID cells in which the major catalytic subunit of the exosome, DIS3, can be rapidly depleted by auxin (Davidson et al 2019). Total RNA was isolated from xrRNA-modified or unmodified DIS3-AID cells treated or not with auxin and qRT-PCR used to assay the levels of MORF4L2 RNA upstream of and downstream from the xrRNA insertion ( Fig.…”