Abstract
SummaryCell-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.
Highlights
The RNA exosome is a multi-subunit, 30/50 exoribonucleasecontaining complex originally discovered as being important for rRNA processing (Mitchell et al, 1997)
Depletion of EXOSC10 or DIS3 Using the Auxin-Inducible Degron System The auxin-inducible degron (AID) system allows the rapid elimination of AID-tagged proteins upon the addition of auxin to cell culture media (Nishimura et al, 2009)
Hygromycin or neomycin resistance markers were incorporated into the cassettes for homology directed repair (HDR) so that biallelic modification could be selected for (Eaton et al, 2018)
Summary
The RNA exosome is a multi-subunit, 30/50 exoribonucleasecontaining complex originally discovered as being important for rRNA processing (Mitchell et al, 1997). It plays a crucial role in the turnover of multiple coding and non-coding (nc) transcript classes (Kilchert et al, 2016; Schmid and Jensen, 2018). Many of these transcripts, such as cryptic unstable transcripts (CUTs) in yeast or promoter upstream transcripts/upstream antisense RNAs (PROMPTs/uaRNAs) in humans, are products of antisense transcription (Flynn et al, 2011; Preker et al, 2008; Wyers et al, 2005). Products of premature cleavage and polyadenylation (PCPA) were revealed as exosome substrates in mouse embryonic stem cells (mESCs) (Chiu et al, 2018)
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