Abstract
SummaryThe exosome plays major roles in RNA processing and surveillance but the in vivo target range and substrate acquisition mechanisms remain unclear. Here we apply in vivo RNA crosslinking (CRAC) to the nucleases (Rrp44, Rrp6), two structural subunits (Rrp41, Csl4) and a cofactor (Trf4) of the yeast exosome. Analysis of wild-type Rrp44 and catalytic mutants showed that both the CUT and SUT classes of non-coding RNA, snoRNAs and, most prominently, pre-tRNAs and other Pol III transcripts are targeted for oligoadenylation and exosome degradation. Unspliced pre-mRNAs were also identified as targets for Rrp44 and Rrp6. CRAC performed using cleavable proteins (split-CRAC) revealed that Rrp44 endonuclease and exonuclease activities cooperate on most substrates. Mapping oligoadenylated reads suggests that the endonuclease activity may release stalled exosome substrates. Rrp6 was preferentially associated with structured targets, which frequently did not associate with the core exosome indicating that substrates follow multiple pathways to the nucleases.
Highlights
Gene expression generates an enormous variety of stable or unstable, protein-coding or non-coding RNA species produced by all three RNA polymerases
Comparison of Targets for Wild-Type and Mutant Forms of Rrp44 To identify targets for the core exosome nuclease Rrp44, we applied in vivo RNA-protein crosslinking (CRAC) (Granneman et al, 2009) to the wild-type protein, or rrp44 mutants carrying point mutations in catalytic residues of the RNB exonuclease domain or PIN endonuclease domain (Figure 1A)
At least two independent experiments were performed in each case and analyzed separately
Summary
Gene expression generates an enormous variety of stable or unstable, protein-coding or non-coding RNA species produced by all three RNA polymerases. A key player in RNA metabolism is the exosome, which participates in 30 end maturation and/or quality control of almost every RNA molecule in the cell. In Saccharomyces cerevisiae, nuclear and cytoplasmic forms of the exosome share the RNase II homolog Rrp44/Dis, which contains two distinct catalytic sites. The RNB domain exhibits 30-50 exonuclease activity, whereas the N-terminal PINc domain plays a dual role in harboring endonuclease activity and tethering Rrp to the core, nine subunit exosome (Lebreton et al, 2008; Lorentzen et al, 2008; Schaeffer et al, 2009; Schneider et al, 2009). In addition to Rrp, the nuclear form of the yeast exosome is associated with a second active 30-50 exonuclease, Rrp (Briggs et al, 1998)
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