Abstract
Background: The exosome complex plays key roles in RNA processing and degradation in Eukaryotes and Archaea. Outstanding structural studies identified multiple pathways for RNA substrates into the exosome in vitro, but identifying the pathway followed by individual RNA species in vivo remains challenging. Methods: We attempted to address this question using RNase protection. In vivo RNA-protein crosslinking (CRAC) was applied to the exosome component Rrp44/Dis3, which has both endonuclease and exonuclease activity. During CRAC, the exosome was purified under native conditions and subjected to RNase digestion, prior to protein denaturation and cDNA cloning. The resulting high-throughput sequence reads were stratified by length of the cDNA sequence. This should reflect RNA fragment lengths, and therefore the RNA region that was protected by exosome binding. We anticipated major read lengths of ~30nt and ~10nt, reflecting the "central channel" and "direct access" routes to the Rrp44 exonuclease active site observed in vitro. Results: Unexpectedly, no clear peak was observed at 30nt, whereas a broad peak was seen around 20nt. The expected ~10nt peak was seen, and showed strong elevation in strains lacking exonuclease activity. Unexpectedly, this peak was suppressed by point mutations in the Rrp44 endonuclease active site. This indicates that the short fragments are degraded by the exonuclease activity of Rrp44, but also suggests that at least some may be generated by endonuclease activity. Conclusions: The absence of 30nt protected fragments may reflect obligatory binding of cofactors at the entrance to the exosome central channel in vivo. The presence of ~20nt fragments apparently indicates an access route not yet reported from in vitro studies. Confident mapping of 10nt reads is challenging, but they are clearly derived from a subset of exosome targets. In particular, pre-rRNA species, which are major exosome targets, are strongly disfavored for the generation of short reads.
Highlights
The exosome nuclease complex in Eukaryotes has a barrel-like structure, with a central channel through which substrate RNAs can be threaded to reach the 3’ exonuclease active site of the RNase II related protein Rrp44 (Dis3)
Length distribution of Rrp44-associated RNAs CRAC was performed on a Rrp44 construct expressed from the endogenous locus and carrying a tripartite C-terminal HTP tag (His - TEV protease cleavage site – 2 copies of the Z-domain 6 of protein A) (Figure 1A)
Plasmid-encoded wildtype Rrp44-HTP expressed from its endogenous promoter was compared to constructs with Rrp44-HTP that lacked exonuclease activity, due to catalytic site point mutation (D551N; Rrp44-exo), or lacked endonuclease activity, due to point mutations at each of the four conserved endonuclease active-site amino acids (D91N, E120Q, D171N, D198N; Rrp44-endo)
Summary
The exosome nuclease complex in Eukaryotes has a barrel-like structure, with a central channel through which substrate RNAs can be threaded to reach the 3’ exonuclease active site of the RNase II related protein Rrp (Dis). Initial functional analyses of the PIN endonuclease activity of Rrp identified only the 7S pre-rRNA and excised 5’ ETS pre-rRNA fragments as targets for cleavage (Lebreton et al, 2008; Schaeffer et al, 2009; Schneider et al, 2009). We previously attempted to identify targets for the PIN domain-associated endonuclease activity by in vivo RNA-protein crosslinking and sequencing of the resulting cDNA products (CRAC) (Figure 1B). The resulting high-throughput sequence reads were stratified by length of the cDNA sequence This should reflect RNA fragment lengths, and the RNA region that was protected by exosome binding. The expected ~10nt peak was seen, and showed strong elevation in strains lacking exonuclease activity This peak was suppressed by point mutations in the Rrp endonuclease active site. Pre-rRNA species, which are major exosome targets, are strongly disfavored for the generation of short reads
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