RNA substrate length as an indicator of exosome interactions in vivo

Wellcome Open Research

article

Clémentine Delan-Forino¹  Claudia Schneider²  David Tollervey¹ 

[1] Wellcome Trust Centre for Cell Biology, University of Edinburgh;Institute for Cell and Molecular Biosciences, Newcastle University

关键词: Exosome;    RNA processing;    RNA degradation;    protein-RNA interaction;    RNA-binding sites;    UV crosslinking;    yeast;   

DOI  :  10.12688/wellcomeopenres.10724.2

学科分类：内科医学

来源: Wellcome

【 摘 要 】

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 exosomein vitro, but identifying the pathway followed by individual RNA speciesin 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 observedin 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 channelin vivo. The presence of ~20nt fragments apparently indicates an access route not yet reported fromin 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.

【 授权许可】

CC BY