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The RNA Exosome Channeling and Direct Access Conformations Have Distinct In Vivo Functions.

ABSTRACT: The RNA exosome is a 3'-5' ribonuclease complex that is composed of nine core subunits and an essential catalytic subunit, Rrp44. Two distinct conformations of Rrp44 were revealed in previous structural studies, suggesting that Rrp44 may change its conformation to exert its function. In the channeling conformation, (Rrp44(ch)), RNA accesses the active site after traversing the central channel of the RNA exosome, whereas in the other conformation, (Rrp44(da)), RNA gains direct access to the active site. Here, we show that the Rrp44(da) exosome is important for nuclear function of the RNA exosome. Defects caused by disrupting the direct access conformation are distinct from those caused by channel-occluding mutations, indicating specific functions for each conformation. Our genetic analyses provide in vivo evidence that the RNA exosome employs a direct-access route to recruit specific substrates, indicating that the RNA exosome uses alternative conformations to act on different RNA substrates.

SUBMITTER: Han J

PROVIDER: S-EPMC5044803 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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The RNA Exosome Channeling and Direct Access Conformations Have Distinct In Vivo Functions.

Han Jaeil J van Hoof Ambro A

Cell reports 20160901 12

The RNA exosome is a 3'-5' ribonuclease complex that is composed of nine core subunits and an essential catalytic subunit, Rrp44. Two distinct conformations of Rrp44 were revealed in previous structural studies, suggesting that Rrp44 may change its conformation to exert its function. In the channeling conformation, (Rrp44(ch)), RNA accesses the active site after traversing the central channel of the RNA exosome, whereas in the other conformation, (Rrp44(da)), RNA gains direct access to the activ ...[more]

PMID: 27653695

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Project description: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.

Dataset Information

The RNA Exosome Channeling and Direct Access Conformations Have Distinct In Vivo Functions.

Publications

The RNA Exosome Channeling and Direct Access Conformations Have Distinct In Vivo Functions.

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