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About the Authors:
Clémentine Delan-Forino
Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
ORCID http://orcid.org/0000-0003-0915-1211
Claudia Schneider
Affiliation: Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, United Kingdom
David Tollervey
* E-mail: [email protected]
Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
ORCID http://orcid.org/0000-0003-2894-2772Abstract
The RNA exosome complex functions in both the accurate processing and rapid degradation of many classes of RNA. Functional and structural analyses indicate that RNA can either be threaded through the central channel of the exosome or more directly access the active sites of the ribonucleases Rrp44 and Rrp6, but it was unclear how many substrates follow each pathway in vivo. We used CRAC (UV crosslinking and analysis of cDNA) in growing cells to identify transcriptome-wide interactions of RNAs with the major nuclear exosome-cofactor Mtr4 and with individual exosome subunits (Rrp6, Csl4, Rrp41 and Rrp44) along the threaded RNA path. We compared exosome complexes lacking Rrp44 exonuclease activity, carrying a mutation in the Rrp44 S1 RNA-binding domain predicted to disfavor direct access, or with multiple mutations in Rrp41 reported to impede RNA access to the central channel in vitro. Preferential use of channel-threading was seen for mRNAs, 5S rRNA, scR1 (SRP) and aborted tRNAs transcripts. Conversely, pre-tRNAs preferentially accessed Rrp44 directly. Both routes participated in degradation and maturation of RNAPI transcripts, with hand-over during processing. Rrp41 mutations blocked substrate passage through the channel to Rrp44 only for cytoplasmic mRNAs, supporting the predicted widening of the lumen in the Rrp6-associated, nuclear complex. Many exosome substrates exhibited clear preferences for a specific path to Rrp44. Other targets showed redundancy, possibly allowing the efficient handling of highly diverse RNA-protein complexes and RNA structures. Both threading and direct access routes involve the RNA helicase Mtr4. mRNAs that are predominately nuclear or cytoplasmic exosome substrates can be distinguished in vivo.
Author summary
In all organisms, diverse classes of RNA perform a wide range of functions, including programing of protein synthesis, transcriptional and post-transcriptional regulation of gene expression and forming key structural and functional components for pre-mRNA splicing, ribosome synthesis and translation. In consequence, control of the quantity and quality of RNA synthesis and maturation is of key importance. In Eukaryotes, the RNA exosome complex...