title: Transcriptome-wide analysis of alternative routes for RNA substrates into the exosome complex.
abstract: 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.
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Clémentine
authors.identifier: 0000-0003-0915-1211
authors.initials: C
authors.lastname: Delan-Forino
authors.affiliation: Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: Claudia
authors.identifier: 0000-0001-5597-2489
authors.initials: C
authors.lastname: Schneider
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: David
authors.identifier: 0000-0003-2894-2772
authors.initials: D
authors.lastname: Tollervey
mesh_terms: D020134:Catalytic Domain / Q000235:genetics; D053487:DEAD-box RNA Helicases / Q000235:genetics*; D063326:Exosome Multienzyme Ribonuclease Complex / Q000235:genetics* / Q000378:metabolism; D055354:Exosomes / Q000235:genetics; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D009687:Nuclear Proteins / Q000235:genetics; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D059467:Transcriptome / Q000235:genetics
chemical_list: C112809:CSL4 protein, S cerevisiae; D009687:Nuclear Proteins; D012333:RNA, Messenger; C111978:SKI6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA; D063326:Exosome Multienzyme Ribonuclease Complex; C542283:DIS3 protein, S cerevisiae; C112610:RRP6 protein, S cerevisiae; C101004:MTR4 protein, S cerevisiae; D053487:DEAD-box RNA Helicases