The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein.
The exosome complex is a key component of the cellular RNA surveillance machinery and is required for normal 3' end processing of many stable RNAs. Exosome activity requires additional factors such as the Ski or TRAMP complexes to activate the complex or facilitate substrate binding. Rrp47p promotes the catalytic activity of the exosome component Rrp6p, but its precise function is unknown. Here we show that recombinant Rrp47p is expressed as an apparently hexameric complex that specifically binds structured nucleic acids. Furthermore, pull-down assays demonstrated that Rrp47p interacts directly with the N-terminal region of Rrp6p that contains the functionally uncharacterized PMC2NT domain. Strains expressing a mutant form of Rrp6p lacking the N-terminal region failed to accumulate Rrp47p at normal levels, exhibited a slow growth phenotype characteristic of rrp47-Delta mutants and showed RNA processing defects consistent with loss of Rrp47p function. These findings suggest Rrp47p promotes Rrp6p activity by facilitating binding via the PMC2NT domain to structural elements within RNA. Notably, characterized Rrp6p substrates such as the 5.8S+30 species are predicted to contain helices at their 3' termini, while others such as intergenic or antisense cryptic unstable transcripts could potentially form extensive double-stranded molecules with overlapping mRNAs.
Stead
Jonathan A
JA
Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
Costello
Joe L
JL
Livingstone
Michaela J
MJ
Mitchell
Phil
P
D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D005095:Exoribonucleases / Q000737:chemistry* / Q000378:metabolism*; D063326:Exosome Multienzyme Ribonuclease Complex; D009687:Nuclear Proteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D026721:RNA 3' End Processing; D012340:RNA, Ribosomal, 5.8S / Q000378:metabolism; D020537:RNA, Small Nucleolar / Q000378:metabolism; D016601:RNA-Binding Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*
D004268:DNA-Binding Proteins; C494789:LRP1 protein, S cerevisiae; D009687:Nuclear Proteins; D012340:RNA, Ribosomal, 5.8S; D020537:RNA, Small Nucleolar; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA; D004247:DNA; D005095:Exoribonucleases; D063326:Exosome Multienzyme Ribonuclease Complex; C112610:RRP6 protein, S cerevisiae
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