title: Reconstitution of Targeted Deadenylation by the Ccr4-Not Complex and the YTH Domain Protein Mmi1.
abstract: Ccr4-Not is a conserved protein complex that shortens the 3' poly(A) tails of eukaryotic mRNAs to regulate transcript stability and translation into proteins. RNA-binding proteins are thought to facilitate recruitment of Ccr4-Not to certain mRNAs, but lack of an in-vitro-reconstituted system has slowed progress in understanding the mechanistic details of this specificity. Here, we generate a fully recombinant Ccr4-Not complex that removes poly(A) tails from RNA substrates. The intact complex is more active than the exonucleases alone and has an intrinsic preference for certain RNAs. The RNA-binding protein Mmi1 is highly abundant in preparations of native Ccr4-Not. We demonstrate a high-affinity interaction between recombinant Ccr4-Not and Mmi1. Using in vitro assays, we show that Mmi1 accelerates deadenylation of target RNAs. Together, our results support a model whereby both RNA-binding proteins and the sequence context of mRNAs influence deadenylation rate to regulate gene expression.
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.
authors.forename: James A W
authors.initials: JAW
authors.lastname: Stowell
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Webster
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.
authors.forename: Alexander
authors.initials: A
authors.lastname: Kögel
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.
authors.forename: Jana
authors.initials: J
authors.lastname: Wolf
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.
authors.forename: Kathryn L
authors.initials: KL
authors.lastname: Shelley
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK. Electronic address: passmore@mrc-lmb.cam.ac.uk.
authors.forename: Lori A
authors.initials: LA
authors.lastname: Passmore
mesh_terms: D000225:Adenine / Q000378:metabolism; D000595:Amino Acid Sequence; D005095:Exoribonucleases / Q000378:metabolism; D046912:Multiprotein Complexes / Q000378:metabolism*; D011061:Poly A / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D011061:Poly A; D005095:Exoribonucleases; D000225:Adenine
keywords: Ccr4-Not; RNA; exonuclease; gene expression; poly(A) tail