Reconstitution of Targeted Deadenylation by the Ccr4-Not Complex and the YTH Domain Protein Mmi1
| Autor: | Alexander Kögel, James A.W. Stowell, Lori A. Passmore, Michael W. Webster, Jana Wolf, Kathryn L. Shelley |
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| Jazyk: | angličtina |
| Předmět: |
0301 basic medicine
Saccharomyces cerevisiae Proteins Polyadenylation exonuclease Protein domain Context (language use) poly(A) tail Saccharomyces cerevisiae Plasma protein binding Biology Article General Biochemistry Genetics and Molecular Biology 03 medical and health sciences Ccr4-Not Protein Domains Schizosaccharomyces Gene expression CCR4-NOT complex Amino Acid Sequence Adenine RNA Translation (biology) Molecular biology Recombinant Proteins 3. Good health Cell biology 030104 developmental biology Multiprotein Complexes Exoribonucleases gene expression Schizosaccharomyces pombe Proteins Poly A Protein Binding |
| Zdroj: | Cell Reports |
| ISSN: | 2211-1247 |
| DOI: | 10.1016/j.celrep.2016.10.066 |
| Popis: | Summary 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. Graphical Abstract Highlights • We purify a fully recombinant S. pombe Ccr4-Not complex • The complex has an intrinsic substrate preference, and both Ccr4 and Caf1 are active • Ccr4-Not stably interacts with the YTH domain RNA-binding protein Mmi1 • Mmi1 accelerates deadenylation of RNAs by Ccr4-Not in a sequence-specific manner Poly(A) tails regulate mRNA stability and translation. Stowell et al. show that the rate of poly(A) tail removal by a fully recombinant Ccr4-Not complex is influenced by both the substrate RNA sequence and an RNA-binding adapter protein, Mmi1. |
| Databáze: | OpenAIRE |
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