title: Structural and biochemical analysis of the assembly and function of the yeast pre-mRNA 3' end processing complex CF I.
abstract: The accuracy of the 3'-end processing by cleavage and polyadenylation is essential for mRNA biogenesis and transcription termination. In yeast, two poorly conserved neighboring elements upstream of cleavage sites are important for accuracy and efficiency of this process. These two RNA sequences are recognized by the RNA binding proteins Hrp1 and Rna15, but efficient processing in vivo requires a bridging protein (Rna14), which forms a stable dimer of hetero-dimers with Rna15 to stabilize the RNA-protein complex. We earlier reported the structure of the ternary complex of Rna15 and Hrp1 bound to the RNA processing element. We now report the use of solution NMR to study the interaction of Hrp1 with the Rna14-Rna15 heterodimer in the presence and absence of 3'-end processing signals. By using methyl selective labeling on Hrp1, in vivo activity and pull-down assays, we were able to study this complex of several hundred kDa, identify the interface within Hrp1 responsible for recruitment of Rna14 and validate the functional significance of this interaction through structure-driven mutational analysis.
authors.affiliation: Department of Chemistry and Biochemistry, University of Washington, Seattle, WA 98195, USA.
authors.forename: Ravi Pratap
authors.initials: RP
authors.lastname: Barnwal
authors.forename: Susan D
authors.initials: SD
authors.lastname: Lee
authors.forename: Claire
authors.initials: C
authors.lastname: Moore
authors.forename: Gabriele
authors.initials: G
authors.lastname: Varani
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008745:Methylation; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D026721:RNA 3' End Processing / Q000235:genetics*; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics*; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D039221:mRNA Cleavage and Polyadenylation Factors / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D046912:Multiprotein Complexes; D050505:Mutant Proteins; D012322:RNA Precursors; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D039221:mRNA Cleavage and Polyadenylation Factors