Rna15 interaction with the A-rich yeast polyadenylation signal is an essential step in mRNA 3'-end formation.
In Saccharomyces cerevisiae, four factors [cleavage factor I (CF I), CF II, polyadenylation factor I (PF I), and poly(A) polymerase (PAP)] are required for maturation of the 3' end of the mRNA. CF I and CF II are required for cleavage; a complex of PAP and PF I, which includes CF II subunits, participates in polyadenylation, along with CF I. These factors are directed to the appropriate site on the mRNA by two sequences: one A-rich and one UA-rich. CF I contains five proteins, two of which, Rna15 and Hrp1, interact with the mRNA through RNA recognition motif-type RNA binding motifs. Previous work demonstrated that the UV cross-linking of purified Hrp1 to RNA required the UA-rich element, but the contact point of Rna15 was not known. We show here that Rna15 does not recognize a particular sequence in the absence of other proteins. However, in complex with Hrp1 and Rna14, Rna15 specifically interacts with the A-rich element. The Pcf11 and Clp1 subunits of CF I are not needed to position Rna15 at this site. This interaction is essential to the function of CF I. A mutant Rna15 with decreased affinity for RNA is defective for in vitro RNA processing and lethal in vivo, while an RNA with a mutation in the A-rich element is not processed in vitro and can no longer be UV cross-linked to the Rna15 subunit assembled into CF I. Thus, the recognition of the A-rich element depends on the tethering of Rna15 through an Rna14 bridge to Hrp1 bound to the UA-rich motif. These results illustrate that the yeast 3' end is defined and processed by a mechanism surprisingly different from that used by the mammalian system.
Gross
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Department of Molecular Biology and Microbiology, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts 02111, USA.
Moore
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D020413:3' Untranslated Regions / Q000096:biosynthesis*; D019943:Amino Acid Substitution; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011061:Poly A / Q000378:metabolism*; D026723:Polyadenylation / Q000502:physiology*; D011485:Protein Binding / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis*; D016601:RNA-Binding Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity / Q000502:physiology; D039221:mRNA Cleavage and Polyadenylation Factors
D020413:3' Untranslated Regions; D005656:Fungal Proteins; C512636:HRP1 protein, S cerevisiae; D046911:Macromolecular Substances; D009687:Nuclear Proteins; C512637:PCF11 protein, S cerevisiae; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; C512638:RNA14 protein, S cerevisiae; C512635:RNA15 protein, S cerevisiae; D011061:Poly A