UMMS Affiliation

Department of Molecular Genetics and Microbiology

Publication Date

5-4-2004

Document Type

Article

Subjects

Base Sequence; Binding Sites; Carrier Proteins; DNA, Fungal; Exoribonucleases; Genes, Fungal; Genetic Complementation Test; Peptide Mapping; *Polyadenylation; Polynucleotide Adenylyltransferase; Protein Structure, Tertiary; RNA, Fungal; RNA, Messenger; RNA-Binding Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Two-Hybrid System Techniques

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

Posttranscriptional maturation of the 3' end of eukaryotic pre-mRNAs occurs as a three-step pathway involving site-specific cleavage, polymerization of a poly(A) tail, and trimming of the newly synthesized tail to its mature length. While most of the factors essential for catalyzing these reactions have been identified, those that regulate them remain to be characterized. Previously, we demonstrated that the yeast protein Pbp1p associates with poly(A)-binding protein (Pab1p) and controls the extent of mRNA polyadenylation. To further elucidate the function of Pbp1p, we conducted a two-hybrid screen to identify factors with which it interacts. Five genes encoding putative Pbp1p-interacting proteins were identified, including (i) FIR1/PIP1 and UFD1/PIP3, genes encoding factors previously implicated in mRNA 3'-end processing; (ii) PBP1 itself, confirming directed two-hybrid results and suggesting that Pbp1p can multimerize; (iii) DIG1, encoding a mitogen-activated protein kinase-associated protein; and (iv) PBP4 (YDL053C), a previously uncharacterized gene. In vitro polyadenylation reactions utilizing extracts derived from fir1 Delta and pbp1 Delta cells and from cells lacking the Fir1p interactor, Ref2p, demonstrated that Pbp1p, Fir1p, and Ref2p are all required for the formation of a normal-length poly(A) tail on precleaved CYC1 pre-mRNA. Kinetic analyses of the respective polyadenylation reactions indicated that Pbp1p is a negative regulator of poly(A) nuclease (PAN) activity and that Fir1p and Ref2p are, respectively, a positive regulator and a negative regulator of poly(A) synthesis. We suggest a model in which these three factors and Ufd1p are part of a regulatory complex that exploits Pab1p to link cleavage and polyadenylation factors of CFIA and CFIB (cleavage factors IA and IB) to the polyadenylation factors of CPF (cleavage and polyadenylation factor).

Rights and Permissions

Citation: Mol Cell Biol. 2004 May;24(10):4196-206.

Related Resources

Link to Article in PubMed

Journal/Book/Conference Title

Molecular and cellular biology

PubMed ID

15121841

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