The formation of polyadenylated 3' termini is essential to the biogenesis of eukaryotic mRNA. Defects in this processing decrease the amount of mRNA available for translation into protein, and thus interfere with normal cell function. Regulation at the level of polyadenylation can affect the amount and type of mRNA synthesized from a transcriptional unit. In this way, it becomes part of a cell's response to external stimuli governing growth, differentiation, and tissue-specific gene expression. For these reasons, it is important to understand the mechanism and regulation of polyadenylation. In mammals, maturation of the mRNA 3' end involves cleavage of precursor and addition of adenylate residues to the new end. This processing has been examined in vivo and in cell-free systems. This research has defined how precursor RNA is cleaved and polyadenylated and what signal sequences on precursor direct the processing. Further progress in characterizing the processing activities is severely limited by the lack of suitable molecular genetics in mammalian systems. However, little is known about 3' end formation in eukaryotes such as the yeast S. cerevisiae, which is more amenable to genetic analysis. The primary goal of this research is a thorough molecular analysis of polyadenylation in yeast using a combination of genetics and biochemistry. The results of these studies will allow us to compare the processing mechanism in yeast to that used in metazoans. Understanding the basic mechanism of polyadenylation will make it feasible to ask how the process is regulated as the physiological state of the cell changes, and how this regulation affects mRNA level globally or specifically.
The aims of this research are: I. Development of a genetic screen to identify the cis-acting RNA sequences and the trans-acting factors necessary for polyadenylation in yeast. Using primary a colorimetric assay, it should be possible to determine the minimal sequences needed for polyadenylation and to screen for mutants which do not recognize a functional polyadenylation signal. The mutant genes can then be identified and cloned by complementary transformation with wild type genes. II. Clarification of the role transcription termination in the formation of the mature 3' end of yeast mRNA. This experiment will use a nuclear run-on assay to detect any transcription beyond the poly(A) addition site. III. Development of an in vitro system in yeast which correctly polyadenylates precursor RNA. The goal of these experiments is to determine the molecular pathway of polyadenylation in yeast, to complement the genetic analysis of signal sequences and trans- acting factors, and to provide an assay for the purification of processing activities from crude extracts.

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National Institute of General Medical Sciences (NIGMS)
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Molecular Biology Study Section (MBY)
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Graber, Joel H; Nazeer, Fathima I; Yeh, Pei-chun et al. (2013) DNA damage induces targeted, genome-wide variation of poly(A) sites in budding yeast. Genome Res 23:1690-703
Ezeokonkwo, Chukwudi; Ghazy, Mohamed A; Zhelkovsky, Alexander et al. (2012) Novel interactions at the essential N-terminus of poly(A) polymerase that could regulate poly(A) addition in Saccharomyces cerevisiae. FEBS Lett 586:1173-8
Schmid, Manfred; Poulsen, Mathias Bach; Olszewski, Pawel et al. (2012) Rrp6p controls mRNA poly(A) tail length and its decoration with poly(A) binding proteins. Mol Cell 47:267-80
Gordon, James M B; Shikov, Sergei; Kuehner, Jason N et al. (2011) Reconstitution of CF IA from overexpressed subunits reveals stoichiometry and provides insights into molecular topology. Biochemistry 50:10203-14
Kuehner, Jason N; Pearson, Erika L; Moore, Claire (2011) Unravelling the means to an end: RNA polymerase II transcription termination. Nat Rev Mol Cell Biol 12:283-94
Ezeokonkwo, Chukwudi; Zhelkovsky, Alexander; Lee, Rosanna et al. (2011) A flexible linker region in Fip1 is needed for efficient mRNA polyadenylation. RNA 17:652-64
Zhao, J; Kessler, M; Helmling, S et al. (1999) Pta1, a component of yeast CF II, is required for both cleavage and poly(A) addition of mRNA precursor. Mol Cell Biol 19:7733-40
Aranda, A; Perez-Ortin, J E; Moore, C et al. (1998) Transcription termination downstream of the Saccharomyces cerevisiae FBP1 [changed from FPB1] poly(A) site does not depend on efficient 3'end processing. RNA 4:303-18
Zhao, J; Kessler, M M; Moore, C L (1997) Cleavage factor II of Saccharomyces cerevisiae contains homologues to subunits of the mammalian Cleavage/ polyadenylation specificity factor and exhibits sequence-specific, ATP-dependent interaction with precursor RNA. J Biol Chem 272:10831-8
Kessler, M M; Henry, M F; Shen, E et al. (1997) Hrp1, a sequence-specific RNA-binding protein that shuttles between the nucleus and the cytoplasm, is required for mRNA 3'-end formation in yeast. Genes Dev 11:2545-56

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