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Yeast Screens Identify the RNA Polymerase II CTD and SPT5 as Relevant Targets of BRCA1 Interaction

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      Abstract

      BRCA1 has been implicated in numerous DNA repair pathways that maintain genome integrity, however the function responsible for its tumor suppressor activity in breast cancer remains obscure. To identify the most highly conserved of the many BRCA1 functions, we screened the evolutionarily distant eukaryote Saccharomyces cerevisiae for mutants that suppressed the G1 checkpoint arrest and lethality induced following heterologous BRCA1 expression. A genome-wide screen in the diploid deletion collection combined with a screen of ionizing radiation sensitive gene deletions identified mutants that permit growth in the presence of BRCA1. These genes delineate a metabolic mRNA pathway that temporally links transcription elongation (SPT4, SPT5, CTK1, DEF1) to nucleopore-mediated mRNA export (ASM4, MLP1, MLP2, NUP2, NUP53, NUP120, NUP133, NUP170, NUP188, POM34) and cytoplasmic mRNA decay at P-bodies (CCR4, DHH1). Strikingly, BRCA1 interacted with the phosphorylated RNA polymerase II (RNAPII) carboxy terminal domain (P-CTD), phosphorylated in the pattern specified by the CTDK-I kinase, to induce DEF1-dependent cleavage and accumulation of a RNAPII fragment containing the P-CTD. Significantly, breast cancer associated BRCT domain defects in BRCA1 that suppressed P-CTD cleavage and lethality in yeast also suppressed the physical interaction of BRCA1 with human SPT5 in breast epithelial cells, thus confirming SPT5 as a relevant target of BRCA1 interaction. Furthermore, enhanced P-CTD cleavage was observed in both yeast and human breast cells following UV-irradiation indicating a conserved eukaryotic damage response. Moreover, P-CTD cleavage in breast epithelial cells was BRCA1-dependent since damage-induced P-CTD cleavage was only observed in the mutant BRCA1 cell line HCC1937 following ectopic expression of wild type BRCA1. Finally, BRCA1, SPT5 and hyperphosphorylated RPB1 form a complex that was rapidly degraded following MMS treatment in wild type but not BRCA1 mutant breast cells. These results extend the mechanistic links between BRCA1 and transcriptional consequences in response to DNA damage and suggest an important role for RNAPII P-CTD cleavage in BRCA1-mediated cancer suppression.

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      Most cited references 57

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      Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

      The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.
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        Cancer susceptibility and the functions of BRCA1 and BRCA2.

        Inherited mutations in BRCA1 or BRCA2 predispose to breast, ovarian, and other cancers. Their ubiquitously expressed protein products are implicated in processes fundamental to all cells, including DNA repair and recombination, checkpoint control of cell cycle, and transcription. Here, I examine what is known about the biological functions of the BRCA proteins and ask how their disruption can induce susceptibility to specific types of cancer.
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          Decapping and decay of messenger RNA occur in cytoplasmic processing bodies.

          A major pathway of eukaryotic messenger RNA (mRNA) turnover begins with deadenylation, followed by decapping and 5' to 3' exonucleolytic decay. We provide evidence that mRNA decapping and 5' to 3' degradation occur in discrete cytoplasmic foci in yeast, which we call processing bodies (P bodies). First, proteins that activate or catalyze decapping are concentrated in P bodies. Second, inhibiting mRNA turnover before decapping leads to loss of P bodies; however, inhibiting turnover at, or after, decapping, increases the abundance and size of P bodies. Finally, mRNA degradation intermediates are localized to P bodies. These results define the flux of mRNAs between polysomes and P bodies as a critical aspect of cytoplasmic mRNA metabolism and a possible site for regulation of mRNA degradation.
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            Author and article information

            Affiliations
            [1 ]Department of Surgery, Duke University Medical Center, Durham, North Carolina, United States of America
            [2 ]Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America
            [3 ]Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, United States of America
            [4 ]Gene and Molecular Therapy Laboratory, Institute of Clinical Physiology, Consiglio Nazionale delle Ricerche (CNR), CNR Research Area Via Moruzzi, Pisa, Italy
            Northwestern University, United States of America
            Author notes
            * To whom correspondence should be addressed. E-mail: benne048@ 123456mc.duke.edu

            Conceived and designed the experiments: AG JM CB. Performed the experiments: CB TW CV TS HP GH AS EM DB AF YM. Analyzed the data: JM CB. Contributed reagents/materials/analysis tools: AG CB HP AG JO. Wrote the paper: AG JM CB.

            Contributors
            Role: Academic Editor
            Journal
            PLoS ONE
            plos
            plosone
            PLoS ONE
            Public Library of Science (San Francisco, USA )
            1932-6203
            2008
            16 January 2008
            : 3
            : 1
            2174531
            18197258
            07-PONE-RA-01638R1
            10.1371/journal.pone.0001448
            (Academic Editor)
            Bennett et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
            Counts
            Pages: 15
            Categories
            Research Article
            Biochemistry/Replication and Repair
            Biochemistry/Transcription and Translation
            Genetics and Genomics/Functional Genomics
            Oncology/Breast Cancer

            Uncategorized

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