Sen1p Performs Two Genetically Separable Functions in Transcription and Processing of U5 Small Nuclear RNA in Saccharomyces cerevisiae by ProQuest

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The Saccharomyces cerevisiae SEN1 gene codes for a nuclear-localized superfamily I helicase. SEN1 is an ortholog of human SETX (senataxin), which has been implicated in the neurological disorders ataxia-ocular apraxia type 2 and juvenile amyotrophic lateral sclerosis. Pleiotropic phenotypes conferred by sen1 mutations suggest that Sen1p affects multiple steps in gene expression. Sen1p is embedded in a protein-protein interaction network involving direct binding to multiple partners. To test whether the interactions occur independently or in a dependent sequence, we examined interactions with the RNA polymerase II subunit Rpb1p, which is required for transcription, and Rnt1p, which is required for 3'-end maturation of many noncoding RNAs. Mutations were identified that impair one of the two interactions without impairing the other interaction. The effects of the mutants on the synthesis of U5 small nuclear RNA were analyzed. Two defects were observed, one in transcription termination and one in 3'-end maturation. Impairment of the Sen1p-Rpb1p interaction resulted in a termination defect. Impairment of the Sen1p-Rnt1p interaction resulted in a processing defect. The results suggest that the Sen1p-Rpb1p and Sen1p-Rnt1p interactions occur independently of each other and serve genetically separable purposes in targeting Sen1p to function in two temporally overlapping steps in gene expression. [PUBLICATION ABSTRACT]

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									Copyright Ó 2010 by the Genetics Society of America
DOI: 10.1534/genetics.109.110031



Sen1p Performs Two Genetically Separable Functions in Transcription and
     Processing of U5 Small Nuclear RNA in Saccharomyces cerevisiae

              Jonathan S. Finkel, Karen Chinchilla, Doris Ursic and Michael R. Culbertson1
                      Laboratories of Genetics and Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706
                                                       Manuscript received September 24, 2009
                                                      Accepted for publication October 23, 2009


                                                             ABSTRACT
                The Saccharomyces cerevisiae SEN1 gene codes for a nuclear-localized superfamily I helicase. SEN1 is an
             ortholog of human SETX (senataxin), which has been implicated in the neurological disorders ataxia-
             ocular apraxia type 2 and juvenile amyotrophic lateral sclerosis. Pleiotropic phenotypes conferred by sen1
             mutations suggest that Sen1p affects multiple steps in gene expression. Sen1p is embedded in a protein–
             protein interaction network involving direct binding to multiple partners. To test whether the interactions
             occur independently or in a dependent sequence, we examined interactions with the RNA polymerase II
             subunit Rpb1p, which is required for transcription, and Rnt1p, which is required for 39-end maturation of
             many noncoding RNAs. Mutations were identified that impair one of the two interactions without
             impairing the other interaction. The effects of the mutants on the synthesis of U5 small nuclear RNA were
             analyzed. Two defects were observed, one in transcription termination and one in 39-end maturation.
             Impairment of the Sen1p–Rpb1p interaction resulted in a termination defect. Impairment of the Sen1p–
             Rnt1p interaction resulted in a processing defect. The results suggest that the Sen1p–Rpb1p and Sen1p–
             Rnt1p interactions occur independently of each other and serve genetically separable purposes in
             targeting Sen1p to function in two temporally overlapping steps in gene expression.




P    ROTEIN–protein interaction networks contribute
       tfhe underlying basis for phenotypic pleiotropy. In
Saccharomyces cerevisiae, global studies suggest that each
                                                                             Suraweera et al. 2009). The yeast and human proteins
                                                                             are strikingly similar in their organization. Some of the
                                                                             human mutations cause changes in the ATP-helicase
protein interacts on average with five other proteins                         domain, whereas others cause changes in the N-terminal
(Grigoriev 2003), leading to a complex network of in-                        region where protein-binding domains reside. Some of
teractions involving at least 16,000 individual protein–                     the clinical differences might be caused by mutations
protein interactions that influence the functions of                          that differentially affect the function of senataxin by dis-
wild-type proteins and the phenotypes of mutants. The                        rupting different protein–protein interactions.
essential S. cerevisiae SEN1 gene codes for a nuclear-                          Sen1p interacts with the C-terminal domain of
localized nucleic acid helicase (DeMarini et al. 1992)                       Rpb1p, the largest subunit of RNA polymerase II (RNAP
that is embedded in a complex network of protein–                            II) (Myer and Young 1998); with Rad2p, a single-strand
protein interactions (Ursic et al. 2004). Furthermore,                       DNA endonuclease required for DNA repair (Habraken
mutations in SEN1 confer pleiotropic phenotypes, in-                         et al. 1993; Prakash and Prakash 2000); with Rnt1p, a
cluding defects in transcription termination, RNA pro-                       double-strand RNA cleavage enzyme involved in 59- or
cessing, and DNA repair (Steinmetz and Brow 1996,                            39-end processing (Elela et al. 1996; Chanfreau et al.
1998; Rasmussen and Culbertson 1998; Steinmetz                               1997; Lamontagne et al. 2000); and with SmD3p
et al. 2001, 2006; Ursic et al. 2004). The study of SEN1                     (Fromont-Racine et al. 1997), a subunit of the hetero-
therefore provides a useful paradigm to examine the                          heptameric Sm complex that assembles small nuclear
impact of protein–protein interactions on mutant phe-             
								
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