Function.
Articles about Human Ssu72
Ssu72 Is an RNA polymerase II CTD phosphatase.
Krishnamurthy S, He X, Reyes-Reyes M, Moore C, Hampsey M. Department of Biochemistry, Division of Nucleic Acids Enzymology, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
Phosphorylation of serine-2 (S2) and serine-5 (S5) of the C-terminal domain (CTD) of RNA polymerase II (RNAP II) is a dynamic process that regulates the transcription cycle and coordinates recruitment of RNA processing factors. The Fcp1 CTD phosphatase catalyzes dephosphorylation of S2-P. Here, we report that Ssu72, a component of the yeast cleavage/polyadenylation factor (CPF) complex, is a CTD phosphatase with specificity for S5-P. Ssu72 catalyzes CTD S5-P dephosphorylation in association with the Pta1 component of the CPF complex, although its essential role in 3' end processing is independent of catalytic activity. Depletion of Ssu72 impairs transcription in vitro, and this defect can be rescued by recombinant, catalytically active Ssu72. We propose that Ssu72 has a dual role in transcription, one as a CTD S5-P phosphatase that regenerates the initiation-competent, hypophosphorylated form of RNAP II and the other as a factor necessary for cleavage of pre-mRNA and efficient transcription termination.
Conserved and specific functions of mammalian ssu72.
St-Pierre B, Liu X, Kha LC, Zhu X, Ryan O, Jiang Z, Zacksenhaus E. Division of Cell and Molecular Biology, Toronto General Research Institute, University Health Network 67 College Street, Room 407, Toronto, Ontario, Canada M5G 2m1.
We describe the cloning and characterization of a human homolog of the yeast transcription/RNA-processing factor Ssu72, following a yeast two-hybrid screen for pRb-binding factors in the prostate gland. Interaction between hSsu72 and pRb was observed in transfected mammalian cells and involved multiple domains in pRb; however, so far, mutual effects of these two factors could not be demonstrated. Like the yeast counterpart, mammalian Ssu72 associates with TFIIB and the yeast cleavage/polyadenylation factor Pta1, and exhibits intrinsic phosphatase activity. Mammals contain a single ssu72 gene and a few pseudogenes. During mouse embryogenesis, ssu72 was highly expressed in the nervous system and intestine; high expression in the nervous system persisted in adult mice and was also readily observed in multiple human tumor cell lines. Both endogenous and ectopically expressed mammalian Ssu72 proteins resided primarily in the cytoplasm and only partly in the nucleus. Interestingly, fusion to a strong nuclear localization signal conferred nuclear localization only in a fraction of transfected cells, suggesting active tethering in the cytoplasm. Suppression of ssu72 expression in mammalian cells by siRNA did not reduce proliferation/survival, and its over-expression did not affect transcription of candidate genes in transient reporter assays. Despite high conservation, hssu72 was unable to rescue an ssu72 lethal mutation in yeast. Together, our results highlight conserved and mammalian specific characteristics of mammalian ssu72.
Role for the Ssu72 C-terminal domain phosphatase in RNA polymerase II transcription elongation.
Reyes-Reyes M, Hampsey M. Department of Biochemistry, Robert Wood Johnson Medical School, 683 Hoes Lane West, Piscataway, NJ 08854, USA.
The RNA polymerase II (RNAP II) transcription cycle is accompanied by changes in the phosphorylation status of the C-terminal domain (CTD), a reiterated heptapeptide sequence (Y(1)S(2)P(3)T(4)S(5)P(6)S(7)) present at the C terminus of the largest RNAP II subunit. One of the enzymes involved in this process is Ssu72, a CTD phosphatase with specificity for serine-5-P. Here we report that the ssu72-2-encoded Ssu72-R129A protein is catalytically impaired in vitro and that the ssu72-2 mutant accumulates the serine-5-P form of RNAP II in vivo. An in vitro transcription system derived from the ssu72-2 mutant exhibits impaired elongation efficiency. Mutations in RPB1 and RPB2, the genes encoding the two largest subunits of RNAP II, were identified as suppressors of ssu72-2. The rpb1-1001 suppressor encodes an R1281A replacement, whereas rpb2-1001 encodes an R983G replacement. This information led us to identify the previously defined rpb2-4 and rpb2-10 alleles, which encode catalytically slow forms of RNAP II, as additional suppressors of ssu72-2. Furthermore, deletion of SPT4, which encodes a subunit of the Spt4-Spt5 early elongation complex, also suppresses ssu72-2, whereas the spt5-242 allele is suppressed by rpb2-1001. These results define Ssu72 as a transcription elongation factor. We propose a model in which Ssu72 catalyzes serine-5-P dephosphorylation subsequent to addition of the 7-methylguanosine cap on pre-mRNA in a manner that facilitates the RNAP II transition into the elongation stage of the transcription cycle.
Functional interactions between the transcription and mRNA 3' end processing machineries mediated by Ssu72 and Sub1.
He X, Khan AU, Cheng H, Pappas DL Jr, Hampsey M, Moore CL. Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Transcription and processing of pre-mRNA are coupled events. By using a combination of biochemical, molecular, and genetic methods, we have found that the phylogenetically conserved transcription factor Ssu72 is a component of the cleavage/polyadenylation factor (CPF) of Saccharomyces cerevisiae. Our results demonstrate that Ssu72 is required for 3' end cleavage of pre-mRNA but is dispensable for poly(A) addition and RNAP II termination. The in vitro cleavage defect caused by depletion of Ssu72 from cells can be rescued by addition of recombinant Ssu72. Ssu72 interacts physically and genetically with the Pta1 subunit of CPF. Overexpression of PTA1 causes synthetic lethality in an ssu72-3 mutant. Moreover, Sub1, which has been implicated in transcription initiation and termination, also interacts with Pta1, and overexpression of SUB1 suppresses the growth and processing defect of a pta1 mutation. Physical interactions of Ssu72 and Sub1 with Pta1 are mutually exclusive. Based on the interactions of Ssu72 and Sub1 with both the Pta1 of CPF and the TFIIB component of the initiation complex, we present a model describing how these novel connections between the transcription and 3' end processing machineries might facilitate transitions in the RNAP II transcription cycle.
Mutational analysis of yeast TFIIB. A functional relationship between Ssu72 and Sub1/Tsp1 defined by allele-specific interactions with TFIIB.
Wu WH, Pinto I, Chen BS, Hampsey M. Department of Biochemistry, Louisiana State University Medical Center, Shreveport, Louisiana 71130, USA.
TFIIB is an essential component of the RNA polymerase II core transcriptional machinery. Previous studies have defined TFIIB domains required for interaction with other transcription factors and for basal transcription in vitro. In the study reported here we investigated the TFIIB structural requirements for transcription initiation in vivo. A library of sua7 mutations encoding altered forms of yeast TFIIB was generated by error-prone polymerase chain reaction and screened for conditional growth defects. Twenty-two single amino acid replacements in TFIIB were defined and characterized. These replacements are distributed throughout the protein and occur primarily at phylogenetically conserved positions. Most replacements have little or no effect on the steady-state protein levels, implying that each affects TFIIB function rather than synthesis or stability. In contrast to the initial sua7 mutants, all replacements, with one exception, have no effect on start site selection, indicating that specific TFIIB structural defects affect transcriptional accuracy. This collection of sua7 alleles, including the initial sua7 alleles, was used to investigate the allele specificity of interactions between ssu72 and sub1, both of which were initially identified as either suppressors (SUB1 2mu) or enhancers (sub1Delta, ssu72-1) of sua7 mutations. We show that the interactions of ssu72-1 and sub1Delta with sua7 are allele specific; that the allele specificities of ssu72 and sub1 overlap; and that each of the sua7 alleles that interacts with ssu72 and sub1 affects the accuracy of transcription start site selection. These results demonstrate functional interactions among TFIIB, Ssu72, and Sub1 and suggest that these interactions play a role in the mechanism of start site selection by RNA polymerase II.
