Function.
Articles
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.
