Ssu72 Is an RNA Polymerase II CTD Phosphatase

Krishnamurthy, Shankarling; He, Xiaohua; Reyes-Reyes, Mariela; Moore, Claire; Hampsey, Michael

doi:10.1016/s1097-2765(04)00235-7

Cited by 215 publications

(259 citation statements)

References 44 publications

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“…Upon binding to Pta1p in CPF, Ssu72p functions as a phosphatase with specificity for the fifth serine of the heptapeptide repeat in the CTD [223,224]. However, this activity is not required for 3′-end processing, but it may affect its role in 5′-end capping [224].…”

Section: Ssu72pmentioning

confidence: 99%

Protein factors in pre-mRNA 3′-end processing

Mandel

Bai

Tong

2007

Cell. Mol. Life Sci.

359

482

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Most eukaryotic mRNA precursors (pre-mRNAs) must undergo extensive processing, including cleavage and polyadenylation at the 3′-end. Processing at the 3′-end is controlled by sequence elements in the pre-mRNA (cis elements) as well as protein factors. Despite the seeming biochemical simplicity of the processing reactions, more than 14 proteins have been identified for the mammalian complex, and more than 20 proteins have been identified for the yeast complex. The 3′-end processing machinery also has important roles in transcription and splicing. The mammalian machinery contains several sub-complexes, including cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage factor I (CF I m ), and cleavage factor II (CF II m ). Additional protein factors include poly(A) polymerase (PAP), poly(A) binding protein (PABP), symplekin, and the C-terminal domain (CTD) of RNA polymerase II largest subunit. The yeast machinery includes cleavage factor IA (CF IA), cleavage factor IB (CF IB), and cleavage and polyadenylation factor (CPF).

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Section: Ssu72pmentioning

confidence: 99%

Protein factors in pre-mRNA 3′-end processing

Mandel

Bai

Tong

2007

Cell. Mol. Life Sci.

359

482

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“…The pellet was rinsed in 0.5 ml of 1 M Tris base without resuspension, dissolved in 100 l of 2ϫ sample buffer (4% SDS, 0.1 M Tris HCl, pH 6.8, 4 mM EDTA, 20% glycerol, 2% 2-mercaptoethanol), and dissociated by heating for 10 min at 37°C. Protein samples corresponding to 0.1-0.2 A 600 units were separated in 8% SDSpolyacrylamide gels, and Western blot analyses were performed as described previously (29). Pho87-3xHA and Pho91-3xHA were assayed by incubation using a 1:3000 dilution of anti-HA antibody followed by incubation with horseradish peroxidase-conjugated anti-mouse antibody.…”

Section: Extract Preparation and Western Blot Analyses-yeastmentioning

confidence: 99%

The Rsp5 E3 Ligase Mediates Turnover of Low Affinity Phosphate Transporters in Saccharomyces cerevisiae

Estrella¹,

Krishnamurthy²,

Timme³

et al. 2008

Journal of Biological Chemistry

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In an effort to identify novel components of the PHO regulon in Saccharomyces cerevisiae, we have isolated and characterized suppressors of the Pho ؊ phenotype associated with deletion of the Pho4 transcriptional activator. Here we report that either a defective form of the Rsp5 E3 ubiquitin ligase or deletion of the End3 component of the endocytic pathway restores growth of the pho4⌬ mutant in the presence of limiting inorganic phosphate (P i ). The spa1-1 suppressor allele of RSP5 encodes a phenylalanine-to-valine replacement at position 748 (F748V) within the catalytic HECT domain of Rsp5. Consistent with suppression due to impaired ubiquitin ligase activity, the heat-sensitive growth defect of the spa1-1 mutant is suppressed either by overexpression of ubiquitin or by osmotic stabilization. Western blot analyses revealed that the cellular levels of the Pho87 and Pho91 low affinity P i are markedly increased in the spa1-1 mutant, yet Pho84 high affinity P i transporter levels are unaffected. Furthermore, Pho87 and Pho91 are ubiquitinated in vivo in an Rsp5-dependent manner, and the Pho ؉ phenotype of the spa1-1 suppressor is dependent upon Pho87 and Pho91. We conclude that turnover of the low affinity P i transporters is initiated by Rsp5-mediated ubiquitination followed by internalization and degradation by the endocytic pathway.The yeast Saccharomyces cerevisiae has evolved an elaborate system to sense, acquire, and store inorganic phosphate (P i ) in response to its availability in the extracellular environment (for review, see Refs. 1 and 2). The PHO system consists of (i) the Pho3, Pho5, Pho11, and Pho12 acid phosphatases that are localized to the periplasmic space, (ii) the Pho8 and Pho13 alkaline phosphatases that are localized to the vacuole and periplasm, respectively, (iii) the high affinity plasma membrane P i transporters Pho84 and Pho89, which are regulated in response to P i availability, and the low affinity, constitutively expressed P i transporters Pho87, Pho90, and Pho91, (iv) Git1, a transporter that scavenges glycerophosphoinositol derived from secreted phosphatidylinositol, thereby replenishing inositol, P i , and glycerol (3), and (v) the PHM proteins that are involved in the synthesis and breakdown of polyphosphate, a storage form of P i in the vacuole (for review, see Ref. 4).The PHO regulon is responsible for scavenging P i and has been most extensively studied with regard to regulation of PHO5 expression. The Pho84 transporter and the Pho80 -Pho85 cyclin/cyclin-dependent kinase are negative regulators of PHO5 transcription, whereas the Pho81 inhibitor of Pho80 -Pho85 and the Pho4 transcriptional activator are positive regulators. In the presence of high external concentrations of P i , Pho85 phosphorylates Pho4, resulting in Pho4 nuclear export via the Msn5 exportin and cytoplasmic retention such that PHO5 is repressed (5). Conversely, when P i concentrations are low, Pho81 inhibits Pho85 activity. Pho4 remains unphosphorylated and has high affinity for the Pse1 importin, resulting i...

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“…In contrast to the four kinases, there are two known CTD phosphatases in yeast, . Biochemical as well as genetic evidence indicates that both proteins may function as CTD phosphatases in the context of transcription (16,19,20,(25)(26)(27)(28)(29)(30)(31).It is the interplay between these CTD kinases and phosphatases that influences which factors associate with transcribing polymerase and when during the transcription cycle such associations occur. As mentioned, the recruitment of capping enzyme early in the transcription cycle is believed to be one consequence of CTD phosphorylation on Ser5 by Kin28 (6,7).…”

mentioning

confidence: 99%

Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II: Novel PhosphoCTD-Associating Proteins in the Yeast Proteome

2004

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CTD kinase I (CTDK-I) of Saccharomyces cerevisiae is required for normal phosphorylation of the C-terminal repeat domain (CTD) on elongating RNA polymerase II. To elucidate cellular roles played by this kinase and the hyperphosphorylated CTD (phosphoCTD) it generates, we systematically searched yeast extracts for proteins that bound to the phosphoCTD made by CTDK-I in vitro. Initially, using a combination of far-western blotting and phosphoCTD affinity chromatography, we discovered a set of novel phosphoCTD-associating proteins (PCAPs) implicated in a variety of nuclear functions. We identified the phosphoCTD-interacting domains of a number of these PCAPs, and in several test cases (namely, Set2, Ssd1, and Hrr25) adduced evidence that phosphoCTD binding is functionally important in vivo. Employing surface plasmon resonance (BIACORE) analysis, we found that recombinant versions of these and other PCAPs bind preferentially to CTD repeat peptides carrying SerPO 4 residues at positions 2 and 5 of each seven amino acid repeat, consistent with the positional specificity of CTDK-I in vitro [Jones, J. C., et al. (2004) J. Biol. Chem. 279, 24957-24964]. Subsequently, we used a synthetic CTD peptide with three doubly phosphorylated repeats (2,5P) as an affinity matrix, greatly expanding our search for PCAPs. This resulted in identification of approximately 100 PCAPs and associated proteins representing a wide range of functions (e.g., transcription, RNA processing, chromatin structure, DNA metabolism, protein synthesis and turnover, RNA degradation, snRNA modification, and snoRNP biogenesis). The varied nature of these PCAPs and associated proteins points to an unexpectedly diverse set of connections between Pol II elongation and other processes, conceptually expanding the role played by CTD phosphorylation in functional organization of the nucleus.The carboxyl-terminal repeat domain (CTD) 1 of the largest subunit of eukaryotic RNA polymerase II (Pol II) comprises tandem repeats of the consensus heptapeptide YSPTSPS. This highly conserved sequence, which is repeated 26 times in yeast and 52 times in mammals, is essential for viability. Phosphorylation of the CTD regulates the activities of the polymerase: only Pol II with an unphosphorylated CTD (Pol IIA) is thought to be able to assemble into preinitiation complexes at the promoter, whereas elongating polymerase has a hyperphosphorylated CTD (Pol IIO) (1,2). The serines at positions 2 and 5 within the heptad repeat represent major sites of phosphorylation during transcription. † Supported by National Institutes of Health Grant GM40505.© 2004 American Chemical Society * To whom correspondence should be addressed. . E-mail: arno@biochem.duke.edu. 1 Abbreviations: CTDK-I, CTD kinase I; Pol II, RNA polymerase II; CTD, C-terminal repeat domain; phosphoCTD, hyperphosphorylated CTD; PCTD, phosphoCTD; PCAP, phosphoCTD-associating protein; PCID, phosphoCTD-interacting domain; SGD, Saccharomyces Genome Database; SPR, surface plasmon resonance; RU, response units. NIH Publi...

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Ssu72 Is an RNA Polymerase II CTD Phosphatase

Cited by 215 publications

References 44 publications

Protein factors in pre-mRNA 3′-end processing

Protein factors in pre-mRNA 3′-end processing

The Rsp5 E3 Ligase Mediates Turnover of Low Affinity Phosphate Transporters in Saccharomyces cerevisiae

Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II: Novel PhosphoCTD-Associating Proteins in the Yeast Proteome

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