Divergent Subunit Interactions among Fungal mRNA 5′-Capping Machineries

Takagi, Toshimitsu; Cho, Eun Jung; Janoo, Rozmin; Polodny, Vladimir; Takase, Yasutaka; Keogh, Michael Christopher; Woo, Sue-Ann; Fresco-Cohen, Lucille D; Hoffman, Charles S.; Buratowski, Stephen

doi:10.1128/ec.1.3.448-457.2002

Cited by 22 publications

(30 citation statements)

References 56 publications

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“…Taken together, the data suggest that a physical connection exists between the capping apparatus and the Cdk9 complex and support a role for Cdk9 in a quality control-an elongation checkpoint-on mRNA synthesis (52). Whereas the other two capping enzymes, Pct1 and the guanylyltransferase Pce1, interact independently with the Rpb1 CTD (51,69) and with Spt5 (54), Pcm1 did not interact in a twohybrid assay with Rpb1, Spt5, or any other component of the capping machinery (Y. Pei and S. Shuman, unpublished observations). In S. cerevisiae, the cap methyltransferase Abd1 can bind directly to the phosphorylated CTD of Pol II in vitro (11,41).…”

Section: Discussionmentioning

confidence: 99%

Cyclin-Dependent Kinase 9 (Cdk9) of Fission Yeast Is Activated by the CDK-Activating Kinase Csk1, Overlaps Functionally with the TFIIH-Associated Kinase Mcs6, and Associates with the mRNA Cap Methyltransferase Pcm1 In Vivo

Pei

Singer

et al. 2006

Molecular and Cellular Biology

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Cyclin-dependent kinase 9 (Cdk9) of fission yeast is an essential ortholog of metazoan positive transcription elongation factor b (P-TEFb), which is proposed to coordinate capping and elongation of RNA polymerase II (Pol II) transcripts. Here we show that Cdk9 is activated to phosphorylate Pol II and the elongation factor Spt5 by Csk1, one of two fission yeast CDK-activating kinases (CAKs). Activation depends on Cdk9 T-loop residue Thr-212. The other CAK-Mcs6, the kinase component of transcription factor IIH (TFIIH)-cannot activate Cdk9. Consistent with the specificities of the two CAKs in vitro, the kinase activity of Cdk9 is reduced ϳ10-fold by csk1 deletion, and Cdk9 complexes from csk1⌬ but not csk1 ؉ cells can be activated by Csk1 in vitro. A cdk9 T212A mutant is viable but phenocopies conditional growth defects of csk1⌬ strains, indicating a role for Csk1-dependent activation of Cdk9 in vivo. A cdk9 T212A mcs6 S165A strain, in which neither Cdk9 nor Mcs6 can be activated by CAK, has a synthetic growth defect, implying functional overlap between the two CDKs, which have distinct but overlapping substrate specificities. Cdk9 forms complexes in vivo with the essential cyclin Pch1 and with Pcm1, the mRNA cap methyltransferase. The carboxyl-terminal region of Cdk9, through which it interacts with another capping enzyme, the RNA triphosphatase Pct1, is essential. Together, the data support a proposed model whereby Cdk9/Pch1-the third essential CDK-cyclin complex described in fission yeasthelps to target the capping apparatus to the transcriptional elongation complex.

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Section: Discussionmentioning

confidence: 99%

Cyclin-Dependent Kinase 9 (Cdk9) of Fission Yeast Is Activated by the CDK-Activating Kinase Csk1, Overlaps Functionally with the TFIIH-Associated Kinase Mcs6, and Associates with the mRNA Cap Methyltransferase Pcm1 In Vivo

Pei

Singer

et al. 2006

Molecular and Cellular Biology

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“…We would emphasize here that orthologous enzymes from different species cannot be presumed to have the same inherent biochemical properties, or to engage in the same repertoire of protein-protein interactions that impact on their enzymatic activity. Indeed, this point is underscored by the demonstration that the orthologous triphosphatase and guanylyltransferase components of the fungal mRNA capping apparatus interact differently with each other, and with the pol II CTD, in S. pombe versus S. cerevisiae (33,44,45). Rather than viewing the clearly demonstrated capacity of S. pombe Fcp1 to act on CTD phosphopeptides in vitro as an aberration, we see S. pombe Fcp1 as being liberated from the dependence on protein-protein contacts (e.g.…”

Section: Discussionmentioning

confidence: 99%

Schizosaccharomyces pombe Carboxyl-terminal Domain (CTD) Phosphatase Fcp1

Hausmann

Erdjument‐Bromage

Shuman

2004

Journal of Biological Chemistry

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Schizosaccharomyces pombe 5 and Ser 2 undergo waves of phosphorylation and dephosphorylation during the transcription cycle, the purpose of which may be to regulate the transition from initiation to elongation modes and to control the recruitment, activity, and egress of the various mRNA-processing machines that act on the nascent transcript (1-3). The dynamic phosphorylation state of the CTD reflects a kinetic balance between the multiple CTD kinase and CTD phosphatase activities found in eukaryotic cells (2, 4 -7).The enzyme Fcp1 is the major protein serine phosphatase responsible for removing phosphates from the CTD (2, 8 -16). Fcp1 orthologs are present in all known eukaryal proteomes, and the enzyme is essential for cell viability in budding and fission yeast (10,15 Fig. 1) and a C-terminal BRCT (BRCA1 C terminus) domain, both of which are essential for Fcp1 phosphatase activity in vivo and in vitro (14,16,19). Although the catalytic contributions made by the BRCT domain are not known, a recent report implicates the BRCT domain in the initial binding of Fcp1 to the phosphorylated CTD (20). A wealth of data implicate the FCPH domain as the seat of the phosphatase active site.A short conserved peptide motif ( 170 DLDQT 174 in S. pombe Fcp1) located near the N-terminal margin of the FCPH domain corresponds to the signature sequence of the DXDX(T/V) family of metal-dependent phosphohydrolases and phosphotransferases (21-23) (Fig. 1). DXDX(T/V) enzymes act via an acylphosphoenzyme intermediate in which the phosphate is linked to the first aspartate in the DXDX(T/V) motif (21, 24 -26). The second aspartate in the DXDX(T/V) motif serves as a general acid catalyst that donates a proton to the leaving group during formation of the acyl-phosphate intermediate. Formation and hydrolysis of the acyl-phosphoenzyme proceed via an associative mechanism through a pentacoordinate phosphorane transition state (27). Insights to the identity of the catalytic functional groups on the enzyme have emerged from crystallographic snapshots of DXDX(T/V) acyl-phosphatases captured at defined stages of the reaction cycle (25,27,28) and from mutational analyses of exemplary DXDX(T/V) family members, including Fcp1.We previously employed site-directed mutagenesis to locate candidate catalytic residues of S. pombe Fcp1 (16,18

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“…31 This Lsm domain is followed by a long C-terminal tail, which may contain another RNAbinding domain involved in tRNA splicing. 31 Lsm12 associates with the transcription factor Stb5, which interacts with the transcriptional repressor Sin3, 49 the mRNA guanylyltransferase Ceg1 of an mRNA capping enzyme complex including the triphosphatase Cet1, 50 and the RNAbinding protein Puf3. Puf3 is a member of the family of pumilio-like Puf repeat containing proteins 51 and functions as transcript-specific regulator of mRNA degradation.…”

Section: Analysis Of Pbp1 Functionsmentioning

confidence: 99%

An Integrative Approach to Gain Insights into the Cellular Function of Human Ataxin-2

Ralser

Albrecht

Nonhoff

et al. 2005

Journal of Molecular Biology

134

145

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Spinocerebellar ataxia type 2 (SCA2) is a hereditary neurodegenerative disorder caused by a trinucleotide expansion in the SCA2 gene, encoding a polyglutamine stretch in the gene product ataxin-2 (ATX2), whose cellular function is unknown. However, ATX2 interacts with A2BP1, a protein containing an RNA-recognition motif, and the existence of an interaction motif for the C-terminal domain of the poly(A)-binding protein (PABC) as well as an Lsm (Like Sm) domain in ATX2 suggest that ATX2 like its yeast homolog Pbp1 might be involved in RNA metabolism. Here, we show that, similar to Pbp1, ATX2 suppresses the petite (pet K ) phenotype of Dmrs2 yeast strains lacking mitochondrial group II introns. This finding points to a close functional relationship between the two homologs. To gain insight into potential functions of ATX2, we also generated a comprehensive protein interaction network for Pbp1 from publicly available databases, which implicates Pbp1 in diverse RNA-processing pathways. The functional relationship of ATX2 and Pbp1 is further corroborated by the experimental confirmation of the predicted interaction of ATX2 with the cytoplasmic poly(A)-binding protein 1 (PABP) using yeast-2-hybrid analysis as well as co-immunoprecipitation experiments. Immunofluorescence studies revealed that ATX2 and PABP co-localize in mammalian cells, remarkably, even under conditions in which PABP accumulates in distinct cytoplasmic foci representing sites of mRNA triage.

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Divergent Subunit Interactions among Fungal mRNA 5′-Capping Machineries

Cited by 22 publications

References 56 publications

Cyclin-Dependent Kinase 9 (Cdk9) of Fission Yeast Is Activated by the CDK-Activating Kinase Csk1, Overlaps Functionally with the TFIIH-Associated Kinase Mcs6, and Associates with the mRNA Cap Methyltransferase Pcm1 In Vivo

Cyclin-Dependent Kinase 9 (Cdk9) of Fission Yeast Is Activated by the CDK-Activating Kinase Csk1, Overlaps Functionally with the TFIIH-Associated Kinase Mcs6, and Associates with the mRNA Cap Methyltransferase Pcm1 In Vivo

Schizosaccharomyces pombe Carboxyl-terminal Domain (CTD) Phosphatase Fcp1

An Integrative Approach to Gain Insights into the Cellular Function of Human Ataxin-2

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