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![RNA polymerase II structure. (a) Side view of the core Pol II crystal structure containing all twelve subunits and displaying the RNA exit channel (bold arrow) and the positioning of the CTD adapted from Armache et al. [71]. Cartoon in the upper right displays the color coding for the Pol II subunits used in the crystal structure. (b) Illustration of the relative length(s) between the CTD in various conformations and the core Pol II adapted from Meinhart et al. [72]. RNA positioning (red) upon exit of the Pol II and the positioning of the DNA template (blue) upstream and downstream of the core Pol II are also displayed. (c) Known modifications possible on the Pol II CTD are displayed. Glycosylation and phosphorylation are mutually exclusive modifications. Structural images of a heptad repeat in the cis- and trans-conformation are also shown [73–75]. G: β-O-linked N-acetylglucosamine [76]; P: O-linked phosphate.](profile/David-Zhang-2/publication/224920065/figure/fig1/AS:203065014657024@1425425880491/RNA-polymerase-II-structure-a-Side-view-of-the-core-Pol-II-crystal-structure.png)
RNA polymerase II structure. (a) Side view of the core Pol II crystal structure containing all twelve subunits and displaying the RNA exit channel (bold arrow) and the positioning of the CTD adapted from Armache et al. [71]. Cartoon in the upper right displays the color coding for the Pol II subunits used in the crystal structure. (b) Illustration of the relative length(s) between the CTD in various conformations and the core Pol II adapted from Meinhart et al. [72]. RNA positioning (red) upon exit of the Pol II and the positioning of the DNA template (blue) upstream and downstream of the core Pol II are also displayed. (c) Known modifications possible on the Pol II CTD are displayed. Glycosylation and phosphorylation are mutually exclusive modifications. Structural images of a heptad repeat in the cis- and trans-conformation are also shown [73–75]. G: β-O-linked N-acetylglucosamine [76]; P: O-linked phosphate.
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The C-terminal domain (CTD) of RNA polymerase II (Pol II) consists of conserved heptapeptide repeats that function as a binding platform for different protein complexes involved in transcription, RNA processing, export, and chromatin remodeling. The CTD repeats are subject to sequential waves of posttranslational modifications during specific stage...
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The C-terminal domain (CTD) of the large subunit of RNA polymerase II is a platform for mRNA processing factors and links gene transcription to mRNA capping, splicing and polyadenylation. Pcf11, an essential component of the mRNA cleavage factor IA, contains a CTD-interaction domain that binds in a phospho-dependent manner to the heptad repeats wit...
Citations
... Furthermore, introducing the Nrd1 truncates resulted in a significant improvement in the diminished RLS observed in the hpr1Δ strain ( Figure S2). In addition, RLS was considerably decreased (Zhang et al., 2012), failed to restore RLS severity in the tho2Δ strain, suggesting that general 3′-end processing factors, Fip1 and Pap1, affect lifespan in a Tho2-dependent manner ( Figure S3). Overall, these results strongly suggested that the interaction of Nrd1 with RNA polymerase II is necessary for the THO complex-dependent lifespan control pathway. ...
The relationship between aging and RNA biogenesis and trafficking is attracting growing interest, yet the precise mechanisms are unknown. The THO complex is crucial for mRNA cotranscriptional maturation and export. Herein, we report that the THO complex is closely linked to the regulation of lifespan. Deficiencies in Hpr1 and Tho2, components of the THO complex, reduced replicative lifespan (RLS) and are linked to a novel Sir2‐independent RLS control pathway. Although transcript sequestration in hpr1Δ or tho2Δ mutants was countered by exosome component Rrp6, loss of this failed to mitigate RLS defects in hpr1Δ. However, RLS impairment in hpr1Δ or tho2Δ was counteracted by the additional expression of Nrd1‐specific mutants that interacted with Rrp6. This effect relied on the interaction of Nrd1, a transcriptional regulator of aging‐related genes, including ribosome biogenesis or RNA metabolism genes, with RNA polymerase II. Nrd1 overexpression reduced RLS in a Tho2‐dependent pathway. Intriguingly, Tho2 deletion mirrored Nrd1 overexpression effects by inducing arbitrary Nrd1 chromatin binding. Furthermore, our genome‐wide ChIP‐seq analysis revealed an increase in the recruitment of Nrd1 to translation‐associated genes, known to be related to aging, upon Tho2 loss. Taken together, these findings underscore the importance of Tho2‐mediated Nrd1 escorting in the regulation of lifespan pathway through transcriptional regulation of aging‐related genes.
... The carboxy-terminal domain (CTD) of the largest subunit of RN A pol ymerase II (RN APII) initiall y plays essential roles in this process. The CTD code, which consists of various combinations of posttranslational modifications of the heptapeptide repeats (YSPTSPS), such as the phosphorylation of specific serine residues, leads to the pertinent recruitment of differ ent pr e-mRNA processing / ma tura tion factors to acti v e genes (6)(7)(8). During transcription initiation phase, S 5 of the CTD is phosphorylated and attracts the capping enzyme (mRN A guanyl yltr ansfer ase; RNGTT), which adds an inverted guanosine moiety to the 5 -end of a nascent transcript by the triphosphatase and guanylyltr ansfer ase activities ( 9 , 10 ). ...
... Expression of Nsp14 was induced for 48 h. Poly(A) + RNPs (Poly(A) + : lanes3,4,7,8) were purified from whole-cell extracts (input: lanes 1, 2, 5, 6) pr epar ed from each cell culture. The samples were analyzed by Western blotting using the antibodies indicated on the right of each panel. ...
To facilitate selfish replication, viruses halt host gene expression in various ways. The nuclear export of mRNA is one such process targeted by many viruses. SARS-CoV-2, the etiological agent of severe acute respiratory syndrome, also prevents mRNA nuclear export. In this study, Nsp14, a bifunctional viral replicase subunit, was identified as a novel inhibitor of mRNA nuclear export. Nsp14 induces poly(A)+ RNA nuclear accumulation and the dissolution/coalescence of nuclear speckles. Genome-wide gene expression analysis revealed the global dysregulation of splicing and 3'-end processing defects of replication-dependent histone mRNAs by Nsp14. These abnormalities were also observed in SARS-CoV-2-infected cells. A mutation introduced at the guanine-N7-methyltransferase active site of Nsp14 diminished these inhibitory activities. Targeted capillary electrophoresis-mass spectrometry analysis (CE-MS) unveiled the production of N7-methyl-GTP in Nsp14-expressing cells. Association of the nuclear cap-binding complex (NCBC) with the mRNA cap and subsequent recruitment of U1 snRNP and the stem-loop binding protein (SLBP) were impaired by Nsp14. These data suggest that the defects in mRNA processing and export arise from the compromise of NCBC function by N7-methyl-GTP, thus exemplifying a novel viral strategy to block host gene expression.
... To gain insights into the status of RNAPII, we assayed levels of RPB1 (also known as POLR2A), the largest subunit of RNAPII, and its phosphorylated forms that mark distinct stages of the transcription cycleinitiation ( phosphorylation of serine 5 of the C-terminal repeat domain sequence, Ser5-p) and elongation ( phosphorylation of serine 2 of the C-terminal repeat domain sequence, Ser2-p) (Zhang et al., 2012). Total RNAPII showed decreased abundance in both G 0 cells and MTs compared to the abundance in MBs (Fig. 1E,F; Fig. S1E-G), as assayed using both western blotting and immunostaining. ...
Adult stem cells persist in mammalian tissues by entering a state of reversible quiescence, referred to as G0, which is associated with low levels of transcription. Using cultured myoblasts and muscle stem cells, we report that in G0, global RNA content and synthesis are substantially repressed, correlating with decreased RNA polymerase II (RNAPII) expression and activation. Integrating RNAPII occupancy and transcriptome profiling, we identify repressed networks and a role for promoter-proximal RNAPII pausing in G0. Strikingly, RNAPII shows enhanced pausing in G0 on repressed genes encoding regulators of RNA biogenesis (such as Ncl, Rps24, Ctdp1), and release of pausing is associated with increased expression of these genes in G1. Knockdown of these transcripts in proliferating cells leads to induction of G0 markers, confirming the importance of their repression in establishment of G0. A targeted screen of RNAPII regulators revealed that knockdown of Aff4 (a positive regulator of elongation) unexpectedly enhances expression of G0-stalled genes and hastens S phase; however, the negative elongation factor (NELF) complex, a regulator of pausing, appears to be dispensable. We propose that RNAPII pausing contributes to transcriptional control of a subset of G0-repressed genes to maintain quiescence and impacts the timing of the G0-G1 transition.
This article has an associated First Person interview with the first authors of the paper.
... The CDK19 module includes cyclin C and Med12L and Med13L. CDK8 and CDK19 are highly conserved serine-threonine kinases that phosphorylate serines 2 and 5 in the heptad repeats of the RNAPII CTD to control transcription pausing and elongation, chromatin remodeling, and RNA processing and export [36][37][38][39]. CDK8 also phosphorylates itself, cyclin C, Med12 and Med13, Mediator middle and head modules, Cyclin H, multiple transcription factors, general transcription factors, the super elongation complex (SEC), chromatin remodelers, and serine 10 of histone H3 (H3S10) [8,9,[40][41][42][43]. ...
... I n budding yeast Saccharomyces cerevisiae, the largest subunit of RNA polymerase II (Pol II) bears 26 tandemly repeating Y 1 S 2 P 3 T 4 S 5 P 6 S 7 heptapeptides 1 . Sequential post-translational modifications of these residues serve as a scaffold to recruit stagespecific protein complexes to the polymerase [2][3][4] . These complexes facilitate multiple processes, including Pol II function through different stages of the transcription cycle, co-transcriptional processing and transport of nascent RNA, remodeling and modification of the underlying chromatin template and even displacement or proteolysis of polymerase that is stalled at sites of DNA damage [2][3][4][5] . ...
... Sequential post-translational modifications of these residues serve as a scaffold to recruit stagespecific protein complexes to the polymerase [2][3][4] . These complexes facilitate multiple processes, including Pol II function through different stages of the transcription cycle, co-transcriptional processing and transport of nascent RNA, remodeling and modification of the underlying chromatin template and even displacement or proteolysis of polymerase that is stalled at sites of DNA damage [2][3][4][5] . ...
... 12 ), priming the CTD for subsequent Ser2 phosphorylation by Ctk1 (CDK12 and CDK13; ref. 13 ). pSer2 marks are critical for productive elongation and recruitment of factors involved in histone modification, co-transcriptional splicing, and transcription termination [2][3][4][5] . This sequential pattern of CTD phosphorylation and stage-specific association of different cellular machines is observed globally at Pol II-transcribed genes. ...
Phosphorylation of the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) governs stage-specific interactions with different cellular machines. The CTD consists of Y1S2P3T4S5P6S7 heptad repeats and sequential phosphorylations of Ser7, Ser5 and Ser2 occur universally at Pol II-transcribed genes. Phosphorylation of Thr4, however, appears to selectively modulate transcription of specific classes of genes. Here, we identify ten new Thr4 kinases from different kinase structural groups. Irreversible chemical inhibition of the most active Thr4 kinase, Hrr25, reveals a novel role for this kinase in transcription termination of specific class of noncoding snoRNA genes. Genome-wide profiles of Hrr25 reveal a selective enrichment at 3ʹ regions of noncoding genes that display termination defects. Importantly, phospho-Thr4 marks placed by Hrr25 are recognized by Rtt103, a key component of the termination machinery. Our results suggest that these uncommon CTD kinases place phospho-Thr4 marks to regulate expression of targeted genes.
... Several nuclear mRNA-binding proteins already bind cotranscriptionally to the mRNA such as the SR-like proteins Nab2 and Npl3 or Tho1 in S. cerevisiae and thus constitute the nuclear mRNP [(1) and references therein]. The recruitment of proteins involved in mRNA processing and mRNP packaging is coordinated with the different phases of transcription by the C-terminal domain (CTD) of Rpb1, the largest subunit of RNAPII (1,8,9). The CTD consists of repeats of the heptapeptide sequence YSPTSPS. ...
The different steps of gene expression are intimately linked to coordinate and regulate this complex process. During transcription, numerous RNA-binding proteins are already loaded onto the nascent mRNA and package the mRNA into a messenger ribonucleoprotein particle (mRNP). These RNA-binding proteins are often also involved in other steps of gene expression than mRNA packaging. For example, TREX functions in transcription, mRNP packaging and nuclear mRNA export. Previously, we showed that the Prp19 splicing complex (Prp19C) is needed for efficient transcription as well as TREX occupancy at transcribed genes. Here, we show that the splicing factor Mud2 interacts with Prp19C and is needed for Prp19C occupancy at transcribed genes in Saccharomyces cerevisiae. Interestingly, Mud2 is not only recruited to intron-containing but also to intronless genes indicating a role in transcription. Indeed, we show for the first time that Mud2 functions in transcription. Furthermore, these functions of Mud2 are likely evolutionarily conserved as Mud2 is also recruited to an intronless gene and interacts with Prp19C in Drosophila melanogaster. Taken together, we classify Mud2 as a novel transcription factor that is necessary for the recruitment of mRNA-binding proteins to the transcription machinery. Thus, Mud2 is a multifunctional protein important for transcription, splicing and most likely also mRNP packaging.
... For the basal transcription in interphase, Kin28/Cdk7 subunit of TFIIH complex phosphorylates CTD at Ser5 (also Ser7) and helps in the preinitiation complex formation, transcription initiation and efficient mRNA capping. During early elongation, Ser2 kinases (Cdk9/Bur1) acts on the pre phosphorylated CTD (pCTD) and help the subsequent progression of transcription and mRNA biogenesis (3)(4)(5). The phosphatases Ssu72, Rtr1 and Fcp1 remove phosphorylated Ser5/Ser7, Ser5, and Ser2 respectively, of CTD during interphase (1,2,6). ...
In eukaryotes, the basal transcription in interphase is orchestrated through the regulation by kinases (Kin28, Bur1 and Ctk1) and phosphatases (Ssu72, Rtr1 and Fcp1) which act through the post-translational modification of CTD (carboxy terminal domain of the largest subunit of RNA Polymerase II). The CTD comprises of repeated Tyr1Ser2Pro3Thr4Ser5Pro6Ser7 motif with potential epigenetic modification sites. Despite the observation of transcription and periodic expression of genes during mitosis with entailing CTD phosphorylation and dephosphorylation, the associated CTD specific kinase/s and its role in transcription remains unknown. Here we have identified Cdc15 as a potential kinase phosphorylating Ser2 as well as Ser5 of CTD for transcription during mitosis in the budding yeast. The phosphorylation of CTD by Cdc15 is independent of any prior Ser phosphorylation/s. The inactivation of Cdc15 causes reduction of global CTD phosphorylation during mitosis and affects the expression of genes whose transcript level peaks during mitosis. Cdc15 also influences the complete transcription of clb2 gene and phosphorylates Ser5 at the promoter and Ser2 towards 3' end of the gene. The observation that Cdc15 could phosphorylate Ser5 as well as Ser2 during transcription in mitosis, is in contrast to the phosphorylation marks put by the kinases in interphase (G1, S and G2), where Cdck7/Kin28 phosphorylates Ser5 at promoter and Bur1/Ctk1 phosphorylates Ser2 at 3' end of the genes.
... In However, the ratio of recovery of CTD Ser5 to total RNA pol II showed a very significant difference between the two diets ( Figure 3C). Since the CTD of RNA pol II must be phosphorylated on Ser5 for release from pausing prior to elongation [41], these data reflect a decrease in the transcriptional rate of this gene after a high fat diet. The pattern for Onecut1 was similar, with RNA pol II most prominent near the 5' end of the gene ( Figure 3D), and with a pronounced decreased ratio (either significant or trending) ...
Objective: Overnutrition can alter gene expression patterns through epigenetic mechanisms that may persist through generations. However, it is less clear if overnutrition, for example a high fat diet, modifies epigenetic control of gene expression in adults, or by what molecular mechanisms, or if such mechanisms contribute to the pathology of the metabolic syndrome. Here we test the hypothesis that a high fat diet?alters hepatic DNA methylation, transcription and gene expression patterns, and explore the contribution of such changes to the pathophysiology of obesity.
Methods: RNA-seq and targeted high-throughput bisulfite DNA sequencing were used to undertake a systematic analysis of the hepatic response to a high fat diet. RT-PCR, chromatin immunoprecipitation and in?vivo knockdown of an identified driver gene, Phlda1, were used to validate the results.
Results: A high fat diet resulted in the hypermethylation and decreased transcription and expression of Phlda1 and several other genes. A subnetwork of genes associated with Phlda1 was identified from an existing Bayesian gene network that contained numerous hepatic regulatory genes involved in lipid and body weight homeostasis. Hepatic-specific depletion of Phlda1 in mice decreased expression of the genes in the subnetwork, and led to increased oil droplet size in standard chow-fed mice, an early indicator of steatosis, validating the contribution of this gene to the phenotype.
Conclusions: We conclude that a high fat diet?alters the epigenetics and transcriptional activity of key hepatic genes controlling lipid homeostasis, contributing to the pathophysiology of obesity.
... The phosphorylation of Ser 5 , primarily by the TFIIH-associated kinase Kin28, enhances the association of CTD with the m7G mRNA capping machinery 16,17 . However, Kin28 also phosphorylates Ser 7 of CTD (only on the prephosphorylated Ser 5 heptad) and the role of this phosphorylation in mRNA transcription remains obscure 2,8,9 . Since Kin28 marks both, the Ser 5 and Ser 7 phosphorylation at the 5′ end of the gene and also the occupancy profile of Ser 5P and Ser 7P overlaps in most of the cases in this region, the role of dual phosphorylation of CTD either in Ceg1 recruitment and subsequent role in mRNA capping cannot be ruled out. ...
... The structure of CTD is very flexible and can adopt multiple conformations. The dynamic phosphorylation patterns of CTD in the transcription cycle undergo significant changes from initiation to termination, however the exact phosphorylation pattern in vivo remains unknown till date 2,9 . It was reported that there may be only a single phosphorylation per heptad repeat (YSPTSPS), however few recent studies suggests a coexistence of Ser 2 and Ser 7 phosphorylation on the same heptad repeat 9 . ...
RNA Polymerase II (RNAPII) uniquely possesses an extended carboxy terminal domain (CTD) on its largest subunit, Rpb1, comprising a repetitive Tyr1Ser2Pro3Thr4 Ser5Pro6Ser7 motif with potential phosphorylation sites. The phosphorylation of the CTD serves as a signal for the binding of various transcription regulators for mRNA biogenesis including the mRNA capping complex. In eukaryotes, the 5 prime capping of the nascent transcript is the first detectable mRNA processing event, and is crucial for the productive transcript elongation. The binding of capping enzyme, RNA guanylyltransferases to the transcribing RNAPII is known to be primarily facilitated by the CTD, phosphorylated at Ser5 (Ser5P). Here we report that the Saccharomyces cerevesiae RNA guanylyltransferase (Ceg1) has dual specificity and interacts not only with Ser5P but also with Ser7P of the CTD. The Ser7 of CTD is essential for the unconditional growth and efficient priming of the mRNA capping complex. The Arg159 and Arg185 of Ceg1 are the key residues that interact with the Ser5P, while the Lys175 with Ser7P of CTD. These interactions appear to be in a specific pattern of Ser5PSer7PSer5P in a tri-heptad CTD (YSPTSPPS YSPTSPSP YSPTSPPS) and provide molecular insights into the Ceg1-CTD interaction for mRNA transcription.
... Kin28/Cdk7-mediated phosphoryla-tion of the CTD also creates a scaffold to recruit factors that act on nascent transcripts, the underlying chromatin, and the CTD itself. Promoter-proximal marks on Ser5 and Ser7 are also thought to ''prime'' the CTD for subsequent modifications that coordinate sequential association of different cellular machines that facilitate transcription (Buratowski, 2009;Corden, 2013;Eick and Geyer, 2013;Perales and Bentley, 2009;Phatnani and Greenleaf, 2006;Zhang et al., 2012). ...
During transcription initiation, the TFIIH-kinase Kin28/Cdk7 marks RNA polymerase II (Pol II) by phosphorylating the C-terminal domain (CTD) of its largest subunit. Here we describe a structure-guided chemical approach to covalently and specifically inactivate Kin28 kinase activity in vivo. This method of irreversible inactivation recapitulates both the lethal phenotype and the key molecular signatures that result from genetically disrupting Kin28 function in vivo. Inactivating Kin28 impacts promoter release to differing degrees and reveals a “checkpoint” during the transition to productive elongation. While promoter-proximal pausing is not observed in budding yeast, inhibition of Kin28 attenuates elongation-licensing signals, resulting in Pol II accumulation at the +2 nucleosome and reduced transition to productive elongation. Furthermore, upon inhibition, global stabilization of mRNA masks different degrees of reduction in nascent transcription. This study resolves long-standing controversies on the role of Kin28 in transcription and provides a rational approach to irreversibly inhibit other kinases in vivo.
