The transcription factor c-Myc amplifies the transcription of many growth-related genes in cancer cells, but its role as an oncogene is not fully understood.
The eukaryotic RNA polymerase II (RNAPII) catalyzes the transcription of all protein encoding genes and is also responsible for the generation of small regulatory RNAs. RNAPII has evolved a unique ...domain composed of heptapeptide repeats with the consensus sequence Tyr1–Ser2–Pro3–Thr4–Ser5–Pro6–Ser7 at the C-terminus (CTD) of its largest subunit (Rpb1). Dynamic phosphorylation patterns of serine residues in CTD during gene transcription coordinate the recruitment of factors to the elongating RNAPII and to the nascent transcript. Recent studies identified threonine 4 and tyrosine 1 as new CTD modifications and thereby expanded the “CTD code”. In this review, we focus on CTD phosphorylation and its function in the RNAPII transcription cycle. We also discuss in detail the limitations of the phosphospecific CTD antibodies, which are used in all studies. This article is part of a Special Issue entitled: RNA Polymerase II Transcript Elongation.
► CTD phosphorylation undergoes dynamic changes in the transcription cycle. ► Phosphorylation of CTD enables and protects factors from binding. ► The CTD phospho-code is complete with Tyr1-P, Ser2-P, Thr4-P, Ser5-P, and Ser7-P.
Gene transcription can be activated by decreasing the duration of RNA polymerase II pausing in the promoter-proximal region, but how this is achieved remains unclear. Here we use a 'multi-omics' ...approach to demonstrate that the duration of polymerase pausing generally limits the productive frequency of transcription initiation in human cells ('pause-initiation limit'). We further engineer a human cell line to allow for specific and rapid inhibition of the P-TEFb kinase CDK9, which is implicated in polymerase pause release. CDK9 activity decreases the pause duration but also increases the productive initiation frequency. This shows that CDK9 stimulates release of paused polymerase and activates transcription by increasing the number of transcribing polymerases and thus the amount of mRNA synthesized per time. CDK9 activity is also associated with long-range chromatin interactions, suggesting that enhancers can influence the pause-initiation limit to regulate transcription.
Co-transcriptional pre-mRNA processing relies on reversible phosphorylation of the carboxyl-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II (RNAP II). In this study, we ...replaced in live cells the endogenous Rpb1 by S2A Rpb1, where the second serines (Ser2) in the CTD heptapeptide repeats were switched to alanines, to prevent phosphorylation. Although slower, S2A RNAP II was able to transcribe. However, it failed to recruit splicing components such as U2AF65 and U2 snRNA to transcription sites, although the recruitment of U1 snRNA was not affected. As a consequence, co-transcriptional splicing was impaired. Interestingly, the magnitude of the S2A RNAP II splicing defect was promoter dependent. In addition, S2A RNAP II showed an impaired recruitment of the cleavage factor PCF11 to pre-mRNA and a defect in 3'-end RNA cleavage. These results suggest that CTD Ser2 plays critical roles in co-transcriptional pre-mRNA maturation in vivo: It likely recruits U2AF65 to ensure an efficient co-transcriptional splicing and facilitates the recruitment of pre-mRNA 3'-end processing factors to enhance 3'-end cleavage.
Getting Access to Low-Complexity Domain Modifications Schüller, Roland; Eick, Dirk
Trends in biochemical sciences (Amsterdam. Regular ed.),
November 2016, 2016-11-00, 20161101, Letnik:
41, Številka:
11
Journal Article
Recenzirano
Low-complexity (LC) domains regulate the aggregation and phase transition of proteins in a modification-dependent manner. The study of LC domain modifications has now become feasible, as shown by ...genetic variants of the carboxy-terminal domain (CTD) of RNA Polymerase II (Pol II) that provide access to the type and position of modifications of a LC domain by mass spectrometry (MS).
In different phases of the transcription cycle, RNA polymerase (Pol) II recruits various factors via its C-terminal domain (CTD), which consists of conserved heptapeptide repeats with the sequence ...Tyr¹-Ser²-Pro³-Thr⁴ -Ser⁵ -Pro⁶ -Ser⁷. We show that the CTD of transcribing yeast Pol II is phosphorylated at Tyr¹, in addition to Ser², Thr⁴, Ser⁵, and Ser⁷. Tyr¹ phosphorylation stimulates binding of elongation factor Spt6 and impairs recruitment of termination factors Nrd1, Pcf11, and Rtt103. Tyr¹ phosphorylation levels rise downstream of the transcription start site and decrease before the polyadenylation site, largely excluding termination factors from gene bodies. These results show that CTD modifications trigger and block factor recruitment and lead to an extended CTD code that explains transcription cycle coordination on the basis of differential phosphorylation of Tyr¹, Ser², and Ser⁵.
The carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) consists of heptad repeats with the consensus motif Y1-S2-P3-T4-S5-P6-S7. Dynamic phosphorylation of the CTD coordinates Pol II ...progression through the transcription cycle. Here, we use genetic and mass spectrometric approaches to directly detect and map phosphosites along the entire CTD. We confirm phosphorylation of CTD residues Y1, S2, T4, S5, and S7 in mammalian and yeast cells. Although specific phosphorylation signatures dominate, adjacent CTD repeats can be differently phosphorylated, leading to a high variation of coexisting phosphosites in mono- and di-heptad CTD repeats. Inhibition of CDK9 kinase specifically reduces S2 phosphorylation levels within the CTD.
•CTD variants make the entire CTD sequence accessible to mass spectrometry analysis•Valid CTD phosphosite mapping is based on a synthetic CTD peptide library•Predominant phosphorylation signatures prevail within neighboring CTD residues•Inhibition of CDK9 strongly reduces Ser2-P phosphorylation in the CTD
A combined genetic and mass spectrometric approach offers comprehensive insights into CTD phosphorylation patterns and changes in CTD heptad-specific phosphorylation states in mammals and yeast.
Phosphorylation of the RNA polymerase II C-terminal domain (CTD) by cyclin-dependent kinases is important for productive transcription. Here we determine the crystal structure of Cdk12/CycK and ...analyse its requirements for substrate recognition. Active Cdk12/CycK is arranged in an open conformation similar to that of Cdk9/CycT but different from those of cell cycle kinases. Cdk12 contains a C-terminal extension that folds onto the N- and C-terminal lobes thereby contacting the ATP ribose. The interaction is mediated by an HE motif followed by a polybasic cluster that is conserved in transcriptional CDKs. Cdk12/CycK showed the highest activity on a CTD substrate prephosphorylated at position Ser7, whereas the common Lys7 substitution was not recognized. Flavopiridol is most potent towards Cdk12 but was still 10-fold more potent towards Cdk9. T-loop phosphorylation of Cdk12 required coexpression with a Cdk-activating kinase. These results suggest the regulation of Pol II elongation by a relay of transcriptionally active CTD kinases.
Drugs for cancer therapy belong to different categories of chemical substances. The cellular targets for the therapeutic efficacy are often not unambiguously identified. Here, we describe the process ...of ribosome biogenesis as a target of a large variety of chemotherapeutic drugs. We determined the inhibitory concentration of 36 chemotherapeutic drugs for transcription and processing of ribosomal RNA by in vivo labeling experiments. Inhibitory drug concentrations were correlated to the loss of nucleolar integrity. The synergism of drugs inhibiting ribosomal RNA synthesis at different levels was studied. Drugs inhibited ribosomal RNA synthesis either at the level of (i) rRNA transcription (e.g. oxaliplatin, doxorubicin, mitoxantrone, methotrexate), (ii) early rRNA processing (e.g. camptothecin, flavopiridol, roscovitine), or (iii) late rRNA processing (e.g. 5-fluorouracil, MG-132, homoharringtonine). Blockage of rRNA transcription or early rRNA processing steps caused nucleolar disintegration, whereas blockage of late rRNA processing steps left the nucleolus intact. Flavopiridol and 5-fluorouracil showed a strong synergism for inhibition of rRNA processing. We conclude that inhibition of ribosome biogenesis by chemotherapeutic drugs potentially may contribute to the efficacy of therapeutic regimens.
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