RNA Polymerase II Elongation Research Articles

Coordinate control of the RNA polymerase II transcription cycle by CDK9-dependent, tripartite phosphorylation of SPT5.

The RNA polymerase II (RNAPII) transcription cycle is regulated throughout its duration by reversible protein phosphorylation. The elongation factor SPT5 contains two regions targeted by cyclin-dependent kinase 9 (CDK9) and previously implicated in promoter-proximal pausing and termination: the linker between KOWx-4 and KOW5 domains and carboxy-terminal repeat (CTR) 1, respectively. Here we show that phosphorylations in the KOWx-4/5 linker, CTR1 and a third region, CTR2, coordinately control pause release, elongation speed and RNA processing. Pausing was increased by mutations preventing CTR1 or CTR2 phosphorylation, but attenuated when both CTRs were mutated. Whereas mutating CTR1 alone slowed elongation and repressed nascent transcription, simultaneous mutation of CTR2 partially reversed both effects. Nevertheless, mutating both CTRs led to aberrant splicing, dysregulated termination and diminished steady-state mRNA levels, and impaired cell proliferation more severely than did either single-CTR mutation. Therefore, tripartite SPT5 phosphorylation times pause release and regulates RNAPII elongation rates positively and negatively to ensure productive transcription and cell viability.

Cockayne Syndrome Linked to Elevated R-Loops Induced by Stalled RNA Polymerase II during Transcription Elongation

Mutations in the Cockayne Syndrome group B (CSB) gene cause cancer in mice, but premature aging and severe neurodevelopmental defects in humans. CSB, a member of the SWI/SNF family of chromatin remodelers, plays diverse roles in regulating gene expression and transcription-coupled nucleotide excision repair (TC-NER); however, these functions do not explain the distinct phenotypic differences observed between CSB-deficient mice and humans. During investigating Cockayne Syndrome-associated genome instability, we uncover an intrinsic mechanism that involves elongating RNA polymerase II (RNAPII) undergoing transient pauses at internal T-runs where CSB is required to propel RNAPII forward. Consequently, CSB deficiency retards RNAPII elongation in these regions, and when coupled with G-rich sequences upstream, exacerbates genome instability by promoting R-loop formation. These R-loop prone motifs are notably abundant in relatively long genes related to neuronal functions in the human genome, but less prevalent in the mouse genome. These findings provide mechanistic insights into differential impacts of CSB deficiency on mice versus humans and suggest that the manifestation of the Cockayne Syndrome phenotype in humans results from the progressive evolution of mammalian genomes.

Age-dependent regulation of ELP1 exon 20 splicing in Familial Dysautonomia by RNA Polymerase II kinetics and chromatin structure.

Familial Dysautonomia (FD) is a rare disease caused by ELP1 exon 20 skipping. Here we clarify the role of RNA Polymerase II (RNAPII) and chromatin on this splicing event. A slow RNAPII mutant and chromatin-modifying chemicals that reduce the rate of RNAPII elongation induce exon skipping whereas chemicals that create a more relaxed chromatin exon inclusion. In the brain of a mouse transgenic for the human FD-ELP1 we observed on this gene an age-dependent decrease in the RNAPII density profile that was most pronounced on the alternative exon, a robust increase in the repressive marks H3K27me3 and H3K9me3 and a decrease of H3K27Ac, together with a progressive reduction in ELP1 exon 20 inclusion level. In HEK 293T cells, selective drug-induced demethylation of H3K27 increased RNAPII elongation on ELP1 and SMN2, promoted the inclusion of the corresponding alternative exons, and, by RNA-sequencing analysis, induced changes in several alternative splicing events. These data suggest a co-transcriptional model of splicing regulation in which age-dependent changes in H3K27me3/Ac modify the rate of RNAPII elongation and affect processing of ELP1 alternative exon 20.

RBM22 regulates RNA polymerase II 5′ pausing, elongation rate, and termination by coordinating 7SK-P-TEFb complex and SPT5

BackgroundSplicing factors are vital for the regulation of RNA splicing, but some have also been implicated in regulating transcription. The underlying molecular mechanisms of their involvement in transcriptional processes remain poorly understood.ResultsHere, we describe a direct role of splicing factor RBM22 in coordinating multiple steps of RNA Polymerase II (RNAPII) transcription in human cells. The RBM22 protein widely occupies the RNAPII-transcribed gene locus in the nucleus. Loss of RBM22 promotes RNAPII pause release, reduces elongation velocity, and provokes transcriptional readthrough genome-wide, coupled with production of transcripts containing sequences from downstream of the gene. RBM22 preferentially binds to the hyperphosphorylated, transcriptionally engaged RNAPII and coordinates its dynamics by regulating the homeostasis of the 7SK-P-TEFb complex and the association between RNAPII and SPT5 at the chromatin level.ConclusionsOur results uncover the multifaceted role of RBM22 in orchestrating the transcriptional program of RNAPII and provide evidence implicating a splicing factor in both RNAPII elongation kinetics and termination control.

DOT1L is a barrier to histone acetylation during reprogramming to pluripotency.

Embryonic stem cells (ESCs) have transcriptionally permissive chromatin enriched for gene activation-associated histone modifications. A striking exception is DOT1L-mediated H3K79 dimethylation (H3K79me2) that is considered a positive regulator of transcription. We find that ESCs are depleted for H3K79me2 at shared locations of enrichment with somatic cells, which are highly and ubiquitously expressed housekeeping genes, and have lower RNA polymerase II (RNAPII) at the transcription start site (TSS) despite greater nascent transcription. Inhibiting DOT1L increases the efficiency of reprogramming of somatic to induced pluripotent stem cells, enables an ESC-like RNAPII pattern at the TSS, and functionally compensates for enforced RNAPII pausing. DOT1L inhibition increases H3K27 methylation and RNAPII elongation-enhancing histone acetylation without changing the expression of the causal histone-modifying enzymes. Only the maintenance of elevated histone acetylation is essential for enhanced reprogramming and occurs at loci that are depleted for H3K79me2. Thus, DOT1L inhibition promotes the hyperacetylation and hypertranscription pluripotent properties.

Hells (LSH) Controls Gene Expression in T-Cell Lymphomas By Regulating RNA Polymerase II with a Bimodal Function

Chromatin modifiers are emerging as determinants of many types of cancer, including anaplastic large cell lymphoma (ALCL), a family of highly heterogeneous T-cell lymphomas for which therapeutic options are still limited. We recently identified the DNA helicase HELLS - a member of the SWI/SNF2 family- as a genetic vulnerability of ALK - ALCLs. HELLS is a multifunctional chromatin remodeler that affects genomic instability. Although its transcriptional function has been suggested, no clues on how HELLS controls transcription are currently available. To explore its transcriptional function in ALCLs, we integrated HELLSChromatin immunoprecipitation-sequencing (ChIP-seq) analysis in the TLBR-2 cell line with RNA-sequencing data TLBR-2 HELLS KD and control cells. Out of 729 genes significantly affected by HELLS KD, 467 genes (64% of the total) resulted directly bound by HELLS. We termed these genes HELLS-direct genes (HDGs). Analysis of HELLS binding profile at HDGs site showed that 67% of HELLS binding sites at HDGs has intragenic localization being associated with promoters, introns, and exons. Gene-set enrichment analysis of HDGs revealed several pathogenic processes associated with deregulated genes including JAK/STAT signaling pathway, inflammation mediated by chemokines, and cytokines signaling pathway. To assess the contribution of HELLS to the transcription of its HDGs, the distribution of RNA-PolymeraseII (RNAPII) was investigated by ChIP-seq in TLBR-2 HELLS KD and control cells. We observed that HELLSdepletion caused a bimodal alteration of RNAPII. On genes belonging to biological processes involved in the dysregulation of circuitries that controls T-cells dependent host interactions (n=195, 40% of HDG promoters), the depletion of HELLS caused a significant impairment of RNAPII recruitment suggesting that HELLS partakes in transcription regulation of these pathways acting as a co-transcriptional factor. Whereas, on the remaining 60% of HDGs (n=272), which are enriched for selective biological functions including cytoskeleton regulation and DNA metabolic processes, we observed a selective loss of RNAPII signal in the regions immediately downstream of the TSS coherent with a potential proximal promoter pausing of RNAPII. Target analysis of phospho-CTD modification ser2P of RNAPII confirmed the RNAPII pausing after HELLS depletion. Stacked RNAPII during elongation drives DNA-RNA hybrids (R-loops) formation and it is a primary source of DNA break accumulation. We hypothesized that HELLS could promote RNAPII elongation by relieving R-loops, thereby decreasing potential obstacles to the transcription machinery. We investigated the effect of HELLS depletion on R-loops accumulation by performing immunofluorescences using the S9.6 antibody to detect R-loops in control and HELLS KD cells. We observed a significant increase in the nuclear intensity of R-loops in several models of ALCL HELLS KD cells. The increased S9.6 signal was accompanied by a different pattern of nuclear Ser2P-RNAPII with clusters adjacent to R-loop structures suggesting that R-loops are associated with stalled transcription. After HELLS KD, we also observed an overall increase in the intensity of the nuclear γH2AX signal - used as a readout of DNA damage accumulation- and a significant increase in the formation of γH2AX foci in all cell lines tested. The increase in γH2AX foci was found in S9.6 positive cells andwasassociated with a distinctive pattern of ser2P-RNAPII. By using reporter-GFP-based assays, we investigated which pathway mediates HELLS ability to repair DNA damage discovering that the loss of HELLS results in decreased NHEJ and MMEJ efficiency (37% and 36% respectively over time). To validate the clinical impact of HELLS in vivo, we interrogated a cohort of 44 ALCL patients using the NCounter platform. Gene expression analysis showed that HELLS was significantly associated with its HDGs showing a positive correlation with 68% of genes and a negative correlation with 32% of genes in ALK -ALCL samples. Our data indicate that i) HELLS works with a bi-modal function in controlling gene expression by regulating both the initiation and the elongation of RNAPII and ii) the different work modalities underline different biological consequences. At the crossroads between transcription and DNA repair, HELLS is as an attractive target for developing novel treatments in hematological malignancies

U1 snRNP increases RNA Pol II elongation rate to enable synthesis of long genes

The expansion of introns within mammalian genomes poses a challenge for the production of full-length messenger RNAs (mRNAs), with increasing evidence that these long AT-rich sequences present obstacles to transcription. Here, we investigate RNA polymerase II (RNAPII) elongation at high resolution in mammalian cells and demonstrate that RNAPII transcribes faster across introns. Moreover, we find that this acceleration requires the association of U1 snRNP (U1) with the elongation complex at 5' splice sites. The role of U1 to stimulate elongation rate through introns reduces the frequency of both premature termination and transcriptional arrest, thereby dramatically increasing RNA production. We further show that changes in RNAPII elongation rate due to AT content and U1 binding explain previous reports of pausing or termination at splice junctions and the edge of CpG islands. We propose that U1-mediated acceleration of elongation has evolved to mitigate the risks that long AT-rich introns pose to transcript completion.

H3K4me3 regulates RNA polymerase II promoter-proximal pause-release

Trimethylation of histone H3 lysine 4 (H3K4me3) is associated with transcriptional start sites and has been proposed to regulate transcription initiation1,2. However, redundant functions of the H3K4 SET1/COMPASS methyltransferase complexes complicate the elucidation of the specific role of H3K4me3 in transcriptional regulation3,4. Here, using mouse embryonic stem cells as a model system, we show that acute ablation of shared subunits of the SET1/COMPASS complexes leads to a complete loss of all H3K4 methylation. Turnover of H3K4me3 occurs more rapidly than that of H3K4me1 and H3K4me2 and is dependent on KDM5 demethylases. Notably, acute loss of H3K4me3 does not have detectable effects on transcriptional initiation but leads to a widespread decrease in transcriptional output, an increase in RNA polymerase II (RNAPII) pausing and slower elongation. We show that H3K4me3 is required for the recruitment of the integrator complex subunit 11 (INTS11), which is essential for the eviction of paused RNAPII and transcriptional elongation. Thus, our study demonstrates a distinct role for H3K4me3 in transcriptional pause-release and elongation rather than transcriptional initiation.

Elongation factor 1 is a component of the ArabidopsisRNA polymerase II elongation complex and associates with a subset of transcribed genes.

Elongation factors modulate the efficiency of mRNA synthesis by RNA polymerase II (RNAPII) in the context of chromatin, thus contributing to implement proper gene expression programmes. The zinc-finger protein elongation factor 1 (ELF1) is a conserved transcript elongation factor (TEF), whose molecular function so far has not been studied in plants. Using biochemical approaches, we examined the interaction of Arabidopsis ELF1 with DNA and histones invitro and with the RNAPII elongation complex invivo. In addition, cytological assays demonstrated the nuclear localisation of the protein, and by means of double-mutant analyses, interplay with genes encoding other elongation factors was explored. The genome-wide distribution of ELF1 was addressed by chromatin immunoprecipitation. ELF1 isolated from Arabidopsis cells robustly copurified with RNAPII and various other elongation factors including SPT4-SPT5, SPT6, IWS1, FACT and PAF1C. Analysis of a CRISPR-Cas9-mediated gene editing mutant of ELF1 revealed distinct genetic interactions with mutants deficient in other elongation factors. Moreover, ELF1 associated with genomic regions actively transcribed by RNAPII. However, ELF1 occupied only c. 33% of the RNAPII transcribed loci with preference for inducible rather than constitutively expressed genes. Collectively, these results establish that Arabidopsis ELF1 shares several characteristic attributes with RNAPII TEFs.

MDC1 maintains active elongation complexes of RNA polymerase II

The role of MDC1 in the DNA damage response has been extensively studied; however, its impact on other cellular processes is not well understood. Here, we describe the role of MDC1 in transcription as a regulator of RNA polymerase II (RNAPII). Depletion of MDC1 causes a genome-wide reduction in the abundance of actively engaged RNAPII elongation complexes throughout the gene body of protein-encoding genes under unperturbed conditions. Decreased engaged RNAPII subsequently alters the assembly of the spliceosome complex on chromatin, leading to changes in pre-mRNA splicing. Mechanistically, the S/TQ domain of MDC1 modulates RNAPII-mediated transcription. Upon genotoxic stress, MDC1 promotes the abundance of engaged RNAPII complexes at DNA breaks, thereby stimulating nascent transcription at the damaged sites. Of clinical relevance, cancer cells lacking MDC1 display hypersensitivity to RNAPII inhibitors. Overall, we unveil a role of MDC1 in RNAPII-mediated transcription with potential implications for cancer treatment.

Approach to Measuring the Effect of PARP1 on RNA Polymerase II Elongation Rates.

The rate of RNA polymerase II (RNAPII) transcriptional elongation plays a critical role in mRNA biogenesis, from transcription initiation to alternative splicing. As RNAPII moves along the DNA, it must read the DNA sequences wrapped up as chromatin. Thus, the structure of chromatin impedes the movement and speed at which RNAPII moves, presenting a crucial regulation to gene expression. Therefore, factors that bind and regulate the structure of chromatin will impact the rate of RNAPII elongation. We previously showed that PARP1 (poly-ADP-ribose polymerase 1) is one of such factors that bind and alter chromatin dynamics. We also showed that its alteration of chromatin structure modulates RNAPII processivity during transcriptional elongation. Here, we aim to understand how PARP1 alters RNAPII elongation kinetics genome wide.

Elongation factor-specific capture of RNA polymerase II complexes

Transcription of protein-coding genes is regulated by dynamic association of co-factors with RNA polymerase II (RNAPII). The function of these factors and their relationship with RNAPII is often poorly understood. Here, we present an approach for elongation-factor-specific mNET capture (ELCAP) of RNAPII complexes for sequencing and mass spectrometry analysis aimed at investigating the function of such RNAPII regulatory proteins. As proof of principle, we apply ELCAP to the RNAPII-associated proteins SCAF4 and SCAF8, which share an essential role as mRNA anti-terminators but have individual roles at the 3' end of genes. Mass spectrometry analysis shows that both SCAF4 and SCAF8 are part of RNAPII elongation complexes containing 3' end processing factors but depleted of splicing components. Importantly, the ELCAP sequencing (ELCAP-seq) profiles of SCAF4- and SCAF8-RNAPII complexes nicely reflect their function as mRNA-anti-terminators and their competing functions at the end of genes, where they prevent or promote transcriptional readthrough.

Distinct role of subunits of the Arabidopsis RNA polymerase II elongation factor PAF1C in transcriptional reprogramming.

Transcript elongation by RNA polymerase II (RNAPII) is dynamic and highly regulated, thereby contributing to the implementation of gene expression programs during plant development or in response to environmental cues. The heterohexameric polymerase-associated factor 1 complex (PAF1C) stabilizes the RNAPII elongation complex promoting efficient transcript synthesis. In addition, PAF1C links transcriptional elongation with various post-translational histone modifications at transcribed loci. We have exposed Arabidopsis mutants deficient in the PAF1C subunits ELF7 or CDC73 to elevated NaCl concentrations to provoke a transcriptional response. The growth of elf7 plants was reduced relative to that of wildtype under these challenging conditions, whereas cdc73 plants exhibited rather enhanced tolerance. Profiling of the transcriptional changes upon NaCl exposure revealed that cdc73 responded similar to wildtype. Relative to wildtype and cdc73, the transcriptional response of elf7 plants was severely reduced in accord with their greater susceptibility to NaCl. The data also imply that CDC73 is more relevant for the transcription of longer genes. Despite the fact that both ELF7 and CDC73 are part of PAF1C the strikingly different transcriptional response of the mutants upon NaCl exposure suggests that the subunits have (partially) specific functions.

Slow RNAPII Transcription Elongation Rate, Low Levels of RNAPII Pausing, and Elevated Histone H1 Content at Promoters Associate with Higher m6A Deposition on Nascent mRNAs.

N6-methyladenosine modification (m6A) fine-tunes RNA fate in a variety of ways, thus regulating multiple fundamental biological processes. m6A writers bind to chromatin and interact with RNA polymerase II (RNAPII) during transcription. To evaluate how the dynamics of the transcription process impact m6A deposition, we studied RNAPII elongation rates in mouse embryonic stem cells with altered chromatin configurations, due to reductions in linker histone H1 content. We found that genes transcribed at slow speed are preferentially methylated and display unique signatures at their promoter region, namely high levels of histone H1, together with marks of bivalent chromatin and low RNAPII pausing. They are also highly susceptible to m6A loss upon histone H1 reduction. These results indicate that RNAPII velocity links chromatin structure and the deposition of m6A, highlighting the intricate relationship between different regulatory layers on nascent mRNA molecules.

Structural basis of nucleosome disassembly and reassembly by RNAPII elongation complex with FACT.

During gene transcription, RNA polymerase II (RNAPII) traverses nucleosomes in chromatin, but the mechanism has remained elusive. Using cryo-electron microscopy, we obtained structures of the RNAPII elongation complex (EC) passing through a nucleosome in the presence of the transcription elongation factors Spt6, Spn1, Elf1, Spt4/5, and Paf1C and the histone chaperone FACT (facilitates chromatin transcription). The structures show snapshots of EC progression on DNA mediating downstream nucleosome disassembly, followed by its reassembly upstream of the EC, which is facilitated by FACT. FACT dynamically adapts to successively occurring subnucleosome intermediates, forming an interface with the EC. Spt6, Spt4/5, and Paf1C form a "cradle" at the EC DNA-exit site and support the upstream nucleosome reassembly. These structures explain the mechanism by which the EC traverses nucleosomes while maintaining the chromatin structure and epigenetic information.

Targeted protein degradation reveals a direct role of SPT6 in RNAPII elongation and termination

SummarySPT6 is a histone chaperone that tightly binds RNA polymerase II (RNAPII) during transcription elongation. However, its primary role in transcription is uncertain. We used targeted protein degradation to rapidly deplete SPT6 in human cells and analyzed defects in RNAPII behavior by a multi-omics approach and mathematical modeling. Our data indicate that SPT6 is a crucial factor for RNAPII processivity and is therefore required for the productive transcription of protein-coding genes. Unexpectedly, SPT6 also has a vital role in RNAPII termination, as acute depletion induced readthrough transcription for thousands of genes. Long-term depletion of SPT6 induced cryptic intragenic transcription, as observed earlier in yeast. However, this phenotype was not observed upon acute SPT6 depletion and therefore can be attributed to accumulated epigenetic perturbations in the prolonged absence of SPT6. In conclusion, targeted degradation of SPT6 allowed the temporal discrimination of its function as an epigenetic safeguard and RNAPII elongation factor.

New connections between ubiquitylation and methylation in the co-transcriptional histone modification network.

Co-transcriptional histone modifications are a ubiquitous feature of RNA polymerase II (RNAPII) transcription, with profound but incompletely understood effects on gene expression. Unlike the covalent marks found at promoters, which are thought to be instructive for transcriptional activation, these modifications occur in gene bodies as a result of transcription, which has made elucidation of their functions challenging. Here we review recent insights into the regulation and roles of two such modifications: monoubiquitylation of histone H2B at lysine 120 (H2Bub1) and methylation of histone H3 at lysine 36 (H3K36me). Both H2Bub1 and H3K36me are enriched in the coding regions of transcribed genes, with highly overlapping distributions, but they were thought to work largely independently. We highlight our recent demonstration that, as was previously shown for H3K36me, H2Bub1 signals to the histone deacetylase (HDAC) complex Rpd3S/Clr6-CII, and that Rpd3S/Clr6-CII and H2Bub1 function in the same pathway to repress aberrant antisense transcription initiating within gene coding regions. Moreover, both of these histone modification pathways are influenced by protein phosphorylation catalyzed by the cyclin-dependent kinases (CDKs) that regulate RNAPII elongation, chiefly Cdk9. Therefore, H2Bub1 and H3K36me are more tightly linked than previously thought, sharing both upstream regulatory inputs and downstream effectors. Moreover, these newfound connections suggest extensive, bidirectional signaling between RNAPII elongation complexes and chromatin-modifying enzymes, which helps to determine transcriptional outputs and should be a focus for future investigation.

Elongin A regulates transcription in vivo through enhanced RNA polymerase processivity

Elongin is an RNA polymerase II (RNAPII)-associated factor that has been shown to stimulate transcriptional elongation in vitro. The Elongin complex is thought to be required for transcriptional induction in response to cellular stimuli and to ubiquitinate RNAPII in response to DNA damage. Yet, the impact of the Elongin complex on transcription in vivo has not been well studied. Here, we performed comprehensive studies of the role of Elongin A, the largest subunit of the Elongin complex, on RNAPII transcription genome-wide. Our results suggest that Elongin A localizes to actively transcribed regions and potential enhancers, and the level of recruitment correlated with transcription levels. We also identified a large group of factors involved in transcription as Elongin A–associated factors. In addition, we found that loss of Elongin A leads to dramatically reduced levels of serine2-phosphorylated, but not total, RNAPII, and cells depleted of Elongin A show stronger promoter RNAPII pausing, suggesting that Elongin A may be involved in the release of paused RNAPII. Our RNA-seq studies suggest that loss of Elongin A did not alter global transcription, and unlike prior in vitro studies, we did not observe a dramatic impact on RNAPII elongation rates in our cell-based nascent RNA-seq experiments upon Elongin A depletion. Taken together, our studies provide the first comprehensive analysis of the role of Elongin A in regulating transcription in vivo. Our studies also revealed that unlike prior in vitro findings, depletion of Elongin A has little impact on global transcription profiles and transcription elongation in vivo.

Nutrient-dependent control of RNA polymerase II elongation rate regulates specific gene expression programs by alternative polyadenylation.

Transcription by RNA polymerase II (RNAPII) is a dynamic process with frequent variations in the elongation rate. However, the physiological relevance of variations in RNAPII elongation kinetics has remained unclear. Here we show in yeast that a RNAPII mutant that reduces the transcription elongation rate causes widespread changes in alternative polyadenylation (APA). We unveil two mechanisms by which APA affects gene expression in the slow mutant: 3' UTR shortening and gene derepression by premature transcription termination of upstream interfering noncoding RNAs. Strikingly, the genes affected by these mechanisms are enriched for functions involved in phosphate uptake and purine synthesis, processes essential for maintenance of the intracellular nucleotide pool. As nucleotide concentration regulates transcription elongation, our findings argue that RNAPII is a sensor of nucleotide availability and that genes important for nucleotide pool maintenance have adopted regulatory mechanisms responsive to reduced rates of transcription elongation.

GSK-3 is an RNA polymerase II phospho-CTD kinase.

We have previously found that UV-induced DNA damage causes hyperphosphorylation of the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), inhibition of transcriptional elongation and changes in alternative splicing (AS) due to kinetic coupling between transcription and splicing. In an unbiased search for protein kinases involved in the AS response to DNA damage, we have identified glycogen synthase kinase 3 (GSK-3) as an unforeseen participant. Unlike Cdk9 inhibition, GSK-3 inhibition only prevents CTD hyperphosphorylation triggered by UV but not basal phosphorylation. This effect is not due to differential degradation of the phospho-CTD isoforms and can be reproduced, at the AS level, by overexpression of a kinase-dead GSK-3 dominant negative mutant. GSK-3 inhibition abrogates both the reduction in RNAPII elongation and changes in AS elicited by UV. We show that GSK-3 phosphorylates the CTD in vitro, but preferentially when the substrate is previously phosphorylated, consistently with the requirement of a priming phosphorylation reported for GSK-3 efficacy. In line with a role for GSK-3 in the response to DNA damage, GSK-3 inhibition prevents UV-induced apoptosis. In summary, we uncover a novel role for a widely studied kinase in key steps of eukaryotic transcription and pre-mRNA processing.