3′ end formation of pre-mRNA and phosphorylation of Ser2 on the RNA polymerase II CTD are reciprocally coupled in human cells

Davidson, Lee; Muniz, Lisa; West, Steven

doi:10.1101/gad.231274.113

Cited by 137 publications

(167 citation statements)

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“…S6E). Neverthless, emerging evidence has shown that CDK12 contains the arginine/serine domain and is involved in RNA processing (32,33). In addition, CDK12 has been implicated in mRNA splicing (15,34,35).…”

Section: Discussionmentioning

confidence: 99%

Heterochromatin remodeling by CDK12 contributes to learning in Drosophila

Pan

Xie

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Dynamic regulation of chromatin structure is required to modulate the transcription of genes in eukaryotes. However, the factors that contribute to the plasticity of heterochromatin structure are elusive. Here, we report that cyclin-dependent kinase 12 (CDK12), a transcription elongation-associated RNA polymerase II (RNAPII) kinase, antagonizes heterochromatin enrichment in Drosophila chromosomes. Notably, loss of CDK12 induces the ectopic accumulation of heterochromatin protein 1 (HP1) on euchromatic arms, with a prominent enrichment on the X chromosome. Furthermore, ChIP and sequencing analysis reveals that the heterochromatin enrichment on the X chromosome mainly occurs within long genes involved in neuronal functions. Consequently, heterochromatin enrichment reduces the transcription of neuronal genes in the adult brain and results in a defect in Drosophila courtship learning. Taken together, these results define a previously unidentified role of CDK12 in controlling the epigenetic transition between euchromatin and heterochromatin and suggest a chromatin regulatory mechanism in neuronal behaviors.A ppropriate regulation of gene transcription is essential throughout the life of an organism. In eukaryotes, DNA and histone octamers are assembled into nucleosomes and further compacted into higher-order structures (1). Dynamic changes in the chromatin architecture affect gene transcription in many aspects of developmental and physiological processes, such as neural plasticity, memory formation, and cognition (2, 3). Eukaryotic genomes are composed of two basic forms, euchromatin and heterochromatin, which are originally characterized by their cytological chromatin packaging levels. Euchromatic regions are generally associated with a relatively open chromatin configuration and mainly contain transcriptionally active genes, whereas heterochromatin is highly compacted and less accessible to the transcriptional machinery (4). Drosophila heterochromatin is mainly localized at the pericentric and subtelomeric regions and enriched for methylation of histone H3 on Lys9 (H3K9me), which provides a docking site for heterochromatin protein 1 (HP1) (5, 6), a highly conserved protein involved in heterochromatin formation (7). Previous studies also show that HP1 and H3K9me associate with a subset of loci on euchromatic regions, and they presumably serve to fine tune the level of gene transcription (8-10). One significant question that comes up is how the chromatin structure is dynamically regulated to impact expression of genes in complex neuronal processes, such as learning and memory. Our earlier study on chromatin domain mapping of chromosome 4 suggests that heterochromatic domains may be regulated by the activities of RNA polymerase II (RNAPII) complexes, which form a "barrier" to prevent heterochromatin spreading and transcriptional gene silencing (11). However, the mechanism underlying such a mode of regulation and the consequence on reprogrammed transcription remain to be investigated. ResultsCyclin-Dependent Kinase 12 ...

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Section: Discussionmentioning

confidence: 99%

Heterochromatin remodeling by CDK12 contributes to learning in Drosophila

Pan

Xie

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…This is consistent with the finding that RNAPII pausing downstream of the 3 0 end of genes is sufficient for Ser2 phosphorylation. 25 Mutational analysis performed in human cells using an RNAPII lacking serine 2 resulted in impaired 3 0 end formation as well as loss in the binding of the 3 0 end factor PCF11. 23 Moreover, RNAPII levels that are maximally phosphorylated on serine 2 tend to peak downstream of the site of polyadenylation.…”

Section: Discussionmentioning

confidence: 99%

“…24 Interestingly, knockdown of CPSF73 also inhibits Ser2p downstream of the B-globin gene. 25 Therefore, the phosphorylation of Ser2 within the CTD most likely stimulates the recruitment of 3 0 end formation factors, which in a positive feedback loop strengthens Ser2 phosphorylation. 25 In mammals, mRNA 3 0 end formation consists of an endonucleolytic cleavage, followed by polyadenylation of the free 3 0 end.…”

Section: Introductionmentioning

confidence: 99%

“…25 Therefore, the phosphorylation of Ser2 within the CTD most likely stimulates the recruitment of 3 0 end formation factors, which in a positive feedback loop strengthens Ser2 phosphorylation. 25 In mammals, mRNA 3 0 end formation consists of an endonucleolytic cleavage, followed by polyadenylation of the free 3 0 end. Transcription terminates often several kb downstream of the poly-A site [26][27][28][29][30] and is mechanistically linked to 3 0 end formation.…”

Section: Introductionmentioning

confidence: 99%

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Localization of RNAPII and 3′ end formation factor CstF subunits onC. elegansgenes and operons

2016

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Transcription termination is mechanistically coupled to pre-mRNA 3 0 end formation to prevent transcription much beyond the gene 3 0 end. C. elegans, however, engages in polycistronic transcription of operons in which 3 0 end formation between genes is not accompanied by termination. We have performed RNA polymerase II (RNAPII) and CstF ChIP-seq experiments to investigate at a genome-wide level how RNAPII can transcribe through multiple poly-A signals without causing termination. Our data shows that transcription proceeds in some ways as if operons were composed of multiple adjacent single genes. Total RNAPII shows a small peak at the promoter of the gene cluster and a much larger peak at 3 0 ends. These 3 0 peaks coincide with maximal phosphorylation of Ser2 within the C-terminal domain (CTD) of RNAPII and maximal localization of the 3 0 end formation factor CstF. This pattern occurs at all 3 0 ends including those at internal sites in operons where termination does not occur. Thus the normal mechanism of 3 0 end formation does not always result in transcription termination. Furthermore, reduction of CstF50 by RNAi did not substantially alter the pattern of CstF64, total RNAPII, or Ser2 phosphorylation at either internal or terminal 3 0 ends. However, CstF50 RNAi did result in a subtle reduction of CstF64 binding upstream of the site of 3 0 cleavage, suggesting that the CstF50/CTD interaction may facilitate bringing the 3 0 end machinery to the transcription complex.

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“…2B) toward Flag-Xrn2. Cdk12 and Cdk13 form complexes with cyclin K and are thought to be Ser2 kinases (Bartkowiak et al 2010) with roles in elongation and 3 ′ end formation (Davidson et al 2014). The amount of each CDK was normalized based on activity toward the Pol II CTD (Supplemental Fig.…”

Section: Xrn2 Is a Cdk Substratementioning

confidence: 99%

P-TEFb regulation of transcription termination factor Xrn2 revealed by a chemical genetic screen for Cdk9 substrates

et al. 2016

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The transcription cycle of RNA polymerase II (Pol II) is regulated at discrete transition points by cyclin-dependent kinases (CDKs). Positive transcription elongation factor b (P-TEFb), a complex of Cdk9 and cyclin T1, promotes release of paused Pol II into elongation, but the precise mechanisms and targets of Cdk9 action remain largely unknown. Here, by a chemical genetic strategy, we identified ∼100 putative substrates of human P-TEFb, which were enriched for proteins implicated in transcription and RNA catabolism. Among the RNA processing factors phosphorylated by Cdk9 was the 5 ′ -to-3 ′ "torpedo" exoribonuclease Xrn2, required in transcription termination by Pol II, which we validated as a bona fide P-TEFb substrate in vivo and in vitro. Phosphorylation by Cdk9 or phosphomimetic substitution of its target residue, Thr439, enhanced enzymatic activity of Xrn2 on synthetic substrates in vitro. Conversely, inhibition or depletion of Cdk9 or mutation of Xrn2-Thr439 to a nonphosphorylatable Ala residue caused phenotypes consistent with inefficient termination in human cells: impaired Xrn2 chromatin localization and increased readthrough transcription of endogenous genes. Therefore, in addition to its role in elongation, P-TEFb regulates termination by promoting chromatin recruitment and activation of a cotranscriptional RNA processing enzyme, Xrn2.

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3′ end formation of pre-mRNA and phosphorylation of Ser2 on the RNA polymerase II CTD are reciprocally coupled in human cells

Cited by 137 publications

References 59 publications

Heterochromatin remodeling by CDK12 contributes to learning in Drosophila

Heterochromatin remodeling by CDK12 contributes to learning in Drosophila

Localization of RNAPII and 3′ end formation factor CstF subunits onC. elegansgenes and operons

P-TEFb regulation of transcription termination factor Xrn2 revealed by a chemical genetic screen for Cdk9 substrates

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