Ssu72 is essential to suppress initiation of cryptic transcription in the 3' end of the RNA polymerase II transcribed regions.
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ABSTRACT:
ORGANISM(S): Schizosaccharomyces pombe
PROVIDER: GSE144603 | GEO | 2020/06/30
REPOSITORIES: GEO
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Project description:Ssu72 is essential to suppress initiation of cryptic transcription in the 3' end of the RNA polymerase II transcribed regions.
| PRJNA604184 | ENA
Project description:In eukaryotes, the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is composed of tandem repeats of the heptapeptide YSPTSPS, which is subjected to reversible phosphorylation at Ser2, Ser5, and Ser7 during the transcription cycle. Dynamic changes in CTD phosphorylation patterns, established by the activities of multiple kinases and phosphatases, are responsible for stage-specific recruitment of various factors involved in RNA processing, histone modification, and transcription elongation/termination. Yeast Ssu72, a CTD phosphatase specific for Ser5 and Ser7, functions in 3'-end processing of pre-mRNAs and in transcription termination of small non-coding RNAs such as snoRNAs and snRNAs. Vertebrate Ssu72 exhibits Ser5- and Ser7-specific CTD phosphatase activity in vitro, but its roles in gene expression and CTD dephosphorylation in vivo remain to be elucidated. To investigate the functions of vertebrate Ssu72 in gene expression, we established chicken DT40 B-cell lines in which Ssu72 expression was conditionally inactivated. Ssu72 depletion in DT40 cells caused defects in 3'-end formation of U2 and U4 snRNAs and GAPDH mRNA. Surprisingly, however, Ssu72 inactivation increased the efficiency of 3'-end formation of non-polyadenylated replication-dependent histone mRNA. Chromatin immunoprecipitation analyses revealed that Ssu72 depletion caused a significant increase in both Ser5 and Ser7 phosphorylation of the Pol II CTD on all genes in which 3'-end formation was affected. These results suggest that vertebrate Ssu72 plays positive roles in 3'-end formation of snRNAs and polyadenylated mRNAs, but negative roles in 3'-end formation of histone mRNAs, through dephosphorylation of both Ser5 and Ser7 of the CTD.
| S-EPMC4148344 | biostudies-literature
Project description:Phosphorylation of the C-terminal domain (CTD) of the large subunit of human RNA polymerase II (Pol II) is regulated during the transcription cycle by the combined action of specific kinases and phosphatases. Pol II enters into the preinitiation complex (PIC) unphosphorylated, but is quickly phosphorylated by Cdk7 during initiation. How phosphatases alter the pattern and extent of CTD phosphorylation at this early stage of transcription is not clear. We previously demonstrated the functional association of an early-acting, magnesium-independent phosphatase with early elongation complexes. Here we show that Ssu72 is responsible for that activity. We found that the phosphatase enters the transcription cycle during the formation of PICs and that Ssu72 is physically associated with very early elongation complexes. The association of Ssu72 with elongation complexes was stable to extensive washing with up to 200 mM KCl. Interestingly, Ssu72 ceased to function on complexes that contained RNA longer than 28 nt. However, when PICs were washed before initiation, the strict cutoff at 28 nt was lost. This suggests that factor(s) are important for the specific regulation of Ssu72 function during the transition between initiation and pausing. Overall, our results demonstrate when Ssu72 can act on early transcription complexes and suggest that Ssu72 may also function in the PIC prior to initiation.
| S-EPMC6430399 | biostudies-literature
Project description:Reversible phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (Pol II) is essential for gene expression control. How altering the phosphorylation of the CTD contributes to gene expression in mammalian systems remains poorly understood. Methods: Primary mouse embryonic fibroblasts, hepatocytes, and embryonic stem cells were isolated from conditional Ssu72 f/f mice. To knockout the mouse Ssu72 gene, we infected the cells with adenoviruses of incorporated luciferase and Cre recombinase, respectively. RNA sequencing, ChIP sequencing, ChIP assay, immunoblot analyses, qRT-PCR assay, and immunostaining were performed to gain insights into the functional mechanisms of Ssu72 loss in Pol II dynamics. Results: Using primary cells isolated from Ssu72 conditional knockout and transgenic mice, we found that mammalian Ssu72-mediated transcriptional elongation rather than polyadenylation or RNA processing contributed to the transcriptional regulation of various genes. Depletion of Ssu72 resulted in aberrant Pol II pausing and elongation defects. Reduced transcriptional elongation efficiency tended to preferentially affect expression levels of actively transcribed genes in a tissue-specific manner. Furthermore, Ssu72 CTD phosphatase seemed to regulate the phosphorylation levels of CTD Ser2 and Thr4 through accurate modulation of P-TEFb activity and recruitment. Conclusions: Our findings demonstrate that mammalian Ssu72 contributes to the transcription of tissue-specific actively transcribed gene expression by regulating reciprocal phosphorylation of Pol II CTD.
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Project description:MicroRNAs (miRNAs) constitute a large family of noncoding RNAs that function as guide molecules in diverse gene silencing pathways. Current efforts are focused on the regulatory function of miRNAs, while little is known about how these unusual genes themselves are regulated. Here we present the first direct evidence that miRNA genes are transcribed by RNA polymerase II (pol II). The primary miRNA transcripts (pri-miRNAs) contain cap structures as well as poly(A) tails, which are the unique properties of class II gene transcripts. The treatment of human cells with alpha-amanitin decreased the level of pri-miRNAs at a concentration that selectively inhibits pol II activity. Furthermore, chromatin immunoprecipitation analyses show that pol II is physically associated with a miRNA promoter. We also describe, for the first time, the detailed structure of a miRNA gene by determining the promoter and the terminator of mir-23a approximately 27a approximately 24-2. These data indicate that pol II is the main, if not the only, RNA polymerase for miRNA gene transcription. Our study offers a basis for understanding the structure and regulation of miRNA genes.
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Project description:We examine the association of the beta-, alpha-, and sigma(70)-subunits of Escherichia coli RNA polymerase (RNAP) and the NusA elongation factor with transcribed regions in vivo by using chromatin immunoprecipitation. RNAP preferentially associates with the promoter-proximal region of several operons, and this preference is particularly pronounced at the lexA-dinF promoter. When cells are grown in exponential phase, little or no sigma(70) is associated with RNAP during early elongation. However, during stationary phase, sigma(70) is retained in a fraction of elongating RNAP complexes throughout the melAB operon. In contrast, sigma(70) is not observed in elongating RNAP complexes at the lacZYA operon during stationary phase. At both operons, NusA associates with RNAP during early elongation, and this association is greatly reduced during stationary phase. These observations suggest that in vivo association of sigma(70) and NusA with elongating RNAP is regulated by growth conditions.
| S-EPMC539717 | biostudies-literature
Project description:Precise regulation of transcription by RNA polymerase II (RNAPII) is critical for organismal growth and development. However, what determines whether an engaged RNAPII will synthesize a full-length transcript or terminate prematurely is poorly understood. Notably, RNAPII is far more susceptible to termination when transcribing non-coding RNAs than when synthesizing protein-coding mRNAs, but the mechanisms underlying this are unclear. To investigate the impact of transcribed sequence on elongation potential, we developed a method to screen the effects of thousands of INtegrated Sequences on Expression of RNA and Translation using high-throughput sequencing (INSERT-seq). We found that higher AT content in non-coding RNAs, rather than specific sequence motifs, drives RNAPII termination. Further, we demonstrate that 5' splice sites autonomously stimulate processive transcription, even in the absence of polyadenylation signals. Our results reveal a potent role for the transcribed sequence in dictating gene output and demonstrate the power of INSERT-seq toward illuminating these contributions.
| S-EPMC9672417 | biostudies-literature
Project description:The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) serves an important role in coordinating stage-specific recruitment and release of cellular machines during transcription. Dynamic placement and removal of phosphorylation marks on different residues of a repeating heptapeptide (YSPTSPS) of the CTD underlies the engagement of relevant cellular machinery. Whereas sequential placement of phosphorylation marks is well explored, genome-wide engagement of phosphatases that remove these CTD marks is poorly understood. In particular, identifying the enzyme that erases phospho-Ser7 (Ser7-P) marks is especially important, because we find that substituting this residue with a glutamate, a phospho-mimic, is lethal. Our observations implicate Ssu72 as a Ser7-P phosphatase. We report that removal of all phospho-CTD marks during transcription termination is mechanistically coupled. An inability to remove these marks prevents Pol II from terminating efficiently and will likely impede subsequent assembly into the pre-initiation complex.
| S-EPMC3318730 | biostudies-literature
Project description:This report investigates the mechanisms by which mammalian cells coordinate DNA replication with transcription and chromatin assembly. In yeast, DNA replication initiates within nucleosome-free regions, but studies in mammalian cells have not revealed a similar relationship. Here, we have used genome-wide massively parallel sequencing to map replication initiation events, thereby creating a database of all replication initiation sites within nonrepetitive DNA in two human cell lines. Mining this database revealed that genomic regions transcribed at moderate levels were generally associated with high replication initiation frequency. In genomic regions with high rates of transcription, very few replication initiation events were detected. High-resolution mapping of replication initiation sites showed that replication initiation events were absent from transcription start sites but were highly enriched in adjacent, downstream sequences. Methylation of CpG sequences strongly affected the location of replication initiation events, whereas histone modifications had minimal effects. These observations suggest that high levels of transcription interfere with formation of pre-replication protein complexes. Data presented here identify replication initiation sites throughout the genome, providing a foundation for further analyses of DNA-replication dynamics and cell-cycle progression.
| S-EPMC3205567 | biostudies-literature
Project description:Interest in the C-terminal domain (CTD) of the RPB1 subunit of the RNA polymerase II (Pol II) has been revived in recent years, owing to its numerous posttranslational modifications and its "phase-separation" properties. A large number of studies have shown that the status of CTD modifications is associated with the activity of Pol II during the transcription cycle. However, because this domain is essential in living cells, the functional requirement of the full CTD for the control of Pol II activity at endogenous mammalian genes has never been addressed directly in living cells. Using an inducible Pol II-degradation system that we previously established, we investigated here the roles of the CTD in the post-initiation control of Pol II. The selective ablation of the RPB1 CTD, post-initiation, at promoter-proximal pause-sites revealed that this domain, and by extension the CTD heptads and their modifications, is functionally neither absolutely required to maintain pausing in the absence of CDK9 activity nor essential for the release of Pol II into productive elongation.
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