Shona Murphy

Serine-7 of the RNA Polymerase II CTD Is Specifically Required for snRNA Gene Expression

RNA polymerase II (Pol II) transcribes genes that encode proteins and noncoding small nuclear RNAs (snRNAs). The carboxyl-terminal repeat domain (CTD) of the largest subunit of mammalian RNA Pol II, comprising tandem repeats of the heptapeptide consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, is required for expression of both gene types. We show that mutation of serine-7 to alanine causes a specific defect in snRNA gene expression. We also present evidence that phosphorylation of serine-7 facilitates interaction with the snRNA gene–specific Integrator complex. These findings assign a biological function to this amino acid and highlight a gene type–specific requirement for a residue within the CTD heptapeptide, supporting the existence of a CTD code.

U1 snRNA associates with TFIIH and regulates transcriptional initiation

Diverse classes of noncoding RNA, including small nuclear RNAs (snRNAs), play fundamental regulatory roles at many stages of gene expression. For example, recent studies have implicated 7SK RNA and components of the splicing apparatus in the regulation of transcriptional elongation. Here we present the first evidence of the involvement of an snRNA in the regulation of transcriptional initiation. We demonstrate that TFIIH, a general transcription initiation factor, specifically associates with U1 snRNA, a core-splicing component. Analysis of the TFIIH-dependent stages of transcription in a reconstituted system demonstrates that U1 stimulates the rate of formation of the first phosphodiester bond by RNA polymerase II. In addition, a promoter-proximal 5′ splice site recognized by U1 snRNA stimulates TFIIH-dependent reinitiation of productive transcription. Our results suggest that U1 snRNA functions in regulating transcription by RNA Polymerase II in addition to its role in RNA processing.

Updating the RNA polymerase CTD code: adding gene-specific layers

The carboxyl-terminal domain (CTD) of RNA polymerase (pol) II comprises multiple tandem repeats with the consensus sequence Tyr(1)-Ser(2)-Pro(3)-Thr(4)-Ser(5)-Pro(6)-Ser(7) that can be extensively and reversibly modified in vivo. CTD modifications orchestrate the interplay between transcription and processing of mRNA. Although phosphorylation of Ser2 (Ser2P) and Ser5 (Ser5P) residues has been described as being essential for the expression of most pol II-transcribed genes, recent findings highlight gene-specific effects of newly discovered CTD modifications. Here, we incorporate these latest findings in an updated review of the currently known elements that contribute to the CTD code and how it is recognized by proteins involved in transcription and RNA maturation. As modification of the CTD has a major impact on gene expression, a better understanding of the CTD code is integral to the understanding of how gene expression is regulated.

Regional and temporal specialization in the nucleus: a transcriptionally‐active nuclear domain rich in PTF, Oct1 and PIKA antigens associates with specific chromosomes early in the cell cycle

PTF (PSE‐binding transcription factor) activates transcription of snRNA and related genes. We investigated its distribution in HeLa nuclei by immunofluorescence, and found it spread throughout the nucleoplasm in small foci. In some cells, PTF is also concentrated in one, or very few, discrete regions (diameter ∼1.3 μm) that appear during G1 phase and disappear in S phase. Oct1, a transcription factor that interacts with PTF, is also enriched in these domains; RNA polymerase II, TBP and Sp1 are also present. Each domain typically contains 2 or 3 transcription ‘factories’ where Br‐UTP is incorporated into nascent transcripts. Accordingly, we have christened this region the Oct1/PTF/transcription (OPT) domain. It colocalizes with some, but not all, PIKA domains. It is distinct from other nuclear domains, including coiled bodies, gemini bodies, PML bodies and the perinucleolar compartment. A small region on chromosome 6 (band 6p21) containing only ∼30 Mbp DNA, and chromosomes 6 and 7, associate with the domain significantly more than other chromosomes. The domains may act like nucleoli to bring particular genes on specific chromosomes together to a region where the appropriate transcription and processing factors are concentrated, thereby facilitating the expression of those genes.

The in vitro transcription of the 7SK RNA gene by RNA polymerase III is dependent only on the presence of an upstream promoter

Deletion analysis was carried out on the human 7SK RNA gene to map regions essential for in vitro transcription by RNA polymerase III. The sequence promoting transcription is located between 37 and 3 bp upstream of the 7SK RNA coding region. RNA polymerase III transcription of adjacent plasmid sequences can be directed by this promoter in the complete absence of the 7SK RNA coding region, indicating that no internal promoter sequences are required. Transcription is terminated by a stretch of T residues, typical of RNA polymerase III transcription. The promoter contains a TATA box at position -25, mutations within which dramatically reduce the efficiency of transcription. Upstream sequences from position -37 to -243 increase the promoters efficiency. The promoter recognized by RNA polymerase III is structurally and functionally similar to the promoter of genes transcribed by RNA polymerase II.

The C-terminal domain of pol II and a DRB-sensitive kinase are required for 3′ processing of U2 snRNA

The human snRNA genes transcribed by RNA polymerase II (e.g. U1 and U2) have a characteristic TATA‐less promoter containing an essential proximal sequence element. Formation of the 3′ end of these non‐polyadenylated RNAs requires a specialized 3′ box element whose function is promoter specific. Here we show that truncation of the C‐terminal domain (CTD) of RNA polymerase II and treatment of cells with CTD kinase inhibitors, including DRB (5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole), causes a dramatic reduction in proper 3′ end formation of U2 transcripts. Activation of 3′ box recognition by the phosphorylated CTD would be consistent with the role of phospho‐CTD in mRNA processing. CTD kinase inhibitors, however, have little effect on initiation or elongation of transcription of the U2 genes, whereas elongation of transcription of the β‐actin gene is severely affected. This result highlights differences in transcription of snRNA and mRNA genes.

Ser7 Phosphorylation of the CTD Recruits the RPAP2 Ser5 Phosphatase to snRNA Genes

Summary The carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II) comprises multiple heptapeptide repeats of the consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Reversible phosphorylation of Ser2, Ser5, and Ser7 during the transcription cycle mediates the sequential recruitment of transcription/RNA processing factors. Phosphorylation of Ser7 is required for recruitment of the gene type-specific Integrator complex to the Pol II-transcribed small nuclear (sn)RNA genes. Here, we show that RNA Pol II-associated protein 2 (RPAP2) specifically recognizes the phospho-Ser7 mark on the Pol II CTD and also interacts with Integrator subunits. siRNA-mediated knockdown of RPAP2 and mutation of Ser7 to alanine cause similar defects in snRNA gene expression. In addition, we show that RPAP2 is a CTD Ser5 phosphatase. Taken together, our results indicate that during transcription of snRNA genes, Ser7 phosphorylation facilitates recruitment of RPAP2, which in turn both recruits Integrator and dephosphorylates Ser5.

P-TEFb is not an essential elongation factor for the intronless human U2 snRNA and histone H2b genes.

Phosphorylation of Ser2 of the heptapeptide repeat of the CTD of mammalian pol II by P‐TEFb is associated with productive elongation of transcription of protein‐coding genes. Here, we show that the CTD of pol II transcribing the human U2 snRNA genes is phosphorylated on Ser2 in vivo and that both the CDK9 kinase and cyclin T components of P‐TEFb are required for cotranscriptional recognition of the 3′ box RNA 3′ end processing signal. However, inhibitors of CDK9 do not affect transcription of the U2 genes, indicating that P‐TEFb functions exclusively as an RNA processing factor in expression of these relatively short, intronless genes. We also show that inhibition of CDK9 does not adversely affect either transcription of an intron‐less, replication‐activated histone H2b gene or recognition of the histone gene‐specific U7‐dependent RNA 3′ end formation signal. These results emphasize that the role of P‐TEFb as an activator of transcription elongation can be separated from its role in RNA processing and that neither function is universally required for expression of mammalian pol II‐dependent genes.

The integrator complex recognizes a new double mark on the RNA polymerase II carboxyl-terminal domain.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II) comprises multiple tandem repeats of the heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. This unusual structure serves as a platform for the binding of factors required for expression of pol II-transcribed genes, including the small nuclear RNA (snRNA) gene-specific Integrator complex. The pol II CTD specifically mediates recruitment of Integrator to the promoter of snRNA genes to activate transcription and direct 3′ end processing of the transcripts. Phosphorylation of the CTD and a serine in position 7 are necessary for Integrator recruitment. Here, we have further investigated the requirement of the serines in the CTD heptapeptide and their phosphorylation for Integrator binding. We show that both Ser2 and Ser7 of the CTD are required and that phosphorylation of these residues is necessary and sufficient for efficient binding. Using synthetic phosphopeptides, we have determined the pattern of the minimal Ser2/Ser7 double phosphorylation mark required for Integrator to interact with the CTD. This novel double phosphorylation mark is a new addition to the functional repertoire of the CTD code and may be a specific signal for snRNA gene expression.

Impact of Alpha Interferon and Ribavirin on the Function of Maturing Dendritic Cells

ABSTRACT Alpha interferon and ribavirin are required in combination to achieve a sustained virological response in the treatment of hepatitis C virus (HCV) infection. Alpha interferon has direct antiviral activity and also enhances HCV-specific T-cell responses. Ribavirin has little direct activity against HCV but reduces hepatic inflammation. It is therefore likely that these drugs in combination have hitherto unidentified immunological effects. In the present study we investigated the effects of alpha interferon and ribavirin on dendritic cell (DC) maturation and cytokine production induced by double-stranded RNA in vitro. Alpha interferon alone enhanced the expression of HLA class I, HLA class II, and CD86 on immature DCs but did not stimulate full DC maturation, which requires the expression of CD83. Alpha interferon enhanced the production of interleukin 12 p70 [IL-12(p70)] and tumor necrosis factor alpha (TNF-α) but had no effect on IL-10 production. In contrast, ribavirin at physiological doses had no effect on DC maturation but markedly suppressed the production of TNF-α, IL-10, and IL-12(p70). The suppression of cytokines by ribavirin cannot be explained by the induction of DC apoptosis or cell death. Quantitative PCR confirmed that cytokine suppression occurs at the level of mRNA. The suppression of IL-12(p70) and TNF-α in maturing DCs may explain the reduction in hepatic inflammation observed during ribavirin monotherapy. Combination alpha interferon-ribavirin therapy may alter the cytokine profile of maturing DCs overall by suppressing IL-10 production but maintaining IL-12(p70) and TNF-α production, a pattern that would favor viral elimination through downstream effects on T cells.