Biomedical subjects

Cyril F Bourgeois

Publications and source records attributed to Cyril F Bourgeois.

6 recordsLinked to original sources

PubMed2007

Alternative RNA splicing complexes containing the scaffold attachment factor SAFB2.

The scaffold attachment factor SAFB1 and its recently discovered homologue SAFB2 might provide an important link between pre-mRNA splicing, intracellular signalling and transcription. Using novel mono-specific antisera, we found endogenous SAFB2 protein has a different spatial distribution from SAFB1 within the nucleus where it is found in much larger nuclear complexes (up to 670 kDa in size), and a distinct pattern of expression in adult human testis. By contrast, SAFB1 protein predominantly exists either as smaller complexes or as a monomeric protein. Our results suggest stable core complexes containing components comprised of SAFB1, SAFB2 and the RNA binding proteins Sam68 and hnRNPG exist in parallel with free SAFB1 protein. We found that SAFB2 protein, like SAFB1, acts as a negative regulator of a tra2beta variable exon. Despite showing an involvement in splicing, we detected no stable interaction between SAFB proteins and SR or SR-related splicing regulators, although these were also found in stable higher molecular mass complexes. Each of the detected alternative splicing regulator complexes exists independently of intact nucleic acids, suggesting they might be pre-assembled and recruited to nascent transcripts as modules to facilitate alternative splicing, and/or they represent nuclear storage compartments from which active proteins are recruited.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Recruitment of TFIIH to the HIV LTR is a rate-limiting step in the emergence of HIV from latency.

Latently infected cells rapidly initiate HIV transcription after exposure to signals that induce NF-kappaB. To investigate the role of TFIIH during HIV reactivation in vivo, we developed a population of Jurkat cells containing integrated, but transcriptionally silent, HIV proviruses. Surprisingly, the HIV promoter in unactivated Jurkat T cells is partially occupied and carries Mediator containing the CDK8 repressive module, TFIID and RNAP II that is hypophosphorylated and confined to the promoter region. Significantly, the promoter is devoid of TFIIH. Upon stimulation of the cells by TNF-alpha, NF-kappaB and TFIIH are rapidly recruited to the promoter together with additional Mediator and RNAP II, but CDK8 is lost. Detailed time courses show that the levels of TFIIH at the promoter fluctuate in parallel with NF-kappaB recruitment to the promoter. Similarly, recombinant p65 activates HIV transcription in vitro and stimulates phosphorylation of the RNAP II CTD by the CDK7 kinase module of TFIIH. We conclude that the recruitment and activation of TFIIH represents a rate-limiting step for the emergence of HIV from latency.

Gene Expression Regulation, Viral↗

PubMed2005

Up-regulation of the ubiquitous alternative splicing factor Tra2beta causes inclusion of a germ cell-specific exon.

We have discovered a new exon of the homeodomain-interacting kinase HipK3 that incorporates a premature stop codon and is included only in the human testis. To investigate this, we tested the effects of transfecting cells with green fluorescent protein fusions of RNA-binding proteins implicated in spermatogenesis using a novel assay based on multi-fraction fluorescence-activated cell sorting (MF-FACS). This allows the effect of a controlled titration of any splicing factor on the splicing of endogenous genes to be studied in vivo. We found that Tra2beta recapitulates testis-specific splicing of endogenous HipK3 in a concentration-dependent manner and binds specifically to a long purine-rich sequence in the novel exon. This sequence was also specifically bound by hnRNP A1, hnRNP H, ASF/SF2 and SRp40, but not by 9G8. Consistent with these observations, in vitro studies showed that this sequence shifts splicing to a downstream 5' splice site within a heterologous pre-mRNA substrate in the presence of Tra2beta, ASF/SF2 and SRp40, whereas hnRNP A1 specifically inhibits this choice. By mutating the purine-rich sequence in the context of the HipK3 gene, we also show that it is the major determinant of Tra2beta- and hnRNP A1-mediated regulation. Tra2 is essential for sex determination and spermatogenesis in flies, and Tra2beta protein was most highly expressed in testis out of six mouse tissues, whereas hnRNP A1 is down-regulated during germ cell development. Therefore, our data imply an evolutionarily conserved role for Tra2 proteins in spermatogenesis and suggest that an elevated concentration of Tra2beta may convert it into a tissue-specific splicing factor.

Alternative Splicing↗

PubMed2004

Broad specificity of SR (serine/arginine) proteins in the regulation of alternative splicing of pre-messenger RNA.

Alternative splicing of pre-messenger RNA (pre-mRNA) is a highly regulated process that allows expansion of the potential of expression of the genome in higher eukaryotes and involves many factors. Among them, the family of the serine- and arginine-rich proteins (SR proteins) plays a pivotal role: it has essential functions during spliceosome assembly and also interacts with RNA regulatory sequences on the pre-mRNA as well as with multiple cofactors. Collectively, SR proteins, because of their capacity to recognize multiple RNA sequences with a broad specificity, are at the heart of the regulation pathways that lead to the choice of alternative splice sites. Moreover, a growing body of evidence shows that the mechanisms of splicing regulation are not limited to the basic involvement of cis- and trans-acting factors at the pre-mRNA level, but result from intricate pathways, initiated sometimes by stimuli that are external to the cell and integrate SR proteins (and other factors) within an extremely sophisticated network of molecular machines associated with one another. This review focuses on the molecular aspects of the functions of SR proteins. In particular, we discuss the different ways in which SR proteins manage to achieve a high level of specificity in splicing regulation, even though they are also involved in the constitutive reaction.

Alternative Splicing↗

PubMed2002

Phosphorylation of the RNA polymerase II carboxyl-terminal domain by CDK9 is directly responsible for human immunodeficiency virus type 1 Tat-activated transcriptional elongation.

Stimulation of transcriptional elongation by the human immunodeficiency virus type 1 Tat protein is mediated by CDK9, a kinase that phosphorylates the RNA polymerase II carboxyl-terminal domain (CTD). In order to obtain direct evidence that this phosphorylation event can alter RNA polymerase processivity, we prepared transcription elongation complexes that were arrested by the lac repressor. The CTD was then dephosphorylated by treatment with protein phosphatase 1. The dephosphorylated transcription complexes were able to resume the transcription elongation when IPTG (isopropyl-beta-D-thiogalactopyranoside) and nucleotides were added to the reaction. Under these chase conditions, efficient rephosphorylation of the CTD was observed in complexes containing the Tat protein but not in transcription complexes prepared in the absence of Tat protein. Immunoblots and kinase assays with synthetic peptides showed that Tat activated CDK9 directly since the enzyme and its cyclin partner, cyclin T1, were present at equivalent levels in transcription complexes prepared in the presence or absence of Tat. Chase experiments with the dephosphorylated elongation transcription complexes were performed in the presence of the CDK9 kinase inhibitor DRB (5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole). Under these conditions there was no rephosphorylation of the CTD during elongation, and transcription through either a stem-loop terminator or bent DNA arrest sequence was strongly inhibited. In experiments in which the CTD was phosphorylated prior to elongation, the amount of readthrough of the terminator sequences was proportional to the extent of the CTD modification. The change in processivity is due to CTD phosphorylation alone, since even after the removal of Spt5, the second substrate for CDK9, RNA polymerase elongation is enhanced by Tat-activated CDK9 activity. We conclude that phosphorylation of the RNA polymerase II CTD by CDK9 enhances transcription elongation directly.

Base Sequence↗

PubMed2002

Spt5 cooperates with human immunodeficiency virus type 1 Tat by preventing premature RNA release at terminator sequences.

The human immunodeficiency virus type 1 (HIV-1) Tat protein activates transcription elongation by stimulating the Tat-activated kinase (TAK/p-TEFb), a protein kinase composed of CDK9 and its cyclin partner, cyclin T1. CDK9 is able to hyperphosphorylate the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase during elongation. In addition to TAK, the transcription elongation factor Spt5 is required for the efficient activation of transcriptional elongation by Tat. To study the role of Spt5 in HIV transcription in more detail, we have developed a three-stage Tat-dependent transcription assay that permits the isolation of active preinitiation complexes, early-stage elongation complexes, and Tat-activated elongation complexes. Spt5 is recruited in the transcription complex shortly after initiation. After recruitment of Tat during elongation through the transactivation response element RNA, CDK9 is activated and induces hyperphosphorylation of Spt5 in parallel to the hyperphosphorylation of the CTD of RNA polymerase II. However, immunodepletion experiments demonstrate that Spt5 is not required for Tat-dependent activation of the kinase. Chase experiments using the Spt5-depleted extracts demonstrate that Spt5 is not required for early elongation. However, Spt5 plays an important role in late elongation by preventing the premature dissociation of RNA from the transcription complex at terminator sequences and reducing the amount of polymerase pausing at arrest sites, including bent DNA sequences. This novel biochemical function of Spt5 is analogous to the function of NusG, an elongation factor found in Escherichia coli that enhances RNA polymerase stability on templates and shows sequence similarity to Spt5.

Cyclin-Dependent Kinase 9↗