Van Trung Nguyen

Bio: Van Trung Nguyen is an academic researcher from École Normale Supérieure. The author has contributed to research in topics: RNA polymerase II & Transcription (biology). The author has an hindex of 16, co-authored 18 publications receiving 2113 citations. Previous affiliations of Van Trung Nguyen include Centre national de la recherche scientifique & French Institute of Health and Medical Research.

7SK small nuclear RNA binds to and inhibits the activity of CDK9/cyclin T complexes

Abstract: The transcription of eukaryotic protein-coding genes involves complex regulation of RNA polymerase (Pol) II activity in response to physiological conditions and developmental cues. One element of this regulation involves phosphorylation of the carboxy-terminal domain (CTD) of the largest polymerase subunit by a transcription elongation factor, P-TEFb, which comprises the kinase CDK9 and cyclin T1 or T2 (ref. 1). Here we report that in human HeLa cells more than half of the P-TEFb is sequestered in larger complexes that also contain 7SK RNA, an abundant, small nuclear RNA (snRNA) of hitherto unknown function2,3. P-TEFb and 7SK associate in a specific and reversible manner. In contrast to the smaller P-TEFb complexes, which have a high kinase activity, the larger 7SK/P-TEFb complexes show very weak kinase activity. Inhibition of cellular transcription by chemical agents or ultraviolet irradiation trigger the complete disruption of the P-TEFb/7SK complex, and enhance CDK9 activity. The transcription-dependent interaction of P-TEFb with 7SK may therefore contribute to an important feedback loop modulating the activity of RNA Pol II.

Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor

Abstract: The positive transcription elongation factor b (P-TEFb) plays a pivotal role in productive elongation of nascent RNA molecules by RNA polymerase II. Core active P-TEFb is composed of CDK9 and cyclin T. In addition, mammalian cell extracts contain an inactive P-TEFb complex composed of four components, CDK9, cyclin T, the 7SK snRNA and the MAQ1/HEXIM1 protein. We now report an in vitro reconstitution of 7SK-dependent HEXIM1 association to purified P-TEFb and subsequent CDK9 inhibition. Yeast three-hybrid tests and gel-shift assays indicated that HEXIM1 binds 7SK snRNA directly and a 7SK snRNA-recognition motif was identified in the central part of HEXIM1 (amino acids (aa) 152–155). Data from yeast two-hybrid and pull-down assay on GST fusion proteins converge to a direct binding of P-TEFb to the HEXIM1 C-terminal domain (aa 181–359). Consistently, point mutations in an evolutionarily conserved motif (aa 202–205) were found to suppress P-TEFb binding and inhibition without affecting 7SK recognition. We propose that the RNA-binding domain of HEXIM1 mediates its association with 7SK and that P-TEFb then enters the complex through association with HEXIM1.

In vivo degradation of RNA polymerase II largest subunit triggered by alpha-amanitin.

Abstract: Alpha-Amanitin is a well-known specific inhibitor of RNA polymerase II (RNAPII) in vitro and in vivo. It is a cyclic octapeptide which binds with high affinity to the largest subunit of RNAPII, RPB1. We have found that in murine fibroblasts exposure to alpha-amanitin triggered degradation of the RPB1 subunit, while other RNAPII subunits, RPB5 and RPB8, remained almost unaffected. Transcriptional inhibition in alpha-amanitin-treated cells was slow and closely followed the disappearance of RPB1. The degradation rate of RPB1 was alpha-amanitin dose dependent and was not a consequence of transcriptional arrest. Alpha-Amanitin-promoted degradation of RPB1 was prevented in cells exposed to actinomycin D, another transcriptional inhibitor. Epitope-tagged recombinant human RPB1 subunits were expressed in mouse fibroblasts. In cells exposed to alpha-amanitin the wild-type recombinant subunit was degraded like the endogenous protein, but a mutated alpha-amanitin-resistant subunit remained unaffected. Hence, alpha-amanitin did not activate a proteolytic system, but instead its binding to mRPB1 likely represented a signal for degradation. Thus, in contrast to other inhibitors, such as actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, which reversibly act on transcription, inhibition by alpha-amanitin cannot be but an irreversible process because of the destruction of RNAPII.

MAQ1 and 7SK RNA Interact with CDK9/Cyclin T Complexes in a Transcription-Dependent Manner

Abstract: Positive transcription elongation factor b (P-TEFb) comprises a cyclin (T1 or T2) and a kinase, cyclin-dependent kinase 9 (CDK9), which phosphorylates the carboxyl-terminal domain of RNA polymerase II. P-TEFb is essential for transcriptional elongation in human cells. A highly specific interaction among cyclin T1, the viral protein Tat, and the transactivation response (TAR) element RNA determines the productive transcription of the human immunodeficiency virus genome. In growing HeLa cells, half of P-TEFb is kinase inactive and binds to the 7SK small nuclear RNA. We now report on a novel protein termed MAQ1 (for menage a quatre) that is also present in this complex. Since 7SK RNA is required for MAQ1 to associate with P-TEFb, a structural role for 7SK RNA is proposed. Inhibition of transcription results in the release of both MAQ1 and 7SK RNA from P-TEFb. Thus, MAQ1 cooperates with 7SK RNA to form a novel type of CDK inhibitor. According to yeast two-hybrid analysis and immunoprecipitations from extracts of transfected cells, MAQ1 binds directly to the N-terminal cyclin homology region of cyclins T1 and T2. Since Tat also binds to this cyclin T1 N-terminal domain and since the association between 7SK RNA/MAQ1 and P-TEFb competes with the binding of Tat to cyclin T1, we speculate that the TAR RNA/Tat lentivirus system has evolved to subvert the cellular 7SK RNA/MAQ1 system.

Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology

Abstract: Molecular chaperones, including the heat-shock proteins (Hsps), are a ubiquitous feature of cells in which these proteins cope with stress-induced denaturation of other proteins. Hsps have received the most attention in model organisms undergoing experimental stress in the laboratory, and the function of Hsps at the molecular and cellular level is becoming well understood in this context. A complementary focus is now emerging on the Hsps of both model and nonmodel organisms undergoing stress in nature, on the roles of Hsps in the stress physiology of whole multicellular eukaryotes and the tissues and organs they comprise, and on the ecological and evolutionary correlates of variation in Hsps and the genes that encode them. This focus discloses that (a) expression of Hsps can occur in nature, (b) all species have hsp genes but they vary in the patterns of their expression, (c) Hsp expression can be correlated with resistance to stress, and (d) species' thresholds for Hsp expression are correlated with levels of stress that they naturally undergo. These conclusions are now well established and may require little additional confirmation; many significant questions remain unanswered concerning both the mechanisms of Hsp-mediated stress tolerance at the organismal level and the evolutionary mechanisms that have diversified the hsp genes.

Non-coding RNA

Abstract: The term non-coding RNA (ncRNA) is commonly employed for RNA that does not encode a protein, but this does not mean that such RNAs do not contain information nor have function. Although it has been generally assumed that most genetic information is transacted by proteins, recent evidence suggests that the majority of the genomes of mammals and other complex organisms is in fact transcribed into ncRNAs, many of which are alternatively spliced and/or processed into smaller products. These ncRNAs include microRNAs and snoRNAs (many if not most of which remain to be identified), as well as likely other classes of yet-to-be-discovered small regulatory RNAs, and tens of thousands of longer transcripts (including complex patterns of interlacing and overlapping sense and antisense transcripts), most of whose functions are unknown. These RNAs (including those derived from introns) appear to comprise a hidden layer of internal signals that control various levels of gene expression in physiology and development, including chromatin architecture/epigenetic memory, transcription, RNA splicing, editing, translation and turnover. RNA regulatory networks may determine most of our complex characteristics, play a significant role in disease and constitute an unexplored world of genetic variation both within and between species.

Exon-intron circular RNAs regulate transcription in the nucleus

Abstract: Noncoding RNAs (ncRNAs) have numerous roles in development and disease, and one of the prominent roles is to regulate gene expression A vast number of circular RNAs (circRNAs) have been identified, and some have been shown to function as microRNA sponges in animal cells Here, we report a class of circRNAs associated with RNA polymerase II in human cells In these circRNAs, exons are circularized with introns 'retained' between exons; we term them exon-intron circRNAs or EIciRNAs EIciRNAs predominantly localize in the nucleus, interact with U1 snRNP and promote transcription of their parental genes Our findings reveal a new role for circRNAs in regulating gene expression in the nucleus, in which EIciRNAs enhance the expression of their parental genes in cis, and highlight a regulatory strategy for transcriptional control via specific RNA-RNA interaction between U1 snRNA and EIciRNAs

The hallmarks of cancer: a long non-coding RNA point of view.

Abstract: With the advent of next generation sequencing methods and progress in transcriptome analysis, it became obvious that the human genome contains much more than just protein-coding genes. In fact, up to 70% of our genome is transcribed into RNA that does not serve as templates for proteins. In this review, we focus on the emerging roles of these long non-coding RNAs (lncRNAs) in the field of tumor biology. Long ncRNAs were found to be deregulated in several human cancers and show tissue-specific expression. Functional studies revealed a broad spectrum of mechanisms applied by lncRNAs such as HOTAIR, MALAT1, ANRIL or lincRNA-p21 to fulfill their functions. Here, we link the cellular processes influenced by long ncRNAs to the hallmarks of cancer and therefore provide an ncRNA point-of-view on tumor biology. This should stimulate new research directions and therapeutic options considering long ncRNAs as novel prognostic markers and therapeutic targets.