Alexandre Akoulitchev

Repression of the human dihydrofolate reductase gene by a non-coding interfering transcript

Alternative promoters within the same gene are a general phenomenon in gene expression. Mechanisms of their selective regulation vary from one gene to another and are still poorly understood. Here we show that in quiescent cells the mechanism of transcriptional repression of the major promoter of the gene encoding dihydrofolate reductase depends on a non-coding transcript initiated from the upstream minor promoter and involves both the direct interaction of the RNA and promoter-specific interference. The specificity and efficiency of repression is ensured by the formation of a stable complex between non-coding RNA and the major promoter, direct interaction of the non-coding RNA with the general transcription factor IIB and dissociation of the preinitiation complex from the major promoter. By using in vivo and in vitro assays such as inducible and reconstituted transcription, RNA bandshifts, RNA interference, chromatin immunoprecipitation and RNA immunoprecipitation, we show that the regulatory transcript produced from the minor promoter has a critical function in an epigenetic mechanism of promoter-specific transcriptional repression.

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.

Role of Ran Binding Protein 5 in Nuclear Import and Assembly of the Influenza Virus RNA Polymerase Complex

ABSTRACT The influenza A virus RNA-dependent RNA polymerase is a heterotrimeric complex of polymerase basic protein 1 (PB1), PB2, and polymerase acidic protein (PA) subunits. It performs transcription and replication of the viral RNA genome in the nucleus of infected cells. We have identified a nuclear import factor, Ran binding protein 5 (RanBP5), also known as karyopherin β3, importin β3, or importin 5, as an interactor of the PB1 subunit. RanBP5 interacted with either PB1 alone or with a PB1-PA dimer but not with a PB1-PB2 dimer or the trimeric complex. The interaction between RanBP5 and PB1-PA was disrupted by RanGTP in vitro, allowing PB2 to bind to the PB1-PA dimer to form a functional trimeric RNA polymerase complex. We propose a model in which RanBP5 acts as an import factor for the newly synthesized polymerase by targeting the PB1-PA dimer to the nucleus. In agreement with this model, small interfering RNA (siRNA)-mediated knock-down of RanBP5 inhibited the nuclear accumulation of the PB1-PA dimer. Moreover, siRNA knock-down of RanBP5 resulted in the delayed accumulation of viral RNAs in infected cells, confirming that RanBP5 plays a biological role during the influenza virus life cycle.

Autocatalytic RNA cleavage in the human |[beta]|-globin pre-mRNA promotes transcription termination

New evidence indicates that termination of transcription is an important regulatory step, closely related to transcriptional interference and even transcriptional initiation. However, how this occurs is poorly understood. Recently, in vivo analysis of transcriptional termination for the human β-globin gene revealed a new phenomenon—co-transcriptional cleavage (CoTC). This primary cleavage event within β-globin pre-messenger RNA, downstream of the poly(A) site, is critical for efficient transcriptional termination by RNA polymerase II. Here we show that the CoTC process in the human β-globin gene involves an RNA self-cleaving activity. We characterize the autocatalytic core of the CoTC ribozyme and show its functional role in efficient termination in vivo. The identified core CoTC is highly conserved in the 3′ flanking regions of other primate β-globin genes. Functionally, it resembles the 3′ processive, self-cleaving ribozymes described for the protein-encoding genes from the myxomycetes Didymium iridis and Physarum polycephalum, indicating evolutionary conservation of this molecular process. We predict that regulated autocatalytic cleavage elements within pre-mRNAs may be a general phenomenon and that functionally it may provide the entry point for exonucleases involved in mRNA maturation, turnover and, in particular, transcriptional termination.

The characterization of the invasion phenotype of uveal melanoma tumour cells shows the presence of MUC18 and HMG‐1 metastasis markers and leads to the identification of DJ‐1 as a potential serum biomarker

Uveal malignant melanoma (UM) is the most frequent primary intraocular tumour in adult humans. Because the survival rate of patients with UM has changed little in the past few decades, a better understanding of the molecular events governing UM development and the identification of markers indicating the potential for metastasis at the time of diagnosis are necessary to design improved and more specific treatments. In this study, we investigated UM tumour development by comparing two recently established UM cultures with different invasion potential by two‐dimensional gel electrophoresis. Protein features expressed differentially were identified by mass spectrometric analysis. Potential markers were assayed in both cultures and in long‐term established UM cell lines (UW‐1, OCM‐1, SP6.5 and 92.1) by Western blotting and their role in invasion analysed using Matrigel membranes. Comparative analysis revealed that UM cultures with low‐ and high‐grade invasion potential differ in their cellular metabolism and, more interestingly, in several cancer‐associated proteins, including those implicated in cell adhesion and migration, proliferation and various oncogenes. Our data indicate a correlation between MUC18 and HMG‐1 expression and the invasiveness of UM cells. We also demonstrate the expression and secretion of DJ‐1 oncoprotein by UM cells. We suggest a possible role for MUC18 and HMG‐1 proteins in UM cell invasion. The secretion of DJ‐1 by UM cells, and the ability to detect this protein in UM patients sera implicate it as a potential noninvasive biomarker for this malignancy.

A genetic code alteration generates a proteome of high diversity in the human pathogen Candida albicans

BackgroundGenetic code alterations have been reported in mitochondrial, prokaryotic, and eukaryotic cytoplasmic translation systems, but their evolution and how organisms cope and survive such dramatic genetic events are not understood.ResultsHere we used an unusual decoding of leucine CUG codons as serine in the main human fungal pathogen Candida albicans to elucidate the global impact of genetic code alterations on the proteome. We show that C. albicans decodes CUG codons ambiguously and tolerates partial reversion of their identity from serine back to leucine on a genome-wide scale.ConclusionSuch codon ambiguity expands the proteome of this human pathogen exponentially and is used to generate important phenotypic diversity. This study highlights novel features of C. albicans biology and unanticipated roles for codon ambiguity in the evolution of the genetic code.

Molecular cross-talk between the transcription, translation, and nonsense-mediated decay machineries

It is widely believed that translation occurs only in the cytoplasm of eukaryotes, but recent results suggest some takes place in nuclei, coupled to transcription. Support for this heterodoxy comes from studies of the nonsense-mediated decay (NMD) pathway; this pathway probably uses ribosomes to proofread messenger RNAs. We find components of the machineries involved in transcription, translation and NMD colocalise, interact and copurify, and that interactions between them are probably mediated by the C-terminal domain of the catalytic subunit of RNA polymerase II. These results are simply explained if the NMD machinery uses nuclear ribosomes to translate – and so proofread – newly made transcripts; then, faulty transcripts and any truncated peptides produced by nuclear translation would be degraded.

Host cell proteins interacting with the 3' end of TGEV coronavirus genome influence virus replication.

n Abstractn n Coronavirus RNA synthesis is performed by a multienzymatic replicase complex together with cellular factors. This process requires the specific recognition of RNA cis-acting signals located at the ends of the viral genome. To identify cellular proteins involved in coronavirus RNA synthesis, transmissible gastroenteritis coronavirus (TGEV) genome ends, harboring essential cis-acting signals for replication, were used as baits for RNA affinity protein purification. Ten proteins were preferentially pulled down with either the 5′ or 3′ ends of the genome and identified by proteomic analysis. Nine of them, including members of the heterogeneous ribonucleoprotein family of proteins (hnRNPs), the poly(A)-binding protein (PABP), the p100 transcriptional co-activator protein and two aminoacyl-tRNA synthetases, showed a preferential binding to the 3′ end of the genome, whereas only the polypyrimidine tract-binding protein (PTB) was preferentially pulled down with the 5′ end of the genome. The potential function of the 3′ end-interacting proteins in virus replication was studied by analyzing the effect of their silencing using a TGEV-derived replicon and the infectious virus. Gene silencing of PABP, hnRNP Q, and glutamyl-prolyl-tRNA synthetase (EPRS) caused a significant 2 to 3-fold reduction of viral RNA synthesis. Interestingly, the silencing of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), initially used as a control gene, caused a 2 to 3-fold increase in viral RNA synthesis in both systems. These data suggest that PABP, hnRNP Q, and EPRS play a positive role in virus infection that could be mediated through their interaction with the viral 3′ end, and that GAPDH has a negative effect on viral infection.n n

Analysis of U1 Small Nuclear RNA Interaction with Cyclin H

TFIIH is a general transcription and repair factor implicated in RNA polymerase II transcription, nucleotide excision repair, and transcription-coupled repair. Genetic defects in TFIIH lead to three distinct inheritable diseases: xeroderma pigmentosa, Cockayne syndrome, and trichothiodystrophy, with xeroderma pigmentosa patients being highly susceptible to skin cancer. Earlier data revealed that the cyclin H subunit of TFIIH associates with U1 small nuclear RNA, a core-splicing component. In addition to its role in RNA processing U1 small nuclear RNA also regulates diverse stages of transcription by RNA polymerase II both in vivo and in vitro, including abortive initiation and re-initiation. Here we identify structural components of U1 and cyclin H implicated in the direct interaction and show how they affect function. Because of unique features of cyclin H we have developed a new methodology for mapping RNA interaction with the full-length cyclin H polypeptide based on electrospray ionization tandem mass spectrometry. We also demonstrate the importance of U1 stem-loops 1 and 2 for the interaction with cyclin H. Functional assays implicate the identified interaction with U1 in regulation of the activity of the cyclin H associated kinase CDK7.

Activation of AP-1-dependent transcription by a truncated translation initiation factor.

ABSTRACT Int6/eIF3e is a highly conserved subunit of eukaryotic translation initiation factor 3 (eIF3) that has also been reported to interact with subunits of the proteasome and the COP9 signalosome. Overexpression of full-length Int6 or a 13-kDa C-terminal fragment, Int6CT, in the fission yeast Schizosaccharomyces pombe causes multidrug resistance that requires the otherwise inessential AP-1 transcription factor Pap1. Here we show for the first time that Int6CT acts to increase the transcriptional activity of Pap1. Microarray hybridization data indicate that Int6CT overexpression resulted in the up-regulation of 67 genes; this expression profile closely matched that of cells overexpressing Pap1. Analysis of the upstream regulatory sequences of these genes showed that the majority contained AP-1 consensus binding sites. Partial defects in ubiquitin-dependent proteolysis have been suggested to confer Pap1-dependent multidrug resistance, but no such defect was seen on Int6CT overexpression. Indeed, none of the previously identified interactions of endogenous Int6 was required for the activation of Pap1 transcription described here. Moreover, Int6CT-induced activation of Pap1-responsive gene expression was independent of the ability of Pap1 to undergo a redox-regulated conformational change which mediates its relocalization to the nucleus and expression of oxidative stress response genes. Int6CT therefore activates Pap1-dependent transcription by a novel mechanism.