The lin-35/Rb and RNAi pathways cooperate to regulate a key cell cycle transition in C. elegans

<p>Abstract</p> <p>Background</p> <p>The Retinoblastoma gene product (Rb) has been shown to regulate the transcription of key genes involved in cell growth and proliferation. Consistent with this, mutations in Rb are associated with numerous types of cancer making it a critical tumour suppressor gene. Its function is conferred through a large multiprotein complex that exhibits a dual function in both activation and repression of gene targets. In <it>C. elegans</it>, the Rb orthologue <it>lin-35 </it>functions redundantly with other transcriptional regulators to appropriately specify both vulval and pharyngeal cell fates.</p> <p>Results</p> <p>In <it>C. elegans </it>the intestinal cells must alter their cell cycle from the mitotic cell divisions typical of embryogenesis to karyokinesis and then endoreplication, which facilitates growth during larval development. While screening for genes that affect the ability of the intestinal cells to appropriately make this cell cycle transition during post-embryonic development, we isolated mutants that either compromise this switch and remain mononucleate, or cause these cells to undergo multiple rounds of nuclear division. Among these mutants we identified a novel allele of <it>lin-35/</it>Rb, while we also found that the components of the synMuv B complex, which are involved in vulval specification, are also required to properly regulate the developmentally-controlled cell cycle transition typical of these intestinal cells during larval development. More importantly, our work uncovered a role for certain members of the pathways involved in RNAi in mediating the efficient transition between these cell cycle programs, suggesting that <it>lin-35</it>/Rb cooperates with these RNAi components. Furthermore, our findings suggest that <it>met-2</it>, a methyltransferase as well as <it>hpl-1 </it>and <it>hpl-2</it>, two <it>C. elegans </it>homologues of the heterochromatin protein HP1 are also required for this transition.</p> <p>Conclusion</p> <p>Our findings are consistent with <it>lin-35</it>/Rb, synMuv and RNAi components cooperating, probably through their additive effects on chromatin modification, to appropriately modulate the expression of genes that are required to switch from the karyokinesis cell cycle to endoreplication; a highly specified growth pathway in the intestinal epithelium. The <it>lin-35/Rb </it>repressor complex may be required to initiate this process, while components of the RNAi machinery positively reinforce this repression.</p

Regulation of host gene expression by HIV-1 TAR microRNAs

Background: The transactivating response (TAR) element of human immunodeficiency virus type 1 (HIV-1) is the source of two functional microRNAs (miRNAs), miR-TAR-5p and miR-TAR-3p. The objective of this study was to characterize the post-transcriptional regulation of host messenger RNAs (mRNAs) relevant to HIV-1 pathogenesis by HIV-1 TAR miRNAs. Results: We demonstrated that TAR miRNAs derived from HIV-1 can incorporate into host effector Argonaute protein complexes, which is required if these miRNAs are to regulate host mRNA expression. Bioinformatic predictions and reporter gene activity assays identified regulatory elements complementary and responsive to miR-TAR-5p and miR-TAR-3p in the 3’ untranslated region (UTR) of several candidate genes involved in apoptosis and cell survival. These include Caspase 8, Aiolos, Ikaros and Nucleophosmin (NPM)/B23. Analyses of Jurkat cells that stably expressed HIV-1 TAR or contained a full-length latent HIV provirus suggested that HIV-1 TAR miRNAs could regulate the expression of genes in T cells that affect the balance between apoptosis and cell survival. Conclusions: HIV-1 TAR miRNAs may contribute to the replication cycle and pathogenesis of HIV-1, by regulating host genes involved in the intricate balance between apoptosis and infected cell, to induce conditions that promote HIV-1 propagation and survival

L’arrimage des sciences humaines du secondaire et du collégial

Depuis 2010, les collèges accueillent les élèves du Renouveau pédagogique. Pour faciliter leur entrée au collégial et optimiser les apprentissages du secondaire dans les cours de sciences humaines, nous avons complété une recherche participative inter-ordres. L’équipe est composée de chercheurs de l’université et du collégial de même que d’enseignants du secondaire et du collégial. Nous présenterons nos résultats issus d’entrevues, de groupes de discussions et de la validation du matériel pédagogique de transition inter-ordres

Novel approaches to study helicases using magnetic tweezers

International audienceHelicases form a universal family of molecular motors that bind and translocate onto nucleic acids. They are involved in essentially every aspect of nucleic acid metabolism: from DNA replication to RNA decay, and thus ensure a large spectrum of functions in the cell, making their study essential. The development of micromanipulation techniques such as magnetic tweezers for the mechanistic study of these enzymes has provided new insights into their behavior and their regulation that were previously unrevealed by bulk assays. These experiments allowed very precise measures of their translocation speed, processivity and polarity. Here, we detail our newest technological advances in magnetic tweezers protocols for high-quality measurements and we describe the new procedures we developed to get a more profound understanding of helicase dynamics, such as their translocation in a force independent manner, their nucleic acid binding kinetics and their interaction with roadblocks

Folding and persistence times of intramolecular G-quadruplexes transiently embedded in a DNA duplex

International audienceG-quadruplex (G4) DNA structures have emerged as important regulatory elements during DNA metabolic transactions. While many in vitro studies have focused on the kinetics of G4 formation within DNA single-strands, G4 are found in vivo in double-stranded DNA regions, where their formation is challenged by the complementary strand. Since the energy of hybridization of Watson-Crick structures dominates the energy of G4 folding, this competition should play a critical role on G4 persistence. To address this, we designed a single-molecule assay allowing to measure G4 folding and persistence times in the presence of the complementary strand. We quantified both folding and unfolding rates of biologically relevant G4 sequences, such as the cMYC and cKIT oncogene promoters, human telomeres and an avian replication origin. We confirmed that G4s are found much more stable in tested replication origin and promoters than in human telomere repeats. In addition, we characterized how G4 dynamics was affected by G4 ligands and showed that both folding rate and persistence time increased. Our assay opens new perspectives for the measurement of G4 dynamics in double-stranded DNA mimicking a replication fork, which is important to understand their role in DNA replication and gene regulation at a mechanistic level

Folding and Persistence Time of Intramolecular G-Quadruplexes Transiently Embedded in a DNA duplex

G-quadruplex (G4) DNA structures have emerged as important regulatory elements during DNA replication, transcription or repair. While many in-vitro studies have focused on the kinetics of G4 formation within DNA single-strands, G4 are found in-vivo in double-stranded DNA regions, where their formation is challenged by pairing between the two complementary strands. Since the energy of hybridization of Watson-Crick structures dominates the energy of G4 folding, this competition should play a critical role on the persistence of G4 in vivo . To address this issue, we designed a single molecule assay allowing measuring G4 folding and persistence while the structure is periodically challenged by the complementary strand. We quantified both the folding rate and the persistence time of biologically relevant G4 structures and showed that the dynamics of G4 formation depends strongly on the genomic location. G4 are found much more stable in promoter regions and replication origins than in telomeric regions. In addition, we characterized how G4 dynamics was affected by G4 ligands and showed that both folding rate and persistence increased. Our assay opens new perspectives for the measurement of G4 dynamics, which is critical to understand their role in genetic regulation

Detection of genetic variation and base modifications at base-pair resolution on both DNA and RNA

International audienceAccurate decoding of nucleic acid variation is critical to understand the complexity and regulation of genome function. Here we use a single-molecule magnetic tweezer (MT) platform to identify sequence variation and map a range of important epigenetic base modifications with high sensitivity, specificity, and precision in the same single molecules of DNA or RNA. We have also developed a highly specific amplification-free CRISPR-Cas enrichment strategy to isolate genomic regions from native DNA. We demonstrate enrichment of DNA from both E. coli and the FMR1 5'UTR coming from cells derived from a Fragile X carrier. From these kilobase-length enriched molecules we could characterize the differential levels of adenine and cytosine base modifications on E. coli, and the repeat expansion length and methylation status of FMR1. Together these results demonstrate that our platform can detect a variety of genetic, epigenetic, and base modification changes concomitantly within the same single molecules

Centromere Binding and a Conserved Role in Chromosome Stability for SUMO-Dependent Ubiquitin Ligases

<div><p>The <i>Saccharomyces cerevisiae</i> Slx5/8 complex is the founding member of a recently defined class of SUMO-targeted ubiquitin ligases (STUbLs). Slx5/8 has been implicated in genome stability and transcription, but the precise contribution is unclear. To characterise Slx5/8 function, we determined genome-wide changes in gene expression upon loss of either subunit. The majority of mRNA changes are part of a general stress response, also exhibited by mutants of other genome integrity pathways and therefore indicative of an indirect effect on transcription. Genome-wide binding analysis reveals a uniquely centromeric location for Slx5. Detailed phenotype analyses of <i>slx5</i>Δ and <i>slx8</i>Δ mutants show severe mitotic defects that include aneuploidy, spindle mispositioning, fish hooks and aberrant spindle kinetics. This is associated with accumulation of the PP2A regulatory subunit Rts1 at centromeres prior to entry into anaphase. Knockdown of the human STUbL orthologue RNF4 also results in chromosome segregation errors due to chromosome bridges. The study shows that STUbLs have a conserved role in maintenance of chromosome stability and links SUMO-dependent ubiquitination to a centromere-specific function during mitosis.</p></div