Mechanistic Analysis of Riboswitch Ligand Interactions Provides Insights into Pharmacological Control over Gene Expression.

Riboswitches are structured RNA elements that regulate gene expression upon binding to small molecule ligands. Understanding the mechanisms by which small molecules impact riboswitch activity is key to developing potent, selective ligands for these and other RNA targets. We report the structure-informed design of chemically diverse synthetic ligands for PreQ1 riboswitches. Multiple X-ray co-crystal structures of synthetic ligands with the Thermoanaerobacter tengcongensis (Tte)-PreQ1 riboswitch confirm a common binding site with the cognate ligand, despite considerable chemical differences among the ligands. Structure probing assays demonstrate that one ligand causes conformational changes similar to PreQ1 in six structurally and mechanistically diverse PreQ1 riboswitch aptamers. Single-molecule force spectroscopy is used to demonstrate differential modes of riboswitch stabilization by the ligands. Binding of the natural ligand brings about the formation of a persistent, folded pseudoknot structure, whereas a synthetic ligand decreases the rate of unfolding through a kinetic mechanism. Single round transcription termination assays show the biochemical activity of the ligands, while a GFP reporter system reveals compound activity in regulating gene expression in live cells without toxicity. Taken together, this study reveals that diverse small molecules can impact gene expression in live cells by altering conformational changes in RNA structures through distinct mechanisms.

Novel approaches to study helicases using magnetic tweezers.

Helicases 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.

G-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.

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

Accurate 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.

The Saccharomyces cerevisiae 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 slx5Δ and slx8Δ 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.

Spindle pole bodies exploit the mitotic exit network in metaphase to drive their age-dependent segregation.

Like many asymmetrically dividing cells, budding yeast segregates mitotic spindle poles nonrandomly between mother and daughter cells. During metaphase, the spindle positioning protein Kar9 accumulates asymmetrically, localizing specifically to astral microtubules emanating from the old spindle pole body (SPB) and driving its segregation to the bud. Here, we show that the SPB component Nud1/centriolin acts through the mitotic exit network (MEN) to specify asymmetric SPB inheritance. In the absence of MEN signaling, Kar9 asymmetry is unstable and its preference for the old SPB is disrupted. Consistent with this, phosphorylation of Kar9 by the MEN kinases Dbf2 and Dbf20 is not required to break Kar9 symmetry but is instead required to maintain stable association of Kar9 with the old SPB throughout metaphase. We propose that MEN signaling links Kar9 regulation to SPB identity through biasing and stabilizing the age-insensitive, cyclin-B-dependent mechanism of symmetry breaking.

Organelle segregation during mitosis: lessons from asymmetrically dividing cells.

Studies on cell division traditionally focus on the mechanisms of chromosome segregation and cytokinesis, yet we know comparatively little about how organelles segregate. Analysis of organelle partitioning in asymmetrically dividing cells has provided insights into the mechanisms through which cells control organelle distribution. Interestingly, these studies have revealed that segregation mechanisms frequently link organelle distribution to organelle growth and formation. Furthermore, in many cases, cells use organelles, such as the endoplasmic reticulum and P granules, as vectors for the segregation of information. Together, these emerging data suggest that the coordination between organelle growth, division, and segregation plays an important role in the control of cell fate inheritance, cellular aging, and rejuvenation, i.e., the resetting of age in immortal lineages.

Notch signalling is required for both dauer maintenance and recovery in C. elegans.

The Notch signalling pathway is conserved among higher metazoans and is used repeatedly throughout development to specify distinct cell fates among populations of equipotent cells. Mounting evidence suggests that Notch signalling may also be crucial in neuronal function in postmitotic, differentiated neurons. Here, we demonstrate a novel role for the canonical Notch signalling pathway in postmitotic neurons during a specialised ;diapause-like' post-embryonic developmental stage in C. elegans called dauer. Our data suggest that cell signalling downstream of the developmental decision to enter dauer leads to the activation of Notch-responding genes in postmitotic neurons. Consistent with this, we demonstrate that glp-1, one of the two C. elegans Notch receptors, and its ligand lag-2 are expressed in neurons during the dauer stage, and both genes are required to maintain this stage in a daf-7/TGFbeta dauer constitutive background. Our genetic data also suggest that a second Notch receptor, lin-12, functions upstream of, or in parallel with, insulin-like signalling components in response to replete growth conditions to promote dauer recovery. Based on our findings, cues associated with the onset of dauer ultimately trigger a glp-1-dependent Notch signalling cascade in neurons to maintain this developmental state. Then, as growth conditions improve, activation of the LIN-12 Notch receptor cooperates with the insulin-like signalling pathway to signal recovery from the dauer stage.

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

BACKGROUND: 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 C. elegans, the Rb orthologue lin-35 functions redundantly with other transcriptional regulators to appropriately specify both vulval and pharyngeal cell fates. RESULTS: In C. elegans 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 lin-35/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 lin-35/Rb cooperates with these RNAi components. Furthermore, our findings suggest that met-2, a methyltransferase as well as hpl-1 and hpl-2, two C. elegans homologues of the heterochromatin protein HP1 are also required for this transition. CONCLUSION: Our findings are consistent with lin-35/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 lin-35/Rb repressor complex may be required to initiate this process, while components of the RNAi machinery positively reinforce this repression.