Pierre Dagenais

Riboswitch structure: an internal residue mimicking the purine ligand

The adenine and guanine riboswitches regulate gene expression in response to their purine ligand. X-ray structures of the aptamer moiety of these riboswitches are characterized by a compact fold in which the ligand forms a Watson–Crick base pair with residue 65. Phylogenetic analyses revealed a strict restriction at position 39 of the aptamer that prevents the G39–C65 and A39–U65 combinations, and mutational studies indicate that aptamers with these sequence combinations are impaired for ligand binding. In order to investigate the rationale for sequence conservation at residue 39, structural characterization of the U65C mutant from Bacillus subtilis pbuE adenine riboswitch aptamer was undertaken. NMR spectroscopy and X-ray crystallography studies demonstrate that the U65C mutant adopts a compact ligand-free structure, in which G39 occupies the ligand-binding site of purine riboswitch aptamers. These studies present a remarkable example of a mutant RNA aptamer that adopts a native-like fold by means of ligand mimicking and explain why this mutant is impaired for ligand binding. Furthermore, this work provides a specific insight into how the natural sequence has evolved through selection of nucleotide identities that contribute to formation of the ligand-bound state, but ensures that the ligand-free state remains in an active conformation.

The ARiBo tag: a reliable tool for affinity purification of RNAs under native conditions

Although RNA-based biological processes and therapeutics have gained increasing interest, purification of in vitro transcribed RNA generally relies on gel-based methods that are time-consuming, tedious and denature the RNA. Here, we present a reliable procedure for affinity batch purification of RNA, which exploits the high-affinity interaction between the boxB RNA and the N-peptide from bacteriophage λ. The RNA of interest is synthesized with an ARiBo tag, which consists of an activatable ribozyme (the glmS ribozyme) and the λBoxB RNA. This ARiBo-fusion RNA is initially captured on Glutathione-Sepharose resin via a GST/λN-fusion protein, and the RNA of interest is subsequently eluted by ribozyme self-cleavage using glucosamine-6-phosphate. Several GST/λN-fusion proteins and ARiBo tags were tested to optimize RNA yield and purity. The optimized procedure enables one to quickly obtain (3 h) highly pure RNA (>99%) under native conditions and with yields comparable to standard denaturing gel-based protocols. It is widely applicable to a variety of RNAs, including riboswitches, ribozymes and microRNAs. In addition, it can be easily adapted to a wide range of applications that require RNA purification and/or immobilization, including isolation of RNA-associated complexes from living cells and high-throughput applications.

Insights into RNA structure and dynamics from recent NMR and X-ray studies of the Neurospora Varkud satellite ribozyme

Despite the large number of noncoding RNAs and their importance in several biological processes, our understanding of RNA structure and dynamics at atomic resolution is still very limited. Like many other RNAs, the Neurospora Varkud satellite (VS) ribozyme performs its functions through dynamic exchange of multiple conformational states. More specifically, the VS ribozyme recognizes and cleaves its stem‐loop substrate via a mechanism that involves several structural transitions within its stem‐loop substrate. The recent publications of high‐resolution structures of the VS ribozyme, obtained by NMR spectroscopy and X‐ray crystallography, offer an opportunity to integrate the data and closely examine the structural and dynamic properties of this model RNA system. Notably, these investigations provide a valuable example of the divide‐and‐conquer strategy for structural and dynamic characterization of a large RNA, based on NMR structures of several individual subdomains. The success of this divide‐and‐conquer approach reflects the modularity of RNA architecture and the great care taken in identifying the independently‐folding modules. Together with previous biochemical and biophysical characterizations, the recent NMR and X‐ray studies provide a coherent picture into how the VS ribozyme recognizes its stem‐loop substrate. Such in‐depth characterization of this RNA enzyme will serve as a model for future structural and engineering studies of dynamic RNAs and may be particularly useful in planning divide‐and‐conquer investigations. WIREs RNA 2017, 8:e1421. doi: 10.1002/wrna.1421

Rational engineering of the Neurospora VS ribozyme to allow substrate recognition via different kissing-loop interactions

The Neurospora VS ribozyme is a catalytic RNA that has the unique ability to specifically recognize and cleave a stem-loop substrate through formation of a highly stable kissing-loop interaction (KLI). In order to explore the engineering potential of the VS ribozyme to cleave alternate substrates, we substituted the wild-type KLI by other known KLIs using an innovative engineering method that combines rational and combinatorial approaches. A bioinformatic search of the protein data bank was initially performed to identify KLIs that are structurally similar to the one found in the VS ribozyme. Next, substrate/ribozyme (S/R) pairs that incorporate these alternative KLIs were kinetically and structurally characterized. Interestingly, several of the resulting S/R pairs allowed substrate cleavage with substantial catalytic efficiency, although with reduced activity compared to the reference S/R pair. Overall, this study describes an innovative approach for RNA engineering and establishes that the KLI of the trans VS ribozyme can be adapted to cleave other folded RNA substrates.

The Health Technology Assessment Unit of the Centre hospitalier universitaire de Sherbrooke (Canada)

The Health Technology Assessment (HTA) Unit of the Centre hospitalier universitaire de Sherbrooke (CHUS) has been created in 2004. A coordinator (middle-level manager) holding a master’s degree from the Universite de Montreal in health technology assessment and an advisor in technology assessment started the development of the unit under the management of the Director of Medical Affairs and Professional Services. By this positioning, the organization allowed the HTA Unit a proximity to clinicians while keeping strategic relations to senior management. To increase credibility among these groups, approval of HTA reports was under the responsibility of the Director of Medical Affairs and Professional Services (a physician and senior manager).

Preparative Separation of Ribonucleoside Monophosphates by Ion-Pair Reverse-Phase HPLC

Structural and dynamic investigations of RNA by nuclear magnetic resonance (NMR) spectroscopy strongly benefit from isotopic-labeling strategies. Among these, nucleotide-specific and site-specific labeling methods can help tremendously in simplifying complex NMR data, while providing unique opportunities for structural investigation of larger RNAs. Such methods generally require separation of individual isotopically labeled ribonucleoside monophosphates prior to their conversion into nucleoside triphosphates and selective incorporation of these nucleoside triphosphates into the RNA. This chapter provides the experimental details for preparative separation of ribonucleoside monophosphates by ion-pair reverse-phase HPLC. It also describes a quick procedure for clean-up and quality control of the individual ribonucleoside monophosphates.

Risk for Osteoporosis and Antiresorptive Therapies in Patients with Crohn's Disease

Background: The risk of osteoporosis is greater in patients with inflammatory bowel disease (IBD) because of several related risk factors such as the prolonged use of corticosteroids, the malabsorption of calcium and vitamin D following small-bowel disease or small-bowel resections, and the inflammatory process resulting from the illness. A reduction in bone mineral density (BMD) has been reported in 30–75% of patients with IBD (Crohns disease or ulcerative colitis). An evaluation of the risk factors of osteoporosis should be performed to identify those in need of antiresorptive drug therapies (ART) and/or calcium and vitamin D supplements. Objective: To determine whether patients with Crohns disease at risk for osteoporosis or already having decreased BMD were identified as such and treated with appropriate therapy. Methods: A cross-sectional study was performed among 54 white patients with Crohns disease who attended a gastroenterology specialized clinic between January and March 2002. All patients with Crohns disease having an outpatient medical visit were asked to participate in the study. Patients who agreed to the study were questioned for Crohns disease activity index (CDAI) and osteoporosis risk factors such as nutrition, lifestyle, and corticosteroid therapy. Serum bone-specific markers and nutritional status were assessed. BMD test of the lumbar vertebrae (L2–L4) and the nondominant hip were assessed with dual-energy X-ray absorptiometry if the participants did not have a BMD test in the year prior to the study. ART and calcium/vitamin D use were assessed through a questionnaire and on community pharmaceutical drug services in the 2 year period prior to the evaluation. Results: Mean age and CDAI severity score were 41.5 years and 145, respectively. None of the patients was evaluated for BMD in the year prior to the study. Osteopenia at the nondominant hip and/or lumbar vertebrae was seen in 29 patients. Eighty-two percent of patients with a T score under −2 SD had not been exposed to ART; 84% of patients with more than 4 risk factors for osteoporosis and a T score less than −1.5 SD had not received ART. Moreover, 88% of patients with a T score less than −1.5 SD and receiving corticosteroid therapy had not been treated with ART. Conclusions: The presence of osteoporosis risk factors and osteopenia had not been recognized and treated in our population. Adequate osteoporosis management should be implemented in patients with Crohns disease to reduce the occurrence of osteoporotic fractures.