Solution structure of the two RNA recognition motifs of hnRNP A1 using segmental isotope labeling: how the relative orientation between RRMs influences the nucleic acid binding topology

Human hnRNP A1 is a multi-functional protein involved in many aspects of nucleic-acid processing such as alternative splicing, micro-RNA biogenesis, nucleo-cytoplasmic mRNA transport and telomere biogenesis and maintenance. The N-terminal region of hnRNP A1, also named unwinding protein 1 (UP1), is composed of two closely related RNA recognition motifs (RRM), and is followed by a C-terminal glycine rich region. Although crystal structures of UP1 revealed inter-domain interactions between RRM1 and RRM2 in both the free and bound form of UP1, these interactions have never been established in solution. Moreover, the relative orientation of hnRNP A1 RRMs is different in the free and bound crystal structures of UP1, raising the question of the biological significance of this domain movement. In the present study, we have used NMR spectroscopy in combination with segmental isotope labeling techniques to carefully analyze the inter-RRM contacts present in solution and subsequently determine the structure of UP1 in solution. Our data unambiguously demonstrate that hnRNP A1 RRMs interact in solution, and surprisingly, the relative orientation of the two RRMs observed in solution is different from the one found in the crystal structure of free UP1 and rather resembles the one observed in the nucleic-acid bound form of the protein. This strongly supports the idea that the two RRMs of hnRNP A1 have a single defined relative orientation which is the conformation previously observed in the bound form and now observed in solution using NMR. It is likely that the conformation in the crystal structure of the free form is a less stable form induced by crystal contacts. Importantly, the relative orientation of the RRMs in proteins containing multiple-RRMs strongly influences the RNA binding topologies that are practically accessible to these proteins. Indeed, RRM domains are asymmetric binding platforms contacting single-stranded nucleic acids in a single defined orientation. Therefore, the path of the nucleic acid molecule on the multiple RRM domains is strongly dependent on whether the RRMs are interacting with each other. The different nucleic acid recognition modes by multiple-RRM domains are briefly reviewed and analyzed on the basis of the current structural informatio

RNA recognition by double-stranded RNA binding domains: a matter of shape and sequence

The double-stranded RNA binding domain (dsRBD) is a small protein domain of 65-70 amino acids adopting an αβββα fold, whose central property is to bind to double-stranded RNA (dsRNA). This domain is present in proteins implicated in many aspects of cellular life, including antiviral response, RNA editing, RNA processing, RNA transport and, last but not least, RNA silencing. Even though proteins containing dsRBDs can bind to very specific dsRNA targets in vivo, the binding of dsRBDs to dsRNA is commonly believed to be shape-dependent rather than sequence-specific. Interestingly, recent structural information on dsRNA recognition by dsRBDs opens the possibility that this domain performs a direct readout of RNA sequence in the minor groove, allowing a global reconsideration of the principles describing dsRNA recognition by dsRBDs. We review in this article the current structural and molecular knowledge on dsRBDs, emphasizing the intricate relationship between the amino acid sequence, the structure of the domain and its RNA recognition capacity. We especially focus on the molecular determinants of dsRNA recognition and describe how sequence discrimination can be achieved by this type of domai

A strong 13C chemical shift signature provides the coordination mode of histidines in zinc-binding proteins

Zinc is the second most abundant metal ion incorporated in proteins, and is in many cases a crucial component of protein three-dimensional structures. Zinc ions are frequently coordinated by cysteine and histidine residues. Whereas cysteines bind to zinc via their unique Sγ atom, histidines can coordinate zinc with two different coordination modes, either Nδ1 or Nε2 is coordinating the zinc ion. The determination of this coordination mode is crucial for the accurate structure determination of a histidine-containing zinc-binding site by NMR. NMR chemical shifts contain a vast amount of information on local electronic and structural environments and surprisingly their utilization for the determination of the coordination mode of zinc-ligated histidines has been limited so far to 15N nuclei. In the present report, we observed that the 13C chemical shifts of aromatic carbons in zinc-ligated histidines represent a reliable signature of their coordination mode. Using a statistical analysis of 13C chemical shifts, we show that 13Cδ2 chemical shift is sensitive to the histidine coordination mode and that the chemical shift difference δ{13Cε1}−δ{13Cδ2} provides a reference-independent marker of this coordination mode. The present approach allows the direct determination of the coordination mode of zinc-ligated histidines even with non-isotopically enriched protein samples and without any prior structural informatio

Bioceramic fabrics improve quiet standing posture and handstand stability in expert gymnasts

Bioceramic fabrics have been claimed to improve blood circulation, thermoregulation and muscle relaxation, thereby also improving muscular activity. Here we tested whether bioceramic fabrics have an effect on postural control and contribute to improve postural stability. In Experiment 1, we tested whether bioceramic fabrics contribute to reduce body-sway when maintaining standard standing posture. In Experiment 2, we measured the effect of bioceramic fabrics on body-sway when maintaining a more instable posture, namely a handstand hold. For both experiments, postural oscillations were measured using a force platform with four strain gauges that recorded the displacements of the center of pressure (CoP) in the horizontal plane. In half of the trials, the participants wore a full-body second skin suit containing a bioceramic layer. In the other half of the trials, they wore a ‘placebo’ second skin suit that had the same cut, appearance and elasticity as the bioceramic suit but did not contain the bioceramic layer. In both experiments, the surface of displacement of the CoP was significantly smaller when participants were wearing the bioceramic suit than when they were wearing the placebo suit. The results suggest that bioceramic fabrics do have an effect on postural control and improve postural stability

Perceived Head-Trunk Angle During Microgravity Produced by Parabolic Flight

International audienceIntroduction: Neck proprioceptors are essential for orienting the head relative to the trunk. However, it has been shown that the available information about the relationship of gravity to different body parts would augment the clues about their relative orientation. In weightlessness , the absence of relevant body position signals from the otoliths and other inertial graviceptors requires the substitution of other sensory information. The aim of the present study was to investigate the ability of humans to accurately locate the head relative to the trunk in microgravity.Methods: Experiments were conducted during two separate sessions: 1) on Earth and 2) during parabolic fl ights. Volunteers were asked to adjust a visual rod until it looked parallel to their head or trunk axis in two different segmental confi gurations: head and trunk aligned or head tilted. Results: There was no effect of microgravity when the head and trunk were aligned. However, when the head was tilted with respect to the trunk, the orientation of the visual rod relative to the head or the trunk (visual egocentric coordinates) was deviated toward the head tilt, although the orientation between the body parts themselves (head-trunk angle) was correctly estimated.Discussion: These results suggested that, in microgravity, the proprioceptive signals from neck muscles seem suf-fi cient to provide accurate head on trunk information. However, the representation of orientation in visual space was modifi ed. This experiment provides evidence for the role of gravity on the visual perception of head-and trunk-based egocentric coordinates

Optimizing HSQC experiment for the observation of exchange broadened signals in RNA–protein complexes

International audienceSites for interaction in protein–RNA complexes are often regions of conformational exchange. Although the study of exchange processes could bring valuable information about the recognition mode between protein and RNA, chemical exchange can be detrimental to the NMR spectra quality, resulting in broad, very weak or even unobservable signals. In the present report, we used CPMG–like experiments to improve HSQC spectra of an RNA–protein complex in fast exchange on the chemical shift time scale. The use of such improvement will allow us to handle the resolution of the three-dimensional structure of the complex by NMR

Ensuring access to high-quality resources reduces the impacts of heat stress on bees

Pollinators are experiencing declines globally, negatively affecting the reproduction of wild plants and crop production. Well-known drivers of these declines include climatic and nutritional stresses, such as a change of dietary resources due to the degradation of habitat quality. Understanding potential synergies between these two important drivers is needed to improve predictive models of the future effects of climate change on pollinator declines. Here, bumblebee colony bioassays were used to evaluate the interactive effects of heat stress, a reduction of dietary resource quality, and colony size. Using a total of 117 colonies, we applied a fully crossed experiment to test the effect of three dietary quality levels under three levels of heat stress with two colony sizes. Both nutritional and heat stress reduced colony development resulting in a lower investment in offspring production. Small colonies were much more sensitive to heat and nutritional stresses than large ones, possibly because a higher percentage of workers helps maintain social homeostasis. Strikingly, the effects of heat stress were far less pronounced for small colonies fed with suitable diets. Overall, our study suggests that landscape management actions that ensure access to high-quality resources could reduce the impacts of heat stress on bee decline.info:eu-repo/semantics/publishedVersio

Metformin-associated lactic acidosis in an intensive care unit

International audienceIntroduction Metformin-associated lactic acidosis (MALA) is aclassic side effect of metformin and is known to be a severedisease with a high mortality rate. The treatment of MALA withdialysis is controversial and is the subject of many case reportsin the literature. We aimed to assess the prevalence of MALA ina 16-bed, university-affiliated, intensive care unit (ICU), and theeffect of dialysis on patient outcome.Methods Over a five-year period, we retrospectively identifiedall patients who were either admitted to the ICU with metforminas a usual medication, or who attempted suicide by metforminingestion. Within this population, we selected patientspresenting with lactic acidosis, thus defining MALA, anddescribed their clinical and biological features.Results MALA accounted for 0.84% of all admissions duringthe study period (30 MALA admissions over five years) and wasassociated with a 30% mortality rate. The only factorsassociated with a fatal outcome were the reason for admissionin the ICU and the initial prothrombin time. Although patientswho went on to haemodialysis had higher illness severity scores,as compared with those who were not dialysed, the mortalityrates were similar between the two groups (31.3% versus28.6%).Conclusions MALA can be encountered in the ICU severaltimes a year and still remains a life-threatening condition.Treatment is restricted mostly to supportive measures, althoughhaemodialysis may possess a protective effect

The relationship between CD4+CD25+CD127- regulatory T cells and inflammatory response and outcome during shock states

International audienceINTRODUCTION: Although regulatory T lymphocytes (Tregs) have a pivotal role in preventing autoimmune diseases and limiting chronic inflammatory conditions, they may also block beneficial immune responses by preventing sterilizing immunity to certain pathogens. METHODS: To determine whether naturally occurring Treg cells have a role in inflammatory response and outcome during shock state we conducted an observational study in two adult ICUs from a university hospital. Within 12 hours of admission, peripheral whole blood was collected for the measurement of cytokines and determination of lymphocyte count. Sampling was repeated at day three, five and seven. Furthermore, an experimental septic shock was induced in adult Balb/c mice through caecal ligation and puncture. RESULTS: Forty-three patients suffering from shock (26 septic, 17 non septic), and 7 healthy volunteers were included. The percentage of Tregs increased as early as 3 days after the onset of shock, while their absolute number remained lower than in healthy volunteers. A similar pattern of Tregs kinetics was found in infected and non infected patients. Though there was an inverse correlation between severity scores and Tregs percentage, the time course of Tregs was similar between survivors and non survivors. No relation between Tregs and cytokine concentration was found. In septic mice, although there was a rapid increase in Treg cells subset among splenocytes, antibody-induced depletion of Tregs before the onset of sepsis did not alter survival. CONCLUSIONS: These data argue against a determinant role of Tregs in inflammatory response and outcome during shock states