Wnt Signaling Interactor WTIP (Wilms Tumor Interacting Protein) Underlies Novel Mechanism for Cardiac Hypertrophy.

BACKGROUND: The study of hypertrophic cardiomyopathy (HCM) can yield insight into the mechanisms underlying the complex trait of cardiac hypertrophy. To date, most genetic variants associated with HCM have been found in sarcomeric genes. Here, we describe a novel HCM-associated variant in the noncanonical Wnt signaling interactor WTIP (Wilms tumor interacting protein) and provide evidence of a role for WTIP in complex disease. METHODS: In a family affected by HCM, we used exome sequencing and identity-by-descent analysis to identify a novel variant in WTIP (p.Y233F). We knocked down WTIP in isolated neonatal rat ventricular myocytes with lentivirally delivered short hairpin ribonucleic acids and in Danio rerio via morpholino injection. We performed weighted gene coexpression network analysis for WTIP in human cardiac tissue, as well as association analysis for WTIP variation and left ventricular hypertrophy. Finally, we generated induced pluripotent stem cell-derived cardiomyocytes from patient tissue, characterized size and calcium cycling, and determined the effect of verapamil treatment on calcium dynamics. RESULTS: WTIP knockdown caused hypertrophy in neonatal rat ventricular myocytes and increased cardiac hypertrophy, peak calcium, and resting calcium in D rerio. Network analysis of human cardiac tissue indicated WTIP as a central coordinator of prohypertrophic networks, while common variation at the WTIP locus was associated with human left ventricular hypertrophy. Patient-derived WTIP p.Y233F-induced pluripotent stem cell-derived cardiomyocytes recapitulated cellular hypertrophy and increased resting calcium, which was ameliorated by verapamil. CONCLUSIONS: We demonstrate that a novel genetic variant found in a family with HCM disrupts binding to a known Wnt signaling protein, misregulating cardiomyocyte calcium dynamics. Further, in orthogonal model systems, we show that expression of the gene WTIP is important in complex cardiac hypertrophy phenotypes. These findings, derived from the observation of a rare Mendelian disease variant, uncover a novel disease mechanism with implications across diverse forms of cardiac hypertrophy.

Current water quality guidelines across North America and Europe do not protect lakes from salinization.

Human-induced salinization caused by the use of road deicing salts, agricultural practices, mining operations, and climate change is a major threat to the biodiversity and functioning of freshwater ecosystems. Yet, it is unclear if freshwater ecosystems are protected from salinization by current water quality guidelines. Leveraging an experimental network of land-based and in-lake mesocosms across North America and Europe, we tested how salinization-indicated as elevated chloride (Cl-) concentration-will affect lake food webs and if two of the lowest Cl- thresholds found globally are sufficient to protect these food webs. Our results indicated that salinization will cause substantial zooplankton mortality at the lowest Cl- thresholds established in Canada (120 mg Cl-/L) and the United States (230 mg Cl-/L) and throughout Europe where Cl- thresholds are generally higher. For instance, at 73% of our study sites, Cl- concentrations that caused a ≥50% reduction in cladoceran abundance were at or below Cl- thresholds in Canada, in the United States, and throughout Europe. Similar trends occurred for copepod and rotifer zooplankton. The loss of zooplankton triggered a cascading effect causing an increase in phytoplankton biomass at 47% of study sites. Such changes in lake food webs could alter nutrient cycling and water clarity and trigger declines in fish production. Current Cl- thresholds across North America and Europe clearly do not adequately protect lake food webs. Water quality guidelines should be developed where they do not exist, and there is an urgent need to reassess existing guidelines to protect lake ecosystems from human-induced salinization.

Freshwater salinisation: a research agenda for a saltier world.

The widespread salinisation of freshwater ecosystems poses a major threat to the biodiversity, functioning, and services that they provide. Human activities promote freshwater salinisation through multiple drivers (e.g., agriculture, resource extraction, urbanisation) that are amplified by climate change. Due to its complexity, we are still far from fully understanding the ecological and evolutionary consequences of freshwater salinisation. Here, we assess current research gaps and present a research agenda to guide future studies. We identified different gaps in taxonomic groups, levels of biological organisation, and geographic regions. We suggest focusing on global- and landscape-scale processes, functional approaches, genetic and molecular levels, and eco-evolutionary dynamics as key future avenues to predict the consequences of freshwater salinisation for ecosystems and human societies.

Shoulder Pain in COVID-19 Survivors Following Mechanical Ventilation.

COVID-19 has caused a certain proportion of patients to be hospitalized in intensive care units (ICU) and may cause musculoskeletal and neurological deficits following intubation and mechanical ventilation. The aim of this study was to quantify and describe the presence of shoulder pain in patients released from hospitals after suffering COVID-19. Patients with positive Apley tests were sent to a physiatrist for a clinical evaluation, ultrasound and electromyography (EMG). This evaluation was completed with a pain scale, joint range and shoulder muscle strength evaluations. Of the one-hundred-sixteen patients, seventy eight entered the respiratory rehabilitation program. Twenty patients were sent to the multidisciplinary shoulder team for positive Apley scratch tests. Of these twenty patients, one had only an EMG, ten had only ultrasounds, seven had an EMG and ultrasound and two did not need complementary tests. The twenty patients were sent to the physical therapist, with all presenting pain and diminished joint range and muscle strength in the affected shoulder. In this context, shoulder pain could be associated with the prone position in the ICU. We suggest time control and position change for patients on mechanical ventilation in a prone position with COVID-19.

Heat Waves Alter Macrophyte-Derived Detrital Nutrients Release under Future Climate Warming Scenarios.

In addition to a rise in global air and water mean temperatures, extreme climate events such as heat waves are increasing in frequency, intensity, and duration in many regions of the globe. Developing a mechanistic understanding of the impacts of heat waves on key ecosystem processes and how they differ from just an increase in mean temperatures is therefore of utmost importance for adaptive management against effects of global change. However, little is known about the impact of extreme events on freshwater ecosystem processes, particularly the decomposition of macrophyte detritus. We performed a mesocosm experiment to evaluate the impact of warming and heat waves on macrophyte detrital decomposition, applied as a fixed increment (+4 °C) above ambient and a fluctuating treatment with similar energy input, ranging from 0 to 6 °C above ambient (i.e., simulating heat waves). We showed that both warming and heat waves significantly accelerate dry mass loss of the detritus and carbon (C) release but found no significant differences between the two heated treatments on the effects on detritus dry mass loss and C release amount. This suggests that moderate warming indirectly enhanced macrophyte detritus dry mass loss and C release mainly by the amount of energy input rather than by the way in which warming was provided (i.e., by a fixed increment or in heat waves). However, we found significantly different amounts of nitrogen (N) and phosphorus (P) released between the two warming treatments, and there was an asymmetric response of N and P release patterns to the two warming treatments, possibly due to species-specific responses of decomposers to short-term temperature fluctuations and litter quality. Our results conclude that future climate scenarios can significantly accelerate organic matter decomposition and C, N, and P release from decaying macrophytes, and more importantly, there are asymmetric alterations in macrophyte-derived detrital N and P release dynamic. Therefore, future climate change scenarios could lead to alterations in N/P ratios in the water column via macrophyte decomposition processes and ultimately affect the structure and function of aquatic ecosystems, especially in the plankton community.

Functionally reversible impacts of disturbances on lake food webs linked to spatial and seasonal dependencies.

Increasing human impact on the environment is causing drastic changes in disturbance regimes and how they prevail over time. Of increasing relevance is to further our understanding on biological responses to pulse disturbances (short duration) and how they interact with other ongoing press disturbances (constantly present). Because the temporal and spatial contexts of single experiments often limit our ability to generalize results across space and time, we conducted a modularized mesocosm experiment replicated in space (five lakes along a latitudinal gradient in Scandinavia) and time (two seasons, spring and summer) to generate general predictions on how the functioning and composition of multitrophic plankton communities (zoo-, phyto- and bacterioplankton) respond to pulse disturbances acting either in isolation or combined with press disturbances. As pulse disturbance, we used short-term changes in fish presence, and as press disturbance, we addressed the ongoing reduction in light availability caused by increased cloudiness and lake browning in many boreal and subarctic lakes. First, our results show that the top-down pulse disturbance had the strongest effects on both functioning and composition of the three trophic levels across sites and seasons, with signs for interactive impacts with the bottom-up press disturbance on phytoplankton communities. Second, community composition responses to disturbances were highly divergent between lakes and seasons: temporal accumulated community turnover of the same trophic level either increased (destabilization) or decreased (stabilization) in response to the disturbances compared to control conditions. Third, we found functional recovery from the pulse disturbances to be frequent at the end of most experiments. In a broader context, these results demonstrate that top-down, pulse disturbances, either alone or with additional constant stress upon primary producers caused by bottom-up disturbances, can induce profound but often functionally reversible changes across multiple trophic levels, which are strongly linked to spatial and temporal context dependencies. Furthermore, the identified dichotomy of disturbance effects on the turnover in community composition demonstrates the potential of disturbances to either stabilize or destabilize biodiversity patterns over time across a wide range of environmental conditions.

Storm impacts on phytoplankton community dynamics in lakes.

In many regions across the globe, extreme weather events such as storms have increased in frequency, intensity, and duration due to climate change. Ecological theory predicts that such extreme events should have large impacts on ecosystem structure and function. High winds and precipitation associated with storms can affect lakes via short-term runoff events from watersheds and physical mixing of the water column. In addition, lakes connected to rivers and streams will also experience flushing due to high flow rates. Although we have a well-developed understanding of how wind and precipitation events can alter lake physical processes and some aspects of biogeochemical cycling, our mechanistic understanding of the emergent responses of phytoplankton communities is poor. Here we provide a comprehensive synthesis that identifies how storms interact with lake and watershed attributes and their antecedent conditions to generate changes in lake physical and chemical environments. Such changes can restructure phytoplankton communities and their dynamics, as well as result in altered ecological function (e.g., carbon, nutrient and energy cycling) in the short- and long-term. We summarize the current understanding of storm-induced phytoplankton dynamics, identify knowledge gaps with a systematic review of the literature, and suggest future research directions across a gradient of lake types and environmental conditions.

Climate warming and heat waves alter harmful cyanobacterial blooms along the benthic-pelagic interface.

In addition to a rise in mean air and water temperatures, more frequent and intense extreme climate events (such as heat waves) have been recorded around the globe during the past decades. These environmental changes are projected to intensify further in the future, and we still know little about how they will affect ecological processes driving harmful cyanobacterial bloom formation. Therefore, we conducted a long-term experiment in 400-L shallow freshwater mesocosms, where we evaluated the effects of a constant +4°C increase in mean water temperatures and compared it with a fluctuating warming scenario ranging from 0 to +8°C (i.e., including heat waves) but with the same +4°C long-term elevation in mean water temperatures. We focused on investigating not only warming effects on cyanobacterial pelagic dynamics (phenology and biomass levels), but also on their recruitment from sediments-which are a fundamental part of their life history for which the response to warming remains largely unexplored. Our results demonstrate that (1) a warmer environment not only induces a seasonal advancement and boosts biomass levels of specific cyanobacterial species in the pelagic environment, but also increases their recruitment rates from the sediments, and (2) these species-specific benthic and pelagic processes respond differently depending on whether climate warming is expressed only as an increase in mean water temperatures or, in addition, through an increased warming variability (including heat waves). These results are important because they show, for the first time, that climate warming can affect cyanobacterial dynamics at different life-history stages, all the way from benthic recruitment up to their establishment in the pelagic community. Furthermore, it also highlights that both cyanobacterial benthic recruitment and pelagic biomass dynamics may be different as a result of changes in the variability of warming conditions. We argue that these findings are a critical first step to further our understanding of the relative importance of increased recruitment rates for harmful cyanobacterial bloom formation under different climate change scenarios.

Allele-Specific Silencing Ameliorates Restrictive Cardiomyopathy Attributable to a Human Myosin Regulatory Light Chain Mutation.

BACKGROUND: Restrictive cardiomyopathy is a rare heart disease associated with mutations in sarcomeric genes and with phenotypic overlap with hypertrophic cardiomyopathy. There is no approved therapy directed at the underlying cause. Here, we explore the potential of an interfering RNA (RNAi) therapeutic for a human sarcomeric mutation in MYL2 causative of restrictive cardiomyopathy in a mouse model. METHODS: A short hairpin RNA (M7.8L) was selected from a pool for specificity and efficacy. Two groups of myosin regulatory light chain N47K transgenic mice were injected with M7.8L packaged in adeno-associated virus 9 at 3 days of age and 60 days of age. Mice were subjected to treadmill exercise and echocardiography after treatment to determine maximal oxygen uptake and left ventricular mass. At the end of treatment, heart, lung, liver, and kidney tissue was harvested to determine viral tropism and for transcriptomic and proteomic analysis. Cardiomyocytes were isolated for single-cell studies. RESULTS: A one-time injection of AAV9-M7.8L RNAi in 3-day-old humanized regulatory light chain mutant transgenic mice silenced the mutated allele (RLC-47K) with minimal effects on the normal allele (RLC-47N) assayed at 16 weeks postinjection. AAV9-M7.8L RNAi suppressed the expression of hypertrophic biomarkers, reduced heart weight, and attenuated a pathological increase in left ventricular mass. Single adult cardiac myocytes from mice treated with AAV9-M7.8L showed partial restoration of contraction, relaxation, and calcium kinetics. In addition, cardiac stress protein biomarkers, such as calmodulin-dependent protein kinase II and the transcription activator Brg1 were reduced, suggesting recovery toward a healthy myocardium. Transcriptome analyses further revealed no significant changes of argonaute (AGO1, AGO2) and endoribonuclease dicer (DICER1) transcripts, and endogenous microRNAs were preserved, suggesting that the RNAi pathway was not saturated. CONCLUSIONS: Our results show the feasibility, efficacy, and safety of RNAi therapeutics directed towards human restrictive cardiomyopathy. This is a promising step toward targeted therapy for a prevalent human disease.

Pathologic gene network rewiring implicates PPP1R3A as a central regulator in pressure overload heart failure.

Heart failure is a leading cause of mortality, yet our understanding of the genetic interactions underlying this disease remains incomplete. Here, we harvest 1352 healthy and failing human hearts directly from transplant center operating rooms, and obtain genome-wide genotyping and gene expression measurements for a subset of 313. We build failing and non-failing cardiac regulatory gene networks, revealing important regulators and cardiac expression quantitative trait loci (eQTLs). PPP1R3A emerges as a regulator whose network connectivity changes significantly between health and disease. RNA sequencing after PPP1R3A knockdown validates network-based predictions, and highlights metabolic pathway regulation associated with increased cardiomyocyte size and perturbed respiratory metabolism. Mice lacking PPP1R3A are protected against pressure-overload heart failure. We present a global gene interaction map of the human heart failure transition, identify previously unreported cardiac eQTLs, and demonstrate the discovery potential of disease-specific networks through the description of PPP1R3A as a central regulator in heart failure.

Charophytes collapse beyond a critical warming and brownification threshold in shallow lake systems.

Charophytes play a critical role for the functioning of shallow lake ecosystems. Although growth of charophytes can be limited by many factors, such as temperature, nutrients and light availability, our understanding about concomitant effects of climate warming and other large-scale environmental perturbations, e.g. increases in humic matter content ('brownification') is still limited. Here we conducted an outdoor mesocosm experiment during 71days with a common charophyte species, Chara vulgaris, along an increasing gradient of temperature and brownification. We hypothesized the growth of C. vulgaris to increase with temperature, but to level off along the combined temperature and brownification gradient when reaching a critical threshold for light limitation via brownification. We show that C. vulgaris increases the relative growth rate (RGR), main and total shoot elongation, as well as number of lateral shoots when temperature and brownification increased by +2°C and+100%, respectively above today's levels. However, the RGR, shoot elongation and number of lateral shoots declined at further increment of temperature and brownification. Macrophyte weight-length ratio decreased with increased temperature and brownification, indicating that C. vulgaris allocate more resources or energy for shoot elongation instead of biomass increase at warmer temperatures and higher brownification. Our study shows that C. vulgaris will initially benefit from warming and brownification but will then decline as a future scenario of increased warming and brownification reaches a certain threshold level, in case of our experiment at +4°C and a 2-fold increase in brownification above today's levels.

Life-history traits buffer against heat wave effects on predator-prey dynamics in zooplankton.

In addition to an increase in mean temperature, extreme climatic events, such as heat waves, are predicted to increase in frequency and intensity with climate change, which are likely to affect organism interactions, seasonal succession, and resting stage recruitment patterns in terrestrial as well as in aquatic ecosystems. For example, freshwater zooplankton with different life-history strategies, such as sexual or parthenogenetic reproduction, may respond differently to increased mean temperatures and rapid temperature fluctuations. Therefore, we conducted a long-term (18 months) mesocosm experiment where we evaluated the effects of increased mean temperature (4°C) and an identical energy input but delivered through temperature fluctuations, i.e., as heat waves. We show that different rotifer prey species have specific temperature requirements and use limited and species-specific temperature windows for recruiting from the sediment. On the contrary, co-occurring predatory cyclopoid copepods recruit from adult or subadult resting stages and are therefore able to respond to short-term temperature fluctuations. Hence, these different life-history strategies affect the interactions between cyclopoid copepods and rotifers by reducing the risk of a temporal mismatch in predator-prey dynamics in a climate change scenario. Thus, we conclude that predatory cyclopoid copepods with long generation time are likely to benefit from heat waves since they rapidly "wake up" even at short temperature elevations and thereby suppress fast reproducing prey populations, such as rotifers. In a broader perspective, our findings suggest that differences in life-history traits will affect predator-prey interactions, and thereby alter community dynamics, in a future climate change scenario.

Climate warming and heat waves affect reproductive strategies and interactions between submerged macrophytes.

Extreme climatic events, such as heat waves, are predicted to increase in frequency and intensity during the next hundred years, which may accelerate shifts in hydrological regimes and submerged macrophyte composition in freshwater ecosystems. Since macrophytes are profound components of aquatic systems, predicting their response to extreme climatic events is crucial for implementation of climate change adaptation strategies. We therefore performed an experiment in 24 outdoor enclosures (400 L) separating the impact of a 4 °C increase in mean temperature with the same increase, that is the same total amount of energy input, but resembling a climate scenario with extreme variability, oscillating between 0 °C and 8 °C above present conditions. We show that at the moderate nutrient conditions provided in our study, neither an increase in mean temperature nor heat waves lead to a shift from a plant-dominated to an algal-dominated system. Instead, we show that species-specific responses to climate change among submerged macrophytes may critically influence species composition and thereby ecosystem functioning. Our results also imply that more fluctuating temperatures affect the number of flowers produced per plant leading to less sexual reproduction. Our findings therefore suggest that predicted alterations in climate regimes may influence both plant interactions and reproductive strategies, which have the potential to inflict changes in biodiversity, community structure and ecosystem functioning.

TRACING CO-REGULATORY NETWORK DYNAMICS IN NOISY, SINGLE-CELL TRANSCRIPTOME TRAJECTORIES.

The availability of gene expression data at the single cell level makes it possible to probe the molecular underpinnings of complex biological processes such as differentiation and oncogenesis. Promising new methods have emerged for reconstructing a progression 'trajectory' from static single-cell transcriptome measurements. However, it remains unclear how to adequately model the appreciable level of noise in these data to elucidate gene regulatory network rewiring. Here, we present a framework called Single Cell Inference of MorphIng Trajectories and their Associated Regulation (SCIMITAR) that infers progressions from static single-cell transcriptomes by employing a continuous parametrization of Gaussian mixtures in high-dimensional curves. SCIMITAR yields rich models from the data that highlight genes with expression and co-expression patterns that are associated with the inferred progression. Further, SCIMITAR extracts regulatory states from the implicated trajectory-evolvingco-expression networks. We benchmark the method on simulated data to show that it yields accurate cell ordering and gene network inferences. Applied to the interpretation of a single-cell human fetal neuron dataset, SCIMITAR finds progression-associated genes in cornerstone neural differentiation pathways missed by standard differential expression tests. Finally, by leveraging the rewiring of gene-gene co-expression relations across the progression, the method reveals the rise and fall of co-regulatory states and trajectory-dependent gene modules. These analyses implicate new transcription factors in neural differentiation including putative co-factors for the multi-functional NFAT pathway.

Local food web management increases resilience and buffers against global change effects on freshwaters.

A major challenge for ecological research is to identify ways to improve resilience to climate-induced changes in order to secure the ecosystem functions of natural systems, as well as ecosystem services for human welfare. With respect to aquatic ecosystems, interactions between climate warming and the elevated runoff of humic substances (brownification) may strongly affect ecosystem functions and services. However, we hitherto lack the adaptive management tools needed to counteract such global-scale effects on freshwater ecosystems. Here we show, both experimentally and using monitoring data, that predicted climatic warming and brownification will reduce freshwater quality by exacerbating cyanobacterial growth and toxin levels. Furthermore, in a model based on long-term data from a natural system, we demonstrate that food web management has the potential to increase the resilience of freshwater systems against the growth of harmful cyanobacteria, and thereby that local efforts offer an opportunity to secure our water resources against some of the negative impacts of climate warming and brownification. This allows for novel policy action at a local scale to counteract effects of global-scale environmental change, thereby providing a buffer period and a safer operating space until climate mitigation strategies are effectively established.

Controlling Harmful Cyanobacteria: Taxa-Specific Responses of Cyanobacteria to Grazing by Large-Bodied Daphnia in a Biomanipulation Scenario.

Lake restoration practices based on reducing fish predation and promoting the dominance of large-bodied Daphnia grazers (i.e., biomanipulation) have been the focus of much debate due to inconsistent success in suppressing harmful cyanobacterial blooms. While most studies have explored effects of large-bodied Daphnia on cyanobacterial growth at the community level and/or on few dominant species, predictions of such restoration practices demand further understanding on taxa-specific responses in diverse cyanobacterial communities. In order to address these questions, we conducted three grazing experiments during summer in a eutrophic lake where the natural phytoplankton community was exposed to an increasing gradient in biomass of the large-bodied Daphnia magna. This allowed evaluating taxa-specific responses of cyanobacteria to Daphnia grazing throughout the growing season in a desired biomanipulation scenario with limited fish predation. Total cyanobacterial and phytoplankton biomasses responded negatively to Daphnia grazing both in early and late summer, regardless of different cyanobacterial densities. Large-bodied Daphnia were capable of suppressing the abundance of Aphanizomenon, Dolichospermum, Microcystis and Planktothrix bloom-forming cyanobacteria. However, the growth of the filamentous Dolichospermum crassum was positively affected by grazing during a period when this cyanobacterium dominated the community. The eutrophic lake was subjected to biomanipulation since 2005 and nineteen years of lake monitoring data (1996-2014) revealed that reducing fish predation increased the mean abundance (50%) and body-size (20%) of Daphnia, as well as suppressed the total amount of nutrients and the growth of the dominant cyanobacterial taxa, Microcystis and Planktothrix. Altogether our results suggest that lake restoration practices solely based on grazer control by large-bodied Daphnia can be effective, but may not be sufficient to control the overgrowth of all cyanobacterial diversity. Although controlling harmful cyanobacterial blooms should preferably include other measures, such as nutrient reductions, our experimental assessment of taxa-specific cyanobacterial responses to large-bodied Daphnia and long-term monitoring data highlights the potential of such biomanipulations to enhance the ecological and societal value of eutrophic water bodies.

The interaction between cyanobacteria and zooplankton in a more eutrophic world.

As blooms of cyanobacteria expand and intensify in freshwater systems globally, there is increasing interest in their ecological effects. In addition to being public health hazards, cyanobacteria have long been considered a poor quality food for key zooplankton grazers that link phytoplankton to higher trophic levels. While past laboratory studies have found negative effects of nutritional constraints and defensive traits (i.e., toxicity and colonial or filamentous morphology) on the fitness of large generalist grazers (i.e., Daphnia), cyanobacterial blooms often co-exist with high biomass of small-bodied zooplankton in nature. Indeed, recent studies highlight the remarkable diversity and flexibility in zooplankton responses to cyanobacterial prey. Reviewed here are results from a wide range of laboratory and field experiments examining the interaction of cyanobacteria and a diverse zooplankton taxa including cladocerans, copepods, and heterotrophic protists from temperate to tropical freshwater systems. This synthesis shows that longer exposure to cyanobacteria can shift zooplankton communities toward better-adapted species, select for more tolerant genotypes within a species, and induce traits within the lifetime of individual zooplankton. In turn, the function of bloom-dominated plankton ecosystems, the coupling between primary producers and grazers, the stability of blooms, and the potential to use top down biomanipulation for controlling cyanobacteria depend largely on the species, abundance, and traits of interacting cyanobacteria and zooplankton. Understanding the drivers and consequences of zooplankton traits, such as physiological detoxification and selective vs. generalist grazing behavior, are therefore of major importance for future studies. Ultimately, co-evolutionary dynamics between cyanobacteria and their grazers may emerge as a critical regulator of blooms.

Rich RNA Structure Landscapes Revealed by Mutate-and-Map Analysis.

Landscapes exhibiting multiple secondary structures arise in natural RNA molecules that modulate gene expression, protein synthesis, and viral infection [corrected]. We report herein that high-throughput chemical experiments can isolate an RNA's multiple alternative secondary structures as they are stabilized by systematic mutagenesis (mutate-and-map, M2) and that a computational algorithm, REEFFIT, enables unbiased reconstruction of these states' structures and populations. In an in silico benchmark on non-coding RNAs with complex landscapes, M2-REEFFIT recovers 95% of RNA helices present with at least 25% population while maintaining a low false discovery rate (10%) and conservative error estimates. In experimental benchmarks, M2-REEFFIT recovers the structure landscapes of a 35-nt MedLoop hairpin, a 110-nt 16S rRNA four-way junction with an excited state, a 25-nt bistable hairpin, and a 112-nt three-state adenine riboswitch with its expression platform, molecules whose characterization previously required expert mutational analysis and specialized NMR or chemical mapping experiments. With this validation, M2-REEFFIT enabled tests of whether artificial RNA sequences might exhibit complex landscapes in the absence of explicit design. An artificial flavin mononucleotide riboswitch and a randomly generated RNA sequence are found to interconvert between three or more states, including structures for which there was no design, but that could be stabilized through mutations. These results highlight the likely pervasiveness of rich landscapes with multiple secondary structures in both natural and artificial RNAs and demonstrate an automated chemical/computational route for their empirical characterization.

Consistent global structures of complex RNA states through multidimensional chemical mapping.

Accelerating discoveries of non-coding RNA (ncRNA) in myriad biological processes pose major challenges to structural and functional analysis. Despite progress in secondary structure modeling, high-throughput methods have generally failed to determine ncRNA tertiary structures, even at the 1-nm resolution that enables visualization of how helices and functional motifs are positioned in three dimensions. We report that integrating a new method called MOHCA-seq (Multiplexed •OH Cleavage Analysis with paired-end sequencing) with mutate-and-map secondary structure inference guides Rosetta 3D modeling to consistent 1-nm accuracy for intricately folded ncRNAs with lengths up to 188 nucleotides, including a blind RNA-puzzle challenge, the lariat-capping ribozyme. This multidimensional chemical mapping (MCM) pipeline resolves unexpected tertiary proximities for cyclic-di-GMP, glycine, and adenosylcobalamin riboswitch aptamers without their ligands and a loose structure for the recently discovered human HoxA9D internal ribosome entry site regulon. MCM offers a sequencing-based route to uncovering ncRNA 3D structure, applicable to functionally important but potentially heterogeneous states.