Base-pair conformational switch modulates miR-34a targeting of Sirt1 mRNA.

MicroRNAs (miRNAs) regulate the levels of translation of messenger RNAs (mRNAs). At present, the major parameter that can explain the selection of the target mRNA and the efficiency of translation repression is the base pairing between the 'seed' region of the miRNA and its counterpart mRNA1. Here we use R1ρ relaxation-dispersion nuclear magnetic resonance2 and molecular simulations3 to reveal a dynamic switch-based on the rearrangement of a single base pair in the miRNA-mRNA duplex-that elongates a weak five-base-pair seed to a complete seven-base-pair seed. This switch also causes coaxial stacking of the seed and supplementary helix fitting into human Argonaute 2 protein (Ago2), reminiscent of an active state in prokaryotic Ago4,5. Stabilizing this transient state leads to enhanced repression of the target mRNA in cells, revealing the importance of this miRNA-mRNA structure. Our observations tie together previous findings regarding the stepwise miRNA targeting process from an initial 'screening' state to an 'active' state, and unveil the role of the RNA duplex beyond the seed in Ago2.

RNA:RNA interaction in ternary complexes resolved by chemical probing.

RNA regulation can be performed by a second targeting RNA molecule, such as in the microRNA regulation mechanism. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) probes the structure of RNA molecules and can resolve RNA:protein interactions, but RNA:RNA interactions have not yet been addressed with this technique. Here, we apply SHAPE to investigate RNA-mediated binding processes in RNA:RNA and RNA:RNA-RBP complexes. We use RNA:RNA binding by SHAPE (RABS) to investigate microRNA-34a (miR-34a) binding its mRNA target, the silent information regulator 1 (mSIRT1), both with and without the Argonaute protein, constituting the RNA-induced silencing complex (RISC). We show that the seed of the mRNA target must be bound to the microRNA loaded into RISC to enable further binding of the compensatory region by RISC, while the naked miR-34a is able to bind the compensatory region without seed interaction. The method presented here provides complementary structural evidence for the commonly performed luciferase-assay-based evaluation of microRNA binding-site efficiency and specificity on the mRNA target site and could therefore be used in conjunction with it. The method can be applied to any nucleic acid-mediated RNA- or RBP-binding process, such as splicing, antisense RNA binding, or regulation by RISC, providing important insight into the targeted RNA structure.

Efficient 3'-pairing renders microRNA targeting less sensitive to mRNA seed accessibility.

MicroRNAs (miRNAs) are short RNAs that post-transcriptionally regulate gene expression by binding to specific sites in mRNAs. Site recognition is primarily mediated by the seed region (nucleotides g2-g8 in the miRNA), but pairing beyond the seed (3'-pairing) is important for some miRNA:target interactions. Here, we use SHAPE, luciferase reporter assays and transcriptomics analyses to study the combined effect of 3'-pairing and secondary structures in mRNAs on repression efficiency. Using the interaction between miR-34a and its SIRT1 binding site as a model, we provide structural and functional evidence that 3'-pairing can compensate for low seed-binding site accessibility, enabling repression of sites that would otherwise be ineffective. We show that miRNA 3'-pairing regions can productively base-pair with nucleotides far upstream of the seed-binding site and that both hairpins and unstructured bulges within the target site are tolerated. We use SHAPE to show that sequences that overcome inaccessible seed-binding sites by strong 3'-pairing adopt the predicted structures and corroborate the model using luciferase assays and high-throughput modelling of 8177 3'-UTR targets for six miRNAs. Finally, we demonstrate that PHB2, a target of miR-141, is an inaccessible target rescued by efficient 3'-pairing. We propose that these results could refine predictions of effective target sites.

Mass Spectrometry of RNA-Binding Proteins during Liquid-Liquid Phase Separation Reveals Distinct Assembly Mechanisms and Droplet Architectures.

Liquid-liquid phase separation (LLPS) of heterogeneous ribonucleoproteins (hnRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility mass spectrometry, and molecular dynamics simulations. We used an LLPS-compatible spider silk domain and pH changes to control the self-assembly of the hnRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with liquid-liquid phase separation. We find that FUS monomers undergo an unfolded-to-globular transition, whereas TDP-43 oligomerizes into partially disordered dimers and trimers. hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over LLPS. The divergent assembly mechanisms revealed by ion mobility mass spectrometry of soluble protein species that exist under LLPS conditions suggest structurally distinct complexes inside liquid droplets that may impact RNA processing and translation depending on biological context.

A robust and versatile method for production and purification of large-scale RNA samples for structural biology.

Recent findings in genome-wide transcriptomics revealed that RNAs are involved in almost every biological process, across all domains of life. The characterization of native RNAs of unknown function and structure is particularly challenging due to their typical low abundance in the cell and the inherent sensitivity toward ubiquitous RNA degrading enzymes. Therefore, robust in vitro synthesis and extensive work-up methods are often needed to obtain samples amenable for biochemical, biophysical, and structural studies. Here, we present a protocol that combines the most recent advances in T7 in vitro transcription methodology with reverse phase ion pairing and ion exchange HPLC purification of RNAs for the production of yield-optimized large-scale samples. The method is easy to follow, robust and suitable for users with little or no experience within the field of biochemistry or chromatography. The complete execution of this method, for example, for production of isotopically labeled NMR samples, can be performed in less than a week.

Secondary structure determination of conserved SARS-CoV-2 RNA elements by NMR spectroscopy.

The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.

Visualizing transient Watson-Crick-like mispairs in DNA and RNA duplexes.

Rare tautomeric and anionic nucleobases are believed to have fundamental biological roles, but their prevalence and functional importance has remained elusive because they exist transiently, in low abundance, and involve subtle movements of protons that are difficult to visualize. Using NMR relaxation dispersion, we show here that wobble dG•dT and rG•rU mispairs in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-populated Watson-Crick-like mispairs that are stabilized by rare enolic or anionic bases. These mispairs can evade Watson-Crick fidelity checkpoints and form with probabilities (10(-3) to 10(-5)) that strongly imply a universal role in replication and translation errors. Our results indicate that rare tautomeric and anionic bases are widespread in nucleic acids, expanding their structural and functional complexity beyond that attainable with canonical bases.

A two-dimensional replica-exchange molecular dynamics method for simulating RNA folding using sparse experimental restraints.

We present a 2D replica exchange protocol incorporating secondary structure information to dramatically improve 3D RNA folding using molecular dynamics simulations. We show that incorporating base-pairing restraints into all-atom, explicit solvent simulations enables the accurate recapitulation of the global tertiary fold for 4 representative RNAs ranging in length from 24 to 68 nt. This method can potentially utilize base-pairing information from a wide variety of experimental inputs to predict complex RNA tertiary folds including pseudoknots, multi-loop junctions, and non-canonical interactions.

One-Pot Production of RNA in High Yield and Purity Through Cleaving Tandem Transcripts.

There is an increasing demand for efficient and robust production of short RNA molecules in both pharmaceutics and research. A standard method is in vitro transcription by T7 RNA polymerase. This method is sequence-dependent on efficiency and is limited to products longer than ~12 nucleotides. Additionally, the native initiation sequence is required to achieve high yields, putting a strain on sequence variability. Deviations from this sequence can lead to side products, requiring laborious purification, further decreasing yield. We here present transcribing tandem repeats of the target RNA sequence followed by site-specific cleavage to obtain RNA in high purity and yield. This approach makes use of a plasmid DNA template and RNase H-directed cleavage of the transcript. The method is simpler and faster than previous protocols, as it can be performed as one pot synthesis and provides at the same time higher yields of RNA.

RNA Dynamics by NMR Spectroscopy.

An ever-increasing number of functional RNAs require a mechanistic understanding. RNA function relies on changes in its structure, so-called dynamics. To reveal dynamic processes and higher energy structures, new NMR methods have been developed to elucidate these dynamics in RNA with atomic resolution. In this Review, we provide an introduction to dynamics novices and an overview of methods that access most dynamic timescales, from picoseconds to hours. Examples are provided as well as insight into theory, data acquisition and analysis for these different methods. Using this broad spectrum of methodology, unprecedented detail and invisible structures have been obtained and are reviewed here. RNA, though often more complicated and therefore neglected, also provides a great system to study structural changes, as these RNA structural changes are more easily defined-Lego like-than in proteins, hence the numerous revelations of RNA excited states.

Observing an Antisense Drug Complex in Intact Human Cells by in-Cell NMR Spectroscopy.

Gaining insight into the uptake, trafficking and target engagement of drugs in cells can enhance understanding of a drug's function and efficiency. However, there are currently no reliable methods for studying untagged biomolecules in macromolecular complexes in intact human cells. Here we have studied an antisense oligonucleotide (ASO) drug in HEK 293T and HeLa cells by NMR spectroscopy. Using a combination of transfection, cryoprotection and dynamic nuclear polarization (DNP), we were able to detect the drug directly in intact frozen cells. Activity of the drug was confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). By applying DNP NMR to frozen cells, we overcame limitations both of solution-state in-cell NMR spectroscopy (e.g., size, stability and sensitivity) and of visualization techniques, in which (e.g., fluorescent) tagging of the ASO decreases its activity. The capability to detect an untagged, active drug, interacting in its natural environment, represents a first step towards studying molecular mechanisms in intact cells.

Efficient Detection of Structure and Dynamics in Unlabeled RNAs: The SELOPE Approach.

The knowledge of structure and dynamics is crucial to explain the function of RNAs. While nuclear magnetic resonance (NMR) is well suited to probe these for complex biomolecules, it requires expensive, isotopically labeled samples, and long measurement times. Here we present SELOPE, a new robust, proton-only NMR method that allows us to obtain site-specific overview of structure and dynamics in an entire RNA molecule using an unlabeled sample. SELOPE simplifies assignment and allows for cost-effective screening of the response of nucleic acids to physiological changes (e.g. ion concentration) or screening of drugs in a high throughput fashion. This single technique allows us to probe an unprecedented range of exchange time scales (the whole µs to ms motion range) with increased sensitivity, surpassing all current experiments to detect chemical exchange. For the first time we could describe an RNA excited state using an unlabeled RNA.

A guide to large-scale RNA sample preparation.

RNA is becoming more important as an increasing number of functions, both regulatory and enzymatic, are being discovered on a daily basis. As the RNA boom has just begun, most techniques are still in development and changes occur frequently. To understand RNA functions, revealing the structure of RNA is of utmost importance, which requires sample preparation. We review the latest methods to produce and purify a variation of RNA molecules for different purposes with the main focus on structural biology and biophysics. We present a guide aimed at identifying the most suitable method for your RNA and your biological question and highlighting the advantages of different methods. Graphical abstract In this review we present different methods for large-scale production and purification of RNAs for structural and biophysical studies.

Visualizing transient low-populated structures of RNA.

The visualization of RNA conformational changes has provided fundamental insights into how regulatory RNAs carry out their biological functions. The RNA structural transitions that have been characterized so far involve long-lived species that can be captured by structure characterization techniques. Here we report the nuclear magnetic resonance visualization of RNA transitions towards 'invisible' excited states (ESs), which exist in too little abundance (2-13%) and for too short a duration (45-250 µs) to allow structural characterization by conventional techniques. Transitions towards ESs result in localized rearrangements in base-pairing that alter building block elements of RNA architecture, including helix-junction-helix motifs and apical loops. The ES can inhibit function by sequestering residues involved in recognition and signalling or promote ATP-independent strand exchange. Thus, RNAs do not adopt a single conformation, but rather exist in rapid equilibrium with alternative ESs, which can be stabilized by cellular cues to affect functional outcomes.

Comprehensive analysis of NMR data using advanced line shape fitting.

NMR spectroscopy is uniquely suited for atomic resolution studies of biomolecules such as proteins, nucleic acids and metabolites, since detailed information on structure and dynamics are encoded in positions and line shapes of peaks in NMR spectra. Unfortunately, accurate determination of these parameters is often complicated and time consuming, in part due to the need for different software at the various analysis steps and for validating the results. Here, we present an integrated, cross-platform and open-source software that is significantly more versatile than the typical line shape fitting application. The software is a completely redesigned version of PINT ( https://pint-nmr.github.io/PINT/ ). It features a graphical user interface and includes functionality for peak picking, editing of peak lists and line shape fitting. In addition, the obtained peak intensities can be used directly to extract, for instance, relaxation rates, heteronuclear NOE values and exchange parameters. In contrast to most available software the entire process from spectral visualization to preparation of publication-ready figures is done solely using PINT and often within minutes, thereby, increasing productivity for users of all experience levels. Unique to the software are also the outstanding tools for evaluating the quality of the fitting results and extensive, but easy-to-use, customization of the fitting protocol and graphical output. In this communication, we describe the features of the new version of PINT and benchmark its performance.

Capturing Excited States in the Fast-Intermediate Exchange Limit in Biological Systems Using 1 H†NMR Spectroscopy.

Changes in molecular structure are essential for the function of biomolecules. Characterization of these structural fluctuations can illuminate alternative states and help in correlating structure to function. NMR relaxation dispersion (RD) is currently the only method for detecting these alternative, high-energy states. In this study, we present a versatile 1 H R1ρ RD experiment that not only extends the exchange timescales at least three times beyond the rate limits of 13 C/15 N R1ρ and ten times for CPMG experiments, but also makes use of easily accessible probes, thus allowing a general description of biologically important excited states. This technique can be used to extract chemical shifts for the structural characterization of excited states and to elucidate complex excited states.

Modulating RNA Alignment Using Directional Dynamic Kinks: Application in Determining an Atomic-Resolution Ensemble for a Hairpin using NMR Residual Dipolar Couplings.

Approaches that combine experimental data and computational molecular dynamics (MD) to determine atomic resolution ensembles of biomolecules require the measurement of abundant experimental data. NMR residual dipolar couplings (RDCs) carry rich dynamics information, however, difficulties in modulating overall alignment of nucleic acids have limited the ability to fully extract this information. We present a strategy for modulating RNA alignment that is based on introducing variable dynamic kinks in terminal helices. With this strategy, we measured seven sets of RDCs in a cUUCGg apical loop and used this rich data set to test the accuracy of an 0.8 µs MD simulation computed using the Amber ff10 force field as well as to determine an atomic resolution ensemble. The MD-generated ensemble quantitatively reproduces the measured RDCs, but selection of a sub-ensemble was required to satisfy the RDCs within error. The largest discrepancies between the RDC-selected and MD-generated ensembles are observed for the most flexible loop residues and backbone angles connecting the loop to the helix, with the RDC-selected ensemble resulting in more uniform dynamics. Comparison of the RDC-selected ensemble with NMR spin relaxation data suggests that the dynamics occurs on the ps-ns time scales as verified by measurements of R(1ρ) relaxation-dispersion data. The RDC-satisfying ensemble samples many conformations adopted by the hairpin in crystal structures indicating that intrinsic plasticity may play important roles in conformational adaptation. The approach presented here can be applied to test nucleic acid force fields and to characterize dynamics in diverse RNA motifs at atomic resolution.

Renal expression of FGF23 and peripheral resistance to elevated FGF23 in rodent models of polycystic kidney disease.

Fibroblast growth factor 23 (FGF23) regulates phosphate homeostasis and is linked to cardiovascular disease and all-cause mortality in chronic kidney disease. FGF23 rises in patients with CKD stages 2-3, but in patients with autosomal dominant polycystic kidney disease, the increase of FGF23 precedes the first measurable decline in renal function. The mechanisms governing FGF23 production and effects in kidney disease are largely unknown. Here we studied the relation between FGF23 and mineral homeostasis in two animal models of PKD. Plasma FGF23 levels were increased 10-fold in 4-week-old cy/+ Han:SPRD rats, whereas plasma urea and creatinine concentrations were similar to controls. Plasma calcium and phosphate levels as well as TmP/GFR were similar in PKD and control rats at all time points examined. Expression and activity of renal phosphate transporters, the vitamin D3-metabolizing enzymes, and the FGF23 co-ligand Klotho in the kidney were similar in PKD and control rats through 8 weeks of age, indicating resistance to FGF23, although phosphorylation of the FGF receptor substrate 2α protein was enhanced. In the kidneys of rats with PKD, FGF23 mRNA was highly expressed and FGF23 protein was detected in cells lining renal cysts. FGF23 expression in bone and spleen was similar in control rats and rats with PKD. Similarly, in an inducible Pkd1 knockout mouse model, plasma FGF23 levels were elevated, FGF23 was expressed in kidneys, but renal phosphate excretion was normal. Thus, the polycystic kidney produces FGF23 but is resistant to its action.

Building a network of ADPKD reference centres across Europe: the EuroCYST initiative.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic inherited kidney disease, affecting an estimated 600 000 individuals in Europe. The disease is characterized by age-dependent development of a multiple cysts in the kidneys, ultimately leading to end-stage renal failure and the need of renal replacement therapy in the majority of patients, typically by the fifth or sixth decade of life. The variable disease course, even within the same family, remains largely unexplained. Similarly, assessing disease severity and prognosis in an individual with ADPKD remains difficult. Epidemiological studies are limited due to the fragmentation of ADPKD research in Europe. METHODS: The EuroCYST initiative aims: (i) to harmonize and develop common standards for ADPKD research by starting a collaborative effort to build a network of ADPKD reference centres across Europe and (ii) to establish a multicentric observational cohort of ADPKD patients. This cohort will be used to study factors influencing the rate of disease progression, disease modifiers, disease stage-specific morbidity and mortality, health economic issues and to identify predictive disease progression markers. Overall, 1100 patients will be enrolled in 14 study sites across Europe. Patients will be prospectively followed for at least 3 years. Eligible patients will not have participated in a pharmaceutical clinical trial 1 year before enrollment, have clinically proven ADPKD, an estimated glomerular filtration rate (eGFR) of 30 mL/min/1.73 m(2) and above, and be able to provide written informed consent. The baseline visit will include a physical examination and collection of blood, urine and DNA for biomarker and genetic studies. In addition, all participants will be asked to complete questionnaires detailing self-reported health status, quality of life, socioeconomic status, health-care use and reproductive planning. All subjects will undergo annual follow-up. A magnetic resonance imaging (MRI) scan will be carried out at baseline, and patients are encouraged to undergo a second MRI at 3-year follow-up for qualitative and quantitative kidney and liver assessments. CONCLUSIONS: The ADPKD reference centre network across Europe and the observational cohort study will enable European ADPKD researchers to gain insights into the natural history, heterogeneity and associated complications of the disease as well as how it affects the lives of patients across Europe.