Bacterial pathogens harbor specialized secretion systems that inject effector proteins into the host cell to establish infection and disease. While many bacterial effectors post-translationally modify proteins to influence host responses, the extent to which effectors modify host RNA is currently unknown. Here we performed RNA-interactome capture (RIC) to isolate effectors bound to host cellular messenger RNA (mRNA) during Legionella pneumophila infection. RIC identified an uncharacterized effector, FadA (Lpw16921), which localized to the host-cell nucleus and interacted with host mRNAs at uracil (U)-rich RNA motifs. FadA exhibited NADPH-oxidase activity that mediated 8-oxo-guanine (oxo8G) modifications of mRNA substrates, resulting in oxidative damage and inhibition of translation. Infection with L. pneumophila harboring wild-type FadA, but not a catalytically inactive mutant, increased oxo8G modifications, suppressed cytokine responses, and promoted bacterial persistence in vivo. Our findings demonstrate the potential for a secreted effector to post-transcriptionally modify host mRNA as a mechanism to promote bacterial virulence.

In the bacterial Csr/Rsm system, non-coding RNAs activate mRNA translation by removing homodimeric Csr/Rsm proteins from ribosome-binding sites of mRNAs. In Pseudomonas protegens, each RsmZ ncRNA sequesters up to five RsmE dimers sequentially and specifically within a narrow affinity range, functioning as a 'protein sponge'. Although the RsmE binding cascade is cooperative, binding of the highest affinity stem-loop RNA in RsmZ (SL2) reduces RNA binding affinity at the second site 10- to 30-fold. This unusual negative cooperativity may facilitate RsmE release from tightly bound mRNA for handover to the non-coding RNA, yet the underlying mechanisms remain unclear. Using Isothermal Titration Calorimetry, NMR spectroscopy and Molecular Dynamics, we reveal an allosteric mechanism resembling a Newton's cradle, coupling the binding at one site to conformational and dynamic changes at the second site, explaining the reduced affinity of the second binding event, and handover of RsmE dimer from mRNA to the ncRNA RsmZ.

Abstract Phosphorylation of intrinsically disordered proteins (IDPs) is essential for regulating biomolecular interactions in many cellular processes. However, a quantitative understanding of how phosphorylation tunes the affinity between highly charged IDPs and nucleic acids is lacking. Here, we show that multi-site phosphorylation of the disordered arginine/serine-rich (RS) domain of the splicing factor SRSF1 acts as an electrostatic rheostat that governs RNA binding. By combining enzymatic phosphorylation, phosphomimetic variants, and chemically synthesised phosphopeptides with single-molecule Förster resonance energy transfer measurements, we reveal the RS domain to be a potent driver of protein–RNA association. Increasing phosphorylation progressively reduces this interaction, and extensive phosphorylation eliminates detectable RNA binding. Remarkably, the binding free energy depends linearly on RS-domain net charge, regardless of whether the charge arises from phosphorylation or acidic residues introduced as phosphomimetics. Together, our findings uncover a quantitative framework for how phosphorylation tunes the interactions of charged IDPs and rationalize why two acidic residues are required to mimic a single phosphorylation event.

An intronic G4C2 repeat expansion in the C9ORF72 gene is the major known cause for Amyotrophic Lateral Sclerosis (ALS), with current evidence for both, loss of function and pathological gain of function disease mechanisms. We screened 96 200 small molecules in C9ORF72 patient iPS neurons for modulation of nuclear G4C2 RNA foci and identified 82 validated hits, including the Brd4 inhibitor JQ1 as well as novel analogs of Spliceostatin-A, a known modulator of SF3B1, the branch point binding protein of the U2-snRNP. Spliceosome modulation by these SF3B1 targeted compounds recruits SRSF1 to nuclear G4C2 RNA, mobilizing it from RNA foci into nucleocytoplasmic export. This leads to increased repeat-associated non-canonical (RAN) translation and ultimately, enhanced cell toxicity. Our data (i) provide a new pharmacological entry point with novel as well as known, publicly available tool compounds for dissection of C9ORF72 pathobiology in C9ORF72 ALS models, (ii) allowing to differentially modulate RNA foci versus RAN translation, and (iii) suggest that therapeutic RNA foci elimination strategies warrant caution due to a potential storage function, counteracting translation into toxic dipeptide repeat polyproteins. Instead, our data support modulation of nuclear export via SRSF1 or SR protein kinases as possible targets for future pharmacological drug discovery.

Protein-RNA interactions underpin many critical biological processes, demanding the development of technologies to precisely characterize their nature and functions. Many such technologies depend upon cross-linking under mild irradiation conditions to stabilize contacts between amino acids and nucleobases; for example, the cross-linking of stable isotope labelled RNA coupled to mass spectrometry (CLIR-MS) method. A deeper understanding of the CLIR-MS workflow is required to maximize its impact for structural biology, particularly addressing the low abundance of cross-linking products and the information content of spatial/geometric restraints reflected by a cross-link. Here, we present a vastly improved CLIR-MS pipeline that features enhanced sample preparation, data acquisition and interpretation. These advances significantly increase the number of detected cross-link products per sample. We demonstrate that the procedure is robust against variation of key experimental parameters, including irradiation energy and temperature. Using this improved protocol on four protein-RNA complexes representing canonical and non-canonical RNA-binding domains, we propose for the first time the distances encoded by protein-RNA cross-links, enabling their use as structural restraints. We also compared the cross-linking of canonical RNA with 4-thiouracil-labeled counterparts, showing slight, but noticeable differences. The improved understanding of protein-RNA cross-links refines their structural interpretation and facilitates the adoption of the method in integrative/hybrid structural biology.

RNA condensation is implicated in the formation of neurotoxic RNA foci in cells affected by genomic expansions of trinucleotide or hexanucleotide repeats. However, the biophysical properties of repeat-expansion RNA condensates are poorly understood. Using CAG repeat-expansion RNA as a model system, we show that these RNA condensates cannot be observed with conventional nuclear magnetic resonance techniques. Therefore, we developed a nuclear magnetic resonance approach, based on water-detected semi-solid magnetization transfer, to detect and characterize RNA condensates in vitro. Our method, termed condensate detection by semi-solid magnetization transfer (CONDENSE-MT), is broadly applicable, highly sensitive and does not require direct observation of the biomolecules of interest. Using CONDENSE-MT, we could obtain dynamic information about RNA condensates, such as the relative amount and the tumbling rate of condensed RNA, the proton–solvent exchange kinetics and the amount of water molecules transiently bound in the condensate. We find that phase separation dramatically decreases molecular tumbling and is driven by heterotypic interactions between RNA and Mg2+. We further show that increasing CAG repeats decreases condensate hydration. The condensation of repeat-containing RNAs can have neurotoxic effects but is challenging to study. Now a NMR approach termed condensate detection by semi-solid magnetization transfer (CONDENSE-MT) can be used to study RNA condensate dynamics, proton–solvent exchange kinetics and condensate hydration.

Liquid-liquid phase separation (LLPS) phenomenon plays a vital role in multiple cell biology processes, providing a mechanism to concentrate biomolecules and promote cellular reactions locally. Despite its significance in biology, there is a lack of conventional techniques suitable for studying biphasic samples in their biologically relevant form. Here, we present a label-free and non-invasive approach to characterize biomolecular condensates termed LLPS REstricted DIFusion of INvisible speciEs (REDIFINE). Relying on diffusion NMR measurements, REDIFINE exploits the exchange dynamics between molecules in the condensed and dispersed phases to determine not only diffusion constants and the fractions in both phases but also the average radius of the condensed droplets and the exchange rate between the phases. Observing proteins, RNAs, water, as well as small molecules, and even assessing the concentrations of biomolecules in both phases, REDIFINE analysis allows a rapid biophysical characterization of multicomponent condensates which is important to understand their functional roles. In comparing multiple systems, REDIFINE reveals that folded RNA-binding proteins form smaller and more dynamic droplets compared to the disordered ones.

Solutions of some proteins phase separate into a condensed state of high protein concentration and a dispersed state of low concentration. Such behavior is observed in living cells for a number of RNA -binding proteins that feature intrinsically disordered domains. It is relevant for cell function via the formation of membraneless organelles and transcriptional condensates. On a basic level, the process can be studied in vitro on protein domains that are necessary and sufficient for liquid -liquid phase separation (LLPS). We have performed distance distribution measurements by electron paramagnetic resonance for 13 sections in an N -terminal domain (NTD) construct of the protein fused in sarcoma (FUS), consisting of the QGSY-rich domain and the RGG1 domain, in the denatured, dispersed, and condensed state. Using 10 distance distribution restraints for ensemble modeling and three such restraints for model validation, we have found that FUS NTD behaves as a random -coil polymer under good -solvent conditions in both the dispersed and condensed state. Conformation distribution in the biomolecular condensate is virtually indistinguishable from the one in an unrestrained ensemble, with the latter one being based on only residue -specific Ramachandran angle distributions. Over its whole length, FUS NTD is slightly more compact in the condensed than in the dispersed state, which is in line with the theory for random coils in good solvent proposed by de Gennes, Daoud, and Jannink. The estimated concentration in the condensate exceeds the overlap concentration resulting from this theory. The QGSY-rich domain is slightly more extended, slightly more hydrated, and has slightly higher propensity for LLPS than the RGG1 domain. Our results support previous suggestions that LLPS of FUS is driven by multiple transient nonspecific hydrogen bonding and 7r - sp 2 interactions.

Phase transitions are important to understand cell dynamics, and the maturation of liquid droplets is relevant to neurodegenerative disorders. We combined NMR and Raman spectroscopies with microscopy to follow, over a period of days to months, droplet maturation of the protein fused in sarcoma (FUS). Our study reveals that the surface of the droplets plays a critical role in this process, while RNA binding prevents it. The maturation kinetics are faster in an agarose-stabilized biphasic sample compared with a monophasic condensed sample, owing to the larger surface-to-volume ratio. In addition, Raman spectroscopy reports structural differences upon maturation between the inside and the surface of droplets, which is comprised of β-sheet content, as revealed by solid-state NMR. In agreement with these observations, a solid crust-like shell is observed at the surface using microaspiration. Ultimately, matured droplets were converted into fibrils involving the prion-like domain as well as the first RGG motif.

Pharmacological modulation of RNA splicing by small molecules is an emerging facet of drug discovery. In this context, the SMN2 splicing modifier SMN-C5 was used as a prototype to understand the mode of action of small molecule splicing modifiers and propose the concept of 5 '-splice site bulge repair. In this study, we combined in vitro binding assays and structure determination by NMR spectroscopy to identify the binding modes of four other small molecule splicing modifiers that switch the splicing of either the SMN2 or the HTT gene. Here, we determined the solution structures of risdiplam, branaplam, SMN-CX and SMN-CY bound to the intermolecular RNA helix epitope containing an unpaired adenine within the G(-2)A(-1)G(+1)U(+2) motif of the 5 '-splice site. Despite notable differences in their scaffolds, risdiplam, SMN-CX, SMN-CY and branaplam contact the RNA epitope similarly to SMN-C5, suggesting that the 5 '-splice site bulge repair mechanism can be generalised. These findings not only deepen our understanding of the chemical diversity of splicing modifiers that target A(-1) bulged 5 '-splice sites, but also identify common pharmacophores required for modulating 5 '-splice site selection with small molecules.

Many biomolecular condensates are enriched in and depend on RNAs and RNA binding proteins (RBPs). So far, only a few studies have addressed the characterization of the intermolecular interactions responsible for liquid-liquid phase separation (LLPS) and the impact of condensation on RBPs and RNAs. Here, we present an approach to study protein-RNA interactions inside biomolecular condensates by applying cross-linking of isotope labeled RNA and tandem mass spectrometry to phase-separating systems (LLPS-CLIR-MS). LLPS-CLIR-MS enables the characterization of intermolecular interactions present within biomolecular condensates at residue-specific resolution and allows a comparison with the same complexes in the dispersed phase. We observe that sequence-specific RBP-RNA interactions present in the dispersed phase are generally maintained inside condensates. In addition, LLPS-CLIR-MS identifies structural alterations at the protein-RNA interfaces, including additional unspecific contacts in the condensed phase. Our approach offers a procedure to derive structural information of protein-RNA complexes within biomolecular condensates that could be critical for integrative structural modeling of ribonucleoproteins (RNPs) in this form.

The SARS-CoV-2 nucleocapsid (N) protein is crucial for virus replication and genome packaging. N protein forms biomolecular condensates both in vitro and in vivo in a process known as liquid-liquid phase separation (LLPS), but the exact factors regulating LLPS of N protein are not fully understood. Here, we show that pH and buffer choice have a profound impact on LLPS of N protein. The degree of phase separation is highly dependent on the pH of the solution, which is correlated with histidine protonation in N protein. Specifically, we demonstrate that protonation of H356 is essential for LLPS in phosphate buffer. Moreover, electrostatic interactions of buffer molecules with specific amino acid residues are able to alter the net charge of N protein, thus influencing its ability to undergo phase separation in the presence of RNA. Overall, these findings reveal that even subtle changes in amino acid protonation or surface charge caused by the pH and buffer system can strongly influence the LLPS behavior, and point to electrostatic interactions as the main driving forces of N protein phase separation. Further, our findings emphasize the importance of these experimental parameters when studying phase separation of biomolecules, especially in the context of viral infections where the intracellular milieu undergoes drastic changes and intracellular pH normally decreases.

Primases are crucial enzymes for DNA replication, as they synthesize a short primer required for initiating DNA replication. We herein present time-resolved nuclear magnetic resonance (NMR) spectroscopy in solution and in the solid state to study the initial dinucleotide formation reaction of archaeal pRN1 primase. Our findings show that the helix-bundle domain (HBD) of pRN1 primase prepares the two substrates and then hands them over to the catalytic domain to initiate the reaction. By using nucleotide triphosphate analogues, the reaction is substantially slowed down, allowing us to study the initial dinucleotide formation in real time. We show that the sedimented protein-DNA complex remains active in the solid-state NMR rotor and that time-resolved P-31-detected cross-polarization experiments allow monitoring the kinetics of dinucleotide formation. The kinetics in the sedimented protein sample are comparable to those determined by solution-state NMR. Protein conformational changes during primer synthesis are observed in time-resolved H-1-detected experiments at fast magic-angle spinning frequencies (100 kHz). A significant number of spectral changes cluster in the HBD pointing to the importance of the HBD for positioning the nucleotides and the dinucleotide.

TRIM25 is an RNA-binding ubiquitin E3 ligase with central but poorly understood roles in the innate immune response to RNA viruses. The link between TRIM25's RNA binding and its role in innate immunity has not been established. Thus, we utilized a multitude of biophysical techniques to identify key RNA-binding residues of TRIM25 and developed an RNA-binding deficient mutant (TRIM25-m9). Using iCLIP2 in virus-infected and uninfected cells, we identified TRIM25's RNA sequence and structure specificity, that it binds specifically to viral RNA, and that the interaction with RNA is critical for its antiviral activity. The RNA-binding ubiquitin E3 ligase TRIM25 plays a critical role in antiviral immunity. Here the authors identify key RNA-binding residues of TRIM25, link RNA binding to antiviral activity, reveal RNA structural and sequence preferences, and investigate binding to the viral genome.

The N-terminal RNA recognition motif domain (RRM1) of polypyrimidine tract binding protein (PTB) forms an additional C-terminal helix α3, which docks to one edge of the β-sheet upon binding to a stem-loop RNA containing a UCUUU pentaloop. Importantly, α3 does not contact the RNA. The α3 helix therefore represents an allosteric means to regulate the conformation of adjacent domains in PTB upon binding structured RNAs. Here we investigate the process of dynamic adaptation by stem-loop RNA and RRM1 using NMR and MD in order to obtain mechanistic insights on how this allostery is achieved. Relaxation data and NMR structure determination of the free protein show that α3 is partially ordered and interacts with the domain transiently. Stem-loop RNA binding quenches fast time scale dynamics and α3 becomes ordered, however microsecond dynamics at the protein-RNA interface is observed. MD shows how RRM1 binding to the stem-loop RNA is coupled to the stabilization of the C-terminal helix and helps to transduce differences in RNA loop sequence into changes in α3 length and order. IRES assays of full length PTB and a mutant with altered dynamics in the α3 region show that this dynamic allostery influences PTB function in cultured HEK293T cells.

Multidimensional NMR spectra are the basis for studying proteins by NMR spectroscopy and crucial for the development and evaluation of methods for biomolecular NMR data analysis. Nevertheless, in contrast to derived data such as chemical shift assignments in the BMRB and protein structures in the PDB databases, this primary data is in general not publicly archived. To change this unsatisfactory situation, we present a standardized set of solution NMR data comprising 1329 2-4-dimensional NMR spectra and associated reference (chemical shift assignments, structures) and derived (peak lists, restraints for structure calculation, etc.) annotations. With the 100-protein NMR spectra dataset that was originally compiled for the development of the ARTINA deep learning-based spectra analysis method, 100 protein structures can be reproduced from their original experimental data. The 100-protein NMR spectra dataset is expected to help the development of computational methods for NMR spectroscopy, in particular machine learning approaches, and enable consistent and objective comparisons of these methods.

The pandemic caused by SARS-CoV-2 has called for concerted efforts to generate new insights into the biology of betacoronaviruses to inform drug screening and development. Here, we establish a workflow to determine the RNA recognition and druggability of the nucleocapsid N-protein of SARS-CoV-2, a highly abundant protein crucial for the viral life cycle. We use a synergistic method that combines NMR spectroscopy and protein-RNA cross-linking coupled to mass spectrometry to quickly determine the RNA binding of two RNA recognition domains of the N-protein. Finally, we explore the druggability of these domains by performing an NMR fragment screening. This workflow identified small molecule chemotypes that bind to RNA binding interfaces and that have promising properties for further fragment expansion and drug development.

RNA-binding proteins (RBPs) are crucial regulators of gene expression, often composed of defined domains interspersed with flexible, intrinsically disordered regions. Determining the structure of ribonucleoprotein (RNP) complexes involving such RBPs necessitates integrative structural modeling due to their lack of a single stable state. In this study, we integrate magnetic resonance, mass spectrometry, and small-angle scattering data to determine the solution structure of the polypyrimidine-tract binding protein 1 (PTBP1/hnRNP I) bound to an RNA fragment from the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). This binding, essential for enhancing the translation of viral RNA, leads to a complex structure that demonstrates RNA and protein compaction, while maintaining pronounced conformational flexibility. Acting as an RNA chaperone, PTBP1 orchestrates the IRES RNA into a few distinct conformations, exposing the RNA stems outward. This conformational diversity is likely common among RNP structures and functionally important. Our approach enables atomic-level characterization of heterogeneous RNP structures.

ABSTRACT Non-physiological alternative splicing patterns are associated with numerous human diseases. Among the strategies developed to treat these diseases, small molecule splicing modifiers are emerging as a new class of RNA therapeutics. The SMN2 splicing modifier SMN-C5 was used as a prototype to understand their mode of action and discover the concept of 5’-splice site bulge repair. However, different small molecules harbouring a similar activity were also identified. In this study, we combined NMR spectroscopy and computational approaches to determine the binding modes of other SMN2 and HTT splicing modifiers at the interface between U1 snRNP and an A -1 bulged 5’-splice site. Our results show that the other splicing modifiers interact with the intermolecular RNA helix epitope containing an unpaired adenine within a G -2 A -1 G +1 U +2 motif, which is essential for their biological activity. We also determined structural models of risdiplam, SMN-CX, and branaplam bound to RNA, and solved the solution structure of the most divergent SMN2 splicing modifier, SMN-CY, in complex with the RNA helix. These findings not only deepen our understanding of the chemical diversity of splicing modifiers that target A -1 bulged 5’-splice sites, but also identify common pharmacophores required for modulating 5’-splice site selection with small molecules.