Tandem RNA Recognition Motifs Research Articles

Thermodynamic coupling of the tandem RRM domains of hnRNP A1 underlie its pleiotropic RNA binding functions.

The functional properties of RNA binding proteins (RBPs) require allosteric regulation through interdomain communication. Despite the importance of allostery to biological regulation, only a few studies have been conducted to describe the biophysical nature by which interdomain communication manifests in RBPs. Here, we show for hnRNP A1 that interdomain communication is vital for the unique stability of its amino-terminal domain, which consists of two RNA recognition motifs (RRMs). These RRMs exhibit drastically different stability under pressure. RRM2 unfolds as an individual domain but remains stable when appended to RRM1. Variants that disrupt interdomain communication between the tandem RRMs show a significant decrease in stability. Carrying these mutations over to the full-length protein for in vivo experiments revealed that the mutations affected the ability of the disordered carboxyl-terminal domain to engage in protein-protein interactions and influenced the protein's RNA binding capacity. Collectively, this work reveals that thermodynamic coupling between the tandem RRMs of hnRNP A1 accounts for its allosteric regulatory functions.

Thermodynamic coupling of the tandem RRM domains of hnRNP A1 underlie its pleiotropic RNA binding functions.

The functional properties of RNA binding proteins (RBPs) require allosteric regulation through interdomain communication. Despite the importance of allostery to biological regulation, only a few studies have been conducted to describe the biophysical nature by which interdomain communication manifests in RBPs. Here, we show for hnRNP A1 that interdomain communication is vital for the unique stability of its amino-terminal domain, which consists of two RNA recognition motifs (RRMs). These RRMs exhibit drastically different stability under pressure. RRM2 unfolds as an individual domain but remains stable when appended to RRM1. Variants that disrupt interdomain communication between the tandem RRMs show a significant decrease in stability. Carrying these mutations over to the full-length protein for in vivo experiments revealed that the mutations affected the ability of the disordered carboxyl-terminal domain to engage in protein-protein interactions and influenced the protein's RNA binding capacity. Collectively, this work reveals that thermodynamic coupling between the tandem RRMs of hnRNP A1 accounts for its allosteric regulatory functions.

Analysis of the inter-domain orientation of tandem RRM domains with diverse linkers: connecting experimental with AlphaFold2 predicted models.

The RNA recognition motif (RRM) is the most prevalent RNA binding domain in eukaryotes and is involved in most RNA metabolism processes. Single RRM domains have a limited RNA specificity and affinity and tend to be accompanied by other RNA binding domains, frequently additional RRMs that contribute to an avidity effect. Within multi-RRM proteins, the most common arrangement are tandem RRMs, with two domains connected by a variable linker. Despite their prevalence, little is known about the features that lead to specific arrangements, and especially the role of the connecting linker. In this work, we present a novel and robust way to investigate the relative domain orientation in multi-domain proteins using inter-domain vectors referenced to a stable secondary structure element. We apply this method to tandem RRM domains and cluster experimental tandem RRM structures according to their inter-domain and linker-domain contacts, and report how this correlates with their orientation. By extending our analysis to AlphaFold2 predicted structures, with particular attention to the inter-domain predicted aligned error, we identify new orientations not reported experimentally. Our analysis provides novel insights across a range of tandem RRM orientations that may help for the design of proteins with a specific RNA binding mode.

The mechanism of UP1 binding and unfolding of human telomeric DNA G-quadruplex

The human telomere contains multiple copies of the DNA sequence d(TTAGGG) which can fold into higher order intramolecular G-quadruplexes and regulate the maintenance of telomere length and chromosomal integrity. The nucleic acid binding protein heteronuclear ribonucleoprotein A1 (hnRNP A1) and its N-terminus proteolytic product UP1 have been shown to efficiently bind and unfold telomeric DNA G-quadruplex. However, the understanding of the molecular mechanism of the UP1 binding and unfolding telomeric G-quadruplexes is still limited. Here, we performed biochemical and biophysical characterizations of UP1 binding and unfolding of human telomeric DNA G-quadruplex d[AGGG(TTAGGG)3], and in combination of systematic site-direct mutagenesis of two tandem RNA recognition motifs (RRMs) in UP1, revealed that RRM1 is responsible for initial binding and unfolding, whereas RRM2 assists RRM1 to complete the unfolding of G-quadruplex. Isothermal titration calorimetry (ITC) and circular dichroism (CD) studies of the interactions between UP1 and DNA G-quadruplex variants indicate that the “TAG” binding motif in Loop2 of telomeric G-quadruplex is critical for UP1 recognition and G-quadruplex unfolding initiation. Together we depict a model for molecular mechanism of hnRNP A1 (UP1) binding and unfolding of the human telomeric DNA G-quadruplex.

Molecular basis of RNA-binding and autoregulation by the cancer-associated splicing factor RBM39

Pharmacologic depletion of RNA-binding motif 39 (RBM39) using aryl sulfonamides represents a promising anti-cancer therapy but requires high levels of the adaptor protein DCAF15. Consequently, novel approaches to deplete RBM39 in an DCAF15-independent manner are required. Here, we uncover that RBM39 autoregulates via the inclusion of a poison exon into its own pre-mRNA and identify the cis-acting elements that govern this regulation. We also determine the NMR solution structures of RBM39’s tandem RNA recognition motifs (RRM1 and RRM2) bound to their respective RNA targets, revealing how RRM1 recognises RNA stem loops whereas RRM2 binds specifically to single-stranded N(G/U)NUUUG. Our results support a model where RRM2 selects the 3’-splice site of a poison exon and the RRM3 and RS domain stabilise the U2 snRNP at the branchpoint. Our work provides molecular insights into RBM39-dependent 3’-splice site selection and constitutes a solid basis to design alternative anti-cancer therapies.

Inter-domain Flexibility of Human Ser/Arg-Rich Splicing Factor 1 Allows Variable Spacer Length in Cognate RNA's Bipartite Motifs.

Ser/Arg-rich splicing factor 1 (SRSF1 or ASF/SF2) is the prototypical member of SR proteins. SRSF1 binds to exonic splicing enhancers, which prompts inclusion of corresponding exons in the mature mRNA. The RNA-binding domain of SRSF1 consists of tandem RNA-recognition motifs (RRM1 and RRM2) separated by a 30 amino acid long linker. In this study, we investigate roles of RRM1, RRM2, and the linker in RNA binding. We find that although both RRMs are crucial to RNA binding, RRM2 plays the dominant role. The linker mildly contributes to RNA binding and remains flexible in the RNA-bound state. Flexibility of the linker allows the RRM1-cognate motif to be either upstream or downstream of the RRM2-cognate motif. In addition, we find that the spacer length between the bipartite motifs varies from 0 to 10 nucleotides. Our binding assays reveal that SRSF1 prefers RNA sequences with shorter spacers and the RRM1-cognate motif being placed upstream. Restrained by nuclear magnetic resonance data, we simulate RNA-bound complexes and demonstrate how tandem RRMs bind to RNA of different spacer lengths and swapped bipartite motifs. We find that when the RRM1-cognate motif is placed downstream, either the RRM1/RRM2 linker needs to be more extended or RNA needs to form a U turn, which may reduce conformational entropy. Our study suggests that the RNA-binding specificity of SRSF1 is broader than traditionally recapitulated by consensus sequences of 7 to 8 nucleotides. Instead, centered on the RRM2-cognate motif, an RNA fragment encompassing 10-nucleotide upstream and downstream should be scrutinized.

The solution structure of Dead End bound to AU-rich RNA reveals an unusual mode of tandem RRM-RNA recognition required for mRNA regulation

Dead End (DND1) is an RNA-binding protein essential for germline development through its role in post-transcriptional gene regulation. The molecular mechanisms behind selection and regulation of its targets are unknown. Here, we present the solution structure of DND1’s tandem RNA Recognition Motifs (RRMs) bound to AU-rich RNA. The structure reveals how an NYAYUNN element is specifically recognized, reconciling seemingly contradictory sequence motifs discovered in recent genome-wide studies. RRM1 acts as a main binding platform, including atypical extensions to the canonical RRM fold. RRM2 acts cooperatively with RRM1, capping the RNA using an unusual binding pocket, leading to an unusual mode of tandem RRM-RNA recognition. We show that the consensus motif is sufficient to mediate upregulation of a reporter gene in human cells and that this process depends not only on RNA binding by the RRMs, but also on DND1’s double-stranded RNA binding domain (dsRBD), which is dispensable for binding of a subset of targets in cellulo. Our results point to a model where DND1 target selection is mediated by a non-canonical mode of AU-rich RNA recognition by the tandem RRMs and a role for the dsRBD in the recruitment of effector complexes responsible for target regulation.

All-Atom Simulations Elucidate the Impact of U2AF2 Cancer-Associated Mutations on Pre-mRNA Recognition.

The U2AF2 splicing factor, made of two tandem RNA recognition motifs (RRMs) joined by a flexible linker, selects the intronic polypyrimidine sequence of premature mRNA, thus ensuring splicing fidelity. Increasing evidence links mutations of key splicing factors, including U2AF2, to a variety of cancers. Nevertheless, the impact of U2AF2 cancer-associated mutations on polypyrimidine recognition remains unclear. Here, we combined extensive (18 μs-long) all-atom molecular dynamics simulations and dynamical network theory analysis (NWA) of U2AF2, in its wild-type form and in the presence of the six most frequent cancer-associated mutations, bound to a poly-U strand. Our results reveal that the selected mutations affect the pre-mRNA binding at two hot spot regions, irrespectively of where these mutants are placed on the distinct U2AF2 domains. Complementarily, NWA traced the existence of cross-communication pathways, connecting each mutation site to these recognition hot spots, whose strength is altered by the mutations. Our outcomes suggest the existence of a structural/dynamical interplay of the two U2AF2's RRMs underlying the recognition of the polypyrimidine tract and reveal that the cancer-associated mutations affect the polypyrimidine selection by altering the RRMs' cooperativity. This mechanism may be shared by other RNA binding proteins hallmarked, like U2AF2, by multidomain architecture and high plasticity.

Molecular insights into binding dynamics of tandem RNA recognition motifs (tRRMs) of human antigen R (HuR) with mRNA and the effect of point mutations in impaired HuR-mRNA recognition

Human antigen R (HuR) is a key regulatory protein with prominent roles in RNA metabolism and post-transcriptional gene regulation. Many studies have shown the involvement of HuR in plethora of human diseases, which are often manifestations of impaired HuR-RNA interactions. However, the inherent complexities of highly flexible protein-RNA interactions have limited our understanding of the structural basis of HuR-RNA recognition. In this study, we dissect the underlying molecular mechanism of interaction between N-terminal tandem RNA-recognition motifs (tRRMs) of HuR and mRNA using molecular dynamics simulation. We have also explored the effect of point mutations (T90A, R97A and R136A) of three reported critical residues in HuR-mRNA binding specificity. Our findings show that N-terminal tRRMs exhibit conformational stability upon RNA binding. We further show that R136A and R97A mutants significantly lose their binding affinity owing to the loss of critical interactions with mRNA. This may be attributed to the larger domain rearrangements in the mutant complexes, especially the β2β3 loops in both the tRRMs, leading to unfavourable conformations and loss of binding affinity. We have identified critical binding residues in tRRMs of HuR, contributing favourable binding energy in mRNA recognition. This study contributes significantly to understand the molecular mechanism of RNA recognition by tandem RRMs and provides a platform to modulate binding affinities through mutations. This may further guide in future structure-based drug-therapies targeting impaired HuR-RNA interactions. Communicated by Ramaswamy H. Sarma

The cooperative binding of TDP-43 to GU-rich RNA repeats antagonizes TDP-43 aggregation.

TDP-43 is a nuclear RNA-binding protein that forms neuronal cytoplasmic inclusions in two major neurodegenerative diseases, ALS and FTLD. While the self-assembly of TDP-43 by its structured N-terminal and intrinsically disordered C-terminal domains has been widely studied, the mechanism by which mRNA preserves TDP-43 solubility in the nucleus has not been addressed. Here, we demonstrate that tandem RNA recognition motifs of TDP-43 bind to long GU-repeats in a cooperative manner through intermolecular interactions. Moreover, using mutants whose cooperativity is impaired, we found that the cooperative binding of TDP-43 to mRNA may be critical to maintain the solubility of TDP-43 in the nucleus and the miscibility of TDP-43 in cytoplasmic stress granules. We anticipate that the knowledge of a higher order assembly of TDP-43 on mRNA may clarify its role in intron processing and provide a means of interfering with the cytoplasmic aggregation of TDP-43.

A synthetic small molecule stalls pre-mRNA splicing by promoting an early-stage U2AF2-RNA complex

Dysregulated pre-mRNA splicing is an emerging Achilles heel of cancers and myelodysplasias. To expand the currently limited portfolio of small-molecule drug leads, we screened for chemical modulators of the U2AF complex, which nucleates spliceosome assembly and is mutated in myelodysplasias. A hit compound specifically enhances RNA binding by a U2AF2 subunit. Remarkably, the compound inhibits splicing of representative substrates and stalls spliceosome assembly at the stage of U2AF function. Computational docking, together with structure-guided mutagenesis, indicates that the compound bridges the tandem U2AF2 RNA recognition motifs via hydrophobic and electrostatic moieties. Cells expressing a cancer-associated U2AF1 mutant are preferentially killed by treatment with the compound. Altogether, our results highlight the potential of trapping early spliceosome assembly as an effective pharmacological means to manipulate pre-mRNA splicing. By extension, we suggest that stabilizing assembly intermediates may offer a useful approach for small-molecule inhibition of macromolecular machines.

VdNop12, containing two tandem RNA recognition motif domains, is a crucial factor for pathogenicity and cold adaption in Verticillium dahliae.

Previous studies have reported the ability of fungi to overwinter in soil or on crop debris under different environmental conditions, but how fungi adapt to chilling is still largely unknown. In this study, we have identified and characterized the RNA binding protein (RBP) (VdNop12) by screening an Agrobacterium tumefaciens-mediated transformation-mediated insertional mutational library of Verticillium dahliae. We determined that this protein was essential to the pathogen for virulence on cotton plants. VdNop12 contains two tandem RNA recognition motif domains, and its orthologs are widely distributed in filamentous fungi. Mutants produced by disruption of VdNop12 showed defects in vegetative growth, conidiation and cell wall integrity. The mutant also showed an increase in sensitivity to low temperature, as compared to the wildtype and complementation strains. Yeast complementation assay showed that VdNop12 could functionally restore the growth phenotype of ΔScNop12 mutant of Saccharomyces cerevisiae at 15°C. We demonstrated that the VdNop12 is localized in the nucleus, and its loss resulted in the downregulated expression of several genes related to cAMP-PKA and MAPK pathways in V. dahliae. Our results demonstrated a crucial role of RBPs in the regulation of morphology, cold adaption, and pathogenic development in V. dahliae.

A splice site-sensing conformational switch in U2AF2 is modulated by U2AF1 and its recurrent myelodysplasia-associated mutation.

An essential heterodimer of the U2AF1 and U2AF2 pre-mRNA splicing factors nucleates spliceosome assembly at polypyrimidine (Py) signals preceding the major class of 3′ splice sites. U2AF1 frequently acquires an S34F-encoding mutation among patients with myelodysplastic syndromes (MDS). The influence of the U2AF1 subunit and its S34F mutation on the U2AF2 conformations remains unknown. Here, we employ single molecule Förster resonance energy transfer (FRET) to determine the influence of wild-type or S34F-substituted U2AF1 on the conformational dynamics of U2AF2 and its splice site RNA complexes. In the absence of RNA, the U2AF1 subunit stabilizes a high FRET value, which by structure-guided mutagenesis corresponds to a closed conformation of the tandem U2AF2 RNA recognition motifs (RRMs). When the U2AF heterodimer is bound to a strong, uridine-rich splice site, U2AF2 switches to a lower FRET value characteristic of an open, side-by-side arrangement of the RRMs. Remarkably, the U2AF heterodimer binds weak, uridine-poor Py tracts as a mixture of closed and open U2AF2 conformations, which are modulated by the S34F mutation. Shifts between open and closed U2AF2 may underlie U2AF1-dependent splicing of degenerate Py tracts and contribute to a subset of S34F-dysregulated splicing events in MDS patients.

Early Metastable Assembly during the Stress-Induced Formation of Worm-like Amyloid Fibrils of Nucleic Acid Binding Domains of TDP-43.

TDP-43 protein travels between the cytosol and the nucleus to perform its nucleic acid binding functions through its two tandem RNA recognition motif domains (TDP-43tRRM). When exposed to various environmental stresses, it forms abnormal aggregates in the cytosol of neurons, which are the hallmarks of amyotrophic lateral sclerosis and other TDP-43 proteinopathies. However, the nature of early structural changes upon stress sensing and the consequent steps during the course of aggregation are not well understood. In this study, we show that under low-pH conditions, mimicking starvation stress, TDP-43tRRM undergoes a conformational opening reaction linked to the protonation of buried ionizable residues and grows into a metastable oligomeric assembly (called the "low-pH form" or the "L form"). In the L form, the protein molecules have disrupted tertiary structure, solvent-exposed hydrophobic patches, and mobile side chains but the native-like secondary structure remains intact. The L form structure is held by weak interactions and has a steep dependence on ionic strength. In the presence of as little as 15 mM KCl, it fully misfolds and further oligomerizes to form a β-sheet rich "β form" in at least two distinct steps. The β form has an ordered, stable structure that resembles worm-like amyloid fibrils. The unstructured regions of the protein gain structure during L ⇌ β conversion. Our results suggest that TDP-43tRRM could function as a stress sensor and support a recent model in which stress sensing during neurodegeneration occurs by assembly of proteins into metastable assemblies that are precursors to the solid aggregates.

ATP is a cryptic binder of TDP-43 RRM domains to enhance stability and inhibit ALS/AD-associated fibrillation

ATP is the universal energy currency for all cells but has cellular concentrations of 2–12 mM, much higher than required for its classic functions. RNA-recognition motif (RRM) constitutes one of the most abundant domains in eukaryotes and most heterogeneous nuclear ribonucleoproteins (hnRNP) contain RRM domains which not only mediate direct interactions with nucleic acids, but whose aggregation/fibrillation is the pathological hallmark of various human diseases. Here, by NMR and molecular docking, ATP has been decoded to bind TDP-43 two tandem RRM domains with distinctive types of interactions, thus resulting in diverse affinities. Most strikingly, the binding of ATP enhances thermodynamic stability of TDP-43 RRM domains and inhibits ALS-/AD-associated fibrillation. Together, ATP is a cryptic binder of RRM-containing proteins which generally safeguards functional phase separation from transforming into pathological aggregation/fibrillation associated with various diseases and ageing. Our study thus reveals a mechanism of ATP to control protein homeostasis by specific binding.

NMR Fragment-Based Screening against Tandem RNA Recognition Motifs of TDP-43.

The TDP-43 is originally a nuclear protein but translocates to the cytoplasm in the pathological condition. TDP-43, as an RNA-binding protein, consists of two RNA Recognition Motifs (RRM1 and RRM2). RRMs are known to involve both protein-nucleotide and protein-protein interactions and mediate the formation of stress granules. Thus, they assist the entire TDP-43 protein with participating in neurodegenerative and cancer diseases. Consequently, they are potential therapeutic targets. Protein-observed and ligand-observed nuclear magnetic resonance (NMR) spectroscopy were used to uncover the small molecule inhibitors against the tandem RRM of TDP-43. We identified three hits weakly binding the tandem RRMs using the ligand-observed NMR fragment-based screening. The binding topology of these hits is then depicted by chemical shift perturbations (CSP) of the 15N-labeled tandem RRM and RRM2, respectively, and modeled by the CSP-guided High Ambiguity Driven biomolecular DOCKing (HADDOCK). These hits mainly bind to the RRM2 domain, which suggests the druggability of the RRM2 domain of TDP-43. These hits also facilitate further studies regarding the hit-to-lead evolution against the TDP-43 RRM domain.

The Folding and Aggregation Energy Landscapes of Tethered RRM Domains of Human TDP-43 Are Coupled via a Metastable Molten Globule-like Oligomer.

Stress-induced misfolding and intraneuronal aggregation of the highly conserved nucleic acid binding protein TDP-43 (transactive response DNA binding protein 43 kDa) and its fragments have been implicated in amyotrophic lateral sclerosis and several other neurodegenerative diseases. However, the physicochemical mechanism of its misfolding from the functional folded state is poorly understood. TDP-43 is a four-domain protein and performs the essential nucleic acid binding function with the help of its two tandem RNA recognition motif domains naturally tethered by a linker (called here the tethered RRM domain of TDP-43 or TDP-43tRRM). Here, we show that the monomeric native form of TDP-43tRRM remains in a pH-dependent and reversible thermodynamic equilibrium with a protonated, nanosized, 40-meric form (the A form). Under the stress-like low-pH condition, the A form becomes predominantly populated. In the A form, protein molecules have restricted dynamics of surface side-chain residues but native-like secondary structure. This self-assembled form possesses a loosely packed core in which the intrinsically disordered and aggregation-prone regions are in the proximity. The A form is metastable and swiftly aggregates into a highly stable amyloid-like protofibrillar form (β form) mediated by the disorder-to-order transition of intrinsically disordered regions upon small environmental perturbations. Interestingly, the A form and the β form are not formed when TDP-43tRRM is bound to DNA, indicating that the nucleic acid binding regions of the protein participate in their formation. Our results reveal how the energy landscapes of folding and aggregation of TDP-43tRRM are coupled by a metastable molten-globule like oligomeric form and modulated by stress-like conditions.

Structural basis of IMP3 RRM12 recognition of RNA

The IMP family of RNA binding proteins, also named as insulin-like growth factor 2 (IGF2) mRNA-binding proteins (IGF2BPs), are highly conserved RNA regulators that are involved in many RNA processing stages, including mRNA stability, localization, and translation. There are three paralogs in the IMP family, IMP1-3, in mammals that all adopt the same domain arrangement with two RNA recognition motifs (RRM) in the N terminus and four KH domains in the C terminus. Here, we report the structure and biochemical characterization of IMP3 RRM12 and its complex with two short RNAs. These structures show that both RRM domains of IMP3 adopt the canonical RRM topology with two α-helices packed on an anti-parallel four stranded β-sheet. The spatial orientation of RRM1 to RRM2 is unique compared with other known tandem RRM structures. In the IMP3 RRM12 complex with RNA, only RRM1 is involved in RNA binding and recognizes a dinucleotide sequence.

Tandem hnRNP A1 RNA recognition motifs act in concert to repress the splicing of survival motor neuron exon 7.

HnRNP A1 regulates many alternative splicing events by the recognition of splicing silencer elements. Here, we provide the solution structures of its two RNA recognition motifs (RRMs) in complex with short RNA. In addition, we show by NMR that both RRMs of hnRNP A1 can bind simultaneously to a single bipartite motif of the human intronic splicing silencer ISS-N1, which controls survival of motor neuron exon 7 splicing. RRM2 binds to the upstream motif and RRM1 to the downstream motif. Combining the insights from the structure with in cell splicing assays we show that the architecture and organization of the two RRMs is essential to hnRNP A1 function. The disruption of the inter-RRM interaction or the loss of RNA binding capacity of either RRM impairs splicing repression by hnRNP A1. Furthermore, both binding sites within the ISS-N1 are important for splicing repression and their contributions are cumulative rather than synergistic.

Cell-type specific role of the RNA-binding protein, NONO, in the DNA double-strand break response in the mouse testes

The tandem RNA recognition motif protein, NONO, was previously identified as a candidate DNA double-strand break (DSB) repair factor in a biochemical screen for proteins with end-joining stimulatory activity. Subsequent work showed that NONO and its binding partner, SFPQ, have many of the properties expected for bona fide repair factors in cell-based assays. Their contribution to the DNA damage response in intact tissue in vivo has not, however, been demonstrated. Here we compare DNA damage sensitivity in the testes of wild-type mice versus mice bearing a null allele of the NONO homologue (Nono gt). In wild-type mice, NONO protein was present in Sertoli, peritubular myoid, and interstitial cells, with an increase in expression following induction of DNA damage. As expected for the product of an X-linked gene, NONO was not detected in germ cells. The Nono gt/0 mice had at most a mild testis developmental phenotype in the absence of genotoxic stress. However, following irradiation at sublethal, 2–4 Gy doses, Nono gt/0 mice displayed a number of indicators of radiosensitivity as compared to their wild-type counterparts. These included higher levels of persistent DSB repair foci, increased numbers of apoptotic cells in the seminiferous tubules, and partial degeneration of the blood-testis barrier. There was also an almost complete loss of germ cells at later times following irradiation, evidently arising as an indirect effect reflecting loss of stromal support. Results demonstrate a role for NONO protein in protection against direct and indirect biological effects of ionizing radiation in the whole animal.