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Folding intermediates mediate both protein folding and the misfolding and aggregation observed in human diseases, including amyotrophic lateral sclerosis (ALS), and are prime targets for therapeutic interventions. In this study, we identified the core nucleus of structure for a folding intermediate in the second RNA recognition motif (RRM2) of the ALS-linked RNA-binding protein, TDP-43 (TAR DNA-binding protein-43), using a combination of experimental and computational approaches. Urea equilibrium unfolding studies revealed that the RRM2 intermediate state consists of collapsed residual secondary structure localized to the N-terminal half of RRM2, while the C-terminus is largely disordered. Steered molecular dynamics simulations and mutagenesis studies yielded key stabilizing hydrophobic contacts that, when mutated to alanine, severely disrupt the overall fold of RRM2. In combination, these findings suggest a role for this RRM intermediate in normal TDP-43 function as well as serving as a template for misfolding and aggregation through the low stability and non-native secondary structure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

SRSF1 governs splicing of over 1500 mRNA transcripts. SRSF1 contains two RNA-recognition motifs (RRMs) and a C-terminal Arg/Ser-rich region (RS). It has been thought that SRSF1 RRMs exclusively recognize single-stranded exonic splicing enhancers, while RS lacks RNA-binding specificity. With our success in solving the insolubility problem of SRSF1, we can explore the unknown RNA-binding landscape of SRSF1. We find that SRSF1 RS prefers purine over pyrimidine. Moreover, SRSF1 binds to the G-quadruplex (GQ) from the ARPC2 mRNA, with both RRMs and RS being crucial. Our binding assays show that the traditional RNA-binding sites on the RRM tandem and the Arg in RS are responsible for GQ binding. Interestingly, our FRET and circular dichroism data reveal that SRSF1 unfolds the ARPC2 GQ, with RS leading unfolding and RRMs aiding. Our saturation transfer difference NMR results discover that Arg residues in SRSF1 RS interact with the guanine base but not other nucleobases, underscoring the uniqueness of the Arg/guanine interaction. Our luciferase assays confirm that SRSF1 can alleviate the inhibitory effect of GQ on gene expression in the cell. Given the prevalence of RNA GQ and SR proteins, our findings unveil unexplored SR protein functions with broad implications in RNA splicing and translation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Fused in sarcoma (FUS) is an abundant RNA-binding protein, which drives phase separation of cellular condensates and plays multiple roles in RNA regulation. The RNA-binding ability of FUS protein is crucial to its cellular function. Here, our molecular simulation study on the FUS-RNA complex provides atomic resolution insights into the observations from biochemical studies and also illuminates our understanding of molecular driving forces that mediate the structure, stability, and interaction of the RNA recognition motif (RRM) and RGG domains of FUS with a stem-loop junction RNA. We observe clear cooperativity and division of labor among the ordered (RRM) and disordered domains (RGG1 and RGG2) of FUS that leads to an organized and tighter RNA binding. Irrespective of the length of RGG2, the RGG2-RNA interaction is confined to the stem-loop junction and the proximal stem regions. On the other hand, the RGG1 interactions are primarily with the longer RNA stem. We find that the C terminus of RRM, which make up the "boundary residues" that connect the folded RRM with the long disordered RGG2 stretch of the protein, plays a critical role in FUS-RNA binding. Our study provides high-resolution molecular insights into the FUS-RNA interactions and forms the basis for understanding the molecular origins of full-length FUS interaction with RNA., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

RNA-binding proteins (RBPs) are structurally and functionally diverse macromolecules with significant involvement in several post-transcriptional gene regulatory processes and human diseases. RNA recognition motif (RRM) is one of the most abundant RNA-binding domains in human RBPs. The unique modular architecture of each RBP containing RRM is crucial for its diverse target recognition and function. Genome-wide study of these structurally conserved and functionally diverse domains can enhance our understanding of their functional implications. In this study, modular architecture of RRM containing RBPs in human proteome is identified and systematically analysed. We observe that 30% of human RBPs with RNA-binding function contain RRM in single or multiple repeats or with other domains with maximum of six repeats. Zinc-fingers are the most frequently co-occurring domain partner of RRMs. Human RRM containing RBPs mostly belong to RNA metabolism class of proteins and are significantly enriched in two functional pathways including spliceosome and mRNA surveillance. Various human diseases are associated with 18% of the RRM containing RBPs. Single RRM containing RBPs are highly enriched in disorder regions. Gene ontology (GO) molecular functions including poly(A), poly(U) and miRNA binding are highly depleted in RBPs with single RRM, indicating the significance of modular nature of RRMs in specific function. The current study reports all the possible domain architectures of RRM containing human RBPs and their functional enrichment. The idea of domain architecture, and how they confer specificity and new functionalities to RBPs, can help in re-designing of modular RRM containing RBPs with re-engineered function., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

TDP-43 is a major pathological protein in sporadic and familial amyotrophic lateral sclerosis (ALS) and mediates mRNA fate. TDP-43 dysfunction leads to causes progressive degeneration of motor neurons, the details of which remain elusive. Elucidation of the molecular mechanisms of RNA binding could enhance our understanding of this devastating disease. We observed the involvement of the glycine-rich (GR) region of TDP-43 in the initial recognition and binding of G-quadruplex (G4)-RNA in conjunction with its RNA recognition motifs (RRM). We performed a molecular dissection of these intramolecular RNA-binding modules in this study. We confirmed that the ALS-linked mutations in the GR region lead to alteration in the G4 structure. In contrast, amino acid substitutions in the GR region alter the protein structure but do not void the interaction with G4-RNA. Based on these observations, we concluded that the structural distortion of G4 caused by these mutations interferes with RRM recruitment and leads to TDP-43 dysfunction. This intramolecular organization between RRM and GR regions modulates the overall G4-binding properties., (© 2023. The Author(s).)

Loss of function of the RNA-binding protein FMRP causes fragile X syndrome, the most common inherited form of intellectual disability and autism spectrum disorders. FMRP is suggested to modulate synaptic plasticity by regulating the synthesis of proteins involved in neuronal and synaptic function; however, the mechanism underlying FMRP mRNA targeting specificity remains unclear. Intriguing recent work published in JBC by Scarpitti and colleagues identifies and characterizes a noncanonical RNA-binding domain that is required for FMRP-mediated translation regulation, shedding light on FMRP function., Competing Interests: Conflicts of interest The author declares that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

Recognition of single-stranded RNA (ssRNA) by RNA recognition motif (RRM) domains is an important class of protein-RNA interactions. Many such complexes were characterized using nuclear magnetic resonance (NMR) and/or X-ray crystallography techniques, revealing ensemble-averaged pictures of the bound states. However, it is becoming widely accepted that better understanding of protein-RNA interactions would be obtained from ensemble descriptions. Indeed, earlier molecular dynamics simulations of bound states indicated visible dynamics at the RNA-RRM interfaces. Here, we report the first atomistic simulation study of spontaneous binding of short RNA sequences to RRM domains of HuR and SRSF1 proteins. Using a millisecond-scale aggregate ensemble of unbiased simulations, we were able to observe a few dozen binding events. HuR RRM3 utilizes a pre-binding state to navigate the RNA sequence to its partially disordered bound state and then to dynamically scan its different binding registers. SRSF1 RRM2 binding is more straightforward but still multiple-pathway. The present study necessitated development of a goal-specific force field modification, scaling down the intramolecular van der Waals interactions of the RNA which also improves description of the RNA-RRM bound state. Our study opens up a new avenue for large-scale atomistic investigations of binding landscapes of protein-RNA complexes, and future perspectives of such research are discussed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (© 2022. The Author(s).)

Acinetobacter baumannii is a Gram-negative pathogen, known to acquire resistance to antibiotics used in the clinic. The RNA-binding proteome of this bacterium is poorly characterized, in particular for what concerns the proteins containing RNA Recognition Motif (RRM). Here, we browsed the A. baumannii proteome for homologous proteins to the human HuR(ELAVL1), an RNA binding protein containing three RRMs. We identified a unique locus that we called AB - Elavl , coding for a protein with a single RRM with an average of 34% identity to the first HuR RRM. We also widen the research to the genomes of all the bacteria, finding 227 entries in 12 bacterial phyla. Notably we observed a partial evolutionary divergence between the RNP1 and RNP2 conserved regions present in the prokaryotes in comparison to the metazoan consensus sequence. We checked the expression at the transcript and protein level, cloned the gene and expressed the recombinant protein. The X-ray and NMR structural characterization of the recombinant AB-Elavl revealed that the protein maintained the typical β 1 α 1 β 2 β 3 α 2 β 4 and three-dimensional organization of eukaryotic RRMs. The biochemical analyses showed that, although the RNP1 and RNP2 show differences, it can bind to AU-rich regions like the human HuR, but with less specificity and lower affinity. Therefore, we identified an RRM-containing RNA-binding protein actually expressed in A. baumannii .

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobe degeneration (FTLD) are two inter-related intractable diseases of motor neuron degeneration. Fused in sarcoma (FUS) is found in cytoplasmic accumulation of ALS and FTLD patients, which readily link the protein with the diseases. The RNA recognition motif (RRM) of FUS has the canonical α-β folds along with an unusual lysine-rich loop (KK-loop) between α1 and β2. This KK-loop is highly conserved among FET family proteins. Another contrasting feature of FUS RRM is the absence of critical binding residues, which are otherwise highly conserved in canonical RRMs. These residues in FUS RRM are Thr286, Glu336, Thr338, and Ser367, which are substitutions of lysine, phenylalanine, phenylalanine, and lysine, respectively, in other RRMs. Considering the importance of FUS in RNA regulation and metabolism, and its implication in ALS and FTLD, it is important to elucidate the underlying molecular mechanism of RNA recognition. In this study, we have performed molecular dynamics simulation with enhanced sampling to understand the conformational dynamics of noncanonical FUS RRM and its binding with RNA. We studied two sets of mutations: one with alanine mutation of KK-loop and another with KK-loop mutations along with critical binding residues mutated back to their canonical form. We find that concerted movement of KK-loop and loop between β2 and β3 facilitates the folding of the partner RNA, indicating an induced-fit mechanism of RNA binding. Flexibility of the RRM is highly restricted upon mutating the lysine residues of the KK-loop, resulting in weaker binding with the RNA. Our results also suggest that absence of the canonical residues in FUS RRM along with the KK-loop is equally important in regulating its binding dynamics. This study provides a significant structural insight into the binding of FUS RRM with its cognate RNA, which may further help in designing potential drugs targeting noncanonical RNA recognition., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Cyp33 is an essential human cyclophilin prolyl isomerase that plays myriad roles in splicing and chromatin remodeling. In addition to a canonical cyclophilin (Cyp) domain, Cyp33 contains an RNA-recognition motif (RRM) domain, and RNA-binding triggers proline isomerase activity. One prominent role for Cyp33 is through a direct interaction with the mixed lineage leukemia protein 1 (MLL1, also known as KMT2A) complex, which is a histone methyltransferase that serves as a global regulator of human transcription. MLL activity is regulated by Cyp33, which isomerizes a key proline in the linker between the PHD3 and Bromo domains of MLL1, acting as a switch between gene activation and repression. The direct interaction between MLL1 and Cyp33 is critical, as deletion of the MLL1-PHD3 domain responsible for this interaction results in oncogenesis. The Cyp33 RRM is central to these activities, as it binds both the PHD3 domain and RNA. To better understand how RNA binding drives the action of Cyp33, we performed RNA-SELEX against full-length Cyp33 accompanied by deep sequencing. We have identified an enriched Cyp33 binding motif (AAUAAUAA) broadly represented in the cellular RNA pool as well as tightly binding RNA aptamers with affinities comparable and competitive with the Cyp33 MLL1-PHD3 interaction. RNA binding extends beyond the canonical RRM domain, but not to the Cyp domain, suggesting an indirect mechanism of interaction. NMR chemical shift mapping confirms an overlapping, but not identical, interface on Cyp33 for RNA and PHD3 binding. This finding suggests RNA can disrupt the gene repressive Cyp33-MLL1 complex providing another layer of regulation for chromatin remodeling by MLL1., Competing Interests: The authors have declared that no competing interests exist.

The human prototypical SR protein SRSF1 is an oncoprotein that contains two RRMs and plays a pivotal role in RNA metabolism. We determined the structure of the RRM1 bound to RNA and found that the domain binds preferentially to a CN motif (N is for any nucleotide). Based on this solution structure, we engineered a protein containing a single glutamate to asparagine mutation (E87N), which gains the ability to bind to uridines and thereby activates SMN exon7 inclusion, a strategy that is used to cure spinal muscular atrophy. Finally, we revealed that the flexible inter-RRM linker of SRSF1 allows RRM1 to bind RNA on both sides of RRM2 binding site. Besides revealing an unexpected bimodal mode of interaction of SRSF1 with RNA, which will be of interest to design new therapeutic strategies, this study brings a new perspective on the mode of action of SRSF1 in cells.

La is an abundant phosphoprotein that protects polymerase III transcripts from 3'-5' exonucleolytic degradation and facilitates their folding. Consisting of the evolutionary conserved La motif (LAM) and two consecutive RNA Recognition Motifs (RRMs), La was also found to bind additional RNA transcripts or RNA domains like internal ribosome entry site (IRES), through sequence-independent binding modes which are poorly understood. Although it has been reported overexpressed in certain cancer types and depletion of its expression sensitizes cancer cells to certain chemotherapeutic agents, its role in cancer remains essentially uncharacterized. Herein, we study the effects of La overexpression in A549 lung adenocarcinoma cells, which leads to increased cell proliferation and motility. Expression profiling of several transcription and translation factors indicated that La overexpression leads to downregulation of global translation through hypophosphorylation of 4E-BPs and upregulation of IRES-mediated translation. Moreover, analysis of La localization after nutrition deprivation of the transfected cells showed a normal distribution in the nucleus and nucleoli. Although the RNA binding capacity of La has been primarily linked to the synergy between the conserved LAM and RRM1 domains which act as a module, we show that recombinant stand-alone LAM can specifically bind a pre-tRNA ligand, based on binding experiments combined with NMR analysis. We propose that LAM RNA binding properties could support the expanding and diverse RNA ligand repertoire of La, thus promoting its modulatory role, both under normal and pathogenic conditions like cancer., Competing Interests: Conflict of interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

hnRNPA2 is a major component of mRNA transport granules in oligodendrocytes and neurons. However, the structural details of how hnRNPA2 binds the A2 recognition element (A2RE) and if this sequence stimulates granule formation by enhancing phase separation of hnRNPA2 has not yet been studied. Using solution NMR and biophysical studies, we find that each of the two individual RRMs retain the domain structure observed in complex with RNA but are not rigidly confined (i.e. they move independently) in solution in the absence of RNA. hnRNPA2 RRMs bind the minimal rA2RE11 weakly but at least, and most likely, two hnRNPA2 molecules are able to simultaneously bind the longer 21mer myelin basic protein A2RE. Upon binding of the RNA, NMR chemical shift deviations are observed in both RRMs, suggesting both play a role in binding the A2RE11. Interestingly, addition of short A2RE RNAs or longer RNAs containing this sequence completely prevents in vitro phase separation of full-length hnRNPA2 and aggregation of the disease-associated mutants. These findings suggest that RRM interactions with specific recognition sequences alone do not account for nucleating granule formation, consistent with models where multivalent protein:RNA and protein:protein contacts form across many sites in granule proteins and long RNA transcripts., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Fragile X syndrome is the most common form of inherited intellectual disability and is caused by a deficiency of the fragile X mental retardation protein (FMRP) in neurons. FMRP regulates the translation of numerous mRNAs within dendritic synapses, but how FMRP recognizes these target mRNAs remains unknown. FMRP has KH0, KH1, KH2, and RGG domains, which are thought to bind to specific RNA recognition elements (RREs). Several studies used high-throughput methods to identify various RREs in mRNAs that FMRP may bind to in vivo. However, there is little overlap in the mRNA targets identified by each study, suggesting that the RNA-binding specificity of FMRP is still unknown. To determine the specificity of FMRP for the RREs, we performed quantitative in vitroRNA binding studies with various constructs of human FMRP. Unexpectedly, our studies show that the KH domains do not bind to the previously identified RREs. To further investigate the RNA-binding specificity of FMRP, we developed a new method called Motif Identification by Analysis of Simple sequences (MIDAS) to identify single-stranded RNA sequences bound by KH domains. We find that the FMRP KH0, KH1, and KH2 domains bind weakly to the single-stranded RNA sequences suggesting that they may have evolved to bind more complex RNA structures. Additionally, we find that the RGG motif of human FMRP binds with a high affinity to an RNAG-quadruplex structure that lacks single-stranded loops, double-stranded stems, or junctions., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

G3BP RNA-binding proteins are important components of stress granules (SGs). Here, we analyze the role of the Drosophila G3BP Rasputin (RIN) in unstressed cells, where RIN is not SG associated. Immunoprecipitation followed by microarray analysis identifies over 550 mRNAs that copurify with RIN. The mRNAs found in SGs are long and translationally silent. In contrast, we find that RIN-bound mRNAs, which encode core components of the transcription, splicing, and translation machinery, are short, stable, and highly translated. We show that RIN is associated with polysomes and provide evidence for a direct role for RIN and its human homologs in stabilizing and upregulating the translation of their target mRNAs. We propose that when cells are stressed, the resulting incorporation of RIN/G3BPs into SGs sequesters them away from their short target mRNAs. This would downregulate the expression of these transcripts, even though they are not incorporated into stress granules., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

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-43 tRRM ). 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-43 tRRM 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-43 tRRM 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.

The autoantigen La protein is an important component of telomerase and a predominantly nuclear phosphoprotein. As a telomerase subunit, La protein associates with the telomerase ribonucleoprotein and influences telomere length. In the reverse transcription, La protein stimulates enzymatic activity and increases repeated addition processivity of telomerase. As nuclear phosphoprotein, La protein binds the 3' poly(U)-rich elements of nascent RNA polymerase III transcripts to facilitate its correct folding and maturation. In this work, we identified a La protein homolog (TbLa) from Trypanosoma brucei (T. brucei). We revealed that TbLa interacts with ribosome-associated protein P34/P37, 40S ribosomal protein SA, and 60S ribosomal subunit L5 in T. brucei. In the interactions between TbLa protein and (P34/P37)/L5/SA, RNA recognition motif (RRM) domain of TbLa was indicated to make the major contribution to the processes. We determined the solution structure of TbLa RRM domain. NMR chemical shift perturbations revealed that the positively charged RNA-binding pocket of TbLa RRM domain is responsible for its interaction with ribosomal and ribosome-associated proteins P37/L5/SA. Furthermore, depletion of TbLa affected the maturation process of 5S rRNA and ribosomal assembly, suggesting TbLa protein might play a significant role in the ribosomal biogenesis pathway in T. brucei. Taken together, our results provide a novel insight and structural basis for better understanding the roles of TbLa and RRM domain in ribosomal biogenesis in T. brucei. DATABASE: Structural data are available in the PDB under the accession number 5ZUH., (© 2019 Federation of European Biochemical Societies.)

Hepatitis B virus (HBV) infects the liver and is a key risk factor for hepatocellular carcinoma. Identification of host factors that support viral replication is important to understand mechanisms of viral replication and to develop new therapeutic strategies. We identified TARDBP as a host factor that regulates HBV. Silencing or knocking out the protein in HBV infected cells severely impaired the production of viral replicative intermediates, mRNAs, proteins, and virions, whereas ectopic expression of TARDBP rescued production of these products. Mechanistically, we found that the protein binds to the HBV core promoter, as shown by chromatin precipitation as well as mutagenesis and protein-DNA interaction assays. Using LC-MS/MS analysis, we also found that TARDBP binds to a number of other proteins known to support the HBV life cycle, including NPM1, PARP1, Hsp90, HNRNPC, SFPQ, PTBP1, HNRNPK, and PUF60. Interestingly, given its key role as a regulator of RNA splicing, we found that TARDBP has an inhibitory role on pregenomic RNA splicing, which might help the virus to export its non-canonical RNAs from the nucleus without being subjected to unwanted splicing, even though mRNA nuclear export is normally closely tied to RNA splicing. Taken together, our results demonstrate that TARDBP is involved in multiple steps of HBV replication via binding to both HBV DNA and RNA. The protein's broad interactome suggests that TARDBP may function as part of a RNA-binding scaffold involved in HBV replication and that the interaction between these proteins might be a target for development of anti-HBV drugs.

Kinetoplastid RNA (kRNA) editing takes place in the mitochondria of kinetoplastid protists and creates translatable mRNAs by uridine insertion/deletion. Extensively edited (pan-edited) transcripts contain quadruplex forming guanine stretches, which must be remodeled to promote uridine insertion/deletion. Here we show that the RRM domain of the essential kRNA-editing factor TbRGG2 binds poly(G) and poly(U) RNA and can unfold both. A region C-terminal to the RRM mediates TbRGG2 dimerization, enhancing RNA binding. A RRM-U4 RNA structure reveals a unique RNA-binding mechanism in which the two RRMs of the dimer employ aromatic residues outside the canonical RRM RNA-binding motifs to encase and wrench open the RNA, while backbone atoms specify the uridine bases. Notably, poly(G) RNA is bound via a different binding surface. Thus, these data indicate that TbRGG2 RRM can bind and remodel several RNA substrates suggesting how it might play multiple roles in the kRNA editing process., (© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Human antigen R (HuR) is a key regulator of cellular mRNAs containing adenylate/uridylate-rich elements (AU-rich elements; AREs). These are a major class of cis elements within 3' untranslated regions, targeting these mRNAs for rapid degradation. HuR contains three RNA recognition motifs (RRMs): a tandem RRM1 and 2, followed by a flexible linker and a C-terminal RRM3. While RRM1 and 2 are structurally characterized, little is known about RRM3. Here we present a 1.9-Å-resolution crystal structure of RRM3 bound to different ARE motifs. This structure together with biophysical methods and cell-culture assays revealed the mechanism of RRM3 ARE recognition and dimerization. While multiple RNA motifs can be bound, recognition of the canonical AUUUA pentameric motif is possible by binding to two registers. Additionally, RRM3 forms homodimers to increase its RNA binding affinity. Finally, although HuR stabilizes ARE-containing RNAs, we found that RRM3 counteracts this effect, as shown in a cell-based ARE reporter assay and by qPCR with native HuR mRNA targets containing multiple AUUUA motifs, possibly by competing with RRM12., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Alternative splicing confers a complexity to the mRNA landscape of apicomplexans, resulting in a high proteomic diversity. The Plasmodium falciparum Ser/Arg-rich protein 1 (PfSR1) is the first protein to be confirmed as an alternative splicing factor in this class of parasitic protists [1]. A recent study [2] showed a purine bias in RNA binding among cognate RNA substrates of PfSR1. Here, we have investigated the role played by the amino-terminal RNA recognition motif (RRM1) of PfSR1 from the solution structure of its complex with ACAUCA RNA hexamer to understand how its mechanism of RNA recognition compares to human orthologs and to the C-terminal RRM. RNA binding by RRM1 is mediated through specific recognition of a cytosine base situated 5' of one or more pyrimidine bases by a conserved tyrosine residue on β 1 and a glutamate residue on the β 4 strand. Affinity is conferred through insertion of a 3' pyrimidine into a positively charged pocket. Retention of fast dynamics and ITC binding constants indicate the complex to be of moderate affinity. Using calorimetry and mapping of NMR chemical shift perturbations, we have also ascertained the purine preference of PfSR1 to be a property of the carboxy terminal pseudo-RRM (RRM2), which binds RNA non-canonically and with greater affinity compared to RRM1. Our findings show conclusive evidence of complementary RNA sequence recognition by the two RRMs, which may potentially aid PfSR1 in binding RNA with a high sequence specificity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Smk1 is a mitogen-activated protein kinase (MAPK) family member in the yeast Saccharomyces cerevisiae that controls the postmeiotic program of spore formation. Ssp2 is a meiosis-specific protein that activates Smk1 and triggers the autophosphorylation of its activation loop. A fragment of Ssp2 that is sufficient to activate Smk1 contains two segments that resemble RNA recognition motifs (RRMs). Mutations in either of these motifs eliminated Ssp2's ability to activate Smk1. In contrast, deletions and insertions within the segment linking the RRM-like motifs only partially reduced the activity of Ssp2. Moreover, when the two RRM-like motifs were expressed as separate proteins in bacteria, they activated Smk1. We also find that both motifs can be cross-linked to Smk1 and that at least one of the motifs binds near the ATP-binding pocket of the MAPK. These findings demonstrate that motifs related to RRMs can directly activate protein kinases.

Cleavage and polyadenylation (C/P) of mRNA is an important cellular process that promotes increased diversity of mRNA isoforms and could change their stability in different cell types. The cleavage stimulation factor (CstF) complex, part of the C/P machinery, binds to U- and GU-rich sequences located downstream from the cleavage site through its RNA-binding subunit, CstF-64. Less is known about the function of the other two subunits of CstF, CstF-77 and CstF-50. Here, we show that the carboxy-terminus of CstF-77 plays a previously unrecognized role in enhancing C/P by altering how the RNA recognition motif (RRM) of CstF-64 binds RNA. In support of this finding, we also show that CstF-64 relies on CstF-77 to be transported to the nucleus; excess CstF-64 localizes to the cytoplasm, possibly via interaction with cytoplasmic RNAs. Reverse genetics and nuclear magnetic resonance studies of recombinant CstF-64 (RRM-Hinge) and CstF-77 (monkeytail-carboxy-terminal domain) indicate that the last 30 amino acids of CstF-77 increases the stability of the RRM, thus altering the affinity of the complex for RNA. These results provide new insights into the mechanism by which CstF regulates the location of the RNA cleavage site during C/P.

The RNA recognition motif (RRM) is the most common RNA binding domain across eukaryotic proteins. It is therefore of great value to engineer its specificity to target RNAs of arbitrary sequence. This was recently achieved for the RRM in Rbfox protein, where four mutations R118D, E147R, N151S, and E152T were designed to target the precursor to the oncogenic miRNA 21. Here, we used a variety of molecular dynamics-based approaches to predict specific interactions at the binding interface. Overall, we have run approximately 50 microseconds of enhanced sampling and plain molecular dynamics simulations on the engineered complex as well as on the wild-type Rbfox·pre-miRNA 20b from which the mutated systems were designed. Comparison with the available NMR data on the wild type molecules (protein, RNA, and their complex) served to establish the accuracy of the calculations. Free energy calculations suggest that further improvements in affinity and selectivity are achieved by the S151T replacement., Competing Interests: The authors have declared that no competing interests exist.

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., (© 2018 Jia et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Long noncoding RNAs (lncRNA) have been associated with various types of cancer; however, the precise role of many lncRNAs in tumorigenesis remains elusive. Here we demonstrate that the cytosolic lncRNA P53RRA is downregulated in cancers and functions as a tumor suppressor by inhibiting cancer progression. Chromatin remodeling proteins LSH and Cfp1 silenced or increased P53RRA expression, respectively. P53RRA bound Ras GTPase-activating protein-binding protein 1 (G3BP1) using nucleotides 1 and 871 of P53RRA and the RRM interaction domain of G3BP1 (aa 177-466). The cytosolic P53RRA-G3BP1 interaction displaced p53 from a G3BP1 complex, resulting in greater p53 retention in the nucleus, which led to cell-cycle arrest, apoptosis, and ferroptosis. P53RRA promoted ferroptosis and apoptosis by affecting transcription of several metabolic genes. Low P53RRA expression significantly correlated with poor survival in patients with breast and lung cancers harboring wild-type p53. These data show that lncRNAs can directly interact with the functional domain of signaling proteins in the cytoplasm, thus regulating p53 modulators to suppress cancer progression. Significance: A cytosolic lncRNA functions as a tumor suppressor by activating the p53 pathway. Cancer Res; 78(13); 3484-96. ©2018 AACR ., (©2018 American Association for Cancer Research.)

One of the multitasking proteins, transactive response DNA-binding protein 43 (tdp43) plays a key role in RNA regulation and the two pathogenic mutations such as D169G and K263E, located at the RNA Recognition Motif (RRM) of tdp43, are reported to cause neurological disorders such as Amyotrophic Lateral Sclerosis and FrontoTemporal Lobar Degeneration. As the exploration of the proteinopathy demands both structural and functional characterizations of mutants, a comparative analysis on the wild type and mutant tdp43 (D169G and K263E) and their complexes with RNA has been performed using computational approaches. Molecular dynamics simulations revealed comparatively stable mutant structures compared to wild type tdp43. Both mutants show lesser binding affinity toward RNA molecule when compared to the wild type tdp43. Some of the observed features, including the increased solvent-accessible surface area, conformational flexibility as well as unfolding of tdp43, and the altered RNA conformation in tp43-RNA complex, reveal the susceptibility of these mutants to induce conformational changes in tdp43 for a possible aggregation in the cytoplasm. Particularly, the enhanced aggregation propensity of both mutants also evidences the higher probability of cytoplasmic aggregation of tdp43 mutants. Hence, the present analysis highlighting the structural and functional aspects of wild and mutant tdp43 will form the basis to gain insight into the proteinopathy of tdp43 and the related structure-based drug discovery. Thus, tdp43 can be used as target to develop novel therapeutic approaches or drug designing.

RRM, or RNA-recognition motif, domains are the largest class of single-stranded RNA binding domains in the human proteome and play important roles in RNA processing, splicing, export, stability, packaging, and degradation. Using a current database of post-translational modifications (PTMs), ProteomeScout, we found that RRM domains are also one of the most heavily modified domains in the human proteome. Here, we present two interesting findings about RRM domain modifications, found by mapping known PTMs onto RRM domain alignments and structures. First, we find significant overlap of ubiquitination and acetylation within RRM domains, suggesting the possibility for ubiquitination-acetylation crosstalk. Additionally, an analysis of quantitative study of ubiquitination changes in response to proteasome inhibition highlights the uniqueness of RRM domain ubiquitination - RRM domain ubiquitination decreases in response to proteasome inhibition, whereas the majority of sites increase. Second, we found conservation of tyrosine phosphorylation within the RNP1 and RNP2 consensus sequences, which coordinate RNA binding - suggesting a possible role for regulation of RNA binding by tyrosine kinase signaling. These observations suggest there are unique regulatory mechanisms of RRM function that have yet to be uncovered and that the RRM domain represents a model system for further studies on understanding PTM crosstalk., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The Fox-1 RNA recognition motif (RRM) domain is an important member of the RRM protein family. We report a 1.8 Å X-ray structure of the free Fox-1 containing six distinct monomers. We use this and the nuclear magnetic resonance (NMR) structure of the Fox-1 protein/RNA complex for molecular dynamics (MD) analyses of the structured hydration. The individual monomers of the X-ray structure show diverse hydration patterns, however, MD excellently reproduces the most occupied hydration sites. Simulations of the protein/RNA complex show hydration consistent with the isolated protein complemented by hydration sites specific to the protein/RNA interface. MD predicts intricate hydration sites with water-binding times extending up to hundreds of nanoseconds. We characterize two of them using NMR spectroscopy, RNA binding with switchSENSE and free-energy calculations of mutant proteins. Both hydration sites are experimentally confirmed and their abolishment reduces the binding free-energy. A quantitative agreement between theory and experiment is achieved for the S155A substitution but not for the S122A mutant. The S155 hydration site is evolutionarily conserved within the RRM domains. In conclusion, MD is an effective tool for predicting and interpreting the hydration patterns of protein/RNA complexes. Hydration is not easily detectable in NMR experiments but can affect stability of protein/RNA complexes., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Å resolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expression., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Every month the editors of Cell Chemical Biology bring you highlights of the most recent chemical biology literature that impressed them with creativity and potential for follow up work. Our August 2016 selection includes a description of hydrogels with self-tunable stiffness that are used to profile lipid metabolites during stems cell differentiation, a look at whether we can find a drug repurposing solution to Zika virus infection, and an engineered RNA recognition motif (RRM)., (Copyright © 2016. Published by Elsevier Ltd.)

RNA recognition motif (RRM) proteins represent an abundant class of proteins playing key roles in RNA biology. We present a joint atomistic molecular dynamics (MD) and experimental study of two RRM-containing proteins bound with their single-stranded target RNAs, namely the Fox-1 and SRSF1 complexes. The simulations are used in conjunction with NMR spectroscopy to interpret and expand the available structural data. We accumulate more than 50 μs of simulations and show that the MD method is robust enough to reliably describe the structural dynamics of the RRM-RNA complexes. The simulations predict unanticipated specific participation of Arg142 at the protein-RNA interface of the SRFS1 complex, which is subsequently confirmed by NMR and ITC measurements. Several segments of the protein-RNA interface may involve competition between dynamical local substates rather than firmly formed interactions, which is indirectly consistent with the primary NMR data. We demonstrate that the simulations can be used to interpret the NMR atomistic models and can provide qualified predictions. Finally, we propose a protocol for 'MD-adapted structure ensemble' as a way to integrate the simulation predictions and expand upon the deposited NMR structures. Unbiased μs-scale atomistic MD could become a technique routinely complementing the NMR measurements of protein-RNA complexes., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)