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.

Unearthing a novel function of SRSF1 in binding and unfolding of RNA G-quadruplexes.

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.

RNA-Binding Peptides Inspired by the RNA Recognition Motif.

ß-Hairpin peptides with RNA-binding sequences mimicking the central two ß-strands of the RNA recognition motif (RRM) protein domain have been observed to bind in a 2:1 fashion to a series of RNA homooligonucleotides in aqueous solution (PBS buffer, pH 7.40) with binding energies (-27 to -35 kJ mol-1) similar to those of full-size protein RRMs. The peptides display mild selectivities with respect to the binding of the different homooligomers. Binding studies in 500 mM magnesium chloride suggest that the complex formation is not predominantly driven by Coulombic attraction. These peptides represent a starting point for further studies of non-Coulombic binding of RNA by peptides and proteins, which is important in the context of contemporary biology, potential therapeutic applications, and prebiotic peptide-RNA interactions.

RRM1 and PAB domains of translation initiation factor eIF4G (Tif4631p) play a crucial role in the nuclear degradation of export-defective mRNAs in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the CBC-Tif4631p-dependent exosomal targeting (CTEXT) complex consisting of Cbc1/2p, Tif4631p and Upf3p promotes the exosomal degradation of aberrantly long 3'-extended, export-defective transcripts and a small group of normal (termed 'special') mRNAs. We carried out a systematic analysis of all previously characterized functional domains of the major CTEXT component Tif4631p by deleting each of them and interrogating their involvement in the nuclear surveillance of abnormally long 3'-extended and export-defective messages. Our analyses show that the N-terminal RNA recognition motif 1 (RRM1) and poly(A)-binding protein (PAB) domains of Tif4631p, spanning amino acid residues, 1-82 and 188-299 in its primary structure, respectively, play a crucial role in degrading these aberrant messages. Furthermore, the physical association of the nuclear exosome with the altered/variant CTEXT complex harboring any of the mutant Tif4631p proteins lacking either the RRM1 or PAB domain becomes abolished. This finding indicates that the association between CTEXT and the exosome is accomplished via interaction between these Tif4631p domains with the major exosome component, Rrp6p. Abolition of interaction between altered CTEXT (harboring any of the RRM1/PAB-deleted versions of Tif4631p) and the exosome further leads to the impaired recruitment of the RNA targets to the Rrp6p subunit of the exosome carried out by the RRM1/PAB domains of Tif4631p. When analyzing the Tif4631p-interacting proteins, we identified a DEAD-box RNA helicase (Dbp2p), as an interacting partner that turned out to be a previously unknown component of CTEXT. The present study provides a more complete description of the CTEXT complex and offers insight into the functional relationship of this complex with the nuclear exosome.

Genome-wide identification of RNA recognition motif (RRM1) in Brassica rapa and functional analysis of RNA-binding protein (BrRBP) under low-temperature stress.

BACKGROUND: The RNA recognition motif (RRM) is primarily engaged in the processing of mRNA and rRNA following gene transcription as well as the regulation of RNA transport; it is critical in preserving RNA stability. RESULTS: In this study, we identified 102 members of the RRM1 gene family in Brassica rapa, which were dispersed across 10 chromosomes with the ninth chromosome being the most extensively distributed. The RRM1 gene family members of Brassica rapa and Arabidopsis thaliana were grouped into 14 subclades (I-XIV) using phylogenetic analysis. Moreover, the results of transcriptome analysis and RT-qPCR indicated that the expression of Brapa05T000840 was upregulated in the cultivars 'Longyou 7' and 'Longyou 99' following exposure to cold stress at a temperature of 4 °C for 24 h. The levels of expression in the leaves and growth cones of the 'Longyou 7' variety were found to be significantly higher than those observed in the 'Longyou 99' variety under conditions of low temperature and NaCl stress. It illustrates the involvement of the RRM1 gene in the physiological response to both low temperature and salt stress. In addition, it was observed that the survival rate of transgenic BrRBP (Brapa05T000840) Arabidopsis thaliana plants was notably higher compared to that of wild-type plants when subjected to varying durations of low temperature treatment. Furthermore, the expression of the BrRBP gene in transgenic plants exhibited an upward trend as the duration of low temperature treatment increased, reaching its peak at 24 h. The in-vivo enzymatic activity of reactive oxygen species-scavenging enzymes were found to be significantly elevated in comparison to wild-type plants, suggesting that the BrRBP gene may enhance the cold tolerance of Arabidopsis thaliana. CONCLUSIONS: This study offers a significant foundation for comprehending the regulation mechanism of the RRM1 gene family in winter Brassica rapa subjected to cold stress, as well as for finding key genes associated with cold resistance.

"Boundary residues" between the folded RNA recognition motif and disordered RGG domains are critical for FUS-RNA binding.

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.

FUS RRM regulates poly(ADP-ribose) levels after transcriptional arrest and PARP-1 activation on DNA damage.

PARP-1 activation at DNA damage sites leads to the synthesis of long poly(ADP-ribose) (PAR) chains, which serve as a signal for DNA repair. Here we show that FUS, an RNA-binding protein, is specifically directed to PAR through its RNA recognition motif (RRM) to increase PAR synthesis by PARP-1 in HeLa cells after genotoxic stress. Using a structural approach, we also identify specific residues located in the FUS RRM, which can be PARylated by PARP-1 to control the level of PAR synthesis. Based on the results of this work, we propose a model in which, following a transcriptional arrest that releases FUS from nascent mRNA, FUS can be recruited by PARP-1 activated by DNA damage to stimulate PAR synthesis. We anticipate that this model offers new perspectives to understand the role of FET proteins in cancers and in certain neurodegenerative diseases such as amyotrophic lateral sclerosis.

In silico identification, characterization, and expression analysis of RNA recognition motif (RRM) containing RNA-binding proteins in Aedes aegypti.

RNA-binding proteins (RBPs) are the proteins that bind RNAs and regulate their functioning. RBPs in mosquitoes are gaining attention due to their ability to bind flaviviruses and regulate their replication and transmission. Despite their relevance, RBPs in mosquitoes are not explored much. In this study, we screened the whole genome of Aedes aegypti, the primary vector of several pathogenic viruses, and identified the proteins containing RNA recognition motif (RRM), the most abundant protein domain in eukaryotes. Using several in silico strategies, a total of 135 RRM-containing RBPs were identified in Ae. aegypti. The proteins were characterized based on their available annotations and the sequence similarity with Drosophila melanogaster. Ae. aegypti RRM-containing RBPs included serine/arginine-rich (SR) proteins, polyadenylate-binding proteins (PABP), heteronuclear ribonucleoproteins (hnRNP), small nuclear ribonucleoproteins (snRNP), splicing factors, eukaryotic initiation factors, transformers, and nucleolysins. Phylogenetic analysis revealed that the proteins and the domain organization are conserved among Ae. aegypti, Bombyx mori, and Drosophila melanogaster. However, the gene length and the intron-exon organization varied across the insect species. Expression analysis of the genes encoding RBPs using publicly available RNA sequencing data for different developmental time points of the mosquito life cycle starting from the ovary and eggs up to the adults revealed stage-specific expression with several genes preferentially expressed in early embryonic stages and blood-fed female ovaries. This is the first database for the Ae. aegypti RBPs that can serve as the reference base for future investigations. Stage-specific genes can be further explored to determine their role in mosquito growth and development with a focus on developing novel mosquito control strategies.

Monovalent Ionic Atmosphere Modulates the Selection of Suboptimal RNA Sequences by Splicing Factors' RNA Recognition Motifs.

The U2AF2 splicing factor is involved in the RNA recognition of the pre-mRNA poly-pyrimidine signaling sequence. This protein contains two RRM domains connected by a flexible linker, which ensure the preferential selection of a poly-uridine sequence over a poly-cytosine one. In this work, all-atom simulations provide insights into the U2AF2 recognition mechanism and on the features underlying its selectivity. Our outcomes show that U2AF2's RNA recognition is driven by cooperative events modulated by RNA-protein and RNA-ion interactions. Stunningly, monovalent ions contribute to mediating the binding of the weakly binding polyC strand, thus contributing to the selection of suboptimal poly-pyrimidine tracts. This finding broadens our understanding of the diverse traits tuning splicing factors' selectivity and adaptability to precisely handle and process diverse pre-mRNA sequences.

saRNA vaccine expressing membrane-anchored RBD elicits broad and durable immunity against SARS-CoV-2 variants of concern.

Several vaccines have been widely used to counteract the global pandemic caused by SARS-CoV-2. However, due to the rapid emergence of SARS-CoV-2 variants of concern (VOCs), further development of vaccines that confer broad and longer-lasting protection against emerging VOCs are needed. Here, we report the immunological characteristics of a self-amplifying RNA (saRNA) vaccine expressing the SARS-CoV-2 Spike (S) receptor binding domain (RBD), which is membrane-anchored by fusing with an N-terminal signal sequence and a C-terminal transmembrane domain (RBD-TM). Immunization with saRNA RBD-TM delivered in lipid nanoparticles (LNP) efficiently induces T-cell and B-cell responses in non-human primates (NHPs). In addition, immunized hamsters and NHPs are protected against SARS-CoV-2 challenge. Importantly, RBD-specific antibodies against VOCs are maintained for at least 12 months in NHPs. These findings suggest that this saRNA platform expressing RBD-TM will be a useful vaccine candidate inducing durable immunity against emerging SARS-CoV-2 strains.

ALS-linked TDP-43 mutations interfere with the recruitment of RNA recognition motifs to G-quadruplex RNA.

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.

The RRM-mediated RNA binding activity in T. brucei RAP1 is essential for VSG monoallelic expression.

Trypanosoma brucei is a protozoan parasite that causes human African trypanosomiasis. Its major surface antigen VSG is expressed from subtelomeric loci in a strictly monoallelic manner. We previously showed that the telomere protein TbRAP1 binds dsDNA through its 737RKRRR741 patch to silence VSGs globally. How TbRAP1 permits expression of the single active VSG is unknown. Through NMR structural analysis, we unexpectedly identify an RNA Recognition Motif (RRM) in TbRAP1, which is unprecedented for RAP1 homologs. Assisted by the 737RKRRR741 patch, TbRAP1 RRM recognizes consensus sequences of VSG 3'UTRs in vitro and binds the active VSG RNA in vivo. Mutating conserved RRM residues abolishes the RNA binding activity, significantly decreases the active VSG RNA level, and derepresses silent VSGs. The competition between TbRAP1's RNA and dsDNA binding activities suggests a VSG monoallelic expression mechanism in which the active VSG's abundant RNA antagonizes TbRAP1's silencing effect, thereby sustaining its full-level expression.

Modular architecture and functional annotation of human RNA-binding proteins containing RNA recognition motif.

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.

Deciphering the RRM-RNA recognition code: A computational analysis.

RNA recognition motifs (RRM) are the most prevalent class of RNA binding domains in eucaryotes. Their RNA binding preferences have been investigated for almost two decades, and even though some RRM domains are now very well described, their RNA recognition code has remained elusive. An increasing number of experimental structures of RRM-RNA complexes has become available in recent years. Here, we perform an in-depth computational analysis to derive an RNA recognition code for canonical RRMs. We present and validate a computational scoring method to estimate the binding between an RRM and a single stranded RNA, based on structural data from a carefully curated multiple sequence alignment, which can predict RRM binding RNA sequence motifs based on the RRM protein sequence. Given the importance and prevalence of RRMs in humans and other species, this tool could help design RNA binding motifs with uses in medical or synthetic biology applications, leading towards the de novo design of RRMs with specific RNA recognition.

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 ß2ß3 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.

Pumping the brakes: A noncanonical RNA-binding domain in FMRP stalls elongating ribosomes.

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.

RNA polymerase II transcription attenuation at the yeast DNA repair gene DEF1 is biologically significant and dependent on the Hrp1 RNA-recognition motif.

Premature transcription termination (i.e. attenuation) is a potent gene regulatory mechanism that represses mRNA synthesis. Attenuation of RNA polymerase II is more prevalent than once appreciated, targeting 10-15% of mRNA genes in yeast through higher eukaryotes, but its significance and mechanism remain obscure. In the yeast Saccharomyces cerevisiae, polymerase II attenuation was initially shown to rely on Nrd1-Nab3-Sen1 termination, but more recently our laboratory characterized a hybrid termination pathway involving Hrp1, an RNA-binding protein in the 3'-end cleavage factor. One of the hybrid attenuation gene targets is DEF1, which encodes a repair protein that promotes degradation of polymerase II stalled at DNA lesions. In this study, we characterized the chromosomal DEF1 attenuator and the functional role of Hrp1. DEF1 attenuator mutants overexpressed Def1 mRNA and protein, exacerbated polymerase II degradation, and hindered cell growth, supporting a biologically significant DEF1 attenuator function. Using an auxin-induced Hrp1 depletion system, we identified new Hrp1-dependent attenuators in MNR2, SNG1, and RAD3 genes. An hrp1-5 mutant (L205S) known to impair binding to cleavage factor protein Rna14 also disrupted attenuation, but surprisingly no widespread defect was observed for an hrp1-1 mutant (K160E) located in the RNA-recognition motif. We designed a new RNA recognition motif mutant (hrp1-F162W) that altered a highly conserved residue and was lethal in single copy. In a heterozygous strain, hrp1-F162W exhibited dominant-negative readthrough defects at several gene attenuators. Overall, our results expand the hybrid RNA polymerase II termination pathway, confirming that Hrp1-dependent attenuation controls multiple yeast genes and may function through binding cleavage factor proteins and/or RNA.

Spontaneous binding of single-stranded RNAs to RRM proteins visualized by unbiased atomistic simulations with a rescaled RNA force field.

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.

Conformational Heterogeneity of RNA Stem-Loop Hairpins Bound to FUS-RNA Recognition Motif with Disordered RGG Tail Revealed by Unbiased Molecular Dynamics Simulations.

RNA-protein complexes use diverse binding strategies, ranging from structurally well-defined interfaces to completely disordered regions. Experimental characterization of flexible segments is challenging and can be aided by atomistic molecular dynamics (MD) simulations. Here, we used an extended set of microsecond-scale MD trajectories (400 µs in total) to study two FUS-RNA constructs previously characterized by nuclear magnetic resonance (NMR) spectroscopy. The FUS protein contains a well-structured RNA recognition motif domain followed by a presumably disordered RGG tail that binds RNA stem-loop hairpins. Our simulations not only provide several suggestions complementing the experiments but also reveal major methodological difficulties in studies of such complex RNA-protein interfaces. Despite efforts to stabilize the binding via system-specific force-field adjustments, we have observed progressive distortions of the RNA-protein interface inconsistent with experimental data. We propose that the dynamics is so rich that its converged description is not achievable even upon stabilizing the system. Still, after careful analysis of the trajectories, we have made several suggestions regarding the binding. We identify substates in the RNA loops, which can explain the NMR data. The RGG tail localized in the minor groove remains disordered, sampling countless transient interactions with the RNA. There are long-range couplings among the different elements contributing to the recognition, which can lead to allosteric communication throughout the system. Overall, the RNA-FUS systems form dynamical ensembles that cannot be fully represented by single static structures. Thus, albeit imperfect, MD simulations represent a viable tool to investigate dynamic RNA-protein complexes.

A noncanonical RNA-binding domain of the fragile X protein, FMRP, elicits translational repression independent of mRNA G-quadruplexes.

Loss of functional fragile X mental retardation protein (FMRP) causes fragile X syndrome, the leading form of inherited intellectual disability and the most common monogenic cause of autism spectrum disorders. FMRP is an RNA-binding protein that controls neuronal mRNA localization and translation. FMRP is thought to inhibit translation elongation after being recruited to target transcripts via binding RNA G-quadruplexes (G4s) within the coding sequence. Here, we directly test this model and report that FMRP inhibits translation independent of mRNA G4s. Furthermore, we found that the RGG box motif together with its natural C-terminal domain forms a noncanonical RNA-binding domain (ncRBD) that is essential for translational repression. The ncRBD elicits broad RNA-binding ability and binds to multiple reporter mRNAs and all four homopolymeric RNAs. Serial deletion analysis of the ncRBD identified that the regions required for mRNA binding and translational repression overlap but are not identical. Consistent with FMRP stalling elongating ribosomes and causing the accumulation of slowed 80S ribosomes, transcripts bound by FMRP via the ncRBD cosediment with heavier polysomes and were present in puromycin-resistant ribosome complexes. Together, this work identifies a ncRBD and translational repression domain that shifts our understanding of how FMRP inhibits translation independent of mRNA G4s.