RNA Granules Research Articles

MtFociCounter for automated single-cell mitochondrial nucleoid quantification and reproducible foci analysis.

Mitochondrial DNA (mtDNA) encodes the core subunits for OXPHOS, essential in near-all eukaryotes. Packed into distinct foci (nucleoids) inside mitochondria, the number of mtDNA copies differs between cell-types and is affected in several human diseases. Currently, common protocols estimate per-cell mtDNA-molecule numbers by sequencing or qPCR from bulk samples. However, this does not allow insight into cell-to-cell heterogeneity and can mask phenotypical sub-populations. Here, we present mtFociCounter, a single-cell image analysis tool for reproducible quantification of nucleoids and other foci. mtFociCounter is a light-weight, open-source freeware and overcomes current limitations to reproducible single-cell analysis of mitochondrial foci. We demonstrate its use by analysing 2165 single fibroblasts, and observe a large cell-to-cell heterogeneity in nucleoid numbers. In addition, mtFociCounter quantifies mitochondrial content and our results show good correlation (R=0.90) between nucleoid number and mitochondrial area, and we find nucleoid density is less variable than nucleoid numbers in wild-type cells. Finally, we demonstrate mtFociCounter readily detects differences in foci-numbers upon sample treatment, and applies to Mitochondrial RNA Granules and superresolution microscopy. mtFociCounter provides a versatile solution to reproducibly quantify cellular foci in single cells and our results highlight the importance of accounting for cell-to-cell variance and mitochondrial context in mitochondrial foci analysis.

Proteomics elucidating physiological and pathological functions of TDP-43.

Trans-activation response DNA binding protein of 43kDa (TDP-43) regulates a great variety of cellular processes in the nucleus and cytosol. In addition, a defined subset of neurodegenerative diseases is characterized by nuclear depletion of TDP-43 as well as cytosolic mislocalization and aggregation. To perform its diverse functions TDP-43 can associate with different ribonucleoprotein complexes. Combined with transcriptomics, MS interactome studies have unveiled associations between TDP-43 and the spliceosome machinery, polysomes and RNA granules. Moreover, the highly dynamic, low-valency interactions regulated by its low-complexity domain calls for innovative proximity labeling methodologies. In addition to protein partners, the analysis of post-translational modifications showed that they may play a role in the nucleocytoplasmic shuttling, RNA binding, liquid-liquid phase separation and protein aggregation of TDP-43. Here we review the various TDP-43 ribonucleoprotein complexes characterized so far, how they contribute to the diverse functions of TDP-43, and roles of post-translational modifications. Further understanding of the fluid dynamic properties of TDP-43 in ribonucleoprotein complexes, RNA granules, and self-assemblies will advance the understanding of RNA processing in cells and perhaps help to develop novel therapeutic approaches for TDPopathies.

Peripheral but critical: Translation of germ granule mRNAs in zebrafish

RNA granule components occupy distinct positions within granules. However, the significance behind this organization is unclear. In this issue of Developmental Cell, Westerich etal. show that the periphery of the zebrafish germ granules promotes mRNA translation while its interior represses it, which is critical for germ cell establishment.

Comparative proteomic analysis of three major extracellular vesicle classes secreted from human primary and metastatic colorectal cancer cells: Exosomes, microparticles, and shed midbody remnants.

Cell-derived extracellular vesicles (EVs) are evolutionary-conserved secretory organelles that, based on their molecular composition, are important intercellular signaling regulators. At least three classes of circulating EVs are known based on mechanism of biogenesis: exosomes (sEVs/Exos), microparticles (lEVs/MPs), and shed midbody remnants (lEVs/sMB-Rs). sEVs/Exos are of endosomal pathway origin, microparticles (lEVs/MPs) from plasma membrane blebbing and shed midbody remnants (lEVs/sMB-Rs) arise from symmetric cytokinetic abscission. Here, we isolate sEVs/Exos, lEVs/MPs, and lEVs/sMB-Rs secreted from human isogenic primary (SW480) and metastatic (SW620) colorectal cancer (CRC) cell lines in milligram quantities for label-free MS/MS-based proteomic profiling. Purified EVs revealed selective composition packaging of exosomal protein markers in SW480/SW620-sEVs/Exos, metabolic enzymes in SW480/SW620-lEVs/MPs, while centralspindlin complex proteins, nucleoproteins, splicing factors, RNA granule proteins, translation-initiation factors, and mitochondrial proteins selectively traffic to SW480/SW620- lEVs/sMB-Rs. Collectively, we identify 39 human cancer-associated genes in EVs; 17 associated with SW480-EVs, 22 with SW620-EVs. We highlight oncogenic receptors/transporters selectively enriched in sEVs/Exos (EGFR/FAS in SW480-sEVs/Exos and MET, TGFBR2, ABCB1 in SW620-sEVs/Exos). Interestingly, MDK, STAT1, and TGM2 are selectively enriched in SW480-lEVs/sMB-Rs, and ADAM15 to SW620-lEVs/sMB-Rs. Our study reveals sEVs/Exos, lEVs/MPs, and lEVs/sMB-Rs have distinct protein signatures that open potential diagnostic avenues of distinct types of EVs for clinical utility.

HnRNPH2 gain-of-function mutations reveal therapeutic strategies and a role for RNA granules in neurodevelopmental disorders.

hnRNPH2-related neurodevelopmental disorder (NDD) is caused by mutations in the HNRNPH2 gene and is associated with substantial challenges, including developmental delay, intellectual disability, growth delay, and epilepsy. There is currently no therapeutic intervention available to those with hnRNPH2-related NDD that addresses its underlying mechanisms. In this issue of the JCI, Korff et al. studied specific gain-of-function mutations associated with hnRNPH2-related NDD, with the help of mouse models that recapitulate key features of the condition in humans. Their work paves the way for therapeutic approaches that aim to reduce the expression of mutant hnRNPH2 and highlights a role for disrupted RNA granules in neurodevelopmental and neurodegenerative disorders.

RNA helicase DDX6 and scaffold protein GW182 in P-bodies promote biogenesis of stress granules.

Two prominent cytoplasmic RNA granules, ubiquitous RNA-processing bodies (PB) and inducible stress granules (SG), regulate mRNA translation and are intimately related. In this study, we found that arsenite (ARS)-induced SG formed in a stepwise process is topologically and mechanically linked to PB. Two essential PB components, GW182 and DDX6, are repurposed under stress to play direct but distinguishable roles in SG biogenesis. By providing scaffolding activities, GW182 promotes the aggregation of SG components to form SG bodies. DEAD-box helicase DDX6 is also essential for the proper assembly and separation of PB from SG. DDX6 deficiency results in the formation of irregularly shaped 'hybrid' PB/SG granules with accumulated components of both PB and SG. Wild-type DDX6, but not its helicase mutant E247A, can rescue the separation of PB from SG in DDX6KO cells, indicating a requirement of DDX6 helicase activity for this process. DDX6 activity in biogenesis of both PB and SG in the cells under stress is further modulated by its interaction with two protein partners, CNOT1 and 4E-T, of which knockdown affects the formation of both PB and also SG. Together, these data highlight a new functional paradigm between PB and SG biogenesis during the stress.

Proteins rather than mRNAs regulate nucleation and persistence of Oskar germ granules in Drosophila

RNA granules are membraneless condensates that provide functional compartmentalization within cells. The mechanisms by which RNA granules form are under intense investigation. Here, we characterize the role of mRNAs and proteins in the formation of germ granules in Drosophila. Super-resolution microscopy reveals that the number, size, and distribution of germ granules is precisely controlled. Surprisingly, germ granule mRNAs are not required for the nucleation or the persistence of germ granules but instead control their size and composition. Using an RNAi screen, we determine that RNA regulators, helicases, and mitochondrial proteins regulate germ granule number and size, while the proteins of the endoplasmic reticulum, nuclear pore complex, and cytoskeleton control their distribution. Therefore, the protein-driven formation of Drosophila germ granules is mechanistically distinct from the RNA-dependent condensation observed for other RNA granules such as stress granules and P-bodies.

Increased G3BP2-Tau interaction in tauopathies is a natural defense against Tau aggregation

Many RNA-binding proteins (RBPs), particularly those associated with RNA granules, promote pathological protein aggregation in neurodegenerative diseases. Here, we demonstrate that G3BP2, a core component of stress granules, directly interacts with Tau and inhibits Tau aggregation. In the human brain, the interaction of G3BP2 and Tau is dramatically increased in multiple tauopathies, and it is independent of neurofibrillary tangle (NFT) formation in Alzheimer's disease (AD). Surprisingly, Tau pathology is significantly elevated upon loss of G3BP2 in human neurons and brain organoids. Moreover, we found that G3BP2 masks the microtubule-binding region (MTBR) of Tau, thereby inhibiting Tau aggregation. Our study defines a novel role for RBPs as a line of defense against Tau aggregation in tauopathies.

Grand challenges in biomolecular condensates: structure, function, and formation

Biomolecular condensates describe concentrated nonstoichiometric assemblies of biomolecules that can form by a range of different mechanisms 1). Biomolecular condensates can arise by phase separation, which in biology involves the demixing of a water-soluble polymer into two co-existing phases: a polymer-dilute phase and a polymer-dense phase. Coacervates describe phase separation mediated by a third element, which may typically be a ligand (such as RNA) to the polymer (such as a protein) that undergoes phase separation. Protein aggregation into amyloids and amorphous aggregates, and the formation of RNA granules, represent other forms of biomolecular condensates. The assembly of proteins and other biomolecules into complexes is a fundamental feature for the execution of biological functions. Biomolecular condensates are a natural variation of the assembly theme. There is an incredible complexity and diversity to how condensates form, are regulated and are structured (reviewed recently in 2)). And there is incredible diversity to how condensates are used by nature to drive biological functions and how when their assemblies go wrong, they can drive disease mechanisms, such as amyloids in neurodegeneration.

A unique mRNA decapping complex in trypanosomes.

Removal of the mRNA 5' cap primes transcripts for degradation and is central for regulating gene expression in eukaryotes. The canonical decapping enzyme Dcp2 is stringently controlled by assembly into a dynamic multi-protein complex together with the 5'-3'exoribonuclease Xrn1. Kinetoplastida lack Dcp2 orthologues but instead rely on the ApaH-like phosphatase ALPH1 for decapping. ALPH1 is composed of a catalytic domain flanked by C- and N-terminal extensions. We show that T. brucei ALPH1 is dimeric in vitro and functions within a complex composed of the trypanosome Xrn1 ortholog XRNA and four proteins unique to Kinetoplastida, including two RNA-binding proteins and a CMGC-family protein kinase. All ALPH1-associated proteins share a unique and dynamic localization to a structure at the posterior pole of the cell, anterior to the microtubule plus ends. XRNA affinity capture in T. cruzi recapitulates this interaction network. The ALPH1 N-terminus is not required for viability in culture, but essential for posterior pole localization. The C-terminus, in contrast, is required for localization to all RNA granule types, as well as for dimerization and interactions with XRNA and the CMGC kinase, suggesting possible regulatory mechanisms. Most significantly, the trypanosome decapping complex has a unique composition, differentiating the process from opisthokonts.

Regulation of RNG105/caprin1 dynamics by pathogenic cytoplasmic FUS and TDP-43 in neuronal RNA granules modulates synaptic loss

In neurodegenerative diseases, the condensation of FUS and TDP-43 with RNA granules in neurons is linked to pathology, including synaptic disorders. However, the effects of FUS and TDP-43 on RNA granule factors remain unclear. Here, using primary cultured neurons from the mouse cerebral cortex, we show that excess cytoplasmic FUS and TDP-43 accumulated in dendritic RNA granules, where they increased the dynamics of a scaffold protein RNG105/caprin1 and dissociated it from the granules. This coincided with reduced levels of mRNA and translation around the granules and synaptic loss in dendrites. These defects were suppressed by non-dissociable RNG105, suggesting that RNG105 dissociation mediated the defects. In contrast to the model where FUS and TDP-43 co-aggregate with RNA granule factors to repress their activity, our findings provide a novel pathogenic mechanism whereby FUS and TDP-43 dissociate RNA scaffold proteins from RNA granules which are required for local translation that regulates synapse formation.

RNA granules: functional compartments or incidental condensates?

RNA granules are mesoscale assemblies that form in the absence of limiting membranes. RNA granules contain factors for RNA biogenesis and turnover and are often assumed to represent specialized compartments for RNA biochemistry. Recent evidence suggests that RNA granules assemble by phase separation of subsoluble ribonucleoprotein (RNP) complexes that partially demix from the cytoplasm or nucleoplasm. We explore the possibility that some RNA granules are nonessential condensation by-products that arise when RNP complexes exceed their solubility limit as a consequence of cellular activity, stress, or aging. We describe the use of evolutionary and mutational analyses and single-molecule techniques to distinguish functional RNA granules from "incidental condensates."

Abstract 104: Gene Regulation By RNA Granule Formation In Atherosclerosis

Background: Atherosclerosis, the leading cause of cardiovascular disease related death, is a chronic inflammatory condition incited by the accumulation of low-density lipoproteins (LDL) and recruitment of macrophages and smooth muscle cells (SMCs) in the arterial intima. Both macrophages and SMCs become lipid laden foam cells that persist in the arterial vasculature, inciting chronic inflammation and plaque formation. A better understanding of the mechanisms underlying this maladaptive inflammatory response may reveal novel therapeutic strategies for intervention. Hypothesis: We hypothesize that the transformation into a foam cell state, a process that requires transcriptional rewiring, is facilitated by stress granules (SGs). SGs are cytoplasmic membrane-less organelles that sequester translation initiation factors, mRNAs, and noncoding RNAs as a result of stress. We previously reported that SG formation is increased in macrophages and vascular smooth muscle cells (SMCs) in plaques of atherosclerotic mice, and can be induced in vitro during cellular cholesterol loading. However, the consequences of SG formation and the transcripts sequestered by these organelles remain unknown Methods and Results: A current limitation in studying SG biology is the lack of in vivo models to track and isolate these dynamic structures. Using CRISPR-Cas9, we generated mice with a 3x-HA-tag on the SG marker G3BP1 in C57BL6 mice, allowing us to identify and purify SGs in vivo and in isolated primary cells. In HA-tagged G3BP1 mice fed western diet, we observe staining for HA-G3BP1 in macrophages and smooth muscle cells localized to the necrotic core and fibrous cap of atherosclerotic plaque. Furthermore, qRT-PCR on lipid laden cells revealed changes in known stress granule markers and transcripts involved in immune regulation. Conclusion: Our data support a role for SGs in repressing the translation of RNA transcripts in lipid-laden foam cells that accumulate in atherosclerotic plaque. Further study of the specific proteins and RNA transcripts sequestered in SGs of foam cells may reveal how they alter gene expression to facilitate cellular phenotypic changes in the plaque.

FMRP phosphorylation and interactions with Cdh1 regulate association with dendritic RNA granules and MEF2-triggered synapse elimination

Fragile X Messenger Ribonucleoprotein (FMRP) is necessary for experience-dependent, developmental synapse elimination and the loss of this process may underlie the excess dendritic spines and hyperconnectivity of cortical neurons in Fragile X Syndrome, a common inherited form of intellectual disability and autism. Little is known of the signaling pathways that regulate synapse elimination and if or how FMRP is regulated during this process. We have characterized a model of synapse elimination in CA1 neurons of organotypic hippocampal slice cultures that is induced by expression of the active transcription factor Myocyte Enhancer Factor 2 (MEF2) and relies on postsynaptic FMRP. MEF2-induced synapse elimination is deficient in Fmr1 KO CA1 neurons, and is rescued by acute (24 h), postsynaptic and cell autonomous reexpression of FMRP in CA1 neurons. FMRP is an RNA binding protein that suppresses mRNA translation. Derepression is induced by posttranslational mechanisms downstream of metabotropic glutamate receptor signaling. Dephosphorylation of FMRP at S499 triggers ubiquitination and degradation of FMRP which then relieves translation suppression and promotes synthesis of proteins encoded by target mRNAs. Whether this mechanism functions in synapse elimination is not known. Here we demonstrate that phosphorylation and dephosphorylation of FMRP at S499 are both necessary for synapse elimination as well as interaction of FMRP with its E3 ligase for FMRP, APC/Cdh1. Using a bimolecular ubiquitin-mediated fluorescence complementation (UbFC) assay, we demonstrate that MEF2 promotes ubiquitination of FMRP in CA1 neurons that relies on activity and interaction with APC/Cdh1. Our results suggest a model where MEF2 regulates posttranslational modifications of FMRP via APC/Cdh1 to regulate translation of proteins necessary for synapse elimination.

ALS Causative Mutations in KIF5A Disrupt Autoinhibition Leading to Toxic Gain of Function (P8-8.002)

<h3>Objective:</h3> The objective of this study was to elucidate the pathogenic mechanisms of ALS causative mutations in motor protein KIF5A. <h3>Background:</h3> KIF5A is a neuronal specific subunit of Kinesin-1, which is a microtubule motor protein that plays roles in axonal transport and cytoskeletal regulation. Mutations in distinct regions of Kinesin-1 lead to a broad range of neurologic diseases. KIF5A enables Kinesin-1 to walk along microtubules and transport various cargos such as mitochondria and RNA granules into the distal axon. ALS-causative mutations in KIF5A affect the structure of the domain of the protein that functions in cargo binding and autoregulation. However, the consequences of ALS-causative mutations on disease pathogenesis are not fully understood. Our central hypothesis was that KIF5A ALS mutations disrupt cargo binding and/or autoregulation leading to neurodegeneration. <h3>Design/Methods:</h3> We generated <i>in vitro</i> models of KIF5A ALS using transient transfection of disease causative variants into neuroblastoma cell lines. We performed immunofluorescence staining and confocal imaging to visualize the distribution of motor proteins KIF5A, KIF5B, and Dynein. We utilized Drosophila S2 cell lines to study mitochondrial motility using live-imaging. <h3>Results:</h3> Whereas wild type (WT) KIF5A displayed relatively homogeneous distribution, ALS mutant KIF5A developed dramatic accumulation within distal neurites. Co-expression of the ALS mutant protein with the WT caused it to accumulate as well. Mitochondria displayed similar mislocalization to distal neurites. No change was seen in the distribution of KIF5B or Dynein. <h3>Conclusions:</h3> We found that ALS mutant KIF5A caused dramatic accumulation of mutant and WT protein as well as mitochondrial within distal neurite tips. These findings suggest that ALS mutation disrupts its autoinhibition causing mislocalization of its cargos through gain of function. These changes in distribution are specific to KIF5A containing motors. Our findings establish dysregulation of KIF5A activity as the underlying pathogenic mechanism for KIF5A ALS. <b>Disclosure:</b> Dr. Brent has received research support from The American Academy of Neurology. Mr. Sterling-Angus has nothing to disclose. Dr. Deng has nothing to disclose.

Cauliflower mosaic virus protein P6 is a multivalent node for RNA granule proteins and interferes with stress granule responses during plant infection.

Biomolecular condensation is a multipurpose cellular process that viruses use ubiquitously during their multiplication. Cauliflower mosaic virus replication complexes are condensates that differ from those of most viruses, as they are nonmembranous assemblies that consist of RNA and protein, mainly the viral protein P6. Although these viral factories (VFs) were described half a century ago, with many observations that followed since, functional details of the condensation process and the properties and relevance of VFs have remained enigmatic. Here, we studied these issues in Arabidopsis thaliana and Nicotiana benthamiana. We observed a large dynamic mobility range of host proteins within VFs, while the viral matrix protein P6 is immobile, as it represents the central node of these condensates. We identified the stress granule (SG) nucleating factors G3BP7 and UBP1 family members as components of VFs. Similarly, as SG components localize to VFs during infection, ectopic P6 localizes to SGs and reduces their assembly after stress. Intriguingly, it appears that soluble rather than condensed P6 suppresses SG formation and mediates other essential P6 functions, suggesting that the increased condensation over the infection time-course may accompany a progressive shift in selected P6 functions. Together, this study highlights VFs as dynamic condensates and P6 as a complex modulator of SG responses.

RNA-Targeting Carbon Dots for Live-Cell Imaging of Granule Dynamics.

It is significant to monitor the different RNA granules dynamics and phase separation process inside cells under various stresses, for example, oxidative stress. The current small-molecule RNA probes work well only in fixed cells and usually encounter problems such as insufficient stability and biocompatibility, and thus a specific RNA-targeting fluorescent nanoprobe thatcan be used in the living cells is urgently desired. Here, the de novo design and microwave-assisted synthesis of a novel RNA-targeting, red-emissive carbon dots (named as M-CDs) are reported by choosing neutral red and levofloxacin as precursors. The as-synthesized M-CDs is water-soluble with a high fluorescence quantum yield of 22.83% and can selectively bind to RNA resulting in an enhanced red fluorescence. More interestingly, such an RNA-targeting, red-emissive M-CDs can be fast internalized into cells within 5s and thus used for real-time imaging the dynamic process of intracellular stress granules under oxidative stress, revealing some characteristics of granules that have not been identified by previously reported RNA and protein biomarkers. This research paves a new pathway for visualizing bulk RNA dynamics and studying phase-separation behaviors in living cells by rational design of the fluorescent carbon dots in terms of structure and functionality.

Role of the RNA-binding protein ZC3H41 in the regulation of ribosomal protein messenger RNAs in trypanosomes

BackgroundTrypanosomes are single-celled eukaryotes that rely heavily on post-transcriptional mechanisms to regulate gene expression. RNA-binding proteins play essential roles in regulating the fate, abundance and translation of messenger RNAs (mRNAs). Among these, zinc finger proteins of the cysteine3histidine (CCCH) class have been shown to be key players in cellular processes as diverse as differentiation, regulation of the cell cycle and translation. ZC3H41 is an essential zinc finger protein that has been described as a component of spliced leader RNA granules and nutritional stress granules, but its role in RNA metabolism is unknown.MethodsCell cycle analysis in ZC3H41- and Z41AP-depleted cells was carried out using 4′,6-diamidino-2-phenylindole staining, microscopic examination and flow cytometry. The identification of ZC3H41 protein partners was done using tandem affinity purification and mass spectrometry. Next-generation sequencing was used to evaluate the effect of ZC3H41 depletion on the transcriptome of procyclic Trypanosoma brucei cells, and also to identify the cohort of mRNAs associated with the ZC3H41/Z41AP complex. Levels of 5S ribosomal RNA (rRNA) species in ZC3H41- and Z41AP-depleted cells were assessed by quantitative reverse transcription-polymerase chain reaction. Surface sensing of translation assays were used to monitor global translation.ResultsWe showed that depletion of the zinc finger protein ZC3H41 resulted in marked cell cycle defects and abnormal cell morphologies. ZC3H41 was found associated with an essential protein, which we named Z41AP, forming a stable heterodimer, and also with proteins of the poly(A)-binding protein 1 complex. The identification of mRNAs associated with the ZC3H41/Z41AP complex revealed that it is primarily composed of ribosomal protein mRNAs, and that binding to target transcripts is diminished upon nutritional stress. In addition, we observed that mRNAs encoding several proteins involved in the maturation of 5S rRNA are also associated with the ZC3H41/Z41AP complex. Finally, we showed that depletion of either ZC3H41 or Z41AP led to the accumulation of 5S rRNA precursors and a decrease of protein translation.ConclusionsWe propose that ZC3H41 and Z41AP play important roles in controlling the fate of ribosomal components in response to environmental cues.Graphical

Multiple functions of stress granules in viral infection at a glance.

Stress granules (SGs) are distinct RNA granules induced by various stresses, which are evolutionarily conserved across species. In general, SGs act as a conservative and essential self-protection mechanism during stress responses. Viruses have a long evolutionary history and viral infections can trigger a series of cellular stress responses, which may interact with SG formation. Targeting SGs is believed as one of the critical and conservative measures for viruses to tackle the inhibition of host cells. In this systematic review, we have summarized the role of SGs in viral infection and categorized their relationships into three tables, with a particular focus on Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. Moreover, we have outlined several kinds of drugs targeting SGs according to different pathways, most of which are potentially effective against SARS-CoV-2. We believe this review would offer a new view for the researchers and clinicians to attempt to develop more efficacious treatments for virus infection, particularly for the treatment of SARS-CoV-2 infection.

Ribosomes in RNA Granules Are Stalled on mRNA Sequences That Are Consensus Sites for FMRP Association.

Local translation in neurons is partly mediated by the reactivation of stalled polysomes. Stalled polysomes may be enriched within the granule fraction, defined as the pellet of sucrose gradients used to separate polysomes from monosomes. The mechanism of how elongating ribosomes are reversibly stalled and unstalled on mRNAs is still unclear. In the present study, we characterize the ribosomes in the granule fraction using immunoblotting, cryogenic electron microscopy (cryo-EM), and ribosome profiling. We find that this fraction, isolated from 5-d-old rat brains of both sexes, is enriched in proteins implicated in stalled polysome function, such as the fragile X mental retardation protein (FMRP) and Up-frameshift mutation 1 homologue. Cryo-EM analysis of ribosomes in this fraction indicates they are stalled, mainly in the hybrid state. Ribosome profiling of this fraction reveals (1) an enrichment for footprint reads of mRNAs that interact with FMRPs and are associated with stalled polysomes, (2) an abundance of footprint reads derived from mRNAs of cytoskeletal proteins implicated in neuronal development, and (3) increased ribosome occupancy on mRNAs encoding RNA binding proteins. Compared with those usually found in ribosome profiling studies, the footprint reads were longer and were mapped to reproducible peaks in the mRNAs. These peaks were enriched in motifs previously associated with mRNAs cross-linked to FMRP in vivo, independently linking the ribosomes in the granule fraction to the ribosomes associated with FMRP in the cell. The data supports a model in which specific sequences in mRNAs act to stall ribosomes during translation elongation in neurons.SIGNIFICANCE STATEMENT Neurons send mRNAs to synapses in RNA granules, where they are not translated until an appropriate stimulus is given. Here, we characterize a granule fraction obtained from sucrose gradients and show that polysomes in this fraction are stalled on consensus sequences in a specific state of translational arrest with extended ribosome-protected fragments. This finding greatly increases our understanding of how neurons use specialized mechanisms to regulate translation and suggests that many studies on neuronal translation may need to be re-evaluated to include the large fraction of neuronal polysomes found in the pellet of sucrose gradients used to isolate polysomes.