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.

RNA granule-clustered mitochondrial aminoacyl-tRNA synthetases form multiple complexes with the potential to fine-tune tRNA aminoacylation.

Mitochondrial RNA metabolism is suggested to occur in identified compartmentalized foci, i.e. mitochondrial RNA granules (MRGs). Mitochondrial aminoacyl-tRNA synthetases (mito aaRSs) catalyze tRNA charging and are key components in mitochondrial gene expression. Mutations of mito aaRSs are associated with various human disorders. However, the suborganelle distribution, interaction network and regulatory mechanism of mito aaRSs remain largely unknown. Here, we found that all mito aaRSs partly colocalize with MRG, and this colocalization is likely facilitated by tRNA-binding capacity. A fraction of human mitochondrial AlaRS (hmtAlaRS) and hmtSerRS formed a direct complex via interaction between catalytic domains in vivo. Aminoacylation activities of both hmtAlaRS and hmtSerRS were fine-tuned upon complex formation in vitro. We further established a full spectrum of interaction networks via immunoprecipitation and mass spectrometry for all mito aaRSs and discovered interactions between hmtSerRS and hmtAsnRS, between hmtSerRS and hmtTyrRS and between hmtThrRS and hmtArgRS. The activity of hmtTyrRS was also influenced by the presence of hmtSerRS. Notably, hmtSerRS utilized the same catalytic domain in mediating several interactions. Altogether, our results systematically analyzed the suborganelle localization and interaction network of mito aaRSs and discovered several mito aaRS-containing complexes, deepening our understanding of the functional and regulatory mechanisms of mito aaRSs.

Activating translation with phase separation.

Ribonucleoprotein granules allow activation of translation to complete mouse spermatogenesis.

Pathological phase transitions in ALS-FTD impair dynamic RNA-protein granules.

The genetics of human disease serves as a robust and unbiased source of insight into human biology, both revealing fundamental cellular processes and exposing the vulnerabilities associated with their dysfunction. Over the last decade, the genetics of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have epitomized this concept, as studies of ALS-FTD-causing mutations have yielded fundamental discoveries regarding the role of biomolecular condensation in organizing cellular contents while implicating disturbances in condensate dynamics as central drivers of neurodegeneration. Here we review this genetic evidence, highlight its intersection with patient pathology, and discuss how studies in model systems have revealed a role for aberrant condensation in neuronal dysfunction and death. We detail how multiple, distinct types of disease-causing mutations promote pathological phase transitions that disturb the dynamics and function of ribonucleoprotein (RNP) granules. Dysfunction of RNP granules causes pleiotropic defects in RNA metabolism and can drive the evolution of these structures to end-stage pathological inclusions characteristic of ALS-FTD. We propose that aberrant phase transitions of these complex condensates in cells provide a parsimonious explanation for the widespread cellular abnormalities observed in ALS as well as certain histopathological features that characterize late-stage disease.

Translation inhibition and suppression of stress granules formation by cisplatin.

Platinum-based antineoplastic drugs, such as cisplatin, are commonly used to induce tumor cell death. Cisplatin is believed to induce apoptosis as a result of cisplatin-DNA adducts that inhibit DNA and RNA synthesis. Although idea that DNA damage underlines anti-proliferative effects of cisplatin is dominant in cancer research, there is a poor correlation between the degree of the cell sensitivity to cisplatin and the extent of DNA platination. Here, we examined possible effects of cisplatin on post-transcriptional gene regulation that may contribute to cisplatin-mediated cytotoxicity. We show that cisplatin suppresses formation of stress granules (SGs), pro-survival RNA granules with multiple roles in cellular metabolism. Mechanistically, cisplatin inhibits cellular translation to promote disassembly of polysomes and aggregation of ribosomal subunits. As SGs are in equilibrium with polysomes, cisplatin-induced shift towards ribosomal aggregation suppresses SG formation. Our data uncover previously unknown effects of cisplatin on RNA metabolism.

RGS4 RNA Secondary Structure Mediates Staufen2 RNP Assembly in Neurons.

RNA-binding proteins (RBPs) act as posttranscriptional regulators controlling the fate of target mRNAs. Unraveling how RNAs are recognized by RBPs and in turn are assembled into neuronal RNA granules is therefore key to understanding the underlying mechanism. While RNA sequence elements have been extensively characterized, the functional impact of RNA secondary structures is only recently being explored. Here, we show that Staufen2 binds complex, long-ranged RNA hairpins in the 3'-untranslated region (UTR) of its targets. These structures are involved in the assembly of Staufen2 into RNA granules. Furthermore, we provide direct evidence that a defined Rgs4 RNA duplex regulates Staufen2-dependent RNA localization to distal dendrites. Importantly, disrupting the RNA hairpin impairs the observed effects. Finally, we show that these secondary structures differently affect protein expression in neurons. In conclusion, our data reveal the importance of RNA secondary structure in regulating RNA granule assembly, localization and eventually translation. It is therefore tempting to speculate that secondary structures represent an important code for cells to control the intracellular fate of their mRNAs.

Persistent mRNA localization defects and cell death in ALS neurons caused by transient cellular stress.

Persistent cytoplasmic aggregates containing RNA binding proteins (RBPs) are central to the pathogenesis of late-onset neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). These aggregates share components, molecular mechanisms, and cellular protein quality control pathways with stress-induced RNA granules (SGs). Here, we assess the impact of stress on the global mRNA localization landscape of human pluripotent stem cell-derived motor neurons (PSC-MNs) using subcellular fractionation with RNA sequencing and proteomics. Transient stress disrupts subcellular RNA and protein distributions, alters the RNA binding profile of SG- and ALS-relevant RBPs and recapitulates disease-associated molecular changes such as aberrant splicing of STMN2. Although neurotypical PSC-MNs re-establish a normal subcellular localization landscape upon recovery from stress, cells harboring ALS-linked mutations are intransigent and display a delayed-onset increase in neuronal cell death. Our results highlight subcellular molecular distributions as predictive features and underscore the utility of cellular stress as a paradigm to study ALS-relevant mechanisms.

Liquid-liquid phase separation underpins the formation of replication factories in rotaviruses.

RNA viruses induce the formation of subcellular organelles that provide microenvironments conducive to their replication. Here we show that replication factories of rotaviruses represent protein-RNA condensates that are formed via liquid-liquid phase separation of the viroplasm-forming proteins NSP5 and rotavirus RNA chaperone NSP2. Upon mixing, these proteins readily form condensates at physiologically relevant low micromolar concentrations achieved in the cytoplasm of virus-infected cells. Early infection stage condensates could be reversibly dissolved by 1,6-hexanediol, as well as propylene glycol that released rotavirus transcripts from these condensates. During the early stages of infection, propylene glycol treatments reduced viral replication and phosphorylation of the condensate-forming protein NSP5. During late infection, these condensates exhibited altered material properties and became resistant to propylene glycol, coinciding with hyperphosphorylation of NSP5. Some aspects of the assembly of cytoplasmic rotavirus replication factories mirror the formation of other ribonucleoprotein granules. Such viral RNA-rich condensates that support replication of multi-segmented genomes represent an attractive target for developing novel therapeutic approaches.

The cytoplasmic SYNCRIP mRNA interactome of mammalian neurons.

SYNCRIP, a member of the cellular heterogeneous nuclear ribonucleoprotein (hnRNP) family of RNA binding proteins, regulates various aspects of neuronal development and plasticity. Although SYNCRIP has been identified as a component of cytoplasmic RNA granules in dendrites of mammalian neurons, only little is known about the specific SYNCRIP target mRNAs that mediate its effect on neuronal morphogenesis and function. Here, we present a comprehensive characterization of the cytoplasmic SYNCRIP mRNA interactome using iCLIP in primary rat cortical neurons. We identify hundreds of bona fide SYNCRIP target mRNAs, many of which encode for proteins involved in neurogenesis, neuronal migration and neurite outgrowth. From our analysis, the stabilization of mRNAs encoding for components of the microtubule network, such as doublecortin (Dcx), emerges as a novel mechanism of SYNCRIP function in addition to the previously reported control of actin dynamics. Furthermore, we found that SYNCRIP synergizes with pro-neural miRNAs, such as miR-9. Thus, SYNCRIP appears to promote early neuronal differentiation by a two-tier mechanism involving the stabilization of pro-neural mRNAs by direct 3'UTR interaction and the repression of anti-neural mRNAs in a complex with neuronal miRISC. Together, our findings provide a rationale for future studies investigating the function of SYNCRIP in mammalian brain development and disease.