Differentiation of lens fiber cells involves a complex interplay of signals from growth factors together with tightly regulated gene expression via transcriptional and post-transcriptional regulators. Various studies have demonstrated that RNA-binding proteins, functioning in ribonucleoprotein granules, have important roles in regulating post-transcriptional expression during lens development. In this study, we examined the expression and localization of two members of the BTG/TOB family of RNA-binding proteins, TOB1 and TOB2, in the developing lens and examined the phenotype of mice that lack Tob1. By RT-PCR, both Tob1 and Tob2 mRNA were detected in epithelial and fiber cells of embryonic and postnatal murine lenses. In situ hybridization showed Tob1 and Tob2 mRNA were most intensely expressed in the early differentiating fibers, with weaker expression in anterior epithelial cells, and both appeared to be downregulated in the germinative zone of E15.5 lenses. TOB1 protein was detected from E11.5 to E16.5 and was predominantly detected in large cytoplasmic puncta in early differentiating fiber cells, often co-localizing with the P-body marker, DCP2. Occasional nuclear puncta were also observed. By contrast, TOB2 was detected in a series of interconnected peri-nuclear granules, in later differentiating fiber cells of the inner cortex. TOB2 did not appear to co-localize with DCP2 but did partially co-localize with an early stress granule marker (EIF3B). These data suggest that TOB1 and TOB2 are involved with different aspects of the mRNA processing cycle in lens fiber cells. In vitro experiments using rat lens epithelial explants treated with or without a fiber differentiating dose of FGF2 showed that both TOB1 and TOB2 were up-regulated during FGF-induced differentiation. In differentiating explants, TOB1 also co-localized with DCP2 in large cytoplasmic granules. Analyses of Tob1-/- mice revealed relatively normal lens morphology but a subtle defect in cell cycle arrest of some cells at the equator and in the lens fiber mass of E13.5 embryos. Overall, these findings suggest that TOB proteins play distinct regulatory roles in RNA processing during lens fiber differentiation.

Ribonucleoprotein (RNP) granules are diverse membrane-less organelles that form through multivalent RNA-RNA, RNA-protein, and protein-protein interactions between RNPs. RNP granules are implicated in many aspects of RNA physiology, but in most cases their functions are poorly understood. RNP granules can be described through four key principles. First, RNP granules often arise because of the large size, high localized concentrations, and multivalent interactions of RNPs. Second, cells regulate RNP granule formation by multiple mechanisms including posttranslational modifications, protein chaperones, and RNA chaperones. Third, RNP granules impact cell physiology in multiple manners. Finally, dysregulation of RNP granules contributes to human diseases. Outstanding issues in the field remain, including determining the scale and molecular mechanisms of RNP granule function and how granule dysfunction contributes to human disease.

Localization of oskar mRNA to the posterior of the Drosophila oocyte is essential for abdominal patterning and germline development. oskar localization is a multi-step process involving temporally and mechanistically distinct transport modes. Numerous cis-acting elements and trans-acting factors have been identified that mediate earlier motor-dependent transport steps leading to an initial accumulation of oskar at the posterior. Little is known, however, about the requirements for the later localization phase, which depends on cytoplasmic flows and results in the accumulation of large oskar ribonucleoprotein granules, called founder granules, by the end of oogenesis. Using super-resolution microscopy, we show that founder granules are agglomerates of smaller oskar transport particles. In contrast to the earlier kinesin-dependent oskar transport, late-phase localization depends on the sequence as well as on the structure of the spliced oskar localization element (SOLE), but not on the adjacent exon junction complex deposition. Late-phase localization also requires the oskar 3\' untranslated region (3\' UTR), which targets oskar to founder granules. Together, our results show that 3\' UTR-mediated targeting together with SOLE-dependent agglomeration leads to accumulation of oskar in large founder granules at the posterior of the oocyte during late stages of oogenesis. In light of previous work showing that oskar transport particles are solid-like condensates, our findings indicate that founder granules form by a process distinct from that of well-characterized ribonucleoprotein granules like germ granules, P bodies, and stress granules. Additionally, they illustrate how an individual mRNA can be adapted to exploit different localization mechanisms depending on the cellular context.

Transposable elements (TEs) are DNA sequences that can transpose and replicate within the genome, leading to genetic changes that affect various aspects of host biology. Evolutionarily, hosts have also developed molecular mechanisms to suppress TEs at the transcriptional and post-transcriptional levels. Recent studies suggest that stress-induced formation of ribonucleoprotein (RNP) granules, including stress granule (SG) and processing body (P-body), can play a role in the sequestration of TEs to prevent transposition, suggesting an additional layer of the regulatory mechanism for TEs. RNP granules have been shown to contain factors involved in RNA regulation, including mRNA decay enzymes, RNA-binding proteins, and noncoding RNAs, which could potentially contribute to the regulation of TEs. Therefore, understanding the interplay between TEs and RNP granules is crucial for elucidating the mechanisms for maintaining genomic stability and controlling gene expression. In this review, we provide a brief overview of the current knowledge regarding the interplay between TEs and RNP granules, proposing RNP granules as a novel layer of the regulatory mechanism for TEs during stress.

RNA granules are dynamic entities controlling the spatiotemporal distribution and translation of RNA molecules. In neurons, a variety of RNA granules exist both in the soma and in cellular processes. They contain transcripts encoding signaling and synaptic proteins as well as RNA-binding proteins causally linked to several neurological disorders. In this review, we highlight that neuronal RNA granules exhibit properties of biomolecular condensates that are regulated upon maturation and physiological aging and how they are reversibly remodeled in response to neuronal activity to control local protein synthesis and ultimately synaptic plasticity. Moreover, we propose a framework of how neuronal RNA granules mature over time in healthy conditions and how they transition into pathological inclusions in the context of late-onset neurodegenerative diseases.

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.\"

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.

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.

Cytoplasmic RNA granules compartmentalize phases of the translation cycle in eukaryotes. We previously reported the localization of oxidized RNA to cytoplasmic foci called oxidized RNA bodies (ORBs) in human cells. We show here that ORBs are RNA granules in Saccharomyces cerevisiae. Several lines of evidence support a role for ORBs in the compartmentalization of no-go decay and ribosome quality control, the translation quality control pathways that recognize and clear aberrant mRNAs, including those with oxidized bases. Translation is required by these pathways and ORBs. Translation quality control factors localize to ORBs. A substrate of translation quality control, a stalled mRNA-ribosome-nascent-chain complex, localizes to ORBs. Translation quality control mutants have altered ORB numbers, sizes or both. In addition, we identify 68 ORB proteins by immunofluorescence staining directed by proteomics, which further support their role in translation quality control and reveal candidate new factors for these pathways.

Herpesviral infection reflects thousands of years of coevolution and the constant struggle between virus and host for control of cellular gene expression. During Kaposi\'s sarcoma-associated herpesvirus (KSHV) lytic replication, the virus rapidly seizes control of host gene expression machinery by triggering a massive RNA decay event via a virally encoded endoribonuclease, SOX. This virus takeover strategy decimates close to 80% of cellular transcripts, reallocating host resources toward viral replication. The host cell, however, is not entirely passive in this assault on RNA stability. A small pool of host transcripts that actively evade SOX cleavage has been identified over the years. One such \"escapee,\" C19ORF66 (herein referred to as Shiftless [SHFL]), encodes a potent antiviral protein capable of restricting the replication of multiple DNA and RNA viruses and retroviruses, including KSHV. Here, we show that SHFL restricts KSHV replication by targeting the expression of critical viral early genes, including the master transactivator protein, KSHV ORF50, and thus subsequently the entire lytic gene cascade. Consistent with previous reports, we found that the SHFL interactome throughout KSHV infection is dominated by RNA-binding proteins that influence both translation and protein stability, including the viral protein ORF57, a crucial regulator of viral RNA fate. We next show that SHFL affects cytoplasmic RNA granule formation, triggering the disassembly of processing bodies. Taken together, our findings provide insights into the complex relationship between RNA stability, RNA granule formation, and the antiviral response to KSHV infection. IMPORTANCE In the past 5 years, SHFL has emerged as a novel and integral piece of the innate immune response to viral infection. SHFL has been reported to restrict the replication of multiple viruses, including several flaviviruses and the retrovirus HIV-1. However, to date, the mechanism(s) by which SHFL restricts DNA virus infection remains largely unknown. We have previously shown that following its escape from KSHV-induced RNA decay, SHFL acts as a potent antiviral factor, restricting nearly every stage of KSHV lytic replication. In this study, we set out to determine the mechanism by which SHFL restricts KSHV infection. We demonstrate that SHFL impacts all classes of KSHV genes and found that SHFL restricts the expression of several key early genes, including KSHV ORF50 and ORF57. We then mapped the interactome of SHFL during KSHV infection and found several host and viral RNA-binding proteins that all play crucial roles in regulating RNA stability and translation. Lastly, we found that SHFL expression influences RNA granule formation both outside and within the context of KSHV infection, highlighting its broader impact on global gene expression. Collectively, our findings highlight a novel relationship between a critical piece of the antiviral response to KSHV infection and the regulation of RNA-protein dynamics.