Cytoplasmic RNP Research Articles

An image-based RNAi screen identifies the EGF.R signaling pathway as a regulator of Imp/ IGF2BP RNP granules.

Biomolecular condensates have recently retained much attention since they provide a fundamental mechanism of cellular organization. Among those, cytoplasmic RNP granules selectively and reversibly concentrate RNA molecules and regulatory proteins, thus contributing to the spatio-temporal regulation of associated RNAs. Extensive in vitro work has unraveled the molecular and chemical bases of RNP granule assembly. The signaling pathways controlling this process in a cellular context are however still largely unknown. Here, we aimed at identifying regulators of cytoplasmic RNP granules characterized by the presence of the evolutionarily conserved IGF2BP/Imp/ZBP1 RNA binding protein. We performed a high-content image-based RNAi screen targeting all Drosophila genes encoding RNA binding proteins, phosphatases and kinases. This led to the identification of dozens of genes regulating the number of Imp+ RNP granules in S2R+ cells, among which components of the MAPK pathway. Combining functional approaches, phospho-mapping and generation of phospho-variants, we further showed that the EGF.R signaling inhibits Imp+ RNP granule assembly through activation of MAPK/Rolled and Imp S15 phosphosite. This work illustrates how signaling pathways can regulate cellular condensate assembly by post-translational modifications of specific components.

Principles of RNA recruitment to viral ribonucleoprotein condensates in a segmented dsRNA virus.

Rotaviruses transcribe 11 distinct RNAs that must be co-packaged prior to their replication to make an infectious virion. During infection, nontranslating rotavirus transcripts accumulate in cytoplasmic protein-RNA granules known as viroplasms that support segmented genome assembly and replication via a poorly understood mechanism. Here, we analysed the RV transcriptome by combining DNA-barcoded smFISH of rotavirus-infected cells. Rotavirus RNA stoichiometry in viroplasms appears to be distinct from the cytoplasmic transcript distribution, with the largest transcript being the most enriched in viroplasms, suggesting a selective RNA enrichment mechanism. While all 11 types of transcripts accumulate in viroplasms, their stoichiometry significantly varied between individual viroplasms. Accumulation of transcripts requires the presence of 3' untranslated terminal regions and viroplasmic localisation of the viral polymerase VP1, consistent with the observed lack of polyadenylated transcripts in viroplasms. Our observations reveal similarities between viroplasms and other cytoplasmic RNP granules and identify viroplasmic proteins as drivers of viral RNA assembly during viroplasm formation.

RNP components condense into repressive RNP granules in the aging brain

Cytoplasmic RNP condensates enriched in mRNAs and proteins are found in various cell types and associated with both buffering and regulatory functions. While a clear link has been established between accumulation of aberrant RNP aggregates and progression of aging-related neurodegenerative diseases, the impact of physiological aging on neuronal RNP condensates has never been explored. Through high-resolution imaging, we uncover that RNP components progressively cluster into large yet dynamic granules in the aging Drosophila brain. We further show that age-dependent clustering is caused by an increase in the stoichiometry of the conserved helicase Me31B/DDX6, and requires PKA kinase activity. Finally, our functional analysis reveals that mRNA species recruited to RNP condensates upon aging exhibit age-dependent translational repression, indicating that co-clustering of selected mRNAs and translation regulators into repressive condensates may contribute to the specific post-transcriptional changes in gene expression observed in the course of aging.

The GNU subunit of PNG kinase, the developmental regulator of mRNA translation, binds BIC-C to localize to RNP granules.

Control of mRNA translation is a key mechanism by which the differentiated oocyte transitions to a totipotent embryo. In Drosophila, the PNG kinase complex regulates maternal mRNA translation at the oocyte-to-embryo transition. We previously showed that the GNU activating subunit is crucial in regulating PNG and timing its activity to the window between egg activation and early embryogenesis (Hara et al., 2017). In this study, we find associations between GNU and proteins of RNP granules and demonstrate that GNU localizes to cytoplasmic RNP granules in the mature oocyte, identifying GNU as a new component of a subset of RNP granules. Furthermore, we define roles for the domains of GNU. Interactions between GNU and the granule component BIC-C reveal potential conserved functions for translational regulation in metazoan development. We propose that by binding to BIC-C, upon egg activation GNU brings PNG to its initial targets, translational repressors in RNP granules.

Electrostatic modulation of hnRNPA1 low-complexity domain liquid-liquid phase separation and aggregation.

Membrane-less organelles and RNP granules are enriched in RNA and RNA-binding proteins containing disordered regions. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a key regulating protein in RNA metabolism, localizes to cytoplasmic RNP granules including stress granules. Dysfunctional nuclear-cytoplasmic transport and dynamic phase separation of hnRNPA1 leads to abnormal amyloid aggregation and neurodegeneration. The intrinsically disordered C-terminal domain (CTD) of hnRNPA1 mediates both dynamic liquid-liquid phase separation (LLPS) and aggregation. While cellular phase separation drives the formation of membrane-less organelles, aggregation within phase-separated compartments has been linked to neurodegenerative diseases. To understand some of the underlying mechanisms behind protein phase separation and LLPS-mediated aggregation, we studied LLPS of hnRNPA1 CTD in conditions that probe protein electrostatics, modulated specifically by varying pH conditions, and protein, salt and RNA concentrations. In the conditions investigated, we observed LLPS to be favored in acidic conditions, and by high protein, salt and RNA concentrations. We also observed that conditions that favor LLPS also enhance protein aggregation and fibrillation, which suggests an aggregation pathway that is LLPS-mediated. The results reported here also suggest that LLPS can play a direct role in facilitating protein aggregation, and that changes in cellular environment that affect protein electrostatics can contribute to the pathological aggregation exhibited in neurodegeneration.

L- Bodies are Novel RNA-Protein Condensates Driving RNA Transport in <i>Xenopus</i> Oocytes

RNP granules are membrane-less compartments within cells, formed by phase separation, that function as regulatory hubs for diverse biological processes. However, the mechanisms by which RNAs and proteins interact to promote RNP granule structure and function in vivo remain unclear. In Xenopus laevis oocytes, maternal mRNAs are transported as large RNPs to the vegetal hemisphere of the developing oocyte, where local translation is critical for proper embryonic patterning. Here, we demonstrate that vegetal transport RNPs represent a new class of cytoplasmic RNP granule, termed Localization-bodies (L-bodies). We show that L-bodies are multiphase RNP granules, containing a dynamic protein-containing phase surrounding a non-dynamic RNA-containing substructure. Our results support a role for RNA as a critical scaffold component within these RNP granules and suggest that cis-elements within localized mRNAs may drive subcellular RNA localization through control over phase behavior.

ELAVL2-directed RNA regulatory network drives the formation of quiescent primordial follicles.

Formation of primordial follicles is a fundamental, early process in mammalian oogenesis. However, little is known about the underlying mechanisms. We herein report that the RNA-binding proteins ELAVL2 and DDX6 are indispensable for the formation of quiescent primordial follicles in mouse ovaries. We show that Elavl2 knockout females are infertile due to defective primordial follicle formation. ELAVL2 associates with mRNAs encoding components of P-bodies (cytoplasmic RNP granules involved in the decay and storage of RNA) and directs the assembly of P-body-like granules by promoting the translation of DDX6 in oocytes prior to the formation of primordial follicles. Deletion of Ddx6 disturbs the assembly of P-body-like granules and severely impairs the formation of primordial follicles, indicating the potential importance of P-body-like granules in the formation of primordial follicles. Furthermore, Ddx6-deficient oocytes are abnormally enlarged due to misregulated PI3K-AKT signaling. Our data reveal that an ELAVL2-directed post-transcriptional network is essential for the formation of quiescent primordial follicles.

Interaction and colocalization of HERMES/RBPMS with NonO, PSF, and G3BP1 in neuronal cytoplasmic RNP granules in mouse retinal line cells.

HERMES, also called RBPMS, is a conserved RNA binding protein with a single RNA recognition motif (RRM) that is abundantly expressed in retinal ganglion cells (RGCs) and in the heart in vertebrates. Here, we identified NonO and PSF as the interacting proteins of HERMES only when the neuronal differentiation of the retinal cell line RGC-5 was induced. Although NonO and PSF are nuclear paraspeckle components, these proteins formed cytoplasmic granules with HERMES in the neurites. G3BP1, a component of stress granules, was also colocalized to the granules, interacting with NonO and HERMES even in the absence of cellular stress. Consistent with a previous report that KIF5 interacts with neuronal granules, the localization of KIF5A overlapped with the cytoplasmic granules in differentiated RGC-5 cells. Thus, our study strongly suggests that the cytoplasmic granule containing HERMES, NonO, PSF, and G3BP1 is a neuronal RNA-protein granule that is transported in neurites during retinal differentiation.

DEAD-Box RNA helicases in animal gametogenesis

This review analyzes and summarizes a current knowledge about a role of RNA helicases in the development and maintenance of gametogenesis of eukaryotic organisms. Here we focused on three representatives of RNA helicase family containing the characteristic motifs in the amino acid sequence (DEAD-box) and carrying substantial and conservative functions in the germinal tissues of various species from drosophila to human. There are such proteins as Vasa/DDX4, BelIe/DDX3 and Spindle-E/TDRD9. They are involved in a wide range of activities that are associated with the regulation of transcription, splicing, nuclear export, and especially with the initiation of translation. The expression of genes required for the gametogenesis appears to be regulated mainly at the translational level. RNA helicases are involved in the formation of cytoplasmic RNP granules and in the implementation of gene RNA-silencing. "DEAD-box RNA helicases" that carry highly homologous central domain, which determines their basic biochemical activity in the ATP-dependent unwinding of short RNA duplexes, perform the essential and non-overlapping functions in the germinal tissues.

Depletion of hnRNP A2/B1 overrides the nuclear retention of the HIV-1 genomic RNA

hnRNP A2 is a cellular protein that is important for nucleocytoplasmic and cytosolic trafficking of the HIV-1 genomic RNA. Both hnRNP A2’s interaction with HIV-1 RNA and its expression levels influence the activities of Rev in mediating nucleocytoplasmic export of the HIV-1 genomic RNA. While the lack of Rev expression during HIV-1 gene expression results in nuclear retention of HIV-1 genomic RNA, we show here by fluorescence in situ hybridization and fractionation studies that the genomic RNA translocates to the cytoplasm when hnRNP A2/B1 are depleted from cells. Polyribosome analyses revealed that the genomic RNA was shunted into a cytoplasmic, dense polyribosomal fraction. This fraction contained several RNA-binding proteins involved in viral gene expression and RNA trafficking but did not contain the translation initiation factor, eIF4G1. Amino acid incorporation into nascent polypeptides in this fraction was also greatly reduced, demonstrating that this fraction contains mRNAs that are poorly translated. These results demonstrate that hnRNP A2/B1 expression plays roles in the nuclear retention of the HIV-1 genomic RNA in the absence of Rev and in the release of the genomic RNA from translationally inactive, cytoplasmic RNP complexes.

Biomass Conversion Inhibitors Furfural and 5-Hydroxymethylfurfural Induce Formation of Messenger RNP Granules and Attenuate Translation Activity in Saccharomyces cerevisiae

Various forms of stress can cause an attenuation of bulk translation activity and the accumulation of nontranslating mRNAs into cytoplasmic messenger RNP (mRNP) granules termed processing bodies (P-bodies) and stress granules (SGs) in eukaryotic cells. Furfural and 5-hydroxymethylfurfural (HMF), derived from lignocellulosic biomass, inhibit yeast growth and fermentation as stressors. Since there is no report regarding their effects on the formation of cytoplasmic mRNP granules, here we investigated whether furfural and HMF cause the assembly of yeast P-bodies and SGs accompanied by translational repression. We found that furfural and HMF cause the attenuation of bulk translation activity and the assembly of cytoplasmic mRNP granules in Saccharomyces cerevisiae. Notably, a combination of furfural and HMF induced the remarkable repression of translation initiation and SG formation. These findings provide new information about the physiological effects of furfural and HMF on yeast cells, and also suggest the potential usefulness of cytoplasmic mRNP granules as a warning sign or index of the deterioration of cellular physiological status in the fermentation of lignocellulosic hydrolysates.

A novel 65 kDa RNA-binding protein in squid presynaptic terminals

A polyclonal antibody (C4), raised against the head domain of chicken myosin Va, reacted strongly towards a 65 kDa polypeptide (p65) on Western blots of extracts from squid optic lobes but did not recognize the heavy chain of squid myosin V. This peptide was not recognized by other myosin Va antibodies, nor by an antibody specific for squid myosin V. In an attempt to identify it, p65 was purified from optic lobes of Loligo plei by cationic exchange and reverse phase chromatography. Several peptide sequences were obtained by mass spectroscopy from p65 cut from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. BLAST analysis and partial matching with expressed sequence tags (ESTs) from a Loligo pealei data bank indicated that p65 contains consensus signatures for the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family of RNA-binding proteins. Centrifugation of post mitochondrial extracts from optic lobes on sucrose gradients after treatment with RNase gave biochemical evidence that p65 associates with cytoplasmic RNP complexes in an RNA-dependent manner. Immunohistochemistry and immunofluorescence studies using the C4 antibody showed partial co-labeling with an antibody against squid synaptotagmin in bands within the outer plexiform layer of the optic lobes and at the presynaptic zone of the stellate ganglion. Also, punctate labeling by the C4 antibody was observed within isolated optic lobe synaptosomes. The data indicate that p65 is a novel RNA-binding protein located to the presynaptic terminal within squid neurons and may have a role in synaptic localization of RNA and its translation or processing.

Compartmentalization Directs Assembly of the Signal Recognition Particle

Many ribonucleoprotein complexes assemble stepwise in distinct cellular compartments, a process that usually involves bidirectional transport of both RNA and proteins between the nucleus and cytoplasm. The biological rationale for such complex transport steps in RNP assembly is obscure. One important example is the eukaryotic signal recognition particle (SRP), a cytoplasmic RNP consisting of one RNA and six proteins. Prior in vivo studies support an "SRP54-late" assembly model in which all SRP proteins, except SRP54, are imported from the cytoplasm to the nucleus to bind SRP RNA. This partially assembled complex is then exported to the cytoplasm where SRP54 binds and forms the SRP holocomplex. Here we show that native SRP assembly requires segregated and ordered binding by its protein components. A native ternary complex forms in vitro when SRP19 binds the SRP RNA prior to binding by SRP54, which approximates the eukaryotic cellular pathway. In contrast, the presence of SRP54 disrupts native assembly of SRP19, such that two RNA-binding loops in SRP19 misfold. These results imply that SRP54 must be sequestered during early SRP assembly steps, as apparently occurs in vivo, for proper assembly of the SRP to occur. Our findings emphasize that spatial compartmentalization provides an additional level of regulation that prevents competition among components and can function to promote native assembly of the eukaryotic SRP.

Efficient Protein Trafficking Requires Trailer Hitch, a Component of a Ribonucleoprotein Complex Localized to the ER in Drosophila

Translational control of localized messenger mRNAs (mRNAs) is critical for cell polarity, synaptic plasticity, and embryonic patterning. While progress has been made in identifying localization factors and translational regulators, it is unclear how broad a role they play in regulating basic cellular processes. We have identified Drosophila trailer hitch (tral) as a gene that is required for the proper secretion of the dorsal-ventral patterning factor Gurken, as well as the vitellogenin receptor Yolkless. Surprisingly, biochemical purification of Tral revealed that it is part of a large RNA-protein complex that includes the translation/localization factors Me31B and Cup as well as the mRNAs for endoplasmic reticulum (ER) exit site components. This complex is localized to subdomains of the ER that border ER exit sites. Furthermore, tral is required for normal ER exit site formation. These findings raise exciting new possibilities for how the mRNA localization machinery could interface with the classical secretory pathway to promote efficient protein trafficking in the cell.

Tap and NXT promote translation of unspliced mRNA.

Tap has been proposed to play a role in general mRNA export and also functions in expression of RNA with retained introns that contain the MPMV CTE (constitutive transport element). Tap forms a functional heterodimer with NXT/p15. We have previously demonstrated that unspliced intron-containing CTE RNA is efficiently exported to the cytoplasm in mammalian cells. Here we show that Tap and NXT proteins function together to enhance translation of proteins from the exported CTE RNA. Pulse chase experiments show that Tap/NXT significantly increases the rate of protein synthesis. Sucrose gradient analysis demonstrates that Tap and NXT efficiently shift the unspliced RNA into polyribosomal fractions. Furthermore, Tap, but not NXT is detected in polyribosomes. Taken together, our results indicate that Tap and NXT serve a role in translational regulation of RNA after export to the cytoplasm. They further suggest that Tap/NXT may play a role in remodeling of cytoplasmic RNP complexes, providing a link between export pathways and cytoplasmic fate.

Sea urchin small RNA ribonucleoprotein particles: identification, synthesis, and subcellular localization during early embryonic development.

Small RNAs in sea urchins were examined in order to characterize developmental changes in their level, subcellular localization, synthesis, and association with proteins and other RNAs. Small RNAs such as the U snRNAs, 5S and 5.8S rRNAs, and 7S RNAs were identified by their mobility on highly cross-linked acrylamide gels. In addition, 7SL and U1 RNAs were identified by northern blot hybridization to cloned human and sea urchin probes, respectively. The level, subcellular localization, and association with proteins or RNA do not change for most small RNAs from fertilization to blastula, even though this is the time when the stored maternal pool of many small RNAs is being supplemented and replaced by embryonically synthesized RNAs. New embryonic synthesis of small RNAs was first detected at the 8-12 hr blastula stage. Although the predicted subsets of the total small RNA pool can be found in the appropriate subcellular compartments, newly synthesized small RNAs have a predominantly cytoplasmic localization: All of the newly synthesized small RNAs were found to be constituents of small RNPs. The RNPs containing newly synthesized small RNAs had sedimentation rates indistinguishable from their maternal counterparts. Thus, on the basis of sedimentation rate, no gross differences could be detected between maternal and embryonic small RNP pools. These small RNPs include a cytoplasmic RNP containing newly synthesized U1 snRNA and the sea urchin signal recognition particle (SRP) containing the 7SL, RNA. We have also identified a small RNP bearing the 5S rRNA which is present in both eggs and embryos. The presence of multiple, abundant, small RNAs and RNPs that are maintained at constant levels in particular subcellular fractions throughout development suggests that small RNAs may be involved in many more cellular activities than have so far been described.

Vaults: Large cytoplasmic RNP's that associate with cytoskeletal elements

Vaults: Large cytoplasmic RNP's that associate with cytoskeletal elements

Prosomes discriminate between mRNA of adenovirus-infected and uninfected HeLa cells

Prosomes are small cytoplasmic RNP complexes associated with repressed mRNA. In in vitro translation, they discriminate between the mRNA of adenovirus-infected HeLa cells and those of uninfected cells grown under normal conditions. Prosomes as well as their RNA constituents interact much more strongly with poly(A) + mRNA of infected cells and inhibit their translation in vitro preferentially. A possible role of prosomes in the differential regulation of translation is discussed.

Prosomes, small cytoplasmic RNP particles, contain glycoproteins

Prosomes, ubiquitous small ribonucleoprotein complexes, were isolated from the cytoplasm of erythropoietic mouse cells induced by Friend leucemia virus. We present evidence that some of the prosomal proteins are glycosylated. Specific reactions with the biotinylated lectins concanavalin agglutinin (Con A), Solanum tuberosum agglutinin (STA) and Limulus polyphemus agglutinin (LPA) indicate that the carbohydrate moieties contain N-acetylneuraminic acid, N-acetylglucosamine and mannosyl- or glucosyl-residues. Glycosylation of prosomal proteins could explain the resistance of prosomes to proteinase K digestion.

Histone H4 mRNA is stored as a small cytoplasmic RNP during the G 2 phase in Physarum polycephalum

In Physarum polycephalum the triggering of histone H4 gene transcription occurs in G2 phase. The rate of synthesis of histone H4 mRNA was measured by in vivo pulse-labeling experiments. We show that it begins to increase in mid-G2. During the second part of G2 it increases approximately 20 fold over its minimum value and reaches a maximum at the end of G2. After entry of the cells in S, histone H4 gene transcription rate begins to decrease and reaches a minimum value in early G2. The histone H4 mRNA which accumulates in G2 is not translated immediately into proteins but is stored in an inactive form until the beginning of the next S phase. Immediately after its transcription the H4 mRNA is transported to the cytoplasm where it is stored and stabilized as an inactive mRNP complex. This was shown by fractionation of cytoplasmic RNP in sucrose gradients and blot hybridization of subcellular fractions.