The chloroplastic DEVH‐box RNA helicase INCREASED SIZE EXCLUSION LIMIT 2 involved in plasmodesmata regulation is required for group II intron splicing

INCREASED SIZE EXCLUSION LIMIT 2 (ISE2) encodes a putative DEVH‐box RNA helicase originally identified through a genetic screening for Arabidopsis mutants altered in plasmodesmata (PD) aperture. Depletion of ISE2 also affects chloroplasts activity, decreases accumulation of photosynthetic pigments and alters expression of photosynthetic genes. In this work, we show the chloroplast localization of ISE2 and decipher its role in plastidic RNA processing and, consequently, PD function. Group II intron‐containing RNAs from chloroplasts exhibit defective splicing in ise2 mutants and ISE2‐silenced plants, compromising plastid viability. Furthermore, RNA immunoprecipitation suggests that ISE2 binds in vivo to several splicing‐regulated RNAs. Finally, we show that the chloroplast clpr2 mutant (defective in a subunit of a plastidic Clp protease) also exhibits abnormal PD function during embryogenesis, supporting the idea that chloroplast RNA processing is required to regulate cell–cell communication in plants.     

Recruitment of the RNA helicase RHAU to stress granules via a unique RNA-binding domain

In response to environmental stress, the translation machinery of cells is reprogrammed. The majority of actively translated mRNAs are released from polysomes and driven to specific cytoplasmic foci called stress granules (SGs) where dynamic changes in protein-RNA interaction determine the subsequent fate of mRNAs. Here we show that the DEAH box RNA helicase RHAU is a novel SG-associated protein. Although RHAU protein was originally identified as an AU-rich element-associated protein involved in urokinase-type plasminogen activator mRNA decay, it was not clear whether RHAU could directly interact with RNA. We have demonstrated that RHAU physically interacts with RNA in vitro and in vivo through a newly identified N-terminal RNA-binding domain, which was found to be both essential and sufficient for RHAU localization in SGs. We have also shown that the ATPase activity of RHAU plays a role in the RNA interaction and in the regulation of protein retention in SGs. Thus, our results show that RHAU is the fourth RNA helicase detected in SGs, after rck/p54, DDX3, and eIF4A, and that its association with SGs is dynamic and mediated by an RHAU-specific RNA-binding domain.     

RNA解旋酶和应激颗粒

应激颗粒（stress granules, SGs）是细胞在环境压力刺激下停止蛋白质翻译后,mRNA与多种细胞蛋白组装而成的胞质颗粒结构.RNA 解旋酶家族作为生物体内普遍存在的一类高度保守的蛋白质酶类,参与了RNA代谢各个环节,近年来其家族成员被陆续发现是一类新的SG重要组分.本文综述了RNA解旋酶参与应激颗粒形成过程,RNA解旋酶家族蛋白的结构和其参与应激颗粒形成的研究进展.     

Modulation of stress granules and P bodies during dicistrovirus infection

Stress granules (SGs) are dynamic cytosolic aggregates composed of ribonucleoproteins that are induced during cellular stress when protein synthesis is inhibited. The function of SGs is poorly understood, but they are thought to be sites for reorganizing mRNA and protein. Several viruses can modulate SG formation, suggesting that SGs have an impact on virus infection. In this study, we have investigated the relationship of SG formation in Drosophila S2 cells infected by cricket paralysis virus (CrPV), a member of the Dicistroviridae family. Despite a rapid shutoff of host translation during CrPV infection, several hallmark SG markers such as the Drosophila TIA-1 and G3BP (RasGAP-SH3-binding protein) homologs, Rox8 and Rin, respectively, do not aggregate in CrPV-infected cells, even when challenged with potent SG inducers such as heat shock, oxidative stress, and pateamine A treatment. Furthermore, we demonstrate that a subset of P body markers become moderately dispersed at late times of infection. In contrast, as shown by fluorescent in situ hybridization, poly(A)(+) RNA granules still form at late times of infection. These poly(A)(+) RNA granules do not contain viral RNA nor do they colocalize with P body markers. Finally, our results demonstrate that the CrPV viral 3C protease is sequestered to SGs under cellular stress but not during virus infection. In summary, we propose that dicistrovirus infection leads to the selective inhibition of distinct SGs so that viral proteins are available for viral processing.     

Inhibition of cytoplasmic mRNA stress granule formation by a viral proteinase

Mammalian cells form dynamic cytoplasmic mRNA stress granules (SGs) in response to environmental stresses including viral infections. SGs are involved in regulating host mRNA function and metabolism, although their precise role during viral infection is unknown. SGs are thought to assemble based on functions of the RNA-binding proteins TIA-1/TIAR or Ras-GAP SH3 domain-binding protein (G3BP). Here, we investigated the relationship between a prototypical plus-strand RNA virus and SGs. Early during poliovirus infection, SG formation is induced, but as infection proceeds this ability is lost, and SGs disperse. Infection resulted in cleavage of G3BP, but not TIA-1 or TIAR, by poliovirus 3C proteinase. Expression of a cleavage-resistant G3BP restored SG formation during poliovirus infection and significantly inhibited virus replication. These results elucidate a mechanism for viral interference with mRNP metabolism and gene regulation and support a critical role of G3BP in SG formation and restriction of virus replication.     

Hsp90 Regulates the Function of Argonaute 2 and Its Recruitment to Stress Granules and P-Bodies

Argonaute proteins are effectors of RNA interference that function in the context of cytoplasmic ribonucleoprotein complexes to regulate gene expression. Processing bodies (PBs) and stress granules (SGs) are the two main types of ribonucleoprotein complexes with which Argonautes are associated. Targeting of Argonautes to these structures seems to be regulated by different factors. In the present study, we show that heat-shock protein (Hsp) 90 activity is required for efficient targeting of hAgo2 to PBs and SGs. Furthermore, pharmacological inhibition of Hsp90 was associated with reduced microRNA- and short interfering RNA-dependent gene silencing. Neither Dicer nor its cofactor TAR RNA binding protein (TRBP) associates with PBs or SGs, but interestingly, protein activator of the double-stranded RNA-activated protein kinase (PACT), another Dicer cofactor, is recruited to SGs. Formation of PBs and recruitment of hAgo2 to SGs were not dependent upon PACT (or TRBP) expression. Together, our data suggest that Hsp90 is a critical modulator of Argonaute function. Moreover, we propose that Ago2 and PACT form a complex that functions at the level of SGs.     

Stress Granule Formation is One of the Early Antiviral Mechanisms for Host Cells Against Coxsackievirus B Infection

Stress granules (SGs) are intracellular granules formed when cellular translation is blocked and have been reported to be involved in a variety of viral infections. Our previous studies revealed that SGs are involved in the coxsackievirus B (CVB) infection process, but the role of SGs in CVB infection has not been fully explored. In this study, we found that CVB type 3 (CVB3) could induce SG formation in the early phase of infection. Results showed that levels of CVB3 RNA and protein were significantly inhibited during the early stage of CVB3 infection by the elevated formation of SGs, while viral RNA and protein synthesis were significantly promoted when SG formation was blocked. Our findings suggest that SG formation is one of the early antiviral mechanisms for host cells against CVB infection.     

Poliovirus unlinks TIA1 aggregation and mRNA stress granule formation

In response to environmental stress and viral infection, mammalian cells form foci containing translationally silenced mRNPs termed stress granules (SGs). As aggregates of stalled initiation complexes, SGs are defined by the presence of translation initiation machinery in addition to mRNA binding proteins. Here, we report that cells infected with poliovirus (PV) can form SGs early that contain T-cell-restricted intracellular antigen 1 (TIA1), translation initiation factors, RNA binding proteins, and mRNA. However, this response is blocked as infection progresses, and a type of pseudo-stress granule remains at late times postinfection and contains TIA but lacks translation initiation factors, mRNA binding proteins, and most polyadenylated mRNA. This result was observed using multiple stressors, including viral infection, oxidative stress, heat shock, and endoplasmic reticulum stress. Multiple proteins required for efficient viral internal ribosome entry site-dependent translation are localized to SGs under stress conditions, providing a potential rationale for the evolution and maintenance of the SG inhibition phenotype. Further, the expression of a noncleavable form of the RasGAP-SH3 domain binding protein in PV-infected cells enables SGs whose constituents are consistent with the presence of stalled 48S translation preinitiation complexes to persist throughout infection. These results indicate that in poliovirus-infected cells, the functions of TIA self-aggregation and aggregation of stalled translation initiation complexes into stress granules are severed, leading to novel foci that contain TIA1 but lack other stress granule-defining components.     

Plant stress granules and mRNA processing bodies are distinct from heat stress granules

Similar to the situation in mammalian cells and yeast, messenger ribonucleo protein (mRNP) homeostasis in plant cells depends on rapid transitions between three functional states, i.e. translated mRNPs in polysomes, stored mRNPs and mRNPs under degradation. Studies in mammalian cells showed that whenever the dynamic exchange of the components between these states is disrupted, stalled mRNPs accumulate in cytoplasmic aggregates, such as stress granules (SGs) or processing bodies (PBs). We identified PBs and SGs in plant cells by detection of DCP1, DCP2 and XRN4, as marker proteins for the 5'-->3' mRNA degradation pathway, and eIF4E, as well as the RNA binding proteins RBP47 and UBP1, as marker proteins for stored mRNPs in SGs. Cycloheximide-inhibited translation, stress treatments and mutants defective in mRNP homeostasis were used to study the dynamic transitions of mRNPs between SGs and PBs. SGs and PBs can be clearly discriminated from the previously described heat stress granules (HSGs), which evidently do not contain mRNPs. Thus, the role of HSGs as putative mRNP storage sites must be revised.     

Activation of Stress Response Pathways Promotes Formation of Antiviral Granules and Restricts Virus Replication

The formation of protein-RNA granules is a part of both natural cellular function (P-bodies and nuclear HNRNPs) and the response to cellular stress (stress granules and ND10 bodies). To better understand the role of stress-induced granules in viral infection, we have studied the ability of cells to restrict poxvirus replication through the formation of antiviral granules (AVGs). Of cells infected with a wild-type poxvirus, a small number spontaneously formed AVGs. In these AVG-positive cells, viral gene expression was inhibited. The addition of compounds that altered RNA helicase activity, induced oxidative stress, or stimulated translation initiation factor phosphorylation significantly increased the number of AVG-positive cells. When AVGs formed, both viral translation and titers were decreased even when host translation persisted. Treatment with the antiviral compound isatin β-thiosemicarbazone (IBT), a compound that was used to treat smallpox infections, induced AVGs, suggesting a role for these structures in the pharmacological inhibition of poxvirus replication. These findings provide evidence that AVGs are an innate host response that can be exogenously stimulated to combat virus infection. Since small molecules are able to stimulate AVG formation, it is a potential target for new antiviral development.     

Identification of a developmental transition in plasmodesmatal function during embryogenesis in Arabidopsis thaliana

Plasmodesmata provide routes for communication and nutrient transfer between plant cells by interconnecting the cytoplasm of adjacent cells. A simple fluorescent tracer loading assay was developed to monitor patterns of cell-to-cell transport via plasmodesmata specifically during embryogenesis. A developmental transition in plasmodesmatal size exclusion limit was found to occur at the torpedo stage of embryogenesis in Arabidopsis; at this time, plasmodesmata are down-regulated, allowing transport of small (approx. 0.5 kDa) but not large (approx. 10 kDa) tracers. This assay system was used to screen for embryo-defective mutants, designated increased size exclusion limit of plasmodesmata (ise), that maintain dilated plasmodesmata at the torpedo stage. The morphology of ise1 and ise2 mutants discussed here resembled that of the wild-type during embryo development, although the rate of their embryogenesis was slower. The ISE1 gene was mapped to position 13 cM on chromosome I using PCR-based biallelic markers. ise2 was found to be allelic to the previously characterized mutant emb25 which maps to position 100 cM on chromosome I. The results presented have implications for intercellular signaling pathways that regulate embryonic development, and furthermore represent the first attempt to screen directly for mutants of Arabidopsis with altered size exclusion limit of plasmodesmata.     

Direct binding of the Alu binding protein dimer SRP9/14 to 40S ribosomal subunits promotes stress granule formation and is regulated by Alu RNA

Stress granules (SGs) are formed in response to stress, contain mRNAs, 40S ribosomal subunits, initiation factors, RNA-binding and signaling proteins, and promote cell survival. Our study describes a novel function of the protein heterodimer SRP9/14 and Alu RNA in SG formation and disassembly. In human cells, SRP9/14 exists assembled into SRP, bound to Alu RNA and as a free protein. SRP9/14, but not SRP, localizes to SGs following arsenite or hippuristanol treatment. Depletion of the protein decreases SG size and the number of SG-positive cells. Localization and function of SRP9/14 in SGs depend primarily on its ability to bind directly to the 40S subunit. Binding of SRP9/14 to 40S and Alu RNA is mutually exclusive indicating that the protein alone is bound to 40S in SGs and that Alu RNA might competitively regulate 40S binding. Indeed, by changing the effective Alu RNA concentration in the cell or by expressing an Alu RNA binding-defective protein we were able to influence SG formation and disassembly. Our findings suggest a model in which SRP9/14 binding to 40S promotes SG formation whereas the increase in cytoplasmic Alu RNA following stress promotes disassembly of SGs by disengaging SRP9/14 from 40S.     

Analysis of stress granule assembly in Schizosaccharomyces pombe

Stress granules (SGs) are cytoplasmic aggregates of RNA and proteins in eukaryotic cells that are rapidly induced in response to environmental stress, but are not seen in cells growing under favorable conditions. SGs have been primarily studied in mammalian cells. The existence of SGs in the fission yeast and the distantly related budding yeast was demonstrated only recently. In both species, they contain many orthologs of the proteins seen in mammalian SGs. In this study, we have characterized these proteins and determined their involvement in the assembly of fission yeast SGs, in particular, the homolog of human G3BP proteins. G3BP interacts with the deubiquitinating protease USP10 and plays an important role in the assembly of SGs. We have also identified Ubp3, an ortholog of USP10, as an interaction partner of the fission yeast G3BP-like protein Nxt3 and required for its stability. Under thermal stress, like their human orthologs, both Nxt3 and Ubp3 rapidly relocalize to cytoplasmic foci that contain the SG marker poly(A)-binding protein Pabp. However, in contrast to G3BP1 and USP10, neither deletion nor overexpression of nxt3(+) or ubp3(+) affected the assembly of fission yeast SGs as judged by the relocalization of Pabp. Similar results were observed in mutants defective in orthologs of SG components that are known to affect SG assembly in human and in budding yeast, such as ataxia-2 and TIA-like proteins. Together, our data indicate that despite similar protein compositions, the underlying molecular mechanisms for the assembly of SGs could be distinct between species.     

Polyamines inhibit the assembly of stress granules in normal intestinal epithelial cells regulating apoptosis

Polyamines regulate multiple signaling pathways and are implicated in many aspects of cellular functions, but the exact molecular processes governed by polyamines remain largely unknown. In response to environmental stress, repression of translation is associated with the assembly of stress granules (SGs) that contain a fraction of arrested mRNAs and are thought to function as mRNA storage. Here we show that polyamines modulate the assembly of SGs in normal intestinal epithelial cells (IECs) and that induced SGs following polyamine depletion are implicated in the protection of IECs against apoptosis. Increasing the levels of cellular polyamines by ectopic overexpression of the ornithine decarboxylase gene decreased cytoplasmic levels of SG-signature constituent proteins eukaryotic initiation factor 3b and T-cell intracellular antigen-1 (TIA-1)-related protein and repressed the assembly of SGs induced by exposure to arsenite-induced oxidative stress. In contrast, depletion of cellular polyamines by inhibiting ornithine decarboxylase with α-difluoromethylornithine increased cytoplasmic eukaryotic initiation factor 3b and TIA-1 related protein abundance and enhanced arsenite-induced SG assembly. Polyamine-deficient cells also exhibited an increase in resistance to tumor necrosis factor-α/cycloheximide-induced apoptosis, which was prevented by inhibiting SG formation with silencing SG resident proteins Sort1 and TIA-1. These results indicate that the elevation of cellular polyamines represses the assembly of SGs in normal IECs and that increased SGs in polyamine-deficient cells are crucial for increased resistance to apoptosis.     

Analysis of RNA associated with P granules in germ cells of C. elegans adults

P granules are cytoplasmic structures of unknown function that are associated with germ nuclei in the C. elegans gonad, and are localized exclusively to germ cells, or germ cell precursors, throughout the life cycle. All the known protein components of P granules contain putative RNA-binding motifs, suggesting that RNA is involved in either the structure or function of the granules. However, no specific mRNAs have been identified within P granules in the gonad. We show here that P granules normally contain a low level of RNA, and describe conditions that increase this level. We present evidence that several, diverse mRNAs, including pos-1, mex-1, par-3, skn-1, nos-2 and gld-1 mRNA, are present at least transiently within P granules. In contrast, actin and tubulin mRNA and rRNA are either not present in P granules, or are present at relatively low levels. We show that pgl-1 and the glh (Vasa-related) gene family, which encode protein components of P granules, do not appear essential for RNA to concentrate in P granules; these proteins may instead function in events that are a prerequisite for RNAs to be transported efficiently from the nuclear surface.