InteractiveFly: GeneBrief

rasputin: Biological Overview | References

Gene name - rasputin

Synonyms - G3BP

Cytological map position - 87F6-87F7

Function - RNA-binding protein

Keywords - component of stress granules - interacts with several protein partners under both stress and non-stress conditions including Caprin, (FMR1 and Lingerer - Sec16, a component of the endoplasmic reticulum exit site, is a Rasputin interactor and stabilizer - a positive regulator of orb in oogenesis - FMR1, Rasputin and Caprin act together with the UBA protein Lingerer to restrict tissue growth

Symbol - rin

FlyBase ID: FBgn0015778

Genetic map position - chr3R:13,647,004-13,652,413

Classification - Nuclear transport factor 2 (NTF2) domain, RRM_G3BP: RNA recognition motif (RRM) in ras GTPase-activating protein-binding protein G3BP1, G3BP2 and similar proteins

Cellular location - cytoplasmic

NCBI link: EntrezGene, Nucleotide, Protein
rin orthologs: Biolitmine

G3BP RNA-binding proteins are important components of stress granules (SGs). This study analyze the role of the Drosophila G3BP Rasputin (RIN) in unstressed cells, where RIN is not SG associated. Immunoprecipitation followed by microarray analysis identifies over 550 mRNAs that copurify with RIN. The mRNAs found in SGs are long and translationally silent. In contrast, it was found that RIN-bound mRNAs, which encode core components of the transcription, splicing, and translation machinery, are short, stable, and highly translated. RIN was shown to be associated with polysomes and evidence was provided for a direct role for RIN and its human homologs in stabilizing and upregulating the translation of their target mRNAs. It is proposed that when cells are stressed, the resulting incorporation of RIN/G3BPs into SGs sequesters them away from their short target mRNAs. This would downregulate the expression of these transcripts, even though they are not incorporated into stress granules (Laver, 2020).

Post-transcriptional regulation (PTR) plays a key role in the control of gene expression in all cell types. PTR is achieved by RNA-binding proteins (RBPs) and small RNAs, such as microRNAs (miRNAs), which act as specificity factors that modulate the interaction of mRNAs with the cellular machinery that localizes, translates, and degrades mRNAs (Laver, 2020).

PTR is particularly important in early animal embryos, where maternally provided mRNAs and proteins control developmental events prior to transcriptional activation of the embryo's genome. In several model animals, including Drosophila, the transfer of control from maternal products to those synthesized by the embryo's own genome -- the maternal-to-zygotic transition (MZT) -- is very rapid, occurring over a matter of hours, and thus facilitating studies of the mechanisms and functions of PTR. For example, it has been shown that the Drosophila RBP Smaug (SMG), which binds to specific stem-loop structures in its target mRNAs to repress their translation and trigger their degradation, is essential for repression and clearance of hundreds of maternal mRNAs and for timely activation of the zygotic genome. SMG is not the only negative regulator of maternal transcripts in Drosophila; additional RBPs (e.g., brain tumor) or miRNAs (e.g., miR-309) function in maternal mRNA clearance (Laver, 2020).

PTR also serves as a rapid response to cellular stress. Under stress conditions, cells shut down translation of many mRNAs while upregulating transcription and/or translation of sets of protein chaperones that maintain basal cellular integrity. Repression occurs, at least in part, in membraneless organelles known as stress granules (SGs). SGs are thought to contain transcripts that are stalled in translation initiation, and recent global analyses have shown that SGs are enriched for long transcripts (Laver, 2020).

An important component of SGs is the RBP G3BP, which is conserved throughout eukaryotes. Mammals have two genes, G3BP1 and G3BP2, whereas in Drosophila there is a single gene, Rasputin (rin). In human cells, G3BPs are necessary for SG formation, and if overexpressed, they are sufficient to induce SGs even in the absence of stress. RIN is necessary for SG formation in the Drosophila S2 tissue culture cell line, and although RIN or G3BP overexpression can induce SGs in human cells, this is not the case in S2 cells (Aguilera-Gomez, 2017). RIN and G3BPs interact with several of the same protein partners under both stress and non-stress conditions; these include Caprin (CAPR), FMRP (FMR1 in Drosophila), and UPA2 (Lingerer [LIG] in Drosophila) (Aguilera-Gomez, 2017; Baumgartner, 2013; Costa, 2013; Jain, 2016; Kedersha, 2016; Markmiller, 2018; Youn, 2018; Laver, 2020).

The roles of RIN/G3BPs in unstressed cells have received less attention than their roles upon stress. Multiple functions have been attributed to G3BPs (reviewed in Alam, 2019), including transcript destabilization and repression (e.g., c-myc, BART, CTNNB1, PMP22, HIV-1, and miR-1), transcript stabilization (e.g., Tau and SART3), subcellular transcript localization (e.g., Twist1), and transcript sequestration into virus- induced foci (e.g., HIV-1). In Drosophila, mutations in the rin gene cause severe defects in oogenesis, mutant females lay few eggs, and those that are laid fail to hatch (Costa, 2013). rin mutations can also result in tissue patterning and growth defects (Baumgartner, 2013; Pazman, 2000). Despite RIN's essential function in the fly life cycle, there have been no analyses of the RIN-bound transcriptome or RIN's global role in gene regulation, nor are the molecular mechanisms that underlie RIN function known (Laver, 2020).

To better understand the function of RIN/G3BP in unstressed cells, a global analyses of the RIN-associated proteome and transcriptome was carried out in early Drosophila embryos. Using an anti-RIN synthetic antibody that was isolated from a phagedisplayed library of fragments antigen binding (Fab), RIN was immunoprecipitated and then mass spectrometry (IP-MS) was carried out to identify RIN's partner proteins. Interactions were found with several RBPs previously shown to interact with G3BP/RIN (e.g., CAPR, FMR1, and LIG), consistent with IP of a biologically relevant RIN-containing complex. RNA-dependent interactions with RIN were found for both small and large ribosomal subunit proteins, suggesting that RIN may be polysome associated, a fact that that was confirmed using polysome gradients. By coimmunoprecipitating RIN together with bound mRNAs followed by microarray analysis, hundreds of in vivo target transcripts were identified in embryos that are characterized by two features: they are short and enriched for a binding motif that was previously identified in vitro. RIN-associated mRNAs are enriched for Gene Ontology (GO) terms for core components of the transcription, splicing, and translation machinery as well as of mitochondria. RIN's endogenous targets in early embryos are more stable and more highly translated than co-expressed unbound transcripts. Their shorter length and higher rates of translation contrast with the behavior of mRNAs associated with SGs. Consistent with a role for RIN as a positive regulator of transcript stability, in rin mutants, the abundance of several highly bound target mRNAs is reduced relative to controls. Using a heterologous RNA-binding domain to tether RIN, G3BP1, or G3BP2 to a luciferase reporter mRNA in S2 tissue culture cells, it was confirmed that RIN/G3BP increases the stability and/or translation of bound transcripts in the absence of stress (Laver, 2020).

The data support a conserved function for G3BP proteins as potentiators of the translation and stability of their target transcripts. It is speculated that stress-dependent recruitment of G3BPs/RIN into SGs may serve as a mechanism to downregulate gene expression both directly, by removing RIN from its endogenous target mRNAs, as well as indirectly, through reduced transcription, splicing, and translation (Laver, 2020).

Two features, short transcript length and the motif bound by the RIN RRM in vitro, are predictive of RIN binding. That short transcripts in general might be more likely to contain the motif is excluded by the fact that RIN's target mRNAs are enriched for the motif compared with length-matched, co-expressed, unbound mRNAs. Thus, it is proposed that each feature separately contributes to RIN target mRNA binding, with shortness playing a greater role than the motif (Laver, 2020).

Although it is unclear how RIN is able to measure transcript length, there is a precedent for the differential behavior and regulation of short versus long mRNAs. For example, in general, short mRNAs are more highly translated than long mRNAs, and this is thought to reflect the fact that short mRNAs have a higher affinity for the cap-binding complex. It has been proposed that this higher affinity is related to the possibility that short mRNAs are able to form a closed-loop structure more readily than longer mRNAs. It is noted that a recent study that calls the closed-loop model into question was unable to test short mRNAs because of technical limitations. Thus, even if long mRNAs do not form a stable closed loop, it remains possible that short mRNAs do (Laver, 2020).

The ribosome-associated protein RACK1 has been shown to be required for the efficient translation of short but not long mRNAs. Although this study has shown that RIN is associated with polysomes in early embryos, Drosophila RACK1 is not on the list of RIN protein interactors and neither is there a significant overlap between the RIN protein interaction network and a set of protein interactors previously identified for RACK1. Thus, the mechanisms by which RIN recognizes short mRNAs may differ from those that have been identified previously (Laver, 2020).

Another striking feature of RIN-bound mRNAs is that they are depleted for SREs, the binding sites of SMG, which destabilizes and translationally represses its target mRNAs. This could suggest that the high translational efficiencies and stability of RIN-target mRNAs is simply a result of a lack of SREs. However, this study has provided evidence that RIN can exert its effects in situations where the SMG protein is not present (i.e., during oogenesis, in embryos older than 3 h, and in S2 cells). Thus, it is concluded that most RIN-bound mRNAs in the early embryo are upregulated directly by RIN and indirectly through a lack of SREs (Laver, 2020).

Because the target mRNAs of RIN are enriched for GO terms related to multiple levels of the core gene expression machinery-transcription, splicing, and translation-RIN may directly potentiate the expression of its bound target transcripts and, in so doing, also indirectly upregulate gene expression globally. As an example of how RIN/G3BP's direct and indirect effects might converge on the same cellular process, the production of cytoplasmic ribosomes is considered. Metabolic labeling with radioactive amino acids has shown that cytoplasmic ribosomal protein (cRP) synthesis increases after fertilization, peaks at 3-4 h, and subsequently decreases. Recent ribosome occupancy-based measurements have confirmed that the translational efficiency of cRP mRNAs increases in early embryos relative to mature oocytes. This study has shown that RIN potentiates target mRNA stability and translation and that cRP mRNAs are highly enriched among RIN's targets; thus, the potentiation of cRP mRNAs is expected to be a direct effect of RIN (Laver, 2020).

cRP mRNAs are also regulated by their conserved 50-terminal oligopyrimidine (50TOP) motifs. Two RBPs, La and Larp1, have been implicated in regulation of the stability and/or translation of 50TOP mRNAs by this motif. This study shows that the Drosophila orthologs of both of these RBPs-LA and LARP-associate with RIN in an RNA-dependent manner. This could reflect the fact that LA, LARP, and RIN co-bind cRP mRNAs and, thus, that this class of mRNAs is subject to multiple direct mechanisms that potentiate its expression. Noteworthy is the fact that, of the RIN target mRNAs, only the cRP transcripts carry 50TOP motifs and, as such, they represent a distinct class of mRNAs (Laver, 2020).

Phospho-Rasputin stabilization by Sec16 is required for stress granule formation upon amino acid starvation

Most cellular stresses induce protein translation inhibition and stress granule formation. Stress granules are well-studied, cytoplasmic reversible, pro-survival stress assemblies where untranslated free RNAs (resulting from protein translation inhibition) are stored and protected together with RNA-binding proteins, translation initiation factors, and the 40S ribosomal subunits. This study used Drosophila S2 cells to investigate the role of G3BP/Rasputin in this process. In contrast to arsenite treatment, where dephosphorylated Ser142 Rasputin is recruited to stress granules, this study found that, upon amino acid starvation, only the phosphorylated Ser142 form is recruited. Furthermore, Sec16, a component of the endoplasmic reticulum exit site, was identified as a Rasputin interactor and stabilizer. Sec16 depletion results in Rasputin degradation and inhibition of stress granule formation. However, in the absence of Sec16, pharmacological stabilization of Rasputin is not enough to rescue the assembly of stress granules. This is because Sec16 specifically interacts with phosphorylated Ser142 Rasputin, the form required for stress granule formation upon amino acid starvation. Taken together, these results demonstrate that stress granule formation is fine-tuned by specific signaling cues that are unique to each stress. These results also expand the role of Sec16 as a stress response protein (Aguilera-Gomez, 2017).

Stress granules are well-studied, cytoplasmic reversible, pro-survival stress assemblies where untranslated free RNAs (resulting from protein translation inhibition) are stored and protected together with RNA-binding proteins, translation initiation factors, and the 40S ribosomal subunits. Stress granule formation has been best investigated in mammalian cells upon different type of stresses, including heat and oxidative stress. This has led to the identification of a number of factors that are essential for their formation, such as the case for Tia-1 and Ras-GAP SH3 domain-binding protein (G3BP1/2, referred to as G3BP hereafter) (Aguilera-Gomez, 2017).

G3BP was first identified in human cells through co-immunoprecipitation with the SH3 domain of RasGAP. However, it has an RNA recognition motif (RRM) toward the C terminus, suggesting that it binds mRNAs. In growing cells, G3BP is normally cytoplasmic; However, after stress induction (especially stress leading to eIF2α phosphorylation), it is not only readily recruited to stress granules but also is necessary for their formation. Furthermore, G3BP drives stress granule formation when overexpressed in the absence of stress. Importantly, G3BP is phosphorylated on Ser149 during basal conditions, but it needs to be dephosphorylated to drive stress granule assembly triggered by arsenite treatment. Taken together, G3BP is critical for stress granule formation when its Ser149 is dephosphorylated and through its binding to Caprin and the 40S ribosomal subunit. Conversely, G3BP is inhibited when binding to peptidase USP10 (Aguilera-Gomez, 2017).

G3BP also appears to have an important role in disease. First, viruses can exploit G3BP. However, G3BP also appears to slow down HIV replication by leading to the sequestration of viral mRNA. Second, G3BP is overexpressed in gastric cancer and bone and lung sarcomas, where it is considered as a marker for poor survival. Strikingly, downregulation of G3BP in cells and in vivo reduces stress granule formation as expected but also tumor invasion and metastasis, showing a clear role for stress granule formation in cancer. Last, G3BP is a target of TDP-43 that is often mutated, mislocalized, and misaccumulated in amyotrophic lateral sclerosis (ALS) (Aguilera-Gomez, 2017).

Stress granules are also formed in Drosophila, for instance, upon heat stress, arsenite exposure, and amino acid starvation of Drosophila S2 cells. However, the mechanism behind their formation upon this latter stress is not completely understood. Interestingly, amino acid starvation leads to the formation of another recently described stress assembly, the Sec bodies that store and protect most of the COPII subunits and the endoplasmic reticulum exit site (ERES) component Sec16 (Zacharogianni, 2014). Importantly, Sec bodies and stress granules are independent structures that are formed at the same time frame of amino acid starvation (Aguilera-Gomez, 2017).

Sec16 is a conserved peripheral membrane protein that tightly localizes and concentrates to the ERES via a domain that has been mapped to a small arginine-rich region upstream of the conserved central domain. It binds nearly all COPII subunits and controls at least two aspects of COPII-coated vesicle dynamics. Sec16 is essential for endoplasmic reticulum (ER) to Golgi transport, especially in Drosophila where the absence of Sec16 results in a severe inhibition of protein exit from the ER (Aguilera-Gomez, 2017).

Interestingly, Sec16 responds to nutrient stress (Zacharogianni, 2011, Zacharogianni, 2014). In this regard, it has been shown that Sec16 is a key factor driving Sec body formation upon amino acid starvation that activates the ER-localized dPARP16. In turn, dPARP16 mono-ADP-ribosylates Sec16 on a conserved sequence close to its C terminus (Aguilera-Gomez, 2016), and Sec16 modification by dPARP16 is enough to elicit Sec body formation. This demonstrates that Sec16 is a stress response protein that plays an important role in the response to amino acid starvation (Aguilera-Gomez, 2017).

This study shows that the phosphorylation state of the G3BP Drosophila ortholog Rasputin (Rin) is differentially required for the formation of stress granules that are formed upon arsenite treatment and amino acid starvation. Whereas stress granule formation upon arsenite treatment requires the non-phosphorylated form of Rin (as is the case for G3BP in mammalian cells), amino acid starvation requires the phosphorylated form. Furthermore, Sec16 was shown to specifically interact with phosphorylated Rin and mediates this differential requirement (Aguilera-Gomez, 2017).

All together, these results provide a link between the protein transport from the ER and protein translation. It also explains the specific requirement of Sec16 for stress granule formation upon amino acid starvation, but not other stresses. Furthermore, it enlarges the scope of Sec16 function at the ER by identifying yet a new role in the response to amino acid starvation (Aguilera-Gomez, 2017).

Stress granules are formed when protein translation initiation is inhibited by cellular stress that leads to elF2α phosphorylation and the accumulation of untranslated mRNAs. Stress granule components start to coalesce through protein-protein interactions mediated by proteins containing regions of low-complexity sequences and displaying multivalence interactions. This process is facilitated by the presence of accumulating free mRNAs. Accordingly, incubation of stressed cells with cycloheximide, which locks ribosomes to the mRNA, blocks stress granule formation, whereas puromycin stimulates their formation. Stress granule formation is also driven by a number of critical factors, including G3BP. In all these respects, the cytoplasmic foci that are formed in Drosophila cells upon amino acid starvation and that are positive for RNA-binding proteins are bona fide stress granules, as they share many features of those found in mammalian cells after arsenite treatment, heat stress, and ER stress. Furthermore, this study shows that Rin, the G3BP Drosophila ortholog, is an essential factor for amino acid starvation-driven stress granules (Aguilera-Gomez, 2017).

However, stress granule formation displays a certain level of heterogeneity. First, some of the signaling cues inducing their formation appear to be different. For instance, although elF2α phosphorylation is required for arsenite-triggered stress granule formation in Drosophila cells, this phosphorylation is not necessary for their formation upon heat stress. Second, mammalian stress granules that are formed upon different stresses appear to have slightly different content, at least in HAP1 cells. Third, stress granules display different material properties. Yeast stress granules possess a solid core made of components that exchange slowly and less dynamically, whereas mammalian stress granules have liquid droplet properties. This is also reflected by the fact that stress granules contain diverse proteomes (Aguilera-Gomez, 2017).

Remarkably, the phosphorylation status of Rin was shown to dictate its differential recruitment to stress granules formed upon arsenite treatment and amino acid starvation. Phosphorylated Rin (on Ser142) is instrumental for stress granule formation upon amino acid starvation, whereas arsenite-driven stress granules require dephosphorylated Rin (as for G3BP in mammalian cells). This is the first example that clearly demonstrates this differential usage. This further documents that stress granules are more complex and variable than previously anticipated and not just a temporal storage of stalled ribonucleoprotein particles (RNPs) (Aguilera-Gomez, 2017).

What determines the use for phosphoRin versus non-phosphoRin in stress granule formation upon different stresses is not fully understood, but the first clue is the discovery that the large hydrophilic ERES protein Sec16 specifically interacts with phosphoRin (Aguilera-Gomez, 2017).

Sec16 adds to the lengthening list of factors modulating stress granule formation via their interaction with G3BP, such as Caprin, TDP43, and Usp10. In addition, YB-1 promotes the translation of G3BP1 mRNA. Sec16 depletion leads to a reduced level of total Rin, but the remaining Rin pool is enough to lead to stress granule formation upon arsenite treatment, heat stress, and ER stress (Aguilera-Gomez, 2017).

Interestingly, Rin level is further reduced when Sec16-depleted cells are amino acid starved. Thus, Sec16 depletion upon amino acid starvation mimics Rin depletion, explaining the inhibition of stress granule formation upon this stress. This suggests that Sec16 protects Rin against proteasome degradation. It is very often the case that proteins in a complex stabilize each other and that when one partner is absent the other is degraded. However, Rin depletion does not affect Sec16 stability. Sec16 has, therefore, an active role in protecting Rin, perhaps by preventing Rin ubiquitination, a signal for degradation through the proteasome (Aguilera-Gomez, 2017).

However, although proteasome inhibition restores Rin level in starved Sec16-depleted cells, this is not enough to recover stress granule formation. This suggests that Sec16 has an additional role. Given that Sec16 specifically interacts with phosphoRin, this interaction appears necessary not only to protect and stabilize phosphoRin but also to facilitate the role of phosphoRin in stress granule formation upon amino acid starvation. It is possible that the Sec16/phosphoRin complex is recruited to stress granules and/or that Sec16 allows Rin to bind another stress granule partner. In any case, this is strictly specific for amino acid starvation (Aguilera-Gomez, 2017).

Indeed, phosphoRin is the form that is specifically required for stress granule formation upon amino acid starvation. Upon arsenite treatment, the dephosphorylated form of Rin is the form required for stress granule formation both in Drosophila (current results and in mammalian cells: Kedersha, 2016, Tourriere, 2003). This is mirrored by the role of Sec16 that is specifically required for stress granule formation upon amino acid starvation, but not upon heat stress, ER stress, and arsenite treatment. Accordingly, Sec16 does not interact with dephosphorylated Rin (Aguilera-Gomez, 2017).

Does Sec16 have a role in Rin phosphorylation? Rin phosphorylation could take place in the cytoplasm independently of Sec16 that would then recognize phosphoRin and stabilize it. Alternatively, Sec16 could contribute to Rin phosphorylation by acting as a scaffold for the kinase required for Rin phosphorylation. This kinase, which is likely to be (hyper-)activated by amino acid starvation, remains to be identified (Aguilera-Gomez, 2017).

Why does amino acid starvation require this specific Sec16/phosphoRin interaction? Why is an ERES component functionally linked to stress granule formation specifically upon this stress? It is likely that this specific interaction elicits the formation of unique stress granules, perhaps storing mRNAs encoding proteins key for survival upon starvation and fitness upon stress relief. In this respect, stress granules formed during amino acid starvation contain the P-body component Tral that is not found in those formed upon heat shock (Jevtov, 2015). Interestingly, Tral has been shown to bind mRNAs encoding COPII subunits, and it is possible that other mRNAs encoding or secretory pathway components may be sequestered and protected inside these stress granules. This would reflect a type of multiplexing that has been observed in neurons. Conversely, as ER-translated mRNAs (possibly encoding secretory proteins required in stress recovery) are proposed to escape sequestration to stress granules , Sec16 interaction with stress granule components might restrict stress granule formation to specific sites away from these mRNAs (Aguilera-Gomez, 2017).

Last, the enrichment of phosphoRin (and the presence of Sec16) in amino acid starvation-driven stress granules might change their material properties and, consequently, their dynamics. This is suggested by the large size of the S142E-positive stress granules and by their poor reversibility. Consequently, the exchange of components with the surrounding cytoplasm might be reduced in phosphoRin-based stress granules when compared to stress granules that depend on the dephosphorylated form of Rin (and G3BP). This is supported by fluorescence recovery after photobleaching (FRAP) experiments. G3BP-positive arsenite-driven stress granules show full recovery in less than 1 s, whereas the recovery of stress granules formed upon amino acid starvation is an order of magnitude slower. The biological relevance of this difference is, however, not fully understood (Aguilera-Gomez, 2017).

Overall, the results presented in this study are in congruence with evidence of the link among protein translation, RNA metabolism, and the secretory pathway. Stress granules are formed in response to ER stress. P-bodies also localize in close proximity to the ER and increase in number in response to ER homeostasis perturbations and in Arf1 yeast mutant. Last, ER-resident proteins are shown to regulate P-body formation in yeast (Aguilera-Gomez, 2017).

The results provide further evidences of the versatility of Sec16. In growing conditions, mammalian Sec16 exists as two isoforms that are both localized to the ERES but have non-redundant functions in humans. Whereas Sec16A is classically required for the ER exit of proteins destined to the Golgi and the plasma membrane, Sec16B specializes in transport to peroxisomes. Furthermore, Sec16 exons have been shown to be alternatively spliced upon T cell activation, and increased expression of the Sec16 isoform containing exon 29 leads to an increased number of ERESs and more efficient COPII transport in activated T cells. In this regard, Sec16 is also specifically phosphorylated by ERK2 upon serum stimulation in mammalian cells, leading to an increase in the number of ERESs and a larger secretory capacity. Sec16 also interacts with LKKR2, albeit in a kinase activity-independent fashion, and with ULK (Atg1) in non-stressed conditions (Aguilera-Gomez, 2017).

Sec16 also plays key roles in the response to stress, for instance, to ER stress where it appears to mediate the Golgi bypass of transmembrane proteins (Piao, 2017), but also to nutrient stress (Zacharogianni, 2011). Amino acid starvation is an interesting stress as it triggers the formation of two stress assemblies in the same time frame, both requiring Sec16 but in two different manners: The first, the MARylation of Sec16 on its C terminus by ER-localized dPARP16, is an event that is enough to trigger the formation of Sec bodies (Aguilera-Gomez, 2016). The second is the Sec16 interaction and stabilization of phosphoRin, leading to the formation of stress granules. Interestingly, neither of these is linked to the Sec16 role in protein exit from the ER or COPII-coated vesicle dynamics (Zacharogianni, 2014; Aguilera-Gomez, 2017 and references therein).

Taken together, this demonstrates the versatility and capacity of the large scaffold protein Sec16 to regulate very diverse cellular processes, many of them pro-survival. Therefore, more Sec16 interactors need to be identified and studied (Aguilera-Gomez, 2017).

Canonical nucleators are dispensable for stress granule assembly in Drosophila intestinal progenitors

Stressed cells downregulate translation initiation and assemble membrane-less foci termed stress granules (SGs). Extensively characterized in cultured cells, the existence of such structures in stressed adult stem cell pools remain poorly characterized. This study reports that Drosophila orthologs of mammalian SG components AGO1, ATX2, CAPRIN, eIF4E, FMRP, G3BP (Rasputin), LIN-28, PABP, and TIAR are enriched in adult intestinal progenitor cells where they accumulate in small cytoplasmic messenger ribonucleoprotein complexes (mRNPs). Treatment with sodium arsenite or rapamycin reorganized these mRNPs into large cytoplasmic granules. Formation of these intestinal progenitor stress granules (IPSGs) depended on polysome disassembly, led to translational downregulation, and was reversible. While canonical SG nucleators ATX2 and G3BP were sufficient for IPSG formation in the absence of stress, neither of them, nor TIAR, either individually or collectively, were required for stress-induced IPSG formation. This work therefore finds that IPSGs do not assemble via a canonical mechanism, raising the possibility that other stem cell populations employ a similar stress-response mechanism (Buddika, 2020).

This study characterized a population of SGs in Drosophila intestinal progenitor cells whose formation does not require the canonical stress granule nucleators needed in other cell types. A model is proposed describing IPSG formation that is based on three main observations: (1) IPSGs are bona fide SGs because they contain mRNAs and their formation can be blocked and reversed, (2) IPSGs are composed of at least nine conserved proteins that are highly expressed and distributed throughout the cytoplasm of progenitor cells prior to stress and that are known to associate with SGs in other cell types, and (3) IPSGs form even in the absence of three components, the Drosophila orthologs of mammalian ATX2, G3BP and TIA1, that are considered to be integral to SG formation in other cells. It is therefore proposed that following acute stresses, pre-existing mRNP particles aggregate together to form mature SGs, bypassing the role of these internally disorganized region (IDR)-rich proteins in nucleating stable cores needed during SG assembly in other cell types. This model indicates that the initial steps of SG assembly are variable and depend upon the cytoplasmic constituency of cells at resting state. Subsequent steps of IPSG assembly might follow the same progression proposed for SGs, including microtubule-dependent fusion of core mRNPs; super-resolution images of IPSGs indicate that they are not uniform but are rather likely composites of fused mRNPs. Since this study observed a complex and partially overlapping pattern of IPSG proteins in the absence of stress, it was hypothesize that mRNPs in unstressed cells are highly dynamic in nature. The pre-existence of such mRNPs may be an adaptation to the harsh intestinal environment and critical for proper epithelial homeostasis, allowing intestinal progenitor cells to rapidly respond to and recover from constant insult. The conservation of the proteins analyzed in this study raises the possibility that SGs in other cell types, including intestinal stem cells in other animals, may form via a pathway similar to Drosophila IPSGs (Buddika, 2020).

In contrast to IPSGs, other known SGs share a common need for three different IDR-containing proteins: ATX2, G3BP1/2 or TIA1/TIAR. These IDR-containing proteins play a critical role in establishing the core structures of nascent SGs that fuse during mature SG formation. Examples of the requirement of these proteins for SG formation include Drosophila G3BP in S2 cells, human G3BP1, either alone or in combination with its paralog G3BP2, in HEK293T, HeLa and U2OS cell types, Drosophila ATX2 and specifically its C-terminal IDR in S2 cells, and the prion-like domains of mammalian TIA1 in cultured COS7 cells. Despite this general requirement, there is also evidence suggesting possible redundancy between these proteins in some contexts. For example, while G3BP1/2 is required for arsenite-induced SG formation, it is not necessary for SG formation following osmotic stress (i.e. after treatment with NaCl or sorbitol) (Kedersha, 2016; Protter, 2016). Furthermore, while loss of ATX2 or TIA1 severely compromises SG assembly in some mammalian and yeast cells, SGs are not completely eliminated. While these data suggest some redundancy between SG nucleators in some contexts, this possibility has not been previously investigated. Since it was observed that ATX2, RIN and ROX8 are co-expressed in intestinal progenitors, this study directly evaluated this potential functional redundancy using combinations of double and triple mutants of atx2, rin and rox8. This rigorous characterization showed that elimination of these integral SG nucleators, either alone or in combination, had little effect on either the size or number of SGs that form after either arsenite- or rapamycin-induced stress. While subtle defects cannot be completely ruled out, the grossly normal appearance of SGs in triple-mutant intestinal progenitors suggests the existence of a non-canonical mechanism for SG formation in these and potentially other cell types (Buddika, 2020).

This study also found that in a complex heterogenous tissue, such as the adult intestine, SGs selectively form in only a subset of cells. This observation may have been previously overlooked because much of the work on SG assembly has been conducted in homogenous cell culture systems rather than in intact tissues. Even the few previous studies on SG formation in Drosophila tissues has found that SG formation occurs uniformly by most cells throughout stressed tissues including, for example, heat-stressed ovarian follicular epithelia and larval imaginal discs, as well as mechanically stressed adult brain. Unlike the adult intestine, however, which is populated by both an active stem cell contingent as well as terminally differentiated cells, these tissues are all relatively homogenous with respect to the differentiation state of resident cell types. This analysis suggests that terminally differentiated intestinal cell types are refractory to SG formation because key RBPs are downregulated during their differentiation. Future studies investigating the molecular basis for this refractory state are medically relevant, since limiting SG formation could prevent the ectopic formation of pathogenic mRNP aggregates thought to underlie neurodegenerative disease (Buddika, 2020).

Embryonic and some somatic stem cell populations are known to maintain low levels of translational activity. This reduced translational activity helps these stem cells maintain an undifferentiated state, while increased translation drives differentiation. For instance, murine embryonic stem cells maintain global low translation during self-renewal, while differentiation proceeds with increased transcript abundance, ribosome loading, and protein synthesis and content. In addition, inhibition of translation by phosphorylation of the eukaryotic translation initiation factor 2α helps maintain low translation in mouse skeletal muscle stem cells; failure to maintain low translation result in loss of quiescence, initiation of the myogenic process and consequent differentiation. Using a microscopy-based OPP-staining approach, this study found that adult Drosophila intestinal progenitors, in contrast, display high levels of protein synthesis even under resting conditions. It is suggested that since increased translation in stem cells is a hallmark of differentiation, intestinal progenitor cells are in a state primed for differentiation that allows ISCs to rapidly proliferate and differentiate in order to replenish cells when needed. Since translation is an energy expensive process, stress responses may divert this energy to more immediate needs. It is proposed that SG formation provides this layer of regulation in intestinal progenitors during episodes of cellular stress. Furthermore, the characteristics of ISCs identified in this study may also be shared by stem cell populations that support other high-turnover adult tissues (Buddika, 2020).

While IPSGs were easily detectable in ex vivo-treated intestines, additional treatments in which adults were either fed chemical stressors or starved for various lengths of time with various vehicles (water, sugar-water or nothing) failed to induce SGs. It is hypothesized that ex vivo treatment induces IPSGs while feeding does not because orally fed chemicals are absorbed by enterocytes and fail to reach basally located and well protected progenitor cells. In addition, it is possible that endogenous IPSGs form transiently or require a dosage of proper duration that this study did not test. One other condition was found that induced small IPSG-like assemblies, namely heat shock. However, the induction was variable, and the treatments led to immediate death, precluding the ability to study them (Buddika, 2020).

There is considerable interest in identifying stem cell-specific factors, and this study shows that ten different RBPs are enriched in intestinal progenitors relative to surrounding differentiated cells. Previous transcriptional profiling analyses identified only one of these, lin-28, as being enriched in stem cells. Consistent with this, the other nine genes display relatively uniform transcript levels in non-differentiated versus differentiated cells. This apparent discrepancy between protein and transcript profiles in differentiated versus progenitor cells suggests active post-transcriptional regulatory mechanisms in intestinal cells. These mechanisms remain largely unexplored due to the lack of tools to profile the translatome relative to the transcriptome specifically in subsets of intestinal cells. Future work focused on developing such tools will likely identify post-transcriptional mechanisms that control stem cell behavior during resting and stressed conditions (Buddika, 2020).

Rasputin functions as a positive regulator of orb in Drosophila oogenesis

The determination of cell fate and the establishment of polarity axes during Drosophila oogenesis depend upon pathways that localize mRNAs within the egg chamber and control their on-site translation. One factor that plays a central role in regulating on-site translation of mRNAs is Orb. Orb is a founding member of the conserved CPEB family of RNA-binding proteins. These proteins bind to target sequences in 3' UTRs and regulate mRNA translation by modulating poly(A) tail length. In addition to controlling the translation of axis-determining mRNAs like grk, fs(1)K10, and osk, Orb protein autoregulates its own synthesis by binding to orb mRNA and activating its translation. Previous studies have shown that Rasputin (Rin), the Drosophila homologue of Ras-GAP SH3 Binding Protein (G3BP), associates with Orb in a messenger ribonucleoprotein (mRNP) complex. Rin is an evolutionarily conserved RNA-binding protein believed to function as a link between Ras signaling and RNA metabolism. This study shows that Orb and Rin form a complex in the female germline. Characterization of a new rin allele shows that rin is essential for oogenesis. Co-localization studies suggest that Orb and Rin form a complex in the oocyte at different stages of oogenesis. This is supported by genetic and biochemical analyses showing that rin functions as a positive regulator in the orb autoregulatory pathway by increasing Orb protein expression. Tandem Mass Spectrometry analysis shows that several canonical stress granule proteins are associated with the Orb-Rin complex suggesting that a conserved mRNP complex regulates localized translation during oogenesis in Drosophila (Costa, 2013).

Previous studies have shown that orb autoregulation promotes localized accumulation of Orb protein in subcellular compartments where its activity is required. To investigate the mechanisms underlying orb autoregulation this study attempted to identify proteins that physically associate with Orb and thus potentially help regulate its expression and/or activity. It has been previously shown that the Drosophila Fragile-X protein (dFMR1) is found in complexes with Orb in Drosophila ovaries and functions to negatively regulate orb accumulation and activity. This study shows that Rin, the Drosophila G3BP homologue, is also associated with Orb in ovaries. However, in contrast to dFMR1, Rin functions as a positive regulatory factor, helping to promote Orb accumulation and activity (Costa, 2013).

The results show that Rin associates with Orb as part of an RNase-resistant complex. The RNase-resistance aspect of this interaction suggests that their association is mediated by protein-protein interactions rather than, or in addition to, binding to the same mRNA species. While it is possible that Orb and Rin interact directly with each other, an equally plausible scenario is that their association is mediated by one or more proteins found in both Orb and Rin immunoprecipitates. For example, mammalian G3BP has been shown to interact directly with Caprin-1, and the fly Caprin protein is found in both Rin and Orb immunoprecipitates. Two other findings would also seem to argue in favor of an indirect, rather than a direct interaction. First the overlap between Rin and Orb, especially in vitellogenic chambers is quite limited. Second, only a small subset of the proteins associated with Rin or Orb are common to both. It is also possible that the initial association between Orb and Rin could depend upon binding to the same target mRNAs and their subsequent interaction could depend upon a short stretch of RNA that is hidden in the complex and protected from RNase activity. In this case, the limited colocalization observed in egg chambers would imply that only a subset of their mRNA targets are in common (Costa, 2013).

GB3Ps in mammals are thought to have two functions. The first is repressing the translation of target mRNAs by mechanisms that depend upon their helicase and RNase activities, while the second is in the assembly of stress granules under conditions of environmental stress such as heat shock or drug treatment. For this reason, it is anticipated that rin, like dfmr1, would function to negatively regulate orb activity and/or expression. However, exactly the opposite result was observed. Instead of suppressing the DV polarity defects in eggs from dominant negative orb, orb mutant (Hd19G orb343/+) females, reducing rin activity increases the frequency of DV defects. Conversely, DV polarity defects are suppressed by providing excess rin activity. While it is clear from the phenotypic effects seen in mutants that rin has multiple functions in oogenesis (and these will be independent of orb), the most plausible explanation for the effects of decreasing and increasing rin activity on DV polarity in Hd19G orb343/+ females is that they arise because of changes in the expression/accumulation of Orb protein. Consistent with this explanation, it was found that the level of Orb protein can be changed by manipulating rin activity. If orb is wild type, reducing the rin dose by half has little if any effect on Orb protein accumulation. However, when orb activity is compromised by the Hd19G orb343/+ combination, heterozygosity for rin results in Orb protein levels that are less than 5% that of wild type. Conversely, increasing rin activity in a wild type background elevates Orb protein levels almost two-fold over wild type, while in background compromised by the Hd19G orb343/+ combination adding extra rin restores Orb protein levels to that of wild type. Taken together, these findings argue that rin functions as a positive regulator of orb. At this point it is not clear how rin might control the accumulation of Orb protein. Since Rin and Orb are associated with each other, the simplest model is that Rin helps activate the translation of orb mRNA and thus functions as a co-factor in orb autoregulation. The substantial reduction in Orb protein levels evident in rin3 ovaries is consistent with this idea. This view would also be supported by the DV polarity defects evident in eggs laid by rin mutant females. However, other less direct models (e.g., rin represses the translation of some factor that inhibits orb mRNA translation or rin is required to stabilize Orb protein) can't be excluded at this time. Likewise, rin may have other targets besides orb in the establishment of DV polarity (Costa, 2013).

Potentially arguing in favor of a role for Rin in the translation of orb mRNA and/or in the activity of Orb protein is the fact that mutations in genes encoding several of the other proteins found in both Orb and Rin immunoprecipitates also show genetic interactions with orb. Thus, dfmr1 and parp suppress the DV polarity defects in eggs laid by Hd19G orb343/+ females, while parp and lig enhance the polarity defects. Previous studies have shown that dfmr1 also exerts its effects, at least in part, by altering the expression of Orb protein. Further connecting the effects of rin and dfmr1 to translation, it was also found that Orb, dFMR1 and Rin fractionate with polysomes in sucrose gradients. If the proteins detected in Orb and Rin immunoprecipitates are part of the same complex rather than different complexes, then the gene dose effects that were observed would suggest that they act coordinately to regulate Orb expression and that the relative balance between positive (e.g., Rin) and negative (e.g., dFMR1) factors in the egg chamber helps set the level of Orb accumulation. Further studies will be required to understand precisely how rin influences Orb protein accumulation and how some of the other factors associated with Rin and Orb like dFMR1, Lig and PABP function in this process (Costa, 2013).

Although these studies implicate rin as a positive regulator of orb, this is clearly not the only role for rin in the ovary. Instead, the phenotypic effects of rin mutations point to a potentially diverse array of functions, not only in gem cells but also in the surrounding somatic follicle cells. For example, the fragmentation of ring canals, the failure to properly disperse the endoreplicated nurse cell chromosomes, and the dumpless phenotype are not observed in orb mutants. Moreover, ring canal and chromatin dispersal defects suggest that rin has functions in nurse cells, which is a compartment that has only little Orb protein. Likewise, the encapsulation defects could be of somatic origin where rin, but not orb is expressed. Further studies will be required to identify the rin regulatory targets in these and potentially other processes (Costa, 2013).

The RNA-binding proteins FMR1, Rasputin and Caprin act together with the UBA protein Lingerer to restrict tissue growth in Drosophila melanogaster

Appropriate expression of growth-regulatory genes is essential to ensure normal animal development and to prevent diseases like cancer. Gene regulation at the levels of transcription and translational initiation mediated by the Hippo and Insulin signaling pathways and by the TORC1 complex, respectively, has been well documented. Whether translational control mediated by RNA-binding proteins contributes to the regulation of cellular growth is less clear. This study has identified Lingerer (Lig), an UBA domain-containing protein, as growth suppressor that associates with the RNA-binding proteins Fragile X mental retardation protein 1 (FMR1) and Caprin (Capr) and directly interacts with and regulates the RNA-binding protein Rasputin (Rin) in Drosophila melanogaster. lig mutant organs overgrow due to increased proliferation, and a reporter for the JAK/STAT signaling pathway is upregulated in a lig mutant situation. rin, Capr or FMR1 in combination as double mutants, but not the respective single mutants, display lig like phenotypes, implicating a redundant function of Rin, Capr and FMR1 in growth control in epithelial tissues. Thus, Lig regulates cell proliferation during development in concert with Rin, Capr and FMR1 (Baumgartner, 2013).

Lig has been identified as a new growth suppressor in eye and wing epithelial tissues. Whereas eyes mutant for lig consist of more ommatidia without cell size defects, eyes overexpressing lig have a reduced cell number due to increased apoptosis and reduced cell cycle progression. lig mutant eyes are sensitive to apoptosis (resulting in a variable phenotype under normal food conditions) but are able to cope with the overgrowth situation when the flies develop under suboptimal growth conditions. Similarly, the reduced eye phenotype of lig overexpressing eyes was partially rescued under suboptimal growth conditions or by expression of DIAP1, suggesting that the starvation response impacts on the apoptosis rates in imaginal discs. However, other indirect effects that might be triggered by starvation cannot be excluded (Baumgartner, 2013).

In addition to these findings, lig mutants have previously been characterized for their behavioral phenotype in the copulation process (Kuniyosh, 2002) and their putative role in neuronal tissues (Kuniyoshi, 2003). Lig is conserved from flies to humans, the human orthologs being ubiquitin associated protein 2 (UBAP2) and ubiquitin associated protein 2 like (UBAP2L). UBAP2 has been identified in a Y2H screen as a direct interaction partner of the zona pellucida 3 (ZP3) protein that is involved in sperm binding and acrosomal exocytosis. UBAP2L has been reported to accumulate at ubiquitin-rich aggregates upon proteasome inhibition in human neuroblastoma tissue culture cells, suggesting that the UBA domain is functional. It is currently unknown whether the Lig orthologs are involved in growth regulation, and no interaction partners have been identified except for ZP3 (Baumgartner, 2013).

Several lines of evidence indicate that Lig interacts with FMR1, Capr and Rin, and via these interactions functions to regulate growth: (1) Lig associated with FMR1 and Rin in an AP-MS experiment, (2) Lig co-localized with FMR1, Capr and Rin, (3) Lig directly interacted with Rin in a Y2H experiment, (4) Lig transcriptionally regulated Rin levels, and (5) FMR1, Capr or rin in combination of double mutants behaved like lig null mutants and (vi) lig downregulation in FMR1, Capr or rin mutant eyes synergistically increased the eye size (Baumgartner, 2013).

The interaction between Lig and Rin, Capr and FMR1, three RNA-binding proteins, and the co-localization with P-body components suggests that Lig regulates the translation and/or stability of specific mRNAs of growth-regulatory genes via FMR1, Capr and Rin function. Indeed, the Drosophila FMR1 and orthologs of Rin are involved in translational regulation of growth-regulatory genes in certain tissues. For example, FMR1 binds bantam miRNA, an inhibitor of the pro-apoptotic gene hid, and regulates cbl, which encodes a component of the EGFR signaling pathway, in germline stem cells. However, bantam miRNA is not regulated by FMR1 in epithelial cells, and Lig was unable to regulate a bantam miRNA reporter. Furthermore, the expression of a pointed transcriptional reporter was unchanged in lig mutant clones, suggesting that cbl regulation by FMR1 is specific to the germline or has only subtle effects in the developing eye. The Rin ortholog G3BP controls myc, CyclinD2, cdk7 and cdk9 mRNA. However, it is not known whether this function is conserved for Rin, and this study did not observe any alterations of Myc protein levels in lig mutant clones. It will be important to identify mRNAs that are regulated by FMR1, Capr and Rin in epithelial tissues during development, and to determine whether Lig mediates specificity for certain mRNAs (Baumgartner, 2013).

To identify the signaling pathway that is regulated by Lig, readouts were used for the Hippo, EGFR, Insulin, Hedgehog, Wnt and JAK/STAT signaling pathways. No alterations of all analyzed pathways were observed except for the highly conserved JAK/STAT signaling pathway. The pathway is composed of four modules: the ligands, Upd, Upd2 and Upd3, the receptor Domeless (Dome), the receptor-associated Janus kinase (JAK) Hopscotch (Hop), and the signal transducer and activator of transcription (STAT) STAT92E. The involvement of Lig in the JAK/STAT signaling pathway leads to a number of assumptions and questions in the context of the current findings. First, the autonomous effect of Lig on the 10xSTAT92E-GFP reporter suggests that Lig regulates the intracellular components (Dome, Hop or STAT92E) or modifiers thereof rather than expression of the ligands, which would result in non-autonomous effects. Second, the physical and genetic interactions of Lig with the mRNA binding proteins FMR1, Capr and Rin raises the question whether Lig directly impacts on the JAK/STAT pathway or whether it modulates the JAK/STAT signaling via FMR1, Capr and Rin. So far, neither option can be excluded. However, it was recently demonstrated that upd and STAT92E mRNAs are targets for posttranscriptional regulation via the miRNA pathway. It will be interesting to determine whether FMR1, Rin or Capr are involved in this process in the case of STAT92E (Baumgartner, 2013).

The data provide evidence that FMR1, Capr and Rin function in a redundant manner in epithelial tissues in growth control, suggesting that they regulate either overlapping sets of mRNAs or different mRNAs encoding proteins with redundant functions. Examples for the former have been described for FMR1, Capr and G3BP, the human ortholog of Rin. In Drosophila, FMR1 cooperates with Capr, and both proteins bind to the same mRNAs frs and CycB. Similarly, G3BP forms a complex with human Caprin and both interact with myc and CycD mRNAs. Both examples suggest a redundant regulation of these targets. There is no direct evidence for the latter possibility. However, G3BP associates with and translationally regulates tau mRNA in neuronal cells. In Drosophila, FMR1 negatively regulates futsch mRNA, and the futsch mutant phenotype is suppressed by overexpression of Tau, suggesting a redundant function of Tau and Futsch (Baumgartner, 2013).

Lig impacts on Rin and slightly on Capr but not on FMR1 levels. However, only FMR1, Capr or rin mutants in combination as double mutants resulted in a lig like phenotype, suggesting that the activity of FMR1 and Capr is altered (probably at the posttranslational level) in a lig mutant situation. AP-MS experiments also revealed DART1 as a physical binding partner of Lig. Arginine methyl transferases are able to methylate RGG motifs and thereby modulate the binding capability to mRNAs. Interestingly, FMR1 contains a conserved RGG domain that can be methylated in Drosophila and humans. In humans, protein methyl transferase 1 (PRMT1), the ortholog of DART1, mediates the arginine methylation of FMR1 to alter its binding affinity to mRNAs. Furthermore, G3BP1, the mouse ortholog of Rin, contains an RGG domain that is methylated by PRMT1 after stimulation of the Wnt signaling pathway to modulate the binding to β-Catenin mRNA. The RGG domain of Rin is weakly conserved and lacks the RGG motifs. It is thus unclear whether Rin can be methylated in the truncated arginine-glycine rich region. Like FMR1 and G3BP, Caprin contains RGG domains, and it was identified as binding partner of PRMT8, which is closely related to PRMT1 at the sequence level. Further experiments are required to resolve whether Lig is involved in a DART1-mediated methylation of FMR1 and Rin under certain conditions, or whether Lig alters the activity of FMR1 and Capr by another mechanism (Baumgartner, 2013).

Lig, FMR1, Rin and Capr have been identified as interactors of Orb in Co-IP experiments, suggesting a complex formation of these proteins. Complex formation has been reported for G3BP and Caprin in human cell lines and for Capr and FMR1 in Drosophila and mouse neurons so far. This study demonstrated that Rin, Capr and FMR1 have a redundant function in the eye, and that they localize in the same subcellular structure in cultured Drosophila cells. This raises the question whether the three RNA-binding proteins Capr, Rin and FMR1 are functionally related only in the eye. Systematic analyses of the phenotypes of double mutant combinations will reveal the tissues in which these RNA-binding proteins exert redundant and non-redundant functions. Furthermore, it will be interesting to determine whether Rin and Capr contribute to phenotypes associated with the FXS (Baumgartner, 2013).

Crystal structure of the Rasputin NTF2-like domain from Drosophila melanogaster

The crystal structure of the NTF2-like domain of the Drosophila homolog of Ras GTPase SH3 Binding Protein (G3BP), Rasputin, was determined at 2.7˚ resolution. The overall structure is highly similar to nuclear transport factor 2: It is a homodimer comprised of a beta-sheet and three alpha-helices forming a cone-like shape. However, known binding sites for RanGDP and FxFG containing peptides show electrostatic and steric differences compared to nuclear transport factor 2. A HEPES molecule bound in the structure suggests a new, and possibly physiologically relevant, ligand binding site (Vognsen, 2012).

Rasputin, the Drosophila homologue of the RasGAP SH3 binding protein, functions in ras- and Rho-mediated signaling

The small GTPase Ras plays an important role in many cellular signaling processes. Ras activity is negatively regulated by GTPase activating proteins (GAPs). It has been proposed that RasGAP may also function as an effector of Ras activity. This study has identified and characterized the Drosophila homologue of the RasGAP-binding protein G3BP encoded by rasputin (rin). rin mutants are viable and display defects in photoreceptor recruitment and ommatidial polarity in the eye. Mutations in rin/G3BP genetically interact with components of the Ras signaling pathway that function at the level of Ras and above, but not with Raf/MAPK pathway components. These interactions suggest that Rin is required as an effector in Ras signaling during eye development, supporting an effector role for RasGAP. The ommatidial polarity phenotypes of rin are similar to those of RhoA and the polarity genes, e.g. fz and dsh. Although rin/G3BP interacts genetically with RhoA, affecting both photoreceptor differentiation and polarity, it does not interact with the gain-of-function genotypes of fz and dsh. These data suggest that Rin is not a general component of polarity generation, but serves a function specific to Ras and RhoA signaling pathways (Pazman, 2000).

Functions of Rasputin orthologs in other species

UBAP2L forms distinct cores that act in nucleating stress granules upstream of G3BP1

Stress granules (SGs) are membraneless organelles that form in eukaryotic cells after stress exposure. Following translation inhibition, polysome disassembly releases 48S preinitiation complexes (PICs). mRNA, PICs, and other proteins coalesce in SG cores. SG cores recruit a dynamic shell, whose properties are dominated by weak interactions between proteins and RNAs. The structure and assembly of SGs and how different components contribute to their formation are not fully understood. Using super-resolution and expansion microscopy, this study found that the SG component UBAP2L and the core protein G3BP1 occupy different domains inside SGs. UBAP2L displays typical properties of a core protein, indicating that cores of different compositions coexist inside the same granule. Consistent with a role as a core protein, UBAP2L is required for SG assembly in several stress conditions. Reverse genetic and cell biology experiments suggest that UBAP2L forms granules independent of G3BP1 and 2 but does not interfere with stress-induced translational inhibition. A model is proposed in which UBAP2L is an essential SG nucleator that acts upstream of G3BP1 and 2 and facilitates G3BP1 core formation and SG assembly and growth (Cirillo, 2020).

RNA-induced conformational switching and clustering of G3BP drive stress granule assembly by condensation

Stressed cells shut down translation, release mRNA molecules from polysomes, and form stress granules (SGs) via a network of interactions that involve G3BP. This study focused on the mechanistic underpinnings of SG assembly. Under non-stress conditions, G3BP adopts a compact auto-inhibited state stabilized by electrostatic intramolecular interactions between the intrinsically disordered acidic tracts and the positively charged arginine-rich region. Upon release from polysomes, unfolded mRNAs outcompete G3BP auto-inhibitory interactions, engendering a conformational transition that facilitates clustering of G3BP through protein-RNA interactions. Subsequent physical crosslinking of G3BP clusters drives RNA molecules into networked RNA/protein condensates. G3BP condensates impede RNA entanglement and recruit additional client proteins that promote SG maturation or induce a liquid-to-solid transition that may underlie disease. It proposed that condensation coupled to conformational rearrangements and heterotypic multivalent interactions may be a general principle underlying RNP granule assembly (Guillen-Boixet, 2020).

CLUH granules coordinate translation of mitochondrial proteins with mTORC1 signaling and mitophagy

Mitochondria house anabolic and catabolic processes that must be balanced and adjusted to meet cellular demands. The RNA-binding protein CLUH (clustered mitochondria homolog) binds mRNAs of nuclear-encoded mitochondrial proteins and is highly expressed in the liver, where it regulates metabolic plasticity. This study shows that in primary hepatocytes, CLUH coalesces in specific ribonucleoprotein particles that define the translational fate of target mRNAs, such as Pcx, Hadha, and Hmgcs2, to match nutrient availability. Moreover, CLUH granules play signaling roles, by recruiting mTOR kinase and the RNA-binding proteins G3BP1 and G3BP2. Upon starvation, CLUH regulates translation of Hmgcs2, involved in ketogenesis, inhibits mTORC1 activation and mitochondrial anabolic pathways, and promotes mitochondrial turnover, thus allowing efficient reprograming of metabolic function. In the absence of CLUH, a mitophagy block causes mitochondrial clustering that is rescued by rapamycin treatment or depletion of G3BP1 and G3BP2. These data demonstrate that metabolic adaptation of liver mitochondria to nutrient availability depends on a compartmentalized CLUH-dependent post-transcriptional mechanism that controls both mTORC1 and G3BP signaling and ensures survival (Pla-Martin, 2020).

Competing protein-RNA interaction networks control multiphase intracellular organization

Liquid-liquid phase separation (LLPS) mediates formation of membraneless condensates such as those associated with RNA processing, but the rules that dictate their assembly, substructure, and coexistence with other liquid-like compartments remain elusive. This study addresses the biophysical mechanism of this multiphase organization using quantitative reconstitution of cytoplasmic stress granules (SGs) with attached P-bodies in human cells. Protein-interaction networks can be viewed as interconnected complexes (nodes) of RNA-binding domains (RBDs), whose integrated RNA-binding capacity determines whether LLPS occurs upon RNA influx. Surprisingly, both RBD-RNA specificity and disordered segments of key proteins are non-essential, but modulate multiphase condensation. Instead, stoichiometry-dependent competition between protein networks for connecting nodes determines SG and P-body composition and miscibility, while competitive binding of unconnected proteins disengages networks and prevents LLPS. Inspired by patchy colloid theory, a general framework is proposed by which competing networks give rise to compositionally specific and tunable condensates, while relative linkage between nodes underlies multiphase organization (Sanders, 2020).

Axonal G3BP1 stress granule protein limits axonal mRNA translation and nerve regeneration

Critical functions of intra-axonally synthesized proteins are thought to depend on regulated recruitment of mRNA from storage depots in axons. This study shows that axotomy of mammalian neurons induces translation of stored axonal mRNAs via regulation of the stress granule protein G3BP1, to support regeneration of peripheral nerves. G3BP1 aggregates within peripheral nerve axons in stress granule-like structures that decrease during regeneration, with a commensurate increase in phosphorylated G3BP1. Colocalization of G3BP1 with axonal mRNAs is also correlated with the growth state of the neuron. Disrupting G3BP functions by overexpressing a dominant-negative protein activates intra-axonal mRNA translation, increases axon growth in cultured neurons, disassembles axonal stress granule-like structures, and accelerates rat nerve regeneration in vivo (Sahoo, 2018).

G3BP-Caprin1-USP10 complexes mediate stress granule condensation and associate with 40S subunits

Mammalian stress granules (SGs) contain stalled translation preinitiation complexes that are assembled into discrete granules by specific RNA-binding proteins such as G3BP. This study shows that cells lacking both G3BP1 and G3BP2 cannot form SGs in response to eukaryotic initiation factor 2alpha phosphorylation or eIF4A inhibition, but are still SG-competent when challenged with severe heat or osmotic stress. Rescue experiments using G3BP1 mutants show that phosphomimetic G3BP1-S149E fails to rescue SG formation, whereas G3BP1-F33W, a mutant unable to bind G3BP partner proteins Caprin1 or USP10, rescues SG formation. Caprin1/USP10 binding to G3BP is mutually exclusive: Caprin binding promotes, but USP10 binding inhibits, SG formation. G3BP interacts with 40S ribosomal subunits through its RGG motif, which is also required for G3BP-mediated SG formation. It is proposed that G3BP mediates the condensation of SGs by shifting between two different states that are controlled by the phosphorylation of S149 and by binding to Caprin1 or USP10 (Kedersha, 2016).


Search PubMed for articles about Drosophila Rasputin

Alam, U. and Kennedy, D. (2019). Rasputin a decade on and more promiscuous than ever? A review of G3BPs. Biochim Biophys Acta Mol Cell Res 1866(3): 360-370. PubMed ID: 30595162

Aguilera-Gomez, A., van Oorschot, M. M., Veenendaal, T. and Rabouille, C. (2016). In vivo vizualisation of mono-ADP-ribosylation by dPARP16 upon amino-acid starvation. Elife 5. PubMed ID: 27874829

Aguilera-Gomez, A., Zacharogianni, M., van Oorschot, M. M., Genau, H., Grond, R., Veenendaal, T., Sinsimer, K. S., Gavis, E. R., Behrends, C. and Rabouille, C. (2017). Phospho-Rasputin stabilization by Sec16 is required for stress granule formation upon amino acid starvation. Cell Rep 20(4): 935-948. PubMed ID: 28746877 

Baumgartner, R., Stocker, H. and Hafen, E. (2013). The RNA-binding proteins FMR1, rasputin and caprin act together with the UBA protein lingerer to restrict tissue growth in Drosophila melanogaster. PLoS Genet 9(7): e1003598. PubMed ID: 23874212

Buddika, K., Ariyapala, I. S., Hazuga, M. A., Riffert, D. and Sokol, N. S. (2020). Canonical nucleators are dispensable for stress granule assembly in Drosophila intestinal progenitors. J Cell Sci 133(10). PubMed ID: 32265270

Cirillo, L., Cieren, A., Barbieri, S., Khong, A., Schwager, F., Parker, R. and Gotta, M. (2020). UBAP2L forms distinct cores that act in nucleating stress granules upstream of G3BP1. Curr Biol 30(4): 698-707 e696. PubMed ID: 31956030

Costa, A., Pazman, C., Sinsimer, K. S., Wong, L. C., McLeod, I., Yates, J., 3rd, Haynes, S. and Schedl, P. (2013). Rasputin functions as a positive regulator of orb in Drosophila oogenesis. PLoS One 8(9): e72864. PubMed ID: 24069162

Guillen-Boixet, J., Kopach, A., Holehouse, A. S., Wittmann, S., Jahnel, M., Schlussler, R., Kim, K., Trussina, I., Wang, J., Mateju, D., Poser, I., Maharana, S., Ruer-Gruss, M., Richter, D., Zhang, X., Chang, Y. T., Guck, J., Honigmann, A., Mahamid, J., Hyman, A. A., Pappu, R. V., Alberti, S. and Franzmann, T. M. (2020). RNA-Induced Conformational Switching and Clustering of G3BP Drive Stress Granule Assembly by Condensation. Cell 181(2): 346-361 e317. PubMed ID: 32302572

Jain, S., Wheeler, J. R., Walters, R. W., Agrawal, A., Barsic, A. and Parker, R. (2016). ATPase-Modulated Stress Granules Contain a Diverse Proteome and Substructure. Cell 164(3): 487-498. PubMed ID: 26777405

Kedersha, N., Panas, M. D., Achorn, C. A., Lyons, S., Tisdale, S., Hickman, T., Thomas, M., Lieberman, J., McInerney, G. M., Ivanov, P. and Anderson, P. (2016). G3BP-Caprin1-USP10 complexes mediate stress granule condensation and associate with 40S subunits. J Cell Biol 212(7): 845-860. PubMed ID: 27022092

Kuniyoshi, H., Baba, K., Ueda, R., Kondo, S., Awano, W., Juni, N. and Yamamoto, D. (2002). lingerer, a Drosophila gene involved in initiation and termination of copulation, encodes a set of novel cytoplasmic proteins. Genetics 162(4): 1775-1789. PubMed ID: 12524348

Kuniyoshi, H., Usui-Aoki, K., Juni, N. and Yamamoto, D. (2003). Expression analysis of the lingerer gene in the larval central nervous system of Drosophila melanogaster. J Neurogenet 17(2-3): 117-137. PubMed ID: 14668197

Laver, J. D., Ly, J., Winn, A. K., Karaiskakis, A., Lin, S., Nie, K., Benic, G., Jaberi-Lashkari, N., Cao, W. X., Khademi, A., Westwood, J. T., Sidhu, S. S., Morris, Q., Angers, S., Smibert, C. A. and Lipshitz, H. D. (2020). The RNA-binding protein Rasputin/G3BP enhances the stability and translation of its target mRNAs. Cell Rep 30(10): 3353-3367. PubMed ID: 32160542

Markmiller, S., Soltanieh, S., Server, K. L., Mak, R., Jin, W., Fang, M. Y., Luo, E. C., Krach, F., Yang, D., Sen, A., Fulzele, A., Wozniak, J. M., Gonzalez, D. J., Kankel, M. W., Gao, F. B., Bennett, E. J., Lecuyer, E. and Yeo, G. W. (2018). Context-dependent and disease-specific diversity in protein interactions within stress granules. Cell 172(3): 590-604. PubMed ID: 29373831

Pazman, C., Mayes, C. A., Fanto, M., Haynes, S. R. and Mlodzik, M. (2000). Rasputin, the Drosophila homologue of the RasGAP SH3 binding protein, functions in ras- and Rho-mediated signaling. Development 127(8): 1715-1725. PubMed ID: 10725247

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Biological Overview

date revised: 27 June 2020

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