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• W3048230065 abstract "•RNA condensation and mRNP recruitment are separable steps in IVSG assembly•ATP availability and ATPase activity regulate IVSG assembly•RNA condensate composition dictates IVSG anatomy Stress granules (SGs) are condensates of mRNPs that form in response to stress. SGs arise by multivalent protein-protein, protein-RNA, and RNA-RNA interactions. However, the role of RNA-RNA interactions in SG assembly remains understudied. Here, we describe a yeast SG reconstitution system that faithfully recapitulates SG assembly in response to trigger RNAs. SGs assembled by stem-loop RNA triggers are ATP-sensitive, regulated by helicase/chaperone activity, and exhibit the hallmarks of maturation observed for SG proteins that phase-separate in vitro. Additionally, the fraction of total RNA that phase-separates in vitro is sufficient to trigger SG formation. However, condensation of NFT1 mRNA, an enriched transcript in this population, can only assemble an incomplete SG. These results suggest that networks of distinct transcripts are required to form a canonical SG and provide a platform for dissecting the interplay between the transcriptome and ATP-dependent remodeling in SG formation. Stress granules (SGs) are condensates of mRNPs that form in response to stress. SGs arise by multivalent protein-protein, protein-RNA, and RNA-RNA interactions. However, the role of RNA-RNA interactions in SG assembly remains understudied. Here, we describe a yeast SG reconstitution system that faithfully recapitulates SG assembly in response to trigger RNAs. SGs assembled by stem-loop RNA triggers are ATP-sensitive, regulated by helicase/chaperone activity, and exhibit the hallmarks of maturation observed for SG proteins that phase-separate in vitro. Additionally, the fraction of total RNA that phase-separates in vitro is sufficient to trigger SG formation. However, condensation of NFT1 mRNA, an enriched transcript in this population, can only assemble an incomplete SG. These results suggest that networks of distinct transcripts are required to form a canonical SG and provide a platform for dissecting the interplay between the transcriptome and ATP-dependent remodeling in SG formation. The assembly of RNA granules, membraneless organelles composed of RNA-binding proteins and non-translating mRNAs, is one of the preeminent examples of biochemical organization in the cytoplasm (Anderson and Kedersha, 2006Anderson P. Kedersha N. RNA granules.J. Cell Biol. 2006; 172: 803-808Crossref PubMed Scopus (795) Google Scholar). RNA granules have been implicated in mRNA processing, degradation, subcellular localization, and translational control (Buchan, 2014Buchan J.R. mRNP granules. Assembly, function, and connections with disease.RNA Biol. 2014; 11: 1019-1030Crossref PubMed Scopus (247) Google Scholar). Stress granules (SGs) represent a conserved class of RNA granules that form in response to stresses that inhibit translation initiation (Protter and Parker, 2016Protter D.S.W. Parker R. Principles and properties of stress granules.Trends Cell Biol. 2016; 26: 668-679Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar). Much of our understanding of the physical chemistry underpinning SG assembly has come from in vitro studies of SG components. Proteomic studies from yeast and mammalian cell lines have found that SGs are enriched in RNA-binding proteins that contain intrinsically disordered regions (IDRs) or low-complexity sequences (LCSs) (Han et al., 2012Han T.W. Kato M. Xie S. Wu L.C. Mirzaei H. Pei J. Chen M. Xie Y. Allen J. Xiao G. McKnight S.L. Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies.Cell. 2012; 149: 768-779Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar; Jain et al., 2016Jain S. Wheeler J.R. Walters R.W. Agrawal A. Barsic A. Parker R. ATPase-modulated stress granules contain a diverse proteome and substructure.Cell. 2016; 164: 487-498Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar; Markmiller et al., 2018Markmiller S. Soltanieh S. Server K.L. Mak R. Jin W. Fang M.Y. Luo E.-C. Krach F. Yang D. Sen A. et al.Context-dependent and disease-specific diversity in protein interactions within stress granules.Cell. 2018; 172: 590-604.e13Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar; Youn et al., 2018Youn J.-Y. Dunham W.H. Hong S.J. Knight J.D.R. Bashkurov M. Chen G.I. Bagci H. Rathod B. MacLeod G. Eng S.W.M. et al.High-density proximity mapping reveals the subcellular organization of mRNA-associated granules and bodies.Mol. Cell. 2018; 69: 517-532.e11Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). The ability of these domains to partition out of solution via liquid-liquid phase separation (LLPS) has suggested a model in which alterations in the valency and/or concentration of SG components can drive SG formation via LLPS (Banani et al., 2017Banani F., S. Lee O., H. Hyman A., A. Rosen K., M. Biomolecular condensates: organizers of cellular biochemsitry.Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298Crossref PubMed Scopus (1348) Google Scholar). Consistent with this model, SG assembly is sensitive to the levels of SG components with IDR/LCS domains. Overexpression of SG proteins with IDRs promotes SG assembly in the absence of stress, while their depletion disrupts SG formation (Anderson and Kedersha, 2008Anderson P. Kedersha N. Stress granules: the Tao of RNA triage.Trends Biochem. Sci. 2008; 33: 141-150Abstract Full Text Full Text PDF PubMed Scopus (741) Google Scholar; Hilliker et al., 2011Hilliker A. Gao Z. Jankowsky E. Parker R. The DEAD-box protein Ded1 modulates translation by the formation and resolution of an eIF4F-mRNA complex.Mol. Cell. 2011; 43: 962-972Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar; Kedersha et al., 2016Kedersha N. Panas M.D. Achorn C.A. Lyons S. Tisdale S. Hickman T. Thomas M. Lieberman J. McInerney G.M. Ivanov P. Anderson P. G3BP-Caprin1-USP10 complexes mediate stress granule condensation and associate with 40S subunits.J. Cell Biol. 2016; 212: 845-860Crossref PubMed Scopus (212) Google Scholar; Matsuki et al., 2013Matsuki H. Takahashi M. Higuchi M. Makokha G.N. Oie M. Fujii M. Both G3BP1 and G3BP2 contribute to stress granule formation.Genes Cells. 2013; 18: 135-146Crossref PubMed Scopus (128) Google Scholar; Takahara and Maeda, 2012Takahara T. Maeda T. Transient sequestration of TORC1 into stress granules during heat stress.Mol. Cell. 2012; 47: 242-252Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Additionally, RNA can act as a scaffold for multiple IDR domain RNA-binding proteins. This scaffolding effect can drive LLPS through an increase in valency and has been observed for proteins and RNAs for several types of RNA granules, including SGs (Lin et al., 2015Lin Y. Protter D.S.W. Rosen M.K. Parker R. Formation and maturation of phase-separated liquid droplets by RNA-binding proteins.Mol. Cell. 2015; 60: 208-219Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar; Maharana et al., 2018Maharana S. Wang J. Papadopoulos K., D. Richter D. Pozniakovsky A. Poser I. Bickle M. Rizk S. Guillen-Boixet J. Franzmann M., T. et al.RNA buffers the phase separation behavior of prion-like RNA binding proteins.Science. 2018; 360: 918-921Crossref PubMed Scopus (322) Google Scholar; Protter et al., 2018Protter D.S.W. Rao B.S. Van Treeck B. Lin Y. Mizoue L. Rosen M.K. Parker R. Intrinsically disordered regions can contribute promiscuous interactions to RNP granule assembly.Cell Rep. 2018; 22: 1401-1412Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Although these approaches have been useful in defining how RNA-protein and protein-protein interactions can drive LLPS of SG components, the promiscuous nature of these in vitro systems, their reliance on crowding agents, and the restriction to examining only one or two proteins at a time has made it difficult to understand how a SG forms without recruiting components of other RNA granules. Recently, specific RNA-RNA interactions have been implicated in the assembly of particular RNA granules, suggesting an alternative route to triggering LLPS (Jain and Vale, 2017Jain A. Vale R.D. RNA phase transitions in repeat expansion disorders.Nature. 2017; 546: 243-247Crossref PubMed Scopus (294) Google Scholar; Langdon et al., 2018Langdon M., E. Qui Y. Ghanbari Niaki A. McLaughlin A., G. Weidmann A., C. Gerbich M., T. Smith A., J. Crutchley M., J. Termini M., C. Weeks M., K. et al.mRNA structure determines specificity of a polyQ-driven phase separation.Science. 2018; 360: 922-927Crossref PubMed Scopus (191) Google Scholar). Interestingly, there is substantial overlap between the SG transcriptome and the fraction of mRNAs with a propensity to phase-separate in vitro under physiological conditions, suggesting that RNA-RNA interactions might contribute to SG assembly (Khong et al., 2017Khong A. Matheny T. Jain S. Mitchell S.F. Wheeler J.R. Parker R. The stress granule transcriptome reveals principles of mRNA accumulation in stress granules.Mol. Cell. 2017; 68: 808-820.e5Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar; Van Treeck et al., 2018Van Treeck B. Protter D.S.W. Matheny T. Khong A. Link C.D. Parker R. RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome.Proc. Natl. Acad. Sci. U S A. 2018; 115: 2734-2739Crossref PubMed Scopus (156) Google Scholar). This set of RNAs is characterized by poor translatability and long coding region/3′ UTR length, consistent with the idea that they can form extended RNA-RNA networks in vivo (Khong et al., 2017Khong A. Matheny T. Jain S. Mitchell S.F. Wheeler J.R. Parker R. The stress granule transcriptome reveals principles of mRNA accumulation in stress granules.Mol. Cell. 2017; 68: 808-820.e5Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Together these results suggest that extended RNA-RNA networks of mRNAs might be capable of triggering SG assembly. Such an idea is attractive, as it would also help explain features of the SG proteome. For instance, translation initiation factors preferentially bind certain RNA sequences (Sen et al., 2015Sen N.D. Zhou F. Ingolia N.T. Hinnebusch A.G. Genome-wide analysis of translational efficiency reveals distinct but overlapping functions of yeast DEAD-box RNA helicases Ded1 and eIF4A.Genome Res. 2015; 25: 1196-1205Crossref PubMed Scopus (71) Google Scholar, Sen et al., 2016Sen N.D. Zhou F. Harris M.S. Ingolia N.T. Hinnebusch A.G. eIF4B stimulates translation of long mRNAs with structured 5′ UTRs and low closed-loop potential but weak dependence on eIF4G.Proc. Natl. Acad. Sci. U S A. 2016; 113: 10464-10472Crossref PubMed Scopus (49) Google Scholar; Zinshteyn et al., 2017Zinshteyn B. Rojas-Duran M.F. Gilbert W.V. Translation initiation factor eIF4G1 preferentially binds yeast transcript leaders containing conserved oligo-uridine motifs.RNA. 2017; 23: 1365-1375Crossref PubMed Scopus (14) Google Scholar); thus, the enrichment of specific transcripts in SGs would help explain the presence of these factors in SGs and their absence from other RNA granules (Zid and O’Shea, 2014Zid B.M. O’Shea E.K. Promoter sequences direct cytoplasmic localization and translation of mRNAs during starvation in yeast.Nature. 2014; 514: 117-121Crossref PubMed Scopus (110) Google Scholar). Similarly, G-quadraplexes assembled from C9ORF72 repeat RNA (G4C2) can build SGs in vivo and in vitro, highlighting that RNAs with the ability to form inter- and intramolecular interactions can promote SG formation (Fay et al., 2017Fay M.M. Anderson P.J. Ivanov P. ALS/FTD-associated C9ORF72 repeat RNA promotes phase transitions in vitro and in cells.Cell Rep. 2017; 21: 3573-3584Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). However, these G4C2-induced SGs can recruit only a subset of SG proteins to foci in cells, suggesting that multiple RNAs might be necessary to fully recapitulate SG composition (Fay et al., 2017Fay M.M. Anderson P.J. Ivanov P. ALS/FTD-associated C9ORF72 repeat RNA promotes phase transitions in vitro and in cells.Cell Rep. 2017; 21: 3573-3584Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The composition of RNA granules also appears to be tuned by passive and active mechanisms. For instance, the ability of different IDRs containing proteins to form distinct LLPS droplets is strongly influenced by the presence of non-IDR proteins independent of their effect of crowding (Protter et al., 2018Protter D.S.W. Rao B.S. Van Treeck B. Lin Y. Mizoue L. Rosen M.K. Parker R. Intrinsically disordered regions can contribute promiscuous interactions to RNP granule assembly.Cell Rep. 2018; 22: 1401-1412Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). This suggests that specificity can be tuned in the cytoplasm by the large number of proteins that can weakly interact with IDR domains and suppress LLPS. Thus, systems that focus only on the phase separation properties of one or two proteins at a time are unlikely to fully recapitulate the behavior that is observed in vivo. Additionally, in vivo SG assembly and disassembly is regulated by chaperones and/or helicases, suggesting that constant ATP-dependent remodeling might play a role in SG assembly (Hondele et al., 2019Hondele M. Sachdev R. Heinrich S. Wang J. Vallotton P. Fontoura B.M.A. Weis K. DEAD-box ATPases are global regulators of phase-separated organelles.Nature. 2019; 573: 144-148Crossref PubMed Scopus (93) Google Scholar; Hilliker et al., 2011Hilliker A. Gao Z. Jankowsky E. Parker R. The DEAD-box protein Ded1 modulates translation by the formation and resolution of an eIF4F-mRNA complex.Mol. Cell. 2011; 43: 962-972Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar; Jain et al., 2016Jain S. Wheeler J.R. Walters R.W. Agrawal A. Barsic A. Parker R. ATPase-modulated stress granules contain a diverse proteome and substructure.Cell. 2016; 164: 487-498Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar; Mateju et al., 2017Mateju D. Franzmann T.M. Patel A. Kopach A. Boczek E.E. Maharana S. Lee H.O. Carra S. Hyman A.A. Alberti S. An aberrant phase transition of stress granules triggered by misfolded protein and prevented by chaperone function.EMBO J. 2017; 36: 1669-1687Crossref PubMed Scopus (172) Google Scholar; Tauber et al., 2020Tauber D. Tauber G. Khong A. Van Treeck B. Pelletier J. Parker R. Modulation of RNA condensation by the DEAD-box protein eIF4A.Cell. 2020; 180: 411-426.e16Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar; Walters et al., 2015Walters R.W. Muhlrad D. Garcia J. Parker R. Differential effects of Ydj1 and Sis1 on Hsp70-mediated clearance of stress granules in Saccharomyces cerevisiae.RNA. 2015; 21: 1660-1671Crossref PubMed Scopus (63) Google Scholar). However, recent work has suggested that ATP can also act as a biological hydrotrope that can dissolve LLPS condensates independent of nucleotide hydrolysis (Patel et al., 2017Patel A. Malinovska L. Saha S. Wang J. Alberti S. Krishnan Y. Hyman A.A. ATP as a biological hydrotrope.Science. 2017; 356: 753-756Crossref PubMed Scopus (306) Google Scholar). Together, these results suggest that the establishment of an in vitro SG assembly system in which the initiating nucleation event could be defined and that recapitulates the ATP-regulated assembly in a native environment would allow a better understanding of how energy interacts with the various RNA-RNA, protein-protein, and RNA-protein networks that drive SG formation. Here, we describe the development of a yeast cytoplasmic extract system that supports SG formation when specific trigger RNAs are added. These in vitro assembled SGs (IVSGs) faithfully recapitulate many of the assembly behaviors of in vivo SGs. In addition to the recruitment of multiple SG markers, formation of IVSGs is disrupted in extracts made from SG-defective strains, suggesting that assembly pathways for IVSGs mirror those of in vivo SGs. The assembly of IVSGs is also sensitive to the energy status of the extract, consistent with the behavior of in vivo SGs. Furthermore, IVSGs display maturation properties reminiscent of in vitro phase-separated proteins. Using this system, we have identified a two-step process for IVSG assembly, with protein-dependent RNA condensation occurring first, followed by recruitment of mRNPs. Interestingly, although a single mRNA condensate can assemble only a partial IVSG, a condensate composed of a mixture of transcripts is sufficient to trigger IVSG formation. This suggests that an ensemble of different mRNA species is necessary to assemble a canonical SG. Together, these data provide framework for understanding how energy and RNA-RNA-mediated events control the assembly of SGs. Previous biochemical studies of SGs have taken two approaches: analysis of the phase separation of individual SG-associated components or characterization of purified SGs. Although proteomic studies of purified SGs have provided insights into the composition of SGs, these SGs are composed of only the “core” components and lack the dynamic, ATP-dependent behaviors that characterize SGs in vivo (Jain et al., 2016Jain S. Wheeler J.R. Walters R.W. Agrawal A. Barsic A. Parker R. ATPase-modulated stress granules contain a diverse proteome and substructure.Cell. 2016; 164: 487-498Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar). In contrast, analyses of the phase separation of the individual protein and/or RNA components of the SG have revealed the role of valency and LCS/IDR domains in driving demixing (Fay et al., 2017Fay M.M. Anderson P.J. Ivanov P. ALS/FTD-associated C9ORF72 repeat RNA promotes phase transitions in vitro and in cells.Cell Rep. 2017; 21: 3573-3584Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar; Jain and Vale, 2017Jain A. Vale R.D. RNA phase transitions in repeat expansion disorders.Nature. 2017; 546: 243-247Crossref PubMed Scopus (294) Google Scholar; Li et al., 2012Li P. Banjade S. Cheng H.-C. Kim S. Chen B. Guo L. Llaguno M. Hollingsworth J.V. King D.S. Banani S.F. et al.Phase transitions in the assembly of multivalent signalling proteins.Nature. 2012; 483: 336-340Crossref PubMed Scopus (933) Google Scholar; Patel et al., 2015Patel A. Lee H.O. Jawerth L. Maharana S. Jahnel M. Hein M.Y. Stoynov S. Mahamid J. Saha S. Franzmann T.M. et al.A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation.Cell. 2015; 162: 1066-1077Abstract Full Text Full Text PDF PubMed Scopus (1004) Google Scholar; Van Treeck et al., 2018Van Treeck B. Protter D.S.W. Matheny T. Khong A. Link C.D. Parker R. RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome.Proc. Natl. Acad. Sci. U S A. 2018; 115: 2734-2739Crossref PubMed Scopus (156) Google Scholar). However, given that these proteins and RNAs exist as components of multivalent mRNPs that are being continuously remodeled by chaperones and helicases in vivo, it has been difficult to define the specific roles of RNAs or proteins in driving SG formation in non-pathological situations. As a result, we have sought to develop an extract system in which SG assembly can be triggered by specific RNAs that recapitulate the dynamic, ATP-regulated behavior observed in vivo. There have been two primary barriers to developing an in vitro SG assembly system: identifying molecules that specifically trigger SG assembly and validating that any assembled structure is a bona fide SG. As the biochemical function of the SG remains unclear, we have focused on four biochemical criteria for assessing whether any assembled structure is a bona fide SG. First, assembly should be dependent on a specific molecular trigger in order to exclude non-specific assembly processes, such as molecular crowding (Lin et al., 2015Lin Y. Protter D.S.W. Rosen M.K. Parker R. Formation and maturation of phase-separated liquid droplets by RNA-binding proteins.Mol. Cell. 2015; 60: 208-219Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar; Molliex et al., 2015Molliex A. Temirov J. Lee J. Coughlin M. Kanagaraj A.P. Kim H.J. Mittag T. Taylor J.P. Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization.Cell. 2015; 163: 123-133Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar). Second, the structures should have the key biochemical markers of SGs and lack the markers for other phase-separated structures (e.g., P bodies) (Buchan et al., 2011Buchan J.R. Yoon J.-H. Parker R. Stress-specific composition, assembly and kinetics of stress granules in Saccharomyces cerevisiae.J. Cell Sci. 2011; 124: 228-239Crossref PubMed Scopus (156) Google Scholar). This will exclude the possibility that the assembled structures are merely non-specifically aggregating mRNPs. Third, extracts from mutant cells defective in SG assembly should fail to support SG assembly in vitro. This ensures that the in vivo SG assembly pathway is the same pathway used to construct SGs in our extract system. Fourth, SGs assembled in vitro should recapitulate the dynamic, energy-dependent behavior observed in vivo (Jain et al., 2016Jain S. Wheeler J.R. Walters R.W. Agrawal A. Barsic A. Parker R. ATPase-modulated stress granules contain a diverse proteome and substructure.Cell. 2016; 164: 487-498Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar). One of the problems with studying SG assembly in vivo is that there are many routes across the phase transition boundary (Van Treeck et al., 2018Van Treeck B. Protter D.S.W. Matheny T. Khong A. Link C.D. Parker R. RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome.Proc. Natl. Acad. Sci. U S A. 2018; 115: 2734-2739Crossref PubMed Scopus (156) Google Scholar). The majority of the in vitro studies of SG components have focused on the role protein-protein interactions and/or RNA-protein interactions in driving phase separation (Lin et al., 2015Lin Y. Protter D.S.W. Rosen M.K. Parker R. Formation and maturation of phase-separated liquid droplets by RNA-binding proteins.Mol. Cell. 2015; 60: 208-219Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar; McGurk et al., 2018McGurk L. Gomes E. Guo L. Mojsilovic-Petrovic J. Tran V. Kalb R.G. Shorter J. Bonini N.M. Poly(ADP-ribose) prevents pathological phase separation of TDP-43 by promoting liquid demixing and stress granule localization.Mol. Cell. 2018; 71: 703-717.e9Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar; Molliex et al., 2015Molliex A. Temirov J. Lee J. Coughlin M. Kanagaraj A.P. Kim H.J. Mittag T. Taylor J.P. Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization.Cell. 2015; 163: 123-133Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar). However, recent work has suggested that RNA can also undergo phase separation directly via RNA-RNA interactions, suggesting that this type of interaction might be a major contributor to RNA granule assembly (Jain and Vale, 2017Jain A. Vale R.D. RNA phase transitions in repeat expansion disorders.Nature. 2017; 546: 243-247Crossref PubMed Scopus (294) Google Scholar). Unfortunately, given the multivalent, dynamic nature of mRNPs, it was unclear whether a single molecular trigger could drive SG formation in vitro. In order to address this problem, we first sought to identify RNA triggers for in vitro assembly of SGs. By leveraging the previous biochemical isolation of yeast SGs for proteomic analysis (Jain et al., 2016Jain S. Wheeler J.R. Walters R.W. Agrawal A. Barsic A. Parker R. ATPase-modulated stress granules contain a diverse proteome and substructure.Cell. 2016; 164: 487-498Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar), we generated an 18,000 × g supernatant that lacked pre-existing SGs yet contained unincorporated and diffuse SG markers and components (Figure 1A). We then tested a variety of in vitro transcribed Cy5-labeled RNAs for their ability to cluster and/or recruit the endogenously expressed SG marker Ded1-GFP from extracts (Figures 1B and S1A). Capped, polyadenylated YFP mRNA did not phase-separate into foci in our extract. Thus, merely adding exogenous RNA is insufficient to trigger RNA condensation and/or SG formation. Similarly, YFP RNA with a 5′ internal ribosome entry site (IRES) also failed to cluster or cause Ded1-GFP to form foci (Figure 1C). This suggested that particular sequence features might be necessary to trigger SG formation in vitro. As recent work on RNA phase separation suggested that arrays of interacting sequences might be necessary in order to trigger condensate formation, we next tested whether arrays of stem loops might promote SG formation in vitro (Jain and Vale, 2017Jain A. Vale R.D. RNA phase transitions in repeat expansion disorders.Nature. 2017; 546: 243-247Crossref PubMed Scopus (294) Google Scholar). Interestingly, insertion of 24xMS2 stem loops into the 3′ UTR of our model YFP transcript was sufficient to support the formation of RNA foci that recruited Ded1-GFP (Figure 1C). Furthermore, an array of the 12xMS2 stem loops alone was sufficient to recapitulate this result, suggesting that the array of stem loops was the key sequence/structural feature driving RNA condensation and Ded1-GFP localization (Figure 1C). In order to test whether the ability of RNAs to cluster and/or recruit Ded1-GFP was a feature common to all repetitive sequences, we also examined the ability of other stem loop arrays and repeat RNAs to form RNA foci that recruit Ded1-GFP. An array of 24xPP7 stem loops was capable of forming RNA foci that recruited Ded1-GFP (Figure 1D). In contrast, an array of 16xU1A stem loops assembled into RNA foci, but these RNA foci failed to recruit Ded1. Thus, not all RNA condensates recruit Ded1. Additionally, we found that although 47xCAG repeats have a known propensity to phase-separate in vitro, the repeat RNA failed to form foci or trigger the formation of Ded1 puncta (Figure 1D). Given that stem loop repeat RNAs exhibit longer half-lives than non-repeat RNA (Svoboda and Di Cara, 2006Svoboda P. Di Cara A. Hairpin RNA: a secondary structure of primary importance.Cell. Mol. Life Sci. 2006; 63: 901-908Crossref PubMed Scopus (119) Google Scholar), one possible concern is that the ability to form a condensate in our system merely reflects the relative stability of the trigger RNAs. Although repeat RNAs (12xMS2, 47xCAG) exhibited higher transcript stability than non-repeat RNA (YFP, IRES-YFP) over longer time periods (Figures S1B–S1C), the RNA levels of all transcripts were similar at 15 min when the reactions were imaged. Thus, the ability of RNAs to cluster and recruit Ded1-GFP in yeast extracts is not a general feature of highly stable RNAs, Cy5-labeled RNA, RNAs containing arrays of stem loops, or highly structured RNAs. Furthermore, the amount of Ded1 protein enrichment in 12xMS2 RNA foci is comparable with that observed in SGs in vivo and SGs isolated by pelleting (Figure 1E). Additionally, compared with the soluble fraction, the 12xMS2 RNA foci were ∼20-fold enriched at lower 12xMS2 concentrations and up to ∼50-fold enriched at higher 12xMS2 concentrations (Figure 1F). These results suggest that our 12xMS2-induced Ded1 foci mimic the enrichment properties of purified and native Ded1-containing SGs. Although our results identified two RNA sequences that are capable of clustering and recruiting Ded1 in extracts, it was unclear whether these sequences merely bound the SG marker, Ded1, or if they nucleated the assembly of a bona fide SG. In order to distinguish between these two possibilities, we tested the ability of 12xMS2 stem loops to specifically recruit other known SG markers in addition to Ded1 and to exclude components of other RNA granules. In order to test whether our exogenous RNA could be triggering the non-specific aggregation of a variety of RNA-binding proteins, some of which are SG components, we assayed the ability of the 12xMS2 RNA to recruit the P body-specific proteins (Dcp2, Dhh1, Pat1) from extracts. Although 12xMS2 RNA was able to form foci in these extracts, the foci failed to recruit Dcp2, and Dhh1 and Pat1 were only minimally recruited to larger 12xMS2 foci (Figures 2A and 2B ). We also found that the highly expressed RNA-binding proteins Pgk1 and Pyk2 were not present in 12xMS2 foci (Figures 2A and 2B; Beckmann et al., 2015Beckmann B.M. Horos R. Fischer B. Castello A. Eichelbaum K. Alleaume A.M. Schwarzl T. Curk T. Foehr S. Huber W. et al.The RNA-binding proteomes from yeast to man harbour conserved enigmRBPs.Nat. Commun. 2015; 6: 10127Crossref PubMed Scopus (227) Google Scholar; Fuller et al., 2020Fuller G.G. Han T. Freeberg M.A. Moresco J.J. Ghanbari Niaki A. Roach N.P. Yates 3rd, J.R. Myong S. Kim J.K. RNA promotes phase separation of glycolysis enzymes into yeast G bodies in hypoxia.eLife. 2020; 9: e48480Crossref PubMed Scopus (17) Google Scholar; Matia-González et al., 2015Matia-González A.M. Laing E.E. Gerber A.P. Conserved mRNA-binding proteomes in eukaryotic organisms.Nat. Struct. Mol. Biol. 2015; 22: 1027-1033Crossref PubMed Scopus (88) Google Scholar). Thus, 12xMS2 RNA triggers the formation of condensates that exclude P body components along with highly expressed RNA-binding proteins. As the SG proteome is affected by the type of stress, we next examined a battery of SG markers representing different cellular functions. 12xMS2 RNA was able to recruit most canonical SG proteins that act at either the 5′ (eIF4E, eIF4G1) or the 3′ (Pab1, Pbp1, Pub1) end of transcripts, suggesting that the assembled structures contain multiple SG markers tha" @default.
• W3048230065 created "2020-08-13" @default.
• W3048230065 creator A5003030236 @default.
• W3048230065 creator A5014701962 @default.
• W3048230065 date "2020-09-01" @default.
• W3048230065 modified "2023-09-28" @default.
• W3048230065 title "An In Vitro Assembly System Identifies Roles for RNA Nucleation and ATP in Yeast Stress Granule Formation" @default.
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• W3048230065 doi "https://doi.org/10.1016/j.molcel.2020.07.017" @default.
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