{"paper_id":"06c53d4d-d9ce-46da-b4bc-e768a767bc68","body_text":"1 \n \nRGG peptide induces the disassembly of disease-relevant FUS and TDP43 \ncondensates  \nMani Garg, Nayana Vinayan, Poulami Ghosh, Kesavardhana Sannula and Purusharth I \nRajyaguru \nDepartment of Biochemistry, Indian Institute of Science \nBangalore 560012 \n \n \nAbstract \nDynamic m embraneless ribonucleoprotein (RNP) condensates regulate different processes \nwithin a cell. The assembly and disassembly of these structures are intricately regulated to \nmaintain cellular homeostasis. Dysregulation of these structures has been  implicated in \nvarious neurodegenerative disorders like Amyotrophic Lateral Sclerosis (ALS)  and \nFrontotemporal Dementia (FTD) . Identifying molecules that can disassemble these toxic \nassemblies is a promising approach to abrogate the associated disease phenotypes  \naugmented by these condensates but is still poorly explored. In this study, we have identified \na role for low -complexity peptide rich in arginine and glycine as a disassembly factor for \nmutant FUS and TDP43  condensates. Deletion of RGG-motif-containing yeast protein Sbp1 \nreduces the disassembly of FUS and TDP43 condensates and increases toxicity. Consistent \nwith that, the expression of Sbp1 in human cells reduced the cytoplasmic condensates of FUS \nand TDP43 mutants  (FUS-P525L and TDP43 lacking nuclear localization signal -NLS) and \nincreased nuclear localization of the FUS -P525L in an RGG -motif dependent manner . In \naccordance with the yeast data, we observed that the  viability of cells expressing FUS-P525L \nimproved upon the expression of Sbp1. In-cell sedimentation assay revealed that purified \nSbp1 could partition FUS-P525L, but not the TDP43 -NLS mutant, from enriched insoluble \ncondensates to soluble fraction. In-vitro sedimentation assay using a two-component purified \nsystem confirmed that partitioning of FUS, but not TDP43, increased to the soluble fraction in \nan RGG-motif-dependent manner. Finally, incubating the cells expressing FUS-P525L and \nTDP43-NLS mutant with RGG-peptide resulted in a reduction of condensate size within the \ncells, suggesting the sufficiency of RGG peptides . Overall, our results identify a role of RGG -\npeptide in disassembling mutant FUS and TDP43 condensates implicated in ALS , projecting \ntheir possible therapeutic role in treating ALS.  \n \n \nKeywords \nNeurodegenerative disorder, amyotrophic lateral sclerosis (ALS), ribonucleoprotein (RNP) \ncondensates, condensate disassembly, intrinsically disordered region (IDR), low -complexity \nsequences (LCSs), RGG motif, Sbp1, FUS, FUS-P525L, TDP43, and TDP43-NLS  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n2 \n \nIntroduction \nCells are decorated with numerous organelles that orchestrate different kinds of functions. \nWhile membrane-bound organelles have been well studied for many years, recent reports \nhighlight the essential role of various membraneless structures in diverse cellular processes. \nThese dynamic and reversible structures contain several RNAs and proteins (therefore called \nribonucleoprotein or RNP condensates). RNP condensates have emerged as major regulatory \nhubs for RNA transcription, splicing, storage, degradation, transport, and translation \nrepression1–8. Stress granules  (SGs) are one of the cytoplasmic RNP condensates that are \nformed in response to various stress conditions (like heat, osmotic, and oxidative stress)3,9,10. \nThese are majorly the sites of mRNA triage where translationally stalled mRNAs are stored \nduring stress conditions6. \nThe assembly and disassembly of SGs are tightly regulated to maintain cellular homeostasis. \nWhile the formation of SGs is essential for many cellular responses2,3,11, a timely disassembly \nis also necessary for maintaining cellular health . The persistent SGs are one of the \ncharacteristic features of various neurological disorders such as amyotrophic lateral sclerosis \n(ALS) and frontotemporal dementia (FTD)12. In such cases, the material properties of the SGs \nshift from a liquid to a more solid state, resulting in the entrapment of proteins like TDP43 and \nFUS, which are nuclear proteins but are observed to be mislocalized to cytoplasmic \ncondensates often induced by specific mutations . Therefore, SGs can act as crucibles for \npathological cellular assemblies , seeding the formation of toxic aggregates. The disease \nphenotypes could be contributed by the gain of function upon cytoplasmic condensate \nformation as well as upon loss of the nuclear function.  A few reports have identified targets \nthat can limit the recruitment and the associated toxicity of TDP43 and FUS to SGs in various \nALS models 13–17. However, the biomolecules that can promote the disassembly  (and not \nclearance) of these structures are poorly explored. Notably, the sporadic nature of most ALS \ncases makes it challenging to identify patients before symptoms arise. Consequently, an \neffective therapeutic strategy would be to focus on slowing disease progression  by targeting \ndisassembly. In this direction, the i dentification of disassembly factors will be beneficial for \nthese conditions where the proteins are already localized to these condensates.  \nIntrinsically disordered region (IDR) containing proteins have been extensively associated with \nthe RNP condensate assembly2,18. Interestingly, apart from being present in abundant \nnumbers (50%) in the proteome of SGs, around 20% of the disassembly -engaged proteins \nwere also depicted to have IDR regions 19. Since these proteins have the tendency to engage \nin several interaction networks, we hypothesized that these could have an important role to \nplay in the disassembly of different RNP condensates. A recent report also highlights the role \nof an IDR -containing protein, Sbp1, from Saccharomyces cerevisiae  in the disassembly of \nprocessing bodies ( PBs, another type of cytoplasmic RNP condensate) 20. Sbp1 is a modular \nprotein having a central RGG/RG repeats rich RGG -motif flanked on either side by RNA -\nrecognition motifs (RRM , Figure 1A ). Apart from its role in translation repression and \ndecapping modulation, it interacts with a core PB-residing protein, Edc3, by its low complexity \nRGG-motif and competes with other Edc3 molecules to disrupt the Edc3:Edc3 self -\ninteraction20–22. The RGG -motif was also reported to be necessary and sufficient for the \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n3 \n \ndisassembly activity, therefore establishing the role of such IDRs in the disassembly of \nphysiological RNP condensates.  \nIn the current report, we explore the role of the RGG-motif sequence in the  disassembly of \nmutant TDP43 and FUS condensates. Our data from Saccharomyces cerevisiae, mammalian \ncells, in-vitro, and ex-vivo experiments propose that  the RGG peptides can directly  \ndisassemble pathological condensates, raising the possibility of a therapeutic role of the RGG \npeptides in treating ALS.  \nResults \n∆sbp1 is defective in the disassembly of TDP43, and FUS condensates \nSaccharomyces cerevisiae has been widely used as a model system to understand various \nneurological disorders23–29. The overexpression of ALS-associated proteins, TDP43 and FUS, is \ntoxic to yeast cells, and multiple reports have identified the modulators of this phenotype23,30–\n36. Overexpression can be carried out by expressing the target protein under a galactose -\ninducible promoter, as has been done for both TDP43 and FUS 33,37. By using this system, we \nwanted to understand the role o f RGG-motif-containing protein Sbp1 in the disassembly of \nTDP43 and FUS condensates. Yeast cells overexpressing TDP43 and FUS under a galactose -\ninducible promoter were incubated in galactose-containing media to induce the expression of \nTDP43/FUS. During this phase, the newly translated TDP43/FUS protein induces stress \n(termed ‘induction’) and accumulates in the cytoplasmic condensates (Figure 1B and E). After \ninduction, the cells were allowed to grow  in glucose-containing media. During this growth \ntime, the protein levels will be reduced because of the inhibition of galactose promoter in \nglucose media. This reduction will lead to  the rescue of cells from stress (termed ‘recovery’) \nand a subsequent decrease in the number of condensates. The amount of protein present in \nthe condensates was then assessed and compared between wild-type and Δsbp1 cells to \nunderstand the role of Sbp1 protei n in the assembly and disassembly of the TDP43/FUS \ncondensates. \nThe induction of TDP43 condensates was first assessed in wild-type and  Δsbp1 cells. The \nfraction of protein present in the condensates was comparable in both backgrounds after \ninduction (Figure 1B and C). However, the fraction of TDP43 protein in condensates during \nrecovery was observed to be more in the Δsbp1 background than in the wild-type cells (Figure \n1B and C).  The dynamics of FUS condensates also followed a similar trend in  the Δsbp1 as \ncompared to the wild-type background (Figure 1E and F). While the fraction of FUS protein \nlocalized to condensates was comparable between wild type and Δsbp1, there was a \nsignificant defect in the disassembly of protein out of condensates during recovery from stress \ninduced by FUS overexpression (Figure 1E and F). These observations highlight the importance \nof Sbp1 in regulating the disassembly of TDP43 and FUS condensates in yeast cells. \nOne of the possible reasons for the disassembly defect in the Δsbp1 background could be \nbecause of an increase in TDP43 and FUS levels. To understand this, the total protein levels \nwere compared for TDP43 and FUS by Western analysis (Figure 1D and G, and Supplementary \nFigure 1A and B) . The relative levels of protein reduction were comparable in different \nbackgrounds for both TDP43 and FUS . Therefore, the defective disassembly could not be \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n4 \n \nattributed to TDP43 and FUS protein accumulation in the Δsbp1 background. Overall, based \non these observations, we propose a role of Sbp1 in the disassembly of TDP43 and FUS \ncondensates in yeast cells. \nTDP43 and FUS overexpression-mediated toxicity increases in Δsbp1 \nTDP43 and FUS overexpression -mediated cytotoxicity have been well-documented in yeast \ncells33–35,37. Cells transformed with a galactose -inducible TDP43/FUS-expressing vector show \ncondensate formation in the cytoplasm, which leads to cytotoxicity. To further understand the \nrole of Sbp1 in TDP4 3 and FUS overexpression-mediated cytotoxicity, we investigated the \ngrowth of TDP43 and FUS overexpressing cells in Δsbp1 background. \nTDP43 and FUS overexpressing cells were significantly defective in their growth compared to \nthe empty vector -transformed cells in both backgrounds, as observed in spot assay  \n(Supplementary Figure 1C and D, and Figure 1H and I). SBP1 deletion led to an aggravation of \nthe growth defect of TDP43 overexpressing cells as compared to the wild-type cells (Figure 1H \nand Supplementary Figure 1C). Similarly, the deletion of SBP1 also increased the sensitivity of \ncells to FUS overexpression (Figure 1I and Supplementary Figure 1D). Based on these results, \nwe conclude that the deletion of SBP1 sensitizes yeast cells to the  overexpression of \nTDP43/FUS. This observation is consistent with defective condensate disassembly observed in \nFigure 1B and E. \nSbp1 expression reduces the mutant TDP43, and FUS condensates in mammalian cells \nIn order to understand the role of Sbp1 as a modulator of TDP43/FUS condensates further, we \naimed to explore the effect of Sbp1 in mammalian cell models. Since there is no homolog of \nSbp1 in the mammalian system , SBP1 from Saccharomyces cerevisiae was cloned into \nmammalian expression constructs. Several disease-relevant mutants of TDP43 and FUS have \nbeen characterized.  TDP43-ΔNLS (lacking nuclear localization signal , Figure 1A ) mutant \nmislocalizes to cytoplasm and forms condensates, which are reported to be toxic and clinically \nrelevant38–40. TDP43-WT was observed to be localized to the nucleus , whereas a significant \nnumber of cells with cytoplasmic condensates were observed when the TDP43-ΔNLS mutant \nwas expressed in HEK293T cells (Figure 2A). The expression of Sbp1 led to a significant increase \nin the cells without condensates of the mutant protein (Figure 2A and B). Interestingly, this \nphenotype was significantly affected when an RGG-motif deletion mutant of Sbp1 was tested \nfor its effect (Figure 2A and B). No change in the nuclear localization was observed for the \nTDP43-WT protein. Further, the Western analysis of TDP4 3-WT and ΔNLS mutant proteins \nwere compared to check the role of Sbp1  or Sbp1ΔRGG in regulating their levels (Figure 2C \nand D). No significant change was observed for any of the proteins in the presence of Sbp1 or \nSbp1ΔRGG. We conclude that Sbp1 reduces the cytoplasmic condensates of ΔNLS mutant of \nTDP43 in mammalian cells in an RGG-motif-dependent manner. \nA similar experiment was also conducted for FUS WT and P525L mutant, where the mutation \nis in the NLS motif of the protein and has been correlated with an aggressive form of juvenile \nALS41 (Figure 1A). Because of this mutation, the protein mislocalizes to the cytoplasm and \nforms cytoplasmic condensates42–44 (Figure 2E). HEK293T cells were co -transfected with \nSbp1/Sbp1ΔRGG and FUS-WT/P525L expressing plasmids and assessed for the condensate s \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n5 \n \n24 hours after transfection. As observed for TDP43 mutants, the expression of Sbp1 led to an \nincrease in cells without FUS-P525L cytoplasmic condensates (Figure 2E and F). Similarly, the \nexpression of the RGG deletion mutant of Sbp1 did not increase the cells with out P525L \ncondensates in a manner comparable to the wild type (Figure 2E and F). This phenotype was \nnot due to any change in  FUS protein levels, as the Western analysis reflected no significant \nchange in FUS protein in any of the conditions (Figure 2G and H). A similar effect was observed \nusing a different construct expressing FLAG-tagged Sbp1, which reduced cells with mutant \nTDP43 and FUS condensates, highlighting that the effect is not due to anomalous behavior \ninduced by the mScarlet tag (Supplementary Figure 2A -D). Overall, we conclude that Sbp1 \nexpression i n mammalian cells significantly reduce s the cytoplasmic condensates of both \nTDP43 and FUS mutant proteins. \nSbp1 expression reduces the defects associated with FUS -P525L overexpression in \nmammalian cells \nApart from inducing the reduction of cells with condensates, the effect of Sbp1 in rescuing the \nnuclear localization of FUS-P525L was also assessed. An analysis of the nuclear to -cytoplasm \nratio of the cells expressing both FUS -P525L and Sbp1 reflected that  Sbp1 expression led to \nan increase in the nuclear localization of the mutant FUS (Figure 3A). Moreover, the RGG-motif \ndeletion mutant was significantly defective in rescuing the nuclear localization defect as \ncompared to the wild type (Figure 3A). No change in the localization of FUS-WT was observed \nin the presence of either Sbp1 or Sbp1ΔRGG. \nOverexpression of FUS has been associated with increased toxicity in HeLa cells45. Considering \nthe deletion of Sbp1 aggravated the growth defects of FUS and TDP43 overexpression in yeast \ncells, we next explore d if the expression of Sbp1 c ould rescue the toxicity of FUS \noverexpressing cells. To assess the cell viability, the number of propidium iodide-positive cells \nwere counted in an incucyte chamber starting after 6 hours of transfection. The \noverexpression of FUS-WT and P525L mutant depicted increased accumulation of dead cells \nas compared to mock and empty vector-transfected cells (Figure 3B and C, and Supplementary \nFigure 3). While not significant, we also observed slightly increased toxicity of FUS-P525L in all \nthe time points of our analysis. Although Sbp1 did not significantly affect the cell viability of \neGFP-transfected cells, we observed a significant reduction in the toxicity of both FUS-WT and \nP525L in the presence of Sbp1 (Figure 3B and C ). This result highlights the role of Sbp1 in  \nsuppressing FUS-overexpression-mediated toxicity in mammalian cells. Therefore, apart from \nreducing the cells with condensates, the expression of Sbp1 reduces the defects associated \nwith the FUS-P525L in mammalian cells. \nSbp1 leads to the disassembly of FUS condensates \nThe reduction in the cells with condensates might result either from the defective assembly \nof TDP43/FUS condensates or from increased disassembly of the pre -formed condensates. \nTherefore, we focused on understanding the mechanism underlying the impact of  Sbp1 on \nmutant FUS and TDP43 condensates.   \nIn this direction, we started by assessing the disassembly activity of Sbp1 on enriched FUS \ncondensates from HEK293T cells by using a modified in-cell sedimentation assay46. Briefly, \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n6 \n \nrecombinant Sbp1 was incubated with the enriched condensates (see methods) of FUS-P525L \nand incubated for 1 hour at 300C (Figure 4A). The supernatant (soluble) and pellet (insoluble) \nfractions were then separated by centrifugation at 18000g for 15 minutes. If Sbp1 affects the \ndisassembly of FUS  mutant condensates, FUS protein will partition more in the supernatant \n(soluble) phase upon incubation with purified Sbp1  compared to the control (buffer) \ncondition. Western analysis was carried out to check protein distributio n in soluble and \ninsoluble fractions. GAPDH, a soluble cytoplasmic protein, did not partition into the pellet \nfraction as expected  (Figure 4B and D) . On the contrary,  FUS-P525L localization to the \ncytoplasmic condensates led to its enrichment to the insoluble pellet fraction  (Figure 4B). \nStrikingly, the incubation of Sbp1 resulted in a significant redistribution of FUS-P525L protein \nto the soluble phase (Figure 4B and C). Such a phenotype was not observed for buffer control. \nInterestingly, when the RGG-motif deletion mutant of Sbp1 was assessed for its disassembly \nactivity on the FUS-P525L mutant,  we observed a significant defect compared to the full -\nlength protein (Figure 4B and C). These observations suggest the RGG-dependent disassembly \nactivity of Sbp1 on the enriched FUS-P525L condensates.  Contrary to the FUS-P525L, the \nenriched TDP43-ΔNLS condensates failed to disassemble in the presence of Sbp1 or \nSbp1ΔRGG using this assay (Figure 4D and E). Therefore, Sbp1 may act differentially on mutant \nTDP43, and FUS condensates inside a cell to induce their disassembly. \nA simple two -component purified system -based sedimentation assay was performed t o \naddress whether Sbp1 could directly affect mutant FUS or TDP43 condensates  (Figure 4F). \nPurified recombinant FUS or TDP43 protein was subjected to phase separation to form the \ncondensates in-vitro (Supplementary Figure 4A-C). Purified Sbp1 or Sbp1 ΔRGG protein was \nthen incubated with these pre-formed condensates to assess their impact. Interestingly, upon \nincubation with Sbp1, there was a significant enrichment of FUS in the supernatant fraction \n(Figure 4G and H). Further, the fraction of FUS protein in the supernatant increased with \nincreasing concentration of Sbp1. Such a phenotype was not observed for the control reaction, \nwhere an equal amount of BSA was incubated with the pre -formed condensates. Moreover, \nas observed for the in-cell sedimentation assay, the extent of partitioning was also significantly \ndefective after incubation with the Sbp1ΔRGG protein (Figure 4G and H). Arginine amino acid \nplays an important role  in protein-protein and protein -RNA interactions by participating in \nmultiple low-affinity interactions such as pi -pi and cation -pi. To test the role of RGG -motif \narginines in disassembling FUS condensates, we used a mutant (AMD, arginine methylation \ndefective) where all the arginines within its RGG -motif were converted to alanine . This \nmutant, like the RGG -motif deletion mutant , was significantly defective in its disassembly \nactivity (Supplementary Figure 4D and E), indicating an important role of the arginine residues. \nSurprisingly, Sbp1 failed to disassemble the in-vitro assembled TDP43 condensates (Figure 4I \nand J). Therefore, with these observations from in-cell and in-vitro sedimentation assays, we \nconclude that Sbp1 directly disassembles FUS condensates, and the RGG-motif is necessary \nfor this activity. Moreover, other cellular factors likely aid the disassembly of mutant TDP43 \ncondensates.   \nRGG-peptides of Sbp1 disassemble TDP43-ΔNLS and FUS-P525L condensates in-vivo \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n7 \n \nThe observations from microscopy analysis and sedimentation assays reflected the \nimportance of the RGG-motif of Sbp1 in disassembling FUS and TDP43 condensates. We next \naimed to understand the sufficiency of RGG -motif in augmenting the disassembly of mutant \nTDP43 and FUS condensates in-vivo using live -cell microscopy analysis. The RGG -motif \npeptides of Sbp1 were added to the culture media of HEK293T cells expressing TDP43-ΔNLS \nor FUS-P525L proteins and observed by live-cell microscopy analysis (Figure 5A and B). We \nobserved a significant reduction in the condensate size of both mutants after 30 min of \npeptide addition (Figures 5C and D). Notably, the condensate size was reduced further after \nanother 30 min of incubation with Sbp1-RGG peptide (Supplementary Figure 5A and B). Such \na reduction was not observed when only the vehicle (solvent control) was added to the media. \nThese observations establish the sufficiency of Sbp1-RGG peptides in disassembling TDP43-\nΔNLS or FUS -P525L condensates. Overall, our results establish the RGG -motif of Sbp1 as a \ndisassembly-inducing peptide for ALS-relevant condensates. \nDiscussion \nIn this study, we identify the low-complexity sequence (LCS) RGG-motif to be important and \nsufficient for the  disassembly of FUS-P525L and TDP43-ΔNLS condensates in-vivo. Our \nexperiments with yeast, mammalian cells, in-vitro reconstituted, and ex-vivo systems establish \nthe involvement of the RGG -motif in regulating the dynamics of these condensates. Our \nconclusion is based on the following observations : 1) Δsbp1 yeast cells are defective in the \ndisassembly of TDP43 and FUS condensates, 2)  TDP43 and FUS overexpression -mediated \ntoxicity in yeast increases in Δsbp1 cells, 3) Heterologous expression of yeast RGG-motif-\ncontaining protein Sbp1 in HEK293T cells increases the number of cells without TDP43-ΔNLS, \nand FUS-P525L cytoplasmic condensates in RGG-motif dependent manner, 4) Sbp1 reduces \nthe FUS overexpression mediated toxicity in mammalian cells  and rescues the nuclear \nlocalization of FUS-P525L, 5) Sbp1 directly disassembles insoluble  FUS-P525L condensates  \nthereby increasing their partitioning to the soluble fraction in RGG-motif dependent manner \nas observed by both modified in-cell and in-vitro sedimentation assays, and 6) RGG-peptide \nderived from  Sbp1 disassemble TDP43-ΔNLS and FUS-P525L condensates in cells when added \nto the media. \nSaccharomyces cerevisiae has served as a simple yet powerful model organism for \nunderstanding the molecular players of various neurological disorders , including ALS 24–29. \nOverexpression of TDP43 and FUS in yeast results in cytoplasmic condensate formation and \nimparts toxicity to the cells 33,37. In agreement with this, we observed TDP43 and FUS -\noverexpression-mediated toxicity and condensate induction in yeast cells (Figure 1). Earlier \nstudies have utilized this system to identify molecular players of the associated cytotoxicity. \nKim et al. iden tified multiple genes that suppress or enhance TDP43 toxicity in yeast when \noverexpressed23. Likewise, specific suppressors and enhancers of cytotoxicity for FUS have \nalso been identified 30,36. While many modifiers have been  identified for TDP43 and FUS \ntoxicity using yeast as a model system, no previous report has explored its potential in the \nidentification of the disassembly factors for disease-relevant condensates. In this study, using \nthe galactose-mediated overexpression system in yeast, we aimed to assess LCS-containing \nfactors for their role in disassembling ALS-relevant condensates of TDP43 and FUS.  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n8 \n \nInspired by the role of Sbp1 RGG-motif in PB disassembly20, we hypothesized that Sbp1 could \ndisassemble TDP43 and FUS condensates . Interestingly, TDP43 and FUS also have long \nstretches of LCS region, w ith FUS having N-terminal QGSY and an  RGG-motif, and TDP43  \nhaving a long C-terminal G-rich region (Figure 1A). Our observations demonstrate a defect in \nthe disassembly of FUS and TDP43 proteins in Δsbp1 cells as compared to the wild-type cells \n(Figure 1). Respective protein levels did not increase in Δsbp1 condition during the stress \nrecovery phase, highlighting that the observed phenotype could not be mediated by a change \nin protein levels. Our observations with the spotting assay analysis report the enhancement \nof TDP43 and FUS toxicity in Δsbp1 cells as compared to wild-type cells (Supplementary Figure \n1C and D, and Figure 1H and I). Our results are supported by the observation that  \noverexpression of Sbp1 suppress es FUS overexpression toxicity in yeast cells 30,36. However, \nthese studies did not assess the underlying mechanism of regulation of toxicity by Sbp1 . \nImportantly, our study characterizes the crucial role of the LCS of Sbp1 in disassembling ALS-\nrelated condensates, a process that was previously unexplored. No such connection has been \nidentified for TDP43 so far. Therefore, our observations establish Sbp1 as a novel and specific \nregulator of mutant TDP43 and FUS condensate disassembly in yeast.  \nBased on our results with yeast cells, we were motivated to check the effect of Sbp1 \nexpression on TDP43 and FUS condensates in mammalian cell models. The cytoplasmic \nmislocalization and aggregate formation are hallmark features of TDP43 in most ALS cases 47. \nEven though present in a lesser number of ALS types, FUS mislocalization , and aggregate \nformation are also well reported 48. Different mutations have been identified in both TDP43 \nand FUS that can enhance the rate of these defects. Overexpression of such mutant forms \nrecapitulates the ALS -related phenotype and has been instrumental in understanding \ndifferent aspects of the disease49,50. In our experiments, we expressed TDP43-ΔNLS and FUS-\nP525L mutants that mislocalized and formed cytoplasmic condensates in HEK293T cells \n(Figure 2). Interestingly, Sbp1 expression significantly reduced the number of cells with \ncytoplasmic condensates of the mutant forms of TDP43 and FUS. The RGG-deletion mutant of \nSbp1 was not effective in a manner comparable to the wild type (Figure 2), suggesting an \nimportant role of the LCS in this activity. It is important to note that the RGG-deletion mutant \ndid reduce cells with mutant condensates to a certain extent, indicating that the RRM domains \ncould also have a role to play. \nThe toxic phenotype of the mutant FUS has been associated with both its cytoplasmic \nmislocalization and condensate formation43,50. Apart from a rescue of the localization of FUS-\nP525L to the cytoplasmic condensates, we observed a change in its localization back to the \nnucleus in the presence of Sbp1 (Figure 3A). This change was also dependent on the Sbp1 \nRGG-motif. Moreover, we observed the suppression of FUS-overexpression-mediated toxicity \nin the presence of Sbp1 (Figure 3B and C). Therefore, on top of rescuing the defects associated \nwith the mutant FUS, Sbp1 significantly improves the cellular fitness of mutant FUS -\noverexpressing cells. These observations open an altogether new direction for exploring \nsimilar LCS -containing disassembly factors for their role in mitigating the effect of toxic \ncondensates. \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n9 \n \nOur experiments further revealed mechanistic insights  into the role of RGG-motif in \nmodulating the cytoplasmic condensates of mutant FUS and TDP43. The modified in-cell and \nin-vitro sedimentation assays depicted the role of low-complexity sequence  as a direct \ndisassembly-inducing factor for the  FUS-P525L condensates (Figure 4). The purified  \nSbp1ΔRGG mutant was as defective as the buffer /BSA control in disassembling the \ncondensates (Figure 4). It is noteworthy that the phase separation of FUS is also depe ndent \non its LCS, including the RGG motifs 51–53. Therefore, it is conceivable that the RGG motif of \nSbp1 interacts with RGG repeats in the FUS LCS to enable disassembly. While the disassembly \nactivity was observed for FUS-P525L condensates, no effect could be seen on TDP43 -ΔNLS \ncondensates (Figure 4) even though ΔNLS condensates were significantly reduced in cells upon \nexpression of Sbp1 in RGG-motif dependent manner. It is likely that the dissolution of mutant \nTDP43 condensates by RGG peptide is accomplished in association with certain cellular factors \n(proteins, RNA, metabolites, etc) that are missing in enriched condensate preparations and in \nthe two-component sedimentation assay system. Alternatively, it is possible that the material \nproperties differ for the enriched or in-vitro assembled TDP43-ΔNLS condensates such that \nSbp1 cannot access the residing molecules for its disa ssembly activity. Understanding the \nmolecular basis underlying the lack of sensitivity of the mutant TDP43 condensates to purified \nSbp1 would be a future endeavour. A recent report identified the proteome of the insoluble \nTDP43 fraction from the brain tissue of TDP -43ΔNLS mice 54. Identifying some intermediate \nplayers from this study that can be further directed to induce the disassembly of toxic TDP43 \ncondensates will be interesting. However, the sensitivity of both FUS-P525L and TDP43-ΔNLS \nto RGG peptides in mammalian cells is encouraging to further explore possible therapeutic \napplications of RGG peptides in ALS. \nDifferent kinds of condensate targeting molecules have been identified , and many of these \nprimarily target the assembly of proteins into  condensates13–17. The list includes many small \nmolecules and a few peptides. Small pl anar compounds, like mitoxantrone , have been \nidentified as affecting both the assembly and disassembly of mutant TDP43 condensate s, \nreducing the cumulative death rate of the primary neurons55. Apart from these, the current \nliterature on peptides targeting TDP43 only reports the degradation-promoting peptides56,57. \nThese peptides were designed to have a TDP43 recognition motif, which is a part of the TDP43 \nprotein having the ability to self-associate. No such peptides are reported for FUS condensates \nto the best of our knowledge. Our report, for the first time, identifies a peptide that functions \nas a genuine disassembly  factor (Figure 5) . Such insight has  opened a new direction for \nexploring the role of LCS peptides as the disassembly factors of other disease-relevant RNP \ncondensates (Figure 6) . Our study provides proof of principle to explore the role of RGG -\npeptides as a therapeutic avenue  for t reating ALS. The use of treatments like ASOs and \ntargeted degradation of aggregated proteins will have an associated drawback as they will not \nbe able to rescue the nuclear functions of TDP43 and FUS. Rescue of the nuclear localization \nof FUS-P525L mutant by Sbp1 in our studies (Figur e 3A) suggests that disassembly-inducing \nmolecules could also promote nuclear relocalization. Therefore , a therapeutic option that \nspecifically targets the disassembly of TDP43/FUS condensates could, in principle, also rescue \nthe nuclear functions. Testing the role of RGG peptides in ALS patient-derived motor neurons \nfor their impact on TDP43/FUS condensates will be a key step in assessing the therapeutic role \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n10 \n \nof RGG peptides. Overall, our results provide an exciting new role of low complexity sequences \nin the disassembly of disease -relevant condensates. These results will motivate  the \nassessment of the potential role of other LCS in condensate disassembly and rescuing toxicity.  \nMaterials and methods \nYeast strain and growth conditions \nThe yeast strains used in this study are listed in Table 1. Strains were grown at 30 0C in yeast \nextract-peptone (YP) medium , and cells with FUS and TDP43 overexpressing plasmids were \nmaintained in synthetic defined (SD) uracil dropout media (SD -Ura-) medium supplemented \nwith 2% raffinose. For secondary culture s, cells were diluted to OD600 0.1 and grown till the \nmid-log phase of OD600 0.4-0.5. For protein induction, the cells were shifted to 2% galactose-\ncontaining media for the mentioned time after the mid-log phase. Recovery experiments were \ncarried out in 2% glucose-containing SD-Ura- media. \nTable 1: List of yeast strains used in this study \nName Genotype Description Source \nyPIR1 MATa his3Δ1 leu2Δ0 met15Δ0 \nura3Δ0 (‘BY4741’) \nWild type (BY4741) yeast \ncells \n58 \nyPIR25 MATa his3D1 leu2 ura3 his3 \nmet15 sbp1∆::KanMX (∆sbp1) \nWild type Saccharomyces \ncerevisiae with SBP1 \ndeletion \nSaccharomyces \ngenome \ndeletion project \nlibrary \n \nYeast spot assays \nCells were grown till the mid-log phase in SD-Ura- media supplemented with raffinose. TDP43 \nand FUS induction were carried out by shifting the cells to 2% galactose-containing media for \n2 and 3 hours, respectively. Post -induction cells were further processed for spotting assays. \nFor spotting assays, cells were serially diluted from 10.0 OD600 to 0.001 OD600 and spotted on \nthe SD-Ura- glucose and galactose-containing plates. After sufficient growth, the images were \nacquired, and the spot area was analyzed as described earlier in Petropavlovskiy et al. 202059. \nPlasmids \nThe list of plasmids used in this study is listed in Table 2. pCEP4 -HIS-SBP1-FLAG was \nconstructed by amplifying HIS-SBP1-FLAG ORF from pPROEx-HIS-SBP1-FLAG construct. The \nprimers were designed using the NEBuilder primer design tool to keep the His and Flag tags \nintact at the N and C -terminus, respectively, and target the amplicon to the BamHI digested \npCEP4 plasmid. Positive clones were confirmed using PCR r eaction, and the expression was \nchecked by transfecting the plasmid in HEK293T cells, followed by Western blotting.  \nTable 2: List of plasmids used in this study \nName Description Source \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n11 \n \npPIR92 pRS316 Empty vector, with URA3 and ampicillin \nresistance genes \nThis study \npPIR74 Plasmid expressing hFUS-YFP under a galactose inducible \npromoter, with URA3 and ampicillin resistance genes \nAddgene \npPIR75 Plasmid expressing hTDP43-GFP under a galactose \ninducible promoter, with URA3 and ampicillin resistance \ngenes \nAddgene \npPIR29 E. coli expression vector for Sbp1 with N-terminal His and \nC-terminal Flag tag, Ampicillin resistance \n22 \npPIR33 pPIR29 with amino acid 125-167 deleted from the Sbp1-\nORF; Sbp1ΔRGG \n22 \npPIR34 pPIR29 with Sbp1R125A, R129A, R131A, R135A, R137A, \nR141A, R145A, R149A, R153A, R155A, R159A, R161A and \nR165A; Sbp1-AMD \n22 \npPIR317 pCEP4, vector with CMV promoter, hygromycin, and \nampicillin resistance genes \nKind gift from Prof. \nK. Somasundaram, \nIISc \npPIR337 pCEP4-His-Sbp1-Flag, pCEP4 expressing N-terminal His \nand C-terminal Flag-tagged SBP1, CMV promoter \nThis study \npPIR308 peGFP-C1, the vector expressing eGFP under a CMV \npromoter \nKind gift from Prof. \nSandeep M \nEswarappa, IISc \npPIR343 peGFP-FUS-WT, the vector expressing eGFP-hFUS-WT \nunder a CMV promoter \nKind gift from Dr. \nDorothee \nDormann, IMB \npPIR344 peGFP-FUS-P525L, the vector expressing eGFP-hFUS-\nP525L mutant under a CMV promoter \nKind gift from Dr. \nDorothee \nDormann, IMB \npPIR313 pDEST eGFP only, the vector expressing eGFP under a \nhybrid CMV and doxycycline-inducible promoter \n40 \npPIR314 pDEST TDP43-WT, pPIR313 with hTDP43 cloned upstream \nof eGFP \n40 \npPIR315 pDEST TDP43-ΔNLS, pPIR313 with hTDP43-ΔNLS cloned \nupstream of eGFP \n40 \npPIR365 pcDNA-mScarlet empty vector Kind gift from Dr. \nJanin \nLautenschlager \npPIR364 pcDNA-Sbp1-mScarlet This study \npPIR373 pPIR364 with amino acids 125-167 (from the Sbp1-ORF \nwhich starts at 22nd amino acid) deleted; Sbp1ΔRGG-\nmScarlet \nThis study \npPIR375 E. coli expression vector pMAL-MBP-TeV-FUS-eGFP-TeV-\nHis \nKind gift from Dr. \nDorothee \nDormann, IMB51 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n12 \n \npPIR376 E. coli expression vector pJ411/TDP-43 (TDP43-TeV-MBP-\nHis) \nKind gift from Dr. \nDorothee \nDormann, IMB17 \npPIR377 E. coli expression vector, expressing His-TEV in a pET-\n24d(+) vector \nKind gift from Dr. \nDorothee \nDormann, IMB51 \n \nMammalian cell cultures \nHEK293T and HeLa cells were maintained in Dulbecco's Modified Eagle Medium  (DMEM) \nsupplemented with 10% FBS and 1X antibacterial -antimycotic solution (complete DMEM). \nMedia was changed every 24 hours for proper growth and split after >90% confluency. \nCultures were checked for Mycoplasma contamination once every month by PCR -based \nmethod. \nTransfection and preparation of mammalian cell samples for microscopy analysis \nFor transfection, Lipofectamine 2000 or 3000 (Thermo ) was used as per the manufacturer’s \nprotocol. The cells were grown, transfected, and processed on coverslips. The samples were \ncollected 24 hours post-transfection, and the cells were fixed with 4% formaldehyde solution \nfor 15-20 minutes. Three washes were given with 1X PBS. For the experiment in Figure 2, the \ncoverslips were directly mounted onto slides with Fluoromount-G containing DAPI and stored \nat 40C until imaging was done. \nFor the experiment in supplementary figure 2 A-D, immunocytochemistry was carried out to \ndetect Sbp1 expression. Briefly, the c ells were permeabilized in 0.25% TritonX100 for 25 \nminutes. Blocking was done for 1.5 -2 hours with a buffer containing 1% BSA and 0.3% \nTritonX100. This was followed by primary antibody (1:200 in blocking buffer) incubation at 40C \novernight in a humidified chamber. Three PBST (PBS + 1% Tween) washes were given the next \nday, and the cells were subjected to secondary antibody (1:300 in blocking buffer) incubation \nfor 2 hours at room temperature. Further, three PBST washes were given, and nuclei were \nstained with DAPI. The coverslips were mounted on slides with Fluoromount-G and stored at \n40C until imaging was done.  \nFor the live -cell peptide uptake assay, cells were grown  and transfected  on 35mm glass -\nbottom dishes, and the plate was directly taken for microscopy. Images were acquired at 63X \nobjective in an incubation chamber with 5% CO2 at 370C. After fixing the fields, the media was \nchanged to a fresh one containing 5μM of the Cy5-labeled peptides (synthesized from \nGenscript). The imaging was done for 60 minutes, with images taken at every 15-minute \ninterval. After 60 minutes, the cells were washed in PBS thrice and resuspended in PBS to \ncheck the uptake of Cy5-labeled peptides. \nMammalian cell viability assay \nThe cell viability assay was performed in the Incucyte chamber. Briefly, after 6 hours of \ntransfections, the cell media was supplemented with 500uM propidium iodide  (PI), and \nimages were acquired every hour to score the number of PI -positive cells. The cell death \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n13 \n \nanalysis was performed using the IncuCyte S3 live-cell analysis instrument (Sartorius), and the \nchange in the number of PI-positive cells (dead cells) in different conditions was plotted in the \ngraph. \nMicroscopy analysis \nAfter the growth in respective media, yeast c ells were centrifuged at 14000 rpm for 15 \nseconds, and pellets were resuspended in 10µl of media. A total of 5µl of the cell suspension \nwas spotted on a coverslip for live cell imaging. The Deltavision Elite microscope system was \nused to acquire all the images. The system was equipped with softWoRx 3.5.1 software \n(Applied Precision, LLC) and an Olympus 100x, oil-immersion 1.4 NA objective. The channel's \nexposure time and transmittance settings were selected depending on protein expression and \nkept the same for all the biological replicates within an experiment. Images were captured as \n512 × 512-pixel files with a CoolSnapHQ camera (Photometrics) using 1 × 1 binning for yeast. \nAll the images were deconvolved using standard softWoRx deconvolution algorithms. ImageJ \nwas used to analyze the data , and the g ranules were counted using the ‘Find Maxima’ tool \nfrom Fiji-ImageJ software. The images were converted to 8 -bit, and the plugin was run. The \nprominence was set from 10-30, and the number of condensates and cells was counted. \nThe microscopy image acquisition  for mammalian cells was performed using the Andor \nDragonfly Confocal Microscope or Leica SP8 Falcon Confocal Microscope (for live cell peptide \nuptake experiment). HEK293T cells were imaged using 63X objective and the exposure time \nand transmittance were adjusted according to the protein expression levels and were kept the \nsame for all the biological replicates within an experiment. Analysis was carried out using Fiji-\nImageJ software.  \nTotal fluorescent intensities or fraction of protein in condensates were calculated by \nmeasuring the CTCF (Corrected Total Cell Fluorescence) values for the ROI. For background \nsubtraction, three regions from the background were selected and the intensity was \ncalculated from all three regions. This was followed by subtraction of background from the \ntotal intensity of ROI (region of interest) using the following formula: \n𝐶𝑇𝐶𝐹 = (𝐴𝑟𝑒𝑎 𝑜𝑓 𝑅𝑂𝐼 ∗ 𝑀𝑒𝑎𝑛 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑅𝑂𝐼) − (𝐴𝑟𝑒𝑎 𝑜𝑓 𝑏𝑎𝑐𝑘𝑔𝑟𝑜𝑢𝑛𝑑\n∗ 𝑀𝑒𝑎𝑛 𝑏𝑎𝑐𝑘𝑔𝑟𝑜𝑢𝑛𝑑 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦) \nFor fraction condensate intensity, the intensity of all condensates from a cell is quantitated \nand divided with that of the total cell intensity. For cells with no condensates in the recovery \nphase, the value was kept at 0. \nFor nuclear: cytoplasm ratio analysis, the CTCF of the nucleus and total cell was quantitated \nby the aforementioned method. The cytoplasm intensity was calculated by subtracting the \nnuclear intensity from that of the total cell intensity. This was followed by calculating the ratio \nof N: C intensity.  To normalize the values ( for cells without condensates and N: C ratio  in \nFigures 2 and 3 ) for the mScarlet expression levels , the respective values were divided with \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n14 \n \nthe mean CTCF mScarlet values. \nExpression and purification of recombinant proteins \nAll the primary cultures of E coli expression strains were cultured in LB and were grown at \n370C overnight in the presence of appropriate antibiotics.  The individual protocols for the \nrecombinant expression and purification of the proteins used in this study are detailed below. \nAfter purification, the elutes were dialyzed against the respective buffers, flash frozen, and \nstored at -800C as 50-100µl aliquots.  \nPurification of MBP-TeV-FUS-eGFP-His \nMBP-TeV-FUS-eGFP-His expressing pMal plasmid was transformed into E. coli BL21 (DE3) \nRosetta competent cells and the cells were  selected on 100µg/µl ampicillin and 50 µg/µl \nchloramphenicol containing LB agar plates. A single colony was inoculated in LB media \n(containing 100µg/µl ampicillin and 50 µg/µl chloramphenicol) and was incubated overnight \nat 370C and 180rpm. A secondary culture was set up from the primary culture and was grown \nto a final OD 600 of 0.8. The cultures were then subjected to a cold shock  by incubating the \nflasks on ice for 15 minutes. This helps in the induction of chaperones that help in preventing \nthe aggregation of FUS. Post-cold shock, the FUS protein was induced with 1mM IPTG and was \nincubated at 100C for 24 hours. Following induction, the cells were pelleted at 4200rpm at 40C \nfor 15 minutes. The cell pellets were stored at -800C. \nFor the Ni -NTA purification of the His tagged FUS, the cells were first resuspended in the \nresuspension buffer containing 50mM NaH 2PO4, pH 8.0, 300mM NaCl, 10mM ZnCl 2, 40mM \nimidazole,4mM beta -mercaptoethanol and 10% glycerol by vortexing. To the completely \nresuspended cells, a final concentration of 1mg/ml RNAase,1mg/ml lysozyme, and 1X PIC was \nadded, followed by 30 minutes of incubation. Cells were lysed by sonication at 40% amplitude \nfor 10 minutes with 10s on and off cycles. The lysate was centrifuged at  15000rpm for 15 \nminutes, and the supernatant was collected in a fresh tube. Ni -NTA resin calibrated with the \nresuspension buffer was added to the supernatant and was incubated for binding for 2 hours \nat 4 0C in a nutator. After binding, the beads were washed thrice with the wash buffer \n(resuspension buffer without glycerol) and eluted with 500mM imidazole. The beads were \nspun down at 1500rpm for 1 minute , and the elute was collected. The FUS elute obtained \nfrom the Ni-NTA purification was then subjected to binding to MBP resin, which was calibrated \nwith the resuspension buffer overnight at 40C in a nutator. After binding, the MBP beads were \nwashed twice with the resuspension buffer and were eluted using resuspension buffer with \n20mM maltose. The eluted proteins were then dialy zed using a buffer containing 20mM \nNaH2PO4, pH 8.1, 150mM NaCl, 5% glycerol, 1mM EDTA, and 1mM DTT. The dialyzed protein \nconcentrations were measured using Bradford , followed by flash freezing and storage at -\n800C51. \nPurification of TDP43-TeV-MBP-His and TeV protease \nTDP43 and TeV protease were both expressed in E coli BL21 (DE3) Rosetta pLys competent \ncells grown in standard LB media. TDP43-TeV-MBP-His was induced with 0.5mM IPTG and was \nincubated overnight at 16 0C. The pellets were resuspended in lysis buffer containing 20mM \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n15 \n \nTris pH 8, 1M NaCl, 10mM imidazole, 10% (v/v) glycerol , and 4mM β-mercaptoethanol and \nwere incubated on ice for 30 minutes with 1mg/ml RNAase,1X PIC and 1mg/ml lysozyme \nfollowed by sonication. The lysate was spun down at 15000rpm for 15 minutes, and the lysate \nwas subjected to binding to Ni -NTA beads. Post binding, the beads were washed thrice with \nbuffer containing 40mM imidazole followed by elution using 300mM imidazole17. \nTeV protease was induced overnight (16h) with 1mM IPTG at 0.6 OD 600 at 200C. The induced \ncell pellets were resuspended in Tris lysis buffer (50mM Tris pH8, 200mM NaCl, 20mM \nimidazole, 10% glycerol, and 4mM b-mercaptoethanol) and were supplemented with 1mg/ml \nRNAase,1mg/ml lysozyme and 1X PIC. The resuspended cells were lysed by sonication. The \nTeV-His in the supernatant was purified by using Ni-NTA beads and washed with the Tris lysis \nbuffer with 1M NaCl. The TeV protease was eluted using 800mM imidazole and was dialyzed \nagainst storage buffer containing 20mM Tris pH 7.4, 150mM NaCl,  20% glycerol, and 2mM \nDTT51. \nPurification of His-Sbp1-Flag, His-Sbp1ΔRGG-FLAG, and His-Sbp1-AMD-Flag \nRecombinant His-Sbp1-Flag, His-Sbp1ΔRGG-FLAG, and His-Sbp1-AMD-Flag transformed in E. \ncoli BL21 cells were induced with 1mM IPTG for  3 hours at 370C. The induced cell pellet was \nresuspended in lysis buffer (300mM NaCl, 50mM NaH2PO4, 1mM DTT, 11ug/ul RNase, 1mg/ml \nlysozyme, 1X PIC, and 10mM Imidazole) followed by sonicati on. Lysate was clarified by \ncentrifugation at 15000rpm for 15 minutes at 4 0C. Clarified lysate was incubated with \nequilibrated Ni-NTA resin for 2 hours at 40C. The beads were washed thrice with wash buffer \n(300mM NaCl and 50mM NaH2PO4) containing increasing concentrations of imidazole at each \nstep (20, 35, and 50mM). Protein was then eluted in elution buffer (50mM NaH2PO4, 300 mM \nNaCl, and 500 mM Imidazole). The protein was dialyzed into dialysis buffer (10mM Tris pH 7.0, \n100 mM NaCl, 10% glycerol, and 1mM dithiothreitol) overnight at 40C. The concentration was \nchecked by Bradford assay, and small protein aliquots were stored at -800C until further usage.  \nIn-cell sedimentation assay \nHEK293T cells (from one T75 flask) transfected with the respective plasmids were collected 24 \nhours post-transfection. The cell lysis was carried out in RIPA buffer (50mM Tris -HCl, 1% NP-\n40, 150mM NaCl, 1mM EDTA, 1mM Na-orthovanadate, 1mM Na-fluoride, 1X PIC, 1mM PMSF \nand RNase Inhibitor) for 30 minutes at 4 0C. Cell debris was removed by a mini -spin at 1000g \nfor 2 minutes at 4 0C. Protein concentration was estimated using Bradford, and 700-800ug of \nlysate was further taken to separate the soluble and insolub le phases from the lysate  by \ncentrifugation at 15000g for 15 minutes at 40C. The supernatant was collected as the soluble \ncytoplasmic fraction in a fresh tube, and the pellet was resuspended in 100ul of RIPA buffer. \nThe disassembly reaction was set up in a fresh tube with 20ul of the pellet fraction and 5uM \nof the purified protein. This was allowed to incubate at 300C for 1 hour and then centrifugated \nat 15000g for 15 minutes at 4 0C to separate the soluble and the pellet fractions. The pellet \nwas resuspended in 2% SDS buffer (2% SDS, 100mM Tris-HCl, pH 7.0) and incubated for 1 hour \nat 30 0C. The resulting solution after that was considered to be the insoluble fraction. All \nsamples were heated at 1000C for 5 minutes in 1X SDS loading dye and were taken ahead for \nWestern analysis. \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n16 \n \nIn-vitro sedimentation assay for FUS and TDP43 \nFor the in-vitro sedimentation assay of FUS, the phase separation of the purified MBP -FUS-\neGFP-His was induced by the cleavage of the MBP tag by the TeV protease. A 50µl phase \nseparation mixture with 1µM FUS in a buffer containing 50mM Tris pH 8, 0.5mM EDTA, and \n1mM DTT w as incubated at 30 0C for 1 hour with the addition of 0.1mg/ml TeV protease 51. \nChange in turbidity after 1 hour indicates phase separation. The test proteins were added at \nthe mentioned concentration to FUS in the phase separation reaction to assess their impact \non phase separation. After 1 hour of phase separation, the samples were centrifuged at \n20000g for 15 minutes, and the soluble and insoluble fractions were separated. The insoluble \n(pellet) fraction was resuspended in 50µl buffer, and equal volumes of the two fractions were \nloaded onto SDS-PAGE gel. CBB staining of the SDS-PAGE gels was carried out, and the ratio of \nFUS in the soluble to the total protein loaded (sum of FUS in the soluble and insoluble) was \ncalculated from the band intensities analyzed by ImageLab software. The same in-vitro \nsedimentation assay protocol was also used to assess the effect of Sbp1 on TDP43 with BSA  \nas the negative control. The only difference in the case of TDP43 was the composition of the \nphase separation buffer. The MBP tag cleavage of TDP43 was carried out in a 50µl reaction \nmix with 1µM TDP43, HEPES buffer containing 20mM HEPES, pH 7.5, 150mM NaCl, and 1mM \nDTT with the addition of 20µg/µl of TeV. \nWestern analysis \nWestern analysis was conducted as per the standard protocol. Blocking was done using 5% \nskim milk powder in TBST. Primary antibody incubation was done overnight at 40C. The blots \nwere incubated at room temperature for 1 hour for secondary antibody and were developed \nusing the Western ECL kit in a BioRad ChemiDoc.  The antibodies used in this study are anti -\nGFP (Biolegend, 902602), anti -Pgk1 (Abcam, Ab113687), anti -Gapdh (Cloud -clone corp., \nMAB932Hu23), anti-mCh ( Abcam, Ab167453), anti-rabbit ( Jackson ImmunoResearch Lab , \n111-035-003), anti -mouse ( Jackson ImmunoResearch Lab , 115-035-003), and anti-rabbit-\nalexafluor568 (Invitrogen, A11011). \nStatistical analysis \nAll the analyses were conducted using the GraphPad  Prism software (version 8.0.2). The \nsignificance was calculated either by unpaired/paired student t -test or 2 -way ANOVA with \nmultiple comparisons. All the figure legends include the specific details of the test used and \nthe p-value considerations. In all the graphs, the error bars represent the standard error of \nthe mean (SEM), and the same color points in a graph depict the data from a single \nexperimental set.  \nAcknowledgment \nWe thank Dr. Dorothee Dormann, Dr. Janin Lautenschlager, Prof. K. Somasundaram, and Prof. \nSandeep M Eswarappa for sharing various plasmids. All the members of the Rajyaguru lab are \nacknowledged for their constant inputs, support, and encouragement.  We thank the Indian \nCouncil of Medical Research (ICMR , grant# IIRPIG-2024-01-00233), Amyotrophic Lateral \nSclerosis (ALS) Association  (Grant#26-SGP-761) and the Department of Biotechnology, \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n17 \n \nGovernment of India (DBT, grant#BT/PR51975/BMS/85/23/2024) for supporting this research; \nthe Department of Science and Technology (DST-FIST) India, and the Indian Institute of Science \n(IISc) for infrastructure and other support. KS acknowledges Indian Council of Medical \nResearch (ICMR , grant# 2021-14148/CMB/ADHOC-BMS) and Department of Biotechnology \n(DBT, grant# BT/PR50450/MED/12/1044/2023). MG tha nks CSIR -UGC and SERB -India, NV \nthanks PMRF, and PG appreciat es GATE for the financial assistance.   Flowchart or model \nfigures were generated from adapted images provided either by Biorender or Servier Medical \nArt (Servier; https://smart.servier.com; licensed under a Creative Commons Attribution 4.0  \nUnported License). \nAuthor contributions \nConceptualization and hypothesis —PIR and MG; Experimental design —PIR, MG, and NV ; \nExperimentation—MG, NV, and PG; Data interpretation—PIR, MG, NV, PG, and KS; Manuscript \nwriting (first draft)—MG, Subsequent draft review and editing—PIR and MG. \nDeclaration of interests \nWe, the authors, have submitted a provisional patent application for the use of RGG-peptides \nin disassembling pathological condensates. \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n18 \n \nFigure Legends \nFigure 1: Δsbp1 cells are defective in the disassembly of TDP43 and FUS condensates in yeast \nand show enhanced toxicity. (A) Schematic representation of TDP43, FUS, and Sbp1 proteins. \nFor Sbp1, the sequence architecture of the RGG-motif is also presented. (B) Representative \nimages for the microscopy analysis of wild-type and ∆sbp1 cells transformed with Gal-TDP43-\nGFP plasmid. After growing to 0.4 OD600, cells were shifted to galactose-containing media for \n3 hours to induce TDP43 expression (stress induction), followed by 4 hours of recovery in \nglucose (stress recovery). Cells were taken for microscopy analysis at both steps. White arrows \nmark the presence of TDP43 condensates. Scale bar=2um. (C) Graph representing the fraction \nof the TDP43 protein present in condensates per cell. Condensate intensities were calculated \nand divided by the total fluorescent intensity of the respective cell. A minimum of 50 cells per \nexperiment were analyzed from 4 independent experiments (n=4) as per formed in B. An \nunpaired t-test was used to calculate the significance. (D) Graph depicting the relative change \nin TDP43 protein levels as compared to the respective induction condition. Significance was \ncalculated using a student -paired t -test analysis (n =8). (E) Representative images for the \nmicroscopy analysis of wild-type and ∆sbp1 cells transformed with Gal-FUS-YFP plasmid. After \ngrowing to 0.4 OD 600, cells were shifted to galactose -containing media for 2 hours to induce \nFUS expression (stress induction), followed by 4 hours of recovery in glucose (stress recovery). \nCells were taken for microscopy analysis at both steps . White arrows mark the presence of \nFUS condensates. Scale bar=5um. (F)  Graph representing the fraction of the FUS protein in \ncondensates per cell. Condensate intensities were calculated and divided by the total \nfluorescent intensity of the respective cell. A minimum of 50 cells per experiment were \nanalyzed from 5 independent experiments as performed in E (n=5). An unpaired t -test was \nused to calculate the significance. (G) Graph depicting the relative change in FUS protein levels \ncompared to the respective induction condition. Significance was calculated using a student-\npaired t-test analysis (n=5). (H and I) Quantitation of the spot area from spot assays of wild -\ntype and Δsbp1 cells transformed with either Gal-TDP43-GFP (H) or Gal-FUS-YFP (I) expressing \nconstructs as performed in Supplementary Figure 1C and D. Values were normalized with \nrespect to their EV controls. Significance was calculated by student-paired t-test analysis (n=6 \nand n=10 for H and I, respectively). Error bars in all graphs represent mean +SEM, and the \nsame color points depict the data from a single experimental set for all the graphs. *, **, ***, \nand **** denote p-value<0.05, <0.01, <0.001, and <0.0001, respectively. \nFigure 2: Sbp1 expression reduces mutant TDP43 and FUS condensates in an RGG -motif-\ndependent manner in mammalian cel ls. (A) Microscopy images of HEK293T cells co -\ntransfected with mScar/Sbp1 -mScar/Sbp1ΔRGG-mScar and different eGFP -TDP43-related \nconstructs. Cells were collected 24 hours after transfection and processed for microscopy \nanalysis. The white arrow marks the presence of cytoplasmic condensates. Scale bar=5um. (B) \nGraph representing the relative  changes in the cells without TDP43 -ΔNLS condensates \n(relative to the mScar transfected cells). The values were also normalized with the respective \nmScarlet fluorescent intens ity expression levels. A paired t -test was used to calculate the \nsignificance value (n=6). (C and D) Western blot analysis (C) and its quantitation (D) \nrepresenting the change in the levels of TDP43 -WT and TDP43 -ΔNLS in the presence of \nmScar/Sbp1-mScar/Sbp1ΔRGG-mScar from 6 independent experiments (n=6). Anti -mCh \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n19 \n \nantibody was used to detect the mScarlet-tagged proteins. Values on the right represent the \nposition of different molecular weight ladder bands in kDa. A student -paired t-test was used \nto calculate the significance value. (E) Microscopy images of HEK293T cells co-transfected with \nmScar/Sbp1-mScar/Sbp1ΔRGG-mScar and eGFP -FUS-WT/P525L constructs. Cells were \nprocessed similarly to the experiment in A . The white arrow marks the presence of \ncytoplasmic condensates. Scale bar=5um. (F) Graph representing the relati ve changes in the \ncells without TDP43 -ΔNLS condensates (relative to the mScar transfected cells). The values \nwere also normalized with the respective mScarlet fluorescent intensity expression levels. A \npaired t-test was used to calculate the significance value (n=5). (G and H) Western blot analysis \n(G) and its quantitation (H) representing the change in the levels of FUS-WT and FUS-P525L in \nthe presence of mScar/Sbp1-mScar/Sbp1ΔRGG-mScar (n=4). Anti-mCh antibody was used to \ndetect the mScarlet-tagged proteins. Values on the right represent the position of different \nmolecular weight ladder bands in kDa. A student -paired t -test was used to calculate the \nsignificance value. Error bars in all graphs represent mean +SEM, and the same color points in \na graph depict the data from a single experimental set.  *, **, ***, and **** denote p -\nvalue<0.05, <0.01, <0.001, and <0.0001, respectively. \nFigure 3: Sbp1 expression reduces overexpression -mediated defects of FUS-P525L in \nmammalian cells. (A) Graph representing the distribution of FUS-P525L protein in nucleus and \ncytoplasm. The fluorescent intensities of the nucleus and cytoplasm were calculated from the \nexperiment as performed in Figure 2E, and the ratio is plotted here. A student -paired t-test \nwas used to calculate the significance value (n=6). (B) Incucyte images (real -time cell death \nanalysis) representing the cellular uptake of propidium i odide (PI) in different conditions in \nHeLa cells. Scale bar=100um. (C) Graph representing the number of propidium iodide (PI) \npositive cells (dead cells) from the experiment as performed in B. The time on the x -axis \nreflects the time-point after transfection in hours. The significance was calculated using 2-way \nANOVA with multiple comparisons (n=4). Error bars in all graphs represent mean +SEM, and \nthe same color points in A depict the data from a single experimental set. *, **, ***, and **** \ndenote p-value<0.05, <0.01, <0.001, and <0.0001, respectively. \nFigure 4: Sbp1, but not Sbp1ΔRGG, disassembles FUS condensates. (A) Schematic depicting \nthe workflow for the in-cell sedimentation assay modified to assess the disassembly activity \nof Sbp1. (B and D) Western analysis of the different fractions from FUS-P525L (B), and TDP43-\nΔNLS (D) in-cell sedimentation assay. The different fraction loadings are as follows: lysate: \n7.5%, cytoplasm: 7.5%, pellet: 15%, soluble: 60%, and insoluble: 60%. GAPDH serves as the \ncontrol for the assay, and ponceau reflects the purified protein added  to the respective \nreaction. Values on the right represent the position of different molecular weight ladder bands \nin kDa. (C and E) Quantitation of the amount of protein in the soluble fraction from \nexperiments as done in B and D. The band intensities wer e calculated using ImageJ, and the \nfraction of protein in the soluble phase was calculated by S/(S+I), where S and I represent the \nprotein in the soluble and the insoluble fractions, respectively. Significance was calculated by \nusing student-paired t-test analysis (n=6 and n=7 for C and E, respectively). (F) Schematic \ndepicting the workflow of the in-vitro sedimentation assay performed to assess the \ndisassembly activity of Sbp1 on the phase -separated FUS and TDP43 condensates. (G) \nCoomassie-stained protein gels depicting the fractionation of FUS to soluble and insoluble \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n20 \n \nphases in the presence of buffer, BSA, Sbp1, Sbp1ΔRGG. The ratio reflects the amount of FUS: \ntest protein taken for the assay. Sbp1ΔRGG and MBP migrate at the same position and hence \nappear as a single band. Values on the left represent the position of different molecular weight \nladder bands in kDa. (H) Quantitation of the fraction of FUS protein present in the soluble \nphase from 7 independent experiments (n=7) as performed in G. Significance was calculated \nby a student-paired t-test analysis. (I) Coomassie-stained protein gels and its quantitation (J) \ndepicting the fractionation of TDP43 to soluble and insoluble phases in the presence of BSA \nand Sbp1. The ratio reflects the amount of TDP43:test protein taken for the assay. The values \non the graph reflect the relative amount of TDP43 protein present in the insoluble fraction. A \nstudent-paired t-test analysis calculated significance (n=3). Error bars represent mean +SEM, \nand the same color points depict the data from a single experimental set. *, **, ***, and **** \ndenote p-value <0.05, <0.01, <0.001, and <0.0001, respectively.  \nFigure 5: Sbp1-RGG peptide disassembles the TDP43-ΔNLS and FUS-P525L condensates. (A) \nSchematic depicting the workflow of the live-cell microscopy with the RGG-peptides. (B) The \nsequence of the peptide used for the experiment as performed in A. 2 extra amino acids at \nthe N-terminal end (M and Q) and 3 amino acids at the C -terminal end (F, N, and G) were \nincluded for better purification of the RGG-peptide. (C) Microscopy images for HEK293T cells \ndepicting the change in the area of TDP43 -ΔNLS condensate (marked by white arrows) after \nincubating with either the vehicle control or Sbp1 -RGG peptides. Scale bar=2um. Graphs on \nthe right depict the quantitation of the condensate area/total cell area from at least 30 cells \nper experiment (n=3). (D) Microscopy images for HEK293T cells depicting the change in the \narea of FUS -P525L condensate (marked by white arrows) after incubating with either the \nvehicle control or Sbp1 -RGG peptides. Scale bar=2um. Graphs on the right depict the \nquantitation of the condensate area/total cell area from at least 40 cells per experiment (n=3). \nSignificance was calculated by paired t-test analysis. **** denotes p-value <0.0001.  \nFigure 6: Schematic depicting the role of RGG peptides as a therapeutic approach to \ndisassemble the toxic cytoplasmic condensates. Green represents mutant proteins, like FUS-\nP525L and TDP43-ΔNLS, that mislocalize to the cytoplasm and form condensates in diseased \nconditions. RGG peptides could have the potential to disassemble these toxic condensates. \nSuch an effect may also result in the restoration of the nuclear localization phenotype. \n \n \n \n \n \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n21 \n \nSupplemental Information \nSupplementary Figures S1-S5. \nSupplementary Figure S1: Δsbp1 cells are defective in the disassembly of TDP43 and FUS \ncondensates in yeast. (A and B)  Western analysis depicting the change in protein levels of \nTDP43 (A) and FUS (B) with respect to the respective induction condition. Quantitation of the \nblots is provided as Figure 1D and G. Values on the right represent the position of different \nmolecular weight ladder bands in kDa. Ponceau served as the loading control. (C and D) Spot \nassays of wild type and Δsbp1 cells transformed with either empty vector (EV) or Gal-TDP43-\nGFP (C) / Gal-FUS-YFP (D) expressing constructs. After growing to 0.4 OD600, cells were shifted \nto galactose -containing media for 2 -3 hours to induce TDP43/ FUS expression. This was \nfollowed by serial dilution of cells and spotting on glucose and galactose -containing SD-Ura- \nmedia plates. Images were taken after 2 -4 days of growth at 300C.  \nSupplementary Figure S2: Sbp1 expression reduces mutant TDP43 and FUS condensates in \nHEK293T cells . (A) Microscopy images of HEK293T cells co -transfected with EV/Sbp1 and \ndifferent eGFP-TDP43-related constructs. Cells were collected 24 hours after transfection and \nprocessed for immuno -cytochemistry analysis to detect Sbp1 using an anti -Sbp1 antibody \n(RFP channel). TDP43 constructs have an N -terminal eGFP-tag that was used to check the \nlocalization through the GFP channel. The white arrow marks the presence of cytoplasmic \ncondensates. Scale bar=5um. (B) Quantitation of the percentage of cells without cytoplasmic \ncondensates from the experiment as performed in A. A student -paired t-test was used to \ncalculate the significance value (n=4). (C) Microscopy images of HEK293T cells co-transfected \nwith EV/Sbp1 and eGFP -FUS-WT/P525L constructs. Cells were collected 24 hours after \ntransfection and processed like the experiment in A. The white arrow marks the presence of \ncytoplasmic condensates. Scale bar=5um. (D) Quantitation of the percentage of cells without \ncytoplasmic condensates from the experiment as performed in C. A student-paired t-test was \nused to calculate the significance value (n=5). Error bars in all the graphs represent mean \n+SEM, and the same color points in a graph depict the data from a single experimental set. ** \nand **** denote p-value <0.01 and <0.0001, respectively. \nSupplementary Figure S3: Sbp1 expression reduces overexpression-mediated defects of FUS-\nP525L in mammalian cells . Incucyte images representing the cellular uptake of propidium \niodide (PI) in different conditions in HeLa cells. Scale bar=100um. The images are part of \nFigures 3B and C. \nSupplementary Figure S4: Sbp1AMD (arginine methylation defective) mutant does not \naffect FUS condensates.  (A) Coomassie -stained protein gel depicting the various purified \nproteins used in this study. (B and C) Coomassie-stained protein gels depicting the partitioning \nof FUS (B) and TDP43 (C) to the insoluble (I) phase after the cleavage of MBP -tag using TeV \nprotease. S denotes the soluble phase. Values on the right represent the position of different \nmolecular weight ladder bands in kDa. (D) Coomassie -stained protein gel depicting the \nfractionation of FUS to soluble and insoluble phases in the presence of Sbp1AMD (mutant \nwhere all arginines within the RGG -motif are covered to alanine, see Figure 1A). The ratio \nreflects the amount of FUS: test protein taken f or the assay. Sbp1AMD and MBP migrate at \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n22 \n \nthe same position and hence appear as a single band. Values on the left represent the position \nof different molecular weight ladder bands in kDa. (E) Quantitation of the fraction of FUS \nprotein present in the soluble phase from 7 independent experiments (n =7) as performed in \nD and Figure 3H. The graph from Figure 3I has been replotted here to include Sbp1AMD values. \nError bars represent mean +SEM, and the same color points in a graph depict the data from a \nsingle experimental set.  *, **, ***, and **** denot e p -value <0.05, <0.01, <0.001, and \n<0.0001, respectively.  \nSupplementary Figure S5: HEK293T cells readily uptake Cy5 -labelled Sbp1-RGG peptide. (A \nand B) Microscopy images for HEK293T cells depicting the change in the area of TDP43-ΔNLS \n(A) and FUS-P525L (B) condensate (marked by white arrows) and the uptake of Cy5 -labelled \npeptide after 60mins of incubation with either the vehicle control or Sbp1-RGG peptides. The \npanel represents the same set of cells as depicted in Figure 4D. Cy5 panel depicts the uptake \nof the peptide. Scale bar=2um. The graph on the right represents the change in the \ncondensate area normalized with the total cell  area after incubation with the Sbp1 -RGG \npeptide (n=3). 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Trends Biochem \nSci 46, 550–563 (2021). \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n26 \n \n54. Dunlop, F., Mason, S., Tsitkanou, S. & Russell, A. P . Proteomic analysis of the TDP-43-associated \ninsoluble fraction from NEFH-TDP-43 mouse brain suggests sustained stress granule formation, \nCLUH granule recruitment and impaired mitochondrial metabolis m. Preprint at \nhttps://doi.org/10.1101/2024.05.23.595607 (2024). \n55. Fang, M. Y . et al. Small-Molecule Modulation of TDP-43 Recruitment to Stress Granules Prevents \nPersistent TDP-43 Accumulation in ALS/FTD. Neuron 103, 802-819.e11 (2019). \n56. Gao, N. et al. TDP-43 specific reduction induced by Di -hydrophobic tags conjugated peptides. \nBioorg Chem 84, 254–259 (2019). \n57. Liu, R. et al. Reducing TDP-43 aggregation does not prevent its cytotoxicity. Acta Neuropathol \nCommun 1, 49 (2013). \n58. Poornima, G., Shah, S., Vignesh, V., Parker, R. & Rajyaguru, P . I. Arginine methylation promotes \ntranslation repression activity of eIF4G-binding protein, Scd6. Nucleic Acids Res gkw762 (2016) \ndoi:10.1093/nar/gkw762. \n59. Petropavlovskiy, A. A., Tauro, M. G., Lajoie, P . & Duennwald, M. L. A Quantitative Imaging-Based \nProtocol for Yeast Growth and Survival on Agar Plates. STAR Protoc 1, 100182 (2020). \n  \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\nWild-type\n∆sbp1\nTDP43-GFP DIC\nInductionRecoveryInductionRecovery\n(B)\nFigure 1\nFraction of TDP43 in condensates\n(C)\n*\n****\nFraction of FUS in condensates\n(F)\n****\n****\n(D)\n(G)\nWild type Ind\nWild type Reco4\nsbp1 Ind\nsbp1 Reco4\nscd6 Ind\nscd6 Reco4\n0.0\n0.5\n1.0\n1.5\nCopy of TDP43 Reco\nRelative Protein Levels\n**** **** ****\nRelative change in TDP43 protein levels\n**** ****\nWT 0\nWT 4\nsbp1_0\nsbp1_4\n0.0\n0.5\n1.0\n1.5\nFUS WT Reco\nRelative change in protein level\n✱ ✱✱\nRelative change in FUS protein levels\n* **\n125-RGGFRGRGGFRGRGGFRGGFRGGYRGGFRGRGNFRGRGGARGG-167\nSbp1\nFUS\nTDP43(A)\n(E)\nWild-type\n∆sbp1\nInductionRecovery\nFUS-YFP DIC\nInductionRecovery\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\nFigure 1: Δsbp1 cells are defective in the disassembly of TDP43 and FUS condensates in yeast and show \nenhanced toxicity. (A) Schematic representation of TDP43, FUS, and Sbp1 proteins. For Sbp1, the sequence \narchitecture of the RGG-motif is also presented. (B) Representative images for the microscopy analysis of \nwild-type and ∆sbp1 cells transformed with Gal-TDP43-GFP plasmid. After growing to 0.4 OD600, cells were \nshifted to galactose-containing media for 3 hours to induce TDP43 expression (stress induction), followed by \n4 hours of recovery in glucose (stress recovery). Cells were taken for microscopy analysis at both steps. \nWhite arrows mark the presence of TDP43 condensates. Scale bar=2um. (C) Graph representing the fraction \nof the TDP43 protein present in condensates per cell. Condensate intensities were calculated and divided by \nthe total fluorescent intensity of the respective cell. A minimum of 50 cells per experiment were analyzed \nfrom 4 independent experiments (n=4) as performed in B. An unpaired t-test was used to calculate the \nsignificance. (D) Graph depicting the relative change in TDP43 protein levels as compared to the respective \ninduction condition. Significance was calculated using a student-paired t-test analysis (n=8). (E) \nRepresentative images for the microscopy analysis of wild-type and ∆sbp1 cells transformed with Gal-FUS-\nYFP plasmid. After growing to 0.4 OD600, cells were shifted to galactose-containing media for 2 hours to \ninduce FUS expression (stress induction), followed by 4 hours of recovery in glucose (stress recovery). Cells \nwere taken for microscopy analysis at both steps. White arrows mark the presence of FUS condensates. \nScale bar=5um. (F) Graph representing the fraction of the FUS protein in condensates per cell. Condensate \nintensities were calculated and divided by the total fluorescent intensity of the respective cell. A minimum \nof 50 cells per experiment were analyzed from 5 independent experiments as performed in E (n=5). An \nunpaired t-test was used to calculate the significance. (G) Graph depicting the relative change in FUS protein \nlevels compared to the respective induction condition. Significance was calculated using a student-paired t-\ntest analysis (n=5). (H and I) Quantitation of the spot area from spot assays of wild-type and Δsbp1 cells \ntransformed with either Gal-TDP43-GFP (H) or Gal-FUS-YFP (I) expressing constructs as performed in \nSupplementary Figure 1C and D. Values were normalized with respect to their EV controls. Significance was \ncalculated by student-paired t-test analysis (n=6 and n=10 for H and I, respectively). Error bars in all graphs \nrepresent mean +SEM, and the same color points depict the data from a single experimental set for all the \ngraphs. *, **, ***, and **** denote p-value<0.05, <0.01, <0.001, and <0.0001, respectively.\nFigure 1\nGalactose\nTDP43: ON\nRelative spot area\n(H)\nBY EV\nBY TDP43\ndelsbp1 EV\ndelsbp1 TDP43\n0.0\n0.5\n1.0\n1.5\nwithout scdt tdp43 ga Copy of Relative GAL\nRelative spot area\n****\n***\n✱✱\n**\n**** ***\nGalactose\nFUS: ON\nRelative spot area\n(I)\nGAL BY EV\nGAL BY FUS\nGAL sbp1 EV\nGAL sbp1 FUS\n0.0\n0.5\n1.0\n1.5\nWithout scd6 Copy of Relative GAL FUS\nRelative growth\n****\n****\n#\n****\n****\n****\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\nFigure 2\n(A)\n(C) (D)\n(E)\nCells without TDP43-ΔNLS \ncondensates (relative to mScar)\n(B)\nmScarlet Sbp1 Sbp1delRGG\n0\n2\n4\n6\n8\n10Relative cells without condensates\n#\n✱\n#\n****\n****\n*\nTDP43-WT TDP43-ΔNLS \nmScar\nSbp1-\nmScar\nSbp1ΔRGG-\nmScar\nmScarlet GFP DAPI MERGE mScarlet GFP DAPI MERGE\nFUS-WT FUS-P525L\nmScar\nSbp1-\nmScar\nSbp1ΔRGG-\nmScar\nmScarlet GFP DAPI MERGE mScarlet GFP DAPI MERGE\nTDP43-\nWT\nTDP43-\nΔNLS \n70\n70\nAnti-mCh\nAnti-GFP\nPonceau\n70Sbp1 (WT or ΔRGG)\nTDP43 (WT or ΔNLS)\nTDP43-\nWT\nTDP43-\nΔNLS \nRelative TDP43 protein levels\nmScar+TDPWT\nSbp1Scar+TDPWT\ndelRGGScar+TDPWT\nmScar+delNLS\nSbp1Scar+delNLS\ndelRGGScar+delNLS\n0.0\n0.5\n1.0\n1.5\n2.0\n2.5\nData without CTF\nRelative change in protein level\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n(F) (G)\nFigure 2: Sbp1 expression reduces mutant TDP43 and FUS condensates in an RGG-motif-dependent \nmanner in mammalian cells. (A) Microscopy images of HEK293T cells co-transfected with mScar/Sbp1-\nmScar/Sbp1ΔRGG-mScar and different eGFP-TDP43-related constructs. Cells were collected 24 hours after \ntransfection and processed for microscopy analysis. The white arrow marks the presence of cytoplasmic \ncondensates. Scale bar=5um. (B) Graph representing the relative changes in the cells without TDP43-ΔNLS \ncondensates (relative to the mScar transfected cells). The values were also normalized with the respective \nmScarlet fluorescent intensity expression levels. A paired t-test was used to calculate the significance value \n(n=6). (C and D) Western blot analysis (C) and its quantitation (D) representing the change in the levels of \nTDP43-WT and TDP43-ΔNLS in the presence of mScar/Sbp1-mScar/Sbp1ΔRGG-mScar from 6 independent \nexperiments (n=6). Anti-mCh antibody was used to detect the mScarlet-tagged proteins. Values on the right \nrepresent the position of different molecular weight ladder bands in kDa. A student-paired t-test was used \nto calculate the significance value. (E) Microscopy images of HEK293T cells co-transfected with mScar/Sbp1-\nmScar/Sbp1ΔRGG-mScar and eGFP-FUS-WT/P525L constructs. Cells were processed similarly to the \nexperiment in A. The white arrow marks the presence of cytoplasmic condensates. Scale bar=5um. (F) Graph \nrepresenting the relative changes in the cells without TDP43-ΔNLS condensates (relative to the mScar \ntransfected cells). The values were also normalized with the respective mScarlet fluorescent intensity \nexpression levels. A paired t-test was used to calculate the significance value (n=5). (G and H) Western blot \nanalysis (G) and its quantitation (H) representing the change in the levels of FUS-WT and FUS-P525L in the \npresence of mScar/Sbp1-mScar/Sbp1ΔRGG-mScar (n=4). Anti-mCh antibody was used to detect the \nmScarlet-tagged proteins. Values on the right represent the position of different molecular weight ladder \nbands in kDa. A student-paired t-test was used to calculate the significance value. Error bars in all graphs \nrepresent mean +SEM, and the same color points in a graph depict the data from a single experimental set. \n*, **, ***, and **** denote p-value<0.05, <0.01, <0.001, and <0.0001, respectively.\nFUS (WT or P525L)\nSbp1 (WT or ΔRGG)\nmScar\n70\n100\n70\nAnti-mCh\nAnti-GFP\nPonceau\nSbp1-\nScar\nSbp1\nΔRGG-\nmScar\n100\nmScar Sbp1 Sbp1delRGG\n0\n5\n10\n15Relative cells without condensates\n#\nRelative cells without condensates\n✱✱\n✱✱\nCells without FUS-P525L \ncondensates (relative to mScar)\n****\n**\n**\nFigure 2\n(H)\nFUS-\nWT\nFUS-\nP525L \nRelative FUS protein levels\nmScar-FUS\nSbp1-FUS\ndelRGG-FUS\nmScar-P525\nSbp1-P525\ndelRGG-P525\n0\n1\n2\n3\n4\nwithout egfpReloaded Relative values\nRelative change in protein level\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\nmScar Sbp1 Sbp1delRGG\n0.0\n0.5\n1.0\n1.5\n2.0\n2.5Mean N:C / Mean Scar\n✱✱\n✱\n✱✱\nNuclear:Cytoplasm (FUS-P525L)\n(A)\n**\n**\n*\nFigure 3\nFigure 3: Sbp1 expression reduces overexpression-mediated defects of FUS-P525L in mammalian cells. (A) \nGraph representing the distribution of FUS-P525L protein in nucleus and cytoplasm. The fluorescent \nintensities of the nucleus and cytoplasm were calculated from the experiment as performed in Figure 2E, \nand the ratio is plotted here. A student-paired t-test was used to calculate the significance value (n=6). (B) \nIncucyte images (real-time cell death analysis) representing the cellular uptake of propidium iodide (PI) in \ndifferent conditions in HeLa cells. Scale bar=100um. (C) Graph representing the number of propidium \niodide (PI) positive cells (dead cells) from the experiment as performed in B. The time on the x-axis reflects \nthe time-point after transfection in hours. The significance was calculated using 2-way ANOVA with multiple \ncomparisons (n=4). Error bars in all graphs represent mean +SEM, and the same color points in A depict the \ndata from a single experimental set. *, **, ***, and **** denote p-value<0.05, <0.01, <0.001, and <0.0001, \nrespectively.\n(B)\n12 24 36 48 12 24 36 48 12 24 36 48 12 24 36 48 12 24 36 48 12 24 36 48 12 24 36 48\n0\n20000\n40000\n60000\n80000\n100000\nTime (hr)\nMOCK\nEGFP+PCEP\nEGFP+SBP\nFUS WT+PCEP\nFUS WT+SBP\nFUS 525+PCEP\nFUS 525+SBP\nCopy of FUS-6hrs\n#\n#\n✱\n✱✱✱\n(C)\n****\n**** *\n***\nNo. of dead cells\nTime (in hours)\npCEP4\nSbp1\nFUS-WT\npCEP4Sbp1\nFUS-P525L\n12 hours 24 hours 36 hours 48 hours\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\nFigure 4\n(B)\n(C)\nSbp1\nSbp1ΔRGG\nIn-vitro \nassembled \ncondensates\nor\nIncubation\n1 hour, 300C\n18000g\n(F) In-vitro sedimentation assay\nAddition of the \npurified proteins\nSupernatant/Soluble\nPellet/Insoluble\n15 minutes\nBuffer\nSbp1\nSbp1RGG\n0\n1\n2\n3\n4Relative S/(S+P)\n✱\n✱✱\nFUS-P525L\nS/(S+I)\n*\n**\n70\n50\n40\n100\nFUS-P525L\nAnti-GFP\nPonceau\nAnti-GAPDH\nGAPDH\nSbp1 (WT or ΔRGG)\nTDP43-ΔNLS 70\n50\n40\nAnti-GFP\nPonceau\nAnti-GAPDH\nGAPDH\nSbp1 (WT or ΔRGG)\n(D)\nSbp1\nSbp1ΔRGG\nEnrichment \nof condensates\nor\nIncubation\n1 hour, 300C\nSupernatant/Soluble\nPellet/Insoluble\n(A) In-cell sedimentation assay\nAddition of 5μM of \nthe purified proteins\n18000g\n15 minutes\nTransfected \nHEK cells\nTDP43-ΔNLS\nS/(S+I)\n(E)\nBuffer\nSbp1\nSbp1RGG\n0.0\n0.5\n1.0\n1.5\n2.0\n2.5Relative S/(S+P)\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n(G)\n1:1 1:2 1:3\nSoluble\n1:1 1:2 1:3\nInsoluble\n+BSA +TeV\n100\n70\n40\nFUS-eGFP\nMBP\nBSA 70\n40\n100\n1:1 1:2 1:3\nSoluble\n1:1 1:2 1:3\nInsoluble\n+Sbp1 +TeV\nFUS-eGFP\nMBP\nSbp1\nFUS-eGFP\nSbp1ΔRGG/MBP\n1:1 1:2 1:3\nSoluble\n1:1 1:2 1:3\nInsoluble\n+Sbp1ΔRGG +TeV\n70\n40\n100\n70\n40\n100\nSbp1TDP43\nMBP\n1:1 1:2 1:3\nSoluble\n1:1 1:2 1:3\nInsoluble\n+Sbp1 +TeV\n70\n40\n100\nBSA\nTDP43\nMBP\n1:1 1:2 1:3\nSoluble\n1:1 1:2 1:3\nInsoluble\n+BSA +TeV\nFigure 4\n(I) (J)\nFigure 4: Sbp1, but not Sbp1ΔRGG, disassembles FUS condensates. (A) Schematic depicting the workflow for the in-\ncell sedimentation assay modified to assess the disassembly activity of Sbp1. (B and D) Western analysis of the \ndifferent fractions from FUS-P525L (B), and TDP43-ΔNLS (D) in-cell sedimentation assay. The different fraction \nloadings are as follows: lysate: 7.5%, cytoplasm: 7.5%, pellet: 15%, soluble: 60%, and insoluble: 60%. GAPDH serves as \nthe control for the assay, and ponceau reflects the purified protein added to the respective reaction. Values on the \nright represent the position of different molecular weight ladder bands in kDa. (C and E) Quantitation of the amount \nof protein in the soluble fraction from experiments as done in B and D. The band intensities were calculated using \nImageJ, and the fraction of protein in the soluble phase was calculated by S/(S+I), where S and I represent the protein \nin the soluble and the insoluble fractions, respectively. Significance was calculated by using student-paired t-test \nanalysis (n=6 and n=7 for C and E, respectively). (F) Schematic depicting the workflow of the in-vitro sedimentation \nassay performed to assess the disassembly activity of Sbp1 on the phase-separated FUS and TDP43 condensates. (G) \nCoomassie-stained protein gels depicting the fractionation of FUS to soluble and insoluble phases in the presence of \nbuffer, BSA, Sbp1, Sbp1ΔRGG. The ratio reflects the amount of FUS: test protein taken for the assay. Sbp1ΔRGG and \nMBP migrate at the same position and hence appear as a single band. Values on the left represent the position of \ndifferent molecular weight ladder bands in kDa. (H) Quantitation of the fraction of FUS protein present in the soluble \nphase from 7 independent experiments (n=7) as performed in G. Significance was calculated by a student-paired t-\ntest analysis. (I) Coomassie-stained protein gels and its quantitation (J) depicting the fractionation of TDP43 to soluble \nand insoluble phases in the presence of BSA and Sbp1. The ratio reflects the amount of TDP43:test protein taken for \nthe assay. The values on the graph reflect the relative amount of TDP43 protein present in the insoluble fraction. A \nstudent-paired t-test analysis calculated significance (n=3). Error bars represent mean +SEM, and the same color \npoints depict the data from a single experimental set. *, **, ***, and **** denote p-value <0.05, <0.01, <0.001, and \n<0.0001, respectively. \n1 2 3\n0.0\n0.5\n1.0\n1.5\n✱✱\n✱ ✱✱✱\n✱✱\n#\n✱✱✱\n✱\n✱✱\n1:1\nS/(S+I)\n1:2 1:3\n(H) **\n*\n**\n**\n* ***\n***\n****\nRelative TDP43 protein in the \ninsoluble fraction\n1:1 1:2 1:3\n1:1 1:2 1:3\n0.0\n0.5\n1.0\n1.5\n2.0\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\nHEK293T\ncells\nCells were \ntransfected with \nLipofectamine \n2000\n24 hours post-\ntransfection\n(A) Live cell microscopy with RGG peptide\nLive cell microscopy \nfor 30-60 minutes\nMedia was \nchanged with the \none containing \n5μM of the RGG-\npeptide\nSbp1-RGG (48 amino acids): \nCy5-MQRGGFRGRGGFRGRGGFRGGFRGGYRGGFRGRGNFRGRGGARGGFNG\n(B) Peptide used for the assay\n(D)\nFigure 5\nFigure 5: Sbp1-RGG peptide disassembles the TDP43-ΔNLS and FUS-P525L condensates. (A) Schematic depicting \nthe workflow of the live-cell microscopy with the RGG-peptides. (B) The sequence of the peptide used for the \nexperiment as performed in A. 2 extra amino acids at the N-terminal end (M and Q) and 3 amino acids at the C-\nterminal end (F, N, and G) were included for better purification of the RGG-peptide. (C) Microscopy images for \nHEK293T cells depicting the change in the area of TDP43-ΔNLS condensate (marked by white arrows) after \nincubating with either the vehicle control or Sbp1-RGG peptides. Scale bar=2um. Graphs on the right depict the \nquantitation of the condensate area/total cell area from at least 30 cells per experiment (n=3). (D) Microscopy \nimages for HEK293T cells depicting the change in the area of FUS-P525L condensate (marked by white arrows) after \nincubating with either the vehicle control or Sbp1-RGG peptides. Scale bar=2um. Graphs on the right depict the \nquantitation of the condensate area/total cell area from at least 40 cells per experiment (n=3). Significance was \ncalculated by paired t-test analysis. **** denotes p-value <0.0001. \n30 mins0 min\nSbp1-RGG\neGFP-FUS-P525L\nVehicle\n30 mins0 min\nSbp1-RGG\n30 mins0 min\nVehicle\nCondensate Area/Total cell area\n0min 30mins\n0.0\n0.1\n0.2\n0.3\n0.4\n0.5\nBuffer P525L 0 and 30\nCondensate Area / Total Cell Area\n#\n0min 30mins\n0.0\n0.1\n0.2\n0.3\n0.4\n0.5\nSbp1RGG P525L 0 and 30\nCondensate Area / Total Cell Area\n#\n**** ****\nCondensate Area/Total cell area\n30 mins0 min\nVehicle\n30 mins0 min\nSbp1-RGG\n0min 30mins\n0.0\n0.2\n0.4\n0.6\n0.8\nsbp1RGG delNLS 0 and  30\nCondensate Area / Total Cell Area\n#\n0min 30mins\n0.0\n0.2\n0.4\n0.6\n0.8\nbuffer delNLS 0 and 30\nCondensate Area / Total Cell Area\n#\n**** ****\n(C) 30 mins0 min\nSbp1-RGG\neGFP-TDP43-ΔNLS\nVehicle\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\nFigure 6\nFigure 6: Schematic depicting the role of RGG peptides as a therapeutic approach to disassemble \nthe toxic cytoplasmic condensates. Green represents mutant proteins, like FUS-P525L and TDP43-\nΔNLS, that mislocalize to the cytoplasm and form condensates in diseased conditions. RGG peptides \ncould have the potential to disassemble these toxic condensates. Such an effect may also result in \nthe restoration of the nuclear localization phenotype.\nAddition of RGG \npeptides\nMutations/stress \nconditions\nProtein mislocalization \nto the cytoplasm and \npresence of cytoplasmic \ncondensates\nDisassembly of the \ncondensates with \nrestoration of the \nnuclear localization\nNuclear \nlocalized \nFUS/TDP43 \nprotein\nUptake of the \npeptide\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\nSupplementary Figures\nS1-S5\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\nSupplementary Figure S1: Δsbp1 cells are defective in the disassembly of TDP43 and FUS condensates in \nyeast. (A and B) Western analysis depicting the change in protein levels of TDP43 (A) and FUS (B) with \nrespect to the respective induction condition. Quantitation of the blots is provided as Figure 1D and G. \nValues on the right represent the position of different molecular weight ladder bands in kDa. Ponceau \nserved as the loading control. (C and D) Spot assays of wild type and Δsbp1 cells transformed with either \nempty vector (EV) or Gal-TDP43-GFP (C) / Gal-FUS-YFP (D) expressing constructs. After growing to 0.4 OD600, \ncells were shifted to galactose-containing media for 2-3 hours to induce TDP43/FUS expression. This was \nfollowed by serial dilution of cells and spotting on glucose and galactose-containing SD-Ura- media plates. \nImages were taken after 2 -4 days of growth at 300C. \nSupplementary Figure S1\nEV\nTDP43\nWild type\n∆sbp1\nGlucose\nTDP43: OFF\nGalactose\nTDP43: ON\nEV\nTDP43\nEV\nFUS\nWild type\n∆sbp1\nGlucose\nFUS: OFF\nGalactose\nFUS: ON\nEV\nFUS\n(C) (D)\n(A) (B)\n100\n75\n100\n75Anti-GFP\nPonceau\nFUS-YFP\nAnti-GFP\nPonceau\nTDP43-GFP\n75\n75\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\nSupplementary Figure S2\n(B)\nPercentage of cells without condensates\nTDP43-ΔNLS + pCEP4\nTDP43-ΔNLS + HSbpF\n0\n20\n40\n60\n80\nCells without nls condensates with HSbpF\nCells without condensates\n#\n****\nFUS-P525L+pcep4\nFUS-P525L+Sbp1\n0\n20\n40\n60\n80\nCells without aggregates\nPercentage of cells without P525 granules\n✱✱\n(D)\nPercentage of cells without condensates\n**\nSupplementary Figure S2: Sbp1 expression reduces mutant TDP43 and FUS condensates in HEK293T \ncells. (A) Microscopy images of HEK293T cells co-transfected with EV/Sbp1 and different eGFP-TDP43-\nrelated constructs. Cells were collected 24 hours after transfection and processed for immuno-\ncytochemistry analysis to detect Sbp1 using an anti-Sbp1 antibody (RFP channel). TDP43 constructs have \nan N-terminal eGFP-tag that was used to check the localization through the GFP channel. The white \narrow marks the presence of cytoplasmic condensates. Scale bar=5um. (B) Quantitation of the \npercentage of cells without cytoplasmic condensates from the experiment as performed in A. A student-\npaired t-test was used to calculate the significance value (n=4). (C) Microscopy images of HEK293T cells \nco-transfected with EV/Sbp1 and eGFP-FUS-WT/P525L constructs. Cells were collected 24 hours after \ntransfection and processed like the experiment in A. The white arrow marks the presence of cytoplasmic \ncondensates. Scale bar=5um. (D) Quantitation of the percentage of cells without cytoplasmic \ncondensates from the experiment as performed in C. A student-paired t-test was used to calculate the \nsignificance value (n=5). Error bars in all the graphs represent mean +SEM, and the same color points in \na graph depict the data from a single experimental set. ** and **** denote p-value <0.01 and <0.0001, \nrespectively.\nTDP43-WT TDP43-ΔNLS TDP43-WT TDP43-ΔNLS \n(A)\nSbp1EV\nGFPRFPDAPIMERGE\n(C)\nFUS-WT FUS-P525L\nSbp1EV\nGFPRFPDAPIMERGE\nFUS-WT FUS-P525L\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\npCEP4\nSbp1\n Mock\neGFP\n12 hours 24 hours 36 hours 48 hours\nSupplementary Figure S3: Sbp1 expression reduces overexpression-mediated defects of FUS-P525L in \nmammalian cells. Incucyte images representing the cellular uptake of propidium iodide (PI) in different \nconditions in HeLa cells. Scale bar=100um. The images are part of Figures 3B and C.\nSupplementary Figure S3\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\n70\n50\n25\n150\n(A)\nS I\nMBP-FUS-eGFP\nFUS-eGFP\nMBP\nTeV\n40\n25\n100\n150\n(B)\n(D)\nSupplementary Figure S4\nSupplementary Figure S4: Sbp1AMD (arginine methylation defective) mutant does not affect FUS \ncondensates. (A) Coomassie-stained protein gel depicting the various purified proteins used in this study. \n(B and C) Coomassie-stained protein gels depicting the partitioning of FUS (B) and TDP43 (C) to the \ninsoluble (I) phase after the cleavage of MBP-tag using TeV protease. S denotes the soluble phase. Values \non the right represent the position of different molecular weight ladder bands in kDa. (D) Coomassie-\nstained protein gel depicting the fractionation of FUS to soluble and insoluble phases in the presence of \nSbp1AMD (mutant where all arginines within the RGG-motif are covered to alanine, see Figure 1A). The \nratio reflects the amount of FUS: test protein taken for the assay. Sbp1AMD and MBP migrate at the same \nposition and hence appear as a single band. Values on the left represent the position of different \nmolecular weight ladder bands in kDa. (E) Quantitation of the fraction of FUS protein present in the \nsoluble phase from 7 independent experiments (n=7) as performed in D and Figure 3H. The graph from \nFigure 3I has been replotted here to include Sbp1AMD values. Error bars represent mean +SEM, and the \nsame color points in a graph depict the data from a single experimental set. *, **, ***, and **** denote \np-value <0.05, <0.01, <0.001, and <0.0001, respectively. \n1:1 1:2 1:3\nSoluble\n1:1 1:2 1:3\nInsoluble\n+Sbp1AMD +TeV\nFUS-eGFP\nSbp1AMD/MBP\n70\n40\n100\nTDP43-MBP\n(C)\n40\n25\n100\nS I\nTDP43\nMBP\nS/(S+I)\n1 2 3\n0.0\n0.5\n1.0\n1.5\nBSA\nSbp1\nSbp1 del RGG\nSbp1 AMD\n✱✱\n✱\n✱\n✱✱✱\n✱✱\n✱✱✱\n#\n✱✱✱\n✱✱✱✱\n✱✱\n1:1 1:2 1:3\n(E) ***\n**\n***\n****\n****\n**\n***\n*\n*\n*\n**\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint \n\nSupplementary Figure S5: HEK293T cells readily uptake Cy5-labelled Sbp1-RGG peptide. (A and B) \nMicroscopy images for HEK293T cells depicting the change in the area of TDP43-ΔNLS (A) and FUS-P525L (B) \ncondensate (marked by white arrows) and the uptake of Cy5-labelled peptide after 60mins of incubation \nwith either the vehicle control or Sbp1-RGG peptides. The panel represents the same set of cells as depicted \nin Figure 4D. Cy5 panel depicts the uptake of the peptide. Scale bar=2um. The graph on the right represents \nthe change in the condensate area normalized with the total cell area after incubation with the Sbp1-RGG \npeptide (n=3). The data plotted is the same as Figure 4D with the addition of a 60 mins time point. Error \nbars represent mean +SEM. Significance was calculated by paired t-test analysis. **** denotes p-value \n<0.0001. \nSupplementary Figure S5\n(B)\n0min 30mins 60mins\n0.0\n0.2\n0.4\n0.6\n0.8Condensate Area / Total Cell Area\n#\n#****\n****\nSbp1-RGG\nCondensate Area/Total cell area\n(A)\n Cy5GFP\nSbp1-RGG\neGFP-TDP43-ΔNLS\nVehicle\n60 mins\nCy5GFP\nSbp1-RGG\neGFP-FUS-P525L\nVehicle\n0min 30mins 60mins\n0.0\n0.1\n0.2\n0.3\n0.4Condensate Area / Total Cell Area\n#\n#\nSbp1 P525L\n#\nSbp1-RGG\nCondensate Area/Total cell area\n****\n****\n****\n60 mins\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.643735doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}