RNP-Granule Assembly via Ataxin-2 Disordered Domains Is Required for Long-Term Memory and Neurodegeneration

Bakthavachalu, Baskar; Huelsmeier, Joern; Sudhakaran, Indulekha P.; Hillebrand, Jens; Singh, Amanjot; Petrauskas, Arnas; Thiagarajan, Devasena; Sankaranarayanan, Mugesh; Mizoue, Laura S.; Anderson, Eric N.; Pandey, Udai Bhan; Ross, Eric D.; VijayRaghavan, K.; Parker, Roy; Ramaswami, Mani

doi:10.1016/j.neuron.2018.04.032

Cited by 104 publications

(137 citation statements)

References 97 publications

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“…Previous studies suggested that the IDLs of adjacent SSB molecules can interact with each other to enhance the cooperativity of ssDNA binding, while the IDL was also proposed to interact with the OB fold Kozlov et al, 2015Kozlov et al, , 2017. Moreover, the central role played by similar low-sequence-complexity, Gln/Gly/Pro-rich IDRs in previously investigated LLPS systems (Bakthavachalu et al, 2018;Mannen et al, 2016;Milovanovic et al, 2018;Sabari et al, 2018) as well as the predicted LLPS propensities suggest a role for the IDL in SSB LLPS ( Fig. 1B-D).…”

Section: Multivalent Interactions Between Ssb Idl Regions Are Requirementioning

confidence: 57%

Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

Harami

Kovács

Pancsa

et al. 2019

Preprint

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Bacterial single stranded (ss) DNA-binding proteins (SSB) are essential for the replication and maintenance of the genome. SSBs share a conserved ssDNA-binding domain, a less conserved intrinsically disordered linker (IDL) and a highly conserved C-terminal peptide (CTP) motif that mediates a wide array of protein-protein interactions with DNA-metabolizing proteins. Here we show that the E. coli SSB protein forms liquid-liquid phase separated condensates in cellular-like conditions through multifaceted interactions involving all structural regions of the protein. SSB, ssDNA and SSBinteracting molecules are highly concentrated within the condensates, whereas phase separation is overall regulated by the stoichiometry of SSB and ssDNA. Together with recent results on subcellular SSB localization patterns, our results point to a conserved mechanism by which bacterial cells store a pool of SSB and SSB-interacting proteins. Dynamic phase separation enables rapid mobilization of this protein pool to protect exposed ssDNA and repair genomic loci affected by DNA damage. RESULTS SSB forms dynamic LLPS condensates in physiologically relevant conditions in vitroDue to the fact that SSB shares several features with hitherto described LLPS drivers, we sought to determine if LLPS could be a yet undiscovered capability of SSB facilitating its multifaceted roles in genome maintenance. We performed a bioinformatics analysis that revealed that E. coli SSB, in particular its IDL region, shows high propensity for LLPS, according to multiple dedicated sequencebased prediction methods (Bolognesi et al., 2016;Lancaster et al., 2014;Vernon et al., 2018) (Fig. 1B-D). This feature of SSB is universally conserved among bacteria, as the majority of analyzed SSBs (72.1 %) harbored by representative bacterial species from 15 large phylogenetic groups show similar LLPS propensities (Tables S1-2; Fig. 1E).In line with its predicted LLPS propensity, purified E. coli SSB (30 µM; SSB concentrations are expressed as those of tetramer molecules throughout the paper) forms an opaque, turbid solution at low NaCl concentration (50 mM) in vitro at room temperature and also at the native temperature of E. coli cells (37°C) ( Fig. 2A-B). The process is reversible as elevation of NaCl concentration to 200 mM leads to immediate loss of turbidity. The Clconcentration in E. coli cells depends on their environment (Schultz et al., 1962); however, while the exact value is not known, it can be assumed to be in a similar range (5-100 mM) as that measured for the cells of mammalian E. coli hosts, whereas the major metabolic anion in bacterial cells is glutamate (Glu; 100 mM) (Bennett et al., 2009). Strikingly, SSB forms a turbid solution even in 200 mM NaGlu ( Fig. 2A-B). (It must be noted that experiments using NaGlu always contained a fixed amount of 20 mM NaCl, for technical reasons.) Investigation of the protein solution by differential interference contrast (DIC) microscopy after diluting SSB into low-salt buffer revealed the formation of regular spherical drop...

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Section: Multivalent Interactions Between Ssb Idl Regions Are Requirementioning

confidence: 57%

Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

Harami

Kovács

Pancsa

et al. 2019

Preprint

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“…Evidence has been accumulating that RNP granules are incubators for the formation of pathogenic protein aggregates associated with neurodegenerative disease. It has been proposed that localization of proteins such as TDP-43, FUS and C9OFR72-encoded dipeptide repeat GR50 in high concentration in stress granules likely in a gel form, promotes their conversion into pathogenic amyloid-like aggregates [78][79][80][81][82].…”

Section: Discussionmentioning

confidence: 99%

“…This was accomplished with the aid of ATXN2, an RNP-protein that is required for RNP-granule formation. Three ALS associated proteins, TDP-43, FUS and C9OFR72-encoded dipeptide repeat GR50, have been shown to co-localize with ATXN2 RNP granules [42,78] in yeast, human or Drosophila S2 cells. Also, either reducing the level of ATXN2 or deleting one of its intrinsically disordered regions both reduced RNP-granule formation and reduced neurodegeneration caused by TDP-43, C9ORF72 dipeptide or FUS [78,[83][84][85].…”

Section: Discussionmentioning

confidence: 99%

“…Three ALS associated proteins, TDP-43, FUS and C9OFR72-encoded dipeptide repeat GR50, have been shown to co-localize with ATXN2 RNP granules [42,78] in yeast, human or Drosophila S2 cells. Also, either reducing the level of ATXN2 or deleting one of its intrinsically disordered regions both reduced RNP-granule formation and reduced neurodegeneration caused by TDP-43, C9ORF72 dipeptide or FUS [78,[83][84][85]. These results help explain why expansions of the polyQ regions in ATXN2 that increase the stability, and thus likely the level of ATXN2, are associated with neurodegenerative disease (susceptibility to ALS and type 2 spinocerebellar ataxia).…”

Section: Discussionmentioning

confidence: 99%

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Calcium-responsive transactivator (CREST) toxicity is rescued by loss of PBP1/ATXN2 function in a novel yeast proteinopathy model and in transgenic flies

Park

Watanabe

et al. 2018

Preprint

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Proteins associated with familial neurodegenerative disease often aggregate in patients' neurons.Several such proteins, e.g. TDP-43, aggregate and are toxic when expressed in yeast. Deletion of the ATXN2 ortholog, PBP1, reduces yeast TDP-43 toxicity, which led to identification of ATXN2 as an amyotrophic lateral sclerosis (ALS) risk factor and therapeutic target. Likewise, new yeast neurodegenerative disease models could facilitate identification of other risk factors and targets.Mutations in SS18L1, encoding the calcium-responsive transactivator (CREST) chromatin-remodeling protein, are associated with ALS. We show that CREST is toxic in yeast and, like the yeast chromatinremodeling factor SWI1, CREST inhibits silencing of FLO genes. Toxicity of CREST is enhanced by the [PIN + ] prion and reduced by deletion of PBP1/ATXN2. CREST forms nuclear and occasionally cytoplasmic foci that stain with an amyloid dye. Overexpression of PBP1 caused considerable CREST co-localization with PBP1 tagged cytoplasmic granules which might promote toxic aggregation of CREST. In accord with the yeast data, we show that the Drosophila ortholog of human ATXN2, dAtx2, is a potent enhancer of CREST toxicity. Down regulation of dAtx2 in flies overexpressing CREST in retinal ganglion cells was sufficient to almost entirely rescue the severe degenerative phenotype induced by human CREST. These results extend the spectrum of ALS associated proteins affected by PBP1/ATXN2, suggesting that therapies targeting ATXN2 may be effective for a wide range of neurodegenerative diseases. Author summaryMutations in the calcium-responsive transactivator (CREST) protein have been shown to cause amyotrophic lateral sclerosis (ALS). Here we show that the human CREST protein expressed in yeast forms nuclear aggregates and is toxic. We also show that ATXN2, previously shown to modify ALS toxicity caused by mutations in the TDP-43 encoding gene, also modifies toxicity of CREST expressed in either yeast or flies. These results extend the spectrum of ALS associated proteins affected by ATXN2, suggesting that therapies targeting ATXN2 may be effective for a wide range of neurodegenerative diseases.

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“…Modulatory regions can also promote LLPS by mediating intermolecular interactions (with proteins, RNA, or DNA). For example, in the memory‐associated ataxin‐2 homolog (Atx2) that accumulates in SGs on arsenite stress, the C‐terminal IDR is the PDR, while the RNA‐binding middle IDR is dispensable for LLPS . Cellular expression of GFP‐Atx2 deletion constructs, however, showed that both IDRs are essential for normal SG formation and long‐term habituation .…”

Section: Regulation By Elements Within the Phase‐separating Proteinmentioning

confidence: 99%

A guide to regulation of the formation of biomolecular condensates

et al. 2020

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RNP-Granule Assembly via Ataxin-2 Disordered Domains Is Required for Long-Term Memory and Neurodegeneration

Cited by 104 publications

References 97 publications

Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

Calcium-responsive transactivator (CREST) toxicity is rescued by loss of PBP1/ATXN2 function in a novel yeast proteinopathy model and in transgenic flies

A guide to regulation of the formation of biomolecular condensates

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