m6A Modified Short RNA Fragments Inhibit Cytoplasmic TLS/FUS Aggregation Induced by Hyperosmotic Stress

Translocated in LipoSarcoma/Fused in Sarcoma (TLS/FUS) is a nuclear RNA binding protein whose mutations cause amyotrophic lateral sclerosis. TLS/FUS undergoes LLPS and forms membraneless particles with other proteins and nucleic acids. Interaction with RNA alters conformation of TLS/FUS, which affects binding with proteins, but the effect of m6A RNA modification on the TLS/FUS–RNA interaction remains elusive. Here, we investigated the binding specificity of TLS/FUS to m6A RNA fragments by RNA pull down assay, and elucidated that both wild type and ALS-related TLS/FUS mutants strongly bound to m6A modified RNAs. TLS/FUS formed cytoplasmic foci by treating hyperosmotic stress, but the cells transfected with m6A-modified RNAs had a smaller number of foci. Moreover, m6A-modified RNA transfection resulted in the cells obtaining higher resistance to the stress. In summary, we propose TLS/FUS as a novel candidate of m6A recognition protein, and m6A-modified RNA fragments diffuse cytoplasmic TLS/FUS foci and thereby enhance cell viability.

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RNA-binding proteins with prion-like domains in health and disease

Biochemical Journal ◽

10.1042/bcj20160499 ◽

2017 ◽

Vol 474 (8) ◽

pp. 1417-1438 ◽

Cited By ~ 152

Author(s):

Alice Ford Harrison ◽

James Shorter

Keyword(s):

Phase Transitions ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Low Complexity ◽

Fused In Sarcoma ◽

Heterogeneous Nuclear Ribonucleoproteins ◽

Health And Disease ◽

Lateral Sclerosis

Approximately 70 human RNA-binding proteins (RBPs) contain a prion-like domain (PrLD). PrLDs are low-complexity domains that possess a similar amino acid composition to prion domains in yeast, which enable several proteins, including Sup35 and Rnq1, to form infectious conformers, termed prions. In humans, PrLDs contribute to RBP function and enable RBPs to undergo liquid–liquid phase transitions that underlie the biogenesis of various membraneless organelles. However, this activity appears to render RBPs prone to misfolding and aggregation connected to neurodegenerative disease. Indeed, numerous RBPs with PrLDs, including TDP-43 (transactivation response element DNA-binding protein 43), FUS (fused in sarcoma), TAF15 (TATA-binding protein-associated factor 15), EWSR1 (Ewing sarcoma breakpoint region 1), and heterogeneous nuclear ribonucleoproteins A1 and A2 (hnRNPA1 and hnRNPA2), have now been connected via pathology and genetics to the etiology of several neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Here, we review the physiological and pathological roles of the most prominent RBPs with PrLDs. We also highlight the potential of protein disaggregases, including Hsp104, as a therapeutic strategy to combat the aberrant phase transitions of RBPs with PrLDs that likely underpin neurodegeneration.

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FUS-ALS mutants alter FMRP phase separation equilibrium and impair protein translation

Science Advances ◽

10.1126/sciadv.abf8660 ◽

2021 ◽

Vol 7 (30) ◽

pp. eabf8660

Author(s):

Nicol Birsa ◽

Agnieszka M. Ule ◽

Maria Giovanna Garone ◽

Brian Tsang ◽

Francesca Mattedi ◽

...

Keyword(s):

Phase Separation ◽

Motor Neurons ◽

Rna Binding ◽

Fragile X ◽

Protein Translation ◽

Fused In Sarcoma ◽

Mental Retardation Protein ◽

Lateral Sclerosis

FUsed in Sarcoma (FUS) is a multifunctional RNA binding protein (RBP). FUS mutations lead to its cytoplasmic mislocalization and cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Here, we use mouse and human models with endogenous ALS-associated mutations to study the early consequences of increased cytoplasmic FUS. We show that in axons, mutant FUS condensates sequester and promote the phase separation of fragile X mental retardation protein (FMRP), another RBP associated with neurodegeneration. This leads to repression of translation in mouse and human FUS-ALS motor neurons and is corroborated in vitro, where FUS and FMRP copartition and repress translation. Last, we show that translation of FMRP-bound RNAs is reduced in vivo in FUS-ALS motor neurons. Our results unravel new pathomechanisms of FUS-ALS and identify a novel paradigm by which mutations in one RBP favor the formation of condensates sequestering other RBPs, affecting crucial biological functions, such as protein translation.

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FUS/TLS undergoes calcium-mediated nuclear egress during excitotoxic stress and is required for Gria2 mRNA processing

10.1101/386870 ◽

2018 ◽

Author(s):

Maeve Tischbein ◽

Desiree M. Baron ◽

Yen-Chen Lin ◽

Katherine V. Gall ◽

John E. Landers ◽

...

Keyword(s):

Neurodegenerative Disease ◽

Rna Binding ◽

Rna Binding Proteins ◽

Physiological Stress ◽

Nucleocytoplasmic Transport ◽

Cellular Stress Response ◽

Calcium Dependent ◽

Fused In Sarcoma ◽

Nuclear Egress ◽

Lateral Sclerosis

AbstractExcitotoxic levels of glutamate represent a physiological stress that is strongly linked to amyotrophic lateral sclerosis (ALS) and other neurological disorders. Emerging evidence indicates a role for neurodegenerative disease linked RNA-binding proteins (RBPs) in the cellular stress response. However, the relationships between excitotoxicity, RBP function and pathology have not been explored. Here, we found that excitotoxicity induced the translocation of select ALS-linked RBPs from the nucleus to the cytoplasm within neurons. RBPs affected by excitotoxicity include TAR DNA-binding protein 43 (TDP-43) and, most robustly, fused in sarcoma/translocated in liposarcoma (FUS/TLS). FUS translocation occurs through a calcium-dependent mechanism and coincides with striking alterations in nucleocytoplasmic transport. Further, glutamate-induced upregulation of Gria2 in neurons was dependent on FUS expression, consistent with a functional role for FUS under excitotoxic stress. These findings reveal a link between prominent factors in neurodegenerative disease, namely excitotoxicity, disease-associated RBPs and nucleocytoplasmic transport.

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Aberrant interaction of FUS with the U1 snRNA provides a molecular mechanism of FUS induced amyotrophic lateral sclerosis

Nature Communications ◽

10.1038/s41467-020-20191-3 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Daniel Jutzi ◽

Sébastien Campagne ◽

Ralf Schmidt ◽

Stefan Reber ◽

Jonas Mechtersheimer ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Motor Neurons ◽

Rna Binding ◽

Muscular Atrophy ◽

Motor Neuron Diseases ◽

Stem Loop ◽

Fused In Sarcoma ◽

Rna Targets ◽

U1 Snrna ◽

Lateral Sclerosis

AbstractMutations in the RNA-binding protein Fused in Sarcoma (FUS) cause early-onset amyotrophic lateral sclerosis (ALS). However, a detailed understanding of central RNA targets of FUS and their implications for disease remain elusive. Here, we use a unique blend of crosslinking and immunoprecipitation (CLIP) and NMR spectroscopy to identify and characterise physiological and pathological RNA targets of FUS. We find that U1 snRNA is the primary RNA target of FUS via its interaction with stem-loop 3 and provide atomic details of this RNA-mediated mode of interaction with the U1 snRNP. Furthermore, we show that ALS-associated FUS aberrantly contacts U1 snRNA at the Sm site with its zinc finger and traps snRNP biogenesis intermediates in human and murine motor neurons. Altogether, we present molecular insights into a FUS toxic gain-of-function involving direct and aberrant RNA-binding and strengthen the link between two motor neuron diseases, ALS and spinal muscular atrophy (SMA).

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FUS inclusions disrupt RNA localization by sequestering kinesin-1 and inhibiting microtubule detyrosination

The Journal of Cell Biology ◽

10.1083/jcb.201608022 ◽

2017 ◽

Vol 216 (4) ◽

pp. 1015-1034 ◽

Cited By ~ 44

Author(s):

Kyota Yasuda ◽

Sarah F. Clatterbuck-Soper ◽

Meredith E. Jackrel ◽

James Shorter ◽

Stavroula Mili

Keyword(s):

Rna Binding ◽

Fibroblast Cell ◽

Rna Localization ◽

Cellular Functions ◽

Cytoplasmic Inclusions ◽

Inclusion Formation ◽

Fused In Sarcoma ◽

Lateral Sclerosis ◽

Tubulin Carboxypeptidase ◽

Dependent Mechanism

Cytoplasmic inclusions of the RNA-binding protein fused in sarcoma (FUS) represent one type of membraneless ribonucleoprotein compartment. Formation of FUS inclusions is promoted by amyotrophic lateral sclerosis (ALS)–linked mutations, but the cellular functions affected upon inclusion formation are poorly defined. In this study, we find that FUS inclusions lead to the mislocalization of specific RNAs from fibroblast cell protrusions and neuronal axons. This is mediated by recruitment of kinesin-1 mRNA and protein within FUS inclusions, leading to a loss of detyrosinated glutamate (Glu)–microtubules (MTs; Glu-MTs) and an inability to support the localization of RNAs at protrusions. Importantly, dissolution of FUS inclusions using engineered Hsp104 disaggregases, or overexpression of kinesin-1, reverses these effects. We further provide evidence that kinesin-1 affects MT detyrosination not through changes in MT stability, but rather through targeting the tubulin carboxypeptidase enzyme onto specific MTs. Interestingly, other pathological inclusions lead to similar outcomes, but through apparently distinct mechanisms. These results reveal a novel kinesin-dependent mechanism controlling the MT cytoskeleton and identify loss of Glu-MTs and RNA mislocalization as common outcomes of ALS pathogenic mutations.

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The RNA-binding protein Fus directs translation of localized mRNAs in APC-RNP granules

The Journal of Cell Biology ◽

10.1083/jcb.201306058 ◽

2013 ◽

Vol 203 (5) ◽

pp. 737-746 ◽

Cited By ~ 102

Author(s):

Kyota Yasuda ◽

Huaye Zhang ◽

David Loiselle ◽

Timothy Haystead ◽

Ian G. Macara ◽

...

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Rna Localization ◽

Polyposis Coli ◽

Fused In Sarcoma ◽

Ribonucleoprotein Complexes ◽

Physiological Processes ◽

Rnp Granules ◽

Lateral Sclerosis

RNA localization pathways direct numerous mRNAs to distinct subcellular regions and affect many physiological processes. In one such pathway the tumor-suppressor protein adenomatous polyposis coli (APC) targets RNAs to cell protrusions, forming APC-containing ribonucleoprotein complexes (APC-RNPs). Here, we show that APC-RNPs associate with the RNA-binding protein Fus/TLS (fused in sarcoma/translocated in liposarcoma). Fus is not required for APC-RNP localization but is required for efficient translation of associated transcripts. Labeling of newly synthesized proteins revealed that Fus promotes translation preferentially within protrusions. Mutations in Fus cause amyotrophic lateral sclerosis (ALS) and the mutant protein forms inclusions that appear to correspond to stress granules. We show that overexpression or mutation of Fus results in formation of granules, which preferentially recruit APC-RNPs. Remarkably, these granules are not translationally silent. Instead, APC-RNP transcripts are translated within cytoplasmic Fus granules. These results unexpectedly show that translation can occur within stress-like granules. Importantly, they identify a new local function for cytoplasmic Fus with implications for ALS pathology.

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The phase separation-dependent FUS interactome reveals nuclear and cytoplasmic function of liquid-liquid phase separation

10.1101/806158 ◽

2019 ◽

Cited By ~ 2

Author(s):

Stefan Reber ◽

Helen Lindsay ◽

Anny Devoy ◽

Daniel Jutzi ◽

Jonas Mechtersheimer ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Rna Binding ◽

Mitochondrial Gene ◽

Liquid Phase Separation ◽

Fused In Sarcoma ◽

Damage Repair ◽

Interaction Partners ◽

Lateral Sclerosis ◽

Liquid Liquid Phase Separation

AbstractLiquid-liquid phase separation (LLPS) of proteins and RNAs has emerged as the driving force underlying the formation of membrane-less organelles. Such biomolecular condensates have various biological functions and have been linked to disease. One of the best studied proteins undergoing LLPS is Fused in Sarcoma (FUS), a predominantly nuclear RNA-binding protein. Mutations in FUS have been causally linked to Amyotrophic Lateral Sclerosis (ALS), an adult-onset motor neuron disease, and LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. In spite of this, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. In order to study the consequences of LLPS on FUS and its interaction partners, we developed a method that allows for the purification of phase separated FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome of FUS, depending on its biophysical state. While non-phase separated FUS interacts mainly with its well-known interaction partners involved in pre-mRNA processing, phase-separated FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, factors with function in mitochondria are strongly enriched with phase-separated FUS, providing a potential explanation for early changes in mitochondrial gene expression observed in mouse models of ALS-FUS. In summary, we present a methodology that allows to investigate the interactome of phase-separating proteins and provide evidence that LLPS strongly shapes the FUS interactome with important implications for function and disease.

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Data Science Issues in Studying Protein–RNA Interactions with CLIP Technologies

Annual Review of Biomedical Data Science ◽

10.1146/annurev-biodatasci-080917-013525 ◽

2018 ◽

Vol 1 (1) ◽

pp. 235-261 ◽

Cited By ~ 24

Author(s):

Anob M. Chakrabarti ◽

Nejc Haberman ◽

Arne Praznik ◽

Nicholas M. Luscombe ◽

Jernej Ule

Keyword(s):

Binding Sites ◽

Data Science ◽

Rna Binding ◽

Positional Distribution ◽

Sequence Motif ◽

Peak Calling ◽

Uv Crosslinking ◽

Rna Fragments ◽

Rna Interaction ◽

Endogenous Interactions

An interplay of experimental and computational methods is required to achieve a comprehensive understanding of protein–RNA interactions. UV crosslinking and immunoprecipitation (CLIP) identifies endogenous interactions by sequencing RNA fragments that copurify with a selected RNA-binding protein under stringent conditions. Here we focus on approaches for the analysis of the resulting data and appraise the methods for peak calling, visualization, analysis, and computational modeling of protein–RNA binding sites. We advocate that the sensitivity and specificity of data be assessed in combination for computational quality control. Moreover, we demonstrate the value of analyzing sequence motif enrichment in peaks assigned from CLIP data and of visualizing RNA maps, which examine the positional distribution of peaks around regulated landmarks in transcripts. We use these to assess how variations in CLIP data quality and in different peak calling methods affect the insights into regulatory mechanisms. We conclude by discussing future opportunities for the computational analysis of protein–RNA interaction experiments.

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Connecting RNA-Modifying Similarities of TDP-43, FUS, and SOD1 with MicroRNA Dysregulation Amidst A Renewed Network Perspective of Amyotrophic Lateral Sclerosis Proteinopathy

International Journal of Molecular Sciences ◽

10.3390/ijms21103464 ◽

2020 ◽

Vol 21 (10) ◽

pp. 3464 ◽

Cited By ~ 1

Author(s):

Jade Pham ◽

Matt Keon ◽

Samuel Brennan ◽

Nitin Saksena

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Rna Splicing ◽

Rna Binding ◽

Rna Binding Proteins ◽

Future Research ◽

Fused In Sarcoma ◽

Traditional Approaches ◽

Microrna Dysregulation ◽

Lateral Sclerosis

Beyond traditional approaches in understanding amyotrophic lateral sclerosis (ALS), multiple recent studies in RNA-binding proteins (RBPs)—including transactive response DNA-binding protein (TDP-43) and fused in sarcoma (FUS)—have instigated an interest in their function and prion-like properties. Given their prominence as hallmarks of a highly heterogeneous disease, this prompts a re-examination of the specific functional interrelationships between these proteins, especially as pathological SOD1—a non-RBP commonly associated with familial ALS (fALS)—exhibits similar properties to these RBPs including potential RNA-regulatory capabilities. Moreover, the cytoplasmic mislocalization, aggregation, and co-aggregation of TDP-43, FUS, and SOD1 can be identified as proteinopathies akin to other neurodegenerative diseases (NDs), eliciting strong ties to disrupted RNA splicing, transport, and stability. In recent years, microRNAs (miRNAs) have also been increasingly implicated in the disease, and are of greater significance as they are the master regulators of RNA metabolism in disease pathology. However, little is known about the role of these proteins and how they are regulated by miRNA, which would provide mechanistic insights into ALS pathogenesis. This review seeks to discuss current developments across TDP-43, FUS, and SOD1 to build a detailed snapshot of the network pathophysiology underlying ALS while aiming to highlight possible novel therapeutic targets to guide future research.

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Cdc48/VCP and Endocytosis Regulate TDP-43 and FUS Toxicity and Turnover

Molecular and Cellular Biology ◽

10.1128/mcb.00256-19 ◽

2019 ◽

Vol 40 (4) ◽

Cited By ~ 4

Author(s):

Guangbo Liu ◽

Aaron Byrd ◽

Amanda N. Warner ◽

Fen Pei ◽

Eman Basha ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Motor Neuron ◽

Rna Binding ◽

Rna Binding Proteins ◽

Dna Binding Protein ◽

Content Type ◽

Fused In Sarcoma ◽

Lateral Sclerosis ◽

Novel Therapeutic ◽

Common Defect

ABSTRACT Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease. TDP-43 (TAR DNA-binding protein 43) and FUS (fused in sarcoma) are aggregation-prone RNA-binding proteins that in ALS can mislocalize to the cytoplasm of affected motor neuron cells, often forming cytoplasmic aggregates in the process. Such mislocalization and aggregation are implicated in ALS pathology, though the mechanism(s) of TDP-43 and FUS cytoplasmic toxicity remains unclear. Recently, we determined that the endocytic function aids the turnover (i.e., protein degradation) of TDP-43 and reduces TDP-43 toxicity. Here, we identified that Cdc48 and Ubx3, a Cdc48 cofactor implicated in endocytic function, regulates the turnover and toxicity of TDP-43 and FUS expressed in Saccharomyces cerevisiae. Cdc48 physically interacts and colocalizes with TDP-43, as does VCP, in ALS patient tissue. In yeast, FUS toxicity also depends strongly on endocytic function but not on autophagy under normal conditions. FUS expression also impairs endocytic function, as previously observed with TDP-43. Taken together, our data identify a role for Cdc48/VCP and endocytic function in regulating TDP-43 and FUS toxicity and turnover. Furthermore, endocytic dysfunction may be a common defect affecting the cytoplasmic clearance of ALS aggregation-prone proteins and may represent a novel therapeutic target of promise.

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