The Solution Structure of FUS Bound to RNA Reveals a Bipartite Mode of RNA Recognition with Both Sequence and Shape Specificity

Loughlin, Fionna E.; Lukavsky, Peter J.; Казеева, Т. Н.; Reber, Stefan; Hock, Eva Maria; Colombo, Martino; Schroetter, Christine von; Pauli, Phillip; Cléry, Antoine; Mühlemann, Oliver; Polymenidou, Magdalini; Ruepp, Marc‐David; Allain, Frédéric H.‐T.

doi:10.1016/j.molcel.2018.11.012

Cited by 177 publications

(271 citation statements)

References 69 publications

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“…Therefore, it is crucial for stabilizing the turn in the backbone observed in our complex. This is reminiscent of other extensions found in the RRM contributing to RNA binding like the -hairpin found in FUS RRM (Loughlin et al 2019) (Fig. 6E) and the fifth -strand of PTB RRM2 (Oberstrass et al 2005) ( Fig.…”

Section: Discussionsupporting

confidence: 53%

“…On the other hand, we know that linear sequence motifs are often insufficient to fully capture RBP binding specificities (Dominguez et al 2018). Specificity may be increased due to contextual features, either in the form of bipartite motifs, such as recently found for FUS (Loughlin et al 2019), preference for a nucleotide composition flanking a high affinity linear motif, or due to the favoring of specific structural motifs adjacent to the linear motif. The fact is that while the RNA binding surfaces of the tandem RRMs are highly conserved within the hnRNPR-like family of proteins, the sequences of the dsRBDs of DND1, RBM46 and ACF1 (APOBEC1 complementation factor) are not that well conserved (Fig.…”

Section: Role Of the Dsrbdmentioning

confidence: 97%

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The solution structure of Dead End bound to AU-rich RNA reveals an unprecedented mode of tandem RRM-RNA recognition required for mRNA regulation

Duszczyk

Wischnewski

Казеева

et al. 2019

Preprint

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A previous version of this manuscript contained an error in the normalization of the luciferase activity in the cell-based assays in Figures 5 and S8. This has been corrected and the text has been updated accordingly. AbstractDead End (DND1) is an RNA-binding protein essential for germline development through its role in posttranscriptional gene regulation. The molecular mechanisms behind selection and regulation of its targets are unknown. Here, we present the solution structure of DND1's tandem RNA Recognition Motifs (RRMs) bound to AU-rich RNA. The structure reveals how an NYAYUNN element is specifically recognized, reconciling seemingly contradictory sequence motifs discovered in recent genome-wide studies. RRM1 acts as a main binding platform, including unusual extensions to the canonical RRM fold.RRM2 acts cooperatively with RRM1, capping the RNA using an unusual binding pocket, leading to an unprecedented mode of tandem RRM-RNA recognition. We show that the consensus motif is sufficient to mediate upregulation of a reporter gene in human cells and that this process depends not only on RNA binding by the RRMs, but also on DND1's double-stranded RNA binding domain (dsRBD), that we show to be dispensable for target binding in cellulo. Our results point to a model where DND1 target selection is mediated by an uncanonical mode of AU-rich RNA recognition by the tandem RRMs and a role for the dsRBD in the recruitment of effector complexes responsible for target regulation.

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Section: Discussionsupporting

confidence: 53%

Section: Role Of the Dsrbdmentioning

confidence: 97%

The solution structure of Dead End bound to AU-rich RNA reveals an unprecedented mode of tandem RRM-RNA recognition required for mRNA regulation

Duszczyk

Wischnewski

Казеева

et al. 2019

Preprint

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“…To test if LLPS-deficient FUS is still functional, we made use of two previously established assays for FUS function. First, we tested if PLD27YS FUS is still capable of autoregulating endogenous FUS mRNA levels when transiently transfected into HeLa cells (Loughlin et al, 2019). While wild type FUS autoregulated endogenous FUS mRNA levels, LLPS-deficient FUS had no effect on endogenous FUS mRNA levels (Fig 4d and e).…”

Section: Llps Is Required For the Association Of Fus With Chromatin Amentioning

confidence: 99%

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

Reber

Lindsay

Devoy

et al. 2019

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Liquid-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.Wang et al., 2018) and the wash volumes applied during the co-IP exceeded the volume applied to analyse the cell lysates in Supplementary Fig. 1b, LLPS of FUS during co-IP conditions is limited to the minimum or even completely prevented. A pulldown of FLAG-eGFP served as control IP. Successful purification of the bait from droplet purifications and co-IPs were verified by SDS-PAGE followed by silver staining or western blotting, respectively ( Supplementary Fig. 2). Proteins and RNAs purified from co-IP and droplet purification experiments were analysed by means of quantitative mass spectrometry or RNA deep sequencing, respectively. Interestingly, the wild type and P525L FUS protein and RNA interactomes (under the same experimental conditions) were mostly identical ( Supplementary Fig. 3), which is why wild type and P525L interactomes from the same experimental conditions were pooled, in order to identify the most robust FUS interactors under LLPS and non-LLPS conditions. We identified 238 proteins interacting with FUS under LLPS conditions and 360 under non-LLPS conditions. 102 proteins were present in both datasets (independent of either biophysical state), resulting in 136 proteins specific to the LLPS condition. The observation that several proteins and RNAs preferentially interacted with FUS under LLPS conditions ( Fig. 1c and Supplementary Table 1) indicates th...

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“…While FUS has been shown to be a relatively promiscuous RNA-binding protein, preference towards GU-rich motifs has been reported in previous CLIP-seq studies 22,38,40,41 , a binding mediated via its ZnF domain 42 . To understand if FUS binding to synaptic RNA targets follows the same modalities as its nuclear targets, we explored the sequence specificity of FUS in the synapse and predicted motifs with HOMER 54 , comparing the FUS peak sequences of cortical synaptoneurosomes and total cortex samples.…”

Section: Resultsmentioning

confidence: 81%

“…Previous studies identified nuclear RNA targets of FUS with different cross-linking immunoprecipitation and high-throughput sequencing (CLIP-seq) approaches 22,37–41 . Collectively, these findings showed that FUS binds mainly introns, without a strong sequence specificity, but a preference for either GU-rich regions 22,38,40,41 , which is mediated via its zinc finger (ZnF) domain, or a stem-loop RNA 37 via its RNA recognition motif 42 . FUS often binds close to alternatively spliced exons, highlighting its role in splicing regulation 22,38,39 .…”

Section: Introductionmentioning

confidence: 94%

Synaptic accumulation of FUS triggers age-dependent misregulation of inhibitory synapses in ALS-FUS mice

Sahadevan

Hembach

Tantardini

et al. 2020

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442 FUS is a primarily nuclear RNA-binding protein with important roles in RNA processing and 45 transport. FUS mutations disrupting its nuclear localization characterize a subset of 46 amyotrophic lateral sclerosis (ALS-FUS) patients, through an unidentified pathological 47 mechanism. FUS regulates nuclear RNAs, but its role at the synapse is poorly understood. 48Here, we used super-resolution imaging to determine the physiological localization of 49 extranuclear, neuronal FUS and found it predominantly near the vesicle reserve pool of 50 presynaptic sites. Using CLIP-seq on synaptoneurosome preparations, we identified 51 synaptic RNA targets of FUS that are associated with synapse organization and plasticity. 52 Synaptic FUS was significantly increased in a knock-in mouse model of ALS-FUS, at 53 presymptomatic stages, accompanied by alterations in density and size of GABAergic 54 synapses. RNA-seq of synaptoneurosomes highlighted age-dependent dysregulation of 55 glutamatergic and GABAergic synapses. Our study indicates that FUS accumulation at the 56 synapse in early stages of ALS-FUS results in synaptic impairment, potentially representing 57 an initial trigger of neurodegeneration.58 59 60 FUS (Fused in sarcoma) is a nucleic acid binding protein involved in several processes of 84 RNA metabolism 1 . Physiologically, FUS is predominantly localized to the nucleus 2 via active 85 transport by transportin (TNPO) 3 and it can shuttle to the cytoplasm by passive diffusion 4,5 . 86In amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), FUS mislocalizes 87 to the cytoplasm where it forms insoluble aggregates 6-8 . In ALS, cytoplasmic mislocalization 88 of FUS is associated with mutations that are mainly clustered in the proline-tyrosine nuclear 89 localization signal (PY-NLS) at the C-terminal site of the protein 9 and lead to mislocalization 90 of the protein to the cytosol. However, in FTD, FUS mislocalization occurs in the absence of 91 mutations 10 . FUS is incorporated in cytoplasmic stress granules 5,11 and undergoes 92 concentration-dependent, liquid-liquid phase separation 12,13 , which is modulated by binding 93 of TNPO and arginine methylation of FUS [14][15][16][17] . This likely contributes to the role of FUS in 94 forming specific identities of ribonucleoprotein (RNP) granules 18,19 and in transporting RNA 95 cargos 20 , which is essential for local translation in neurons 21 . 96 Despite the central role of FUS in neurodegenerative diseases, little is known about its 97 function in specialized neuronal compartments, such as synapses. FUS was shown to 98 mediate RNA transport 20 and is involved in stabilization of RNAs that encode proteins with 99 important synaptic functions 22 , such as GluA1 and SynGAP1 23,24 . While the presence of FUS 100 protein in synaptic compartments has been confirmed, its exact subsynaptic localization is 101 debated. Diverging results described the presence of FUS at the pre-synapses in close 102 proximity to synaptic vesicles 25-27 , but also in dendritic...

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The Solution Structure of FUS Bound to RNA Reveals a Bipartite Mode of RNA Recognition with Both Sequence and Shape Specificity

Cited by 177 publications

References 69 publications

The solution structure of Dead End bound to AU-rich RNA reveals an unprecedented mode of tandem RRM-RNA recognition required for mRNA regulation

The solution structure of Dead End bound to AU-rich RNA reveals an unprecedented mode of tandem RRM-RNA recognition required for mRNA regulation

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

Synaptic accumulation of FUS triggers age-dependent misregulation of inhibitory synapses in ALS-FUS mice

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