An Integrative Study of Protein-RNA Condensates Identifies Scaffolding RNAs and Reveals Players in Fragile X-Associated Tremor/Ataxia Syndrome

摘要

class="head no_bottom_margin" id="sec1title">IntroductionProteins and RNAs coalesce in large phase-separated condensates that are implicated in several cellular processes (, ).Among the most studied condensates are ribonucleoprotein (RNP) granules that assemble in liquid-like cellular compartments composed of RNA-binding proteins (RBPs) (, ) that are in dynamic exchange with the surrounding environment (). RNP granules, such as processing bodies and stress granules (SGs), are evolutionarily conserved from yeast to human (, , ) and contain constitutive protein components, such as G3BP1 (yeast: Nxt3), TIA1 (Pub1), and TIAR (Ngr1) (). Several granule-forming RBPs are prone to form amyloid aggregates upon amino acid mutations (, ) that induce a transition from a liquid droplet to a solid phase (). This observation has led to the proposal that a liquid-to-solid phase transition is a mechanism of cellular toxicity () in diseases, such as amyotrophic lateral sclerosis (ALS) () and myotonic dystrophy ().All the components of molecular complexes need to be physically close to each other to perform their functions. One way to achieve this, while keeping selectivity in a crowded cell, is to use platform or scaffold molecules that piece together components of a complex or a pathway. Indeed, RBPs are known to act as scaffolding elements promoting RNP assembly through protein-protein interactions (PPIs) (href="#bib3" rid="bib3" class=" bibr popnode">Banani et al., 2017); yet, protein-RNA interactions (PRIs) also play a role in the formation of condensates. Recent work based on G3BP1 pull-down indicates that 10% of the human transcripts can assemble into SGs (href="#bib33" rid="bib33" class=" bibr popnode">Khong et al., 2017). If distinct RNA species are present in the condensates, a fraction of them could be involved in mediating RBP recruitment. In this regard, we previously observed that different RNAs act as scaffolds for RNP complexes (href="#bib61" rid="bib61" class=" bibr popnode">Ribeiro et al., 2018), which indicates that specific transcripts might promote the formation of RNP condensates.Combining PPI and PRI networks revealed by enhanced cross-linking and immunoprecipitation (eCLIP) (href="#bib75" rid="bib75" class=" bibr popnode">Van Nostrand et al., 2016) and mass spectrometric analysis of SGs (href="#bib29" rid="bib29" class=" bibr popnode">Jain et al., 2016), we identified a class of transcripts that bind to a large number of proteins and, therefore, qualify as potential scaffolding elements. In agreement with recent literature reports, we found that UTRs have a particularly strong potential to bind proteins in RNP granules, especially when they contain specific homo-nucleotide repeats (href="#bib63" rid="bib63" class=" bibr popnode">Saha and Hyman, 2017). In support of this observation, several diseases, including myotonic dystrophy (MD) and a number of ataxias (spinocerebellar ataxia [SCA]), have been reported to be linked to expanded trinucleotide repeats that trigger the formation of intranuclear condensates in which proteins are sequestered and functionally impaired. Specifically, expanded RNA repeats lead to RNA-mediated condensate formation in DM1 (href="#bib49" rid="bib49" class=" bibr popnode">Mooers et al., 2005), SCA8 (href="#bib52" rid="bib52" class=" bibr popnode">Mutsuddi et al., 2004), and SCA10 (href="#bib79" rid="bib79" class=" bibr popnode">White et al., 2010).By understanding the characteristics of RNAs involved in RNP assembly, we aim to unveil the molecular details of specific human diseases. Indeed, the appearance of RNP condensates, often called inclusions or foci, is not only linked to ALS, Huntington’s disease, and MD but also other diseases, such as fragile X-associated tremor/ataxia syndrome (FXTAS) (href="#bib71" rid="bib71" class=" bibr popnode">Tassone et al., 2004, href="#bib66" rid="bib66" class=" bibr popnode">Sellier et al., 2017). The onset and development of FXTAS is currently explained by two main mechanisms (href="#bib6" rid="bib6" class=" bibr popnode">Botta-Orfila et al., 2016): (1) RNA-mediated recruitment of proteins attracted by CGG trinucleotide repeats in the 5′ UTR of fragile X mental retardation protein (FMR1) RNA and (2) aggregation of repeat-associated non-AUG (RAN) polyglycine peptides translated from the FMR1 5′ UTR (FMRpolyG) (href="#bib74" rid="bib74" class=" bibr popnode">Todd et al., 2013). Previous work indicates that FMR1 inclusions contain specific proteins, such as HNRNP A2/B1, MBNL1, LMNA, and INA (href="#bib27" rid="bib27" class=" bibr popnode">Iwahashi et al., 2006). Also, FMRpolyG peptides (href="#bib66" rid="bib66" class=" bibr popnode">Sellier et al., 2017) have been found in the inclusions, together with CUGBP1, KHDRBS1, and DGCR8 that are involved in splicing regulation and mRNA transport regulation of microRNA regulation (href="#bib64" rid="bib64" class=" bibr popnode">Sellier et al., 2010, href="#bib65" rid="bib65" class=" bibr popnode">Sellier et al., 2013). Although KHDRBS1 does not bind physically (href="#bib64" rid="bib64" class=" bibr popnode">Sellier et al., 2010), its protein partner DGCR8 interacts with CGG repeats (href="#bib65" rid="bib65" class=" bibr popnode">Sellier et al., 2013), indicating that sequestration is a process led by a pool of proteins that progressively attract other networks.Notably, CGG repeats contained in the FMR1 5′ UTR are of different lengths (the most common allele in Europe being of 30 repeats). At over 200 repeats, methylation and silencing of the FMR1 gene block FMRP protein expression (href="#bib74" rid="bib74" class=" bibr popnode">Todd et al., 2013). The premutation range (55–200 CGG repeats) is instead accompanied by appearance of foci that are the typical hallmark of FXTAS (href="#bib74" rid="bib74" class=" bibr popnode">Todd et al., 2013). These foci are highly dynamic and behave as RNP condensates that phase separate in the nucleus forming inclusions (href="#bib71" rid="bib71" class=" bibr popnode">Tassone et al., 2004). Although long lived, they rapidly dissolve upon tautomycin treatment, which indicates liquid-like behavior (href="#bib68" rid="bib68" class=" bibr popnode">Strack et al., 2013).The lability of FMR1 inclusions, which impedes their biochemical characterization (href="#bib47" rid="bib47" class=" bibr popnode">Mitchell et al., 2013, href="#bib41" rid="bib41" class=" bibr popnode">Marchese et al., 2016), complicates the identification of RBPs involved in FXTAS. As shown in previous studies of RNP networks (href="#bib14" rid="bib14" class=" bibr popnode">Cirillo et al., 2017, href="#bib42" rid="bib42" class=" bibr popnode">Marchese et al., 2017), computational methods can be exploited to identify key partners of RNA molecules. New contributions from other research areas are needed, especially because FXTAS pathological substrate is still under debate and there is still insufficient knowledge of targets for therapeutic intervention (href="#bib74" rid="bib74" class=" bibr popnode">Todd et al., 2013, href="#bib66" rid="bib66" class=" bibr popnode">Sellier et al., 2017). Here, we propose an integrative approach to identify new markers based on the properties of PRI networks and characteristics of scaffolding RNAs.