FUS binds the CTD of RNA polymerase II and regulates its phosphorylation at Ser2
Howard Hughes Medical Institute. Genes & development
(Impact Factor: 10.8).
12/2012; 26(24):2690-5. DOI: 10.1101/gad.204602.112
Mutations in the RNA-binding protein FUS (fused in sarcoma)/TLS have been shown to cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS), but the normal role of FUS is incompletely understood. We found that FUS binds the C-terminal domain (CTD) of RNA polymerase II (RNAP2) and prevents inappropriate hyperphosphorylation of Ser2 in the RNAP2 CTD at thousands of human genes. The loss of FUS leads to RNAP2 accumulation at the transcription start site and a shift in mRNA isoform expression toward early polyadenylation sites. Thus, in addition to its role in alternative RNA splicing, FUS has a general function in orchestrating CTD phosphorylation during RNAP2 transcription.
Available from: Kathleen Anne Burke
- "Additionally, more than a dozen related sarcomas and leukemias are caused by chromosomal translocations fusing the LC domain of FUS or that of two other human paralogs , RNA-binding protein EWS and TATA-binding protein-associated factor 2N (product of the TAF15 gene), to one of several DNA-binding domains, forming strong transcriptional activators (Riggi et al., 2007). Transcriptional activation by FUS LC may be due to the ability of phase-separated forms of FUS to bind the C-terminal domain of DNA directed RNA polymerase II subunit RPB1 (the large subunit of RNA polymerase II) (Kwon et al., 2013; Schwartz et al., 2012). Although the macroscopic behavior of protein-rich RNP granules and disease-associated inclusions has begun to be elucidated by microscopy both in vitro and in vivo (Brangwynne et al., 2009), the structural and mechanistic details of the protein domains as free monomers, in granule assemblies, and in disease aggregates remain uncharacterized. "
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ABSTRACT: Phase-separated states of proteins underlie ribonucleoprotein (RNP) granules and nuclear RNA-binding protein assemblies that may nucleate protein inclusions associated with neurodegenerative diseases. We report that the N-terminal low-complexity domain of the RNA-binding protein Fused in Sarcoma (FUS LC) is structurally disordered and forms a liquid-like phase-separated state resembling RNP granules. This state directly binds the C-terminal domain of RNA polymerase II. Phase-separated FUS lacks static structures as probed by fluorescence microscopy, indicating they are distinct from both protein inclusions and hydrogels. We use solution nuclear magnetic resonance spectroscopy to directly probe the dynamic architecture within FUS liquid phase-separated assemblies. Importantly, we find that FUS LC retains disordered secondary structure even in the liquid phase-separated state. Therefore, we propose that disordered protein granules, even those made of aggregation-prone prion-like domains, are dynamic and disordered molecular assemblies with transiently formed protein-protein contacts.
Molecular cell 10/2015; 60(2). DOI:10.1016/j.molcel.2015.09.006 · 14.02 Impact Factor
- "Recombinant versions of FUS-GFP were purified using the baculovirus expression system (Hoell et al., 2011; Schwartz et al., 2012). Cells were lysed in resuspension buffer (50 mM Tris-HCl, 1 M KCl, 5% glycerol, 0.1% CHAPS, 1 mM DTT, pH 7.4), and FUS-GFP was captured using nickel-nitrilotriacetic acid (Ni-NTA) resin (QIAGEN) and eluted with elution buffer (resuspension buffer + 250 mM imidazole). "
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ABSTRACT: Many proteins contain disordered regions of low-sequence complexity, which cause aging-associated diseases because they are prone to aggregate. Here, we study FUS, a prion-like protein containing intrinsically disordered domains associated with the neurodegenerative disease ALS. We show that, in cells, FUS forms liquid compartments at sites of DNA damage and in the cytoplasm upon stress. We confirm this by reconstituting liquid FUS compartments in vitro. Using an in vitro "aging" experiment, we demonstrate that liquid droplets of FUS protein convert with time from a liquid to an aggregated state, and this conversion is accelerated by patient-derived mutations. We conclude that the physiological role of FUS requires forming dynamic liquid-like compartments. We propose that liquid-like compartments carry the trade-off between functionality and risk of aggregation and that aberrant phase transitions within liquid-like compartments lie at the heart of ALS and, presumably, other age-related diseases. VIDEO ABSTRACT.
Copyright © 2015 Elsevier Inc. All rights reserved.
Cell 08/2015; 162(5):1066-1077. DOI:10.1016/j.cell.2015.07.047 · 32.24 Impact Factor
- "that FUS can affect CTD phosphorylation in vitro (Schwartz et al. 2012). Thus, nascent RNA would stimulate fibrous assembly of FUS, which in turn would attenuate CTD phosphorylation and suppress transcriptional activity in vivo. "
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ABSTRACT: More than half of all human genes produce prematurely terminated polyadenylated short mRNAs. However, the underlying mechanisms remain largely elusive. CLIP-seq (cross-linking immunoprecipitation [CLIP] combined with deep sequencing) of FUS (fused in sarcoma) in neuronal cells showed that FUS is frequently clustered around an alternative polyadenylation (APA) site of nascent RNA. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing) of RNA polymerase II (RNAP II) demonstrated that FUS stalls RNAP II and prematurely terminates transcription. When an APA site is located upstream of an FUS cluster, FUS enhances polyadenylation by recruiting CPSF160 and up-regulates the alternative short transcript. In contrast, when an APA site is located downstream from an FUS cluster, polyadenylation is not activated, and the RNAP II-suppressing effect of FUS leads to down-regulation of the alternative short transcript. CAGE-seq (cap analysis of gene expression [CAGE] combined with deep sequencing) and PolyA-seq (a strand-specific and quantitative method for high-throughput sequencing of 3' ends of polyadenylated transcripts) revealed that position-specific regulation of mRNA lengths by FUS is operational in two-thirds of transcripts in neuronal cells, with enrichment in genes involved in synaptic activities.
© 2015 Masuda et al.; Published by Cold Spring Harbor Laboratory Press.
Genes & development 05/2015; 29(10):1045-57. DOI:10.1101/gad.255737.114 · 10.80 Impact Factor
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