Prion-like domains in RNA binding proteins are essential for building subnuclear paraspeckles

Hennig, Sven; Kong, Geraldine; Mannen, Taro; Sadowska, Agata; Kobelke, Simon; Blythe, Amanda; Knott, Gavin J.; Iyer, K. Swaminathan; Ho, Diwei; Newcombe, Estella A.; Hosoki, Kana; Goshima, Naoki; Kawaguchi, Tetsuya; Hatters, Danny M.; Trinkle-Mulcahy, Laura; Hirose, Tetsuro; Bond, Charles S.; Fox, Archa H.

doi:10.1083/jcb.201504117

Cited by 301 publications

(337 citation statements)

References 43 publications

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“…Because RBM14 was purified from E. coli, it may lack posttranslational modifications necessary for interaction with EBER2 (Discussion). This notion is supported by the previous observation that phosphorylation of the C-terminal region of RBM14 is essential for protein-protein interactions (22).…”

Section: Resultssupporting

confidence: 77%

“…They are also essential for the formation of paraspeckles, which are subnuclear bodies consisting of more than 30 proteins that form on the long ncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) (18)(19)(20). Interestingly, RRM proteins not only play roles in overlapping cellular processes, but can also act in concert with each other, as they exhibit physical interaction (21,22).Here, we isolated ribonucleoprotein (RNP) complexes containing both EBER2 and PAX5 with the goal of identifying the protein(s) that bridges their interaction. As in our EBER2 CHART experiments (3), we used an antisense oligonucleotide (ASO) complementary to one of the accessible sites in EBER2 for selection, followed by immunoprecipitation using anti-PAX5 antibodies; the selected RNP complexes were subsequently analyzed by mass spectrometry.…”

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confidence: 99%

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EBV noncoding RNA EBER2 interacts with host RNA-binding proteins to regulate viral gene expression

Lee

Yario

Gao

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Epstein-Barr virus (EBV) produces a highly abundant noncoding RNA called EBV-encoded RNA 2 (EBER2) that interacts indirectly with the host transcription factor paired box protein 5 (PAX5) to regulate viral latent membrane protein 1/2 (LMP1/2) gene expression as well as EBV lytic replication. To identify intermediary proteins, we isolated EBER2-PAX5-containing complexes and analyzed the protein components by mass spectrometry. The top candidates include three host proteins splicing factor proline and glutamine rich (SFPQ), non-POU domaincontaining octamer-binding protein (NONO), and RNA binding motif protein 14 (RBM14), all reported to be components of nuclear bodies called paraspeckles. In vivo RNA-protein crosslinking indicates that SFPQ and RBM14 contact EBER2 directly. Binding studies using recombinant proteins demonstrate that SFPQ and NONO associate with PAX5, potentially bridging its interaction with EBER2. Similar to EBER2 or PAX5 depletion, knockdown of any of the three host RNA-binding proteins results in the up-regulation of viral LMP2A mRNA levels, supporting a physiologically relevant interaction of these newly identified factors with EBER2 and PAX5. Identification of these EBER2-interacting proteins enables the search for cellular noncoding RNAs that regulate host gene expression in a manner similar to EBER2.noncoding RNA | Epstein-Barr virus | paraspeckle | RNA binding protein E pstein-Barr virus (EBV)-encoded RNA 2 (EBER2) is one of two highly abundant nuclear noncoding RNAs (ncRNAs) expressed during both latent and lytic infection of human B cells by the gamma herpesvirus EBV (1). Previous genome-wide location analysis of EBER2 using capture hybridization analysis of RNA targets (CHART) (2) revealed that EBER2 binds to the socalled terminal repeat (TR) regions of the double-stranded EBV genome (3). After DNA circularization to form the latent episome, these repeats are located in the first intron of the viral transcripts encoding latent membrane protein (LMP) 2A and 2B that are generated by alternative promoter use (4). Until recently, only the RNA chaperone La was known to interact with EBER2 through the stretch of uridylates at its 3′ end (5). Prompted by chromatin colocalization of EBER2 and the host transcription factor paired box protein 5 (PAX5) at the TR regions (6), we showed that PAX5 associates with EBER2 as well (3). However, negative results in electrophoretic mobility shift assays (EMSAs) using recombinantly expressed EBER2 and PAX5 suggested that the interaction between the ncRNA and host transcription factor might be bridged by an intermediate protein(s).Within EBER2 are two accessible regions available for hybridization with complementary nucleic acids based on ribonuclease H sensitivity (3). One of these sites (Fig. 1A, Top) engages in RNA-RNA interactions with nascent transcripts from the TR regions, thus facilitating the recruitment and accumulation of PAX5 at this locus (3). Loss of either EBER2 or PAX5 binding to the TR regions results in up-regulation of the nearby LMP1, 2A,...

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Section: Resultssupporting

confidence: 77%

mentioning

confidence: 99%

EBV noncoding RNA EBER2 interacts with host RNA-binding proteins to regulate viral gene expression

Lee

Yario

Gao

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Recently, low complexity domains in paraspeckle-localized proteins were implicated in the formation of this sub-nuclear body. 63 Although not yet demonstrated, it is likely that CB formation involves similar physical events. The process has been shown to be temperature-dependent, 64 for example, while p80-coilin, in common with other proteins shown to drive phase transitions, can self-associate, 25 binds RNA, 65,66 and contains a central low complexity region.…”

Section: Physical Properties Of Nucleoli and Cbsmentioning

confidence: 99%

The Cajal body and the nucleolus: “In a relationship” or “It's complicated”?

Trinkle-Mulcahy

Sleeman

2016

RNA Biology

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From their initial identification as 'nucleolar accessory bodies' more than a century ago, the relationship between Cajal bodies and nucleoli has been a subject of interest and controversy. In this review, we seek to place recent developments in the understanding of the physical and functional relationships between the 2 structures in the context of historical observations. Biophysical models of nuclear body formation, the molecular nature of CB/nucleolus interactions and the increasing list of joint roles for CBs and nucleoli, predominantly in assembling ribonucleoprotein (RNP) complexes, are discussed.

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“…Several groups have shown that multivalent proteins enriched with highly disordered domains are capable of forming liquid droplets in vitro that display many characteristic features of NBs, including fusion and fission events. 86,87,90,91,145 It has also been suggested that separation of nucleoli, paraspeckles 146 and Nuage bodies formed by DEAD-box helicase 4 (Ddx4) protein 86,147 is driven by these disordered domains-containing proteins. Coilin and its homologs are highly disordered but Arabidopsis thaliana-derived coilin is stabilized upon snRNA binding leading to multimerization in vitro, 114 which can be assumed to contribute to CB nucleation.…”

Section: Proteinmentioning

confidence: 99%

Specific genomic cues regulate Cajal body assembly

2016

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The assembly of specialized sub-nuclear microenvironments known as nuclear bodies (NBs) is important for promoting efficient nuclear function. In particular, the Cajal body (CB), a prominent NB that facilitates spliceosomal snRNP biogenesis, assembles in response to genomic cues. Here, we detail the factors that regulate CB assembly and structural maintenance. These include the importance of transcription at nucleating gene loci, the grouping of these genes on human chromosomes 1, 6 and 17, as well as cell cycle and biochemical regulation of CB protein function. We also speculate on the correlation between CB formation and RNA splicing levels in neurons and cancer. The timing and location of these specific molecular events is critical to CB assembly and its contribution to genome function. However, further work is required to explore the emerging biophysical characteristics of CB assembly and the impact upon subsequent genome reorganization.

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Prion-like domains in RNA binding proteins are essential for building subnuclear paraspeckles

Abstract: Paraspeckles are mammalian subnuclear bodies built on a long noncoding RNA and are enriched in RNA binding proteins with prion-like domains; two of these proteins, RBM14 and FUS, use these domains to hold paraspeckles together.

Cited by 301 publications

References 43 publications

EBV noncoding RNA EBER2 interacts with host RNA-binding proteins to regulate viral gene expression

EBV noncoding RNA EBER2 interacts with host RNA-binding proteins to regulate viral gene expression

The Cajal body and the nucleolus: “In a relationship” or “It's complicated”?

Specific genomic cues regulate Cajal body assembly

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