HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-Mediated Innate Immune Response.

The DNA-mediated innate immune response underpins anti-microbial defenses and certain autoimmune diseases. Here we used immunoprecipitation, mass spectrometry, and RNA sequencing to identify a ribonuclear complex built around HEXIM1 and the long non-coding RNA NEAT1 that we dubbed the HEXIM1-DNA-PK-paraspeckle components-ribonucleoprotein complex (HDP-RNP). The HDP-RNP contains DNA-PK subunits (DNAPKc, Ku70, and Ku80) and paraspeckle proteins (SFPQ, NONO, PSPC1, RBM14, and MATRIN3). We show that binding of HEXIM1 to NEAT1 is required for its assembly. We further demonstrate that the HDP-RNP is required for the innate immune response to foreign DNA, through the cGAS-STING-IRF3 pathway. The HDP-RNP interacts with cGAS and its partner PQBP1, and their interaction is remodeled by foreign DNA. Remodeling leads to the release of paraspeckle proteins, recruitment of STING, and activation of DNAPKc and IRF3. Our study establishes the HDP-RNP as a key nuclear regulator of DNA-mediated activation of innate immune response through the cGAS-STING pathway.

Role of RNA binding proteins of the Drosophila behavior and human splicing (DBHS) family in health and cancer.

RNA-binding proteins (RBPs) play crucial roles in the functions and homoeostasis of various tissues by regulating multiple events of RNA processing including RNA splicing, intracellular RNA transport, and mRNA translation. The Drosophila behavior and human splicing (DBHS) family proteins including PSF/SFPQ, NONO, and PSPC1 are ubiquitously expressed RBPs that contribute to the physiology of several tissues. In mammals, DBHS proteins have been reported to contribute to neurological diseases and play crucial roles in cancers, such as prostate, breast, and liver cancers, by regulating cancer-specific gene expression. Notably, in recent years, multiple small molecules targeting DBHS family proteins have been developed for application as cancer therapeutics. This review provides a recent overview of the functions of DBHS family in physiology and pathophysiology, and discusses the application of DBHS family proteins as promising diagnostic and therapeutic targets for cancers.

SFPQ regulates the accumulation of RNA foci and dipeptide repeat proteins from the expanded repeat mutation in C9orf72.

The expanded GGGGCC repeat mutation in the C9orf72 gene is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The expansion is transcribed to sense and antisense RNA, which form RNA foci and bind cellular proteins. This mechanism of action is considered cytotoxic. Translation of the expanded RNA transcripts also leads to the accumulation of toxic dipeptide repeat proteins (DPRs). The RNA-binding protein splicing factor proline and glutamine rich (SFPQ), which is being increasingly associated with ALS and FTD pathology, binds to sense RNA foci. Here, we show that SFPQ plays an important role in the C9orf72 mutation. Overexpression of SFPQ resulted in higher numbers of both sense and antisense RNA foci and DPRs in transfected human embryonic kidney (HEK) cells. Conversely, reduced SPFQ levels resulted in lower numbers of RNA foci and DPRs in both transfected HEK cells and C9orf72 mutation-positive patient-derived fibroblasts and lymphoblasts. Therefore, we have revealed a role of SFPQ in regulating the C9orf72 mutation that has implications for understanding and developing novel therapeutic targets for ALS and FTD.This article has an associated First Person interview with the first author of the paper.

Insight into the Tumor Suppression Mechanism from the Structure of Human Polypyrimidine Splicing Factor (PSF/SFPQ) Complexed with a 30mer RNA from Murine Virus-like 30S Transcript-1.

Human polypyrimidine-binding splicing factor (PSF/SFPQ) is a tumor suppressor protein that regulates the gene expression of several proto-oncogenes and binds to the 5'-polyuridine negative-sense template (5'-PUN) of some RNA viruses. The activity of PSF is negatively regulated by long-noncoding RNAs, human metastasis associated in lung adenocarcinoma transcript-1 and murine virus-like 30S transcript-1 (VL30-1). PSF is a 707-amino acid protein that has a DNA-binding domain and two RNA recognition motifs (RRMs). Although the structure of the apo-truncated PSF is known, how PSF recognizes RNA remains elusive. Here, we report the 2.8 Å and 3.5 Å resolution crystal structures of a biologically active truncated construct of PSF (sPSF, consisting of residues 214-598) alone and in a complex with a 30mer fragment of VL30-1 RNA, respectively. The structure of the complex reveals how the 30mer RNA is recognized at two U-specific induced-fit binding pockets, located at the previously unrecognized domain-swapped, inter-subunit RRM1 (of the first subunit)-RRM2 (of the second subunit) interfaces that do not exist in the apo structure. Thus, the sPSF dimer appears to have two conformations in solution: one in a low-affinity state for RNA binding, as seen in the apo-structure, and the other in a high-affinity state for RNA binding, as seen in the sPSF-RNA complex. PSF undergoes an all or nothing transition between having two or no RNA-binding pockets. We predict that the RNA binds with a high degree of positive cooperativity. These structures provide an insight into a new regulatory mechanism that is likely involved in promoting malignancies and other human diseases.

Molecular Modelling of NONO and SFPQ Dimerization Process and RNA Recognition Mechanism.

NONO and SFPQ are involved in multiple nuclear processes (e.g., pre-mRNA splicing, DNA repair, and transcriptional regulation). These proteins, along with NEAT1, enable paraspeckle formation, thus promoting multiple myeloma cell survival. In this paper, we investigate NONO and SFPQ dimer stability, highlighting the hetero- and homodimer structural differences, and model their interactions with RNA, simulating their binding to a polyG probe mimicking NEAT1guanine-rich regions. We demonstrated in silico that NONO::SFPQ heterodimerization is a more favorable process than homodimer formation. We also show that NONO and SFPQ RRM2 subunits are primarily required for protein-protein interactions with the other DBHS protomer. Simulation of RNA binding to NONO and SFPQ, beside validating RRM1 RNP signature importance, highlighted the role of ß2 and ß4 strand residues for RNA specific recognition. Moreover, we demonstrated the role of the NOPS region and other protomer's RRM2 ß2/ß3 loop in strengthening the interaction with RNA. Our results, having deepened RNA and DBHS dimer interactions, could contribute to the design of small molecules to modulate the activity of these proteins. RNA-mimetics, able to selectively bind to NONO and/or SFPQ RNA-recognition site, could impair paraspeckle formation, thus representing a first step towards the discovery of drugs for multiple myeloma treatment.

Nuclear RNA foci from C9ORF72 expansion mutation form paraspeckle-like bodies.

The GGGGCC (G4C2) repeat expansion mutation in the C9ORF72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Transcription of the repeat and formation of nuclear RNA foci, which sequester specific RNA-binding proteins, is one of the possible pathological mechanisms. Here, we show that (G4C2) n repeat RNA predominantly associates with essential paraspeckle proteins SFPQ, NONO, RBM14, FUS and hnRNPH and colocalizes with known paraspeckle-associated RNA hLinc-p21. As formation of paraspeckles in motor neurons has been associated with early phases of ALS, we investigated the extent of similarity between paraspeckles and (G4C2) n RNA foci. Overexpression of (G4C2)72 RNA results in their increased number and colocalization with SFPQ-stained nuclear bodies. These paraspeckle-like (G4C2)72 RNA foci form independently of the known paraspeckle scaffold, the long non-coding RNA NEAT1 Moreover, the knockdown of SFPQ protein in C9ORF72 expansion mutation-positive fibroblasts significantly reduces the number of (G4C2) n RNA foci. In conclusion, (G4C2) n RNA foci have characteristics of paraspeckles, which suggests that both RNA foci and paraspeckles play roles in FTD and ALS, and implies approaches for regulation of their formation.

SFPQ associated with a co-activator for PPARγ, HELZ2, regulates key nuclear factors for adipocyte differentiation.

We recently isolated a novel co-activator of peroxisome proliferator-activated receptor γ, helicase with zinc finger 2 (HELZ2). HELZ2 null mice were resistant to diet-induced obesity and NAFFL/NASH, and HELZ2 was phosphorylated at tyrosine residues. In order to find a factor related to HELZ2, we analyzed products co-immunoprecipitated with phosphorylated HELZ2 by mass spectrometry analyses. We identified proline- and glutamine-rich (SFPQ) as a protein associating with tyrosine-phosphorylated HELZ2. The knockdown of SFPQ in 3T3-L1 cells downregulated mRNA levels of transcription factors including Krox20, Cebpß, and Cebpδ: key factors for early-stage adipocyte differentiation. In addition, knockdown of SFPQ inhibited 3T3-L1 cell differentiation to mature adipocytes. These findings demonstrated that SFPQ associating with HELZ2 is an important novel transcriptional regulator of adipocyte differentiation.

PSF functions as a repressor of hypoxia-induced angiogenesis by promoting mitochondrial function.

BACKGROUND: Abnormal neovascularization is the most common cause of blindness, and hypoxia alters tissue metabolism, function, and morphology. HIF-1α, the transcriptional activator of VEGF, has intricate mechanisms of nuclear translocation and activation, but its signal termination mechanisms remain unclear. METHODS: We investigated the role of polypyrimidine tract-binding protein-associated splicing factor (PSF) in cellular energy production, migration, and proliferation by targeting HIF-1α in vivo and in vitro PSF plasmids were transfected with liposome 2000 transfection reagent. Young C57/BL6J mice were kept in a hyperoxia environment, followed by indoor air, resulting in oxygen-induced retinopathy. Oxygen-induced retinopathy (OIR) animals were randomly divided into three groups: OIR group, OIR + vector group (OIR cubs treated with rAAV vector) and OIR + PSF group (OIR cubs treated with rAAV-PSF). Age-matched C57/BL6J mice were used as controls and exposed to constant normoxic conditions. The animals were executed and their pupils were subjected to subsequent experiments. The metabolic spectrum was analyzed by Seahorse XFe96 flux analyzer, and OCR and extracellular acidification rate were quantified at the same time. RESULTS: PSF ameliorated retinal neovascularization and corrected abnormal VEGF expression in mice with oxygen-induced retinopathy and reduced intra-retinal neovascularization in Vldlr - / - mice. PSF reprogrammed mitochondrial bioenergetics and inhibited the transition of endothelial cells after hypoxia, suggesting its involvement in pathological angiogenesis.Ectopic PSF expression inhibited hypoxia-induced HIF-1α activation in the nucleus by recruiting Hakai to the PSF/HIF-1α complex, causing HIF-1α inhibition. PSF knockdown increased hypoxia-stimulated HIF-1α reactions. These hypoxia-dependent processes may play a vital role in cell metabolism, migration, and proliferation. Thus, PSF is a potential treatment target in neovascularization-associated ophthalmopathy. CONCLUSION: This is the first study showing that PSF inhibits HIF-1α via recruitment of Hakai, modulates mitochondrial oxidation and glycolysis, and downregulates VEGF expression under hypoxia. We propose a new HIF-1 α/Hakai regulatory mechanism that may play a vital role in the pathogenesis of neovascularization in ophthalmopathy. PSF-Hakai-HIF-1α signaling pathway under hypoxia condition. Schematic diagram showing that the PSF-Hakai-HIF-1α signaling pathway. Under hypoxia condition, PSF-Hakai complex regulate HIF-1α signaling, thus inhibiting downstream target gene VEGF, cell metabolism and angiogenesis eventually. Video Abstract: Detailed information of Materials and Methods.

Aberrant interaction between FUS and SFPQ in neurons in a wide range of FTLD spectrum diseases.

Fused in sarcoma (FUS) is genetically and clinicopathologically linked to frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). We have previously reported that intranuclear interactions of FUS and splicing factor, proline- and glutamine-rich (SFPQ) contribute to neuronal homeostasis. Disruption of the FUS-SFPQ interaction leads to an increase in the ratio of 4-repeat tau (4R-tau)/3-repeat tau (3R-tau), which manifests in FTLD-like phenotypes in mice. Here, we examined FUS-SFPQ interactions in 142 autopsied individuals with FUS-related ALS/FTLD (ALS/FTLD-FUS), TDP-43-related ALS/FTLD (ALS/FTLD-TDP), progressive supranuclear palsy, corticobasal degeneration, Alzheimer's disease, or Pick's disease as well as controls. Immunofluorescent imaging showed impaired intranuclear co-localization of FUS and SFPQ in neurons of ALS/FTLD-FUS, ALS/FTLD-TDP, progressive supranuclear palsy and corticobasal degeneration cases, but not in Alzheimer's disease or Pick's disease cases. Immunoprecipitation analyses of FUS and SFPQ revealed reduced interactions between the two proteins in ALS/FTLD-TDP and progressive supranuclear palsy cases, but not in those with Alzheimer disease. Furthermore, the ratio of 4R/3R-tau was elevated in cases with ALS/FTLD-TDP and progressive supranuclear palsy, but was largely unaffected in cases with Alzheimer disease. We concluded that impaired interactions between intranuclear FUS and SFPQ and the subsequent increase in the ratio of 4R/3R-tau constitute a common pathogenesis pathway in FTLD spectrum diseases.

Dido3-dependent SFPQ recruitment maintains efficiency in mammalian alternative splicing.

Alternative splicing is facilitated by accessory proteins that guide spliceosome subunits to the primary transcript. Many of these splicing factors recognize the RNA polymerase II tail, but SFPQ is a notable exception even though essential for mammalian RNA processing. This study reveals a novel role for Dido3, one of three Dido gene products, in alternative splicing. Binding of the Dido3 amino terminus to histones and to the polymerase jaw domain was previously reported, and here we show interaction between its carboxy terminus and SFPQ. We generated a mutant that eliminates Dido3 but preserves other Dido gene products, mimicking reduced Dido3 levels in myeloid neoplasms. Dido mutation suppressed SFPQ binding to RNA and increased skipping for a large group of exons. Exons bearing recognition sequences for alternative splicing factors were nonetheless included more efficiently. Reduced SFPQ recruitment may thus account for increased skipping of SFPQ-dependent exons, but could also generate a splicing factor surplus that becomes available to competing splice sites. Taken together, our data indicate that Dido3 is an adaptor that controls SFPQ utilization in RNA splicing. Distributing splicing factor recruitment over parallel pathways provides mammals with a simple mechanism to regulate exon usage while maintaining RNA splicing efficiency.

Exploitation of nuclear functions by human rhinovirus, a cytoplasmic RNA virus.

Protein production, genomic RNA replication, and virion assembly during infection by picornaviruses like human rhinovirus and poliovirus take place in the cytoplasm of infected human cells, making them the quintessential cytoplasmic pathogens. However, a growing body of evidence suggests that picornavirus replication is promoted by a number of host proteins localized normally within the host cell nucleus. To systematically identify such nuclear proteins, we focused on those that appear to re-equilibrate from the nucleus to the cytoplasm during infection of HeLa cells with human rhinovirus via quantitative protein mass spectrometry. Our analysis revealed a highly selective re-equilibration of proteins with known mRNA splicing and transport-related functions over nuclear proteins of all other functional classes. The multifunctional splicing factor proline and glutamine rich (SFPQ) was identified as one such protein. We found that SFPQ is targeted for proteolysis within the nucleus by viral proteinase 3CD/3C, and a fragment of SFPQ was shown to migrate to the cytoplasm at mid-to-late times of infection. Cells knocked down for SFPQ expression showed significantly reduced rhinovirus titers, viral protein production, and viral RNA accumulation, consistent with SFPQ being a pro-viral factor. The SFPQ fragment that moved into the cytoplasm was able to bind rhinovirus RNA either directly or indirectly. We propose that the truncated form of SFPQ promotes viral RNA stability or replication, or virion morphogenesis. More broadly, our findings reveal dramatic changes in protein compartmentalization during human rhinovirus infection, allowing the virus to systematically hijack the functions of proteins not normally found at its cytoplasmic site of replication.

SFPQ and Tau: critical factors contributing to rapid progression of Alzheimer's disease.

Dysfunctional RNA-binding proteins (RBPs) have been implicated in several neurodegenerative disorders. Recently, this paradigm of RBPs has been extended to pathophysiology of Alzheimer's disease (AD). Here, we identified disease subtype specific variations in the RNA-binding proteome (RBPome) of sporadic AD (spAD), rapidly progressive AD (rpAD), and sporadic Creutzfeldt Jakob disease (sCJD), as well as control cases using RNA pull-down assay in combination with proteomics. We show that one of these identified proteins, splicing factor proline and glutamine rich (SFPQ), is downregulated in the post-mortem brains of rapidly progressive AD patients, sCJD patients and 3xTg mice brain at terminal stage of the disease. In contrast, the expression of SFPQ was elevated at early stage of the disease in the 3xTg mice, and in vitro after oxidative stress stimuli. Strikingly, in rpAD patients' brains SFPQ showed a significant dislocation from the nucleus and cytoplasmic colocalization with TIA-1. Furthermore, in rpAD brain lesions, SFPQ and p-tau showed extranuclear colocalization. Of note, association between SFPQ and tau-oligomers in rpAD brains suggests a possible role of SFPQ in oligomerization and subsequent misfolding of tau protein. In line with the findings from the human brain, our in vitro study showed that SFPQ is recruited into TIA-1-positive stress granules (SGs) after oxidative stress induction, and colocalizes with tau/p-tau in these granules, providing a possible mechanism of SFPQ dislocation through pathological SGs. Furthermore, the expression of human tau in vitro induced significant downregulation of SFPQ, suggesting a causal role of tau in the downregulation of SFPQ. The findings from the current study indicate that the dysregulation and dislocation of SFPQ, the subsequent DNA-related anomalies and aberrant dynamics of SGs in association with pathological tau represents a critical pathway which contributes to rapid progression of AD.

IGFBP-3 interacts with NONO and SFPQ in PARP-dependent DNA damage repair in triple-negative breast cancer.

Women with triple-negative breast cancer (TNBC) are generally treated by chemotherapy but their responsiveness may be blunted by DNA double-strand break (DSB) repair. We previously reported that IGFBP-3 forms nuclear complexes with EGFR and DNA-dependent protein kinase (DNA-PKcs) to modulate DSB repair by non-homologous end-joining (NHEJ) in TNBC cells. To discover IGFBP-3 binding partners involved in chemoresistance through stimulation of DSB repair, we analyzed the IGFBP-3 interactome by LC-MS/MS and confirmed interactions by coimmunoprecipitation and proximity ligation assay. Functional effects were demonstrated by DNA end-joining in vitro and measurement of γH2AX foci. In response to 20 µM etoposide, the DNA/RNA-binding protein, non-POU domain-containing octamer-binding protein (NONO) and its dimerization partner splicing factor, proline/glutamine-rich (SFPQ) formed complexes with IGFBP-3, demonstrated in basal-like TNBC cell lines HCC1806 and MDA-MB-468. NONO binding to IGFBP-3 was also shown in a cell-free biochemical assay. IGFBP-3 complexes with NONO and SFPQ were blocked by inhibiting EGFR with gefitinib or DNA-PKcs with NU7026, and by the PARP inhibitors veliparib and olaparib, which also reduced DNA end-joining activity and delayed the resolution of the γH2AX signal (i.e. inhibited DNA DSB repair). Downregulation of the long noncoding RNA in NHEJ pathway 1 (LINP1) by siRNA also blocked IGFBP-3 interaction with NONO-SFPQ. These findings suggest a PARP-dependent role for NONO and SFPQ in IGFBP-3-dependent DSB repair and the involvement of LINP1 in the complex formation. We propose that targeting of the DNA repair function of IGFBP-3 may enhance chemosensitivity in basal-like TNBC, thus improving patient outcomes.

The role of the protein-RNA recognition code in neurodegeneration.

MicroRNAs are small endogenous RNAs that pair and bind to sites on mRNAs to direct post-transcriptional repression. However, there is a possibility that microRNAs directly influence protein structure and activity, and this influence can be termed post-translational riboregulation. This conceptual review explores the literature on neurodegenerative disorders. Research on the association between neurodegeneration and RNA-repeat toxicity provides data that support a protein-RNA recognition code. For example, this code explains why hnRNP H and SFPQ proteins, which are involved in amyotrophic lateral sclerosis, are sequestered by the (GGGGCC)n repeat sequence. Similarly, it explains why MNBL proteins and (CTG)n repeats in RNA, which are involved in myotonic dystrophy, are sequestered into RNA foci. Using this code, proteins involved in diseases can be identified. A simple protein BLAST search of the human genome for amino acid repeats that correspond to the nucleotide repeats reveals new proteins among already known proteins that are involved in diseases. For example, the (CAG)n repeat sequence, when transcribed into possible peptide sequences, leads to the identification of PTCD3, Rem2, MESP2, SYPL2, WDR33, COL23A1, and others. After confirming this approach on RNA repeats, in the next step, the code was used in the opposite manner. Proteins that are involved in diseases were compared with microRNAs involved in those diseases. For example, a reasonable correspondence of microRNA 9 and 107 with amyloid-ß-peptide (Aß42) was identified. In the last step, a miRBase search for micro-nucleotides, obtained by transcription of a prion amino acid sequence, revealed new microRNAs and microRNAs that have previously been identified as involved in prion diseases. This concept provides a useful key for designing RNA or peptide probes.

Identification of conserved, primary sequence motifs that direct retrovirus RNA fate.

Precise stoichiometry of genome-length transcripts and alternatively spliced mRNAs is a hallmark of retroviruses. We discovered short, guanosine and adenosine sequence motifs in the 5'untranslated region of several retroviruses and ascertained the reasons for their conservation using a representative lentivirus and genetically simpler retrovirus. We conducted site-directed mutagenesis of the GA-motifs in HIV molecular clones and observed steep replication delays in T-cells. Quantitative RNA analyses demonstrate the GA-motifs are necessary to retain unspliced viral transcripts from alternative splicing. Mutagenesis of the GA-motifs in a C-type retrovirus validate the similar downregulation of unspliced transcripts and virion structural protein. The evidence from cell-based co-precipitation studies shows the GA-motifs in the 5'untranslated region confer binding by SFPQ/PSF, a protein co-regulated with T-cell activation. Diminished SFPQ/PSF or mutation of either GA-motif attenuates the replication of HIV. The interaction of SFPQ/PSF with both GA-motifs is crucial for maintaining the stoichiometry of the viral transcripts and does not affect packaging of HIV RNA. Our results demonstrate the conserved GA-motifs direct the fate of retrovirus RNA. These findings have exposed an RNA-based molecular target to attenuate retrovirus replication.

Acute hepatotoxicity of 2' fluoro-modified 5-10-5 gapmer phosphorothioate oligonucleotides in mice correlates with intracellular protein binding and the loss of DBHS proteins.

We reported previously that a 2' fluoro-modified (2' F) phosphorothioate (PS) antisense oligonucleotides (ASOs) with 5-10-5 gapmer configuration interacted with proteins from Drosophila behavior/human splicing (DBHS) family with higher affinity than PS-ASOs modified with 2'-O-(2-methoxyethyl) (2' MOE) or 2',4'-constrained 2'-O-ethyl (cEt) did. Rapid degradation of these proteins and cytotoxicity were observed in cells treated with 2' F PS-ASO. Here, we report that 2' F gapmer PS-ASOs of different sequences caused reduction in levels of DBHS proteins and hepatotoxicity in mice. 2' F PS-ASOs induced activation of the P53 pathway and downregulation of metabolic pathways. Altered levels of RNA and protein markers for hepatotoxicity, liver necrosis, and apoptosis were observed as early as 24 to 48 hours after a single administration of the 2' F PS-ASO. The observed effects were not likely due to the hybridization-dependent RNase H1 cleavage of on- or potential off-target RNAs, or due to potential toxicity of 2' F nucleoside metabolites. Instead, we found that 2' F PS-ASO associated with more intra-cellular proteins including proteins from DBHS family. Our results suggest that protein-binding correlates positively with the 2' F modification-dependent loss of DBHS proteins and the toxicity of gapmer 2' F PS-ASO in vivo.

The Emerging Role of the RNA-Binding Protein SFPQ in Neuronal Function and Neurodegeneration.

RNA-binding proteins (RBPs) are a class of proteins known for their diverse roles in RNA biogenesis, from regulating transcriptional processes in the nucleus to facilitating translation in the cytoplasm. With higher demand for RNA metabolism in the nervous system, RBP misregulation has been linked to a wide range of neurological and neurodegenerative diseases. One of the emerging RBPs implicated in neuronal function and neurodegeneration is splicing factor proline- and glutamine-rich (SFPQ). SFPQ is a ubiquitous and abundant RBP that plays multiple regulatory roles in the nucleus such as paraspeckle formation, DNA damage repair, and various transcriptional regulation processes. An increasing number of studies have demonstrated the nuclear and also cytoplasmic roles of SFPQ in neurons, particularly in post-transcriptional regulation and RNA granule formation. Not surprisingly, the misregulation of SFPQ has been linked to pathological features shown by other neurodegenerative disease-associated RBPs such as aberrant RNA splicing, cytoplasmic mislocalization, and aggregation. In this review, we discuss recent findings on the roles of SFPQ with a particular focus on those in neuronal development and homeostasis as well as its implications in neurodegenerative diseases.

Crystal structure of a SFPQ/PSPC1 heterodimer provides insights into preferential heterodimerization of human DBHS family proteins.

Members of the Drosophila behavior human splicing (DBHS) protein family are nuclear proteins implicated in many layers of nuclear functions, including RNA biogenesis as well as DNA repair. Definitive of the DBHS protein family, the conserved DBHS domain provides a dimerization platform that is critical for the structural integrity and function of these proteins. The three human DBHS proteins, splicing factor proline- and glutamine-rich (SFPQ), paraspeckle component 1 (PSPC1), and non-POU domain-containing octamer-binding protein (NONO), form either homo- or heterodimers; however, the relative affinity and mechanistic details of preferential heterodimerization are yet to be deciphered. Here we report the crystal structure of a SFPQ/PSPC1 heterodimer to 2.3-Å resolution and analyzed the subtle structural differences between the SFPQ/PSPC1 heterodimer and the previously characterized SFPQ homodimer. Analytical ultracentrifugation to estimate the dimerization equilibrium of the SFPQ-containing dimers revealed that the SFPQ-containing dimers dissociate at low micromolar concentrations and that the heterodimers have higher affinities than the homodimer. Moreover, we observed that the apparent dissociation constant for the SFPQ/PSPC1 heterodimer was over 6-fold lower than that of the SFPQ/NONO heterodimer. We propose that these differences in dimerization affinity may represent a potential mechanism by which PSPC1 at a lower relative cellular abundance can outcompete NONO to heterodimerize with SFPQ.

Exploitation of nuclear protein SFPQ by the encephalomyocarditis virus to facilitate its replication.

The encephalomyocarditis virus (EMCV) is a single-stranded RNA virus that induces sudden death, diabetes, myocarditis and nervous disorders in non-human primates. The rapid development of xenografts such as heart transplantation from pig to human raises the issue of EMCV safety in human cells. SFPQ, a proline and glutamine rich splicing factor that participates in diverse molecular functions including paraspeckle formation, microRNA synthesis and transcription regulation, is known to regulate host innate immune response to viruses. However, the role of SFPQ in EMCV infection remains unclear. Here we reported that the SFPQ was essential for EMCV replication. Depletion of SFPQ impaired EMCV production, while forced expression of SFPQ promoted viral replication. Mechanistically, loss of SFPQ affected the transcription profile of host mitochondria pathway related genes. In addition, cellular SFPQ was exploited by EMCV and accumulated in cytoplasm and it interacted with eukaryotic initiation factors and ribosomal proteins to facilitate internal ribosome entry site (IRES)-dependent translation of EMCV protein. Altogether, our work discovered host SFPQ as a new target to inhibit EMCV replication and infection.

Dynamic paraspeckle component localisation during spermatogenesis.

Expression profiles and subcellular localisations of core Drosophila behaviour/human splicing (DBHS) proteins (PSPC1, SFPQ and NONO) and NEAT1, a long noncoding RNA (lncRNA), are investigated in developing and adult mouse testes. Core DBHS proteins are markers for the distinct subnuclear domain termed paraspeckles, while a long NEAT1 isoform scaffold facilitates paraspeckle nucleation. Paraspeckles contain many proteins (>40) and are broadly involved in RNA metabolism, including transcriptional regulation by protein sequestration, nuclear retention of A-to-I edited RNA transcripts to regulate translation and promoting survival during cellular stress. Immunohistochemistry reveals cell-specific profiles for core DBHS paraspeckle protein expression, indicating their functional diversity. PSPC1 is an androgen receptor co-activator, and it is detected in differentiating Sertoli cell nuclei from day 15 onwards, as they develop androgen responsiveness. PSPC1 is nuclear in the most mature male germ cell type present at each age, from foetal to adult life. In adult mouse testes, PSPC1 and SFPQ are present in Sertoli cells, spermatocytes and round spermatids, while the NEAT1 lncRNA appears in the punctate nuclear foci delineating paraspeckles only within Leydig cells. Identification of NEAT1 in the cytoplasm of spermatogonia and spermatocytes must reflect non-paraspeckle-related functions. NONO was absent from germ cells but nuclear in Sertoli cells. Reciprocal nuclear profiles of PSPC1 and γ-H2AX in spermatogenic cells suggest that each performs developmentally regulated roles in stress responses. These findings demonstrate paraspeckles and paraspeckle-related proteins contribute to diverse functions during testis development and spermatogenesis.