Structural basis of the zinc-induced cytoplasmic aggregation of the RNA-binding protein SFPQ.

SFPQ is a ubiquitous nuclear RNA-binding protein implicated in many aspects of RNA biogenesis. Importantly, nuclear depletion and cytoplasmic accumulation of SFPQ has been linked to neuropathological conditions such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Here, we describe a molecular mechanism by which SFPQ is mislocalized to the cytoplasm. We report an unexpected discovery of the infinite polymerization of SFPQ that is induced by zinc binding to the protein. The crystal structure of human SFPQ in complex with zinc at 1.94 Å resolution reveals intermolecular interactions between SFPQ molecules that are mediated by zinc. As anticipated from the crystal structure, the application of zinc to primary cortical neurons induced the cytoplasmic accumulation and aggregation of SFPQ. Mutagenesis of the three zinc-coordinating histidine residues resulted in a significant reduction in the zinc-binding affinity of SFPQ in solution and the zinc-induced cytoplasmic aggregation of SFPQ in cultured neurons. Taken together, we propose that dysregulation of zinc availability and/or localization in neuronal cells may represent a mechanism for the imbalance in the nucleocytoplasmic distribution of SFPQ, which is an emerging hallmark of neurodegenerative diseases including AD and ALS.

Rapid purification method for human SFPQ by implementing zinc-induced polymerization.

Splicing factor proline- and glutamine-rich (SFPQ) is an RNA-binding protein, playing significant roles in gene regulation and subnuclear body formation. Our recent serendipitous discovery showed that SFPQ binds zinc directly and forms an infinite polymer that is induced by zinc binding to the protein. The zinc-induced reversible polymerization has led us to exploit this property to develop a rapid purification strategy for SFPQ without the use of affinity tags. In combination with the variation of ionic strength for salting-out of SFPQ, the reversible zinc-induced precipitation of SFPQ reduced the purification time required to obtain pure SFPQ to a single day. The purified protein was subjected to the previously reported crystallization condition. The resulting crystals diffracted to 2.22 Å resolution, confirming the quality of SFPQ purified with this new rapid purification strategy.

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.

SFPQ•NONO and XLF function separately and together to promote DNA double-strand break repair via canonical nonhomologous end joining.

A complex of two related mammalian proteins, SFPQ and NONO, promotes DNA double-strand break repair via the canonical nonhomologous end joining (c-NHEJ) pathway. However, its mechanism of action is not fully understood. Here we describe an improved SFPQ•NONO-dependent in vitro end joining assay. We use this system to demonstrate that the SFPQ•NONO complex substitutes in vitro for the core c-NHEJ factor, XLF. Results are consistent with a model where SFPQ•NONO promotes sequence-independent pairing of DNA substrates, albeit in a way that differs in detail from XLF. Although SFPQ•NONO and XLF function redundantly in vitro, shRNA-mediated knockdown experiments indicate that NONO and XLF are both required for efficient end joining and radioresistance in cell-based assays. In addition, knockdown of NONO sensitizes cells to the interstrand crosslinking agent, cisplatin, whereas knockdown of XLF does not, and indeed suppresses the effect of NONO deficiency. These findings suggest that each protein has one or more unique activities, in addition to the DNA pairing revealed in vitro, that contribute to DNA repair in the more complex cellular milieu. The SFPQ•NONO complex contains an RNA binding domain, and prior work has demonstrated diverse roles in RNA metabolism. It is thus plausible that the additional repair function of NONO, revealed in cell-based assays, could involve RNA interaction.

Splicing factor proline/glutamine-rich is a novel autoantigen of dermatomyositis and associated with anti-melanoma differentiation-associated gene 5 antibody.

OBJECTIVE: Anti-MDA5 antibody positive dermatomyositis (DM) and clinically amyopathic DM (CADM) often develop into rapidly progressive interstitial lung disease, but their pathogenesis remains unclear. We observed that sera from DM/CADM patients immunoprecipitated a common 110 kDa polypeptide. We investigated this autoantigen and its clinical significance. METHODS: Autoantibodies were screened in 333 patients with various connective tissue diseases (CTDs) and 20 healthy controls (HCs) by immunoprecipitation with [35S]methionine-labeled HeLa cells. Immunoabsorbent column chromatography was used to purify the reactive autoantigen which was subsequently analyzed by peptide mass fingerprinting. RESULTS: Anti-110 kDa antibody was detected in sera from 27 DM/CADM patients, but not in sera from other CTD patients or HCs. All patients with anti-110 kDa antibody had anti-MDA5 antibody. The maximum KL-6 levels in anti-110 kDa antibody-positive patients were higher than in anti-110 kDa antibody-negative patients, and all anti-MDA5-antibody-positive patients who showed the recurrence of DM/CADM were anti-110 kDa antibody-positive. The corresponding autoantigen was identified as splicing factor proline/glutamine-rich protein (SFPQ). In some cases, anti-SFPQ antibody was detected at diagnosis (early-detected group), but in other cases, it appeared during the disease course (delayed-detected group). The diagnosis timing of DM/CADM showed seasonal patterns according to the timing of anti-SFPQ antibody appearance. Specifically, 77% (10/13) of patients were diagnosed between August and October in the early-detected group, while 57% (8/14) of patients were diagnosed between January and March in the delayed-detected group. CONCLUSIONS: Some anti-MDA5 antibody-positive patients had an antibody to SFPQ, which is known to play a role in innate immune responses. Anti-SFPQ antibody may be involved in the chronic disease course of DM/CADM. The diagnosis timing of DM/CADM in anti-MDA5 antibody-positive patients showed seasonal patterns according to the timing of anti-SFPQ antibody appearance. These findings may provide new insights into the pathogenesis of DM/CADM.

SFPQ Is an FTO-Binding Protein that Facilitates the Demethylation Substrate Preference.

The fat mass and obesity-associated protein (FTO) is the first identified demethylase of the internal RNA modification N6-methyladenosine (m6A), which also exhibits demethylation activity toward N6,2'-O-dimethyladenosine (m6Am) and N1-methyladenosine (m1A). Demethylation of m6A at specific sites on target transcripts is a key enzymatic function of FTO that modulates diverse physiological and/or pathological processes. However, how FTO selects target RNA and whether additional interaction proteins facilitate this process remain elusive. Herein, via the genetically encoded and site-specific photocrosslinking strategy, we identified the major RNA-binding protein SFPQ as a direct interaction partner of FTO. Our study showed that FTO and SFPQ were located in close proximity throughout the transcriptome and that overexpression of SFPQ led to the demethylation of adjacent m6As, likely through recruiting FTO to these specific RNA sites. These results uncovered a new layer of regulation mechanism that may assist FTO to gain substrate specificity.

A new crystal structure and small-angle X-ray scattering analysis of the homodimer of human SFPQ.

Splicing factor proline/glutamine-rich (SFPQ) is an essential RNA-binding protein that is implicated in many aspects of nuclear function. The structures of SFPQ and two paralogs, non-POU domain-containing octamer-binding protein and paraspeckle component 1, from the Drosophila behavior human splicing protein family have previously been characterized. The unusual arrangement of the four domains, two RNA-recognition motifs (RRMs), a conserved region termed the NonA/paraspeckle (NOPS) domain and a C-terminal coiled coil, in the intertwined dimer provides a potentially unique RNA-binding surface. However, the molecular details of how the four RRMs in the dimeric SFPQ interact with RNA remain to be characterized. Here, a new crystal structure of the dimerization domain of human SFPQ in the C-centered orthorhombic space group C2221 with one monomer in the asymmetric unit is presented. Comparison of the new crystal structure with the previously reported structure of SFPQ and analysis of the solution small-angle X-scattering data revealed subtle domain movements in the dimerization domain of SFPQ, supporting the concept of multiple conformations of SFPQ in equilibrium in solution. The domain movement of RRM1, in particular, may reflect the complexity of the RNA substrates of SFPQ. Taken together, the crystal and solution structure analyses provide a molecular basis for further investigation into the plasticity of nucleic acid binding by SFPQ in the absence of the structure in complex with its cognate RNA-binding partners.

Vault RNAs partially induces drug resistance of human tumor cells MCF-7 by binding to the RNA/DNA-binding protein PSF and inducing oncogene GAGE6.

BACKGROUND: Vault is the largest nonicosahedral cytosolic nucleoprotein particle, which is widely involved in induction of chemoresistance and lead to failure in long-term chemotherapy. Vault contains three different major vault proteins (MVPs) and four vault RNAs paralogues (vtRNAs, vtRNA1-1, vtRNA1-2, vtRNA1-3 and vtRNA2-1). Disruption of the MVPs do not induce hypersensitivity while expression of vtRNAs contributes to cells' drug resistance, indicates that vtRNAs, but not MVPs play an important role in causing drug resistance. Polypyrimidine tract binding protein associated splicing factor (PSF) contributes to cell sensitivity to chemotherapy by its transcriptional activity, promotes us to figure out its potential association with vtRNAs. METHODS: We investigate the interaction between PSF and vtRNAs by electrophoretic mobility shift assays (EMSA) and RNA-immunoprecipitation (IP), and showed the binding between PSF and vtRNAs. Chromatin Immunoprecipitation (ChIP) was performed to detect the effects of vtRNAs on the interaction of PSF with GAGE6 promoter. The role of vtRNAs on chemoresistance in MCF-7 was detected by CCK-8 and EdU staining. The independent role of vtRNAs with MVP is detected by MVP or vtRNAs knockdown. RESULTS: The complex with vtRNA1-1 releases PSF, allowing transcription of GAGE6 to proceed. Then we showed that induction of GAGE6 caused drug resistance by promoting cell proliferation and colony formation in soft agar. Ectopic expression of shRNA targets to vtRNA1-1 further confirmed the role of vtRNA1-1 in regulating PSF transcriptional activity independent with the expression of MVP. By vtRNA1-1 or MVP knockdown, it is revealed that vtRNA1-1 caused chemoresistance independent of MVP. Furthermore, knockdown of GAGE6 does not cause drug resistance, indicates the GAGE6 is directly involved in cell proliferation, but not the drug resistance. CONCLUSION: These results suggest that vtRNAs regulates cell proliferation, drug resistance, and possibly other physiological processes of humans, by complex formation with PSF.