Paraspeckle subnuclear bodies depend on dynamic heterodimerisation of DBHS RNA-binding proteins via their structured domains
2022; Elsevier BV; Volume: 298; Issue: 11 Linguagem: Inglês
10.1016/j.jbc.2022.102563
ISSN1083-351X
AutoresPei Wen Lee, A.C. Marshall, Gavin J. Knott, Simon Kobelke, Luciano G. Martelotto, Hyun‐Jung Cho, Paul J. McMillan, Mihwa Lee, Charles S. Bond, Archa H. Fox,
Tópico(s)RNA and protein synthesis mechanisms
ResumoRNA-binding proteins of the DBHS (Drosophila Behavior Human Splicing) family, NONO, SFPQ, and PSPC1 have numerous roles in genome stability and transcriptional and posttranscriptional regulation. Critical to DBHS activity is their recruitment to distinct subnuclear locations, for example, paraspeckle condensates, where DBHS proteins bind to the long noncoding RNA NEAT1 in the first essential step in paraspeckle formation. To carry out their diverse roles, DBHS proteins form homodimers and heterodimers, but how this dimerization influences DBHS localization and function is unknown. Here, we present an inducible GFP-NONO stable cell line and use it for live-cell 3D-structured illumination microscopy, revealing paraspeckles with dynamic, twisted elongated structures. Using siRNA knockdowns, we show these labeled paraspeckles consist of GFP-NONO/endogenous SFPQ dimers and that GFP-NONO localization to paraspeckles depends on endogenous SFPQ. Using purified proteins, we confirm that partner swapping between NONO and SFPQ occurs readily in vitro. Crystallographic analysis of the NONO-SFPQ heterodimer reveals conformational differences to the other DBHS dimer structures, which may contribute to partner preference, RNA specificity, and subnuclear localization. Thus overall, our study suggests heterodimer partner availability is crucial for NONO subnuclear distribution and helps explain the complexity of both DBHS protein and paraspeckle dynamics through imaging and structural approaches. RNA-binding proteins of the DBHS (Drosophila Behavior Human Splicing) family, NONO, SFPQ, and PSPC1 have numerous roles in genome stability and transcriptional and posttranscriptional regulation. Critical to DBHS activity is their recruitment to distinct subnuclear locations, for example, paraspeckle condensates, where DBHS proteins bind to the long noncoding RNA NEAT1 in the first essential step in paraspeckle formation. To carry out their diverse roles, DBHS proteins form homodimers and heterodimers, but how this dimerization influences DBHS localization and function is unknown. Here, we present an inducible GFP-NONO stable cell line and use it for live-cell 3D-structured illumination microscopy, revealing paraspeckles with dynamic, twisted elongated structures. Using siRNA knockdowns, we show these labeled paraspeckles consist of GFP-NONO/endogenous SFPQ dimers and that GFP-NONO localization to paraspeckles depends on endogenous SFPQ. Using purified proteins, we confirm that partner swapping between NONO and SFPQ occurs readily in vitro. Crystallographic analysis of the NONO-SFPQ heterodimer reveals conformational differences to the other DBHS dimer structures, which may contribute to partner preference, RNA specificity, and subnuclear localization. Thus overall, our study suggests heterodimer partner availability is crucial for NONO subnuclear distribution and helps explain the complexity of both DBHS protein and paraspeckle dynamics through imaging and structural approaches. NONO (Non-POU domain-containing octamer-binding protein), SFPQ (splicing factor proline and glutamine rich), and PSPC1 (paraspeckle component protein 1) comprise the three proteins of the DBHS (Drosophila behavior human splicing) protein family in complex vertebrates. NONO, SFPQ, and PSPC1 share 50% sequence identity and typically exist as dimers (1Myojin R. Kuwahara S. Yasaki T. Matsunaga T. Sakurai T. Kimura M. et al.Expression and functional significance of mouse paraspeckle protein 1 on Spermatogenesis1.Biol. Reprod. 2004; 71: 926-932Crossref PubMed Scopus (36) Google Scholar, 2Fox A.H. Bond C.S. Lamond A.I. P54nrb forms a heterodimer with PSP1 that localizes to paraspeckles in an.Mol. Biol. Cell. 2005; 16: 5304-5315Crossref PubMed Scopus (188) Google Scholar, 3Passon D.M. Lee M. Fox A.H. Bond C.S. Crystallization of a paraspeckle protein PSPC1-NONO heterodimer.Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2011; 67: 1231-1234Crossref PubMed Scopus (15) Google Scholar) (Fig. 1). The core conserved region of DBHS proteins is composed of tandem RNA recognition motif (RRM) domains, a NOPS (NONA/ParaSpeckle) domain (an Interpro defined domain that is only found in this protein family), and an extended coiled-coil and is responsible for dimerization and interaction with RNA (4Knott G.J. Bond C.S. Fox A.H. The DBHS proteins SFPQ, NONO and PSPC1: a multipurpose molecular scaffold.Nucl. Acids Res. 2016; 44: 3989-4004Crossref PubMed Scopus (170) Google Scholar). 3D atomic structures of this core region have revealed an extensive dimer interface between monomers, supporting the notion that these proteins are obligate dimers (3Passon D.M. Lee M. Fox A.H. Bond C.S. Crystallization of a paraspeckle protein PSPC1-NONO heterodimer.Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2011; 67: 1231-1234Crossref PubMed Scopus (15) Google Scholar, 5Knott G.J. Chong Y.S. Passon D.M. Liang X. Deplazes E. Conte M.R. et al.Structural basis of dimerization and nucleic acid binding of human DBHS proteins NONO and PSPC1.Nucl. Acids Res. 2022; 50: 522-535Crossref PubMed Scopus (8) Google Scholar, 6Lee M. Sadowska A. Bekere I. Ho D. Gully B.S. Lu Y. et al.The structure of human SFPQ reveals a coiled-coil mediated polymer essential for functional aggregation in gene regulation.Nucl. Acids Res. 2015; 43: 3826-3840Crossref PubMed Scopus (97) Google Scholar, 7Huang J. Casas Garcia G.P. Perugini M.A. Fox A.H. Bond C.S. Lee M. Crystal structure of a SFPQ/PSPC1 heterodimer provides insights into preferential heterodimerization of human DBHS family proteins.J. Biol. Chem. 2018; 293: 6593-6602Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In HeLa cells, the majority of DBHS proteins are NONO-SFPQ heterodimers, with a smaller pool of NONO-PSPC1 heterodimers (2Fox A.H. Bond C.S. Lamond A.I. P54nrb forms a heterodimer with PSP1 that localizes to paraspeckles in an.Mol. Biol. Cell. 2005; 16: 5304-5315Crossref PubMed Scopus (188) Google Scholar). However, little is known about the structural and dynamic constraints of different partner choice for DBHS heterodimers and the subsequent functional roles of different DBHS dimers. DBHS proteins are implicated in numerous aspects of gene regulation and expression, such as transcriptional regulation, splicing regulation, RNA transport, pri-miRNA processing, miRNA targeting, and DNA repair, with these functions correlated with their presence at different subnuclear and subcellular locations (reviewed in (4Knott G.J. Bond C.S. Fox A.H. The DBHS proteins SFPQ, NONO and PSPC1: a multipurpose molecular scaffold.Nucl. Acids Res. 2016; 44: 3989-4004Crossref PubMed Scopus (170) Google Scholar)). One such example of DBHS function in the nucleus is their essential role in the formation of subnuclear bodies termed paraspeckles (8Fox A.H. Lam Y.W. Leung A.K.L. Lyon C.E. Andersen J. Mann M. et al.Paraspeckles: a novel nuclear domain.Curr. Biol. 2002; 12: 13-25Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). The structural backbone of paraspeckles is a long noncoding RNA, named NEAT1 (Nuclear Paraspeckle Assembly Transcript 1) (9Clemson C.M. Hutchinson J.N. Sara S.A. Ensminger A.W. Fox A.H. Chess A. et al.An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure of paraspeckles.Mol. Cell. 2009; 33: 717-726Abstract Full Text Full Text PDF PubMed Scopus (1089) Google Scholar, 10Sasaki Y.T.F. Ideue T. Sano M. Mituyama T. Hirose T. MENε/β noncoding RNAs are essential for structural integrity of nuclear paraspeckles.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 2525-2530Crossref PubMed Scopus (470) Google Scholar, 11Chen L.-L. Carmichael G.G. Altered nuclear retention of mRNAs containing inverted repeats in human embryonic stem cells: functional role of a nuclear noncoding RNA.Mol. Cell. 2009; 35: 467-478Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). Upon NEAT1 transcription, DBHS proteins bind the RNA, extensively coating it through oligomerization, conferring increased stability to the RNA and forming a ribonucleoprotein (RNP) particle (6Lee M. Sadowska A. Bekere I. Ho D. Gully B.S. Lu Y. et al.The structure of human SFPQ reveals a coiled-coil mediated polymer essential for functional aggregation in gene regulation.Nucl. Acids Res. 2015; 43: 3826-3840Crossref PubMed Scopus (97) Google Scholar, 12Yamazaki T. Hirose T. The building process of the functional paraspeckle with long non-coding RNAs.Front. Biosci. Elite. 2015; 7: 1-47Crossref PubMed Scopus (20) Google Scholar). Approximately, 50 NEAT1-DBHS RNPs are then arranged into a single mature paraspeckle via liquid-liquid phase separation (LLPS) mediated by the low complexity region of the accessory protein FUS, as well as recruitment of over 40 additional RNA-binding proteins to the paraspeckle (13Naganuma T. Nakagawa S. Tanigawa A. Sasaki Y.F. Goshima N. Hirose T. Alternative 3'-end processing of long noncoding RNA initiates construction of nuclear paraspeckles.EMBO J. 2012; 31: 4020-4034Crossref PubMed Scopus (305) Google Scholar, 14West J.A. Mito M. Kurosaka S. Takumi T. Tanegashima C. Chujo T. et al.Structural, super-resolution microscopy analysis of paraspeckle nuclear body organization.J. Cell Biol. 2016; 214: 817-830Crossref PubMed Scopus (199) Google Scholar). Paraspeckles are found in various mammalian cultured cells and tissues, including primary and transformed cell lines (8Fox A.H. Lam Y.W. Leung A.K.L. Lyon C.E. Andersen J. Mann M. et al.Paraspeckles: a novel nuclear domain.Curr. Biol. 2002; 12: 13-25Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 15Sunwoo H. Dinger M.E. Wilusz J.E. Amaral P.P. Mattick J.S. Spector D.L. MEN ε/β nuclear-retained non-coding RNAs are up-regulated upon muscle differentiation and are essential components of paraspeckles.Genome Res. 2009; 19: 347-359Crossref PubMed Scopus (520) Google Scholar, 16Prasanth K.V. Prasanth S.G. Xuan Z. Hearn S. Freier S.M. Bennett C.F. et al.Regulating gene expression through RNA nuclear retention.Cell. 2005; 123: 249-263Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar), with the exception of embryonic stem cells (11Chen L.-L. Carmichael G.G. Altered nuclear retention of mRNAs containing inverted repeats in human embryonic stem cells: functional role of a nuclear noncoding RNA.Mol. Cell. 2009; 35: 467-478Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). Organization of proteins into paraspeckles can enhance the efficiency of paraspeckle protein functions in pri-miRNA RNA processing (17Jiang L. Shao C. Wu Q.-J. Chen G. Zhou J. Yang B. et al.NEAT1 scaffolds RNA-binding proteins and the microprocessor to globally enhance pri-miRNA processing.Nat. Struct. Mol. Biol. 2017; 24: 816-824Crossref PubMed Scopus (141) Google Scholar). A variety of cellular stresses, including hypoxia and viral infection, lead to an increase in paraspeckle size and abundance, and this paraspeckle induction correlates with cancer progression of some cancer types (18McCluggage F. Fox A.H. Paraspeckle nuclear condensates: global sensors of cell stress?.Bioessays. 2021; 432000245Crossref PubMed Scopus (31) Google Scholar). Recently paraspeckles were mapped at the ultrastructural level in fixed cells using super-resolution structured illumination microscopy (SIM), revealing an unusual core-shell molecular organization (14West J.A. Mito M. Kurosaka S. Takumi T. Tanegashima C. Chujo T. et al.Structural, super-resolution microscopy analysis of paraspeckle nuclear body organization.J. Cell Biol. 2016; 214: 817-830Crossref PubMed Scopus (199) Google Scholar). FISH against different parts of NEAT1, combined with immunofluorescence against paraspeckle proteins revealed the central part of the 23 kb NEAT1 RNA is found in the core of the paraspeckle, along with SFPQ, NONO, PSPC1, and FUS. In contrast, the 5′ and 3′ ends of NEAT1 and TARDBP proteins are found in the shell of the paraspeckle. Several other proteins (RBM14 and BRG1) bridge the two zones. Paraspeckles can either be single, approximately spherical units of 360 nm diameter, or chains of paraspeckle units, with a constant 360 nm width and varying length, up to 2 microns (14West J.A. Mito M. Kurosaka S. Takumi T. Tanegashima C. Chujo T. et al.Structural, super-resolution microscopy analysis of paraspeckle nuclear body organization.J. Cell Biol. 2016; 214: 817-830Crossref PubMed Scopus (199) Google Scholar, 19Souquere S. Beauclair G. Harper F. Fox A. Pierron G. Highly ordered spatial organization of the structural long noncoding NEAT1 RNAs within paraspeckle nuclear bodies.Mol. Biol. Cell. 2010; 21: 4020-4027Crossref PubMed Scopus (162) Google Scholar, 20Wang Y. Hu S.-B. Wang M.-R. Yao R.-W. Wu D. Yang L. et al.Genome-wide screening of NEAT1 regulators reveals cross-regulation between paraspeckles and mitochondria.Nat. Cell Biol. 2018; 20: 1145-1158Crossref PubMed Scopus (106) Google Scholar). Given that paraspeckles are very dynamic structures, forming rapidly in response to stress and disassembling just as rapidly under different conditions (indeed in less than 30 min in some cases (21Mao Y.S. Sunwoo H. Zhang B. Spector D.L. Direct visualization of the Co-transcriptional assembly of a nuclear body by noncoding RNAs.Nat. Cell Biol. 2011; 13: 95-101Crossref PubMed Scopus (373) Google Scholar)), it is important to complement these fixed cell super resolution observations with live cell studies. In this study, we have used cellular paraspeckle imaging experiments and in vitro methods to characterize DBHS protein dimers. We generated a HeLa stable cell line with inducible GFP-NONO to examine its targeting to paraspeckles and perform live cell 3D-SIM imaging of NONO-labeled paraspeckles, revealing how they twist and form in real time. We show that NEAT1 and SFPQ are both involved in targeting NONO to paraspeckles, but to different extents, revealing a critical role for SFPQ in targeting the NONO-SFPQ heterodimer to distinct nuclear locations. Given the key role of the NONO-SFPQ heterodimer, we also perform in vitro studies to show rapid partner swapping of NONO and SFPQ homodimers, with a preference for the heterodimer state. Finally, we describe the NONO-SFPQ crystal structure and how key differences could explain the heterodimerization preference. Of the three DBHS proteins, PSPC1, was named Paraspeckle Protein Component 1, as it was the first paraspeckle marker protein and it has been extensively used in prior live cell paraspeckle studies (8Fox A.H. Lam Y.W. Leung A.K.L. Lyon C.E. Andersen J. Mann M. et al.Paraspeckles: a novel nuclear domain.Curr. Biol. 2002; 12: 13-25Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). However, as PSPC1 is in fact dispensable for paraspeckle formation, we generated a tool for live cell imaging of paraspeckles with fluorescently tagged NONO, an essential paraspeckle protein component. We therefore generated a HeLaGFP-NONO stable cell line that constitutively expresses the Tet-On 3G transactivator and contains a genomically integrated PTRE3G–tagGFP–NONO plasmid (hereafter termed GFP-NONO) that expresses GFP-NONO only in the presence of Doxycycline (Dox, Fig. 2A). Dox concentrations above 25 ng/ml induced an appropriately low level of exogenous GFP-NONO that was consistently less abundant than that of the endogenous NONO (Fig. 2B). Furthermore, the expression level of endogenous NONO was unaffected by the small and stable expression of GFP-NONO (Fig. 2B). As a result, we used 50 ng/ml Dox for the remainder of the study. Of note, extending the duration of Dox treatment to 48 h did not result in excess GFP-NONO nor adverse autoregulatory effects on endogenous NONO levels (Fig. 2C). To observe the stability of GFP-NONO, we induced expression for 24 h, then analyzed NONO levels over the following 48 h, observing a detectable amount of GFP-NONO 24 h after Dox removal but not after 48 h (Fig. 2D). We next verified that the induced GFP-NONO was appropriate for use as a clear marker for paraspeckles. Fixed and induced HeLaGFP-NONO cells displayed bright and distinct subnuclear puncta, as marked by the green fluorescence of GFP-NONO (Fig. 2E, arrows). The identity of paraspeckles was confirmed by colocalization of endogenous NONO (Fig. 2, F and G, left panels) with FISH against the long noncoding RNA, NEAT1 (Fig. 2, F and G, arrows show paraspeckles, Fig. S2 shows additional representative images). We next tested if the Dox-induced GFP-NONO displayed similar behavior to endogenous NONO with respect to colocalization with NEAT1 in paraspeckles. Figure 2, F and G, right panels show that, indeed, as with endogenous NONO, the low levels of Dox-induced GFP-NONO also colocalize with NEAT1 in an identical manner. Line scans through individual paraspeckles confirm no change in localization behavior between endogenous NONO (Fig. 2G, green, left) and GFP-NONO (Fig. 2G, green, right) with respect to paraspeckles/NEAT1 colocalization (Fig. 2G, red). 3D-SIM imaging of the samples also showed clear colocation of GFP-NONO and NEAT1 (volume view in Fig. S1). Taken together, the generated HeLaGFP-NONO cells were confirmed as an appropriate tool for studying paraspeckles in living cells, as they express low levels of exogenous GFP-NONO marking paraspeckles, as defined by colocalization with NEAT1. Given the many roles and nuclear locations of DBHS proteins, we sought to examine the temporal localization of GFP-NONO after its translation in the cytosol prior to its recruitment to paraspeckles. Live cell time-lapse fluorescence imaging of HeLaGFP-NONO cells was performed to observe localization changes of nascent GFP-NONO over a period of 24 h upon induction with Dox (Fig. 3A). Cells were counterstained with Hoechst 33342 nucleic acid dye to easily mark nuclei prior to the experiment. Images were taken every 30 min and fluorescence from GFP-NONO was first detected at the 12th hour of induction, followed by increasing fluorescence signal over time (Fig. 3, A and B, arrows show paraspeckles). The first detectable GFP-NONO fluorescence appears to be within paraspeckles, and with continued induction with Dox, the emerging fluorescently green nuclear foci persisted, enlarged, and increased in numbers over time. At the 18th hour of induction, bright and distinct paraspeckles were readily visible, hence indicating that 18 h of induction is sufficient for expressing the amount of GFP-NONO that can provide for clear observation of paraspeckle localization and behavior. Further induction till the 24th hour gave bright and distinct paraspeckles that are reminiscent of the ones obtained previously (Fig. 2, E and F). Thus, nascent GFP-NONO molecules directly accumulate at paraspeckles, without first transiting or becoming enriched in any other clearly discernible subcellular structure. Since GFP-NONO has been proved to be a reliable marker for paraspeckles, it was of interest to observe the enlargement of paraspeckles over time. Proteasome inhibition via treatment with the drug MG132 can induce enlargement and elongation of paraspeckles (22Hirose T. Virnicchi G. Tanigawa A. Naganuma T. Li R. Kimura H. et al.NEAT1 long noncoding RNA regulates transcription via protein sequestration within subnuclear bodies.Mol. Biol. Cell. 2014; 25: 169-183Crossref PubMed Scopus (322) Google Scholar). To observe any MG132-mediated changes in appearance of GFP-NONO–labeled paraspeckles over time, we performed time-lapse fluorescence imaging of Dox-induced HeLaGFP-NONO cells over a period of 12 h upon treatment with 1 μM of MG132 (Fig. 3C). Marked enlargement of paraspeckles was observed, although at this resolution, it was not possible to distinguish elongation from increased paraspeckle numbers (Fig. 3, C and D). To gain deeper insights into the growth, elongation and formation of paraspeckles, we performed 3D-SIM time-lapse fluorescence imaging on MG132-treated cells over a period of 2 h, according to the timeline illustrated in Figure 4A. Due to the larger number of exposures required to reconstruct a 3D-SIM dataset, the number of images acquired was minimized to reduce phototoxicity and photobleaching. This enhanced visualization of paraspeckles in 3D allowed us to see, for the first time, how paraspeckles can twist and grow over time. Figure 4B shows a representative MG132-treated cell captured over time with 3D-SIM. Unexpectedly, apparent MG132-mediated elongation of paraspeckles could not be observed in this instance, perhaps due to the short time course of imaging. However, several cells revealed the initial appearance of newly formed GFP-NONO–labeled paraspeckles followed by their gradual growth and enlargement through the rest of the live-imaging experiment (Fig. 4B, arrow shows newly formed paraspeckle). With 3D rendering as shown in Figure 4C, visualizations of newly formed paraspeckles (in yellow) and an adjacent existing paraspeckle (in green) were obtained. Imaging of additional live cells allowed the monitoring of changes in individual paraspeckles over the 2 h time course (Fig. S3). Overall, this imaging reveals the extent with which individual paraspeckles change shape and conformation in this short time frame. These studies reveal the dynamics of paraspeckle growth in line with their role as immediate stress responsive structures. We next examined how the individual ablation of the key NONO-associated molecules, NEAT1, SFPQ, and PSPC1, using siRNAs, would alter the localization of GFP-NONO. To validate effective siRNA-mediated knockdown (KD) of SFPQ or PSPC1, Western Blotting was conducted, confirming markedly decreased expression of each protein in HeLaGFP-NONO cells treated with the respective siRNAs (Fig. 5C). Real-time quantitative PCR performed on HeLaGFP-NONO cells treated with NEAT1 siRNA showed more than 70% efficiency in knocking down NEAT1 (Fig. 5D). The siRNA-mediated resultant changes in GFP-NONO localization were examined via microscopy on fixed induced cells. In line with previously reported studies (10Sasaki Y.T.F. Ideue T. Sano M. Mituyama T. Hirose T. MENε/β noncoding RNAs are essential for structural integrity of nuclear paraspeckles.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 2525-2530Crossref PubMed Scopus (470) Google Scholar), cells treated with NEAT1 siRNA showed loss of paraspeckles as observed by NEAT1 FISH (Fig. 5A, central panel), although it should be noted that some residual NEAT1 foci were sometimes apparent in the NEAT1 siRNA-treated cells, but these foci were markedly reduced in size compared with control cells (Fig. 5A). When NEAT1 was knocked down, the background GFP-NONO fluorescence within the nucleus also increased relative to control cells (Fig. 5A), with a diffuse distribution of nuclear GFP-NONO. Thus, ablation of NEAT1 and paraspeckles by NEAT1 siRNA resulted in redistribution of GFP-NONO to the rest of the nucleoplasm. It was previously shown that SFPQ KD results in reduced levels of NEAT1 RNA and the disassembly of paraspeckles (10Sasaki Y.T.F. Ideue T. Sano M. Mituyama T. Hirose T. MENε/β noncoding RNAs are essential for structural integrity of nuclear paraspeckles.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 2525-2530Crossref PubMed Scopus (470) Google Scholar). KD of SFPQ in the HeLaGFP-NONO cells indeed reduced paraspeckles, as seen by diminished NEAT1 foci (Fig. 5A central panel). However, in the absence of both SFPQ and paraspeckles, GFP-NONO exhibited a distinctly different localization compared to paraspeckle reduction alone; in this case, numerous NONO-labeled nuclear aggregates were observed (Fig. 5A). The randomly assorted GFP-NONO foci are more numerous (Fig. 5B, upper plot) and smaller than the GFP-NONO paraspeckles observed in the scrambled control (Fig. 5B, lower plot). Finally, PSPC1 KD had little effect on GFP-NONO localization either inside or outside paraspeckles, as GFP-NONO localization was unchanged in the absence of PSPC1 (Fig. 5A). Given the altered localization of NONO in the absence of SFPQ, but not PSPC1 (Fig. 5A), we wondered if this was indicative of dynamic switching of DBHS proteins into different dimer configurations that then influenced subnuclear localization. Of note, recombinant SFPQ and NONO core DBHS regions expressed in Escherichia coli purify as heterodimers in vitro (6Lee M. Sadowska A. Bekere I. Ho D. Gully B.S. Lu Y. et al.The structure of human SFPQ reveals a coiled-coil mediated polymer essential for functional aggregation in gene regulation.Nucl. Acids Res. 2015; 43: 3826-3840Crossref PubMed Scopus (97) Google Scholar), suggesting that a heterodimer of NONO-SFPQ is the more favored state over either homodimeric SFPQ or NONO. In agreement with this, analytical ultracentrifugation experiments show that the apparent dissociation constant of this NONO-SFPQ heterodimer is ∼3-fold lower than that of the SFPQ homodimer (7Huang J. Casas Garcia G.P. Perugini M.A. Fox A.H. Bond C.S. Lee M. Crystal structure of a SFPQ/PSPC1 heterodimer provides insights into preferential heterodimerization of human DBHS family proteins.J. Biol. Chem. 2018; 293: 6593-6602Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). To investigate this further, we purified homodimeric hexahistidine-tagged SFPQ (H6SFPQ) and untagged homodimeric NONO to test for their ability to swap dimerization partner in vitro (Fig. 6). In nickel-affinity pull-down experiments, we observed that when 1:1 mixtures of the two homodimers were loaded onto a column, it was the heterodimer that eluted (Fig. 6A). We then characterized a complex of SFPQ and a maltose-binding protein fusion of NONO (MBP-NONO) using analytical size-exclusion chromatography (Fig. 6B). The complex of SFPQ and MBP-NONO elutes between the two homodimeric peaks, confirming that they are able to readily exchange partner to form a stable heterodimer. Thus, incubating SFPQ and NONO homodimers shows DBHS proteins rapidly exchange dimerization partner in vitro. Taken together, these data could be used to support the notion that the NONO-SFPQ heterodimer is the favored combination both in vitro and inside cells. While 3D atomic structures for the three human DBHS protein homodimers (Protein Data Bank [PDB] IDs 4wii, 5ifm, 5ifn) and two heterodimers, NONO-PSPC1 (3sde) and SFPQ-PSPC1 (5wpa), have been published previously (3Passon D.M. Lee M. Fox A.H. Bond C.S. Crystallization of a paraspeckle protein PSPC1-NONO heterodimer.Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2011; 67: 1231-1234Crossref PubMed Scopus (15) Google Scholar, 6Lee M. Sadowska A. Bekere I. Ho D. Gully B.S. Lu Y. et al.The structure of human SFPQ reveals a coiled-coil mediated polymer essential for functional aggregation in gene regulation.Nucl. Acids Res. 2015; 43: 3826-3840Crossref PubMed Scopus (97) Google Scholar, 7Huang J. Casas Garcia G.P. Perugini M.A. Fox A.H. Bond C.S. Lee M. Crystal structure of a SFPQ/PSPC1 heterodimer provides insights into preferential heterodimerization of human DBHS family proteins.J. Biol. Chem. 2018; 293: 6593-6602Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 23Knott G.J. Lee M. Passon D.M. Fox A.H. Bond C.S. Caenorhabditis elegans NONO-1: insights into DBHS protein structure, architecture, and function.Protein Sci. 2015; 24: 2033-2043Crossref PubMed Scopus (18) Google Scholar), the predominance of the NONO-SFPQ heterodimer in cells and its essential role in paraspeckle formation prompted us to determine its structure, completing the repertoire of structures for all six human DBHS dimer combinations. The X-ray crystal structure of a heterodimer containing the core DBHS region of SFPQ (residues 276–535) and NONO (residues 53–312) was determined by molecular replacement using data to 2.3 Å in space group P3121 (7lrq; Table S1). The asymmetric unit contains a single heterodimer with 2-fold noncrystallographic rotational symmetry, as described for the other two DBHS heterodimers (3Passon D.M. Lee M. Fox A.H. Bond C.S. Crystallization of a paraspeckle protein PSPC1-NONO heterodimer.Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2011; 67: 1231-1234Crossref PubMed Scopus (15) Google Scholar, 7Huang J. Casas Garcia G.P. Perugini M.A. Fox A.H. Bond C.S. Lee M. Crystal structure of a SFPQ/PSPC1 heterodimer provides insights into preferential heterodimerization of human DBHS family proteins.J. Biol. Chem. 2018; 293: 6593-6602Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) (Fig. 6, C–E). The extensive dimerization interface is composed of interactions between the RRM2 domain of one partner and the NOPS domain of the other, which wraps around the outside of its respective partner RRM2, followed by an antiparallel right-handed coiled-coil formed by the α6 helices at the C terminus of each partner. In addition, the RRM1 domains pack against one another via their α2 helices—an interaction likely involved in determining their relative orientations. We compared the NONO-SFPQ structure with the NONO homodimer (PDB 5ifm) and the NONO-PSPC1 heterodimer (PDB 3sde) and noted that although the overall domain architecture is the same, superposition highlights conformational differences in the coiled-coil, NOPS domain, and RRM1 domain, which may contribute to their partner preference, RNA specificity, and subnuclear localization. Previous studies suggest structural plasticity in the NOPS domain and coiled-coil part of the dimerization domain is associated with conformational changes in specific residues at the dimer interface. This structural plasticity is readily apparent in the crystal of the NONO homodimer, where six independent NONO homodimers were observed (5Knott G.J. Chong Y.S. Passon D.M. Liang X. Deplazes E. Conte M.R. et al.Structural basis of dimerization and nucleic acid binding of human DBHS proteins NONO and PSPC1.Nucl. Acids Res. 2022; 50: 522-535Crossref PubMed Scopus (8) Google Scholar), and is also indicated fr
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