RCOR2 is compartmentalized at nuclear speckles
While examining published RCOR2 immunofluorescence studies performed on murine brain slices or neuronal primary cultures, we noticed an intriguing subcellular distribution of RCOR2 showing a nuclear punctate pattern [10, 11]. We wondered whether this nuclear distribution could be detected in different cell lines. After testing different antibodies, we selected a Prestige-validated, Protein-Atlas recommended, anti-RCOR2 antibody (Millipore-Sigma, #HPA021638) since when validating its specificity, it preferentially detected a band with the predicted RCOR2 molecular weight (Additional file 1: Fig. S1A) and recognized overexpressed Myc-tagged RCOR2 both by Western blot (Additional file 1: Fig. S1B) and immunofluorescence (Additional file 1: Fig. S1C). In addition, its immunofluorescent signal disappeared when pre-incubated with its peptide antigen, but not with a non-specific histone H3 peptide (Additional file 1: Fig. S1D). Furthermore, it detected the loss of endogenous RCOR2 when performing knock down experiments both by Western blot and immunofluorescence (Additional file 1: Fig. S1E and F).
We therefore carried out RCOR2 immunostaining on PC-12, N2A, HEK293T, HT22, MEF and mESC cells. In all cases, a nuclear enrichment was observed with different ratios of clustered and dispersed nucleoplasmic patterns (Fig. 1A), confirming that RCOR2 is distributed in the nucleus as puncta with differences in size and abundance depending on the cell type. This observation raised the possibility that RCOR2 may be recruited to some type of nuclear body or chromatin condensate. To test this, we carried out double immunofluorescence labeling of RCOR2 and protein markers for nucleoli (nucleophosmin), Cajal bodies (coilin), pericentric heterochromatin (Heterochromatin protein 1α, HP1α; and H3K9me3). To label nuclear speckles, we used a monoclonal antibody originally described and widely used to recognize the SC35/SRSF2 protein. However, it was recently shown that the actual target of this antibody corresponds to the serine arginine repetitive matrix protein 2 (SRRM2) and it can also detect serine and arginine splicing factor 7 (SRSF7), two bona fide nuclear speckle residents [14].
As expected, RCOR2 showed an intranuclear punctate distribution, enriched at the interchromatin space since it was excluded from regions with intense Hoechst staining in several cell types (Fig. 1A, B). RCOR2 puncta were excluded from nuclear territories occupied by nucleoli, Cajal bodies, and chromocenters (pericentric heterochromatin), as showed by nucleophosmin, coilin, and H3K9me3/HP1α co-staining, respectively (Fig. 1B–E). Quantitation of RCOR2 colocalization with these nuclear bodies showed less than 8.5% of its fluorescence signal overlapping them (Fig. 1G), and no correlation between their fluorescence intensity profiles (Fig. 1B–E). Conversely, when we analyzed the co-staining between RCOR2 and nuclear speckles by SRRM2 immunofluorescence, we found a strong correlation of their intensities and about 74% of RCOR2 signals overlapping SRRM2 territories in HT22 cells (Fig. 1F, G). We also observed that transiently expressed HA-RCOR2 mimicked the punctate pattern in HeLa cells (Fig. 1H). Furthermore, this localization was specific for this member of the RCOR family, as RCOR1 did not exhibit this pattern (Fig. 1I). Overall, our data showed that a large fraction of RCOR2 localizes to nuclear speckles.
RCOR2 is recruited to nuclear speckles in the mouse brain
We wondered next whether RCOR2 recruitment to nuclear speckles could be a broader phenomenon. We analyzed the RCOR2/SRRM2 colocalization in different cell types. While nuclear speckle particles looked similar both in HEK293T cells and PC12 cells, the former depicted RCOR2 puncta whose particles were smaller and more disperse than in PC12 cells (Fig. 2A, B). Notably, RCOR2 recruitment to nuclear speckles was much stronger in PC12 than in HEK293T cells, since colocalization with SRRM2 was only partial on the latter, suggesting its recruitment is a cell type dependent process. Nevertheless, RCOR2-FLAG overexpression on PC12 cells imitated the endogenous RCOR2 distribution whose puncta colocalized with SRRM2 (Fig. 2C). This colocalization of RCOR2 and SRRM2 was also observed in mouse brain, as detected in mouse striatum tissue slices (Fig. 2D and Additional file 5: Video S1). Similar results were observed in prefrontal-cortex slices (Additional file 2: Fig. S2A).
RCOR2 localizes to the core of nuclear speckles
Nuclear speckles display a structure defined by an inner core where SRRM2 and SON locate, and this center is coated by a second layer containing RNAs such as Malat1 and U1 whose periphery is surrounded by poly-(A)-RNAs and other factors [15]. This feature inspired us to determine at which region of the nuclear speckle architecture RCOR2 is recruited. To this end, we examined the colocalization between RCOR2 and SRRM2 at high resolution by Airyscan confocal microscopy with a 2D super-resolution acquisition mode, which can achieve ~ 120 nm resolution in XY planes [16]. We detected a strong colocalization between both marks (Fig. 3A), indicating that RCOR2 recruitment occurs at the core region of nuclear speckles.
Previous studies have shown that nuclear speckle components can be co-extracted from nuclear fractions upon fractionating them with hypertonic buffers [17]. Thereby, we hypothesized that if RCOR2 is actually interacting with nuclear speckle core-components, we could detect their co-extraction and interactions. Thus, we first separated cytosol and nuclei from HT22 cells, and then sequentially extracted nuclear fractions by incubating the nuclei with increasing salt concentrations steps (Additional file 2: Fig. S2B). We found RCOR2 distributed in two main subnuclear fractions, the first one was extracted between 250 and 350 mM NaCl and the second one resisted all the salt-induced extractions, remaining enriched in the chromatin pellet (Fig. 3B). Supporting the idea that RCOR2 belongs to the nuclear speckles, we observed that SRRM2, SON and SRSF7 were co-extracted with RCOR2 at fractions between 250 and 350 mM NaCl (Fig. 3B). In addition, we performed co-immunoprecipitation experiments starting from the nuclear RCOR2 peak and, in conditions where HP1ɑ, which does not interact with RCOR2, was absent; we were able to detect SON as part of the RCOR2 immunocomplex, suggesting RCOR2 forms complexes with a speckle core component in the extracted fractions (Fig. 3C). Finally, we decided to test if RCOR2 interactions with nuclear speckle components could also be detected when performing co-immunoprecipitation assays starting from HT22 and N2A whole-cell protein extracts. In this way, RCOR2 immunoprecipitation effectively precipitated its bona fide interactor LSD1 and SRSF7 in HT22 cells (Fig. 3D) and HEK293T cells (Additional file 2: Fig. S2C), while it co-precipitated SRRM2 in N2A cells (Fig. 3E). Altogether these data suggest that the RCOR2 is a core component of nuclear speckles.
RCOR2 associates with nuclear speckles inside its core region stabilized by an RNA component
As recently shown, SRRM2 forms the core region of nuclear speckles [14], while both a subset of polyadenylated pre-mRNAs and exon-junction processing complexes are enriched at the periphery [18, 19]. To further inquire about the position of RCOR2 in the nuclear speckles, we established an immuno-RNA FISH protocol that enabled us to perform a triple fluorescence labeling of RCOR2, SRRM2 and poly-adenylated RNA (poly(A)-RNA). Previous studies suggested that poly(A)-RNAs are distributed between nuclear speckles and the nucleoplasmic space [22]. Super-resolution acquisition mode of Airyscan confocal microscopy enabled us to observe that RCOR2, SRRM2, and poly(A)-RNAs colocalized at nuclear speckles, with poly(A)-RNAs decorating their periphery, and forming a fiber-like structures, which seemed to connect different speckles (Fig. 4A). Both RCOR2 and SRRM2 appeared to be in close proximity with poly(A)-RNA, as no space was observed between them (Fig. 4A, B).
Considering the sub-compartmentalized layers of nuclear speckles [15], some components, such as the stress response protein Gadd45 are recruited in an RNA-dependent manner [20], while others, such as PRPF40B are not [21]. To test whether RNA molecules stabilize RCOR2 at nuclear speckles, we analyzed RCOR2 localization after partial digestion of RNA. Here, we pre-treated permeabilized cells with DNAse-free RNAse A before fixation and then carried out triple labeling of RCOR2, SRRM2, and poly(A) RNAs to correlate RCOR2 levels with remaining RNA content in cells of the same samples. Under control conditions, the permeabilization and subsequent mock treatment did not change the subnuclear distribution of RCOR2 and SRRM2 (Additional file 2: Fig. S2D). However, under RNAse A treatment, cells that lost more than 80% of their poly(A)-RNAs showed around 60% decrease RCOR2 fluorescence intensity (Fig. 4C and D). Also, SRRM2 fluorescence intensity dropped around 40%, indicating a partial RNA-dependence on its nuclear speckle localization when analyzing it with in situ internal RNA degradation controls. These data suggest that RCOR2 localization within nuclear speckles depends on RNA or is stabilized by RNA.
Accordingly, we tested if RCOR2 is an RNA-binding protein by subjecting native, chromatin-free cell extracts of HT22 cells to poly(A)-RNA pull-down using oligo-dT-conjugated beads. In conditions where GAPDH, that does not bind RNA, was not pulled down, we detected strong enrichment of RCOR2 in the beads, pulling down more than 30% of the input protein (Fig. 4E), indicating that RCOR2 binds poly(A)-RNA in cells. Finally, since we observed close proximity and even colocalization between RNA and RCOR2, we tested if RCOR2 can interact with non-coding RNAs that are enriched in the second layer of nuclear speckles by native RNA immunoprecipitation (RIP) assays. In conditions where no DNA was amplified by qPCR, we detected specific interactions between RCOR2 and the speckle RNAs MALAT1 and 7SK (Fig. 4F). To further confirm the specificity of this interaction, we compared our results with HDAC2 RIP as another nuclear protein whose interaction with RCOR2 is neglectable [4]. As shown in Fig. 4F, none of the tested RNAs was pulled down with anti-HDAC2 antibody. In addition, HOTAIR, another nuclear LncRNA, was not enriched by RCOR2 antibodies (Fig. 4F). Therefore, RCOR2 specifically interacts with nuclear speckle RNAs from their second layer and its localization is impaired after in situ RNA degradation.
RCOR2 behaves as a core component of nuclear speckle
The morphology of nuclear speckles changes significantly with the inhibition of transcription [22]. Consistently, variations in nuclear speckle sizes result from speckle fusion and over-recruitment of nucleoplasmic factors. Our previous data showing that RCOR2 localizes in the nuclear speckle core prompted us to test if it could react to this cellular stress as other core components do. To this end, we treated HeLa cells with actinomycin D, which blocks transcriptional elongation. As expected, nuclear speckles became bigger and rounder (Fig. 5A, B, F and Additional file 3: Fig. S3A). RCOR2 remained recruited to these bodies, as observed by its colocalization with SRRM2 (Fig. 5B, E). Thus, RCOR2 responded as a core nuclear speckle component when exposed to this treatment.
We also tested the effect of isoginkgetin, a biflavonoid that inhibits the recruitment of the U4/U6/U5 snRNP complex into the spliceosome, by blocking its assembly at an early stage of the splicing cycle [23]. The exposure of cells to low and high concentrations of isoginkgetin caused a decrease in nuclear speckle sizes (Fig. 5C, F and Additional file 3: Fig. S3B). Meanwhile, the signal for RCOR2 remained similar to controls and colocalizing with SRRM2 (Fig. 5C, E and Additional file 3: Fig. S3B). This data suggests that RCOR2 behaves as a core component of nuclear speckles when cells are exposed to chemicals that inhibit spliceosome assembly. We finally tested tubercidin to induce disassembly of the nuclear speckles. This adenosine analog was can displace poly(A)-RNA from the nucleus causing release of some splicing factors from nuclear speckles, stress granule formation and mRNA export defects [24, 25]. In our model, tubercidin caused a partial dispersion of SRRM2 to the nucleoplasm and cytoplasm, with some recruitment of this splicing factor to cytoplasmic granules (Fig. 5D). Surprisingly, RCOR2 maintained a clustered distribution, being still colocalizing with nuclear SRRM2. Altogether, these data indicate that RCOR2 behaves as a nuclear speckle core component even under stress conditions that challenge speckle morphology and integrity.
RCOR2 stabilizes the morphology of nuclear speckles
To determine if RCOR2 localization within the nuclear speckle core is of functional significance, we perturbed RCOR2 levels inside cells. First, we knocked down RCOR2 with siRNA in HeLa cells and achieved a significant decrease in RCOR2 mRNA and protein levels without affecting other nuclear speckle components (Fig. 6A–C). The latter finding indicates that RCOR2 is not regulating cellular levels of the core components. We then asked if RCOR2 could affect the morphology of nuclear speckles by analyzing the staining pattern of their core component SON. Indeed, cells lacking RCOR2 depicted bigger nuclear speckles compared to control condition (Fig. 6D). Second, we transiently overexpressed RCOR2 in HeLa cells and found smaller nuclear speckles compared to the control condition (Fig. 6E).
To confirm these observations, we analyzed the area of SON particles under each condition after segmenting the images to identify single particles from each sample (Additional file 4: Fig. S4). We sorted the distribution of the population area values dividing it into quintiles and this allowed us to detect that RCOR2 knockdown led to a significant increase in the area of nuclear speckles coming from quintiles comprising the bigger speckles from each sample (Fig. 6F). Accordingly, we observed that RCOR2 overexpression led to a significant decrease on size of speckles from all the quintiles analyzed. In parallel, we defined two functions to complement our analyses: speckle-elongation (1 – width/height) and speckle-circularity (2•π•equivalent disk radius/polygonal length) that allowed us to study their changes when modulating RCOR2 levels in HeLa cells. RCOR2 knock down produced a significant increase in nuclear speckle elongation when analyzing the Top 50% values from each condition, yet no significant changes were observed when comparing circularity (Fig. 6H). Consistently, RCOR2 overexpression significantly produced rounder and less-elongated nuclear speckles (Fig. 6I). These observations together suggest that RCOR2 levels regulate the size and morphology of nuclear speckles.