Splicing factor dynamics suggests that accumulation in speckles is due to inefficient recruitment to pre-mRNA.

<p>(A) FRAP analysis of SRSF1 and U2AF65 splicing factors fused to EYFP show that their kinetics of association to speckles are not modified after TSA treatment. For this experiment, C33A cells stably expressing the corresponding EYFP-fusion protein were used. Similar results were obtained for other splicing factors transiently transfected in HeLa cells (not shown). We show a representative cell for each group. The circle shows the speckle area where the laser pulse was applied, and where fluorescence recovery was measured afterwards. Fluorescence intensity is expressed relative to the fluorescence prior photobleaching. The length of TSA treatment was 4 h for U2AF65 and 6 h for SRSF1. The curves are averages from 10 to 15 cells. (B) Direct interaction between splicing factors and histones is detectable but not affected by TSA treatment. Interaction between the mCherry-tagged splicing factor SRSF1 and the EGFP-tagged histone H2B was analyzed by FLIM-FRET technique. A representative cell expressing both tagged proteins and its FRET efficiency map is showed. Also, the FRET efficiency map of a representative cell expressing EGFP-H2B and mCherry-C1 empty plasmid is shown as a control (NO FRET). The pixels histogram shows average FRET efficiency distributions for Control (7 cells) and TSA-treated cells (6 cells). Peaks of FRET are marked by colored arrowheads, indicating interaction between these two proteins. Nuclear regions associated to these distinct FRET populations are also marked by colored arrowheads in the E _FRET map. (C) Nucleoplasmic interaction between splicing factors is not impaired by TSA treatment. The interaction between EGFP-U1-70K and mCherry-SRSF1, a known interaction pair <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048084#pone.0048084-Ellis1" target="_blank">[51]</a> was assessed by FLIM-FRET in Control and TSA-treated cells. Upper panels: control cells, transfected with mCherry instead of mCherry-SRSF1, show no FRET signal in either speckles or nucleoplasm. Lower panels: FRET efficiency between the two splicing factors is intermediate in nucleoplasm (green) with stronger (red) areas in speckles (marked with white arrowheads in control cells). (D) Proposed model to explain how relaxation of chromatin structure affects splicing factor distribution. Nucleoplasmic free splicing factors are in dynamic equilibrium with splicing factors in speckles (<i>a</i>). Free splicing factors can also interact with other splicing factors independently of splicing (<i>b</i>), these complexes being found both in nucleoplasm and speckles. However, what we call nucleoplasmic fraction involves also splicing factors briefly interacting with other molecules (such as histones) through abundant low affinity interactions (<i>c</i>) and splicing factors recruited to RNA and engaged in productive spliceosome assembly (<i>d</i>). For model simplicity, in <i>c</i> we only included interactions with histones and in <i>d</i> we only depicted co-transcriptional splicing. The red lines and arrows symbolize the proposed system response after TSA treatment: decreased efficiency in recruitment cause an excess of free splicing factors that is buffered by the speckles compartment.</p

Histone acetylation affects distribution of several splicing factors involved in constitutive and alternative splicing.

<p>(A) Several splicing factors tested show accumulation in nuclear speckles after a 6-hour TSA treatment of HeLa cells, including many SR proteins (SRSF2, SRSF1, SRSF3) and proteins of the U1 and U2 complexes (U1 70K, U1A, U2AF65). Top row shows control cells and bottom row shows cells treated with 0.2 ng/µl TSA. Some enlarged nuclear granules containing splicing factors in TSA-treated cells are marked by yellow arrowheads. Scale bars, 5 µm. (B) TSA treatment affects histone but not splicing factor lysine acetylation. HEK-293T cells were transfected with plasmids coding for the indicated tagged proteins (lanes 5–10) or not transfected (lanes 1–4). After one day, cells were incubated for 6 hours with or without 1 µM TSA, lysed and either histones were extracted (lanes 1–2) or proteins immunoprecipitated with antibodies against T7 tag (lanes 3–10). Purified proteins were analyzed by Western blot using an antibody that recognizes acetylated lysines (αAcLys, upper panels). As loading controls, proteins were also detected with antibodies against histone H3 or T7 tag (lower panels). In lanes 1 and 2, the two bands for the αAcLys correspond to histones H3 and H4, showing a dramatic increase of acetylation in both histones upon TSA treatment. In lanes 7 and 8, cells transfected with T7-SRSF2 were treated with the proteasome inhibitor MG132 (see text). (C) Total transcription is not affected after 6 h-TSA treatment. Global levels of nascent RNA in untreated and TSA-treated HeLa cells were measured by <i>in vivo</i> 5-FU incorporation and immunostaining with an antibody specific for 5-FU nucleotide. A representative field for each condition is shown. Brighter spots correspond to ribosomal RNA in nucleoli. Scale bars, 10 µm. Quantification of the 5-FU signal in individual cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048084#s3" target="_blank">Materials and Methods</a>) shows no significant change in transcription in TSA-treated vs. Control cells (<i>n</i> = 15 and 10 respectively). In contrast, treatment with the P-TEFb inhibitor DRB (<i>n</i> = 10 for each condition) and with the general transcription inhibitor actinomycin D (<i>n</i> = 5 for Control and 6 for ActD) causes a clear decrease in 5-FU incorporation, as expected from transcriptional inhibition.(D) Time course of EYFP-SRSF3 distribution in HeLa cells treated with 0.2 ng/µl TSA. A representative nucleus is shown. Images were acquired every 30 minutes. Yellow arrowheads point to a single speckle where accumulation of the tagged-SR protein is observed. Scale bars, 5 µm.</p

Chromatin relaxation affects spliceosome recruitment to nascent RNA and binding of splicing factors to ncRNAs.

<p>Binding of U2AF65 to RNA was assessed by iCLIP in 293-Flp cells, as a measure of spliceosome recruitment in Control cells or cells treated with 1 µM TSA for 2 h (before splicing factor accumulation in speckles is apparent) and 6 h. (A) TSA treatment causes an impairment of U2AF65 recruitment to the 3′ splice sites. Total U2AF65 counts in exon-intron (E-I) and intron-exon (I-E) junctions for all transcripts are plotted in a window of +/−50 nt from the junctions. The graph shows that iCLIP consistently captures U2AF65 at the expected location on the 3′ splice site (I-E junction), where only non-specific binding is detected at the E-I junction. The curve for 6 h TSA treatment (green) shows a reduction with respect to the control (blue) and 2 h TSA treatment (red). (B) A fraction of the genes show sensitivity to TSA treatment at 2 hours. Each junction was analyzed separately, and for each junction genes included in the analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048084#s3" target="_blank">Materials and Methods</a>) were divided into categories of increased (Up), decreased (Down) or unchanged (less than 2-fold change in any direction) U2AF65 counts in a region of +/−100 nt around the corresponding junction. E-I junction was used as a control, assuming that variations in these counts (non-specific) were random. Analysis of changes in the junctions of individual genes after 2 h TSA treatment shows that almost 20% of the genes tend to have less U2AF65 binding in I-E junctions (and only 5% have increased binding). As a control, E-I junction shows less than 10% of the genes with decreased binding and 7% with increased binding. Distributions for the two junctions are significantly different (asterisk, chi-squared test for independence between fold-change categories and type of junction, <i>p</i> = 0.00032). (C) U2AF65 binding to lincRNAs increase upon TSA treatment. Distribution of lincRNA iCLIP counts in the three libraries, discriminating by the abundant MALAT1 and NEAT1 nuclear-retained ncRNAs and all the other lincRNAs. Inset: magnification of the binding to MALAT1 and other lincRNAs, excluding NEAT1. (D) U2AF65 increase binding to specific sites in MALAT1 and NEAT1. UCSC browser window showing a portion of MALAT1 (top) and NEAT1 (bottom) genes and the U2AF65 iCLIP counts for Control, 2 h and 6 h TSA-treated cells (complete genes with binding sequence info can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048084#pone.0048084.s005" target="_blank">Fig. S5</a>). Orange boxes mark regions with peaks appearing at 2 h TSA and not further increasing, or even decreasing, at 6 h (MALAT1 peaks). Green boxes mark regions with peaks gradually increasing to their maximum at 6 h TSA (NEAT1 peaks). (E) Co-immunofluorescence analysis on untreated HEK-293 cells (top row) and cells treated with 1 µM TSA for 2 h (middle) or 6 h (bottom). For each condition, a high magnification view of speckle and paraspeckle compartments is shown (right panels). In untreated cells, the paraspeckles marker PSP1 does not colocalize with U2AF65 speckles, but after 6 h TSA co-localization of PSP1 and U2AF65 is observed. 2 h TSA treatment leads to an intermediate state were paraspeckles and U2AF65 granules are starting to be associated. Nuclei were stained with DAPI. Scale bars, 10 µm.</p

Membrane potential depolarization and TSA treatment of neuroblastoma cells cause splicing factors accumulation in speckles.

<p>(A) N2a cells were transiently transfected with plasmids encoding SRSF2 or SRSF1 splicing factors fused to EGFP, along with a plasmid encoding histone H3 fused to mCherry. After one day, cells were either treated for 6 hours with 0.2 ng/µl TSA (TSA), 60 mM KCl (DEPOL) or left untreated (CONTROL). Enlarged nuclear speckles in N2a cells containing the splicing factors in DEPOL or TSA-treated cells are marked by yellow arrowheads. Scale bars, 5 µm. (B) Analysis of intensity profile of EGFP-SRSF2 and mCherry-H2B signals across a line (yellow dotted lines) for a representative cell for each experimental condition. SRSF2 profile shows higher and wider peaks in DEPOL and TSA cells, corresponding to enlargement of nuclear speckles. No intensity changes are observed for mCherry-H2B profiles. The drop in mCherry-H2B signal at the SRSF2 peaks is typical of inter-chromatin granules. Scale bars, 5 µm. (C) Statistical analysis of splicing factor enrichment in speckles. Signal of EGFP-SRSF2 (top) and EGFP-SRSF1 (bottom) in speckles increases both in response to depolarization and TSA treatments. Intensity of splicing factor in all speckles of a focal plane was calculated for individual cells using automatic threshold and particle analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048084#s3" target="_blank">Materials and Methods</a>). The total integrated density of speckles particles was normalized by total integrated density of the cell. For EGFP-SRSF2, 10 cells (Control), 8 cells (Depol) and 11 cells (TSA) were analyzed. For EGFP-SRSF1, 10 cells (Control), 7 cells (Depol) and 10 cells (TSA) were analyzed. * means significant differences between treated and control cells, using Mann-Whitney U test (p = 0.023 in Depol and 0.0044 in TSA for EGFP-SRSF2; p = 0.036 in Depol and 0.031 in TSA for EGFP-SRSF1).</p

Analysis of endogenous SRSF2 distribution shows that histone acetylation causes splicing factor depletion from nucleoplasm.

<p>(A) Immunostaining for endogenous SRSF2 in HeLa cells untreated or treated with TSA (0.2 ng/µl, 6 hours). DNA was stained with DAPI. Visualization of endogenous SRSF2 distribution suggests depletion from nucleoplasm after TSA treatment. Scale bars, 10 µm. (B) Total levels of endogenous SRSF2 do not change upon TSA treatment. Box plot showing similar distribution of SRSF2/DAPI intensities in individual untreated or TSA-treated HeLa cells (26 cells of each type). The SRSF2/DAPI ratio is NOT significantly different between CONTROL and TSA cells (Mann-Whitney U test: p = 0.23; Kolmogov-Smirnov test: p = 0.501). (C) TSA treatment causes depletion of nucleoplasmic SRSF2 fraction. Nucleoplasmic intensity was calculated in the same cells as (B) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048084#s3" target="_blank">Materials and Methods</a>). Box plot shows nucleoplasmic/total SRSF2 fractions in CONTROL and TSA cells, which further supports the observation of SRSF2 depletion from nucleoplasm after TSA treatment. The nucleoplasmic SRSF2 fraction is significantly reduced in TSA-treated cells compared to control cells (marked by asterisk; Mann-Whitney U test: <i>p</i> = 0.0017; Kolmogov-Smirnov test: <i>p</i> = 0.018). (D) High resolution imaging analysis of SRSF2 distribution and speckle internal structure in untreated and TSA-treated cells. Control and TSA-treated cells were fixed, immunostained for SRSF2 and analyzed by SIM/OMX microscopy. Upper panels: a representative cell in each condition is shown, where depletion of diffuse SRSF2 in nucleoplasm and concentration in speckles is apparent after TSA treatment. Lower panels: enlarged view of an individual speckle (yellow boxes) from cells in each experimental condition. Scale bars, 5 µm. (E) HP1α knockdown has similar effect than TSA treatment on endogenous SRSF2 distribution. HeLa cells were transfected with control siRNAs or siRNAs against HP1α. After two days, the cells were fixed and stained for endogenous SRSF2. Depletion of nucleoplasmic SRSF2 staining and accumulation in speckles is evidenced, similar to what is seen after TSA treatment (A). Scale bars, 5 µm.</p