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W4313238906 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Internal ribosome entry sites (IRESs) drive translation initiation during stress. In response to hypoxia, (lymph)angiogenic factors responsible for tissue revascularization in ischemic diseases are induced by the IRES-dependent mechanism. Here, we searched for IRES trans-acting factors (ITAFs) active in early hypoxia in mouse cardiomyocytes. Using knock-down and proteomics approaches, we show a link between a stressed-induced nuclear body, the paraspeckle, and IRES-dependent translation. Furthermore, smiFISH experiments demonstrate the recruitment of IRES-containing mRNA into paraspeckle during hypoxia. Our data reveal that the long non-coding RNA Neat1, an essential paraspeckle component, is a key translational regulator, active on IRESs of (lymph)angiogenic and cardioprotective factor mRNAs. In addition, paraspeckle proteins p54nrb and PSPC1 as well as nucleolin and RPS2, two p54nrb-interacting proteins identified by mass spectrometry, are ITAFs for IRES subgroups. Paraspeckle thus appears as a platform to recruit IRES-containing mRNAs and possibly host IRESome assembly. Polysome PCR array shows that Neat1 isoforms regulate IRES-dependent translation and, more widely, translation of mRNAs involved in stress response. Editor's evaluation The paper reports that the long non-coding RNA Neat1 (nuclear paraspeckle assembly transcript 1) is required for IRES Internal Ribosome Entry Site)-mediated mRNA translation activity. Neat1 is required for the activity of many cellular IRESs during the stress response in angiogenesis and/or cardio-protection. The authors conclude that nuclear paraspeckles serve as areas where cellular IRESes acquire ITAFs (IRES trans-activating factors. The findings of this paper have practical implications beyond a single subfield and the methods, data, and analyses broadly support the claims with only minor weaknesses. https://doi.org/10.7554/eLife.69162.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Cell stress triggers major changes in the control of gene expression at the transcriptional and post-transcriptional levels. One of the main responses to stress is the blockade of global translation allowing cells to save energy. This process results from inactivating the canonical cap-dependent mechanism of translation initiation (Holcik and Sonenberg, 2005). However, translation of specific mRNAs is maintained or even increased during stress via alternative mechanisms of translation initiation. One of these mechanisms involves internal ribosome entry sites (IRES), structural elements mostly present in the 5’ untranslated regions of specific mRNAs, which drive the internal recruitment of ribosomes onto mRNA and promote cap-independent translation initiation (Godet et al., 2019). Hypoxia, or the lack of oxygen, is a major stress in pathologies such as cancer and cardiovascular diseases (Pouysségur et al., 2006). In particular, in ischemic heart failure disease, coronary artery branch occlusion exposes cardiac cells to hypoxic conditions. The cell response to hypoxia induces angiogenesis and lymphangiogenesis to reperfuse the stressed tissue with new vessels and allow cell survival (Morfoisse et al., 2014; Pouysségur et al., 2006; Tatin et al., 2017). The well-known response to hypoxia is the transcriptional induction of specific genes under the control of the hypoxia-induced factors 1 and 2 (HIF1, HIF2) (Hu et al., 2003; Koh et al., 2011). However, we have recently reported that most mRNAs coding (lymph)angiogenic growth factors are induced at the translatome level in hypoxic cardiomyocytes (Hantelys et al., 2019). Expression of these factors allows the recovery of functional blood and lymphatic vasculature in ischemic diseases, including myocardial infarction (Tatin et al., 2017; Ylä-Herttuala and Baker, 2017). The mRNAs of the major (lymph)angiogenic growth factors belonging to the fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) families all contain IRESs that are activated in early hypoxia (Morfoisse et al., 2014; Hantelys et al., 2019). IRES-dependent translation is regulated by IRES trans-acting factors (ITAFs) that are in most cases RNA-binding proteins acting as positive or negative regulators. A given ITAF can regulate several IRESs, while a given IRES is often regulated by several ITAFs (Godet et al., 2019), depending on the cell type or physiology. This has led to the concept of IRESome, a multi-partner ribonucleic complex allowing ribosome recruitment onto the mRNA via the IRES. ITAFs often exhibit several functions in addition to their ability to control translation. Many of them play a role in alternative splicing, transcription, ribosome biogenesis or RNA stability (Godet et al., 2019). Clearly, a large part of ITAFs are nuclear proteins able to shuttle between nucleus and cytoplasm. Previous data have also shown that a nuclear event is important for cellular IRES activity, leading to the hypothesis of IRESome formation in the nucleus (Ainaoui et al., 2015; Semler and Waterman, 2008; Stoneley et al., 2000). Interestingly, several ITAFs are components of a nuclear body, the paraspeckle, formed in response to stress (Choudhry et al., 2015; Fox et al., 2002). These ITAFs include several hnRNPs, as well as major paraspeckle proteins such as P54nrb nuclear RNA binding (P54nrb/NONO) and splicing factor proline and glutamine-rich (SFPQ/PSF). P54nrb and SFPQ belong to the family of Drosophila melanogaster behavior and human splicing (DBHS) proteins whose third member is the paraspeckle protein C1 (PSPC1). P54nrb and SFPQ are essential for paraspeckle formation while PSPC1 is not. These three DBHS proteins are known to interact with each other and function in heteroduplexes (Fox et al., 2005; Lee et al., 2015; Passon et al., 2012). In addition, P54nrb and SFPQ interact with the long non-coding RNA (lncRNA) Neat1 (nuclear enriched abundant transcript 1), that constitutes the skeleton of the paraspeckle (Clemson et al., 2009; Sunwoo et al., 2009). This lncRNA, a paraspeckle essential component, is present as two isoforms Neat1_1 and Neat1_2 whose sizes in mouse are 3.2 and 20.8 kilobases, respectively (Sunwoo et al., 2009). Its transcription is induced during hypoxia by HIF2 and promotes paraspeckle formation (Choudhry et al., 2015). Neat1 is overexpressed in many cancers (Yang et al., 2017). Recently, its induction by hypoxia has been shown in cardiomyocytes where it plays a role in cell survival (Kenneweg et al., 2019). According to previous reports, paraspeckle is able to control gene expression via the retention of edited mRNAs and transcription factors (Hirose et al., 2014; Imamura et al., 2014; Prasanth et al., 2005). In 2017, Shen et al. have also shown that the paraspeckle might inhibit translation by sequestering p54nrb and SFPQ which are ITAFs of the MYC IRES (Shen et al., 2017). In this study, we were interested in finding new ITAFs responsible for activating (lymph)angiogenic factor mRNA IRESs in HL-1 cardiomyocytes, during early hypoxia. We have previously shown that the two paraspeckle proteins p54nrb and hnRNPM are ITAFs, activators of the FGF1 IRES during myoblast differentiation (Ainaoui et al., 2015). This incited us to investigate the potential role of the paraspeckle and of Neat1 in the control of IRES-dependent translation in hypoxic cardiomyocytes. We show here that Neat1 expression and paraspeckle formation correlate with the activation of the FGF1 IRES during hypoxia, in cardiomyocytes and breast cancer cells. The knock-down of p54nrb, PSPC1 or Neat1 generates a decrease in FGF1 IRES activity and in endogenous FGF1 expression. Furthermore, our data revealed that IRES-containing mRNA is colocalized with Neat1 in paraspeckle during hypoxia. By quantitative mass spectrometry analysis of the p54nrb interactome, we identified two additional ITAFs able to control the FGF1 IRES activity: nucleolin and ribosomal protein RPS2. Analysis of IRESs in the knock-down experiments showed that p54nrb and PSPC1 are activators of several but not all IRESs of (lymph)angiogenic and cardioprotective factor mRNAs whereas Neat1 appears as a strong activator of all the cellular IRESs tested. These data suggest that the paraspeckle, via Neat1 and several protein components would be the site of IRESome assembly in the nucleus. In addition, a polysome PCR array reveals that Neat1 affects the translation of most IRES-containing mRNAs and of several mRNA families involved in hypoxic response, angiogenesis and cardioprotection. Results FGF1 IRES activation during hypoxia correlates with paraspeckle formation and with Neat1 induction in different cell types In order to analyze the regulation of IRES activity during hypoxia, HL-1 cardiomyocytes were transduced with the ‘Lucky Luke’ bicistronic lentivector validated in our previous reports, containing the renilla luciferase (LucR) and firefly luciferase (LucF) genes separated by the FGF1 IRES (Video 1, Figure 1A). In this construct, the first cistron LucR is expressed in a cap-dependent manner and the second cistron LucF is under the control of the IRES. The ratio LucF/LucR reflects the IRES activity. Figure 1 with 3 supplements see all Download asset Open asset FGF1 IRES activation during hypoxia correlates with Neat1 induction and paraspeckle formation. (A) Schema depicting the Lucky Luke bicistronic construct and HL-1 cells transduced by a lentivector carrying the transgene. The LucF/LucR ratio indicates the IRES activity. (B) Activity of the human FGF1 IRES in HL-1 cardiomyocytes at 4 hr, 8 hr, or 24 hr of hypoxia normalized to normoxia. The corresponding luciferase values are presented in Figure 1—figure supplement 1, Supplementary file 1. (C) Detection of endogenous mouse FGF1 by capillary Simple Western in normoxic and hypoxic (2 hr) cardiomyocytes. The curve corresponds to the chemiluminescence signal detected with FGF1 antibody. A numerical blot is represented. Below the blot is shown the quantification of FGF1 normalized to total proteins and to control gapmer. Total proteins are detected by a dedicated channel in capillary Simple Western. The full raw unedited gel is provided in Figure 1—figure supplement 1 (Figure 1—figure supplement 1—source data 1). (D) HL-1 cells were subjected to normoxia (0 hr) or to hypoxia during 4 hr, 8 hr, and 24 hr. Neat1 and Neat1_2 expression was analyzed by droplet digital PCR (Primer sequences in Supplementary file 2). RNA expression is normalized to the normoxia time point. (E) Schema depicting the Neat1 mouse gene and the Neat1_1 and Neat1_2 RNA isoform carrying a poly(A) tail or a triple helix, respectively. Black arrowheads represent FISH probes against Neat1 and Neat1_2 (sequences in Supplementary file 2). (F–K) Neat1 (F) or Neat1_2 (I) FISH labeling in HL-1 cardiomyocytes in normoxia or at 4 hr, 8 hr, and 24 hr of 1% O2. DAPI staining is represented in blue and Neat1 or Neat1_2 cy3 labeling in red. Nuclei are delimited by dotted lines. Scale bar = 10 µm. Larger fields are presented in Figure 1—figure supplement 2. (G and J) Quantification of Neat1 (G) or Neat1_2 (J) foci per cell by automated counting (ImageJ). (H and K) Percentage of cell harboring at least one focus of Neat1 (H) or Neat1_2 (K); Histograms correspond to means ± standard deviation, with Mann-Whitney (n=12) (B) or one-way ANOVA (G-H, n=269–453) and (J-K, n=342–499); **p<0.01, ***<0.001, ****p<0.0001. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Beating HL-1 cardiomyocytes (Enlargement 40X). Mouse atrial HL-1 cardiomyocytes exhibit a beating phenotype when cultured in Claycomb medium at high density (Claycomb et al., 1998). This phenotype was required to obtain all the data described in the present study. LucR and LucF activities were measured in HL-1 cells subjected to hypoxia for 4 hr, 8 hr, or 24 hr (Figure 1—figure supplement 1, Supplementary file 1). These conditions were exactly the same as that used in our previous report providing evidence of IRES activation by hypoxia (Hantelys et al., 2019). We previously showed in the same report that eIF2α is phosphorylated after 4 hr of hypoxia, while no change in 4E-BP1 phosphorylation is observed. The polysome/monosome ratio indicated that global protein synthesis decreases in these conditions (Hantelys et al., 2019). Those data allowed us to conclude that IRES activities are not negatively affected by eIF2α phosphorylation. Here, we showed that both luciferase activities increase after 4 hr of hypoxia and decreased at 24 hr. However, LucF increased more than LucR (2.5 times versus 1.5 times, respectively). Thus the ratio LucF/LucR revealed a significant activation of the FGF1 IRES in early hypoxia, correlated to induction of endogenous FGF1 as previously shown (Hantelys et al., 2019; Figure 1B and C, Figure 1—figure supplement 1). Neat1 and Neat1_2 expression in cells was measured by reverse transcription and droplet digital PCR (RT ddPCR), showing an increase of Neat1 and Neat1_2 at 4 hr with a peak of expression of Neat1 at 8 hr of hypoxia, while the peak of expression of Neat1_2 was observed after 4 hr of hypoxia (Figure 1D). The same data were also obtained by classical RT-qPCR (data not shown), in agreement with our previous report showing Neat1 induction by hypoxia in HL-1 cells (Hantelys et al., 2019). In parallel, paraspeckle formation was studied by fluorescent in situ hybridization (FISH) targeting the non-coding RNA Neat1, considered as the main marker of paraspeckles. The fluorescent probes targeted either the common part of the two isoforms Neat1_1 and Neat 1_2, or only the large isoform Neat1_2 (Figure 1E). After 4 hr of hypoxia, the number of foci increased and reached 2 foci per cell on average, while the number of cells containing at least one focus shifted from 20% to 70% (Figure 1F–K, Figure 1—figure supplement 2). This was observed with both Neat1 and Neat1_2 probes. The values observed at 4 hr did not change after 8 hr and 24 hr of hypoxia with the Neat1 probe (Figure 1F–H). In contrast, the number of foci containing Neat1_2 decreased after longer times of hypoxia: at 8 hr and 24 hr, the number of foci per cell reached 1 and 0.5 while only 50% and 40% of the cells contained at least one focus, respectively (Figure 1I–K). Surprisingly, Neat1_2 was detected in the cytoplasm in normoxia and after 24 hr of hypoxia (Figure 1I, Figure 1—figure supplement 2). These data revealed that FGF1 IRES activation correlates with increased Neat1 expression and paraspeckle formation after 4 hr of hypoxia in HL-1 cardiomyocytes. To determine whether such a correlation also occurs in other cell types, similar experiments were performed in a mouse breast tumor cell line 67NR (Figure 1—figure supplement 3). In these cells, known to be more resistant to hypoxia, Neat1 increased only after 24 hr of hypoxia. In particular, we observed a strong and significant induction of Neat1_2 (Figure 1—figure supplement 3B). As regards the IRES activity (LucF/LucR ratio), it also increased after 24 hr of hypoxia (Figure 1—figure supplement 3C). These data indicate that the correlation between Neat1_2 isoform induction and IRES activation under hypoxia exists in different cell types. LncRNA Neat1 knock-down drastically affects the FGF1 IRES activity and endogenous FGF1 expression To determine whether Neat1 could have a role in the regulation of FGF1 IRES activity, we depleted HL-1 for this non-coding RNA using locked nucleic acid (LNA) gapmers, antisense modified oligonucleotides described for their efficiency in knocking-down nuclear RNAs. HL-1 cells transduced with the bicistronic vector were transfected with a pool of gapmers targeting Neat1 and with a control gapmer (Supplementary file 2). The knock-down efficiency was measured by smiFISH (single molecule inexpensive FISH) and ddPCR and showed a decrease in the number of paraspeckles, correlated to the decrease of Neat1 RNA, which shifted from 5 to 2 foci per cell (Figure 2A–B, Figure 2—figure supplement 1A; Tsanov et al., 2016). In these experiments performed in normoxia, the number of paraspeckles was high (almost 5 foci per cell), suggesting that cells were already stressed by the gapmer treatment, before being submitted to hypoxia. Alternatively, it could also be explained by the high sensitivity of the smiFISH method used here, whereas paraspeckles were detected by FISH in Figure 1. To evaluate the IRES activity, the ratio LucF/LucR was measured in normoxia or after 4 hr of hypoxia, revealing that the IRES activity decreased by two times upon Neat1 depletion (Figure 2C, Supplementary file 3). This effect was also observed on endogenous FGF1 protein expression, measured by capillary Simple Western, which decreased by three times (Figure 2D, Figure 2—figure supplement 2). Figure 2 with 6 supplements see all Download asset Open asset LncRNA Neat1 knock-down drastically affects the FGF1 IRES activity and endogenous FGF1 expression. (A) SmiFISH imaging of Neat1 knock-down by a pool of LNA gapmers targeting both isoforms (Sequences in Supplementary file 2C). Cells were treated during 48 hr with the gapmers. Scale bar = 10 µm. (B) Neat1 foci counting per cell for the control gapmer and Neat1 LNA gapmer pool, using unpaired two-tailed student t-test with n=249 for control and 187 for Neat1 LNA gapmer. (C) FGF1 IRES activities in HL-1 cells transduced with Lucky Luke bicistronic reporter and treated with gapmer Neat1 or control during normoxia or hypoxia (1% O2). Histograms correspond to means ± standard deviation of the mean. Non-parametric Mann-Whitney test was performed with n=9. *p<0.05, ***<0.001, ****p<0.0001. The mean has been calculated with nine cell culture biological replicates, each of them being already the mean of three technical replicates (27 technical replicates in total). Detailed values of biological replicates are presented in Supplementary file 3. (D) Detection of endogenous mouse FGF1 by capillary Simple Western. The curve corresponds to the chemiluminescence signal detected with FGF1 antibody. A numerical blot is represented. Below the blot is shown the quantification of FGF1 normalized to total proteins and to control gapmer. The source data of the capillary Simple Western are provided in Figure 2—figure supplement 2. Total proteins are detected by a dedicated channel in capillary Simple Western. Neat1_2 knock-down was then performed to evaluate the contribution of the long Neat1 isoform. Also, the FGF1 IRES activity decreased following Neat1_2 depletion, however less importantly than with the knock-down of the two isoforms (Figure 2—figure supplement 3), suggesting an involvement of both Neat1 isoforms. Capillary Western experiments indicated a slight increase of eIF2α phosphorylation upon Neat1_2 depletion (Figure 2—figure supplement 4). It was not sufficient to block global translation, as shown by the renilla luciferase activity (Supplementary file 3, page 2). Furthermore, we have shown in a previous report that the FGF1 IRES activity increases in hypoxia in conditions of strong eIF2α phosphorylation. FGF1 half-life was superior to 24 hr and was not affected by Neat1 knock-down (Figure 2—figure supplements 5–6). All these arguments indicate that the significant decrease of FGF1 IRES activity and of endogenous FGF1 expression observed in Figure 2 does not result from eIF2α phosphorylation or decrease in FGF1 half-life, and probably results from Neat1 depletion. This suggested that Neat1 might regulate FGF1 mRNA translation, directly or indirectly. The IRES-containing mRNA is colocalized with Neat1 during hypoxia The effect of Neat1 on FGF1 IRES activity suggested an interaction (direct or indirect) between these two RNAs. SmiFISH experiments were performed with two sets of 48 primary probes targeting Neat1 or the bicistronic mRNA, respectively. As a control, we also used a bicistronic construct with a hairpin instead of the IRES. The two secondary probes were coupled to different fluorophores to detect Neat1 and the bicistronic mRNA separately and look for a putative colocalization (Figure 3). Data clearly show that the IRES containing bicistronic mRNA is colocalized with Neat1 and that this colocalization significantly increases during hypoxia, which is not the case for the hairpin control (Figure 3C and D). These data suggested that the IRES-containing mRNA is recruited into paraspeckles during hypoxia. Figure 3 Download asset Open asset IRES-containing mRNA is colocalized with Neat1 in hypoxic HL-1 cells. Cells were transduced with lentivectors carrying bicistronic Lucky Luke constructs with the FGF1 IRES or a hairpin (control), subjected or not to 4 hr hypoxia. SmiFISH experiments were performed. (A) SmiFISH images showing the bicistronic mRNA carrying the FGF1 IRES (green) colocalized with Neat1 RNA (red) in hypoxia condition. Two representative cells are presented. Scale bars are 3 µm for higher panels, 4 µm for lower panesl and 1 µm for zoomed images of colocalized spots. (B) Quantification of colocalized spots per cell (n=30). Unpaired two-tailed Student T-test was performed. Paraspeckle proteins P54nrb and PSCP1, but not SFPQ, are ITAFs of the FGF1 IRES The correlation between paraspeckle formation and FGF1 IRES activation, together with the probable recruitment of IRES-containing mRNA into paraspeckles during hypoxia, incited us to study the role of other paraspeckle components in the control of IRES activity. Three major paraspeckle proteins were chosen, the DBHS proteins, SFPQ, p54nrb and PSPC1 (Figure 4A). SFPQ and p54nrb have been previously described for their ITAF function (Ainaoui et al., 2015; Cobbold et al., 2008; Lampe et al., 2018; Sharathchandra et al., 2012; Shen et al., 2017). In particular, p54nrb regulates the FGF1 IRES activity during myoblast differentiation (Ainaoui et al., 2015). Figure 4 with 4 supplements see all Download asset Open asset Paraspeckle proteins p54nrb and PSCP1, but not SFPQ, are ITAFs of the FGF1 IRES. (A) Schema of paraspeckle and DBHS proteins. (B–D) FGF1 IRES activity upon knock-down of SFPQ (B), P54nrb (C) or PSPC1 (D) in HL-1 cell (Figure 4—figure supplement 1—source data 1) transduced with Lucky Luke bicistronic reporter during normoxia or hypoxia was measured as in Figure 2. Cells were harvested 72 hr after siRNA treatment. The IRES activity values have been normalized to the control siRNA. Histograms correspond to means ± standard deviation of the mean, with a non-parametric Mann-Whitney test with n=9; *p<0.05, ***<0.001. The mean has been calculated with nine cell culture biological replicates, each of them being already the mean of three technical replicates (27 technical replicates in total). Detailed values of biological replicates are presented in Supplementary file 3, Supplementary file 4, Supplementary file 5. (E and F) Capillary Simple Western detection of endogenous FGF1 protein with P54nrb (E) or PSPC1 (F) knock-down. Source data of capillary Simple Western are presented in Figure 4—figure supplement 2 (Figure 4—figure supplement 2—source data 1). HL-1 cells transduced by the ‘Lucky Luke’ bicistronic construct were transfected with siRNA smartpools targeting each of the three proteins. The knock-down efficiency was checked by capillary Simple Western, classical Western, or RT qPCR (Figure 4—figure supplement 1). SFPQ knock-down did not affect the IRES activity (Figure 4B, Supplementary file 4). In contrast, we observed a decrease in IRES activity with p54nrb and PSPC1 knock-down, both in normoxia and in hypoxia (Figure 4C–DSupplementary file 4, Supplementary file 5), despite a knock-down efficiency below 50%. p54nrb and PSPC1 knock-down also inhibited the expression of endogenous FGF1 protein (Figure 3E–F, Figure 4—figure supplement 2). FGF1 half-life was not altered by siRNA treatment, indicating a translational control (Figure 4—figure supplements 3–4). These data confirmed the ITAF role of p54nrb in HL-1 cardiomyocyte, and indicated that PSPC1 is also an ITAF of the FGF1 IRES. The ability of three paraspeckle components, Neat1, p54nrb and PSPC1, to regulate the FGF1 IRES activity, together with the colocalization of the bicistronic mRNA with Neat1 observed in Figure 3, led us to the hypothesis that the paraspeckle might be involved in the control of IRES-dependent translation. P54nrb interactome in normoxic and hypoxic cardiomyocytes The moderate effect of p54nrb or PSPC1 depletion on FGF1 IRES activity, possibly due to the poor efficiency of knock-down (>50%), also suggested that other proteins may be involved. Previous data from the literature support the hypothesis that the IRESome is a multi-partner complex. In order to identify other members of this complex, we analysed the p54nrb interactome in HL-1 cell nucleus and cytoplasm using a label-free quantitative mass spectrometry approach. For this purpose, cells were transduced by a lentivector expressing an HA-tagged p54nrb (Figure 5A). After cell fractionation (Figure 5B and Figure 5—figure supplement 1A and B), protein complexes from normoxic and hypoxic cells were immunoprecipitated with anti-HA antibody. Immunoprecipitated interacting proteins (three to four biological replicates for each group) were isolated by SDS-PAGE, in-gel digested with trypsin and analyzed by nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS), leading to the identification and quantification of 2013 proteins (Supplementary file 7). To evaluate p54nrb interaction changes, pairwise comparisons based on MS intensity values were performed for each quantified protein between the four groups, cytoplasmic and nuclear complexes from cells subjected to normoxia or hypoxia (Figure 5C). Enriched proteins were selected based on their significant protein abundance variations between the two compared group (fold-change (FC) >2 and<0.5, and Student t test p<0.05) (see STAR Method for details) (Figure 5D–E and Figure 5—figure supplement 1). Globally, the HA-tag capture revealed an enrichment of hnRNP proteins in nucleus and of ribosomal proteins in the cytoplasm (Figure 5—figure supplement 1C and D). In nucleus P54nrb interacted with itself (endogenous mouse Nono), PSPC1 and SFPQ, as well as with other paraspeckle components: in total P54nrb interaction was identified with 22 proteins among 40 paraspeckle components listed in previous reports (Table 1; Naganuma et al., 2012; Yamamoto et al., 2021). Six of these paraspeckle components exhibit an ITAF function (FUS, hnRNPA1, hnRNPK, hnRNPM, hnRNPR, and SFPQ Figure 5—figure supplement 1, Table 1). Two additional ITAFs interact with p54: hnRNPC and hnRNPI (Godet et al., 2019). Figure 5 with 2 supplements see all Download asset Open asset P54nrb interactome in normoxic and hypoxic cardiomyocytes. (A) Experimental workflow: p54nrb-HA transduced HL-1 cells were subjected to normoxia or hypoxia, then nucleus and cytoplasm fractionation was performed and extracts were immunoprecipitated using anti-HA antibody. Enriched interacting proteins were identified by using a label-free quantitative mass spectrometry approach. (B) Western blot of fractionation experiment of HL-1 cells in normoxia and hypoxia. Histone H3 was used as a nuclear control and GAPDH as a cytoplasm control. The dotted line delineates two different blots of the same fractionation experiment. (C) Schema of the four pairwise comparisons submitted to statistical analysis. (D and E) Volcano plots showing proteins enriched (bold black) and significantly enriched (after elimination of false-positive hits from quantitation of low-intensity signals) in the nucleus for hypoxia (purple) versus normoxia (red) (D) or in the cytoplasm for hypoxia (green) versus normoxia (E). An unpaired bilateral student t-test with equal variance was used. Enrichment significance thresholds are represented by an absolute log2-transformed fold-change (FC) greater than 1 and a -log10-transformed (p-value) greater than 1.3. Details are provided in Supplementary file 7. Table 1 The p54 interactome includes 22 among 40 proteins described as paraspeckle components. The paraspeckle components listed in the reports by Naganuma et al., 2012 and by Yamamoto et al., 2021 is presented here with their ITAF function and their presence in the p54nrb interactome. Their belonging to class I, II, or III of the paraspeckle proteins is indicated. Class I proteins are essential for paraspeckle formation. NameAlternative nameClassITAFPresence in p54nrb MS-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-43IINoNoUBAP2LIIIANoYesZNF335TARDBPIIIBNoYes As regards cytoplasmic proteins, we identified RPS25, a ribosomal protein previously described as an ITAF for many IRESs (Figure 5—figure supplement 1A; Hertz et al., 2013). Interestingly, p54nrb also interacted with RPS5, RPS18 and RPS19, and other RPs, mainly from the small ribosomal subunit. Only few proteins were" @default.
W4313238906 created "2023-01-06" @default.
W4313238906 date "2021-05-18" @default.
W4313238906 modified "2023-09-27" @default.
W4313238906 title "Decision letter: Long non-coding RNA Neat1 and paraspeckle components are translational regulators in hypoxia" @default.
W4313238906 doi "https://doi.org/10.7554/elife.69162.sa1" @default.
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