Lnc RNA ‐dependent nuclear stress bodies promote intron retention through SR protein phosphorylation
2019; Springer Nature; Volume: 39; Issue: 3 Linguagem: Inglês
10.15252/embj.2019102729
ISSN1460-2075
AutoresKensuke Ninomiya, Shungo Adachi, Tohru Natsume, Junichi Iwakiri, Goro Terai, Kiyoshi Asai, Tetsuro Hirose,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle29 November 2019free access Source DataTransparent process LncRNA-dependent nuclear stress bodies promote intron retention through SR protein phosphorylation Kensuke Ninomiya Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Shungo Adachi orcid.org/0000-0001-5232-411X Molecular Profiling Research Center, National Institute for Advanced Industrial Science and Technology (AIST), Tokyo, Japan Search for more papers by this author Tohru Natsume Molecular Profiling Research Center, National Institute for Advanced Industrial Science and Technology (AIST), Tokyo, Japan Search for more papers by this author Junichi Iwakiri Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Japan Search for more papers by this author Goro Terai Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Japan Search for more papers by this author Kiyoshi Asai Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Japan Search for more papers by this author Tetsuro Hirose Corresponding Author [email protected] orcid.org/0000-0003-1068-5464 Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Kensuke Ninomiya Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Shungo Adachi orcid.org/0000-0001-5232-411X Molecular Profiling Research Center, National Institute for Advanced Industrial Science and Technology (AIST), Tokyo, Japan Search for more papers by this author Tohru Natsume Molecular Profiling Research Center, National Institute for Advanced Industrial Science and Technology (AIST), Tokyo, Japan Search for more papers by this author Junichi Iwakiri Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Japan Search for more papers by this author Goro Terai Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Japan Search for more papers by this author Kiyoshi Asai Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Japan Search for more papers by this author Tetsuro Hirose Corresponding Author [email protected] orcid.org/0000-0003-1068-5464 Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Author Information Kensuke Ninomiya1, Shungo Adachi2, Tohru Natsume2, Junichi Iwakiri3, Goro Terai3, Kiyoshi Asai3 and Tetsuro Hirose *,1 1Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan 2Molecular Profiling Research Center, National Institute for Advanced Industrial Science and Technology (AIST), Tokyo, Japan 3Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Japan *Corresponding author. Tel: +81 11 706 5071; Fax: +81 11 706 7540; E-mail: [email protected] EMBO J (2020)39:e102729https://doi.org/10.15252/embj.2019102729 See also: S Erhardt & G Stoecklin (February 2020) PDFDownload PDF of article text and main figures.AM PDF Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract A number of long noncoding RNAs (lncRNAs) are induced in response to specific stresses to construct membrane-less nuclear bodies; however, their function remains poorly understood. Here, we report the role of nuclear stress bodies (nSBs) formed on highly repetitive satellite III (HSATIII) lncRNAs derived from primate-specific satellite III repeats upon thermal stress exposure. A transcriptomic analysis revealed that depletion of HSATIII lncRNAs, resulting in elimination of nSBs, promoted splicing of 533 retained introns during thermal stress recovery. A HSATIII-Comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) analysis identified multiple splicing factors in nSBs, including serine and arginine-rich pre-mRNA splicing factors (SRSFs), the phosphorylation states of which affect splicing patterns. SRSFs are rapidly de-phosphorylated upon thermal stress exposure. During stress recovery, CDC like kinase 1 (CLK1) was recruited to nSBs and accelerated the re-phosphorylation of SRSF9, thereby promoting target intron retention. Our findings suggest that HSATIII-dependent nSBs serve as a conditional platform for phosphorylation of SRSFs by CLK1 to promote the rapid adaptation of gene expression through intron retention following thermal stress exposure. Synopsis Membraneless nuclear bodies are formed on architectural ncRNAs by selective sequestration of RNA-binding proteins. Here, nuclear stress bodies (nSBs) are shown to function as platform for efficient SRSF splicing factor phosphorylation to modulate intron retention during stress recovery. nSBs promote retention of 533 introns during thermal stress recovery. nSBs contain 141 proteins, especially RNA-binding proteins involved in splicing, processing and export of mRNAs. CLK1 kinase is recruited to nSBs during stress recovery to efficiently phosphorylate the sequestrated SRSFs. CLK1 phosphorylation of SRSF9 promotes rapid accumulation of intron-retaining pre-mRNAs in the nucleus during thermal stress recovery. Introduction Long noncoding RNAs (lncRNAs) have recently been recognized as fundamental regulators of gene expression, but their mechanisms of action remain largely unknown. Among the tens of thousands of human lncRNAs, a subset of architectural RNAs (arcRNAs) function as structural scaffolds of membrane-less subnuclear organelles or nuclear bodies (NBs; Chujo et al, 2016). NBs are usually located in inter-chromatin spaces in the highly organized nucleus and consist of specific factors that function in various nuclear processes (Banani et al, 2017). ArcRNAs sequestrate sets of specific RNA-binding proteins to initiate building of specific NBs near transcription sites (Clemson et al, 2009; Chujo et al, 2016). Nuclear stress bodies (nSBs) were primarily reported 30 years ago (Mahl et al, 1989) as primate-specific NBs that respond to thermal and chemical stresses. The assembly of nSBs is initiated alongside HSF1-dependent transcription of the primate-specific highly repetitive satellite III (HSATIII) lncRNA (Jolly et al, 1999) and heat shock-induced HSF1 aggregation (Jolly et al, 1997). HSATIII lncRNAs are transcribed from pericentromeric HSATIII repeated arrays on several human chromosomes (Denegri et al, 2002; Jolly et al, 2002). These arrays are usually located in heterochromatinic regions that are transcriptionally silent; however, upon exposure to thermal stress, they are rapidly euchromatinized and produce HSATIII lncRNAs (Biamonti & Vourc'h, 2010). HSATIII lncRNAs are retained on chromosomes near their own transcription sites for several hours, even after stress removal, and recruit various RNA-binding proteins, including Scaffold attachment factor B (SAFB), HNRNPM, SRSF1, SRSF7, and SRSF9 (Weighardt et al, 1999; Denegri et al, 2001; Metz et al, 2004), as well as specific chromatin-remodeling factors (Kawaguchi et al, 2015; Hussong et al, 2017) and transcription factors (Jolly et al, 2004), which results in the assembly of nSBs. SRSF1, SRSF7, and SRSF9 are serine and arginine-rich (SR) pre-mRNA splicing factors (SRSFs) that contain one or two RNA recognition motifs and a signature RS domain, an intrinsically disordered stretch of multiple SR (in part, SP or SK) dipeptides, near the C-terminus (Long & Caceres, 2009; Shepard & Hertel, 2009). The RS domain is required for protein–protein interactions with other SRSFs or RS domain-harboring proteins (Xiao & Manley, 1997). In addition, the functions of SRSFs in pre-mRNA splicing and mRNA export are modulated through phosphorylation of the RS domain (Cao et al, 1997; Graveley, 2000; Huang et al, 2004). Notably, the phosphorylation state of the RS domain is dynamically changed in response to environmental transitions, such as thermal stress or circadian changes in body temperature (Guil & Caceres, 2007; Preussner et al, 2017). Upon exposure to thermal stress, protein phosphatase 1 is activated to de-phosphorylate the RS domains of SR proteins, leading to modulation of a wide variety of pre-mRNA splicing events (Shi & Manley, 2007). Concomitantly, CDC2-like kinases 1 and 4 (CLK1 and CLK4), members of the nuclear SR protein kinase family, are induced upon thermal stress exposure to promote re-phosphorylation of SRSFs after stress removal (Ninomiya et al, 2011). These reports raise the intriguing possibility that nSBs are involved in the regulatory network of thermal stress-responsive pre-mRNA splicing by concentrating or sequestering SRSFs; however, the function of nSBs remains poorly investigated. Here, we performed a genome-wide transcriptomic analysis of HeLa cells following knockdown of HSATIII lncRNAs to deplete nSBs and found that HSATIII mainly promotes intron retention by hundreds of genes. Further investigation revealed that nSBs accelerate intron retention specifically during recovery from thermal stress. We also explored the protein composition of nSBs using chromatin isolation by Comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) and an antisense oligonucleotide (ASO) of HSATIII lncRNA as a stable core component of nSBs. This analysis identified 141 proteins as putative nSB components in HeLa cells. Among them, we focused on CLK1, a SR protein kinase that is specifically recruited to nSBs during thermal recovery to re-phosphorylate SRSFs. Notably, we found that nSBs accelerate the rate of re-phosphorylation of SRSFs after stress removal. Phosphorylated SRSFs then promote retention of the HSATIII target introns. Based on our findings, we propose that nSBs serve as a platform to control the temperature-dependent phosphorylation states of selected SRSFs to regulate intron retention by hundreds of pre-mRNAs. The transcriptomic and subcellular proteomic analyses described here unveil the molecular events occurring in nSBs, which have remained enigmatic for the 30 years since their discovery. Results HSATIII lncRNAs facilitate intron retention in hundreds of mRNAs during stress recovery To investigate the role of nSBs in stress-dependent gene expression, particularly splicing regulation, we analyzed global transcriptomic changes in HSATIII-depleted HeLa cells (Fig 1A). Defective formation of two nSB subpopulations (Aly et al, 2019) was confirmed in HSATIII ASO-treated (HSATIII KD) cells exposed to thermal stress at 42°C for 2 h and recovery at 37°C for 1 h (Fig 1B and C). We confirmed that HSATIII depletion did not affect the integrity of other nuclear bodies such as nuclear speckles and paraspeckles (Fig EV1A–E). Subsequently, RNA sequencing (RNA-seq) was used to compare the transcriptomes of control and HSATIII KD cells. To efficiently detect changes in splicing patterns, nuclear poly(A)+ RNAs were prepared from the isolated nuclei of control and HSATIII KD cells during the thermal stress recovery phase, and the expression levels of each annotated exon and intron were compared (Fig 1A). The expression levels of 533 introns in 434 genes and 3 exons in three genes were significantly decreased upon HSATIII knockdown (HSATIII KD/control < 0.5; Fig 1D and E). Changes in the expression levels of exons were hardly detectable, with a few exceptions (Figs 1E and EV1F). In addition, the expression levels of 17 introns in 17 genes and 4 exons in four genes were significantly increased upon HSATIII knockdown (HSATIII KD/control > 2; Fig 1C and D). For example, HSATIII knockdown reduced the expression levels of introns 2 and 3 of TAF1D (Fig 1F, blue bars), and decreased and increased the expression levels of intron 2 (blue bar) and exon 3 (red bar) of DNAJB9, respectively (Fig 1G). Expression of the complete transcript of DNAJB9 was increased in HSATIII KD cells, suggesting that nSBs may also affect expression through HSATIII-independent mechanisms. These findings suggest that HSATIII lncRNAs mainly promote intron retention of pre-mRNAs during cell recovery from thermal stress. Figure 1. HSATIII lncRNAs control intron retention of a specific set of genes A. Outline of the screening for HSATIII-regulated genes during thermal stress recovery. HeLa cells were transfected with a HSATIII ASO (HSATIII KD) or HSATIII sense oligonucleotide (control), exposed to thermal stress. Nuclear polyA(+) RNAs were analyzed by next-generation sequencing (NGS). NGS data have been deposited in the DDBJ Sequence Read Archive (DRA) (accession number: DRA007304). B. HSATIII ASO-mediated depletion of nSBs. Thermal stress-exposed HeLa cells (42°C for 2 h and recovery for 1 h at 37°C) were visualized by HSATIII-FISH and immunofluorescence using an anti-SAFB antibody or anti-HNRNPM antibody. The nuclei were stained with DAPI. Scale bar: 10 μm. C. qRT–PCR validation of HSATIII knockdown. The graph shows the qRT–PCR level of HSATIII RNAs in control and HSATIII knockdown cells under three conditions: 37°C, 42°C for 2 h, and thermal stress followed by recovery at 37°C for 1 h (Recovery). Expression levels were calculated as ratios to GAPDH mRNA and were normalized to the levels in control cells under thermal stress conditions (42°C for 2 h). Data are shown as the mean ± SD (n = 3). HSATIII RNAs ratios (%) are indicated. D. MA plot (log2 fold change over the average expression level) of all detected introns in HSATIII KD and control cells (n = 3). The introns with significant changes in their expression levels (adjusted P-value < 0.01) are shown as green dots. Among these introns, up-regulated (log2 fold change > 1) and down-regulated (log2 fold change < −1) introns are shown with magenta and light blue dots, respectively. Genes and introns that were experimentally validated in subsequent experiments are indicated as gene symbol-i (intron) #. Total numbers of introns and genes utilized were 144,769 and 32,623, respectively. E. The numbers of introns and exons that were affected by HSATIII knockdown (fold change > 2). F, G. Examples of RNA-seq read maps of HSATIII target RNAs. Affected introns and exons are indicated by blue (down-regulated upon HSATIII knockdown) and magenta (up-regulated) boxes. H. Cumulative frequency curves of the lengths of adjacent exons of 399 HSATIII-up-regulated internal introns. The lengths of whole annotated internal introns are shown as a reference. Source data are available online for this figure. Source Data for Figure 1 [embj2019102729-sup-0006-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Effect of HSATIII knockdown on other nuclear bodies and gene expression A, B. Normal assembly of nuclear speckles upon HSATIII knockdown. 16 h after HSATIII ASO transfection, HeLa cells were exposed to thermal stress (42°C for 2 h followed by recovery for 1 h at 37°C) and stained using anti-SRSF2 antibody with the HSATIII-FISH probe (A) or anti-SRSF3 antibody (B). Nuclei were stained with DAPI. Scale bar: 10 μm. C. Normal assembly of paraspeckles upon HSATIII knockdown. 16 h after HSATIII ASO transfection, HeLa cells were exposed to thermal stress and stained using NEAT1 ASOs and anti-SFPQ antibody as paraspeckle markers. Scale bar: 10 μm. D, E. Box plot of total area of nuclear speckles (D) and paraspeckles (E) in each nucleus. Nuclear speckles, paraspeckles, and nuclei areas are defined by binarized images of SRSF2, NEAT1, and DAPI, respectively. Mean is indicated by X (n = 39 (SRSF2, control), 44 (SRSF2, HSATIII KD), 30 (NEAT1, control), 50 (NEAT1, HSATIII KD) nuclei). The first and third quartiles are the ends of the box, the median is indicated with the vertical line in the box, and the minimum and maximum are the ends of the whiskers. The outliers are indicated with open circles. P-values (Mann–Whitney U-test) are shown above the graphs. F. MA plot (log2 fold change over average expression level) of all detected exons (n = 3). Significant change (green), significant and > 2-fold increase (magenta), and decrease (light blue). Total numbers of exons and genes utilized are 162,989 and 32,623, respectively. Source data are available online for this figure. Download figure Download PowerPoint We noticed that the sizes of the exons adjacent to HSATIII-targeted introns tended to be longer than the average size of exons (Fig 1H, see also Fig EV5F). To avoid sampling bias caused by the first and the last exons, which tend to be longer than internal exons, we compared the sizes of the exons adjacent to 399 HSATIII-targeted internal introns with those adjacent to 175,923 whole annotated human internal introns. Overall, 8.8% of the exons upstream and 10.3% of the exons downstream of HSATIII-targeted introns were longer than 1 kb (Fig 1H). By contrast, only 0.7% of exons adjacent to the whole annotated internal introns were longer than 1 kb, confirming that the exons adjacent to HSATIII-targeted introns tended to be longer than the average exon size. A gene ontology analysis of 434 genes in which intron retention was promoted by HSATIII revealed a significant enrichment (FDR < 0.05) of genes associated with multiple functions, including DNA/RNA metabolism, biosynthesis, stress response, and cell cycle (Table EV1). Only 57 and 30 of the 533 HSATIII-targeted introns were previously identified as retained introns (also referred to as detained introns) in the human ENCODE database and a human glioblastoma cell line, respectively (Table EV2, discussed later; Boutz et al, 2015; Braun et al, 2017). HSATIII lncRNAs promote intron retention during thermal stress recovery To validate the RNA-seq data, we used CLK1 intron 3 and randomly selected 10 introns that were affected by HSATIII knockdown (10 down-regulated (fold change < −2) and 1 up-regulated (fold change > 2); mean expression level > 1e2) (Fig 1D) for quantitative RT–PCR (qRT–PCR) to analyze their levels in total RNAs from control and HSATIII KD cells. The levels of the intron-retaining pre-mRNAs were examined under three conditions: normal (37°C), thermal stress at 42°C for 2 h, and thermal stress at 42°C for 2 h followed by recovery at 37°C for 1 h (Fig 2A). For all down-regulated introns examined, the levels of intron retention were comparable in control and HSATIII KD cells during thermal stress, but were significantly lower in HSATIII KD cells than in control cells during the recovery phase (Fig 2A, upper panel). Notably, we recognized two distinct classes of intron retention during temperature transition: Class 1 introns were retained at substantial levels under normal conditions, excised during thermal stress, and re-accumulated during stress recovery; and class 2 introns were mostly excised under normal and thermal stress conditions, but retained during stress recovery (Fig 2A). Intron 1 of PFKP was an exceptional up-regulated intron that was retained during stress recovery of HSATIII KD cells (Fig 2A). A qRT–PCR analysis of subcellularly fractionated nuclear and cytoplasmic RNAs confirmed that all of the intron-retaining pre-mRNAs mentioned above were retained in the nucleus (Fig 2B), suggesting that mRNA export is prevented by the intron retention. In contrast to the marked effect on the levels of intron-retaining pre-mRNAs, HSATIII knockdown scarcely affected the levels of the cognate intron-removed (spliced) mRNAs. As exceptions, the levels of the spliced DNAJB9, CLK1, and PFKP mRNAs were significantly higher (CLK1, DNAJB9) or lower (PFKP) in HSATIII KD cells than in control cells (Fig 2A, lower panel). Figure 2. HSATIII lncRNAs control intron retention by regulating splicing A. Validation of HSATIII target introns by qRT–PCR. The graphs show the relative amounts of the intron-retaining (IR) (upper) and spliced (lower) forms in control and HSATIII knockdown cells under three conditions: 37°C (normal), 42°C for 2 h (thermal stress), and thermal stress followed by recovery at 37°C for 1 h. Expression levels were calculated as the ratio of each RNA to GAPDH mRNA and were normalized to the levels in control cells under normal conditions (37°C). Data are shown as the mean ± SD (n = 3); *P < 0.05 (Sidak's multiple comparison test). B. Nuclear localization of the intron-retaining RNAs. The relative amounts of intron-retaining RNAs in the nuclear and cytoplasmic fractions were quantified by qRT–PCR and are represented as the ratio (% of the total). GAPDH mRNA and U1 snRNA were used as cytoplasmic and nuclear controls, respectively. Data are shown as the mean ± SD (n = 3). C. Overview of the qRT–PCR analysis of newly synthesized RNA within 1 h after thermal stress removal. CHX, cycloheximide; EU, 5-ethynyl uridine. D, E. The levels of HSATIII target introns in newly synthesized RNAs within 1 h after thermal stress removal, as determined by qRT–PCR. The graphs show the changes in the expression levels of the intron-retaining (IR) (D) and spliced (E) forms. Expression levels were calculated as the ratio of each RNA to GAPDH mRNA and were normalized to the level in the control cells. Data are shown as the mean ± SD (n = 3); *P < 0.05 (multiple t-test modified by Holm–Sidak's method). Download figure Download PowerPoint Next, the levels of the intron-retaining pre-mRNAs and the spliced mRNAs were measured in newly synthesized nascent RNA pools captured by pulse-labeled RNAs with ethynyl uridine (see Fig 2C and Materials and Methods). As shown in Fig 2D and E, with the exception of PFKP, the levels of the intron-retaining pre-mRNAs were lower in HSATIII KD cells than in control cells. By contrast, with the exceptions of EP400 and PFKP, the levels of the spliced mRNAs were significantly higher in HSATIII KD cells than in control cells. Overall, these data suggest that HSATIII promotes intron retention by suppressing splicing of newly synthesized transcripts. HSATIII affects the kinetics of accumulation of intron-retaining pre-mRNAs during thermal recovery Among the retained introns regulated by HSATIII, a subpopulation of the CLK1 mRNA reportedly localizes in the nucleus as a partially unspliced pre-mRNA that retains introns 3 and 4 (Fig 3A; Duncan et al, 1995; Ninomiya et al, 2011). Excision of retained introns is induced by thermal and osmotic stresses, neuronal activity, and inhibition of CLK1 kinase activity to produce mature mRNAs (Ninomiya et al, 2011; Mauger et al, 2016). We also detected another spliced isoform of the CLK1 mRNA produced by skipping of exon 4, which is committed to nonsense-mediated mRNA decay (Fig 3A). Consequently, we examined the effect of HSATIII knockdown on thermal stress-responsive excision of the retained introns of the CLK1 pre-mRNA at several time points using semi-quantitative RT–PCR. As reported previously (Ninomiya et al, 2011), the retained introns were excised to form the mature mRNA in both control and HSATIII KD cells after a 2-h thermal stress exposure (Fig 3B, 42°C, 2 h). In control cells, the level of the intron 3 and 4-retaining CLK1 pre-mRNA was restored within 1 h after stress removal (Fig 3B, lanes 6–10, and C). Notably, this process was markedly delayed in HSATIII KD cells, in which restoration of the original level of the intron 3 and 4-retaining CLK1 pre-mRNA took longer than 4 h (Fig 3B, lanes 1–5, and C). Figure 3. The HSATIII lncRNA is necessary and sufficient to promote nuclear intron retention Splicing isoforms of the CLK1 pre-mRNA. The retained introns are indicated by red lines. An asterisk indicates the position of the premature termination codon in the nonsense-mediated mRNA decay-targeted isoform. Time course analysis of the splicing pattern of CLK1 pre-mRNAs in control and HSATIII knockdown (HSATIII KD) cells by semi-quantitative RT–PCR. Arrows indicate the positions of PCR primers. The GAPDH mRNA was used as an internal control. Quantification of the data shown in (B). Data are shown as the mean ± SD (n = 3); *P < 0.05 (Sidak's multiple comparison test). Thermal stress-induced expression of HSATIII in CHO (His9) cells. The cells were visualized by HSATIII-FISH (green), and the nuclei were stained with DAPI (blue). Scale bar: 10 μm. Ectopic HSATIII-induced nSB assembly in CHO (His9) cells. The nSBs (yellow arrowheads) were visualized by HSATIII-FISH and immunofluorescence using an anti-SRSF1 antibody. The nuclei were stained with DAPI. Scale bar: 10 μm. Time course analysis of the splicing pattern of Chinese hamster Clk1 pre-mRNAs in control (CHO) and CHO (His9) cells by semi-quantitative RT–PCR. Arrows indicate the positions of PCR primers. The GAPDH mRNA was used as an internal control. Quantification of the data shown in (F). Data are shown as the mean ± SD (n = 3); *P < 0.05 (Sidak's multiple comparison test). The effect of HSATIII knockdown on pooling of intron-retaining RNAs in control (CHO) and CHO (His9) cells (42°C for 2 h and recovery for 2 h at 37°C). Arrows indicate the positions of PCR primers. Source data are available online for this figure. Source Data for Figure 3 [embj2019102729-sup-0007-SDataFig3.pdf] Download figure Download PowerPoint Like CLK1, TAF1D has two HSATIII target introns (Fig 1E) that border a cassette-type exon 3 (Fig EV2A). Semi-quantitative RT–PCR confirmed that the partially unspliced TAF1D pre-mRNA retaining introns 2 and 3 underwent splicing upon thermal stress and was rapidly re-accumulated after stress removal in control cells (Fig EV2B). However, this re-accumulation was markedly delayed in HSATIII KD cells (Fig EV2B). In addition, we also examined the levels of the DNAJB9 mRNA, another intron-retaining RNA that harbors a single HSATIII target intron (Fig EV2A), and found that intron retention was detectable in control cells but not HSATIII KD cells (Fig EV2B). We also confirmed that the thermal stress-responsive alternative splicing of the HSP105 and TNRC6a mRNAs reported previously (Yasuda et al, 1995; Yamamoto et al, 2016) was barely affected by HSATIII knockdown (Fig EV2C), indicating selectivity of target pre-mRNAs by HSATIII lncRNAs. Click here to expand this figure. Figure EV2. Effect of HSATIII knockdown on intron retention of TAF1D and DNAJB9 pre-mRNAs Splicing isoforms of TAF1D and DNAJB9. The retained introns are indicated by red lines. An asterisk indicates the position of the premature termination codon (PTC) in the NMD-targeted isoform. Time course analysis of splicing pattern of TAF1D and DNAJB9 pre-mRNAs in control and HSATIII knockdown (HSATIII KD) cells by semi-quantitative RT–PCR. Arrows indicate the positions of PCR primers. GAPDH mRNA was used as internal control. Specificity of the HSATIII-dependent splicing control. Semi-quantitative RT–PCR validation of the splicing pattern of newly synthesized RNAs within 1 h after thermal stress removal (see also Fig 2C). Arrows indicate the positions of PCR primers. CLK1 and GAPDH pre-mRNAs were used as positive and internal controls, respectively. Source data are available online for this figure. Download figure Download PowerPoint To further support the proposal that HSATIII lncRNAs promote intron retention, we used a gain-of-function approach using a somatic hybrid Chinese hamster ovary (CHO) cell line possessing human chromosome 9 (CHO (His9)) (Tanabe et al, 2000). Since HSATIII is primate-specific, these lncRNAs are only expressed from human chromosome 9 in CHO (His9) cells upon thermal stress and form nSB-like granules with hamster SRSF1 (Fig 3D and E). A time course analysis of the splicing pattern of the endogenous hamster Clk1 mRNA revealed that re-accumulation of the intron-retaining pre-mRNA after stress removal was markedly faster in CHO (His9) cells than in control CHO cells (Fig 3F and G). Notably, HSATIII knockdown abolished the acceleration effect in CHO (His9) cells (Fig 3H). Taken together, these data suggest that HSATIII lncRNAs are required and sufficient for acceleration of intron retention of the CLK1 pre-mRNA during stress recovery, and this process was likely acquired in primate species. Multiple SRSFs and SR-related proteins interact with HSATIII in nSBs To examine the molecular mechanism of intron retention by HSATIII lncRNAs, we attempted to comprehensively identify the proteins associated with HSATIII, which likely correspond to nSB components. ChIRP was employed to pull down the ribonucleoprotein (RNP) complexes of HSATIII using a biotinylated ASO (Fig 4A). Because HSATIII lncRNAs consist of multiple GGAAU repetitive sequences (Valgardsdottir et al, 2005; Jarmuz et al, 2007), HSATIII RNP complexes could be efficiently captured by a single biotinylated HSATIII ASO consisting of four tandem repeats of ATTCC. ChIRP was carried out using formaldehyde-crosslinked HeLa cells that were treated at 42°C for 2 h followed by recovery at 37°C for 1 h. A RT–PCR analysis revealed efficient (> 10%) and specific precipitation of HSATIII with the HSATIII ASO from thermally stressed cells (Fig 4B). As a negative control, neither the NEAT1 nuclear lncRNA nor the GAPDH mRNA was precipitated with the HSATIII ASO (Fig 4B). Silver staining of the coprecipitated proteins in a SDS–PAGE gel identified multiple bands that were strongly detected in the stressed cells, but were only detected as faint background signals in the control samples (Fig 4C). A liquid chromatography–mass spectrometry analysis revealed that 141 proteins, most of which that have not yet been reported as nSB components, were specifically coprecipitated with HSATIII lncRNAs from the stressed cells (Table EV3). Most of these proteins are likely RNA-binding proteins that possess canonical RNA-binding domains. A gene ontology analysis revealed significant enrichment of proteins functionally asso
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