NF45/NF90‐mediated rDNA transcription provides a novel target for immunosuppressant development
2021; Springer Nature; Volume: 13; Issue: 3 Linguagem: Inglês
10.15252/emmm.202012834
ISSN1757-4684
AutoresHsiang‐i Tsai, Xiaobin Zeng, Longshan Liu, Shengchang Xin, Yingyi Wu, Zhanxue Xu, Huanxi Zhang, Liu Gan, Zirong Bi, Dandan Su, Min Yang, Yijing Tao, Changxi Wang, Jing Crystal Zhao, John Eriksson, Wenbin Deng, Fang Cheng, Hongbo Chen,
Tópico(s)RNA regulation and disease
ResumoArticle8 February 2021Open Access Source DataTransparent process NF45/NF90-mediated rDNA transcription provides a novel target for immunosuppressant development Hsiang-i Tsai Hsiang-i Tsai orcid.org/0000-0002-4233-1428 School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, ChinaThese authors contributed equally to this work Search for more papers by this author Xiaobin Zeng Xiaobin Zeng orcid.org/0000-0002-3111-9202 Center Lab of Longhua Branch and Department of Infectious Disease, Shenzhen People's Hospital, 2nd Clinical Medical College of Jinan University, Shenzhen, China Guangdong Provincial Key Laboratory of Regional Immunity and Diseases, Medicine School of Shenzhen University, Shenzhen, ChinaThese authors contributed equally to this work Search for more papers by this author Longshan Liu Longshan Liu Organ Transplant Centerm, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, ChinaThese authors contributed equally to this work Search for more papers by this author Shengchang Xin Shengchang Xin State Key Laboratory of Coordination Chemistry, Institute of Chemistry and Biomedical Sciences, School of Life Sciences, Nanjing University, Nanjing, China Search for more papers by this author Yingyi Wu Yingyi Wu School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, China Search for more papers by this author Zhanxue Xu Zhanxue Xu School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, China Search for more papers by this author Huanxi Zhang Huanxi Zhang Organ Transplant Centerm, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Gan Liu Gan Liu School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, China Search for more papers by this author Zirong Bi Zirong Bi Organ Transplant Centerm, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Dandan Su Dandan Su School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, China Search for more papers by this author Min Yang Min Yang School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, China Search for more papers by this author Yijing Tao Yijing Tao School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, China Search for more papers by this author Changxi Wang Changxi Wang Organ Transplant Centerm, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Jing Zhao Jing Zhao State Key Laboratory of Coordination Chemistry, Institute of Chemistry and Biomedical Sciences, School of Life Sciences, Nanjing University, Nanjing, China Search for more papers by this author John E Eriksson John E Eriksson Cell Biology, Biosciences, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland Search for more papers by this author Wenbin Deng Corresponding Author Wenbin Deng [email protected] School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, China Search for more papers by this author Fang Cheng Corresponding Author Fang Cheng [email protected] School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, China Search for more papers by this author Hongbo Chen Corresponding Author Hongbo Chen [email protected] orcid.org/0000-0002-0954-5600 School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, China Search for more papers by this author Hsiang-i Tsai Hsiang-i Tsai orcid.org/0000-0002-4233-1428 School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, ChinaThese authors contributed equally to this work Search for more papers by this author Xiaobin Zeng Xiaobin Zeng orcid.org/0000-0002-3111-9202 Center Lab of Longhua Branch and Department of Infectious Disease, Shenzhen People's Hospital, 2nd Clinical Medical College of Jinan University, Shenzhen, China Guangdong Provincial Key Laboratory of Regional Immunity and Diseases, Medicine School of Shenzhen University, Shenzhen, ChinaThese authors contributed equally to this work Search for more papers by this author Longshan Liu Longshan Liu Organ Transplant Centerm, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, ChinaThese authors contributed equally to this work Search for more papers by this author Shengchang Xin Shengchang Xin State Key Laboratory of Coordination Chemistry, Institute of Chemistry and Biomedical Sciences, School of Life Sciences, Nanjing University, Nanjing, China Search for more papers by this author Yingyi Wu Yingyi Wu School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, China Search for more papers by this author Zhanxue Xu Zhanxue Xu School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, China Search for more papers by this author Huanxi Zhang Huanxi Zhang Organ Transplant Centerm, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Gan Liu Gan Liu School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, China Search for more papers by this author Zirong Bi Zirong Bi Organ Transplant Centerm, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Dandan Su Dandan Su School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, China Search for more papers by this author Min Yang Min Yang School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, China Search for more papers by this author Yijing Tao Yijing Tao School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, China Search for more papers by this author Changxi Wang Changxi Wang Organ Transplant Centerm, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Jing Zhao Jing Zhao State Key Laboratory of Coordination Chemistry, Institute of Chemistry and Biomedical Sciences, School of Life Sciences, Nanjing University, Nanjing, China Search for more papers by this author John E Eriksson John E Eriksson Cell Biology, Biosciences, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland Search for more papers by this author Wenbin Deng Corresponding Author Wenbin Deng [email protected] School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, China Search for more papers by this author Fang Cheng Corresponding Author Fang Cheng [email protected] School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, China Search for more papers by this author Hongbo Chen Corresponding Author Hongbo Chen [email protected] orcid.org/0000-0002-0954-5600 School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, China Search for more papers by this author Author Information Hsiang-i Tsai1, Xiaobin Zeng2,3, Longshan Liu4, Shengchang Xin5, Yingyi Wu1, Zhanxue Xu1, Huanxi Zhang4, Gan Liu1, Zirong Bi4, Dandan Su1, Min Yang1, Yijing Tao1, Changxi Wang4, Jing Zhao5, John E Eriksson6,7, Wenbin Deng *,1, Fang Cheng *,1 and Hongbo Chen *,1 1School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen, China 2Center Lab of Longhua Branch and Department of Infectious Disease, Shenzhen People's Hospital, 2nd Clinical Medical College of Jinan University, Shenzhen, China 3Guangdong Provincial Key Laboratory of Regional Immunity and Diseases, Medicine School of Shenzhen University, Shenzhen, China 4Organ Transplant Centerm, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China 5State Key Laboratory of Coordination Chemistry, Institute of Chemistry and Biomedical Sciences, School of Life Sciences, Nanjing University, Nanjing, China 6Cell Biology, Biosciences, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland 7Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland *Corresponding author. Tel: +86 15071561390; E-mail: [email protected] *Corresponding author. Tel: +86 18123846151; E-mail: [email protected] *Corresponding author. Tel: +86 15889353410; E-mail: [email protected] EMBO Mol Med (2021)13:e12834https://doi.org/10.15252/emmm.202012834 PDFDownload PDF of article text and main figures. 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 Herein, we demonstrate that NFAT, a key regulator of the immune response, translocates from cytoplasm to nucleolus and interacts with NF45/NF90 complex to collaboratively promote rDNA transcription via triggering the directly binding of NF45/NF90 to the ARRE2-like sequences in rDNA promoter upon T-cell activation in vitro. The elevated pre-rRNA level of T cells is also observed in both mouse heart or skin transplantation models and in kidney transplanted patients. Importantly, T-cell activation can be significantly suppressed by inhibiting NF45/NF90-dependent rDNA transcription. Amazingly, CX5461, a rDNA transcription-specific inhibitor, outperformed FK506, the most commonly used immunosuppressant, both in terms of potency and off-target activity (i.e., toxicity), as demonstrated by a series of skin and heart allograft models. Collectively, this reveals NF45/NF90-mediated rDNA transcription as a novel signaling pathway essential for T-cell activation and as a new target for the development of safe and effective immunosuppressants. Synopsis This study reveals NFAT-NF45/NF90-mediated rDNA transcription as a key regulating axis in modulating T cell activation. Targeting ribosome biogenesis could be a novel immunosuppressive therapy for organ transplantation. NF45/NF90 protein complex positively regulates rDNA transcription by directly binding to rDNA gene promoter. Upon T cell activation, NAFT translocates to nucleolus and interacts with NF45/NF90 to cooperatively promote rDNA transcription. Knockdown of NF45/NF90 or inhibiting rDNA transcription using a polymerase I inhibitor CX5461 suppresses T cell activation. CX5461 treatment prolongs the survival of mouse skin or heart allografts even more than the most commonly used suppressant FK506. The paper explained Problem T cells are highly activated and play a central role for transplant rejection. Two potent immunosuppressants, cyclosporin A, and FK506, block the calcineurin-NFAT-dependent T-cell activation in organ transplantation. However, the serious side effects limit the clinical use of both drugs. Clinically applicable strategies for inhibiting T-cell activation through alternative regulatory mechanisms need to be developed to achieve long-term graft tolerance. The heterodimeric nuclear complex NF45/NF90 binds to an NFAT response element in the IL-2 promoter and regulates IL-2 activation in Jurkat T cells. The current study explores whether and how NF45/NF90 complex mediate T-cell activation in experimental transplantation. Results Here, we show that the NF45/NF90 complex is preferentially localized in the nucleoli and directly binds to an ARRE2-like sequence in rDNA promoter. Upon T-cell activation, NFAT translocates from cytoplasm to the nucleolus and cooperatively promotes rDNA transcription by interacting with NF45/90. NF45/NF90 knockdown significantly suppresses rDNA transcription and T-cell activation, indicating NF45/NF90-mediated rDNA transcription plays a key role in the classical calcineurin-NFAT T-cell activation pathway. Excitingly, a specific rRNA synthesis inhibitor CX5461 shows a more dramatic inhibition effect on T-cell activation and leads to a longer survival time of skin and heart allografts than the most commonly used calcineurin inhibitor FK506. Impact Our study offers insights into the role and molecular mechanism of NFAT and NF45/NF90 in modulating T-cell activation. We propose that preventing rDNA transcription and ribosome biogenesis may be a promising strategy to prevent organ transplant rejection. Introduction The inhibition of T-cell activation is crucial for both the prevention of organ transplantation rejection and graft-versus-host disease (GVHD) that accompanies allogeneic hematopoietic stem cell transplantation, as well as in the treatment of certain autoimmune diseases (Ichiki et al, 2006; Coghill et al, 2011; Petrelli & Van Wijk, 2016; Szyska & Na, 2016). The calcineurin-nuclear factor of activated T cells (NFAT) binding inhibitors cyclosporine A (CsA) and tacrolimus (FK506) have proven highly effective at suppressing T-cell response to allografts and are among the most widely used immunosuppressive drugs to significantly prolong graft survival and reduce patient morbidity (Monostory, 2018). The use of these immunosuppressive agents has also been reported in a variety of autoimmune diseases (Kovarik & Burtin, 2003). Despite their widespread application in the clinic, calcineurin inhibitors have been the cause of myriad side effects, including nephrotoxicity, chronic kidney damage, and post-transplant malignancies (Group et al, 2018). This may be the result of the general inhibition of calcineurin activity, which plays other biologically important roles besides NFAT activation. The discovery of novel mechanisms of early T-cell activation that obviate the inhibition of calcineurin is therefore of great value in the search for safer and more efficient immunosuppressive agents. The Ca2+-calcineurin-NFAT signaling pathway is a master regulator of T-cell proliferation and activation (Okeefe et al, 1992). Five NFAT family members have been identified, namely NFATc1 (also known as NFAT2), NFATc2 (NFAT1), NFATc3 (NFAT4), NFATc4 (NFAT3), and TonEBP (tonicity-responsive enhancer [TonE] binding protein, or NFAT5), among them. NFATC1, NFATc2, and NFATc3 are well-known to play important roles in T-cell activation (Timmerman et al, 1997; Macian, 2005; Lee et al, 2018). During an adaptive immune response, phosphatase calcineurin dephosphorylates NFAT within T cells. The dephosphorylated NFAT then translocate from the cytoplasm to the nucleus, where they bind directly to antigen response recognition element (ARRE2) within the interleukin-2 (IL-2) enhancer region. This in turn induces IL-2 gene transcription (Jain et al, 1993), an essential cytokine for the clonal proliferation and activation of T lymphocytes (Broere & van Eden, 2019). The calcineurin inhibitors CsA and FK506 prevent the calcineurin-driven dephosphorylation of NFAT, thereby inhibiting NFAT nuclear accumulation, IL-2 expression, and the downstream functions of effector T cells. The nuclear factors NF45 and NF90 were originally isolated in activated Jurkat cells as a heterodimeric complex (NF45/NF90) binding specifically to the ARRE2 enhancer element of the IL-2 promoter (Kao et al, 1994). Both NF45 and NF90 have an N-terminal "domain-associated with zinc fingers" (DZF) that resembles template-free nucleotidyltransferases and mediates the heterodimerization of NF45/NF90 through a structurally conserved interface (Wolkowicz & Cook, 2012). Moreover, NF90 has one nuclear localization signal (NLS) domain and two double-stranded RNA binding domains (dsRBDs) in the C-terminal region (Wen et al, 2014), which confer binding to highly structured RNAs (Parker et al, 2001). In mammals, the NF45/NF90 protein complex is expressed in a wide variety of tissues (Zhao et al, 2005) and participates in numerous cellular functions (Shim et al, 2002), including cell cycle regulation (Guan et al, 2008), transcription activation (Kiesler et al, 2010), translational control (Castella et al, 2015), DNA damage response (Shamanna et al, 2011), microRNA (miRNA) biogenesis (Masuda et al, 2013), and viral infection (Idda et al, 2019). However, the specific role of NF45/NF90 in the regulation of T-cell activation has not been well established. In this study, a novel mechanism for the regulation of rDNA transcription in the nucleolus is presented, in which the NF45/NF90 complex plays a key role by interacting with the upstream binding factor (UBF) and promoting the recruitment of RNA Pol I to rDNA promoter regions. We found that the NF45/NF90 regulation of rRNA transcription positively regulates T-cell activation. Suppressing rDNA transcription using the Pol I inhibitor CX5461 significantly inhibited IL-2 secretion, reduced the proliferation of T lymphocytes, and enhanced the survival of mouse skin and heart allografts. This mechanism constitutes a novel therapeutic strategy for immunosuppression. Results NF45 and NF90 are nucleolar proteins that positively regulate rDNA transcription Mass spectrometry has been used as a diagnostic tool to confirm the presence of both NF45 and NF90 in the nucleolar proteome (Wandrey et al, 2015). In the present study, the nucleolar enrichment of endogenous NF45 and NF90 proteins was confirmed by subcellular fractionation, and an aggregate morphology was observed to colocalize in the nucleolar markers fibrillarin and nucleolin, indicating that NF45 and NF90 are indeed nucleolar proteins compared to nucleoplasm (lamin B1 is a nucleoplasm marker; Fig 1A and Appendix Fig S1A). The primary function of a nucleolus is ribosome biogenesis (including the transcription of rRNAs from rDNAs), the folding, processing, and modification of rRNAs, as well as the assembly of major ribosomal proteins. Intriguingly, the NF45/NF90 heterodimer was recently revealed as a novel regulator of ribosome biogenesis. A luciferase reporter containing the rDNA promoter was therefore used to assess the relationship between NF45/NF90 and rRNA synthesis. Inhibition of NF45/NF90 by shRNA lentivirus downregulated the luciferase signal, whereas ectopic expression of NF45/NF90 enhanced luciferase activity, suggesting that NF45/NF90 is a positive regulator of rDNA synthesis (Fig 1B and C). Figure 1. NF45/NF90 is a nucleolar protein complex that positively regulates rDNA transcription A. Confocal images of HeLa cells stained with NF45/NF90 antibodies and antibodies against nucleolin and fibrillarin. Scale bar, 10 μm. B. HEK293 cells were infected with lentiviruses containing negative control shRNA (shNC) or NF45/NF90 specific shRNA (shNF45#1, shNF45#2, shNF90#1, and shNF90#2) for 24 h and then transfected with rDNA-Luc. Luciferase activity was measured after a further 24 h (n = 3). Western blots show the protein levels of NF45 and NF90. C. Empty vector (EV), FLAG-NF45 plasmid (NF45), or FLAG-NF90 plasmid (NF90) were transfected into HEK293 cells for 24 h, and then, rDNA-Luc was cotransfected into the cells. Luciferase activity was measured after a further 24 h (n = 3). Western blots show the protein levels of FLAG-NF45 and FLAG-NF90. D. The line indicates the position of qPCR primer on the 45S pre-rRNA. E, F. The qPCR analysis of 45S pre-rRNA from HEK293 cells treated with indicated shRNAs (E) and plasmids (F) for 24 h. Western blots show the expression levels of NF45, NF90, FLAG-NF45, and FLAG-NF90. (n = 3) Data information: In all panels, bars and error bars represent mean ± SD. Statistical analysis by unpaired Student t-test (C, F). One-way ANOVA analysis followed by Tukey's test (B, E). (*P < 0.05, ***P < 0.001). Exact P values are reported in Appendix Table S3. Source data are available online for this figure. Source Data for Figure 1 [emmm202012834-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint Transcription of the 45S rRNA precursor, 45S pre-rRNA (comprised of the externally transcribed spacers 5′-ETS and 3′-ETS), is a rate-limiting step in ribosome biogenesis, which is followed by the cleavage of 45S pre-rRNA into several smaller rRNAs (18S, 5.8S and 28S rRNAs) (Tiku & Antebi, 2018). We therefore designed primers to target the relevant regions of 45S pre-rRNA in order to measure its rate of transcription (Fig 1D). Consistent with the data from the rDNA promoter-driven luciferase assay, real-time PCR analysis showed that silencing NF45 and NF90 significantly slowed down pre-rRNA transcription, and upregulation of NF45/NF90 dramatically strengthened the process (Fig 1E and F). Silencing NF45/NF90 also suppressed nucleolar FUrd (fluorine-conjugated UTP analogue) incorporation (Appendix Fig S1B), further supporting the regulatory role of NF45/NF90 in rRNA synthesis. It is known that within the NF45/NF90 heterodimer complex, NF90 contains a DZF motif (NF45 binding domain) and an NLS motif, as well as two RBD domains (RNA binding domains) which can directly bind RNA or DNA (Appendix Fig S2A). In order to investigate the involvement of the individual domains in rRNA synthesis, we inactivated the DZF and RBD domains using various point mutations and truncated the NLS motif to prevent its location in the nucleolus (Appendix Fig S2A–D). Interestingly, the DZF mutant completely reversed the effect of NF90 on transcription of pre-rRNA, indicating that binding to NF45 supports the NF90 regulation of rRNA synthesis (Appendix Fig S2E). Importantly, the NLS mutant also lost the ability to upregulate 45S pre-rRNA levels, demonstrating that the nucleolar localization of NF90 is required for moderating rRNA synthesis (Appendix Fig S2E). Surprisingly, the RBD mutant also resulted in the obvious translocation of NF90 from the nucleolus to the nucleoplasm and downregulated 45S pre-rRNA levels by 12- and 7.7-fold compared to NF90 and the empty vector, respectively (Appendix Fig S2E), suggesting that the RBD mutant of NF90 not only has a lower binding ability to rDNA and rRNA, but also serves as a dominant negative mutant to interfere with wild-type NF45/NF90 functions in the nucleolus. NF45/NF90 directly binds rDNA promoters by recognizing ARRE2-like sequence and regulates RNA Pol I transcription machinery To examine the protein occupancies of NF45 and NF90 at the rDNA loci, chromatin immunoprecipitation (ChIP) experiments were performed (Fig 2A). NF45 and NF90 binding were enhanced by approximately 3- to 5.2-fold and 3.8- to 8.7-fold respectively, compared with the IgG control (Fig 2B), suggesting that NF45/NF90 might regulate rDNA transcription through direct binding to the rDNA loci, especially the rDNA promoter region (H42.9). Since the NF45/NF90 complex has been shown to recognize upstream ARRE2 in the human IL-2 promoter (Shi et al, 2007b), the previous report revealed that ARRE-2 site in the IL-2 promoter contains three conserved regions, including a 5'purine NFAT binding site, a AP-1 binding site in the core region, and a 3'purine sequences (Nirula et al, 1997). We analyzed the promoter of rDNA gene and found that there was a similar ARRE2 region (named ARRE2-like sequence) at nucleotide positions −1,830 to −1,757 from the transcription start site (Fig 2C). As expected, an electrophoretic mobility shift assay (EMSA) and luciferase assay confirmed that the ARRE2-like elements at the rDNA promoter loci are required for NF90 interaction and regulation of rDNA promoter activity (Fig 2D and E). Figure 2. NF45/NF90 preferentially binds to the promoter region of the rDNA gene loci by directly recognizing the ARRE2-like sequence Schematic illustration of a single human rDNA gene repeat and positions of the primers used for ChIP. In Jurkat cells, a ChIP assay was performed with control IgG, NF45, and NF90 antibodies, and then, the precipitated DNA was analyzed using qPCR with the aforementioned primers. The relative rDNA fold enrichment was normalized to control IgG treatment (n = 3). The human rDNA promoter region contains one ARRE2-like sequence. The red letters in gray shaded area indicate the probe sequences used in panel D. Gray shaded area indicates the upstream control element. Blue color indicates the core promoter region. EMSA assay. Lane 1) Biotin-labeled rDNA probe. Lane 2) Biotin-labled rDNA probe + nuclear protein. Lane 3) Biotin-labled rDNA probe + nuclear protein + 100-fold molar excess of biotin-unlabeled specific competitor. Lane 4) Biotin-labled rDNA probe + nuclear protein + 100-fold molar excess of biotin-unlabeled mutant competitor. Lane 5) Biotin-labled rDNA probe + nuclear protein + NF90 antibody. Lane 6) Biotin-labled rDNA probe + nuclear protein + NF45 antibody. The details were described in Materials and Methods section. The relative luciferase activity of HEK293T cells transfected with empty vector (EV) or FLAG-NF90 (NF90) in combination with rDNA-Luc wild-type (WT) or mutated rDNA-Luc reporter plasmid (Mutant) for 24 h. (n = 3) Western blot shows FLAG-NF90 protein level. Data information: In all panels, bars and error bars represent mean ± SD. Statistical analysis by unpaired Student t-test (E) (***P < 0.001). Exact P values are reported in Appendix Table S3. Source data are available online for this figure. Source Data for Figure 2 [emmm202012834-sup-0003-SDataFig2.zip] Download figure Download PowerPoint The regulatory mechanism of rDNA transcription requires the synergistic transaction of UBF followed by promoter selectivity factor 1 (SL1) in order to recruit RNA polymerase I (Pol I) to sit on the rDNA promoter (Fig 3A) (Friedrich et al, 2005). Interestingly, in a co-immunoprecipitation (co-IP) assay, NF45/NF90 had a stronger binding affinity with UBF than a panel of other major nucleolar proteins including S5, L9, B23, and C23 (Fig 3B and C). As UBF activity correlates with phosphorylation and ubiquitination (Zhang et al, 2011), the NF45 and NF90 proteins in HEK293 cells were silenced in order to test their involvement in UBF post-translational modification. In separate experiments, both NF45 and NF90 knockdown decreased the expression level of total UBF upon serum stimulation (Fig 3D), indicating NF45/NF90 is important for UBF stability. Expectedly, ubiquitination assay found NF90 knockdown caused a K48-linked ubiquitination degradation of UBF protein (Appendix Fig S3A–C). Since UBF activates Pol I to promote rDNA transcription, RNA pol I levels were also examined upon depletion of NF45/NF90. A relative downregulation of RNA pol I expression in shNF45/NF90 cells was observed (Fig 3D), further suggesting that the NF45/NF90 complex positively regulates UBF to recruit RNA pol I to sit on the rDNA promoter. Figure 3. NF45/NF90 affects rDNA transcription as a positive regulatory factor by recruiting and regulating UBF1 activity The regulatory mechanism of the rDNA transcription. Firstly, UBF1 binds to the upstream control element (UCE) of rDNA promoter to recruit SL1, then SL1 recruits Pol I for rDNA transcription. Co-IP analysis of nucleolar proteins with NF90 in HEK293 cells transfected with EV, and the following plasmids: GFP-S5, GFP-L9, GFP-B23, GFP-UBF, and GFP-C23. * represents the target protein. Co-IP analysis of endogenous UBF with NF45 and NF90 in HEK293 cells. HEK293 cells were infected by lentivirus containing NF45-specific shRNA or NF90-specific shRNA and then stimulated in serum for 0, 3, and 6 h. Cell lysates were prepared to analyze the expression levels of the indicated proteins by Western blot. ChIP analysis was used to determinate the binding ability of NF45 and NF90 to rDNA promoters in HeLa cells stimulated with serum for 0, 3 and 6 h. (n = 3) ChIP analysis of NF45 and NF90 binding to the H42.9 loci in HeLa cells with the treatment of CX5461 or DMSO for 2 h. (n = 3) Confocal images of HeLa cells treated with CX5461 for 2 h showing NF45 and NF90 colocalized with fibrillarin. Scale bar, 10 μm. Data information: In all panels, bars and error bars represent mean ± SD. Statistical analysis by unpaired Student t-test (F). One-way ANOVA analysis followed by Tukey's test (E) (**P < 0.01, ***P < 0.001). Exact P values are reported in Appendix Table S3. Source data are available online for this figure. Source Data for Figure 3 [emmm202012834-sup-0004-SDataFig3.pdf] Download figure Download PowerPoint Previous studies have shown that rDNA transcription can be activated in serum-deprived cells when re-subjected to serum stimulation. To investigate the effects of rRNA transcription on NF45/NF90 localization and function, a ChIP assay was performed. The binding ability of NF45/NF90 to the rDNA promoter was significantly increased following serum stimulation (Fig 3E). CX5461 (Drygin et al, 2011), a highly specific Pol I inhibitor that can suppress rDNA transcription by preventing Pol I-specific transcription initiation factors binding to the rDNA promoter to determine the relationship of NF45/NF90 localization and rDNA transcription, was also investigated. In agreement with our hypothesis, the binding ability of NF45/NF90 to the rDNA loci was markedly decreased following treatment with CX5461 (Fig 3F). Interestingly, immunofluorescence analysis revealed a strong translocation of NF45 and NF90 from the nucleolus to the nucleoplasm following CX5461 treatment (Fig 3G), suggesting that the subcellular location of NF45/NF90 is associated with the Pol I-driven transcription machinery. NF45/NF90-mediated rDNA transcription contributes to calcineurin-NFAT-mediated T-cell activation in vitro NF45/NF90 was originally isolated in activated Jurkat T cells and is believed to be involved in T-cell activation, but the specific mechanism has yet to be fully elucidated (Kao
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