RNA-binding protein RBM28 can translocate from the nucleolus to the nucleoplasm to inhibit the transcriptional activity of p53

Abstract

RNA-binding protein RBM28 (RBM28), as a nucleolar component of spliceosomal small nuclear ribonucleoproteins, is involved in the nucleolar stress response. Whether and how RBM28 regulates tumor progression remains unclear. Here, we report that RBM28 is frequently overexpressed in various types of cancer and that its upregulation is associated with a poor prognosis. Functional and mechanistic assays revealed that RBM28 promotes the survival and growth of cancer cells by interacting with the DNA-binding domain of tumor suppressor p53 to inhibit p53 transcriptional activity. Upon treatment with chemotherapeutic drugs (e.g., adriamycin), RBM28 is translocated from the nucleolus to the nucleoplasm, which is likely mediated via phosphorylation of RBM28 at Ser122 by DNA checkpoint kinases 1 and 2 (Chk1/2), indicating that RBM28 may act as a nucleolar stress sensor in response to DNA damage stress. Our findings not only reveal RBM28 as a potential biomarker and therapeutic target for cancers but also provide mechanistic insights into how cancer cells convert stress signals into a cellular response linking the nucleolus to regulation of the tumor suppressor p53. RNA-binding protein RBM28 (RBM28), as a nucleolar component of spliceosomal small nuclear ribonucleoproteins, is involved in the nucleolar stress response. Whether and how RBM28 regulates tumor progression remains unclear. Here, we report that RBM28 is frequently overexpressed in various types of cancer and that its upregulation is associated with a poor prognosis. Functional and mechanistic assays revealed that RBM28 promotes the survival and growth of cancer cells by interacting with the DNA-binding domain of tumor suppressor p53 to inhibit p53 transcriptional activity. Upon treatment with chemotherapeutic drugs (e.g., adriamycin), RBM28 is translocated from the nucleolus to the nucleoplasm, which is likely mediated via phosphorylation of RBM28 at Ser122 by DNA checkpoint kinases 1 and 2 (Chk1/2), indicating that RBM28 may act as a nucleolar stress sensor in response to DNA damage stress. Our findings not only reveal RBM28 as a potential biomarker and therapeutic target for cancers but also provide mechanistic insights into how cancer cells convert stress signals into a cellular response linking the nucleolus to regulation of the tumor suppressor p53. The nucleolus is associated with the regulation of several major physiological cellular processes including ribosome assembly, cell mitosis, stress responses, and ribonucleoprotein complex generation (1Nousbeck J. Spiegel R. Ishida-Yamamoto A. Indelman M. Shani-Adir A. Adir N. Lipkin E. Bercovici S. Geiger D. van Steensel M.A. Steijlen P.M. Bergman R. Bindereif A. Choder M. Shalev S. et al.Alopecia, neurological defects, and endocrinopathy syndrome caused by decreased expression of RBM28, a nucleolar protein associated with ribosome biogenesis.Am. J. Hum. Genet. 2008; 82: 1114-1121Google Scholar). Recent landmark proteomic studies have led to the discovery of more than 4500 nucleolus-associated proteins, and 70% of nucleolar proteins have functions unrelated to the production of ribosome subunits, including the regulation of cell cycle progression, DNA damage sensing and repair, genomic organization, nuclear architecture establishment, and global gene expression, suggesting that nucleolar functions might be significantly broader than previously thought (2Orsolic I. Jurada D. Pullen N. Oren M. Eliopoulos A.G. Volarevic S. The relationship between the nucleolus and cancer: Current evidence and emerging paradigms.Semin. Cancer Biol. 2016; 37-38: 36-50Google Scholar). Recently, the nucleolus has gained attention for its novel role in the regulation of cellular stress (3Liu Y. Deisenroth C. Zhang Y. RP–MDM2–p53 pathway: Linking ribosomal biogenesis and tumor surveillance.Trends Cancer. 2016; 2: 191-204Google Scholar). Nucleolar stress is emerging as a new concept and is characterized by various stressor-induced impairments in nucleolar morphology and function that ultimately lead to disturbances in cell homeostasis through the activation of p53 or other stress signaling pathways (4Yang K. Yang J. Yi J. Nucleolar stress: Hallmarks, sensing mechanism and diseases.Cell Stress. 2018; 2: 125-140Google Scholar). The following elements are described as hallmarks of nucleolar stress: a wide range of stimuli as stressors cause disturbances in ribosome biogenesis, nucleoplasmic translocation of nucleolar proteins, morphological alterations in nucleolar stress, impaired rRNA transcription and processing, and activation of p53 signaling and involvement of p53-independent stress signaling (4Yang K. Yang J. Yi J. Nucleolar stress: Hallmarks, sensing mechanism and diseases.Cell Stress. 2018; 2: 125-140Google Scholar). Theoretically, nucleoli do not possess defined structural barriers and thus may not have a system akin to nuclear transport machinery (such as importins, a nuclear pore complex, and exportins); proteins that localize to nucleoli can potentially traffic in and out of the nucleolus in a relatively free manner (5Kodiha M. Banski P. Stochaj U. Computer-based fluorescence quantification: A novel approach to study nucleolar biology.BMC Cell Biol. 2011; 12: 25Google Scholar). Present studies have shown that the translocation of nucleolar proteins in nucleolar stress signaling mainly relies on specific interactions with binding sites within nucleolar components, which raises the question of the identity of the molecular mechanism involved in protein nucleolar targeting. Posttranslational modifications (PTMs) of nucleolar proteins are also speculated to play a major role in stress-induced changes in protein localization. For instance, during nucleolar oxidation, NPM1 undergoes S-glutathionylation on cysteine 275, which triggers the dissociation of NPM1 from nucleolar nucleic acids, resulting in the nucleoplasmic translocation and activation of p53 (6Yang K. Wang M. Zhao Y.Z. Sun X.X. Yang Y. Li X. Zhou A.W. Chu H.L. Zhou H. Xu J.R. Wu M. Yang J. Yi J. A redox mechanism underlying nucleolar stress sensing by nucleophosmin.Nat. Commun. 2016; 7: 13599Google Scholar). RNA-binding protein (RBP) is a general term for a class of proteins that bind to RNA. RBPs are highly conserved across species and play key roles in maintaining gene expression homeostasis. Mounting evidence has shown that RBPs are involved in various important cellular processes, such as cell transport, localization, differentiation, and metabolism by regulating RNA splicing, polyadenylation, mRNA stability, mRNA localization, and translation through interactions with coding and noncoding RNAs and other proteins (7Qin H. Ni H.W. Liu Y.C. Yuan Y.Q. Xi T. Li X.M. Zheng L.F. RNA-binding proteins in tumor progression.J. Hematol. Oncol. 2020; 13: 90Google Scholar). A recent global survey of proteins that are UV cross-linkable to RNA revealed a large number of both canonical and noncanonical RBPs (8Xiao R. Chen J.-Y. Liang Z. Luo D. Chen G. Lu Z.J. Chen Y. Zhou B. Li H. Du X. Yang Y. San M. Wei X. Liu W. Lécuyer E. et al.Pervasive chromatin-RNA binding protein interactions enable RNA-based regulation of transcription.Cell. 2019; 178: 107-121.e18Google Scholar). Recent studies also found that RBPs have some nontraditional functions, such as participating in gene transcription regulation and acting as sensors for cellular stress signals through p53 activation (3Liu Y. Deisenroth C. Zhang Y. RP–MDM2–p53 pathway: Linking ribosomal biogenesis and tumor surveillance.Trends Cancer. 2016; 2: 191-204Google Scholar, 8Xiao R. Chen J.-Y. Liang Z. Luo D. Chen G. Lu Z.J. Chen Y. Zhou B. Li H. Du X. Yang Y. San M. Wei X. Liu W. Lécuyer E. et al.Pervasive chromatin-RNA binding protein interactions enable RNA-based regulation of transcription.Cell. 2019; 178: 107-121.e18Google Scholar). Several studies have provided evidence to link known cancer drivers to abnormal expression or altered activity of RBPs; however, the extent to which RBPs act on cancer progression remains unclear, and further exploration of RBP functions and their regulatory networks may provide new molecular targets for cancer therapy. RBPs are well documented to be subjected to a variety of PTMs including acetylation, phosphorylation, methylation, and ubiquitination (9Pereira B. Billaud M. Almeida R. RNA-binding proteins in cancer: Old players and new actors.Trends Cancer. 2017; 3: 506-528Google Scholar). PTMs alter RBP binding properties, functions, or subcellular localization. Notably, the PTMs of RNA-binding elements in RBPs are particularly prominent (7Qin H. Ni H.W. Liu Y.C. Yuan Y.Q. Xi T. Li X.M. Zheng L.F. RNA-binding proteins in tumor progression.J. Hematol. Oncol. 2020; 13: 90Google Scholar). Abnormal modification of these sites may be one of the main factors leading to RBP dysfunction in tumors (7Qin H. Ni H.W. Liu Y.C. Yuan Y.Q. Xi T. Li X.M. Zheng L.F. RNA-binding proteins in tumor progression.J. Hematol. Oncol. 2020; 13: 90Google Scholar), and many studies have shown that abnormal expression or mutation in the RNA- binding motif family plays a very important role in the occurrence and metastasis of tumors (10Sutherland L.C. Rintala-Maki N.D. White R.D. Morin C.D. RNA binding motif (RBM) proteins: A novel family of apoptosis modulators?.J. Cell. Biochem. 2005; 94: 5-24Google Scholar). For example, RBM10 activates key proliferative signaling pathways, including the epidermal growth factor receptor, mitogen-activated protein kinase, and phosphatidylinositol-3-kinase (PI3K)-Akt pathway and inhibits apoptotic pathways in lung adenocarcinoma (11Sun X.N. Jia M.Q. Sun W. Feng L. Gu C.D. Wu T.H. Functional role of RBM10 in lung adenocarcinoma proliferation.Int. J. Oncol. 2019; 54: 467-478Google Scholar). RBM14 promotes radioresistance in glioblastoma by regulating DNA damage repair and cell differentiation (12Yuan M. Eberhart C.G. Kai M. RNA binding protein RBM14 promotes radio-resistance in glioblastoma by regulating DNA repair and cell differentiation.Oncotarget. 2014; 5: 2820-2826Google Scholar). RBM28, as a nucleolar component of spliceosomal small nuclear ribonucleoproteins (snRNPs) may also play roles in cancer progression by involving in the nucleolar stress response. According to bioinformatics analysis, head and neck squamous cell carcinoma and lung squamous cell carcinoma share common splicing alterations, nearly half of which are associated with RBM28 (13Sebestyen E. Singh B. Minana B. Pages A. Mateo F. Pujana M.A. Valcarcel J. Eyras E. Large-scale analysis of genome and transcriptome alterations in multiple tumors unveils novel cancer-relevant splicing networks.Genome Res. 2016; 26: 732-744Google Scholar). The binding motifs of RBM28 were significantly enriched in the 3′UTR of nonsmall cell lung cancer patient’s platelet RNAs (14Best M.G. Sol N. In 't Veld S.G.J.G. Vancura A. Muller M. Niemeijer A.L.N. Fejes A.V. Fat L.A.T.K. In 't Veld A.E.H. Leurs C. Le Large T.Y. Meijer L.L. Kooi I.E. Rustenburg F. Schellen P. et al.Swarm intelligence-enhanced detection of non-small-cell lung cancer using tumor-educated platelets.Cancer Cell. 2017; 32: 238-252Google Scholar). However, whether and how RBM28 regulates tumor progression remain unclear. In this report, we found that Chk1 and Chk2 phosphorylate RBM28 at S122 upon DNA damage, promoting RBM28 nucleolar–nucleoplasmic translocation, where RBM28 directly binds to p53 to suppress the transcriptional activity of p53. To explore the potential cancer driver genes in the RNA-binding motif family, we analyzed gene dependency scores in 903 cancer cell lines from the DepMap database and found that RBM28 is one of the highest cancer-dependent genes (Fig. 1A). Interestingly, as shown in the Clinical Proteomic Tumor Analysis Consortium proteome database, RBM28 protein levels were upregulated in a variety of cancers, such as colon cancer, clear cell renal cell carcinoma, uterine corpus endometrial carcinoma, ovarian cancer, breast cancer, and lung adenocarcinoma (Fig. 1B). According to The Cancer Genome Atlas transcriptome data and patient survival information, a high mRNA level of RBM28 was associated with a poor prognosis in multiple cancer types, including breast cancer, uterine corpus endometrial carcinoma, clear cell renal cell carcinoma, lung adenocarcinoma, hepatocellular carcinoma, and all types of sarcomas (Fig. 1C). These results suggest that RBM28 may act as an oncogene in various cancers. To investigate the role of RBM28 in cancer progression, we constructed constitutive HCT116 and U2OS cell lines with RBM28 KO using CRISPR-Cas9 technology. The knockout efficiency was determined by Western blot (Fig. 2A). According to 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and RBM28 KO significantly the of both HCT116 and U2OS cells (Fig. and RBM28 KO significantly the growth of as determined using of HCT116 cells in (Fig. and which was by the that RBM28 KO decreased the cell determined by using these (Fig. These results that RBM28 plays a role in the growth of cancer we how RBM28 promotes cell an in RBM28 KO U2OS which expression with upregulated and of and pathway analysis that pathways were significantly enriched in RBM28 KO cells cells (Fig. A from global transcriptome analysis that p53 target genes were upregulated upon RBM28 KO (Fig. These results were by that RBM28 and RBM28 and decreased the expression of and (Fig. and further the of RBM28 on p53 using as the According to assay and RBM28 activity and the binding of p53 to the in U2OS cells (Fig. and which is the protein by was upregulated in p53 cells but not p53 suggesting that the of RBM28 on p53 target genes was (Fig. these results that RBM28 may the transcriptional activity of p53 by the binding of p53 to its target gene In in the by of RBM28 was in HCT116 cells than that in HCT116 as shown in indicating that RBM28 has other p53. is with the DepMap data results that RBM28 is for cancer both in p53 and cell as shown in these results that p53 is one of the of RBM28 to cell Upon DNA damage, at and phosphorylation at are in p53 activation Y. Lu Wu Wang Chen Y.Q. Sun J. Y. Zhou W. Zheng Wu M. of p53 at by the expression of Cell. Biol. 26: Scholar, K. p53 functions through p53 Cell. Biol. 2019; Scholar). However, of RBM28 not p53 at phosphorylation at or the p53 level with or treatment (Fig. RBM28 KO not the of mRNA (Fig. that RBM28 may the transcriptional activity of p53 by the binding of p53 to its target gene as shown (Fig. and we that RBM28 may bind to p53. RBM28 with p53 at their levels (Fig. and this using RBM28 and p53 was not by the A indicating that the of RBM28 with p53 is (Fig. we to the for the between RBM28 and p53. A of of p53 and RBM28 were and was (Fig. A and The DNA-binding domain of p53 was and to bind to RBM28 (Fig. Interestingly, of RBM28, 1 to to and to with p53 (Fig. further several of RBM28 by (Fig. The between RBM28 and p53 was not by of (Fig. These results revealed that RBM28 may with the DNA-binding domain of p53 through multiple The nucleolus is a major in of the stress response S. S. The nucleolus Cell. Scholar). Nucleolar stress many nucleolar to in the that to be from the nucleolus to the (4Yang K. Yang J. Yi J. Nucleolar stress: Hallmarks, sensing mechanism and diseases.Cell Stress. 2018; 2: 125-140Google Scholar). that RBM28 is a nucleolar protein (Fig. p53 as a transcription factor in the nucleoplasm, we speculated whether RBM28 might nucleolar–nucleoplasmic translocation upon cellular stress. a of as shown in we found that nucleoplasmic of RBM28 significantly upon treatment with in cancer such as and K. G. C. in cancer and Biol. Scholar, B. S. the of the to cells from J. Cancer. Scholar). The DNA damage by was determined by the detection of expression (Fig. In response to the was the nucleoplasm, where with (Fig. the assay that the binding of p53 and RBM28 was in response to in a manner (Fig. These findings revealed that RBM28 translocated from the nucleolus to the nucleoplasm, where with upon The DNA damage response is a pathway involved in many processes of activation A. The DNA damage to play with Cell. Scholar), the that RBM28 is by some key through modification in the DNA damage an and a Chk1 nucleoplasmic of RBM28 in HCT116 cells upon treatment (Fig. both Chk1 and Chk2 also nucleoplasmic of RBM28 in HCT116 cells upon treatment (Fig. and as and also suppress the activity of both Chk1 and Chk2 in DNA repair in 2019; Scholar), indicating that the nucleolar–nucleoplasmic translocation of RBM28 by we analyzed the of RBM28 and found potential phosphorylation Ser122 and to a phosphorylation the Xu Zhou Zhang Y. Y. D. Y. on the of phosphorylation sites in 2020; Scholar). to and to of these phosphorylation sites were Interestingly, only the RBM28 was in the other were the (Fig. indicating that the phosphorylation of RBM28 at S122 may be to the subcellular of using the phosphorylation of RBM28 was with or (Fig. and only the the phosphorylation of RBM28 (Fig. that S122 is the key for phosphorylation by Notably, S122 not the of RBM28 with (Fig. we a specific and found that S122 phosphorylation of RBM28 was by Chk1 or Chk2 in cells with their (Fig. treatment was found to phosphorylation (Fig. using an assay that the nucleoplasmic of RBM28, but not the RBM28 was upon with or (Fig. the RBM28 the nucleoplasmic of RBM28 by (Fig. These results revealed that may phosphorylate S122 of RBM28 to nucleolar–nucleoplasmic translocation of RBM28 upon In this report, as in chemotherapeutic drugs (e.g., to phosphorylate S122 of RBM28, promoting the translocation of RBM28 from the nucleolus to the nucleoplasm, where RBM28 with the DNA-binding domain of p53 to inhibit p53 transcriptional activity. Our findings provide mechanistic insights into how cancer cells convert stress signals into a cellular response linking the nucleolus to regulation of the tumor suppressor p53. a nucleolar component of spliceosomal small nuclear ribonucleoproteins RBM28 is involved in and RNA motifs and an of between and A. M. Bindereif A. RBM28 protein is a specific nucleolar component of the spliceosomal Scholar). A function mutation in RBM28 was to neurological defects, and endocrinopathy syndrome (1Nousbeck J. Spiegel R. Ishida-Yamamoto A. Indelman M. Shani-Adir A. Adir N. Lipkin E. Bercovici S. Geiger D. van Steensel M.A. Steijlen P.M. Bergman R. Bindereif A. Choder M. Shalev S. et al.Alopecia, neurological defects, and endocrinopathy syndrome caused by decreased expression of RBM28, a nucleolar protein associated with ribosome biogenesis.Am. J. Hum. Genet. 2008; 82: 1114-1121Google Scholar, R. Shalev A. E. Y. syndrome caused by RBM28 A novel of J. Scholar). A mutation in the domain of its was found to growth and defects, suggesting a role for RBM28 in ribosome biogenesis T. Zhang J. The molecular for syndrome revealed by the large ribosomal 2016; Scholar). Here, we determined for the that RBM28 may act as an oncogene to growth of cancer as RBM28 significantly activates the p53 pathway and inhibits cancer cell survival and and that high RBM28 expression a poor prognosis in cancer These findings that RBM28 may be a potential biomarker and therapeutic target for cancers. between the nucleolus and cellular stress were on the that the nucleolus in regulating the of p53 (4Yang K. Yang J. Yi J. Nucleolar stress: Hallmarks, sensing mechanism and diseases.Cell Stress. 2018; 2: 125-140Google Scholar). The that the nucleolus plays a role in regulating cellular stress at of the p53 activation by nucleolar mainly involved in changes in the in which the nucleolus is a sensor for cellular stress, with stress-induced nucleoplasmic translocation of nucleolar proteins, such as NPM1 and p53 activation (6Yang K. Wang M. Zhao Y.Z. Sun X.X. Yang Y. Li X. Zhou A.W. Chu H.L. Zhou H. Xu J.R. Wu M. Yang J. Yi J. A redox mechanism underlying nucleolar stress sensing by nucleophosmin.Nat. Commun. 2016; 7: 13599Google Scholar, S. Nucleolar protein p53 in response to ribosomal Scholar). The other on a where the of ribosomal proteins mainly and is in mRNA with on their translocation L. Volarevic S. Oren M. p53 and ribosome biogenesis stress: The 2014; Scholar). In this report, upon nucleolar stress, such as the we a novel mechanism of RBM28 on p53 where RBM28 is translocated to the from the which is mediated by phosphorylation of RBM28 at S122 by These findings that RBM28 may act as a nucleolar stress sensor in response to DNA damage stress. as the of the is an tumor suppressor gene that activates the transcription of multiple target genes of the Scholar). The common and are in the DNA-binding that this of p53 is for tumor Yang M. J. E. B. T. A. B. et processes the of in Genet. 2018; Scholar). In this we found that nucleoplasmic translocation of RBM28 is for its binding to which be by chemotherapeutic drugs (e.g., may provide a novel for p53 cancer to some that the may be to the of cancer cells with p53 to Notably, Chk1 has as an target in tumors but not p53 cancers, p53 can for the of cell cycle regulation of as in Scholar, Z. J. P. Li Wang G. H.L. Wang et Chk1 cancer cells to cancer J. Cancer. Scholar). Here, we found that a Chk1 the translocation of RBM28 from the nucleolar to the nucleoplasm, which in to the of RBM28 on p53. In this cancer cells with RBM28 levels may be more to Chk1 suggesting that RBM28 may as a biomarker for in cancer with p53. 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Li and Y. W. X. L. X. and Y. W. X. J. Li and Y. W. X. L. X. L. and Y. W. X. R. T. and Y. W. T. K. and Y. W. T. K. and with and

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