Generation of mice deficient in RNA-binding motif protein 3 (RBM3) and characterization of its role in innate immune responses and cell growth

https://doi.org/10.1016/j.bbrc.2011.06.038Get rights and content

Abstract

The activation of innate immune responses is critical to host defense against microbial infections, wherein nucleic acid-sensing pattern recognition receptors recognize DNA or RNA from viruses or bacteria and activate downstream signaling pathways. In a search for new DNA-sensing molecules that regulate innate immune responses, we identified RNA-binding motif protein 3 (RBM3), whose role has been implicated in the regulation of cell growth. In this study, we generated Rbm3-deficient (Rbm3-/-) mice to study the role of RBM3 in immune responses and cell growth. Despite evidence for its interaction with immunogenic DNA in a cell, no overt phenotypic abnormalities were found in cells from Rbm3-/- mice for the DNA-mediated induction of cytokine genes. Interestingly, however, Rbm3-/- mouse embryonic fibroblasts (MEFs) showed poorer proliferation rates as compared to control MEFs. Further cell cycle analysis revealed that Rbm3-/- MEFs have markedly increased number of G2-phase cells, suggesting a hitherto unknown role of RBM3 in the G2-phase control. Thus, these mutant mice and cells may provide new tools with which to study the mechanisms underlying the regulation of cell cycle and oncogenesis.

Highlights

► We identified RNA-binding motif protein 3 (RBM3) as CpG-B DNA-binding protein. ► RBM3 translocates from the nucleus to the cytoplasm and co-localized with CpG-B DNA. ► We newly generated Rbm3-deficient (Rbm3-/-) mice. ► DNA-mediated cytokine gene induction was normally occured in Rbm3-/- cells. Rbm3-/- MEFs showed poorer proliferation rate and increased number of G2-phase cells.

Introduction

Nucleic acids from viruses or bacteria potently activate immune responses through nucleic acid-sensing pattern recognition receptors (PRRs), namely, membrane-bound Toll-like receptors (TLRs) such as TLR3, TLR7 and TLR9, and cytosolic receptors, which include retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), DNA-dependent activator of IRFs (DAI) [1], [2], [3], [4]. The hallmark of the activation of these receptors is the induction of genes encoding type-I IFN and proinflammatory cytokine gene expression [1], [2], [3], [4]; however, the detailed signaling pathways and mechanisms of gene activation following activation of these receptors still remain elusive. Currently, the DNA-sensing system remains less well known than the RNA-sensing system, perhaps suggesting a more complicated system, and there is evidence for an as yet unknown cytosolic DNA sensor(s) that activates the signaling pathway for proinflammatory cytokine genes [3], [4], [5], [6]. It has also been known that TLR9 (and also TLR7) needs to translocate from the endoplasmic reticulum to endosomes/lysosomes upon stimulation [7], [8], [9], [10], [11], a process which requires Unc-93 homolog B1 (UNC93B1) is involved in the translocation [7], [9], [11]. How the trafficking signal is activated and regulated still remains to be clarified, and it is therefore possible that another DNA-sensing molecule participates in the regulation of the entire TLR9 signaling process.

To gain new insights into the complexity of the DNA-sensing mechanisms in the cell, we searched for immunogenic DNA-binding proteins and identified RNA-binding motif protein 3 (RBM3). RBM3 was originally described as a nuclear protein with one RNA recognition motif [12]. The Rbm3 gene is located on chromosome X of mice (also Xp11.23 in humans) and is ubiquitously expressed in a variety of cell types but is expressed at relatively high levels in cancer cell lines [13], [14]. Because of its potential role in oncogenesis, RBM3 has been studied in relation to cell growth activity and viability. It has been reported that the suppression of RBM3 expression by siRNA knockdown in cancer cell lines results in an inhibition of cell proliferation and increases susceptibility to anti-cancer drugs, whereas the overexpression of RBM3 promotes cell proliferation [14], [15]. Thus, although these observations are intriguing, the physiological role of RBM3 in cell growth control still remains unclear. To study the role of RBM3 in immunity and cell growth, we newly generated Rbm3-deficient (Rbm3-/-) mice. Our results indicate that RBM3 is not involved in the regulation of nucleic acid-mediated cytokine gene induction, but it does play a critical role in cell cycle regulation. We discuss our findings in terms of the utility of these mutant mice for the analysis of innate immune signaling, cell cycle, and oncogenesis.

Section snippets

Generation of Rbm3-deficient mice

Genomic DNA containing Rbm3 gene was amplified by polymerase chain reaction (PCR) from 129/Sv genome DNA. An Rbm3-targeting construct that replaces exon 3–6 with a phosphoglycerate kinase promoter-driven neomycin-resistant gene was transfected into E14–1 ES cells. Homologous recombinants were injected into C57BL/6 blastocysts. The resulting chimera mice were intercrossed heterozygous F1 progenies to obtain Rbm3-/- mice as described previously [16]. C57BL/6J mice were purchased from CLEA Japan.

Screening for CpG-B ODN-binding protein

Identification of RBM3 as an immunogenic DNA-associated protein

We first sought to identify proteins involved in immunogenic DNA recognition systems and performed biochemical screening by the cell-free protein display (CFPD) method [17] using purified mRNAs from dendritic cells (DCs) differentiated from bone marrow cells cultured with the Flt3-ligand (Supplementary Fig. S1A). In this screening, we identified RBM3 as the most prominent protein that binds to CpG-B DNA, a TLR9 agonist (Supplementary Fig. S1B). Previously, RBM3 was studied in the context of

Discussion

In this paper, we report on the generation of mice deficient in RBM3 (Rbm3-/- mice) and our initial results on the role of this protein in the context of innate immune responses and cell cycle regulation. We newly identified RBM3 as a binding protein for CpG-B DNA that activates TLR9 (Fig. 1A) and adduced evidence that RBM3 accumulates in lysosomes, where TLR9 signaling is reported to occur [8], [10], upon CpG-B DNA stimulation. However, our analysis of cDCs and pDCs from these mice revealed

Note added in proof

Currently, we have no evidence for an interaction of RBM3 with TLR9 in CpG-B stimulated cells.

Acknowledgments

We thank ZOEGENE Co., Ltd., for CFPD, M. Shishido, R. Takeda, and M. Taniguchi for technical assistance, and K. Atarashi for invaluable advice. This work was supported in part by a Grant-In-Aid for Scientific Research on Innovative Areas, and Global Center of Excellence Program ‘Integrative Life Science Based on the Study of Biosignaling Mechanisms’ from the Ministry of Education, Culture, Sports, Science.

References (29)

  • T. Tamura et al.

    The IRF family transcription factors in immunity and oncogenesis

    Annu. Rev. Immunol.

    (2008)
  • M.K. Choi et al.

    A selective contribution of the RIG-I-like receptor pathway to type I interferon responses activated by cytosolic DNA

    Proc. Natl. Acad. Sci. USA

    (2009)
  • S.E. Ewald et al.

    The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor

    Nature

    (2008)
  • K. Honda et al.

    Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction

    Nature

    (2005)
  • Cited by (26)

    • Time-resolved proteome and transcriptome of paraquat-induced pulmonary fibrosis

      2022, Pulmonary Pharmacology and Therapeutics
      Citation Excerpt :

      It promotes the stability and translation of rapidly-degraded mRNAs such as those for the expression of interleukin-8 (IL-8), cyclooxygenase 2 (COX-2), and vascular endothelial growth factor (VEGF) [40]. RBM3 modulates the cell cycle during the G2/M transition [40,41] and regulates cell stem maintenance through β-catenin signaling [42]. RBM3 also inhibits apoptosis by repressing poly ADP-ribose polymerase (PARP) cleavage [43], suppressing caspase expression, and inducing apoptosis regulator Bcl-2 [44,45].

    • RBM3 Increases Cell Survival but Disrupts Tight Junction of Microvascular Endothelial Cells in Acute Lung Injury

      2021, Journal of Surgical Research
      Citation Excerpt :

      RBM3 can promote cell activity and protect cells from death caused by serum starvation.4 RBM3 has also been found to be involved in the regulation of skeletal muscle growth and innate immune response.5 A recent study reported that RBM3 could mediate synapse regeneration, and may have protective effects in neurodegenerative diseases.6

    • Role of RBM3 in the regulation of cell proliferation in hepatocellular carcinoma

      2020, Experimental and Molecular Pathology
      Citation Excerpt :

      Emerging evidence suggests that RBM3 plays a vital role in the regulation of cell proliferation. Rbm3-deficient mouse embryonic fibroblasts (MEFs) reveals more restricted proliferation rates as compared to control MEFs (Matsuda et al., 2011). RBM3 regulates neural stem cell proliferation under hypoxia (Yan et al., 2019) and maternal cold stress (Xia et al., 2018).

    • RNA Binding Proteins in Intestinal Epithelial Biology and Colorectal Cancer

      2018, Trends in Molecular Medicine
      Citation Excerpt :

      It binds to RNAs via its RRM domain and alters the secondary structure of the RNA, affecting the access of mRNA initiation factor to the ribosome subunit [124], which modulates the potential activity of kinases in tumors. RBM3 deficient mice show no overt phenotype or growth changes and are fertile [125]. RBM3 overexpression in HCT116 and DLD1 colon cancer cells increases proliferation and engenders chemotherapy resistance.

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    2

    Present address: Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan.

    View full text