Research ArticleThe cold-inducible RNA-binding protein migrates from the nucleus to cytoplasmic stress granules by a methylation-dependent mechanism and acts as a translational repressor
Introduction
RNA-binding proteins are key components in the post-transcriptional regulation of gene expression. These proteins often contain well-conserved RNA-binding domains mediating RNA contact but also auxiliary domains involved in protein–protein interactions and subcellular targeting (see [1] for review). Proteins of the heterogenous nuclear ribonucleoprotein (hnRNP) family have been originally described as nuclear RNA-binding factors involved in different steps of RNA processing within the nucleus (reviewed in [2]). The original observation by Dreyfuss and co-workers that several hnRNPs constantly shuttle between the nucleus and the cytoplasm opened the way to the discovery of cytoplasmic functions for a subset of hnRNPs [3]. Indeed, some of these proteins are involved in various aspects of RNA metabolism in the cytoplasm such as nuclear/cytoplasmic transport, RNA stability, RNA localization and translation. CIRP/hnRNP A18 is a RNA-binding factor composed of a N-terminal RNA Recognition Motif (RRM) and a C-terminal region containing several repeats of the RGG motif. Originally, CIRP has been described as a nuclear protein whose synthesis is induced by mild hypothermic shock in fibroblastic cells [4]. The cirbp gene is constitutively expressed in male murine germ cell [5] and at least three CIRP homologs are abundantly expressed in Xenopus oocytes [6]. These two cell types are naturally exposed to temperatures lower than the body temperature. Therefore, it has been proposed that CIRP might be crucial for RNA metabolism upon cellular cold shock. However, microarray analysis revealed that CIRP is expressed in a large variety of tissues and cells both from human and murine origins [7]. Other data demonstrated that CIRP synthesis is enhanced in response to UV irradiation [8] as well as upon hypoxia [9]. Therefore, it appears that CIRP is induced by a wide variety of cellular stresses which might reflect a broader role of this protein than originally suspected.
CIRP is detected in the nucleus of various human and mouse cell lines. However, the xCIRP2 Xenopus homolog is mainly detected in the oocyte cytoplasm. The analysis of interspecies heterokaryons revealed that xCIRP2 was capable of nucleocytoplasmic shuttling. Moreover, cytoplasmic accumulation of xCIRP2 seems conditioned by arginine methylation as overexpression of the arginine N-methyltransferase 1 xPRMT1 induces xCIRP2 accumulation in the cytoplasm [10]. CIRP is also detected in the cytoplasm of human spermatid cells [5] and is translocated in the cytoplasm of colorectal carcinoma RKO cells upon UV exposure [11]. CIRP proteins could thus play different roles both in the nucleus and the cytoplasm. At the nuclear level, CIRP might play an important role in cold-induced suppression of cell growth [4]. In the cytoplasm, the association of xCIRP2 with the RNA-binding protein ElrA might regulate the length of the poly(A) tail of specific mRNAs [12]. In addition, CIRP has been shown to be involved in mRNA stabilization in human RKO cells upon genotoxic stress [11], [13].
Several studies demonstrate that the protein synthesis is tightly regulated by environmental stress in eukaryotic cells. The vast majority of mRNA becomes translationally silent whereas the translation of a few specific stress-induced transcripts is enhanced. These cellular stresses are usually sub-classified into “cytoplasmic” or “endoplasmic reticulum” (ER) stresses based on the different transcript species induced by these stresses which result either in protein denaturation in the cytoplasm or inhibition of protein synthesis by Ca++ mobilization and proteins misfolding in the ER [14]. In response to both cytoplasmic and ER stresses, untranslated mRNAs are sequestered in cytoplasmic foci, called stress granules (SGs), in which several RNA-binding proteins co-migrate. Among these proteins are found several RNA-binding proteins such as TIA-1/TIAR [15], HuR [16], FMRP [17], Staufen [18], CPEBP [19], FUSE-BPs [20] and hnRNP A1 [21] and ZBP1 [22] as well as enzymes such as the RasGAP-associated endoribonuclease G3BP [23], the endonuclease PRM1 [24] and the APOBEC cytidine deaminase [25]. The formation of these granules seems to require TIA-1 and can be induced either by the phosphorylation of the initiation factor eiF2α [26] or by eiF2α phosphorylation-independent inhibition of ribosome recruitment [27]. To date, SGs appear as genuine triage centers that sort, remodel and export specific mRNPs for re-initiation, decay or storage [28].
Based on the observations that CIRP seems to be involved in various cellular stress responses [8], [9], [29], we investigated the effect of cytoplasmic and ER stresses on CIRP synthesis and subcellular localization. We demonstrate that CIRP synthesis remains unchanged upon oxidative stress. However, a significant proportion of CIRP migrates from the nucleus to the cytoplasm in response to arsenite and accumulates in SGs. CIRP is also recruited in such structures upon other cytoplasmic stresses (heat shock and osmotic pressure) and upon ER stresses. The migration of CIRP into SGs is independent of TIA-1 and can be mediated either by a functional RRM or the RGG domain. However, the methylation of arginine residues in the RGG motif is necessary for CIRP nuclear exit and subsequent recruitment into SGs. Finally, RNA-tethering experiments revealed that CIRP down-regulates mRNA translation and that this activity is mediated by the carboxyl-terminal RGG-enriched domain.
Section snippets
Materials
Enzymes were purchased from Invitrogen and Roche. Oligonucleotides were purchased from Sigma. The synthetic peptide used to immunize rabbits was purchased from Eurogentec. Cell culture media were purchased from Invitrogen.
DNA constructs
DNA constructs used to express EYFP hybrid proteins were generated by introducing PCR amplified fragments corresponding to mouse CIRP full-length, partial or mutated sequences between EcoRI and BamHI sites of the pEYFP-C1 plasmid (Clontech). The expressed fragments of CIRP are
CIRP migrates into stress granules upon various cytoplasmic stresses
It has been demonstrated that the gene encoding CIRP is up-regulated in response to various cellular stresses in different species ranging from fish to man. Sodium arsenite is considered as a prototype compound inducing cytoplasmic stress associated with a general blockade of protein synthesis [14] and the mobilization of a wide range of RNA-binding proteins into cytoplasmic granules [37]. Therefore, we determined whether arsenite modified the synthesis and/or the cellular distribution of CIRP.
Discussion
Previous reports described the induction of CIRP expression upon stresses such as cold shock [41], hypoxia [9], and UV irradiation [8]. While cold shock does not modify CIRP nuclear localization ([42] and Supplementary data 3), UV triggers CIRP translocation from the nucleus to the cytoplasm [11]. Here, we show that oxidative stress does not modify CIRP synthesis but induces its migration from the nucleus to the cytoplasm and its further recruitment into cytoplasmic foci unambiguously
Acknowledgments
We thank J.C. Marine for providing us with the pBABEpuro retroviral vector, L. Paillard for the tethering constructs, D. Weil for RFP-Dcp1a construct, J. Tazi for G3BP-GFP, and P. Anderson for the MEF TIAR−/− and the MEF TIA-1−/− cells. This work was funded by the EC Biotech Program (QLK3-2000-00721), the Fund for Medical Scientific Research (Belgium, grant 2.4.511.00.F), and the “Actions de Recherches Concertées” (grants 00-05/250 and AV.06/11-345). F. De Leeuw is supported by a Ph.D. FRIA
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2022, Journal of Biological ChemistryA proteomics approach identifies novel resident zebrafish Balbiani body proteins Cirbpa and Cirbpb
2022, Developmental BiologyCitation Excerpt :Cirbp proteins are highly expressed in the gonads, particularly in the testis in response to cold stress (Nishiyama et al., 1998). In cells stressed by cold or other insults, Cirbp expression increases, and the protein shuttles from its usual location in the nucleus to stress granules in the cytoplasm (De Leeuw et al., 2007; Liao et al., 2017). Like the Bb, stress granules are membraneless, RNA-rich structures, and have been shown to have a phase-separated liquid-like outer layer and a more solid aggregate core (Wheeler et al., 2017).
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The last two authors equally contributed to the work.