Cold-induced RNA-binding Protein Genes Research Articles

A Cold-Inducible RNA Binding Protein Gene from Acanthopagrus schlegelii: Molecular Characterization, Expression, and Association with Apoptosis to Low-Temperature Stress

In this study, the complete cDNA sequence (1552 bp) of the cold-inducible RNA binding protein gene (cirbp gene) was successfully cloned from the liver in Acanthopagrus schlegelii (initial weight: 15.0 ± 2.3 g). Results showed that Ascirbp (cirbp gene from A. schlegelii) gene has 24 phosphorylation sites, no signal peptide, and no transmembrane helix structure. AsCIRBP, with a molecular weight of 18.84 ku and an isoelectric point of 9.04 was a stable protein that encodes 182 amino acids. Subcellular localization analysis of this protein showed that it was located in the nucleus. Sequence alignment results showed that the AsCIRBP amino acid sequences of various fishes including black porgy were highly conserved, especially the RNA recognition motif (RRM). Those results of real-time quantitative PCR (qRT-PCR) demonstrated that Ascirbp gene was specifically expressed in the liver tissue of black porgy and its expression was significantly increased under cold stress or cold acclimation. The RNA interference experiment results showed that Ascirbp-dsRNA could suppress the expression of Ascirbp gene in the liver of black porgy through intraperitoneal injection. After silencing the expression of Ascirbp gene, RNAi groups were more severely damaged in the structure of the liver tissue and more prone to apoptosis under cold stress than control groups. The results of the study on the linkage between Ascirbp gene expression and mitochondrial apoptosis pathways showed that changes in the expression of the Ascirbp gene had a significant effect on the expression of key genes of apoptosis. The most striking result from silencing the expression of the Ascirbp gene was that expressions of the bcl-2 and apaf-1 gene in the liver of black porgy decreased significantly, which can block the normal apoptotic process. After the disruption of the normal apoptotic process, the expressions of p53, bax, cyto-c, caspase-9, caspase-3, diablo, and caspase-1 gene were significantly affected. These results suggest that Ascirbp gene can inhibit apoptosis and protect tissue structure in the liver tissue of black porgy at low temperatures.

Downregulation of CIRP Prone Cells to Oxidative Injury via Regulating Nrf2 Signaling Pathway.

Cold-inducible RNA-binding protein (CIRP) is a cellular stress-response protein, whose expression can be induced by a variety of stress conditions. Our previous study showed that intracellular CIRP is a protective factor against cellular oxidative stress and silencing of CIRP gene prone cells to apoptosis. However, the underlying mechanism remains unknown. The present study was aimed at investigating the possible mechanisms underlying the protective role of CIRP in oxidative stress injury. Herein, we used HEK293T cells as our cell model to investigate the relation between CIRP and the possible antioxidant pathways by using the latest genetic silencing technologies. Our results showed that silencing CIRP by using SaiRNA-based genetic silencing tool leads to the downregulation of Nrf2 and Nrf2-regulated antioxidant genes in HEK293T cells. Taken together, our study identified the antioxidant Nrf2 signaling pathway as a downstream target of CIRP, and silencing CIRP may prone cells to apoptosis by downregulating the Nrf2 antioxidant pathway in response to oxidative injury.

Transcriptome analysis reveals molecular mechanisms responsive to acute cold stress in the tropical stenothermal fish tiger barb (Puntius tetrazona)

BackgroundTropical stenothermal fish exhibit special tolerance and response to cold stress. However current knowledge of the molecular mechanisms response to cold stress in aquatic ectotherms is largely drawn from eurythermal or extreme stenothermal species. The tiger barb Puntius tetrazona is a tropical stenothermal fish, with great popularity in aquarium trade and research.ResultsTo investigate the response mechanism of P. tetrazona to low temperature, fish were exposed to increasing levels of acute cold stress. Histopathological analysis showed that the brain, gill, liver and muscle tissues appeared serious damage after cold stress (13 °C). Brain, gill, liver and muscle tissues from control (CTRL) groups (27 °C) and COLD stress groups (13 °C) of eight-month fish (gender-neutral) were sampled and assessed for transcriptomic profiling by high-throughput sequencing. 83.0 Gb of raw data were generated, filtered and assembled for de novo transcriptome assembly. According to the transcriptome reference, we obtained 392,878 transcripts and 238,878 unigenes, of which 89.29% of the latter were annotated. There were 23,743 differently expressed genes (DEGs) been filtered from four pairs of tissues (brain, gill, liver and muscle) between these cold stress and control groups. These DEGs were mainly involved in circadian entrainment, circadian rhythm, biosynthesis of steroid and fatty acid. There were 64 shared DEGs between the four pairs of groups, and five were related to ubiquitylation/deubiquitylation. Our results suggested that ubiquitin-mediated protein degradation might be necessary for tropical stenothermal fish coping with acute cold stress. Also, the significant cold-induced expression of heat shock 70 kDa protein (HSP70) and cold-induced RNA-binding protein (CIRBP) was verified. These results suggested that the expression of the molecular chaperones HSP70 and CIRBP in P. tetrazona might play a critical role in coping with acute cold stress.ConclusionsThis is the first transcriptome analysis of P. tetrazona using RNA-Seq technology. Novel findings about tropical stenothermal fish under cold stress (such as HSP70 and CIRBP genes) are presented here. This study contributes new insights into the molecular mechanisms of tropical stenothermal species response to acute cold stress.

Cold-inducible RNA-binding protein mediates airway inflammation and mucus hypersecretion through a post-transcriptional regulatory mechanism under cold stress

Acute or chronic cold exposure exacerbates chronic inflammatory airway diseases, such as chronic obstructive pulmonary disease (COPD) and asthma. Cold-inducible RNA-binding protein (CIRP) is a cold-shock protein and is induced by various environmental stressors, such as hypothermia and hypoxia. In this study, we showed that CIRP gene and protein levels were significantly increased in patients with COPD and in rats with chronic airway inflammation compared with healthy subjects. Similarly, inflammatory cytokine production and MUC5AC secretion were up-regulated in rats following cigarette smoke inhalation. Cold temperature-induced CIRP overexpression and translocation were shown to be dependent on arginine methylation in vitro. CIRP overexpression promoted stress granule (SG) assembly. In the cytoplasm, the stability of pro-inflammatory cytokine mRNAs was increased through specific interactions between CIRP and mediator mRNA 3⿲-UTRs; these interactions increased the mRNA translation, resulting in MUC5AC overproduction in response to cold stress. Conversely, CIRP silencing and a methyltransferase inhibitor (adenosine dialdehyde) promoted cytokine mRNA degradation and inhibited the inflammatory response and mucus hypersecretion. These findings indicate that cold temperature can induce an airway inflammatory response and excess mucus production via a CIRP-mediated increase in mRNA stability and protein translation.

Construction of pECFP-C1- CIRP and its effect on cold-induced apoptosis in mouse NIH/3T3 cells

Cold-inducible RNA-binding protein (CIRP) is suggested to be involved in protecting cells from the damage caused by cold stress. In this study, the recombinant plasmid, pECFP-C1-CIRP, was constructed and transfected into NIH/3T3 cells by lipofectamineTM 2000. The fluorescence microscopy, real-time polymerase chain reaction (RT-PCR) and Western blot detection were done for transient and stable expression assays. CIRP gene was successfully subcloned into eukaryotic expression vector of pECFP-C1, confirmed by restrictive enzyme digestion analysis and DNA sequencing. Fluorescent microscopy study showed that green fluorescence was generated from NIH/3T3 cell. The results of RT-PCR indicated that the CIRP mRNA was expressed in NIH/3T3 cells, while the results of Western blot detection further indicated that the CIRP protein was overexpressed in NIH/3T3 cells. Stable expression in cells transfected with pECFP-C1-CIRP was achieved by selection in a G418-containing medium for 2 weeks. We performed experiments of cold-induced apoptosis under cold treatment of 4, 14 and 24°C. The results of the cell apoptosis detected by fluorescence microscopy indicated that the apoptosis level was reduced in pECFP-C1-CIRP stably expressed group due to overexpression of CIRP. This study suggested that CIRP played a role in protecting cells from cold-induced apoptosis. Key words : Cold inducible RNA-binding protein, NIH/3T3 cell, transfection, cold-induced apoptosis.

Overexpression of CIRP may reduce testicular damage induced by cryptorchidism

To investigate the protective effect of overexpression of cold-inducible RNA-binding protein (CIRP) on testicular damage induced by cryptorchidism. Male BALB/c mice were made surgically cryptorchid and CIRP gene was transferred into the cryptorchid testis by in vivo electroporation. Seven or ten days after electroporation, the expression of CIRP, p53 and Fas mRNA and protein were analyzed by reverse-transcription polymerase chain reaction (RT-PCR) and immunoblotting, respectively. Meanwhile, Histopathological changes were observed by light microscope, and flow cytometry was used to detect testicular cell apoptosis. Testicular weights after transfection with pVAX1-CIRP or pVAX1 were 0.083+/-0.005 g and 0.065+/-0.004 g, respectively, on day 7(P < 0.05) and 0.078+/-0.004 g and 0.052+/-0.007 g, on day 10 (P < 0.05). Testicular cell apoptosis after transfection with pVAX1-CIRP or pVAX1 were 9.8+/-1.1 % and 20.7+/-1.3 %, respectively, on day 7 (P < 0.01) and 10.4+/-0.9 % and 27.5+/-1.2 %, on day 10 (P < 0.01). In addition, the expression of CIRP mRNA and protein in the testes transfected with pVAX1-CIRP were both increased (P < 0.05) at each indicated time point. Meanwhile, the expression of p53 was decreased on day 7 (P < 0.05) and Fas was decreased on day 10(P < 0.05). Overexpression of CIRP may reduce testicular damage induced by cryptorchidism by down-regulating the levels of p53 and Fas.

Cloning and characterization of amphibian cold inducible RNA-binding protein

Gene expression of cold inducible RNA-binding protein (CIRP) was examined in the frog. In Xenopus laevis, expression of CIRP (XCIRP) was observed in both brain and liver at 24°C. Circadian expression of XCIRP was observed in brain. Expression of XCIRP in brain was induced by cold treatment and gradually decreased to the control level at 24°C, but no significant changes were observed in liver. Employing the sequence of murine CIRP, bullfrog ( Rana catesbeiana) CIRP gene was cloned. The bullfrog CIRP gene, designated BFCIRP, was 706 bp in length and encoded a putative protein of 164 amino acid residues. The deduced protein contained one consensus sequence of RNA-binding domain (CS-RBD) and a glycine rich domain (GRD). The amino acid sequence of BFCIRP was 78.4% identical to XCIRP. Expression of BFCIRP in brain was stronger in winter than that in summer. These findings suggest that BFCIRP expression in brain may link to hibernation.

Cloning and characterization of human CIRP (cold-inducible RNA-binding protein) cDNA and chromosomal assignment of the gene.

Cold stress induces in microorganisms the synthesis of several proteins that are involved in various cellular processes such as transcription, translation and recombination. Recently, the cold-inducible RNA-binding protein (Cirp) was found to be induced in rodent cells by mild cold stress (32 degrees C). Cirp consists of an N-terminal RNA-binding domain and a C-terminal Gly-rich domain, and plays an essential role in cold-induced suppression of cell proliferation. We report here the cloning of a cDNA encoding an 18-kDa protein with 95.3% identity in an amino-acid sequence to that of mouse Cirp. The human CIRP gene has been mapped to the chromosomal locus 19p13.3 by fluorescence in-situ hybridization. CIRP mRNA is constitutively expressed in all cell lines examined, including K562, HepG2, NC65, HeLa, T24, and NEC8 cells. In all of them, the levels of CIRP mRNA and protein were increased within 12 h after a temperature down-shift from 37 degrees C to 32 degrees C. These results demonstrated that CIRP is a cold-shock protein in human cells. Identification of CIRP may provide a clue to the regulatory mechanisms of cold responses in human cells.