Cold-inducible RNA-binding protein (CIRP) and RNA-binding motif protein 3 (RBM3) are two evolutionarily conserved RNA-binding proteins that are transcriptionally upregulated in response to low temperature. Featuring an RNA-recognition motif (RRM) and an arginine–glycine-rich (RGG) domain, these proteins display many similarities and specific disparities in the regulation of numerous molecular and cellular events. The resistance to serum withdrawal, endoplasmic reticulum stress, or other harsh conditions conferred by RBM3 has led to its reputation as a survival gene. Once CIRP protein is released from cells, it appears to bolster inflammation, contributing to poor prognosis in septic patients. A variety of human tumor specimens have been analyzed for CIRP and RBM3 expression. Surprisingly, RBM3 expression was primarily found to be positively associated with the survival of chemotherapy-treated patients, while CIRP expression was inversely linked to patient survival. In this comprehensive review, we summarize the evolutionary conservation of CIRP and RBM3 across species as well as their molecular interactions, cellular functions, and roles in diverse physiological and pathological processes, including circadian rhythm, inflammation, neural plasticity, stem cell properties, and cancer development.

The cold-inducible RNA-binding motif protein 3 (RBM3) is involved in the protection of neurons in hypoxic-ischemic and neurodegenerative disorders. RBM3 belongs to a small group of proteins whose synthesis increases during hypothermia while global protein production is slowed down. To investigate the molecular mechanisms underlying RBM3 action, we subjected hippocampal organotypic slice cultures from RBM3 knockout mice to various stressors and found exuberant signaling of the endoplasmic reticulum (ER) stress pathway PRKR-like ER kinase (PERK)-eukaryotic translation initiation factor 2α (eIF2α)-CCAAT/enhancer-binding protein homologous protein (CHOP) as compared with wild-type mice. Further, blocking RBM3 expression in human embryonic kidney HEK293 cells by specific small interfering RNAs increased phosphorylation of PERK and eIF2α, whereas overexpression of RBM3 prevented PERK-eIF2α-CHOP signaling during ER stress induced by thapsigargin or tunicamycin. RBM3 did not affect expression of the ER stress sensor immunoglobulin binding protein/GRP78. However, based on affinity purification coupled with mass spectrometry, coimmunoprecipitation, and proximity ligation assay, we revealed that nuclear factor 90 (NF90) is a novel protein interactor of PERK and that this interaction is essential for RBM3-mediated regulation of PERK activity, which requires an RNA-dependent interaction. In conclusion, our data provide evidence for a central role of RBM3 in preventing cell death by inhibiting the PERK-eIF2α-CHOP ER stress pathway through cooperation with NF90.

Hypoxic ischemia (HI) is an acute brain threat across all age groups. Therapeutic hypothermia ameliorates resulting injury in neonates but its side effects prevent routine use in adults. Hypothermia up-regulates a small protein subset that includes RNA-binding motif protein 3 (RBM3), which is neuroprotective under stressful conditions. Here we show how RBM3 stimulates neuronal differentiation and inhibits HI-induced apoptosis in the two areas of persistent adult neurogenesis, the subventricular zone (SVZ) and the subgranular zone (SGZ), while promoting neural stem/progenitor cell (NSPC) proliferation after HI injury only in the SGZ. RBM3 interacts with IGF2 mRNA binding protein 2 (IMP2), elevates its expression and thereby stimulates IGF2 release in SGZ but not SVZ-NSPCs. In summary, we describe niche-dependent regulation of neurogenesis after adult HI injury via the novel RBM3-IMP2-IGF2 signaling pathway.

Neural stem cells (NSCs) reside physiologically in a hypoxic niche to maintain self-renewal and multipotency. Whereas mild hypoxia is known to promote NSC proliferation, severe hypoxia in pathological conditions exerts the reverse effect. The multi-functional RNA-binding protein RBM3 is abundant in NSCs and can be regulated by hypoxic exposure. Although RBM3 has been shown to accelerate cell growth in many cell types, whether and how it affects NSC proliferation in hypoxic environment remains largely unknown. In this study, we tested how RBM3 regulates cell proliferation under hypoxia in C17.2 mouse NSC cell line and in primary mouse NSCs from both the forebrain of postnatal day 0 (P0) mice and the subgranular zone (SGZ) of adult mice. Our results demonstrated that RBM3 expression was highly sensitive to hypoxia, and NSCs were arrested in G0/G1 phase by 5, 2.5, and 1% O2 treatment. When we overexpressed RBM3, hypoxia-induced cell cycle arrest in G0/G1 phase was relieved and more cell transit into S phase was observed. Furthermore, cell viability under hypoxia was also increased by RBM3. In contrast, in RBM3-depleted primary NSCs, less BrdU-incorporated cells were detected, indicating exacerbated cell cycle arrest in G1 to S phase transition. Instead, overexpressed RBM3 significantly increased proliferation ratio in primary NSCs. Our findings indicate RBM3 as a potential target to maintain the proliferation capacity of NSCs under hypoxia, which can be important in NSC-based therapies of acute brain injury and chronic neurodegenerative diseases.

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins. Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection. Video LinkThe video component of this article can be found at

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