Abstract
RNA-binding proteins (RBPs) have important roles in transcription, pre-mRNA processing/transport, mRNA degradation, translation, and non-coding RNA processing, among others. RBPs that are expressed in response to cold stress, such as Cirp and Rbm3, could regulate RNA stability and translation in hibernating mammals that reduce their body temperatures from 37 °C to as low as 0–5 °C during torpor bouts. RBPs including Cirp, Rbm3, and stress-inducible HuR translocate from the nucleus to stabilize mRNAs in the cytoplasm, and thereby could regulate which mRNA transcripts are protected from degradation and are translated, versus stored, for future protein synthesis or degraded by nucleases during cell stress associated with metabolic rate depression. This is the first study to explore the transcriptional/translational regulation, and subcellular localization of cold-inducible RBPs in a model hibernator, the 13-lined ground squirrel (Ictidomys tridecemlineatus). Cirp protein levels were upregulated in liver, skeletal muscle, and brown adipose tissue throughout the torpor-arousal cycle whereas Rbm3 protein levels stayed constant or decreased, suggesting an important role for Cirp, but likely not Rbm3, in the hibernator stress response. Increased cytoplasmic localization of Cirp in liver and muscle and HuR in liver during torpor, but no changes in the relative levels of Rbm3 in the cytoplasm, emphasizes a role for Cirp and possibly HuR in regulating mRNA processing during torpor. This study informs our understanding of the natural adaptations that extreme animals use in the face of stress, and highlight natural stress response mediators that could be used to bolster cryoprotection of human organs donated for transplant.
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Supplemental Fig. 1
HuR protein sequence of the experimentally validated human (Homo sapiens) HuR proteins (36 kDa and 38 kDa, sourced from UniProt database) and the computationally predicted 13-lined ground squirrel (Ictidomys tridecemlineatus) sequence from NCBI database. A) Sequence alignment of 36 kDa and 38 kDa human HuR proteins with 36 kDa I. tridecemlineatus HuR protein indicates the incorporation of an additional exon in the N-terminal end of the human HuR protein. B) Alignment of the human HuR protein (38 kDa) with the genome-predicted 38 kDa 13-lined ground squirrel HuR protein showing 94.05% sequence similarity between human and ground squirrel HuR proteins. An asterisk (*) indicates perfect alignment, a colon (:) indicates strongly similar amino acids and a period (.) indicates conservation between weakly similar groups. Lettering with the color red indicates small and hydrophobic amino acids, blue indicates acidic amino acids, magenta indicates basic amino acids, green indicates hydroxyl, sulfhydryl, amine and glycine. The region in yellow highlight signifies the N-terminal domain present in the 38 kDa HuR protein that is not present in the 36 kDa isoform. (PNG 1332 kb)
Supplemental Fig. 2
Relative cold-inducible RNA-binding protein levels in cytoplasmic (cyt) and nuclear (nuc) fractions of liver (LIV), muscle (MUS) and brown adipose tissue (BAT), comparing euthermic (EC) and torpid (LT) 13-lined ground squirrels. (a) Histogram shows mean standardized expression levels of Cirp (18 kDa and 28 kDa), HuR, and Rbm3 (± S.E.M., n = 4 independent protein isolations from different animals). (b) Representative nuclear Western blots show two different samples (n = 2) of the total n = 4 for each time point. Data were analyzed using a Student’s t test, where cytoplasmic and nuclear levels were analyzed separately. An asterisk above the LT time point indicates a significant difference from the EC value (p < 0.05) (PNG 3406 kb)
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Logan, S.M., Storey, K.B. Cold-inducible RNA-binding protein Cirp, but not Rbm3, may regulate transcript processing and protection in tissues of the hibernating ground squirrel. Cell Stress and Chaperones 25, 857–868 (2020). https://doi.org/10.1007/s12192-020-01110-3
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DOI: https://doi.org/10.1007/s12192-020-01110-3