An Anoxia‐Tolerant Cell Line, AL4, Provides New Insight into Small Non‐Coding RNA Biology

Abstract

Small non‐coding RNAs (ncRNAs) can rapidly, reversibly, and specifically regulate gene expression. These characteristics make them compelling candidates for regulating gene expression in anoxia‐tolerant vertebrates, as survival of anoxia requires dramatic yet reversible changes in physiology. Recent small ncRNA sequencing in anoxia‐tolerant vertebrates revealed groups of candidate small ncRNAs for supporting anoxia tolerance. The expression of mitosRNAs, small ncRNAs derived from the mitochondria, was particularly striking in the most anoxia‐tolerant vertebrate, embryos of the annual killifish Austrofundulus limnaeus, and inspired further study. Cell culture is a particularly useful tool for detailed study of small ncRNA biology, due to the ease of subcellular imaging and chemical and genetic manipulation. Therefore, we aimed to establish and characterize an anoxia‐tolerant cell line from A. limnaeus to use as a tool to probe the biology of small ncRNAs of interest. The AL4 cell line was established from embryonic tissue of A. limnaeus using a tissue explant method. Proteomic phenotyping suggested a neuroepithelial identity. The cells can survive for over 40 d without oxygen or replenishment of growth medium, compared to only 3 d of anoxic survival for green monkey kidney epithelial cells (Vero) and mouse neuroblastoma cells (N2A). Additionally, AL4 cells accumulate lactate during anoxia, which is consistent with whole embryos, as well as anoxia‐sensitive mammals. AL4 express many of the same small noncoding RNAs as the whole annual killifish embryos, including anoxia‐responsive mitosRNAs. Known mammalian stress‐responsive small ncRNAs also respond to anoxia in AL4. Therefore AL4 both reflects the biology of whole embryos and can be used in comparative studies with anoxia‐sensitive mammalian species. We used the cell line to investigate highly anoxia‐responsive mitosRNAs derived from mitochondrial tRNA cysteine. We found that these mitosRNAs are enriched in the mitochondria, particularly near the nucleus, and appear to be present in the cytoplasm as well. They also appear to be de novo transcribed in the mitochondria during anoxia. This body of work provides the first evidence for mitochondrial de novo transcription and cytoplasmic localization of mitosRNAs in response to stress. The establishment of the AL4 cell line provides a unique tool for investigating cellular mechanisms of vertebrate anoxia tolerance, including mitosRNAs, and has the potential to transform our understanding of the role of oxidative metabolism in cell biology.Support or Funding InformationThis work was supported by NSF GRFP DGE‐1057604 to Claire L. Riggs and NSF IOS‐1354549 to Jason E. Podrabsky.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.