Abstract
Numerous miRNAs have been detected in mitochondria, which play important roles in many physiological and pathophysiological processes. However, the dynamic changes of miRNA distribution in mitochondria and their mechanisms in reactive oxygen species (ROS)-induced endothelial injury remain unclear. Therefore, miRNA levels in whole cells and mitochondria of H2O2-treated endothelial cells were analyzed by small RNA sequencing in the present study. The results showed that H2O2 significantly reduced the relative mitochondrial distribution of dozens of miRNAs in human umbilical vein endothelial cells (HUVECs). Among the high-abundance miRNAs, miR-301a-3p has the most significant changes in the redistribution between cytosol and mitochondria confirmed by absolute quantitative polymerase chain reaction (qPCR). To unravel the mechanism of miR-301a-3p distribution in mitochondria, RNA pull-down followed by label-free quantitative proteomic analysis was performed, and RNA-binding protein Musashi RNA binding protein 2 (MSI2) was found to specifically bind to miR-301a-3p. Western blotting and immunofluorescence colocalization assay showed that MSI2 was located in mitochondria of various cell types. H2O2 significantly downregulated MSI2 expression in whole endothelial cells, promoted the distribution of MSI2 in cytosol and decreased its distribution in the mitochondria. Moreover, overexpression of MSI2 increased the mitochondrial distribution of miR-301a-3p, whereas inhibition of MSI2 decreased its distribution in mitochondria. Thus, MSI2 might be responsible for the distribution of miR-301a-3p between cytosol and mitochondria in endothelial cells. Our findings revealed for the first time that MSI2 was involved in the regulation of miRNA distribution in mitochondria and provided valuable insight into the mechanism of mitochondrial distribution of miRNAs.
Highlights
It is believed that miRNAs regulate mRNA stability and translation primarily in the cytosol (Gebert and MacRae, 2019)
The results showed that a mitochondrial protein, translocase of outer mitochondrial membrane 40 (Tomm40), was strongly enriched while two cytosolic proteins, β-actin and ribosomal protein S9 (RPS9) were undetected in the mitochondrial fraction (Supplementary Figure 1A)
The results showed that H2O2 treatment significantly reduced the relative mitochondrial distribution of dozens of miRNAs in human umbilical vein endothelial cells (HUVECs) (Figure 2C)
Summary
It is believed that miRNAs regulate mRNA stability and translation primarily in the cytosol (Gebert and MacRae, 2019). A growing body of evidence indicated that miRNAs are distributed in various subcellular regions, such as nucleus, processing bodies, endoplasmic reticulum and mitochondria (Leung, 2015; Trabucchi and Mategot, 2019). Subcellular distribution of miRNAs is indicative of regulatory role of miRNA in various cellular processes and it profoundly influenced many pathophysiological processes (Trabucchi and Mategot, 2019). Hundreds of mature miRNAs have been detected in mitochondria of various types of cells and tissues (Barrey et al, 2011; Sripada et al, 2012a). Many miRNAs located in mitochondria have been found to regulate many general and mitochondrial-specific biological processes (Borralho et al, 2014). The expression profile of miRNAs in mitochondria was dynamically changed under different pathophysiological conditions (Wang et al, 2015)
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