Abstract
MicroRNAs (miRNAs) are small non-coding RNAs that have well-characterized roles in cytoplasmic gene regulation, where they act by binding to mRNA transcripts and inhibiting their translation (i.e. post-transcriptional gene silencing, PTGS). However, miRNAs have also been implicated in transcriptional gene regulation and alternative splicing, events that are restricted to the cell nucleus. Here we performed nuclear-cytoplasmic fractionation in a mouse endothelial cell line and characterized the localization of miRNAs in response to hypoxia using small RNA sequencing. A highly diverse population of abundant miRNA species was detected in the nucleus, of which the majority (56%) was found to be preferentially localized in one compartment or the other. Induction of hypoxia resulted in changes in miRNA levels in both nuclear and cytoplasmic compartments, with the majority of changes being restricted to one location and not the other. Notably, the classical hypoxamiR (miR-210-3p) was highly up-regulated in the nuclear compartment after hypoxic stimulus. These findings reveal a previously unappreciated level of molecular complexity in the physiological response occurring in ischemic tissue. Furthermore, widespread differential miRNA expression in the nucleus strongly suggests that these small RNAs are likely to perform extensive nuclear regulatory functions in the general case.
Highlights
The canonical view of microRNA function is that these small RNA molecules typically repress gene expression by targeting the mRNA 3′ untranslated region (3′ UTR) in order to induce post-transcriptional gene silencing (PTGS)[1]
To investigate the extent to which mature miRNAs are present in the nucleus, we performed high throughput sequencing of small RNAs in nuclear and cytoplasmic fractions derived from endothelial cells after the induction of hypoxia
We show for the first time, that specific miRNAs are differentially expressed in a subcellular location restricted manner in response to hypoxic stimulus. These findings suggest that miRNAs likely execute nuclear functions in addition to their cytoplasmic PTGS activity, and reveal a previously unappreciated level of complexity in the cellular response to hypoxia
Summary
The canonical view of microRNA (miRNA) function is that these small RNA molecules typically repress gene expression by targeting the mRNA 3′ untranslated region (3′ UTR) in order to induce post-transcriptional gene silencing (PTGS)[1]. We have previously shown that short hairpin RNA (shRNA)-mediated transcriptional activation of the Vegfa promoter is of therapeutic benefit in murine models of hindlimb ischemia[9] and myocardial infarction[10] These observations suggested that shRNAs may function by mimicking endogenous miRNAs. To date, intracellular miRNA localization has been largely overlooked, and only a handful of publications have performed global miRNA analysis in nuclear and cytoplasmic fractions. We show for the first time, that specific miRNAs are differentially expressed in a subcellular location restricted manner in response to hypoxic stimulus These findings suggest that miRNAs likely execute nuclear functions in addition to their cytoplasmic PTGS activity, and reveal a previously unappreciated level of complexity in the cellular response to hypoxia
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