Expression of the miR-302/367 microRNA cluster is regulated by a conserved long non-coding host gene

Abstract

MicroRNAs (miRNAs) are important regulators of cellular functions. MiR-302/367 is a polycistronic miRNA cluster including miR-302b/c/a/d (collectively termed miR-302s) and miR-367. The cluster is located in the intron of a non-coding host gene. MiR-302s have been shown to repress mRNAs required for differentiation and to induce pluripotency in somatic cells. The stem cell specific transcription factors OCT4, SOX2 and Nanog drive miR-302s expression, however, the reported expression in human and mice indicates a more complex transcriptional regulation. Here we investigate the transcriptional control and the processing of the miR-302 host gene. The murine miR-302 host gene is alternatively spliced, polyadenylated and exported from the nucleus. The regulatory sequences extend at least 2 kb upstream of the transcription start side and contain several conserved binding sites for both transcriptional activators and repressors. Reporter constructs with different upstream regions revealed a significant influence of the more distant regulatory sequences in pluripotent stem cells. The gene structure and regulatory elements like binding sites for activating and repressing transcriptional regulators, splice, and polyadenylation signals are highly conserved between mouse and human. So far, no miR-302 independent function has been annotated for the miR-302 host gene and we hypothesize that the complex and differential regulation of the miRNA transcription and processing might the reason for its conservation. Thus, regulation or micro-RNA expression might be a so far less recognized function of non-coding RNA genes. Author SummaryNon-coding RNAs constitute a large part of the mammalian genome. Interestingly, some long non-coding RNAs (lncRNA) are transcribed and processed in the same way as mRNAs but lack an open reading frame. Here we give evidence that a so far less recognized function of such lncRNAs could be to supply microRNAs with the complex transcriptional control and processing required for their intricate expression. As an example, we analyzed the regulatory sequences of the miR-302/367 host gene. MiR-302/367 is a microRNA cluster involved in the regulation of stem cells and cellular differentiation. We show here that the regulatory region is much more complex than anticipated, a complexity that can not be conferred alone by any of the stem cell specific transcription factors which were so far associated with the expression of miR-302/367.

Transgenic Mice and Pluripotent Stem Cells Express EGFP under the Control of miR-302 Promoter

“…Some miRNAs regulate gene expression levels in the stem cells, such as the miR-302 cluster. MiR-302s are located in the first intron of the host gene, which is classified as a non-coding RNA (Rahimi et al, 2018). The expression of this gene is driven by Pol II RNA polymerase, and its regulatory sequence is more complex than a simple promoter for miRNA expression.…”

“…Their order within the cluster represents their placement within the gene. These miRNAs share the same promoter and are generated from the same primary transcript (Barroso-delJesus et al, 2008;Rahimi et al, 2018). All five miRNAs are produced from an intron sequence.…”

Isolation of cancer stem cells by selection for miR-302 expressing cells

“…Murine miR-302 host RNA is a non-coding transcript of unknown function. The RNA is spliced, poly-adenylated and exported from the nucleus (Rahimi et al, 2018) and thus might be used to introduce a coding sequence to be translated. In order to test this, we introduced the fused coding sequences of egfp-neo into the second exon of the miR-302 host RNA under the control of the PGK promoter ( figure S3C).…”

“…The miR-302 cluster processes and produces five miRNAs including miR-367 from the first intron of the non-coding host transcript (Barroso-delJesus et al, 2008). The primary host RNA includes two exons in mouse (Rahimi et al, 2018). The miRNAs share the same seed sequence, which is also shared with miR-290-295 in mice and miR-373 in humans.…”

“…In human ES cells, expression of the miR-302 cluster is conferred by 525 bp immediately upstream of the transcription start site (Barroso-delJesus et al, 2008;Barroso-delJesus et al, 2009). In mouse, we have shown that a highly conserved regulatory sequence of at least 2.1 kb, is involved in miR-302 gene regulation (Rahimi et al, 2018).…”