An in silico screen for myogenic long noncoding RNAs (lncRNAs) revealed nine lncRNAs that are upregulated more than 10-fold in myotubes versus levels in myoblasts. One of these lncRNAs, MyoD upstream noncoding (MUNC, also known as DRR eRNA ), is encoded 5 kb upstream of the transcription start site of MyoD, a myogenic transcription factor gene. MUNC is specifically expressed in skeletal muscle and exists as in unspliced and spliced isoforms, and its 5= end overlaps with the cis-acting distal regulatory region (DRR) of MyoD. Small interfering RNA (siRNA) of MUNC reduced myoblast differentiation and specifically reduced the association of MyoD to the DRR enhancer and myogenin promoter but not to another MyoD-dependent enhancer. Stable overexpression of MUNC from a heterologous promoter increased endogenous MyoD, Myogenin, and Myh3 (myosin heavy chain, [MHC] gene) mRNAs but not the cognate proteins, suggesting that MUNC can act in trans to promote gene expression but that this activity does not require an induction of MyoD protein. MUNC also stimulates the transcription of other genes that are not recognized as MyoD-inducible genes. Knockdown of MUNC in vivo impaired murine muscle regeneration, implicating MUNC in primary satellite cell differentiation in the animal. We also discovered a human MUNC that is induced during differentiation of myoblasts and whose knockdown decreases differentiation, suggesting an evolutionarily conserved role of MUNC lncRNA in myogenesis. Although MUNC overlaps with the DRR enhancer, our results suggest that MUNC is not a classic cisacting enhancer RNA (e-RNA) acting exclusively by stimulating the neighboring MyoD gene but more like a promyogenic lncRNA that acts directly or indirectly on multiple promoters to increase myogenic gene expression.

The androgen receptor (AR) stimulates and represses gene expression to promote the initiation and progression of prostate cancer. Here we report that androgen represses the miR-99a/let7c/125b-2 cluster through AR and anti-androgen drugs block the androgen-repression of the miRNA cluster. AR directly binds to the host gene of the miR-99a/let7c/125b-2 cluster, LINC00478. Expression of the cluster is repressed or activated by chromatin remodelers EZH2 or JMJD3 in the presence or absence of androgen, respectively. Bioinformatics analysis reveals a significant enrichment of targets of miR-99a, let-7c and miR-125b in androgen-induced gene sets, suggesting that downregulation of the miR-99a/let7c/125b-2 cluster by androgen protects many of their target mRNAs from degradation and indirectly assists in the gene induction. We validated the hypothesis with twelve potential targets of the miR-99a/let7c/125b-2 cluster induced by androgen: nine out of the twelve mRNAs are downregulated by the microRNA cluster. To ascertain the biological significance of this hypothesis we focused on IGF1R, a known prostate cancer growth factor that is induced by androgen and directly targeted by the miR-99a/let7c/125b-2 cluster. The androgen-induced cell proliferation is ameliorated to a similar extent as anti-androgen drugs by preventing the repression of the microRNAs or induction of IGF1R in androgen-dependent prostate cancer cells. Expression of a microRNA-resistant form of IGF1R protects these cells from inhibition by the miR-99a/let7c/125b-2 cluster. These results indicate that a thorough understanding of how androgen stimulates prostate cancer growth requires not only an understanding of genes directly induced/repressed by AR but also of genes indirectly induced by AR through the repression of key microRNAs.

MyoD Upstream Noncoding RNA (MUNC), initiates in the Distal Regulatory Region enhancer of (DRR), and is formally classified as an enhancer RNA (DRR). MUNC is required for optimal myogenic differentiation, induces specific myogenic transcripts (, , and) and has a functional human homolog. The vast majority of eRNAs are believed to act primarily on their neighboring genes (1) (2), making it likely that MUNC action is dependent on the induction of RNA. Surprisingly, MUNC overexpression in C2C12 cells induces many myogenic transcripts in the complete absence of MyoD protein. Genome wide analysis shows that while many genes are regulated by MUNC in a MyoD dependent manner, there is a set of genes that is regulated by MUNC, both upwards and downwards, independent of MyoD. MUNC and MyoD even appear to act antagonistically on certain transcripts. Deletion mutagenesis shows that there are at least two independent functional sites on the MUNC lncRNA, exon 1 being more active than exon 2, with very little activity from the intron. Thus although MUNC is an eRNA of, it is also a -acting lncRNA whose sequence, structure and co-operating factors that include but are not limited to MyoD, determine the regulation of many myogenic genes.

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