Transcriptional Gene Silencing in Mammalian Cells by MicroRNAs That Target Gene Promoters

A rich history exists for RNA-based regulation of gene transcription. It was reported more than a decade ago that RNA is capable of inducing DNA methylation and transcriptional gene silencing in plants. It was subsequently shown that small RNAs are involved in the establishment of heterochromatic regions of the yeast genome. More recently it has been demonstrated that small duplex RNAs designed to be complementary to gene promoters are potent regulators of gene transcription in mammalian cells. Potent and robust transcriptional regulation by designed small RNAs suggests the existence of endogenous mechanisms that facilitate recognition of gene promoters by small RNAs in mammalian cells. microRNAs (miRNAs) are endogenous small RNAs that regulate gene expression post transcriptionally through base complementarity to target sequences within 3’ UTRs of mRNA transcripts. In this body of work I test the hypothesis that miRNAs can also recognize sequences within gene promoters using two alternative approaches.
In the first approach I computationally evaluate the potential for miRNAs to recognize gene promoters by performing a genome-wide survey of putative miRNA target sites within promoter sequences. In the second approach I use the well characterized human progesterone receptor (PR) gene as a model to experimentally validate that miRNAs possess the ability to regulate transcription in a cell culture system. Upon completion of this work I found that gene promoters are significantly enriched for miRNA target sites. Furthermore, the frequency of miRNA target sites within promoter sequences is comparable to their frequency within 3’ UTRs. I experimentally screened multiple miRNAs predicted to target the PR gene promoter, identified several that were capable of inhibiting transcription of the PR gene, and characterize the mechanism of transcriptional silencing. miRNAs have been understood to regulate gene expression at the post transcriptional level through recognition of 3’ UTRs within mRNA transcripts. My study extends miRNA function to recognition of sequences within gene promoters. Sequence specific recognition of gene promoters by miRNAs may complement protein transcription factors. In addition, the ability of small RNAs to rapidly evolve specificity for new sequences would have evolutionary advantages.