Transcription Activation by Micrornas: From Mechanism to Application

For decades, microRNAs (miRNAs) have been canonically viewed as post-transcriptional repressors that direct mRNA degradation and translational repression through sequence-specific target recognition. Accumulating evidence has revealed that miRNAs also regulate gene transcription, functioning as either transcriptional repressors or, more recently, activators. These discoveries raise a fundamental question: what determines whether miRNAs activate or repress gene transcription? Addressing this question requires defining where and how miRNAs interact with chromatin. However, accurate mapping of chromatin-associated miRNA target sites has remained a longstanding technical challenge because miRNAs are short, present at low abundance in the nucleus, and difficult to capture on chromatin. Consequently, the mechanisms underlying miRNA-mediated transcriptional regulation remain far less understood than their canonical post-transcriptional functions.

To address this challenge, this dissertation presents a strategy for mapping chromatin-associated miRNA target sites, enabling mechanistic investigation of miRNA-mediated transcriptional regulation. Building upon recent advances in epitranscriptomics, we uncovered a previously unrecognized mechanism in which promoter-targeting miRNAs cooperate with the m⁶A-dependent chromatin regulatory machinery to activate endogenous gene transcription. These findings establish a direct connection between two major regulators of gene expression—RNA modifications and miRNAs—in transcriptional regulation, explaining how miRNAs selectively activate endogenous gene transcription. Finally, we translate these mechanistic insights into the rational design of transcription-activating miRNAs that selectively activate endogenous therapeutic genes while minimizing off-target effects, establishing a framework for programmable RNA-based gene activation and RNA therapeutics.