MicroRNAs (miRNAs) are a class of ∼18-24 nucleotide endogenous RNA molecules that broadly impact cellular gene expression programs by regulating the translational efficiency and/or stability of hundreds of messenger RNAs. miRNA biogenesis is a highly regulated process. It starts in the nucleus, where miRNAs are transcribed by RNA polymerase II as long primary transcripts. Within these transcripts, sequences containing the miRNAs form ∼60-80 nucleotide hairpin structures that are excised by the nuclear enzyme Drosha. These excised hairpins, known as pre-miRNAs, are transported to the cytoplasm, where they undergo further processing by the endonuclease Dicer to produce the mature miRNAs. While transcription has a central role in controlling miRNA expression, increasing evidence suggests that post-transcriptional regulation of miRNA biogenesis is critical for appropriate miRNA expression. In this thesis, I present evidence that demonstrates dynamic post-transcriptional regulation of miRNA biogenesis at multiple levels.

First, evidence is provided that expression of a human miRNA, miR-29b, is regulated at the level of miRNA stability in HeLa cells. Its expression during the cell cycle in HeLa cells is limited to mitosis when it is stabilized.

Second, identification and characterization of a cis-regulatory motif in this unstable miRNA is described. This hexanucleotide motif is a transferable nuclear import element for 21-23 nucleotide small RNAs but does not promote fast decay of the small RNA to which it is attached. These findings demonstrate that, despite their small size, miRNAs also have cis-regulatory elements that control their post-transcriptional behaviors such as stability and subcellular localization. Moreover, these results also suggest that miRNAs might have unconventional nuclear functions.

Finally, evidence is provided that cell-cell contact globally activates miRNA biogenesis at multiple levels in culture cells. Increased cell-cell contact is associated with globally enhanced processing of miRNA primary transcripts by Drosha and an increased efficiency of the formation of the RNA-induced silencing complex. These findings uncover a critical parameter necessary for accurate analysis of miRNAs in cell culture settings, establish a tractable system for the study of regulated miRNA biogenesis, and inform our understanding of the events that globally influence miRNA expression in physiologic and pathophysiologic states.