The regulation of let-7 microRNA biogenesis in early embryogenesis

microRNAs (miRNAs) are small non-protein-coding RNAs that silence gene expression post-transcriptionally and have critical functions in development, tissue homeostasis and in the pathogenesis of many diseases. They carry out gene silencing by targeting complementary sequences in messenger RNAs (mRNAs) in the context of a ribonucleoprotein particle named RISC (for RNA induced silencing complex). The mature miRNA contained within RISC is a ~21 nucleotide single stranded RNA that must be processed in two RNase III-catalyzed reactions. In animals, our precise understanding of miRNA funtion in vivo has been hindered by the fact that animal miRNAs do not basepair with perfect complementarity to their miRNA targets. However, much insight into miRNA function has been gained by the study of their spatio-temporal expression. Of particular interest is that the majority of known mouse miRNAs are not detectable during early embryogenesis but are robustly induced in later in gestation, during the time of tissue differentiation. Intriguingly, there is a broad down regulation of miRNAs in poorly differentiated tumors; thus it is a critical long-term goal to understand whether the global absence of miRNAs in cancer reflects the reversion to an embryonic-like cellular state. Studies described herein show that the abundance of many miRNAs, including the let-7 family of tumor suppressor miRNAs, are regulated post-transcriptionally both in murine development and in cancer. Furthermore, RNase III-mediated processing of let-7 is blocked by the embryonic stem (ES) cell-specific RNA binding protein Lin28. While the exact mechanism of the Lin28-mediated processing block is not known, a popular model predicts that Lin28 binds to the short hairpin precursor let-7 (pre-let-7) and recruits the terminal uridyl transferase, TUT4, leading to oligo-uridylation and turnover of pre-let-7. Directly testing this hypothesis is technically challenging, in part, because short-hairpin miRNA precursors (pre-miRNAs) are low in abundance. We developed a novel and powerful technique to detect pre-miRNAs utilizing high-throughput DNA sequencing. Unexpectedly, we observed that pre-let-7 is uridylated in differentiated cells, which lack Lin28; furthermore, many pre-miRNAs are uridylated in vivo, likely through a Lin-28-independent pathway