| MicroRNAs (miRNAs) are a diverse and evolutionarily conserved class of non-coding RNAs that play a multitude of roles in many branches of eukaryotic biology. The regulation of miRNAs is dynamically controlled both spatially and temporally, and the expression of miRNAs can be modulated at the level of transcription or at points downstream of the miRNA maturation process. A relevant example of post-transcriptional miRNA regulation is the blockade of let-7 precursor miRNAs by Lin28 in embryonic stem cells. This pathway, which is initiated by the small RNA-binding protein Lin28, recruits the terminal uridyl transferase (TUTase) Zcchc11 to add a non-templated oligouridine tail to the miRNAs 3' end, and signals it for degradation by the cytoplasmic exonuclease Dis3l2. The Lin28/let-7 axis is essential for development and metabolic homeostasis, and is reactivated in a subset of human cancers. This thesis describes the biochemical mechanism underlying Lin28-mediated degradation of let-7, as well as a novel role for Zcchc11 and the related TUTase Zcchc6 in targeting mature developmental miRNAs in a Lin28-independent manner.;As shown in Chapter 2, we uncovered the mechanism through which the multi-domain protein Zcchc11 recognizes pre-let-7 and Lin28. This work used in vitro biochemical techniques to monitor the activity of Zcchc11 mutants and uncovered residues that are required to form the ternary complex of Zcch11, Lin28, and let-7. This work also found the highly similar TUTase Zcchc6 to function redundantly with Zcchc11 in vitro and in vivo..;Chapter 3 describes the discovery of an intrinsic sequence preference for Zcchc6 and Zcchc11 towards single stranded mature miRNAs. This sequence motif, which is necessary and sufficient to mediate TUTase-miRNA binding and monouridylation, was surprisingly found only in developmental miRNAs that preferentially target Hox genes. Bioinformatic approaches and knockdown experiments in cultured cells showed that indeed these TUTases preferentially target the motif-containing miRNAs in vivo. Surprisingly, after observing a specific TUTase-mediated loss of uridylation, these same miRNAs underwent a proportional increase of non-templated uridylation. Furthermore, both Zcchc6 and Zcch11 were found to be absent from nearly all adult tissues and were potently down-regulated during an in vitro model of differentiation, suggesting that their activity is primarily relevant in early stages of development.;Considered together, these data refine the mechanism of known TUTase activity and also expand the roles for TUTases in development. This work should be useful in the future to assist in the development of small molecule inhibitors against this relevant oncogenic pathway, and to elucidate the myriad levels of gene regulation during development. |
