Person:

Thornton, James Edward

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.

Pluripotent embryonic stem cells (ESCs) have a shortened cell cycle that enables their rapid proliferation. The ESC-specific miR-290 and miR-302 microRNA families promote proliferation whereas let-7 microRNAs inhibit self-renewal and promote cell differentiation. Lin28 suppresses let-7 expression in ESCs. Here, to gain further insight into mechanisms controlling ESC self-renewal we explore the molecular and cellular role of the let-7 target Trim71 (mLin41). We show that Trim71 associates with Argonaute2 (Ago2) and microRNAs and represses expression of Cdkn1a, a cyclin-dependent kinase inhibitor that negatively regulates the G1–S transition. We identify protein domains required for Trim71 association with Ago2, localization to P-bodies, and for repression of reporter mRNAs. Trim71 knockdown prolongs the G1 phase of the cell cycle and slows ESC proliferation, a phenotype that was rescued by depletion of Cdkn1a. Thus, we demonstrate Trim71 is a factor that facilitates the G1–S transition to promote rapid ESC self-renewal.