Author(s)

Shuang Wu

Date Approved

7-2016

Document Type

Dissertation

Degree Name

PhD in Cell and Molecular Biology

Department

Cell Biology

College

Graduate School of Biomedical Sciences

First Advisor

Hristo Houbaviy, PhD

Committee Member 1

Eric Moss, PhD

Committee Member 2

Dimitri Pestov, PhD

Committee Member 3

Randy Strich, PhD

Committee Member 4

Sergei Borukhov, PhD

Subject(s)

Embryonic Development; Gene Expression; Stem Cells; MicroRNAs; Gene Expression Regulation; Argonaute Proteins

Disciplines

Cell Biology | Developmental Biology | Genetic Processes | Medical Cell Biology | Medicine and Health Sciences

Abstract

Animal embryogenesis is a highly orchestrated process involving many geneexpression programs that determine specific fates of individual cells during development. Gene expression can be regulated at different stages such as at transcription initiation or mRNA stability, and microRNA-mediated gene regulation is a crucial pathway on the regulation of mRNA stability. microRNAs, or miRNAs, are small, conserved non-coding RNAs that negatively regulate gene expression post-transcriptionally. The mouse miR-290-295 cluster and its human homolog miR-371-373 are stem cell-specific miRNA clusters, and their expression can only be observed in early embryos, primordial germ cells, germ cell compartments of adult testis and embryonic stem cell lines. The miR-290-295 cluster consists of seven homologous precursor miRNA (pre-miRNA) hairpins, and small RNA sequencing data suggested seed diversification among the hairpins. It is not a priori clear whether this diversification is also conserved in the human miR-371-373 cluster, which contains only three pre-RNA hairpins, i.e., whether the two clusters are functionally equivalent. In the first half of this dissertation, using luciferase reporter data we were able to assign functional miRNAs in both miR-290-295 and miR-371-373 clusters. Subsequently, with seed-specific reporters, we demonstrated that the mouse diversified 3p seeds are also conserved and functional in human. The combination of seed sequences deduced from small RNA sequencing data in embryonic stem cells and our functional data 10 indicate the essentially functional equivalence of the two clusters. Together with the pre-miRNA sequences from other placental species we have proposed a model for the evolution of this eutherian-specific miRNA cluster family, which reveals the feature of gradual addition of new miRNA seeds that increases seed diversification across different species.

After establishing this functional equivalence relationship between the two clusters, it is important to know what targets these two clusters regulate and whether the targets are also conserved. Silencing of miRNA targets in animals is mediated by the Argonaute (Ago) proteins. They directly bind miRNA and mRNA, and an Ago partner protein G W J 82 (TNRC6 in mammals), which recruits mRNA destabilization machinery to induce mRNA decay. Targets of miR-290-295 and miR-371-373 have already been identified by a biochemical method called iCLIP (individual-nucleotide resolution UV cross-linking and immunoprecipitation), however due to the active degradation mediated by GWI 82 proteins, it is likely that the iCLIP method has missed some bona fide targets that are less abundant. In the second half of this dissertation, our initial goal was to disrupt the Ago-GW182 interaction to stabilize targets so that we can identify authentic ones that have been missed by iCLIP. However, we unexpectedly discovered that GW182-binding-deficient mutants, but not wild-type proteins, were unable to silence multiple adiacent targets efficiently. Further investigation using flow cytometry and the study of cooperativity using Hill coefficients, indicate that Ago-G182 is crucial for silencing cooperativity, which is a completely novel role for GW182 proteins. Together, the work presented herein furthers our understanding in miRNA evolution as well as their mechanism of action.

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