Dissecting Microrna Requirements In Early Human Embryonic Development
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DICER1 is the enzyme responsible for cleaving double-stranded RNAs (dsRNAs) into functionally mature ~20-24 nucleotide (nt) long microRNAs (miRNAs). miRNAs are the effectors of an RNA-induced gene silencing system that functions at the post-transcriptional and post-translational levels, and are essential for mouse development. Due to maternal Dicer1 contribution in the early stages of embryonic development, the exact timing for miRNA requirement has not yet been established. Furthermore, precise miRNA functional studies in early embryonic development have been lacking since miRNA knockout studies are hindered due to redundancy in the miRNA network. Thus, miRNA function in early embryonic development remains elusive. To address this knowledge gap, we set out to knockout DICER1 in human ESCs (hESCs) and assess human-specific miRNA requirements in the primed state. We report that DICER1 is essential in hESCs unlike in mouse embryonic stem cells (mESCs), and that this likely reflects a unique requirement for DICER1 in primed versus naïve pluripotency. Additionally, we designed an inducible DICER1 system to bypass the lethality of DICER1 loss in hESCs and enable the generation of homozygous mutants. A targeted mature miRNA rescue screen identified members of the miRNA-302-367 and miRNA-371-373 clusters, but surprisingly not miRNA-17-92, as having pro-survival functions in hESCs. Since it bypasses the common issue of redundancy in the miRNA network, our screening platform is particularly suited to dissect the roles of individual miRNAs and miRNA clusters in early human development and hESC differentiation.
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2017
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V2
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Cell & Developmental Biology
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Doctor of Philosophy
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Government Document
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Attribution-NonCommercial-NoDerivatives 4.0 International
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dissertation or thesis