Consequences of miRNA misregulation on embryonic development and aging Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/2z10wv66v

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  • microRNAs (miRNAs), ~21-24 nucleotide-long RNAs that post-transcriptionally regulate gene expression, have rapidly become one of the most extensively studied mechanisms of the past decade. Since their discovery as temporal regulators of post-embryonic development in C. elegans, miRNAs have been functionally implicated in almost every cellular process investigated to date. miRNAs are integral to the complex biological processes of embryonic development and aging. In this research, we sought to determine whether misregulation of miRNAs could be responsible for eliciting adverse effects during these two distinct developmental stages. First, to uncover the potential role of miRNAs in teratogenicity, we investigated whether miRNAs were involved in regulation of retinoic acid (RA) induced vertebrate axis defects. Global miRNA expression profiling revealed that RA exposure suppressed the expression of miR-19 family members during zebrafish somitogenesis. Bioinformatics analyses predict that miR-19 targets cyp26a1, a key RA detoxifying enzyme, and a physiological reporter assay confirmed that cyp26a1 is a bona fide target of miR-19. Transient knockdown of miR-19 phenocopied RA-induced body axis defects. In gain-of-function studies, exogenous miR-19 rescued the axis defects caused by RA exposure. Our findings indicate that the teratogenic effects of RA exposure result, in part, from repression of miR-19 and the subsequent misregulation of cyp26a1. This highlights a previously unidentified role of miR-19 in facilitating vertebrate axis development. Next, to explore whether age-related changes in miRNAs trigger deficits in regeneration capacity, we performed mRNA and small RNA sequencing on regenerating and non-regenerating caudal fin tissue from aged, adult and juvenile zebrafish. An unbiased approach identified cbx7 as the most abundant transcript with significantly increased expression in regenerative-competent adult and juvenile tissue and decreased expression in regenerative-compromised aged tissue. While cbx7 is a known regulator of aging, this is the first report of its role in tissue regeneration. A computational approach was used to discover mRNAs expressed during regeneration, which are potential targets of the significantly expressed miRNAs in regenerating tissue. miR-21 was one of the most abundant and significantly increased miRNAs in regenerating tissue and exhibited an aberrant age-dependent expression profile. Bioinformatics predicts miR-21 to target the 3' UTR of cbx7 and a reporter assay confirmed that miR-21 targets cbx7 in vivo. Transient knockdown of miR-21 inhibited tissue regeneration, suggesting a role for miRNA mediated regulation of cbx7 during regeneration. These findings reveal a novel, age-dependent regenerative function of cbx7 and emphasize the importance of miR-21 as a master regulator of vertebrate regenerative responses. This research, when combined, underscores the negative consequences misregulation of miRNAs has on embryonic development and aging.
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