Date of Award
Doctor of Philosophy (PhD)
Development in animals requires precise and coordinated changes in gene expression. This genetic remodeling is achieved through extensive regulatory networks of proteins and RNAs that function together to specify new cell fates and patterns. One developmental event heavily reliant on these regulatory networks is the maternal-to-zygotic transition (MZT), a universal step in metazoan embryogenesis in which a fertilized oocyte is reprogrammed into a pluripotent embryo. The earliest stages of the MZT are governed by maternally inherited gene products, which are required for cellular functions in the initially transcriptionally silent embryo. To shift developmental control to the zygote, these maternal mRNAs are massively degraded through multiple posttranscriptional mechanisms. The RNA modification, N6-methyladenosine (m6A) has been proposed as a master regulator of mRNA decay during developmental transitions, but the direct effects of this pathway on maternal transcript clearance remain unclear. To determine whether m6A facilitates gene expression changes during the MZT, I employed zebrafish embryos as a model system to dissect the contributions of RNA methylation and its reader proteins to maternal transcript fate. Through transcriptome analysis and reporter assays, I found that m6A controls maternal mRNA degradation by promoting deadenylation. To understand how RNA methylation fits into the framework of known decay pathways, I compared transcripts co-targeted by m6A and miR-430, a microRNA that controls mRNA clearance in zebrafish. This revealed that these mechanisms function independently but additively to promote mRNA degradation, reflecting that methylation modulates transcript abundance in concert with known regulators. To disentangle the roles of the Ythdf proteins that mediate the effects of m6A on mRNA, I generated zebrafish genetic mutants of Ythdf1, Ythdf2, and Ythdf3. Through transcriptomic and phenotypic analysis of these mutants, I determined that global maternal mRNA clearance, zygotic genome activation, and development proceed normally in the absence of any one reader. This revealed that individual Ythdf protein have limited effects on the removal of methylated maternal mRNAs during the MZT. To test if this restricted impact of single Ythdf loss stems from functional redundancy between the readers, I produced double mutants of Ythdf2 and Ythdf3. Double Ythdf deletion prevents female gonad development, indicating that these factors exert overlapping activities during oogenesis. Finally, to fully establish functionally redundancy, I created triple Ythdf mutants, which were larval lethal. I observed this same phenotype in zebrafish lacking the methylases that add m6A to mRNA, indicating that RNA methylation is essential for developmental viability. Together, this work provides insight into the contributions of the m6A modification and its Ythdf effectors to maternal mRNA clearance, and establishes how these key regulators coordinate the gene expression changes that underlie embryonic reprogramming.
Kontur, Cassandra, "RNA Methylation and Ythdf Readers in Posttranscriptional Regulation and Development" (2021). Yale Graduate School of Arts and Sciences Dissertations. 68.