Thesis Dissertation Abstract: Context Dependent Regulation of Dicer Function During Oocyte-to-Embryo Transition

Nick Alexander Newkirk (Advisor: Swathi Arur, PhD)

          Over two decades ago, Dicer was discovered as the primary enzyme responsible for the generation of small RNAs known as the microRNAs (miRNAs) and short interfering RNAs (siRNAs). Spatiotemporal regulation of these Dicer-dependent small-RNAs affects many aspects of biology, including aging, disease progression, fertility, and cancer. Interestingly, a distinct population of Dicer-dependent small RNAs, known as the endogenous siRNAs (endo-siRNAs), are uniquely abundant in the germ cells and gametes, and are critical for normal fertility. Work to understand these unique small RNAs in C. elegans, where small RNA function was first identified, has amplified our overall understanding of endo-siRNA function, including the revelation that the Dicer helicase domain has an exclusive regulatory effect on developmental endo-siRNAs. Despite this revelation, the mechanisms regulating Dicer helicase domain activity and subsequent endo-siRNA biogenesis are enigmatic due to the responsibility of Dicer’s helicase domain in performing multiple dynamic functions.

In this thesis, using Caenorhabditis elegans, I uncovered a regulatory role for the arginine-rich GRARR sequence of Dicer helicase domain motif VI, based around potential arginine methylation of this domain. I find that mutation to conserved arginine residues of the GRARR sequence disrupts the oocyte-to-embryo transition with defects manifesting primarily during anaphase I. Furthermore, these mutations also result in the loss of maternal 26G endo-siRNAs, while miRNAs remain unperturbed. I find that in vitro synthetic asymmetric di-methylated Dicer helicase (R495) peptides promote interaction with ERI-5, a tandem-Tudor protein within the ERI Complex (ERIC) that is necessary for 26G endo-siRNA biogenesis. This was the first evidence that Dicer methylation could potentiate interaction with ERI-5 and the ERI complex to promote sexually dimorphic 26G siRNA production. Using genetic analysis, I illustrate that the Dicer-ERIC interaction potentiates 26G endo-siRNA production, as deletion of ERI-5 phenocopies Dicer GRARR mutants. Strikingly, these defects mirror those of Dicer-phosphorylated catalytic domain mutants and when arginine methylation and serine phosphorylation are modeled in AlphaFold, this suggested that distinct Dicer domains may operate in a coordinated manner to modulate structural conformation and effector recruitment. The totality of the findings here have identified a context dependent phosphorylation-to-methylation Dicer post-translational switch controlling engagement with ERI-5 of the ERIC to ensure proper small RNA biogenesis and fertility.

Advisory Committee:

• Swathi Arur, PhD, Chair
• Mark Bedford, PhD
• Xiaodong Cheng, PhD
• Sharon Dent, PhD
• Michael Galko, PhD

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