Specialized cellular identity requires unique protein expression control. Such expression control in gametes and
reproductive neurons ensures fertility. By studying cell- and tissue-level genetic code regulation, we discovered
multiple links to reproductive biology. Specifically, we found that in both flies (Drosophila) and humans, the germ
cells of the testis but not the ovary upregulate numerous mRNAs that are enriched in rarely used codons. We
then revealed that rare codons restrict the ribosomal protein RpL10Aa to the fly testis to ensure fertility. Few
other fly cell types express a rare codon reporter outside of the testis, with an additional example being most
types of neurons of the brain. From genetic screening, we identified the conserved mRNA binding proteins Not3
and Orb2 as critical regulators of rare codon-dependent expression in both germ cells of the testis and in neurons
throughout the brain. Notably, Orb2 has two main functions- ensuring spermatogenesis and control of mating
behavior in reproductive neurons of the brain. Thus, our data are revealing important rare codon regulation of
reproduction in both male gametes and neurons (including those neurons that control reproduction). In this
proposal, we capitalize on our model and robust preliminary data to find new rare codon-dependent regulation
in the testis and brain, and to define its function in fertility and reproductive behavior. The significance of our
proposed work lies in the conservation of our findings in human testis biology and the impact on reproductive
biology. Further significance is in the conservation of the new regulators of rare codon expression to be studied
(Not 3 and Orb2). Our studies are innovative because we will study testis/neuron biology and reproduction in a
new way, from the perspective of tissue-specific interpretation of the redundant code. In Aim1, we will identify
the role of testis- and neuron-specific tRNA levels and mRNA stability on rare codon expression. To answer this
question, we will determine the impact of rare codon tRNA levels, rates of mRNA decay, and the CCR4-Not de-
adenylation complex on rare codon expression. We will compare each of these mRNA translation inputs in the
testis vs. ovary and neurons vs. neural progenitors. In Aim 2, we will define the mechanism of Orb2 rare codon
regulation in the testis and neurons. To do so, in the testis and neurons we will identify critical Orb2 domains
required for rare codon expression, determine the role of rare codons in mRNA/Orb2 interactions, and identify
rare codon-enriched Orb2 targets involved in fertility and reproductive behavior. In Aim 3, we will define the
function of specific rare codon-derived proteins in the testis and neurons. To do so, we will further our mechanistic
study of the testis-specific RpL10Aa in gametogenesis and fertility and use candidate genetics and ribosome
profiling to find new examples where rare codons direct expression and/or function, focusing on testis-specific
expression in gametogenesis/fertility, and male neuron-specific expression in reproductive behavior (courtship).
Together, our proposed work will reveal new insight into how an ancient feature of life, the redundancy of the
genetic code, regulates testes and neuron protein expression in the context of reproduction.