Nuclear RNAs play key roles in the regulation of transcription, splicing, and RNA modifications; processes that have widespread impacts on cellular function. RNA binding proteins (RBPs) modulate RNA function by altering the composition, stability, or protein association of target RNAs. Although cell-specific nuclear RNA regulation has been observed, the underlying mechanisms and functional consequences have remained unclear. The goal of this research is to determine how two male germ cell-specific RBPs (ADAD1 and ADAD2) regulate nuclear RNA biogenesis and the functional impact this regulation has on spermatogenesis. This research builds upon studies supported by my NIH postdoctoral fellowship. High-throughput RNA sequencing (RNA- seq) analysis of a male infertile mutant Adad1 model demonstrated substantial changes in the expression of a class of nuclear RNAs (small nucleolar RNA - snoRNAs) in spite of virtually no changes in gene expression or isoform abundance. snoRNAs guide nucleotide modification in other RNAs, particularly ribosomal RNAs (rRNAs). Improper rRNA modification leads to ribosome malfunction and abnormal translation, a process that is tightly controlled in the male germ cell. This, combined with ADAD1 nuclear localization, suggests that ADAD1 plays a critical role in snoRNA biogenesis or regulation, as may its closely-related homologue ADAD2. The proposed research will occur in two phases, a mentored (I) and an independent phase (II) and will test the hypothesis that ADADs impact translation by regulating the biogenesis of snoRNAs in male germ cells. To address the hypothesis, this research will: 1) establish the impact of Adad1 mutation on rRNA modification and translation (phase I); 2) demonstrate whether ADAD1 and 2 associate with snoRNAs or snoRNA-regulating proteins (phase II); and 3) determine the impact of Adad1 or 2 ablation on snoRNA abundance and germ cell development (phase I) using a combination of biochemical analysis, RNA-seq, and novel genetic models. Ultimately, this work will define the connection between ADADs, snoRNAs and germ cell translation thus informing on the mechanisms by which nuclear RNAs affect male fertility.
Translation is regulated in part by nuclear RNAs. Germ cells are particularly sensitive to aberrations in translational control, with defects often leading to infertility. Infertility affects one in seven couples, roughly half of these cases being a result o male factor defects. Of these, nearly half again are idiopathic, with the molecular underpinnings of infertility undefined. The work outlined in this proposal will inform on the mechanisms of nuclear RNA regulation and how this affects male germ cell translation, providing insight into potential causes of male infertility.
