ADAD1 and ribosome biogenesis in the post-meiotic male germ cell - PROJECT SUMMARY Due to their unique biology, post-meiotic male germ cells are highly reliant upon translation regulation. One of the main mediators of translation regulation is RNA binding proteins (RBPs), many of which remain to be described. ADAD1 is a testis-specific RNA binding protein expressed in post-meiotic round spermatids and it is required for male fertility, although its molecular mechanism is still unknown. Preliminary data from the Snyder laboratory shows that ADAD1 loss leads to a decrease in ribosomal RNA (rRNA) modifications and a decrease in the maturation of snoRNAs, non-coding RNAs that template site- specific rRNA modifications. In addition, Adad1 mutants have reduced ribosome association of many post- meiotic germ cell transcripts including those encoding ribosome proteins and ribosome biogenesis factor proteins as well as transcripts necessary for fertility. This signature suggests ADAD1 is involved in proper ribosome assembly and function via snoRNA-mediated rRNA modification. This proposal will test the hypothesis that ADAD1 impacts the translation of post-meiotic transcripts required for male fertility by acting as a male germ cell specific ribosome biogenesis factor through two aims. Aim 1 will define ADAD1 RNA and protein interactors through use of a myc-tagged Adad1 allele. To shed light on ADAD1’s molecular mechanism, myc-immunoprecipitation will be followed by RNA sequencing or mass spectrometry. In Aim 2, ribosome protein composition and ribosome associated proteins will be quantified in round spermatids from Adad1 mutant mice using quantitative mass spectrometry on polysome purified ribosomes. This will, for the first time, define the round spermatid’s ribosome proteome and interactome as well as determine ADAD1’s influence on ribosome protein and ribosome associated protein composition. Together, these studies will characterize the role of ADAD1 in post-meiotic germ cell ribosome biogenesis, with a special focus on ribosome composition. Although previously thought to be invariant, recent studies have shown that ribosome protein and RNA composition can vary between cells as well as within a single cell. This is particularly relevant to male germ cells, which are highly reliant on translation regulation. Through completion of the proposed aims, this work will shed light on the post-meiotic male germ cell’s ribosome assembly and the molecular mechanism of ADAD1, informing on the role of both in male fertility. In addition, the enclosed training plan has been designed to enhance my reproductive biology knowledge and computational skillset with the aim to become a principal investigator studying reproduction at a research university. My training will include analysis of RNA sequencing and proteomics data, formalized computation courses, internal / external conferences on reproduction, and attending the intensive six-week reproduction course, Frontiers in Reproduction. Training will take place at Rutgers University under the guidance of my sponsor, Dr. Elizabeth Snyder and members of my committee, Drs. Anthony, Mickolajczyk, and Singson.
