Role of Long Non-Coding RNAs in the Ovulatory Process

TITLE: Role of Long non-coding RNAs in the Ovulatory Process. ABSTRACT Women’s reproductive health plays a pivotal role in regulating overall well-being. Unfortunately, about 12% of reproductive-age women experience infertility. One of the most common causes of infertility is anovulation or oligo-ovulation, supporting the paramount role of the ovulatory process in female reproductive success. Although a few key ovulatory mediators induced by LH have been established, new and innovative research techniques continue to discover new players in the ovulatory process. Using RNA sequencing, we have identified several long non-coding RNAs (lncRNAs, transcripts >200 nucleotides) that are upregulated during the periovulatory period in mouse granulosa cells. In past, these lncRNAs were considered as junk DNA and believed to be transcriptional noise and functionless. But, in recent years, evidence has accumulated showing that lncRNAs are widely expressed, are tissue-specific, and have key roles in gene regulation by interacting with DNA, RNA, and proteins. However, the role of lncRNAs in the ovary and specifically the ovulatory process has not been well explored. From our novel RNA sequencing data, we have found 22 lncRNAs transcripts that are increased in granulosa cells collected at 4h and 12h after human chorionic gonadotropin (hCG: LH analog) administration. Amongst these 22 lncRNAs, Gm12648, a 656-nucleotide lncRNA was the most highly induced by hCG (160- fold). Although very little is known about Gm12648, it is located directly upstream of cyclin dependent kinase inhibitor 2A which encodes the cell-cycle inhibitor p16 in the mouse. Moreover, the RNA-RNA interactome shows that Gm12648 can interact with transcription factors (TFs) that are essential during the ovulatory process. Thus, it is possible that Gm12648 may exert some regulatory control of genes and TFs and thereby possibly contribute to ovarian cell fate during the periovulatory period. Similarly, other hCG upregulated lncRNAs could also exert their regulatory role on adjacent protein-coding genes. Thus, our goal is to identify the top 5 most highly LH/hCG- regulated candidate lncRNAs and investigate the function of these lncRNAs in the ovary, specifically their role in the ovulatory process. To achieve our goal, mouse ovaries, granulosa cells, and COCs collected across the periovulatory period will be utilized to characterize the expression of lncRNAs that were discovered in our preliminary study (Aim 1), determine the target genes of these lncRNAs in the granulosa cells using a RNA-seq approach after knocking down the expression of selected highly expressed lncRNAs (Aim 2), and begin to elucidate the function of lncRNAs by characterizing the proteins coded by the target genes, and identify post- translational modifications such as phosphorylation, acetylation, methylation, and ubiquitination (Aim 3) in the ovulation process. We will use a high-impact, translational mouse model in this study. The proposed project will lay a foundation for future studies examining lncRNA function in the human ovary. These discoveries will further improve our understanding of the ovulatory process which provides a fundamental platform to improve infertility treatments and/or to develop the contraceptive measures that specifically target ovulation inhibition.