(340) CircRNA biogenesis and role during periovulatory follicular development.

V. Praveen Chakravarthi, Wei-Ting Hung, Sumedha Gunewardena, and Lane K. Christenson.

Department of Cell Biology and Physiology, University of Kansas Medical Center, 3075 HLSIC, 3901 Rainbow Blvd., Kansas City, KS 66160, USA

Abstract Text: CircularRNA (circRNA) act as key regulators of gene expression and play a vital role in many biological functions. The role of circRNA in ovarian follicular development has yet to be studied. In the present study, we analyzed granulosa circRNA expression by high throughput total RNA sequencing 46h after eCG (0 h-post hCG) or 4 h post-hCG (eCG followed by hCG). Transcripts were then analyzed for back splice junctions and then 1000 bp of intronic sequence immediately adjacent to the back splice was interrogated for RNA binding protein motifs. Gonadotropin stimulation resulted in the identification of 44,883 circRNA originating from 7712 genes. Based on having >100 counts per million (cpm) in at least three of six samples we detected 1658 circRNA originating from 1077 genes. Ingenuity Pathway Analysis of the differential expressed transcripts at 0 and 4h post-hCG showed upregulation of different circRNA involved in the abnormal morphology of egg cylinder and arrest in the growth of embryos. Comparison of circRNA vs linear RNA at 0 and 4h post-hCG showed enrichment of circRNA related to the downstream signaling of FSH, Beta estradiol, ESR1, and TGFB1 pathways. Differentially expressed circRNA were subjected to circInteractome analysis to identify the circRNA interacting proteins and miRNA. CircRNA interacting proteins (AGO2, IGFBP1, ELF4A3, HUR, PTB, FMNRP, and DGCR8) and circRNA/miRNA/mRNA (circMast4/miR-323-3p/Egfr,circMast4/miR-361-3p/Cyp19a1, circMast4/miR877/Sult1e1, circMast4 /miR-647/Runx2, circMast4/miR-155/Smad1, circKif24/miR-296-5p/Cyp11a1, circVan/miR-326/Fshr, circmllt10/miR-647/Runx2) networks play a crucial role in ovarian follicular development were identified. Screening of circRNA biogenesis-associated RNA binding proteins in ovarian cells demonstrated elevated presence of these 8 of these RBPs in granulosa cells. One of these RBPs, KH RNA binding domain containing, signal transduction associated 1 (KHDRBS1) was highly expressed in ovarian granulosa cells and found in have high binding probability with differentially expressed circRNA by RBPmap and Cross-linking immunoprecipitation analysis. Previous studies indicated complete KO of KHDRBS1 rendered female mice sub-fertile. In conclusion, we observed robust circRNA biogenesis in ovarian granulosa cells immediately after the LH surge and identified KHDRBS1 as a possible key RBP involved in this gonadotrophin induced circRNA biogenesis