Detecting Potentially Conserved CircRNAs in the Estrogen Pathway and in Mammary Glands

Paige Ryan

Mentor: Dr. Markus Hoffmann, Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center.

Date/Time: August 21st, 2025 at 2:00 PM.

Abstract: Circular RNAs (circRNAs) are a type of long noncoding RNA (lncRNAs) that undergo a back-splicing event to create a circular structure. Due to this loop formation, circRNAs do not have a 5’ cap or 3’ polyadenylation tail, making them less prone to degradation (Hwang & Kim, 2024). The stability of circRNAs makes them a useful research focus to explore pathological processes in estrogen signaling and breast cancer (Liu et al., 2019). This study, in collaboration with Professor Markus List and Professor Priscilla Furth at the Technical University of Munich, sought to investigate potentially conserved circRNAs present in the mammary gland across a variety of species. The data analyzed was publicly available total RNA-sequencing data gathered from the Gene Expression Omnibus (GEO) [Bovine: GSE133333; Canine: GSE137825, GSE203106, GSE203589, GSE219045; Goat: ERR9390120, ERR9390121; Human: GSE138734, GSE190615; Mouse: GSE219045; Rat: GSE60539, GSE219045; Sheep: GSE71424, SRR10245467-72]. First, circRNAs were identified from the sequencing data using the nf-core/circRNA pipeline (Digby et al., 2023; Hoffmann et al., 2023). This pipeline incorporates multiple tools such as CIRCexplorer2, circRNA_finder, DCC, and others to identify back-splice junctions. These tools use STAR to detect chimeric reads, areas in the genome that do not align linearly, to identify back-splice junctions. Quantification is also completed by psirc, which combines both linear and circular transcripts to better determine the expression levels of the identified circRNAs. Once circRNAs were identified in each organism, the circtools conservation module (Kulkarni, Dieterich, & Jakobi, 2025) was utilized to identify conserved circRNAs across species. The conservation module uses the UCSC LiftOver tool to select the surrounding exons of the back-splice junction and identify homologous regions in other species (Kulkarni, Dieterich, & Jakobi, 2025). After LiftOver, the conservation module uses MAFFT, a multiple sequence alignment, to find sequence similarity between the homologous regions. Based on sequence similarity, a conservation score was calculated for each organism, and a phylogenetic tree was produced to visualize the degree of conservation (Kulkarni, Dieterich, & Jakobi, 2025). Lastly, the potentially conserved circRNAs were confirmed in databases such as circAtlas 3.0 (Wu, Zhao, & Zhang, 2024) and circBase (Glažar, Papavasileiou, & Rajewsky, 2014). The circtools conservation module identified 16 circRNAs that had conservation across all five species. Of these, 12 were validated in circAtlas. CircRNAs that were validated were hsa-DBT_0010, hsa-DOCK7_0018, hsa-DOCK7_0044, hsa-FGGY_0006, hsa-NFIA_0004, hsa-NPHP4_0009, hsa-NPHP4_0004, hsa-NPHP4_0022, hsa-PATJ_0020, hsa-PATJ_0016, hsa-RBP7_0001, hsa-SLC35A3_0007. The circRNAs that could not be validated were in PATJ gene in regions chr1:61762858|61771626, chr1:61791353|61801769, chr1:61856030|61927829, and chr1:61899583|61901459. For the potentially conserved results identified in other species, some were found in circAtlas, such as sus-DOCK7_0034 and sus-RBP7_0001. While no functional data currently exists for sus-DOCK7_0034 and sus-RBP7_0001, both host genes play important biological roles. One study showed RBP7 was expressed in adipose tissue in pigs, regulating fat cell formation and metabolism (Ahn, Suh, & Lee, 2019). In addition, the fatty acid-binding domains were found to be highly conserved between pigs, humans, rats, and mice (Ahn, Suh, & Lee, 2019). In humans, RBP7 also plays a role in adipogenesis and has been identified as a tumor suppressor in breast cancer (Yu et al., 2024). The precise role of DOCK7 in pigs is still unclear, but there is some suggestion that it may play a role in growth-related traits, such as body weight and fat deposition (Yang et al., 2019). In humans, DOCK7 primarily regulates cell movement and contributes to differentiation. DOCK7 has recently been shown to work with myosin VI to promote coordinated migration of mammary epithelial cells, a process that plays a key role in tumor invasion (Menin et al., 2023). The parallels between pig and human functions in RBP7 and DOCK7 highlight the importance of investigating circRNAs in various organisms to better understand their potential regulatory roles. Other regions yielded no results in databases or found potential isoforms that need experimental validation. Some limitations to consider are that the circtools conservation module does not identify circRNAs in other species; it only identifies potentially homologous regions. It does not identify back-splice junctions or any other features to confirm circRNA presence. Without experimental validation, we cannot be sure that these regions contain any circRNAs. Furthermore, the conservation module currently only supports input files from DCC-formatted files, whereas the circRNA pipeline integrates multiple circRNA detection tools. This limits the conservation analysis to circRNAs detected by DCC alone, potentially overlooking conserved circRNAs detected by other methods. In summary, we identified multiple circRNAs that are potentially conserved between several species. These findings highlight the need for further investigation and experimental validation to confirm the presence and functional roles of conserved circRNAs. Identifying conserved circRNAs allows us to pinpoint ones that may be of interest to investigate further. Additionally, it can help gain insight into processes involved in estrogen signaling and breast cancer that are not well understood in humans. These findings can help refine future experimental targets and enhance understanding of circRNA function across species.