# N6-Methyladenosine (m6A)-Circular RNA Pappalysin 1 (circPAPPA) from Cashmere Goats: Identification, Regulatory Network and Expression Potentially Regulated by Methylation in Secondary Hair Follicles Within the First Intron of Its Host Gene

## TL;DR

This study identifies and characterizes an m6A-modified circular RNA, circPAPPA, in cashmere goats, linking its expression to methylation and potential roles in hair follicle development.

## Contribution

The study identifies m6A-modified circPAPPA in cashmere goat secondary hair follicles and links its expression to intron methylation of the host gene.

## Key findings

- circPAPPA contains at least four m6A modification sites in secondary hair follicles of cashmere goats.
- circPAPPA is predominantly localized in the cytoplasm of SHF stem cells and is derived from the PAPPA gene.
- The first intron methylation of the PAPPA gene is likely involved in the dynamic expression of m6A-circPAPPA.

## Abstract

Cashmere goats are an important livestock species in the agricultural and pastoral areas of northern China. Cashmere is one of the main products of cashmere goats. It is derived from secondary hair follicles (SHFs) in the skin tissue of cashmere goats. However, the precise molecular mechanism of the growth of cashmere fibers is still not fully understood. In this study, we characterized the N6-methyladenosine (m6A)-modified circPAPPA and confirmed that circPAPPA contained at least four m6A modification sites in the SHFs of cashmere goats, including m6A-450/456, m6A-852, m6A-900, and m6A-963. The m6A-modified circPAPPA exhibits higher expression in the cytoplasm of SHF stem cells of cashmere goats in comparison to the nucleus. Based on bioinformatics analysis, m6A-modified circPAPPA may play multiple functional roles in SHF development and the growth of cashmere through potential regulatory networks mediated by miRNAs and RNA-binding proteins. First, intron methylation of the host gene (PAPPA) of m6A-modified circPAPPA is most likely significantly involved in the dynamic expression of m6A-modified circPAPPA in the SHFs of cashmere goats.

N6-methyladenosine (m6A) is one of the most abundant modifications in eukaryotic RNA molecules and mediates the functional exertion of RNA molecules. We characterized the circPAPPA and validated its potential m6A modification sites in secondary hair follicles (SHFs) of cashmere goats. Furthermore, we generated integrated regulatory networks of the circPAPPA along with enrichment analysis of signaling pathways. We also explored the potential relationship of circPAPPA expression with the first intron methylation of the PAPPA gene in SHFs of cashmere goats. Host source analysis revealed that circPAPPA is derived from the complete exon 2 of the PAPPA gene, spliced in reverse orientation, and predominantly localized in the cytoplasm of SHF stem cells in cashmere goats. The circPAPPA was verified to contain at least four m6A modification sites in SHFs of cashmere goats, including m6A-450/456, m6A-852, m6A-900, and m6A-963. The generated regulatory network indicated complex and diverse regulatory relationships of m6A-circPAPPA with its putative regulatory molecules, including miRNAs, mRNAs, and proteins. Enrichment analysis of signaling pathways showed that m6A-circPAPPA might play multiple functional roles in the growth and development of SHF in cashmere goats through the putative regulatory network mediated by its target miRNAs and regulatory proteins. The first intron methylation of the PAPPA gene most likely is significantly involved in the dynamic expression of m6A-circPAPPA in cashmere goat SHFs. Results from this study provided novel information to elucidate the biological roles and functional regulatory pathways of m6A-circPAPPA in SHF development and the growth of cashmere goat fiber.