Animal growth and development are regulated by long non-coding RNAs (lncRNAs). However, the functions of lncRNAs in regulating cashmere fineness are poorly understood. To identify the key lncRNAs that are related to cashmere fineness in skin, we have collected skin samples of Liaoning cashmere goats (LCG) and Inner Mongolia cashmere goats (MCG) in the anagen phase, and have performed RNA sequencing (RNA-seq) approach on these samples. The high-throughput sequencing and bioinformatics analyses identified 437 novel lncRNAs, including 93 differentially expressed lncRNAs. We also identified 3084 differentially expressed messenger RNAs (mRNAs) out of 27,947 mRNAs. Gene ontology (GO) analyses of lncRNAs and target genes in cis show a predominant enrichment of targets that are related to intermediate filament and intermediate filament cytoskeleton. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, sphingolipid metabolism is a significant pathway for lncRNA targets. In addition, this is the first report to reveal the possible lncRNA–mRNA regulatory network for cashmere fineness in cashmere goats. We also found that lncRNA XLOC_008679 and its target gene, KRT35, may be related to cashmere fineness in the anagen phase. The characterization and expression analyses of lncRNAs will facilitate future studies on the potential value of fiber development in LCG.

N⁶-methyladenosine (m⁶A) is the most abundant modification in linear RNA molecules. Over the last few years, interestingly, many circRNA molecules are also found to have extensive m⁶A modification sites with temporal and spatial specific expression patterns. To date, however, little information is available concerning the expression profiling and functional regulatory characteristics of m⁶A modified circRNAs (m⁶A-circRNAs) in secondary hair follicles (SHFs) of cashmere goats. In this study, a total of fifteen m⁶A-circRNAs were identified and characterized in the skin tissue of cashmere goats. Of these, six m⁶A-circRNAs were revealed to have significantly higher expression in skin at anagen compared with those at telogen. The constructed ceRNA network indicated a complicated regulatory relationship of the six anagen up-regulated m⁶A-circRNAs through miRNA mediated pathways. Several signaling pathways implicated in the physiological processes of hair follicles were enriched based on the potential regulatory genes of the six anagen up-regulated m⁶A-circRNAs, such as TGF-beta, axon guidance, ribosome, and stem cell pluripotency regulatory pathways, suggesting the analyzed m⁶A-circRNAs might be essentially involved in SHF development and cashmere growth in cashmere goats. Further, we showed that four m⁶A-circRNAs had highly similar expression trends to their host genes in SHFs of cashmere goats including m⁶A-circRNA-ZNF638, -TULP4, -DNAJB6, and -CAT. However, the expression patterns of two m⁶A-circRNAs (m⁶A-circRNA-STAM2 and -CAAP1) were inconsistent with the linear RNAs from their host genes in the SHFs of cashmere goats. These results provide novel information for eluci-dating the biological function and regulatory characteristics of the m⁶A-circRNAs in SHF development and cashmere growth in 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.

Cashmere goats are widely distributed in the cold, arid and semi-arid remote regions of northern China. Their main economic value is the production of precious cashmere fibers. The differentiation of second hair follicle (SHF) stem cells into hair follicle lineages plays a crucial role in SHF regeneration as well as in the morphogenesis and growth of cashmere fibers; however, its precise molecular mechanism is still unclear. In this study, we found that N⁶-methyladenosine (m⁶A)-circHECA recruiting FUS promoted the differentiation of SHF stem cells into hair follicle lineages through stabilizing FOXM1 mRNA in SHF stem cells, thereby activating the NOTCH pathway in cashmere goats. Furthermore, we confirmed that the m⁶A modification of circHECA is required for the FUS/FOXM1-mediated NOTCH signaling pathway to facilitate the differentiation of SHF stem cells into hair follicle lineages via transfecting circHECA m⁶A-deficient mutants. Our results contribute to elucidating the functional mechanism of m⁶A-circHECA in the differentiation of SHF stem cells into hair follicle lineages in cashmere goats.