Super-Enhancer-Associated circRHOQ Regulates Intramuscular Fat Deposition by Modulating the miR-5093/IRF5 Axis

Circular RNAs (circRNAs) play a crucial role in post-transcriptional regulation during the development and physiology of mammals. However, the role of superenhancer (SE)-associated circRNAs (SE-circRNAs) in adipogenesis remains largely unexplored. In this study, RNA and H3K27ac chromatin immunoprecipitation sequencing analyses of yak muscle tissues with varying IMF contents were performed, and 81 differentially expressed SE-circRNAs were identified in the high IMF (intramuscular fat) group. Among them, circRHOQ was notably associated with SEs, with H3K27ac signals enriched at its genomic locus. Enhancer activity assays identified two key regulatory regions (chr9:49134487–49135239 and chr9:49136018–49136614) with significant transcriptional activation. Treatment with JQ-1 suppressed circRHOQ expression, confirming that circRHOQ is an SE-driven regulatory circRNA. Functional assays validated that circRHOQ is a cytoplasmic circRNA that promotes yak intramuscular preadipocyte adipogenic differentiation, as indicated by the increased lipid accumulation, triacylglycerol content, and adipogenic gene expression. Mechanistically, circRHOQ acts as a competing endogenous RNA by binding to miR-5093, thereby derepressing its target IRF5 and promoting adipogenesis. Dual-luciferase assays validated the direct interactions among circRHOQ, miR-5093, and IRF5. This study revealed that circRHOQ promotes yak IMF development via the SE-circRHOQ/miR-5093/IRF5 axis, thus providing a basis for further investigation into IMF deposition and improvement of high-altitude livestock breeds.在哺乳动物的发育与生理过程中，环状RNA（Circular RNAs, circRNAs）在转录后调控层面发挥关键作用。然而，超级增强子（superenhancer, SE）关联的环状RNA（SE-circRNAs）在脂肪生成过程中的功能，目前仍有待深入探究。本研究针对不同肌内脂肪（intramuscular fat, IMF）含量的牦牛肌肉组织，开展了RNA测序与H3K27ac染色质免疫共沉淀测序分析，并在高IMF组中鉴定得到81个差异表达的SE-circRNAs。其中，circRHOQ与SEs存在显著关联，其基因组位点富集有H3K27ac信号。增强子活性实验鉴定出两个关键调控区域（chr9:49134487–49135239与chr9:49136018–49136614），二者具备显著的转录激活活性。使用JQ-1进行处理可抑制circRHOQ的表达，由此证实circRHOQ是一类由SE驱动的调控性环状RNA。功能实验验证显示，circRHOQ属于胞质环状RNA，可促进牦牛肌内前体脂肪细胞的成脂分化，具体表现为脂滴积累增多、三酰甘油含量升高以及成脂相关基因表达上调。从机制层面来看，circRHOQ可作为内源竞争RNA（competing endogenous RNA, ceRNA）结合miR-5093，由此解除其靶基因IRF5的表达抑制，进而促进脂肪生成。双荧光素酶报告实验验证了circRHOQ、miR-5093与IRF5三者之间的直接相互作用。本研究揭示，circRHOQ通过SE-circRHOQ/miR-5093/IRF5轴促进牦牛IMF的沉积与发育，为后续探究IMF沉积机制以及改良高海拔畜禽品种提供了理论依据。