Divergent Regulatory Roles of Transcriptional Variants of the Chicken LDB3 Gene in Muscle Shaping

LIM domain binding 3 (LDB3) serves as a striated muscle-specific Z-band alternatively spliced protein that plays an important role in mammalian skeletal muscle development, but its regulatory role and molecular mechanism in avian muscle development are still unclear. In this study, we reanalyzed RNA sequencing data sets of 1415 samples from 21 chicken tissues published in the NCBI GEO database. First, three variants (LDB3-X, LDB3-XN1, and LDB3-XN2) generated by alternative splicing of the LDB3 gene were identified in chicken skeletal muscle, among which LDB3-XN1 and LDB3-XN2 are novel variants. LDB3-X and LDB3-XN1 are derived from exon skipping in chicken skeletal muscle at the E18-D7 stage and share three LIM domains, but LDB3-XN2 lacks a LIM domain. Our results preliminarily suggest that the formation of three variants of LDB3 is regulated by RBM20. The three splice isomers have divergent functions in skeletal muscle according to in vitro and in vivo assays. Finally, we identified the mechanism by which different variants play different roles through interactions with IGF2BP1 and MYHC, which promote the proliferation and differentiation of chicken myoblasts, in turn regulating chicken myogenesis. In conclusion, this study revealed the divergent roles of three LDB3 variants in chicken myogenesis and muscle remodeling and demonstrated their regulatory mechanism through protein–protein interactions.LIM结构域结合蛋白3（LIM domain binding 3, LDB3）是一种横纹肌特异性表达的Z盘（Z-band）可变剪接（alternative splicing）蛋白，在哺乳动物骨骼肌发育中发挥关键调控作用，但目前其在禽类肌肉发育中的调控功能与分子机制仍未明确。本研究对NCBI GEO数据库中已发表的21种鸡组织共1415份样本的RNA测序（RNA sequencing）数据集进行了重新分析。首先，我们在鸡骨骼肌中鉴定出LDB3基因经可变剪接产生的三种变体（LDB3-X、LDB3-XN1及LDB3-XN2），其中LDB3-XN1与LDB3-XN2为新型变体。LDB3-X与LDB3-XN1均来源于鸡骨骼肌E18-D7阶段的外显子跳跃（exon skipping）事件，二者共享三个LIM结构域（LIM domains），而LDB3-XN2则缺失一个LIM结构域。本研究结果初步表明，LDB3的三种变体的形成受RBM20调控。通过体外（in vitro）与体内（in vivo）实验证实，这三种剪接异构体（splice isoforms）在骨骼肌中具有功能分化。最后，我们阐明了不同变体通过与胰岛素样生长因子2 mRNA结合蛋白1（IGF2BP1）和肌球蛋白重链（MYHC）相互作用发挥不同功能的分子机制：上述相互作用可促进鸡成肌细胞（myoblasts）的增殖与分化，进而调控鸡肌发生（myogenesis）过程。综上，本研究揭示了三种LDB3变体在鸡肌发生与肌肉重塑中的功能分化，并明确了其通过蛋白质相互作用（protein–protein interactions）实现调控的分子机制。