The Role And Molecular Mechanisms Of Alternative Splicing Variants Within LDB3 Gene Underlying Skeletal Muscle Development In Chicken

LDB3 is a highly conserved PDZ-LIM protein among different species,which plays an important role in maintaining the stability of muscle fibers in drosophila,zebrafish and mice.Three splicing variants within LDB3 gene were identified in mouse skeletal muscle,a short one(LDB3-C)and two longer ones(named LDB3-An and LDB3-B,respectively).The current research is mainly focused on the effects of different splicing variants of LDB3 gene and its coding region mutations on muscle development in mammals,but the function of different splicing variants of LDB3 gene in chickens has not been reported.In this study,adenoviral over-expression vectors and specific interference fragments of different splicing variants of LDB3 gene were constructed and synthesized,and then infected and transfected into chicken primary myoblasts respectively.The functions of different splicing variants in chicken primary cell were analyzed.On the other hand,IP and RNA-seq were used to analyze the regulation mechanisms of different splicing variants that perform different functions.In addition the adenovirus-coated over-expression vectors of different splicing variants were injected into chicken gastrocnemius muscle to explore the effects of different splicing variants of LDB3 gene on chicken muscle development.The contents and results of the study are as follows:1.Identification of different splices of chicken LDB3 geneThe 5 ’end and 3’ end of LDB3 gene were amplified by RACE from the samples of AA broiler and Lu’s chicken at E12 and 1W stages.According to the results of RACE,three splicing isoforms were cloned and named LDB3-A,LDB3-B and LDB3-C.LDB3-A and LDB3-B were identified as alternative splicing with development by reverse transcription PCR and WB.The results showed that there were three splicing variants of LDB3 in skeletal muscle of different breeds.The subcellular localization of LDB3 in primary chicken myoblasts showed that LDB3-A in primary chicken myoblasts showed that LDB3-A was mainly expressed in cytoplasm,LDB3-B and LDB3-C were expressed in nucleus and cytoplasm.2.Different splicing variants of LDB3 gene regulate the proliferation and differentiation of primary chicken myoblastsThrough over-expression and interference of different splicing variants,it was found that LDB3-A could promote the proliferation and inhibit the differentiation of chicken primary myoblasts,LDB3-B could inhibit the proliferation and promote differentiation of chicken primary myoblasts,and LDB3-C could promote the proliferation and differentiation of primary chicken myoblasts.3.The effect of different splices of LDB3 gene on muscle atrophyDifferent splicing variants of LDB3 gene were packaged by adenovirus,and the adenovirus was injected into gastrocnemius muscle of chicks at 1 day and 7 days old,and gastrocnemius muscle was collected on the 13 th day.The effects of different splicing variants on the diameter of muscle fibers and the ratio of fast to slow muscles were detected by HE staining.The results showed that compared with the control group,LDB3-A could inhibit muscle atrophy and significantly increase muscle fiber diameter,while the over-expression of LDB3-B and LDB3-C could promote muscle atrophy and significantly reduce muscle fiber diameter.4.Different splicing variants of LDB3 gene regulate sarcomere formationThe effects of different splices of LDB3 gene on sarcomere and its internal mechanism were studied by using q PCR,WB,IF and protein Co-IP in vitro.The results showed that LDB3-A could inhibit the expression of ACTN2 protein and reduce the number of sarcomere in chicken primary myoblasts.LDB3-B and LDB3-C could significantly increase the number of sarcomere in chicken primary myoblasts by promoting the expression of ACTN2 protein.2.RNA-seq and protein Co-IP mass spectrometry were used to identify 5.molecular regulation mechanism of different splicing variantsof LDB3 geneThrough transcriptome and mass spectrometry sequencing of three splices of over-expressed LDB3 gene,a large number of differential genes and binding proteins related to muscle development were identified.The important thing is that the expression trend of 1497 differential genes after over-expression of LDB3-An and LDB3-B is opposite.The results of mass spectrometry sequencing showed that the binding proteins and related pathways of the three splice bodies of LDB3 gene were highly similar,but they played different functions in muscle development through different regulation ways.To sum up,through the over-expression of LDB3 splicing variants in chicken primary myoblasts and 1-day-old chicks and a series of experiments,this study revealed the function and regulatory mechanism of different splicing variants of LDB3 gene in chicken primary myoblasts and muscle development.