Joint Multi-omics Identification And Analysis Of Key CncRNAs And Their Encoded Micropeptides For Muscle Development And Differentiation And Their Regulatory Roles

Skeletal myogenesis is a very complex and sophisticated process involving the activation of various factors and cellular processes.Satellite cells are essential for skeletal muscle myogenic differentiation and regeneration,and their functions are regulated by multiple genes and pathways.As research continues,a novel RNA molecule with both non-coding RNA regulatory functions and protein-coding functions,namely coding and non-coding RNA（cncRNA）,has come to our attention.In order to further investigate the specific functions played by cncRNA coding products,bovine skeletal muscle satellite cells at two different stages of proliferation（GM）and second day of differentiation（DM2）were used as the target of the study,and the combined high-throughput histological sequencing technologies such as transcriptome（RNA-seq）,translatome（RNC-seq）,proteome（LC-MS/MS）and micro-peptidome sequencing were used to carry out multi omics and multi-level joint comparative analysis,which would screen out the latent cncRNAs and their encoded micropeptide proteomes in bovine myocytes,and to detect their specific effects on the differentiation process of adult muscle by q RT-PCR,Western Blot and EdU,in order to provide a new way to understand the presence of cncRNAs in the bovine organism.1.Expression characteristics and trend analysis of various molecular genes in bovine muscle satellite cells at different molecular levels..In this study,RNA,RNC and protein samples of bovine skeletal muscle satellite cells from GM and DM2 were sequenced and analyzed by LC-MS/MS,RNA-seq,and RNC-seq techniques,which showed that 4196 quantitative proteins with two unique peptides,17340 quantitative transcripts,and 21,812 transcripts potentially bound to ribosomes were identified in each of the three groups.Bioinformatics analysis of the identified differential data showed that many genes had different expression trends in three different omics,and some genes had opposite expression trends in mRNA transcription level and ribosomal translation level.This universal result showed that the expression trends of the same gene were significantly different at different expression levels,and also confirmed the transcriptional regulation,post transcriptional regulation Translation regulation and post-translational regulation play a more important role in the process of gene function than its expression.It also shows the importance of transcription / translation regulators.At the same time,the statistical results of small peptide omics showed that 834 small peptides were identified at 36-18kda;176 small peptides were identified at 18k-0kda,indicating that this small peptide is common in the body and may play an important regulatory or functional role,which provides an endogenous basis for our subsequent screening and identification of functional active molecules（such as micro peptides）.2.Screening and validation of latent cncRNAs and the small peptides they encode in bovine muscle satellite cells.Firstly,the transcriptome data were compared with the translational group data,and a total of 35289 transcripts co-expressed in transcriptome and translational group were screened,including 455 lncRNAs and 2315 un-know RNAs,and the above data were constructed as cncRNA dataset.The cncRNA virtual protein database was obtained by ORF prediction of cncRNA dataset.The 46 cncRNAs with potential coding ability were selected after searching the database by comparing with the total proteins,and the micro-peptides generated by cncRNA coding were selected from them according to the expression amount of each histological data to construct overexpression vectors and cell models,and the effects on myogenic cells after upregulation of the study targets were detected by q RT-PCR,EdU cell proliferation assay and Western Blot technique.The results revealed that upregulation of METRG.27141.2,MSTRG.13302.1,MSTRG.3197.1 and MSTRG.3774.1 had no significant effect on the proliferation and differentiation of bovine myosatellite cells.To further investigate the conserved nature of the encoded small peptides among different species and their effects on other myogenic cells,MSTRG.3197.1 and MSTRG.3774.1 were selected for transfer into C2C12 cells and found that up-regulation of MSTRG.3197.1 expression promoted cell proliferation and differentiation,while up-regulation of MSTRG.3774.1 expression promoted cell The above results tentatively confirmed the existence of cncRNA and encoded small peptides in bovine body,and the screened small peptides had certain effects on the myogenic differentiation of skeletal muscle.3.Screening lncRNAs with potential coding ability as well as to functionally validate the proteins produced by their codes.By comparing and analyzing the transcriptome and translatome data,lncRNAs that were differentially expressed in different periods of bovine muscle satellite cells and might have translation products were screened.The coding potential of lncRNAs was predicted by CPC,a total of 8 lncRNAs with coding ability were finally screened,from which 4 lncRNA-encoded proteins were selected as targets and relevant overexpression vectors and cell models were constructed.After that,q RT-PCR,EdU cell proliferation assay,Western Blot and other techniques were used to detect the effect of upregulated target expression on different myogenic cells.The results showed that upregulation of ENSBTAT00000084270,MEG3 and LOC101905509 expression had no significant effect on bovine myosatellite cells,and upregulation of ENSBTAT00000081974 expression could inhibit the proliferation of bovine myosatellite cells.After that,we also transferred ENSBTAT00000081974 and MEG3 into mouse myogenic cells and found that the upregulation of ENSBTAT00000081974 expression level significantly inhibited cell proliferation and differentiation both at mRNA level and protein level,while the upregulation of MEG3 expression had no significant effect on proliferation and differentiation.The above experiments tentatively confirmed the coding function of some lncRNAs,and found that some lncRNA-encoded micro-peptides had a certain effect on myogenic differentiation.In summary,this study aims to identify and characterize cncRNAs in cattle by combined RNA-seq,RNC-seq and protein profiling,obtain cncRNA datasets in cattle,and carry out functional validation of some cncRNAs in order to deeply analyze the mode of action of cncRNAs from another level,broaden and deepen the theoretical understanding of cncRNA function and mechanism of action,screen and identify important micro-peptides or small molecule proteins in cattle muscle development,and lay the theoretical foundation for in-depth analysis of the mechanism of muscle growth and development,and promote the process of genetic improvement of beef breeds.The aim is to deepen the theoretical understanding of cncRNA function and mechanism of action,to screen and identify important micro-peptides or small molecule proteins in the process of bovine muscle development,and to lay the theoretical foundation for deepening the analysis of muscle growth and development mechanism and promoting the process of genetic improvement of beef breed.