- Moreover, 16 mRNAs, 5 miRNAs, 3 lncRNAs, and 5 circRNAs were co-differentially expressed in both types of tissue.. - We suspected that these co-differentially expressed genes were involved in IMF-deposition in the yak. - Additionally, LPL, ACADL, SCD, and FASN, which were previously shown to be associated with the IMF content, were identified in the competing endogenous RNA (ceRNA) regulatory network that was constructed on the basis of the IMF deposition-related genes. - 3 State Key Laboratory of Hulless Barley and Yak Germplasm Resources and Genetic Improvement, the Tibet Academy of Agricultural and Animal Husbandry Science , Lhasa, Tibet 850000, People ’ s Republic of China Full list of author information is available at the end of the article. - Keywords: Bos grunniens, Intramuscular fat content, Transcriptome, Co-differentially expressed transcripts, ceRNA. - The intramuscular fat (IMF) content in livestock is posi- tively correlated with various aspects of meat quality, such as tenderness, flavor, and juiciness, and as such is one of the key traits related to consumer preference.. - Studies have found the heritability of the IMF content to range from 0.47 to 0.53 [4–6]. - The yak (Bos grunniens), one of the ruminants that live in the Qinghai-Tibet Plateau and adjacent areas, is well adapted to the high-altitude environments. - Many recent studies on the mechanism of IMF deposition in cattle have already revealed some of the genes that are involved in the IMF deposition-regulating pathway [18].. - The one-by-one identification of the potential regulatory genes in the yak would undoubtedly be like trying to find a needle in a haystack. - Moreover, previous studies have showed that the IMF content varies even between breeds of the same species [19] and between different development stages [20]. - Until now, a global analysis of the molecular mechanism of IMF deposition in yak has not been previously per- formed. - Therefore, the elucidation of the differences in the whole transcriptomes related to IMF deposition at different development stages of the yak is essential for interpreting the function of the DEGs. - In this study, the IMF contents in and 7.5-year-old yaks were determined, and the whole-transcriptome profiles of the LD muscle and its adjacent intermuscular adipose tissues (AA) in the 0.5- and 2.5-year-old yaks were ob- tained to compare the DEGs in these two tissues. - Finally, we constructed a comprehensive com- peting endogenous RNA (ceRNA) network on the basis of the co-DEGs between the LD and AA tissues to high- light the genes that are most likely to be involved with the IMF trait in yaks.. - Intramuscular fat contents of the longissimus dorsi muscle in yaks of different ages. - The IMF content of the LD increased along with the de- velopment of the yaks from 0.5 to 7.5 years of age. - Com- pared with the IMF content in the 0.5-year-old yaks, that in the 2.5-year-old animals was significantly higher (p <. - 0.05), and this age group also showed the fastest LD fat deposition of the yaks. - Finally, 39,853 circRNAs were identi- fied in the yak (Table S3), of which 17,211 and 9616 were found to be LD tissue specific and AA tissue spe- cific, respectively (Table S4).. - Differentially expressed mRNAs during intramuscular fat deposition. - Fig 1 The dynamics in the live weight (a) and the intramuscular fat (IMF) content (b) across and 7.5-year-old of age.. - Similarly, after comparison of the data between the 0.5- and 2.5-year-old AA tissues, 72 DEGs were obtained, of which 39 were upregulated and 33 were downregulated (Fig. - These results indicated that these 16 DEGs may have a role in the regulation of IMF- deposition development.. - Total lncRNAs and differentially expressed lncRNAs during intramuscular fat deposition. - To reveal the potential functions of the 4142 identified lncRNAs in IMF deposition, three independent algo- rithms—antisense (mRNA sequence complementarity), cis (genomic location), and trans (expression correlation). - were performed to predict the target genes of the lncRNAs. - Four differentially expressed lncRNAs (DELs) (2 up- regulated and 2 down-regulated) and 9 DELs (8 up-. - 2 Differentially expressed mRNAs during LD (a) and AA (b) tissues development, respectively. - 3 KEGG pathway analysis for differentially expressed mRNAs in LD (a) and AA (b), respectively. - Table 1 The co-differentially expressed genes between LD and AA tissues. - ID Differentially expressed genes in LD aging Differentially expressed genes in AA aging. - regulated and 1 down-regulated) were identified in the LD and AA tissues, respectively (Table 2). - As a prelimin- ary exploration of the functional implications of the DELs across genomes, we investigated whether lncRNAs were co-regulated with the DEGs during IMF- deposition. - We also identified 223 differentially expressed cir- cRNAs (DECs. - 125 upregulated and 98 downregulated) in the LD tissue (Fig. - Construction of the ceRNA coregulatory network. - On the basis of the data of the co- differentially expressed mRNA, lncRNA, circRNA, and miRNA transcripts, we obtained the mRNA-miRNA, lncRNA-miRNA, and circRNA-miRNA pairs, combined them with the lncRNA-mRNA pairs, and then con- structed the integrated ceRNA network. - Validation of the RNA-seq results was carried out using the quantitative reverse-transcription polymerase chain reaction (RT-qPCR) for 3 DEGs (LPL, SIRT1 and PRKCA), 3 DEMs (miR-122-x, miR-381-y, and miR-499- y), 2 DELs (TCONS-00016416,and TCONS and 2 DECs (Circ_040844, and Circ_053707). - Table 2 Differentially expressed lncRNAs of 0.5-year-old LD vs 2.5-year-old LD. - lncRNA ID 0.5-LD 2.5-LD log2(FC) P-value FDR Differentially expressed co-target. - Table 3 Differentially expressed lncRNAs of 0.5-year-old AA vs 2.5-year-old AA. - lncRNA ID 0.5-AA 2.5-AA log2(FC) P-value FDR Differentially expressed co-target. - 4 Differentially expressed miRNAs and circRNAs during LD and AA development, respectively. - (a and b) differentially expressed miRNAs. - (c and d) differentially expressed circRNAs. - The results indicated the high reproducibility and reliability of the gene ex- pression profiles obtained in our study.. - As is widely known, the IMF content is one of the poly- genic traits in animal that is an important determinant of meat quality characteristics. - Given the importance of IMF on the economics of livestock production, the clari- fication of the molecular mechanisms underlying IMF deposition holds great significance. - In this study, to iden- tify genes that are related to IMF deposition in the yak, we first measured the IMF content in LD of and 7.5-year-old yaks, whereupon it was found that the IMF content was deposited quickly from 0.5 to 2.5 years. - Of these, 16 DEGs were co-differentially expressed in both tissues, many of which had known functions in lipid. - ACADL, one of the rate-limiting enzymes in fatty acid beta- oxidation, has been identified as candidate function gene in IMF deposition in chicken [26]. - As a major class of the noncoding RNAs, lncRNAs could act as key regulators in many biological and pathological processes via trans, cis, and antisense activ- ities. - The lncRNA Jpx acts both in trans and in cis to activate X specific transcripts expression in mouse Table 4 The co-differentially expressed miRNAs in LD and AA. - ID Differentially expressed miRNAs in LD Differentially expressed miRNAs in AA. - Table 5 The co-differentially expressed circRNAs in LD and AA. - ID Differentially expressed circRNAs in LD Differentially expressed circRNAs in AA. - On the basis of these results, we suspected that one of the main roles of these 3 lncRNAs is to regulate the IMF deposition in yak, and further highlighting of. - Interestingly, 5 DEMs and 5 DECs were found to be co-differentially expressed during muscle and adipose tissue develop- ment. - Therefore, we speculate that these three subnetworks may play a key role in the regulation of IMF deposition.. - The present study provides a comprehensive landscape of the differences in the whole- transcriptome profiles of LD and AA tissues between two developmental stages in. - 6 The validation of the mRNA and miRNA RNA-seq results. - We identified 16 DEGs related to lipid biosynthesis that were co-differentially expressed in the two tissues during development, including ACACB, ACADL, ELOVL7, SIRT1, FASN, LPL, PRKCA, and SCD. - miRNAs, and 5 circRNAs that were co-differentially expressed in the two tissues may play a crucial role in regulating IMF deposition. - On the basis of the co- differently expressed transcripts, we constructed a ceRNA regulatory network which contained 10 DEGs, 5 DEMs, 5 DECs, 3 DELs, and 29 relationships. - 7 The validation of the lncRNA and circRNA RNA-seq results. - (a and b) the validation of the lncRNAs expression in LD and AA tissues. - the validation of the circRNAs expression in LD and AA tissues. - miR-122-x-TCONS-00061798-PRKCA, and miR-499-y- TCONS-00084092-LPL) may play a crucial role in the regulation of IMF deposition. - Be- tween Oct 21st and 22 nd, 2017, all yaks were stunned with a captive bolt pistol (Cash 8000 Model Stunner, 0.22 calibre, 4.5 grain cartridge) to ameliorate the suffer- ing of the animals prior to their humane killing, follow- ing which exsanguination via a transverse incision of the neck was carried out in the slaughterhouse of Zang Jia Mao Niu Co, Ltd. - Analysis of the intramuscular fat content. - The IMF content of the 12 LD samples were determined according to the standard Soxhlet extraction method [41, 42]. - Total RNA isolation, sequencing and raw data analysis On the basis of the IMF content results, total RNA was isolated from LD and AA samples excised from the 0.5- and 2.5-year-old yak with TRIzol reagent (Invitrogen, CA, USA). - Scientific, DE, USA), Bio-Photometer (Eppendorf, Ham- burg, Germany) and 1% agarose gel electrophoresis were used to measure the quantity and quality of the extrac- tion total RNA. - The annotated and unannotated transcripts were obtained using Cufflinks after reconstruction of the transcripts from our RNA-seq data. - Those transcripts that were predicted to have coding potential by two or all of the above three tools named as candi- date set of novel protein-coding transcripts, whereas those without coding potential were named as novel lncRNAs. - 0.05 were then assigned as differentially expressed transcripts.. - Gene ontology enrichment and KEGG pathway analyses GO annotation and KEGG enrichment analyses were conducted to annotate the potential function of the genes. - GO enrichment analysis was carried out with the GOseq R package, and the GO terms of the DEGs were assessed using Fisher’s exact test. - Prediction of lncRNA and miRNA targets and construction of the ceRNA network. - On the basis of this theory, we constructed a co-expression ceRNA network related to the regulation of IMF depos- ition in the yak. - For the cis-acting prediction, the locations of the paired lncRNAs and mRNAs in the genomic of yak were calculated, and the genes within 10 kb of the lncRNAs were named as cis-acting regulatory targets.. - For trans-acting regulatory targets, the expression of the lncRNA was determined to be not related with the loca- tion of the mRNA but co-expressed with it. - To evaluate the reliability of the transcript expression data obtained by RNA-Seq. - Overview of the data for RNA sequencing.. - Overview of the data for small RNA sequencing.. - Differentially expressed genes during LD and AA development, and GO enrichment and KEGG pathway analysis for differential expression genes.. - The targets prediction of lncRNAs and the differentially expressed lncRNAs during LD and AA development.. - Differentially expressed miRNAs during LD and AA development, and KEGG pathway analysis for their target genes.. - Differentially expressed circRNAs during LD and AA development, and KEGG pathway analysis for their target genes.. - DECs: Differentially expressed circular ribonucleic acids;. - DEGs: Differentially expressed genes. - DELs: Differentially expressed long noncoding ribonucleic acids. - DEMs: Differentially expressed micro-ribonucleic acids. - 31902153), and the Program of the National Beef Cattle and Yak Industrial Technology System (No. - The funding body had no role in the design of the study, sample collection, data analysis, and manuscript writing.. - The accession numbers of RNA-seq data of the 12 samples are SAMN14599609, SAMN14599610, SAMN14599611, SAMN14599612, SAMN14599613, SAMN14599614, SAMN14599615, SAMN14599616, SAMN14599617, SAMN14599618, SAMN14599619, SAMN14599620, respectively. - involvement in the crosstalk between skeletal muscle and adipose tissue.. - A splice site single nucleotide polymorphism of the fatty acid binding protein 4 gene appears to be associated with intramuscular fat deposition in longissimus muscle in australian cattle. - Role of lipids in the MAPK signaling pathway
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