New insights into transcriptome variation during cattle adipocyte adipogenesis by direct RNA sequencing

Authors: PENG, LINGWEI - Huazhong Agricultural University; ZHANG, XIAOLIAN - Huazhong Agricultural University; DU, YUQIN - Huazhong Agricultural University; LI, FAN - Huazhong Agricultural University; HAN, JIAZHENG - Huazhong Agricultural University; LIU, OUJIN - Huazhong Agricultural University; DAI, SHOULU - Huazhong Agricultural University; ZHANG, XIANG - Huazhong Agricultural University; Liu, Ge - George; YANG, LIGUO - Huazhong Agricultural University; ZHOU, YANG - Huazhong Agricultural University

Submitted to: iScience
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/24/2023
Publication Date: 8/28/2023
Citation: Peng, L., Zhang, X., Du, Y., Li, F., Han, J., Liu, O., Dai, S., Zhang, X., Liu, G., Yang, L., Zhou, Y. 2023. New insights into transcriptome variation during cattle adipocyte adipogenesis by direct RNA sequencing. iScience. 26(10):107753. https://doi.org/10.1016/j.isci.2023.107753.
DOI: https://doi.org/10.1016/j.isci.2023.107753

Interpretive Summary: Comprehensive analyses of transcriptomes will benefit our understanding of genetic bases for complex traits. We compared direct RNA sequencing using long reads to PCR-amplified cDNA sequencing using either long or short reads. We found that direct RNA sequencing outperformed other methods, providing more accurate results and complex mechanisms. These results fill our knowledge gaps and provide the foundation for incorporating new transcriptome insights into the future animal breeding program. Farmers, scientists, and policy planners who need to improve animal health and production based on genome-enable animal selection will benefit from this study.

Technical Abstract: Introduction: As one of the most widely concerned topics, adipogenesis is essential to both the public health of humans and the meat quality of livestock. However, the adipogenesis mechanism was possibly misled and lost important factors by previous indirect sequencing methods. Objectives: To reveal advantages of direct RNA sequencing (DRS) and apply it to uncover adipogenesis mechanisms. Methods: DRS together with PCR-amplified cDNA long-read sequencing and short-read sequencing were performed for the primary adipocyte (PA) and the differentiated adipocyte (DA) of cattle, which was followed by bioinformatic analyses. Results: We obtained 68,124 high-quality transcripts with information on alternative splicing, poly(A) length, and mRNA modification using DRS. Through comprehensive analyses, we proved that DRS was advantageous in avoiding artificial transcripts and questionable exitrons when compared to traditional strategies containing PCR amplification. The transcript numbers of adipogenesis genes were expanded in adipocytes because of alternative splicing, which led to regulation mechanisms far more complex than ever known. We detected 891 differentially expressed genes (DEGs) and 1800 differentially expressed transcripts. However, 62.78% of transcripts of DEGs were not significantly differentially expressed, and 248 transcripts showed opposite changing directions with their genes during adipogenesis. Interestingly, in terms of the poly(A) tail, we observed that it became globally shorter in DA than in PA and had a weak negative correlation with gene/transcript expression levels. As shown in gene FADS2, it produced five different transcripts through alternative splicing, and their expression levels decreased as their length of poly(A) tail increased. Moreover, the mRNA modifications (m5C and m6A) were preferred to locate in regions of the gene body, 3’UTR, exon-intron junctions, and multiple exon genes, which implied their potential regulations in gene expression and alternative splicing. Conclusion: Overall, our study identified highly accurate novel transcripts of cattle adipocytes using DRS and showed more complex adipogenesis mechanisms.