Journal of Integrative Agriculture ›› 2022, Vol. 21 ›› Issue (5): 1444-1456.DOI: 10.1016/S2095-3119(21)63826-1

所属专题：动物科学合辑Animal Science

• • 上一篇下一篇

收稿日期:2020-12-26 接受日期:2021-08-30 出版日期:2022-05-01 发布日期:2021-08-30

The expression, function, and coding potential of circular RNA circEDC3 in chicken skeletal muscle development

WEI Yuan-hang*, ZHAO Xi-yu*, SHEN Xiao-xu, YE Lin, ZHANG Yao, WANG Yan, LI Di-yan, ZHU Qing, YIN Hua-dong

Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, P.R.China

Received:2020-12-26 Accepted:2021-08-30 Online:2022-05-01 Published:2021-08-30
About author:WEI Yuan-hang, E-mail: weiyuanhang@stu.sicau.edu.cn; ZHAO Xi-yu, E-mail: 739803368@qq.com; Correspondence YIN Hua-dong, Tel: +86-28-82621602, E-mail: yinhuadong@sicau.edu.cn * These authors contributed equally to this study.
Supported by:
This research was funded by the Sichuan Science and Technology Program, China (2018JY0488, 2016NYZ0050 and 2016NZ0104).

摘要/Abstract

摘要：

本研究旨在探讨circEDC3对鸡骨骼肌卫星细胞SMSCs增殖、分化和凋亡的调控功能，从而揭示circEDC3在鸡骨骼肌发育中的作用。我们构建了circEDC3的小干扰RNA (siRNA) 及过表达载体(pCD2.1-circEDC3)来调控体外培养的鸡原代骨骼肌卫星细胞中circEDC3的表达水平，通过运用Quantitative Real-Time PCR (qPCR)，Western Blot (WB)，Cell counting kit 8 (CCK-8)，5-Ethynyl-2’-Deoxyuridine (EdU)，flow cytometry，以及immunofluorescence等功能分析方法，检测发现circEDC3能抑制SMSCs增殖、分化相关基因的表达，阻滞细胞周期进程，降低增殖细胞比率，抑制分化相关蛋白的表达，抑制肌管形成，但对SMSCs的凋亡没有明显影响。circRNA通常可以通过靶向微小RNA (miRNAs) 来调控靶基因的表达，然而我们发现circEDC3并未直接靶向肌肉发育相关的miRNAs。此外有研究表明，circRNA可通过直接编码蛋白来调节骨骼肌发育，为了进一步探索circEDC3调控鸡骨骼肌发育的潜在机制，我们对circEDC3的编码能力进行了预测。通过对circEDC3序列信息进行分析，我们发现circEDC3在物种间 (鸡、人、小鼠、大鼠、猪) 保守，且具有不同的开放阅读框、内部核糖体进入位点 (IRES) 和N⁶-甲基腺苷 (m⁶A) 基序，表明circEDC3满足编码蛋白质的前提条件，具备一定程度的编码能力，但这仍需进一步的研究论证。总的来说，我们的研究发现了一个在物种间保守的环状RNA circEDC3，通过分析circEDC3的序列信息，预测该circRNA具有一定的蛋白质编码潜力，通过功能分析试验证明circEDC3是一种新的鸡肌肉发育的负调节因子，提示circEDC3可作为肉鸡分子育种的一个重要候选靶标，为肉鸡的育种改良提供新的切入点。

Abstract: As an emerging class of non-coding transcripts, circular RNAs (circRNAs) are proved to participate in the complex process of myogenesis in diverse species. A previous study has identified circular RNA EDC3 (circEDC3) as a typical covalently closed circular RNA abundant in chicken skeletal muscle. This study found that circEDC3 is a conservative circular RNA and performed functional analysis to investigate the role of circEDC3 in chicken muscle growth. The results indicated that circEDC3 could inhibit (P<0.05) chicken skeletal muscle satellite cells (SMSCs) proliferation and differentiation but had no significant influence on SMSCs apoptosis. Additionally, bioinformatics analysis showed that circEDC3 had promising coding potential. The open reading frames (ORF) were found in circEDC3 in this study. Furthermore, this study predicted that circEDC3 had internal ribosome entry sites (IRES) and N6-methyladenosine (m6A) motifs in different species, implying that circEDC3 might be translatable. This study revealed that circEDC3 might be a negative regulator in chicken muscle development and suggested it has protein-coding potential in different species.

Key words: circEDC3 , chicken , SMSCs , myogenesis , coding potential

. [J]. Journal of Integrative Agriculture, 2022, 21(5): 1444-1456.

WEI Yuan-hang, ZHAO Xi-yu, SHEN Xiao-xu, YE Lin, ZHANG Yao, WANG Yan, LI Di-yan, ZHU Qing, YIN Hua-dong. The expression, function, and coding potential of circular RNA circEDC3 in chicken skeletal muscle development[J]. Journal of Integrative Agriculture, 2022, 21(5): 1444-1456.

参考文献

Anantharaman V, Aravind L. 2004. Novel conserved domains in proteins with predicted roles in eukaryotic cell-cycle regulation, decapping and RNA stability. BMC Genomics, 5, 45.
Basselduby R S, Olson E N. 2006. Signaling pathways in skeletal muscle remodeling. Annual Review of Biochemistry, 75, 19–37.
Buckingham M, Bajard L, Chang T H, Daubas P, Hadchouel J, Meilhac S M, Montarras D, Rocancourt D, Relaix F. 2003. The formation of skeletal muscle: From somite to limb. Journal of Anatomy, 202, 59–68.
Capel B, Swain A, Nicolis S, Hacker A, Walter M, Koopman P, Goodfellow P, Lovell-Badge R. 1993. Circular transcripts of the testis-determining gene Sry in adult mouse testis. Cell, 73, 1019–1030.
Cesana M, Cacchiarelli D, Legnini I, Santini T, Sthandier O, Chinappi M, Tramontano A, Bozzoni I. 2011. A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA. Cell, 147, 358–369.
Chen B, Yu J, Guo L, Byers M, Wang Z, Chen X, Xu H, Nie Q. 2019. Circular RNA circHIPK3 promotes the proliferation and differentiation of chicken myoblast cells by sponging miR-30a-3p. Cells, 8, 177.
Chen C Y, Sarnow P. 1995. Initiation of protein synthesis by the eukaryotic translational apparatus on circular RNAs. Science, 268, 415–417.
Chen L L, Yang L. 2015. Regulation of circRNA biogenesis. Rna Biology, 12, 381–388.
Chen X, Ouyang H, Wang Z, Chen B, Nie Q. 2018. A novel circular RNA generated by FGFR2 gene promotes myoblast proliferation and differentiation by sponging miR-133a-5p and miR-29b-1-5p. Cells, 7, 199.
Cocquerelle C, Mascrez B, Hétuin D, Bailleul B. 1993. Mis-splicing yields circular RNA molecules. Faseb Journal, 7, 155–160.
Conn S J, Pillman K A, Toubia J, Conn V M, Salmanidis M, Phillips C A, Roslan S, Schreiber A W, Gregory P A, Goodall G J. 2015. The RNA binding protein quaking regulates formation of circRNAs. Cell, 6, 1125–1134.
Dong S, Li C, Zenklusen D, Singer R, Jacobson A, He F. 2007. YRA1 autoregulation requires nuclear export and cytoplasmic Edc3p-mediated degradation of its pre-mRNA. Molecular Cell, 25, 559–573.
Esteller M. 2011. Non-coding RNAs in human disease. Nature Reviews Genetics, 12, 861–874.
Guarnerio J, Bezzi M, Jeong J C, Paffenholz S V, Berry K, Naldini M M, Lococo F, Tay Y, Beck A H, Pandolfi P P. 2016. Oncogenic role of fusion-circRNAs derived from cancer-associated chromosomal translocations. Cell, 165, 289–302.
Han S, Cui C, Wang Y, He H, Liu Z, Shen X, Chen Y, Li D, Zhu Q, Yin H. 2019a. Knockdown of CSRP3 inhibits differentiation of chicken satellite cells by promoting TGF-β/Smad3 signaling. Gene, 707, 36–43.
Han S, Cui C, Wang Y, He H, Yin H. 2019b. FHL3 negatively regulates the differentiation of skeletal muscle satellite cells in chicken. 3 Biotech, 9, 1–8.
Hansen T B, Jensen T I, Clausen B H, Bramsen J B, Finsen B, Damgaard C K, Kjems J R. 2013. Natural RNA circles function as efficient microRNA sponges. Nature, 495, 384–388.
Hayakawa T, Shiraishi J I, Ohta Y. 2019. Effects of in ovo vitamin D3 injection on subsequent growth of broilers. The Journal of Poultry Science, 56, 220–223.
Jeck W R, Sharpless N E. 2014. Detecting and characterizing circular RNAs. Nature Biotechnology, 32, 453–461.
Jeck W R, Sorrentino J A, Wang K, Slevin M K, Burd C E, Liu J, Marzluff W F, Sharpless N E. 2013. Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA, 19, 141–157.
Kshirsagar M, Parker R. 2004. Identification of Edc3p as an enhancer of mRNA decapping in Saccharomyces cerevisiae. Genetics, 166, 729–739.
Legnini I, Timoteo G D, Rossi F, Morlando M, Briganti F, Sthandier O, Fatica A, Santini T, Andronache A, Wade M. 2017. Circ-ZNF609 is a circular RNA that can be translated and functions in myogenesis. Molecular Cell, 66, 22–37.
Li S, Mason C E. 2014. The pivotal regulatory landscape of RNA modifications. Annual Review of Genomics & Human Genetics, 15, 127–150.
Li Y, Dilworth F J. 2016. Compacting chromatin to ensure muscle satellite cell quiescence. Cell Stem Cell, 18, 162–164.
Li Y, Zheng Q, Bao C, Li S, Guo W, Zhao J, Chen D, Gu J, He X, Huang S. 2015a. Circular RNA is enriched and stable in exosomes: A promising biomarker for cancer diagnosis. Cell Research, 25, 981–984.
Li Z, Huang C, Bao C, Chen L, Lin M, Wang X, Zhong G, Yu B, Hu W, Dai L. 2015b. Exon-intron circular RNAs regulate transcription in the nucleus. Nature Structural & Molecular Biology, 22, 256–264.
Memczak S, Jens M, Elefsinioti A, Torti F, Krueger J, Rybak A, Maier L, Mackowiak S D, Gregersen L H, Munschauer M. 2013. Circular RNAs are a large class of animal RNAs with regulatory potency. Nature, 495, 333–338.
Moresi V, Marroncelli N, Adamo S. 2015. New insights into the epigenetic control of satellite cells. World Journal of Stem Cells, 7, 945–955.
Ouyang H, Chen X, Li W, Li Z, Nie Q, Zhang X. 2018a. Circular RNA circSVIL promotes myoblast proliferation and differentiation by sponging miR-203 in chicken. Frontiers in Genetics, 9, 172.
Ouyang H, Chen X, Wang Z, Yu J, Jia X, Li Z, Luo W, Abdalla B A, Jebessa E, Nie Q. 2018b. Circular RNAs are abundant and dynamically expressed during embryonic muscle development in chickens. DNA Research, 25, 71–86.
Pamudurti N R, Bartok O, Jens M, Ashwalfluss R, Stottmeister C, Ruhe L, Hanan M, Wyler E, Perezhernandez D, Ramberger E. 2017. Translation of CircRNAs. Molecular Cell, 66, 9–21.
Pelletier J, Sonenberg N. 1988. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature, 334, 320–325.
Pesti G M. 1995. Nutrient requirements of poultry. Animal Feed Science and Technology, 56, 177–178.
Salzman J, Chen R E, Olsen M N, Wang P L, Brown P O, Moran J V. 2013. Cell-type specific features of circular RNA expression. Plos Genetics, 9, e1003777.
Sanger H L, Klotz G, Riesner D, Gross H J, Kleinschmidt A K. 1976. Viroids are single-stranded covalently closed circular RNA molecules existing as highly base-paired rod-like structures. Proceedings of the National Academy of Sciences of the United States of America, 73, 3852–3856.
Shen X, Liu Z, Cao X, He H, Han S, Chen Y, Cui C, Zhao J, Li D, Wang Y. 2019. Circular RNA profiling identified an abundant circular RNA circTMTC1 that inhibits chicken skeletal muscle satellite cell differentiation by sponging miR-128-3p. International Journal of Biological Sciences, 15, 2265–2281.
Shin J, Mcfarland D C, Strasburg G M, Velleman S G. 2013. Function of death-associated protein 1 in proliferation, differentiation, and apoptosis of chicken satellite cells. Muscle & Nerve, 48, 777–790.
Sung K H, Tae-Young H, Yeong-Min Y. 2016. Melatonin-mediated intracellular insulin during 2-Deoxy-D-glucose treatment is reduced through autophagy and EDC3 protein in insulinoma INS-1E cells. Oxidative Medicine and Cellular Longevity, 2016, 1–11.
Venø M T, Hansen T B, Venø S T, Clausen B H, Kjems J. 2015. Spatio-temporal regulation of circular RNA expression during porcine embryonic brain development. Genome Biology, 16, 245.
Wang Y, Wang Z. 2015. Efficient backsplicing produces translatable circular mRNAs. RNA, 21, 172–179.
Wang X, Zhao B S, Roundtree I A, Lu Z, Han D, Ma H, Weng X, Chen K, Shi H, He C. 2015. N6-methyladenosine modulates messenger RNA translation efficiency. Cell, 6, 1388–1399.
Wei X, Li H, Yang J, Hao D, Dong D, Huang Y, Lan X, Plath M, Lei C, Lin F. 2017. Circular RNA profiling reveals an abundant circLMO7 that regulates myoblasts differentiation and survival by sponging miR-378a-3p. Cell Death and Disease, 8, e3153.
Weingarten-Gabbay S, Eliaskirma S, Nir R, Gritsenko A A, Sternginossar N, Yakhini Z, Weinberger A, Segal E. 2016. Comparative genetics. Systematic discovery of cap-independent translation sequences in human and viral genomes. Science, 351, 240–240.
Xia X, Li X, Li F, Wu X, Zhang M, Zhou H, Huang N, Yang X, Xiao F, Liu D. 2019. A novel tumor suppressor protein encoded by circular AKT3 RNA inhibits glioblastoma tumorigenicity by competing with active phosphoinositide-dependent Kinase-1. Molecular Cancer, 18, 1–16.
Yang Y, Fan X, Mao M, Song X, Wu P, Zhang Y, Jin Y, Yang Y, Chen L, Wang Y. 2017. Extensive translation of circular RNAs driven by N6-methyladenosine. Cell Research, 27, 626–641.
Yin H, Zhang S, Gilbert E R, Siegel P B, Zhu Q, Wong E A. 2014. Expression profiles of muscle genes in postnatal skeletal muscle in lines of chickens divergently selected for high and low body weight. Poultry Science, 93, 147–154.
Yue B, Wang J, Ru W, Wu J, Cao X, Yang H, Huang Y, Lan X, Lei C, Huang B. 2020. The circular RNA circHUWE1 sponges the miR-29b-AKT3 axis to regulate myoblast development. Molecular Therapy Nucleic Acids, 19, 1086–1097.
Yue Y, Liu J, He C. 2015. RNA N6-methyladenosine methylation in post-transcriptional gene expression regulation. Genes & Development, 13, 1343–1355.
Zaphiropoulos G P. 1997. Exon skipping and circular RNA formation in transcripts of the human cytochrome P-450 2C18 gene in epidermis and of the rat androgen binding protein gene in testis. Molecular and Cellular Biology, 17, 2985–2993.
Zhang M, Huang N, Yang X, Luo J, Zhang N. 2018. A novel protein encoded by the circular form of the SHPRH gene suppresses glioma tumorigenesis. Oncogene, 37, 1805–1814.
Zhao J, Lee E E, Kim J, Yang R, Wang R C. 2019. Transforming activity of an oncoprotein-encoding circular RNA from human papillomavirus. Nature Communications, 10, 2300.
Zhao J, Shen X, Cao X, He H, Yin H. 2020. HDAC4 regulates the proliferation, differentiation and apoptosis of chicken skeletal muscle satellite cells. Animals, 10, 84.
Zhao J, Wu J, Xu T Y, Yang Q C, He J H, Song X F. 2018. IRESfinder: Identifying RNA internal ribosome entry site in eukaryotic cell using framed k-mer features. Journal of Genetics and Genomics, 7, 403–406.
Zheng X, Chen L, Zhou Y, Wang Q, Zheng Z, Xu B, Wu C, Zhou Q, Hu W, Wu C. 2019. A novel protein encoded by a circular RNA circPPP1R12A promotes tumor pathogenesis and metastasis of colon cancer via Hippo-YAP signaling. Molecular Cancer, 18, 47.

相关文章 0

编辑推荐

Metrics