Abstract：Quaking (QKI) is a member of RNA-binding protein family, and involved in the regulation of cell proliferation, differentiation and apoptosis. In order to explore the biological functions of QKI family genes in goat (Capra hircus) muscle development, Jianzhou big-eared goat was used as the research object in present study. The coding sequences of goat QKI family genes were obtained by TA cloning and analyzed by bioinformatics. qRT-PCR and Western blot were used to detect the expression of QKI family genes in different tissues (brain, heart, liver, spleen, lung, kidney, longissimus dorsi, semimembranosus, semitendinosus, gastrocnemius, psoas major, and adductor) of goats (3 d after birth) and developmental stages (embryonic 90 d, 105 d, 135 d; and 3 d, 150 d after birth) of the semimembranosus muscle, and their expression during the proliferation and differentiation of goat skeletal muscle satellite cells (SMSCs). The results showed that the complete coding sequences of goat QKI-5 (GenBank No. MW651981), QKI-6 (GenBank No. MW651982), and QKI-7 (GenBank No. MW651983) genes were obtained by amplification, with the length of 1 026, 978, and 960 bp, which encoding 341, 325, and 319 amino acids, respectively. Nucleotide sequence alignment revealed that QKI-5, QKI-6, and QKI-7 were highly conserved among species, and had the closest phylogenetic relationship with sheep (Ovis aries) and cattle (Bos taurus). The secondary and tertiary structures of goat QKI-5, QKI-6, and QKI-7 protein were mainly composed of α-helix and random coil, and there was a common conserved functional domain, KH (K homology) domain. The results of qRT-PCR showed that the expression of QKI-5, QKI-6, and QKI-7 genes in goat skeletal muscle tissues was significantly higher than that in other tissues (P<0.05); In the semimembranosus muscle of different developmental stages, QKI-5, QKI-6, and QKI-7 showed a trend of increasing first and then decreasing, with the highest expression at 3 d after birth (P<0.05). Western blot results showed that the expression of QKI-5 protein was only detected in goat skeletal muscle tissue, and mainly expressed after birth. Furthermore, QKI-5, QKI-6, and QKI-7 genes showed increasing expression tendency during the proliferation and differentiation of goat SMSCs, and reached the peak at 7 d after differentiation (P<0.05). The present study cloned and obtained the complete coding sequences of goat QKI-5, QKI-6, and QKI-7 genes, and revealed their high expression in goat skeletal muscle tissues and increasing expression patterns during proliferation and differentiation of goat SMSCs, indicating that QKI family genes might play an important role in goat skeletal muscle development. The present study provides basic data for further investigation of molecular mechanism on goat muscle development regulated by QKI.
[1] 张杰. 2009. NF-κB信号通路对RNA结合蛋白QKI的转录调控及其在骨骼肌中的功能研究[D]. 硕士学位论文, 第四军医大学, 导师: 卢兹凡, pp. 1-66. (Zhang J.2009. Transcriptional regulation of RNA binding protein QKI by NF-κB and elucidation of its function in skeletal muscle[D]. Thesis for M.S., Fourth Military Medical University, Supervisor: Lu Z F, pp. 1-66.) [2] Artzt K, Wu J I.2010. STAR trek: An introduction to STAR family proteins and review of quaking (QKI)[J]. Advances in Experimental Medicine and Biology, 693(1): 1-24. [3] Biedermann B, Hotz H R, Ciosk R.2010. The Quaking family of RNA-binding proteins: Coordinators of the cell cycle and differentiation[J]. Cell Cycle, 9(10): 1929-1933. [4] Bismuth K, Relaix F.2010. Genetic regulation of skeletal muscle development[J]. Experimental Cell Research, 316(18): 3081-3086. [5] Bonnet A, Lambert G, Ernest S, et al.2017. Quaking RNA-binding proteins control early myofibril formation by modulating tropomyosin[J]. Developmental Cell, 42(5): 527-541. [6] Braun T, Gautel M.2011. Transcriptional mechanisms regulating skeletal muscle differentiation, growth and homeostasis[J]. Nature Reviews. Molecular cell biology, 12(6): 349-361. [7] Callis T E, Chen J F, Wang D Z.2007. MicroRNAs in skeletal and cardiac muscle development[J]. DNA and Cell Biology, 26(4): 219-225. [8] Chen R, Lei S, Jiang T, et al.2020. Roles of lncRNAs and circRNAs in regulating skeletal muscle development[J]. Acta Physiologica, 228(2): e13356. [9] Darbelli L, Richard S.2016. Emerging functions of the quaking RNA-binding proteins and link to human diseases[J]. Wiley Interdisciplinary Reviews. RNA, 7(3): 399-412. [10] Dayton W R, Weber W J, White M E, et al.2013. Role of estrogen receptor-alpha (ESR1) and the type 1 insulin-like growth factor receptor (IGFR1) in estradiol-stimulated proliferation of cultured bovine satellite cells[J]. Domestic Animal Endocrinology, 44(1): 36-45. [11] Guo J, Liu H B, Sun C, et al.2019. MicroRNA-155 promotes myocardial infarction-induced apoptosis by targeting RNA-binding protein QKI[J]. Oxidative Medicine and Cellular Longevity, 5: 4579806. [12] Guo W, Shi X, Liu A, et al.2011. RNA binding protein QKI inhibits the ischemia/reperfusion-induced apoptosis in neonatal cardiomyocytes[J]. Cellular Physiology and Biochemistry, 28(4): 593-602. [13] Hall M P, Nagel R J, Fagg W S, et al.2013. Quaking and PTB control overlapping splicing regulatory networks during muscle cell differentiation[J]. RNA, 19(5): 627-638. [14] Livak K J, Schmittgen T D.2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method[J]. Methods, 25(4): 402-408. [15] Kondo T, Furuta T, Mitsunaga K, et al.1999. Genomic organization and expression analysis of the mouse qkI locus[J]. Mammalian Genome, 10(7): 662-669. [16] Legnini I, Di Timoteo G, Rossi F, et al.2017. Circ-ZNF609 is a circular RNA that can be translated and functions in myogenesis[J]. Molecular Cell, 66(1): 22-37. [17] Li D, Zhan S, Wang Y, et al.2015. Role of microRNA-101a in the regulation of goat skeletal muscle satellite cell proliferation and differentiation[J]. Gene, 572(2): 198-204. [18] Montoya-Flores D, Mora O, Tamariz E, et al.2012. Ghrelin stimulates myogenic differentiation in a mouse muscle satellite cell line and in primary cultures of bovine myoblasts[J]. Journal of Animal Physiology and Animal Nutrition, 96(4): 725-738. [19] Saccomanno L, Loushin C, Jan E, et al.2001. The STAR protein QKI-6 is a translational repressor[J]. Proceedings of the National Academy of Sciences of the USA, 96(22): 12605-12610. [20] Suiko T, Kobayashi K, Aono K, et al.2016. Expression of quaking RNA-Binding protein in the adult and developing mouse retina[J]. PLOS ONE, 11(5): e0156033. [21] Valverde R, Edwards L, Regan L.2008. Structure and function of KH domains[J]. The FEBS Journal, 275(11): 2712-2726. [22] Wu J I, Reed R B, Grabowski P J, et al.2002. Function of quaking in myelination: Regulation of alternative splicing[J]. Proceedings of the National Academy of Sciences of the USA, 99(7): 4233-4238. [23] Wu Y, Li Z, Yang M, et al.2017. MicroRNA-214 regulates smooth muscle cell differentiation from stem cells by targeting RNA-binding protein QKI[J]. Oncotarget, 8(12): 19866-19878. [24] Yang G, Lu X, Wang L, et al.2011. E2F1 and RNA binding protein QKI comprise a negative feedback in the cell cycle regulation[J]. Cell Cycle, 10(16): 2703-2713. [25] Yu F, Jin L, Yang G, et al.2014. Post-transcriptional repression of FOXO1 by QKI results in low levels of FOXO1 expression in breast cancer cells[J]. Oncology Reports, 31(3): 1459-1465.
