摘要
In mammals, the testis-specific serine/threonine kinase (TSSK) is essential for spermatogenesis and male fertility. TSSK4 belongs to the family of the testis-specific serine/threonine-protein kinase (TSSK), with a crucial role in spermatogenesis. This study aimed to analyze the variable spliceosome of the TSSK4 gene in the yak for understanding the regulatory function of the TSSK4 spliceosome in yak testis development using PCR amplification and cloning techniques. The GST pull-down was used for pulling down the protein interacting with TSSK4, and then the protein interacting with TSSK4 was identified using LC–MS/MS. The results of the PCR amplification demonstrated multiple bands of the TSSK4 gene in the yak. The cloning and sequencing yielded a total of six alternative spliceosomes, which included only two alternative spliceosomes before sexual maturity and four alternative spliceosomes after sexual maturity. The sub-cells of the alternative spliceosomes were found to localize in the nucleus before sexual maturity and in the cytoplasm after sexual maturity. The LC–MS/MS analysis of the alternative spliceosome with the highest expression after sexual maturity yielded a total of 223 interacting proteins. The enrichment analysis of the 223 interacting proteins revealed these proteins participate in biological processes, cell composition, and molecular functions. The KEGG analysis indicated that the TSSK4-interacting protein participates in the estrogen signaling pathways, tight junctions, endoplasmic reticulum protein processing, and other signaling pathways. This study cloned the six alternative spliceosomes of the TSSK4 gene laying the foundation for studying the function of each spliceosome in the future.
摘要译文
在哺乳动物中，睾丸特异性丝氨酸/苏氨酸激酶（TSSK）对于精子发生和男性生育至关重要。 TSSK4属于睾丸特异性丝氨酸/苏氨酸蛋白激酶（TSSK）的家族，在精子发生中起着至关重要的作用。这项研究旨在分析YAK中TSSK4基因的可变剪接体，以了解使用PCR扩增和克隆技术在YAK睾丸开发中TSSK4剪接体的调节功能。 GST下拉用于拉下与TSSK4相互作用的蛋白质，然后使用LC-MS/MS鉴定与TSSK4相互作用的蛋白质相互作用。 PCR扩增的结果表明牛中TSSK4基因的多个频带。克隆和测序总共产生了六个替代剪接体，其中包括性成熟之前的两个替代剪接体和性成熟后的四个替代剪接体。发现替代剪接体的子细胞在性成熟前和性成熟后的细胞质中定位在细胞核中。性成熟后具有最高表达的替代剪接体的LC – MS/MS分析产生了总共223种相互作用的蛋白质。对223种相互作用蛋白的富集分析揭示了这些蛋白质参与生物学过程，细胞组成和分子功能。 KEGG分析表明，TSSK4相互作用蛋白参与雌激素信号通路，紧密连接，内质网蛋白加工和其他信号通路。这项研究将TSSK4基因的六个替代剪接体克隆为研究未来每个剪接体功能的基础。
Xingdong Wang;Jie Pei;Lin Xiong;Shaoke Guo;Mengli Cao;Yandong Kang;Pengjia Bao;Xiaoyun Wu;Min Chu;Chunnian Liang;Ping Yan;Xian Guo. Identification of the TSSK4 Alternative Spliceosomes and Analysis of the Function of the TSSK4 Protein in Yak (Bos grunniens)[J]. Animals, 2022,12(11): 1380