藏绵羊BOLL的分子特征及其在睾丸中的表达调控与功能分析

Molecular Characterization of Tibetan Sheep BOLL and Its Expression Regulation and Functional Analysis in Testis

【目的】BOLL作为一个RNA结合蛋白,可通过与其他分子相互作用而在精子发生过程中发挥不可或缺的作用。研究旨在分析藏绵羊BOLL的序列特征及其在睾丸中的表达与分布模式,进而探究其表达调控与潜在的生物学功能,以期为进一步解读BOLL在绵羊精子发生中的作用机制提供必要的思路和分子见解。【方法】选取3月龄(性成熟前)、1岁龄(性成熟)和3岁龄(成年)3个关键发育阶段各8只健康的雄性藏绵羊。以睾丸组织总RNA为模板,采用RT-PCR技术克隆藏绵羊BOLL的完整编码序列(coding sequence,CDS)区;通过相关生物信息学软件对BOLL的序列与结构特征及其互作蛋白进行分析;采用实时荧光定量PCR(quantitative real time PCR,qRT-PCR),Western blot和免疫荧光染色法对BOLL在3个发育阶段睾丸组织中的表达和免疫定位特征进行检测;基于课题组前期有关藏绵羊睾丸组织全转录组测序数据,借助相关数据库进行绵羊BOLL的竞争性内源RNA(competing endogenous RNA,ceRNA)调控网络和功能注释分析,并采用qRT-PCR和双荧光素酶报告实验对其表达特征及靶向关系进行验证。【结果】藏绵羊BOLL CDS区全长为888 bp,可编码295个氨基酸,含有由81个氨基酸残基组成的RRM结构域(靠近N端)和25个氨基酸残基组成的DAZ重复基序。绵羊BOLL在不同哺乳物种间(特别是山羊、川南山地黄牛和牦牛)具有高度的序列同源性和进化保守性。BOLL蛋白与10个雄性生殖细胞发育相关蛋白质分子间存在潜在的相互作用。随着年龄的增加,在藏绵羊睾丸中BOLL mRNA的表达持续上调,而其蛋白的表达先上调后下调。BOLL蛋白主要存在于性成熟后(1岁龄和3岁龄)睾丸内的精子细胞中,也在精母细胞和整个发育阶段的精原细胞中有少量分布。qRT-PCR结果显示,与3月龄相比,在1岁龄和3岁龄睾丸中微小RNAs(microRNAs,miRNAs)oar-miR-127-5p、oar-miR-382-5p和oar-miR-760-3p的表达量极显著下调(P<0.01),而长链非编码RNAs(long noncoding RNAs,lncRNAs)LOC105602204、LOC105603195和LOC105616228以及环状RNAs(circular RNAs,circRNAs)circ-ECT2L和circ-SPHKAP的表达量极显著上调(P<0.01)。oar-miR-127-5p和oar-miR-760-3p明显降低了BOLL 3′UTR野生型的荧光素酶活性,并且oar-miR-760-3p明显降低了野生型Circ-ECT2L和野生型LOC105616228报告基因的荧光素酶活性。【结论】报道了藏绵羊睾丸中BOLL的分子结构特征、表达规律及潜在的表达调控作用。BOLL主要在藏绵羊减数分裂后的圆形和长形精子细胞中表达,并且其表达受到oar-miR-127-5p和oar-miR-760-3p的直接靶向负调控以及oar-miR-760-3p介导的circRNA Circ-ECT2L和lncRNA LOC105616228的正向调控,进而可能与下游信号分子相互作用,以参与调节绵羊精子细胞向成熟精子分化。

【Objective】 As a RNA-binding protein, BOLL exerts integral roles during spermatogenesis through interactions with other molecules. The present study was aimed to analyze the sequence characteristics of Tibetan sheep BOLL and its expression and distribution patterns in testis which, in turn, explored the regulation and potential biological function of BOLL expression, hoping to offer much needed perspective and molecular insight for deciphering the mechanism of BOLL during sheep spermatogenesis in the future. 【Method】Eight healthy male Tibetan sheep from each of three key developmental stages, including 3 months old(sexually immature), 1 year old(sexually mature) and 3 years old(adult), were selected. The full-length coding sequence(CDS) region of Tibetan sheep BOLL was cloned by real time PCR(RT-PCR) using total RNA from the right testis samples;the sequence and structural signatures of BOLL and its interacting proteins were analyzed via relevant bioinformatics software;the expression and immunolocalization characteristics of BOLL in testicular tissues at three developmental stages were assessed with quantitative RT-PCR(qRT-PCR), Western blot, and immunofluorescence staining;the competing endogenous RNA(ceRNA) regulatory network and GO functional annotation for ovine BOLL were investigated with the aid of related databases, based on previous data of an integrative analysis of transcriptional profiles from Tibetan sheep testicular tissues by our group, and their expression patterns and targeting relationships were verified by qRT-PCR and dual luciferase reporter assay, respectively. 【Result】 The full CDS region of Tibetan sheep BOLL was 888 bp in length, capable of encoding 295 amino acids which contained a RRM domain of 81 amino acids(near the N-terminal) and a DAZ repeat motif of 25 amino acids. Ovine BOLL exhibited high sequence homology and evolutionary conservation with other mammals, especially for goat, cattle, and yak. There was the potential interaction between sheep BOLL protein and 10 proteins associated with the development of male germ cells. With increasing age, mRNA expression of BOLL was consistently up-regulated in Tibetan sheep testes, while protein expression of which was up-regulated followed by down-regulation. BOLL protein predominantly existed in spermatids from post-pubertal(1 year old and 3 years old) testes, and small amounts were present in spermatocytes, as well as spermatogonia in testes throughout development stages. The q RT-PCR results showed that compared with 3 months old, the expression levels of microRNAs(miRNAs) oar-miR-127-5 p、oar-miR-382-5 p and oar-miR-760-3 p exhibited an extremely significant reduction(P<0.01), while the expression levels for long noncoding RNAs(lnc RNAs) LOC105602204, LOC105603195 and LOC105616228, as well as for circular RNAs(circRNAs) circ-ECT2 L and circ-SPHKAP exhibited an extremely significant increase in 1 year old and 3 years old(P<0.01). oar-miR-127-5 p and oar-miR-760-3 p significantly decreased the luciferase activity of BOLL 3′-UTR wild-type, and oar-miR-760-3 p significantly decreased the luciferase activities of wild-type Circ-ECT2 L and wild-type LOC105616228 reporter genes. 【Conclusion】 This study was the first to report the molecular characteristics and expression patterns of BOLL gene and regulation of its expression in Tibetan sheep testis. BOLL was predominantly expressed in the post-meiotic round and elongating spermatids of Tibetan sheep, and the expression of which was directly targeted and negatively regulated by oar-miR-127-5 p and oar-miR-760-3 p;and was positively regulated by oar-miR-760-3 p-mediated circRNA Circ-ECT2 L and lncRNA LOC105616228. In turn, which might may interact with downstream signaling molecules to participate in the differentiation of ovine spermatids into mature spermatozoa.