TDRD6 mediates early steps of spliceosome maturation in primary spermatocytes

Very little is known about splicing and its regulation in germ cells, particularly during meiosis. This paper describes the role of a male germ cell-specific protein, Tudor containing protein 6 (TDRD6), in assembly of the spliceosome in spermatocytes. We show that in spermatocytes, TDRD6 interacts with the key protein methyl transferase of the splicing pathway PRMT5. PRMT5 methylates arginines in substrate proteins. In a methylation dependent manner, TDRD6 also associates with spliceosomal core protein SmB in the absence of RNA, thus before an RNP-type spliceosome has been assembled. In Tdrd6-/- primary spermatocytes, PRMT5's association with SmB and the arginine dimethylation of SmB are much reduced. Abrogation of arginine methylation impaired the assembly of spliceosomes and the presence of the spliceosomal RNA U5 is aberrantly increased. These deficiencies in spliceosome maturation correlated with decreased numbers of Cajal bodies and gems involved in later stages, i.e. nuclear snRNP maturation. To reveal functional consequences of these deficiencies, transcriptome analysis of primary spermatocytes showed high numbers of splicing defects such as aberrant usage of intron and exons as well as aberrant representation of splice junctions upon TDRD6 loss. This study reveals a novel function of TDRD6 in spliceosome maturation and mRNA splicing in spermatocytes. Examination of splicing defects in isolated diplotene cells of 20dpp Tdrd6-/- vs. Tdrd6+/- testes pooled from at least 4 mice by deep sequencing in duplicate using Illumina® HiSeq 2500.目前学界对生殖细胞中的剪接过程及其调控机制，尤其是减数分裂阶段的相关研究仍十分匮乏。本研究阐明了雄性生殖细胞特异性蛋白含Tudor结构域蛋白6（Tudor containing protein 6, TDRD6）在精母细胞（spermatocyte）剪接体（spliceosome）组装过程中的作用。我们证实，在精母细胞中，TDRD6可与剪接通路中的关键蛋白精氨酸甲基转移酶PRMT5（PRMT5）相互结合；PRMT5能够对底物蛋白中的精氨酸残基实施甲基化修饰。在甲基化依赖的调控模式下，TDRD6还可在RNA缺失的状态下，即核糖核蛋白（ribonucleoprotein, RNP）型剪接体组装完成前，与剪接体核心蛋白SmB（SmB）结合。在Tdrd6基因敲除（Tdrd6-/-）的初级精母细胞（primary spermatocyte）中，PRMT5与SmB的结合水平以及SmB的精氨酸二甲基化修饰程度均显著降低。精氨酸甲基化的缺失会损害剪接体的组装，且剪接体RNA U5（U5 snRNA）的异常积累量显著升高。上述剪接体成熟缺陷，与参与后期阶段（即细胞核内小核核糖核蛋白（small nuclear ribonucleoprotein, snRNP）成熟过程）的卡哈尔体（Cajal body）和Gem小体（Gem body）的数量减少密切相关。为明确这些缺陷带来的功能影响，我们对初级精母细胞开展了转录组（transcriptome）分析，结果显示在TDRD6缺失后，会出现大量剪接异常事件，包括内含子（intron）与外显子（exon）的异常使用以及剪接接头（splice junction）的异常呈现。本研究揭示了TDRD6在精母细胞剪接体成熟及信使RNA（messenger RNA, mRNA）剪接过程中的全新功能。本研究采用Illumina® HiSeq 2500测序平台，对至少4只小鼠混合的出生后20天（20 days postpartum, 20dpp）的Tdrd6基因敲除（Tdrd6-/-）与杂合型（Tdrd6+/-）睾丸分离得到的双线期细胞（diplotene cell）进行了两次重复的深度测序（deep sequencing），以检测相关剪接缺陷。