Supplementary Material for: Molecular Characterization of the <i>DDX3Y</i> Gene and Its Homologs in Cattle

<i>DDX3Y</i> (also known as <i>DBY</i>) is a member of the DEAD box protein family, which is involved in ATP-dependent RNA unwinding, needed in a variety of cellular processes including splicing, ribosome biogenesis and RNA degradation. In the human, <i>DDX3Y </i>is located in the <i>AZFa</i> interval in the Y chromosome. Deletion of the <i>AZFa</i> region has been shown to disrupt spermatogenesis, causing subfertility and infertility in otherwise healthy men. Here, we report the characterization of the bovine (b) <i>DDX3Y</i> gene and its homologs <i>DDX3X </i>and <i>PL10</i>. We found 2 transcripts for the b<i>DDX3Y</i> (b<i>DDX3Y</i>-L and -S), which correspond to the long and short transcripts of the human <i>DDX3Y</i> and mouse <i>Ddx3y</i> gene. The 2 transcripts are identical except for a 3-bp (AGT) insertion at the position of nt 2025 and an expanded 3′UTR (nt 2155–2769) in b<i>DDX3Y</i>-L. The b<i>DDX3Y</i>-S encodes a peptide of 660 amino acids (aa), while the b<i>DDX3Y</i>-L encodes a peptide of 661 aa as the result of an additional serine (S) insertion at the position of aa 634. Both b<i>DDX3Y</i> isoforms contain the conserved DEAD-box motif. The b<i>DDX3Y </i>is composed of 17 exons. The homologous gene on the X chromosome, b<i>DDX3X</i>, is highly conserved to the Y-copy at mRNA (83%) and protein (88%) levels as well as in the genomic structure. The autosomal copy, b<i>PL10</i>, mapped on BTA15, is a processed pseudogene with a similarity of 88.1% to b<i>DDX3Y</i> and 93.7% to b<i>DDX3X</i> mRNA, suggesting that <i>PL10</i> is a retroposon of <i>DDX3X</i>. RT-PCR analyses showed that b<i>DDX3Y</i>-L, -S, b<i>DDX3X</i> and b<i>PL10</i> were all widely expressed with predominant expression in testis and brain. Testicular section in situ hybridization revealed that sense and anti-sense RNAs of b<i>DDX3Y</i>-L, -S, and b<i>DDX3X</i> were expressed in interstitial cells. These results together with the finding that the pseudogene b<i>PL10</i> is transcriptionally active in this study provide a basis for further investigating the <i>DDX3</i> gene function in spermatogenesis, male fertility and gene evolution in mammals.

<i>DDX3Y</i>（又称<i>DBY</i>）属于DEAD盒蛋白家族（DEAD box protein family）成员，可参与ATP依赖的RNA解旋过程，该过程参与包括剪接、核糖体生物发生及RNA降解在内的多种细胞生理活动。在人类基因组中，<i>DDX3Y</i>定位于Y染色体的<i>AZFa</i>区间。研究表明，删除<i>AZFa</i>区域会破坏精子发生过程，导致健康男性出现生育力低下乃至不育。本研究对牛源<i>DDX3Y</i>（b<i>DDX3Y</i>）基因及其同源基因<i>DDX3X</i>与<i>PL10</i>进行了鉴定与表征。

研究发现牛<i>DDX3Y</i>存在两种转录本，即b<i>DDX3Y</i>-L与b<i>DDX3Y</i>-S，分别对应人类<i>DDX3Y</i>及小鼠<i>Ddx3y</i>基因的长、短转录本。两种转录本仅存在两处差异：一是在核苷酸位点nt 2025处存在3bp（AGT）的插入，二是b<i>DDX3Y</i>-L具有更长的3'非翻译区（3'UTR，nt 2155–2769）。b<i>DDX3Y</i>-S编码含660个氨基酸（aa）的肽链，而b<i>DDX3Y</i>-L因在氨基酸位点aa 634处多了一个丝氨酸（S）插入，编码含661个氨基酸的肽链。两种牛<i>DDX3Y</i>剪接异构体均含有保守的DEAD-box基序。牛<i>DDX3Y</i>由17个外显子组成。

位于X染色体的同源基因b<i>DDX3X</i>，在mRNA水平（同源性83%）、蛋白质水平（同源性88%）以及基因组结构上均与Y染色体拷贝高度保守。定位于牛15号染色体（BTA15）的常染色体同源拷贝b<i>PL10</i>是一个经过加工的假基因，其与b<i>DDX3Y</i>的序列同源性为88.1%，与b<i>DDX3X</i> mRNA的同源性为93.7%，提示<i>PL10</i>是<i>DDX3X</i>的逆转座子。

逆转录PCR（RT-PCR）分析显示，b<i>DDX3Y</i>-L、b<i>DDX3Y</i>-S、b<i>DDX3X</i>及b<i>PL10</i>均呈广泛表达，且在睾丸与大脑中表达丰度最高。睾丸组织原位杂交结果显示，b<i>DDX3Y</i>-L、b<i>DDX3Y</i>-S及b<i>DDX3X</i>的正义链与反义链RNA均在间质细胞中表达。

本研究中发现假基因b<i>PL10</i>具有转录活性，结合上述实验结果，可为进一步研究<i>DDX3</i>基因在哺乳动物精子发生、雄性生育力及基因进化中的功能提供理论基础。