Abstract

There is a need to understand the molecular basis of testes under Non-Obstructive Azoospermia (NOA), a state of failed spermatogenesis. There has been a lack of attention to the transcriptome at the level of alternatively spliced mRNAs (iso-mRNAs) and the mechanism of gene expression regulation. Hence, we aimed to establish a reliable iso-mRNA profile of NOA-testes, and explore molecular mechanisms – especially those related to gene expression regulation. We sequenced mRNAs from testicular samples of donors with complete spermatogenesis (control samples) and a failure of spermatogenesis (NOA samples). We identified differentially expressed genes and their iso-mRNAs via standard NGS data analyses. We then listed these iso-mRNAs hierarchically based on the extent of consistency of differential quantities across samples and groups, and validated the lists via RT-qPCRs (for 80 iso-mRNAs). In addition, we performed extensive bioinformatic analysis of the splicing features, domains, interactions, and functions of differentially expressed genes and iso-mRNAs. Many top-ranking down-regulated genes and iso-mRNAs, i.e., those down-regulated more consistently across the NOA samples, are associated with mitosis, replication, meiosis, cilium, RNA regulation, and post-translational modifications such as ubiquitination and phosphorylation. Most down-regulated iso-mRNAs correspond to full-length proteins that include all expected domains. The predominance of alternative promoters and termination sites in these iso-mRNAs indicate their gene expression regulation via promoters and UTRs. We compiled a new, comprehensive list of human transcription factors (TFs) and used it to identify TF-'TF gene’ interactions with potential significance in down-regulating genes under the NOA condition. The results indicate that RAD51 suppression by HSF4 prevents SP1-activation, and SP1, in turn, could regulate multiple TF genes. This potential regulatory axis and other TF interactions identified in this study could explain the down-regulation of multiple genes in NOA-testes. Such molecular interactions may also have key regulatory roles during normal human spermatogenesis.

摘要

有必要了解非阻塞性无精子症 (NOA) 下睾丸的分子基础，这是一种精子发生失败的状态。在可变剪接 mRNA (iso-mRNA) 和基因表达调控机制的水平上，人们一直缺乏对转录组的关注。因此，我们旨在建立可靠的 NOA 睾丸 iso-mRNA 图谱，并探索分子机制——尤其是那些与基因表达调控相关的机制。我们对精子发生完全（对照样本）和精子发生失败（NOA 样本）的供体睾丸样本中的 mRNA 进行了测序。我们通过标准 NGS 数据分析。然后，我们根据样本和组间差异量的一致性程度，分层列出这些 iso-mRNA，并通过以下方法验证列表RT-qPCR（针对 80 种 iso-mRNA）。此外，我们对差异表达基因和 iso-mRNA 的剪接特征、结构域、相互作用和功能进行了广泛的生物信息学分析。许多排名靠前的下调基因和 iso-mRNA，即那些在 NOA 样本中更一致地下调的基因，与有丝分裂、复制、减数分裂、纤毛、RNA 调节以及泛素化和磷酸化等翻译后修饰有关. 大多数下调的 iso-mRNA 对应于包含所有预期结构域的全长蛋白质。这些 iso-mRNA 中替代启动子和终止位点的优势表明它们的基因表达调控通过启动子和 UTR。我们编制了一份新的、全面的人类转录因子 (TF) 列表，并用它来识别在 NOA 条件下对下调基因具有潜在意义的 TF-“TF 基因”相互作用。结果表明，HSF4 对 RAD51 的抑制阻止了 SP1 的激活，而 SP1 反过来又可以调节多个 TF 基因。这个潜在的调控轴和本研究中发现的其他 TF 相互作用可以解释 NOA 睾丸中多个基因的下调。这种分子相互作用在正常人类精子发生过程中也可能具有关键的调节作用。