Elucidating the molecular basis of lncRNA evolution for mammalian dosage compensation

摘要 遗传性别决定和性染色体在动物和植物中都已进化。在哺乳动物中， 男性为 XY，女性为 XX。 Y染色体变性和X染色体数量差异 染色体会导致男性和女性细胞之间X连锁基因产物的剂量不平衡。解决 这个问题，异配物种中进化出了各种剂量补偿机制。在哺乳动物中， 在 X 失活过程中，雌性体内两条 X 染色体之一的转录沉默。当X-失活时 作为性染色体表观遗传调控的范例已被广泛研究，它在很大程度上受到控制 长非编码 RNA (lncRNA) 在剂量补偿进化中的作用仍然难以捉摸 机制。一般来说，人类 lncRNA 影响早期发育并影响人类疾病。然而， lncRNA是否代表转录的“副产品”或基因调控中的必需生物分子，例如X- 失活，一直处于争论之中。功能性lncRNA及其进化的实验模型 RNA介导的机制很少。 我们的研究重点是一组具有 X 失活功能的 lncRNA 基因。这个lncRNA簇 基因在 X 染色体上同时进化，从祖先的蛋白质编码基因到 RNA 功能的假生成和获得，与 X 失活沿着 真兽类和有袋类哺乳动物的分歧。我们最近的数据表明，在这个基因中 在小鼠基因组中，lncRNA Jpx 激活 lncRNA Xist 并充当分子开关 小鼠X-失活。我们还通过比较小鼠和人类同源物报告了功能性 尽管整体序列和 RNA 结构存在差异，但 Jpx 在 X 失活中的保守性。但问题 关于 1) 哪些序列变异是 X 失活 lncRNA 功能的决定因素，2) 有哪些 X-失活所必需的 lncRNA 的调控特征，3) 驱动 X- 中 Xic 位点功能的因素 染色体沉默，尚未解决。这些是与剂量演变直接相关的问题 哺乳动物的补偿。在这个项目中，我们将 (1) 确定 Jpx lncRNA 的区域序列基序 对于其在 X 失活中的功能至关重要，(2) 确定 Jpx lncRNA 的反式和顺式活性是否相同 对于 X 失活中的功能是必需的，并且 (3) 了解 Xic 在染色体沉默中的功能 以及通过将 Xic 序列整合到常染色体中对其顺式和反式活性的要求。拟议的 预计研究将揭示 lncRNA 在 X 失活中的分子特征， 这不仅有助于我们了解 lncRNA 进化，还有助于了解剂量补偿 哺乳动物的进化。

ABSTRACT Genetic sex determination and sex chromosomes have evolved in both animals and plants. In mammals, males are XY and females are XX. Degeneration of the Y chromosome and the difference in the number of X- chromosome would lead to dose imbalance of X-linked gene products between male and female cells. To resolve this problem, various dosage compensation mechanisms have evolved in heterogametic species. In mammals, one of the two X chromosomes is transcriptionally silenced in females during X-inactivation. While X-inactivation has been extensively studied as a paradigm for epigenetic regulation of sex chromosomes, it is largely controlled by long noncoding RNAs (lncRNAs) that remain elusive for their role in the evolution of dosage compensation mechanisms. In general, human lncRNAs impact early development and affect human disease. However, whether lncRNAs represent “byproducts” of transcription or essential biomolecules in gene regulation, e.g. X- inactivation, has been under continuous debate. Experimental models for functional lncRNA and evolution of RNA-mediated mechanisms are scarce. Our research focuses on a cluster of lncRNA genes functional for X-inactivation. This cluster of lncRNA genes have evolved concomitantly on the X chromosome, from ancestral protein-coding genes through pseudogenization and gain of RNA functions, coincidental with the evolution of X-inactivation along the divergence of eutherian and marsupial mammals. Our recent data have demonstrated that, within this gene cluster in the mouse genome, the lncRNA Jpx activates lncRNA Xist and functions as a molecular switch for mouse X-inactivation. We have also reported, by comparing mouse and human homologs, functional conservation of Jpx in X-inactivation despite overall sequence and RNA structural divergence. But questions regarding 1) what sequence variations are determinant of lncRNA function for X-inactivation, 2) are there regulatory features of lncRNA essential for X-inactivation, 3) what drives the function of Xic locus in X- chromosome silencing, are unresolved. These are questions directly related to the evolution of dosage compensation in mammals. In this project, we will (1) determine the regional sequence motifs of Jpx lncRNA that are essential for its function in X-inactivation, (2) determine whether both trans and cis activities of Jpx lncRNA are necessary for its function in X-inactivation, and (3) understand the function of Xic in chromosome silencing and the requirement of its cis and trans activities by integrating the Xic sequence into an autosome. The proposed research is anticipated to uncover insights into the molecular features of lncRNA for function in X-inactivation, which will contribute to our understanding about not only lncRNA evolution, but also dosage compensation evolution in mammals.