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进化了。在哺乳动物身上， 男性为XY，女性为XX。Y染色体的退化和X-染色体数目的差异 染色体会导致X连锁基因产物在雄性和雌性细胞之间的剂量不平衡。要解决 在这个问题上，各种剂量补偿机制已经在异性恋物种中进化出来。在哺乳动物身上， 在X染色体失活的过程中，两条X染色体中的一条在雌性体内转录沉默。而X-失活 作为性染色体表观遗传调控的范例被广泛研究，它在很大程度上是受控制的 通过长的非编码RNA(LncRNAs)，它们在剂量补偿的进化中的作用仍然难以捉摸 机械装置。一般来说，人类的lncRNA影响早期发育并影响人类疾病。然而， 无论lncRNAs是转录的“副产物”还是基因调控中的基本生物分子，例如X- 失活，一直处于持续的争论中。功能性lncRNA的实验模型及其进化 RNA介导的机制很少。 我们的研究集中在一组与X失活有关的lncRNA基因上。这一簇lncRNA 基因在X染色体上伴随着进化，从祖先的蛋白质编码基因到 RNA功能的伪基因化和获得，与X失活的进化是一致的 真兽类和有袋类哺乳动物的分化。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，并作为分子开关 鼠标X-失活。我们还报道，通过比较老鼠和人类的同源物，功能 JPX在X-失活中的保守性，尽管总序列和RNA结构存在差异。但问题是 关于1)哪些序列变异是决定X失活的lncRNA功能的因素，2)有哪些 X-失活所必需的lncRNA的调控特征，3)是什么驱动了X-CIC基因座的功能。 染色体沉默，是未解决的。这些问题与剂量的演变直接相关。 哺乳动物的补偿。在本项目中，我们将(1)确定JPX lncRNA的区域序列基序 对于其在X失活中的作用是必不可少的，(2)确定JPX lncRNA的反式和顺式活性 以及(3)了解XIC在染色体沉默中的作用 以及通过将XIC序列整合到常染色体中来满足其顺式和反式活性的要求。建议数 预计研究将揭示lncRNA在X失活中的作用的分子特征， 这不仅有助于我们理解lncRNA的进化，也有助于我们理解剂量补偿。 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.