Role of dyskerin in oral epithelial homeostasis

描述（由申请人提供）：候选人致力于口腔健康相关的研究，以补充他在口腔颌面病理学方面的培训。他正在申请K08指导临床科学家发展奖，以便为他的研究活动提供保护时间，并在转化研究方法方面进行教学培训。在研究期间，他将获得正式的指导，掌握新的技能，学习最前沿的方法，并直接受益于世界级研究人员的指导和专业知识。在博士的杰出指导下。Anil Rustgi和Sarah Millar，以及多元化咨询委员会的经验和见解，职业发展奖将确保Alawi博士能够继续他的专业发展，并实现他成为一名独立研究者的目标。口腔鳞状上皮细胞经历一个明确的分化程序。为此，一些遗传疾病，包括x连锁先天性角化不良（DC），影响口腔黏膜组织。x连锁DC是由DKC1 （dyskerin）基因突变引起的。口腔白斑是最常见的临床表现之一，这些肿瘤前病变在儿童和青少年时期的出现表明正常dyskerin功能的丧失破坏了口腔上皮的稳态。然而，其机制仍有待阐明。Dyskerin是结合以H/ACA二级结构为特征的小非编码RNA分子的核糖核蛋白的生物发生所必需的。正是在这种能力下，dyskerin有助于端粒酶活性和前体rRNA加工。然而，尽管这两种细胞过程在有丝分裂期间都受到抑制，但我们已经表明，dyskerin在有丝分裂期间表达高峰，该蛋白定位于有丝分裂口腔角化细胞的不同亚细胞结构，并且dyskerin功能的急性丧失触发G2/M阻滞并导致具有多极纺锤体的非典型有丝分裂的积累。我们最近还证明，dyskerin缺失降低了H/ACA小核仁rna衍生的microrna （miRNA）及其相应前体的水平。MicroRNAs通过调控转录后基因的表达，包括有丝分裂所必需的基因，在维持正常细胞稳态中发挥着关键作用。在这个假设驱动的提案中，我们将使用新的形态学，生化，功能和遗传学方法来确定dyskerin及其同源RNA对口腔上皮稳态的关键调节机制。特别是，我们将(1)确定dyskerin定位在有丝分裂中的作用和机制，(2)确定dyskerin在转录后基因表达中的作用，(3)使用小鼠x -连锁DC模型确定dyskerin突变对口腔上皮的体内影响。通过阐明dyskerin的新功能，确定其相关RNA的作用，并通过体外实验与体内研究相补充，这些研究将带来丰富的新知识，可能导致口腔白斑和x连锁DC的新治疗策略。

DESCRIPTION (provided by applicant): The candidate has dedicated himself to a career in oral health-related research to complement his training in oral and maxillofacial pathology. He is applying for a K08 Mentored Clinical Scientist Development Award to facilitate protected time for the pursuit of his research activities together with didactic training in Translational Research methodology. During the course of the award period, he will obtain formal instruction, acquire new skills, learn cutting-edge methodologies, and directly benefit from the guidance and expertise of world-class investigators. Under the outstanding mentorship of Drs. Anil Rustgi and Sarah Millar, and the experience and insights of a diverse Advisory Committee, the Career Development Award will ensure that Dr. Alawi can continue his professional development and achieve his goal of becoming an independent investigator. Oral squamous epithelial cells undergo a well-defined differentiation program. To that end, several genetic diseases, including X-linked dyskeratosis congenita (DC), affect the oral mucosal tissues. X-linked DC is caused by mutations in the DKC1 (dyskerin) gene. Oral leukoplakia is one of the most common clinical manifestations, and the appearance of these preneoplastic lesions during childhood and adolescence indicates that loss of normal dyskerin function disrupts oral epithelial homeostasis. However, the mechanisms remain to be elucidated. Dyskerin is required for the biogenesis of ribonucleoproteins that incorporate small non-coding RNA molecules characterized by the H/ACA secondary structure. It is in this capacity that dyskerin contributes to telomerase activity and precursor rRNA processing. However, while both of these cellular processes are repressed during mitosis, we have shown that dyskerin expression peaks during mitosis, the protein localizes to distinct sub-cellular structures in mitotic oral keratinocytes, and acute loss of dyskerin function triggers G2/M arrest and leads to the accumulation of atypical mitoses with multi-polar spindles. We also recently demonstrated that dyskerin depletion reduces the levels of a subset of H/ACA small nucleolar RNA-derived microRNAs (miRNA) and their corresponding precursors. MicroRNAs play critical roles in the maintenance of normal cell homeostasis through regulation of post-transcriptional gene expression, including of genes essential for mitosis. In this hypothesis-driven proposal, we will use novel morphological, biochemical, functional and genetic approaches to determine the mechanisms by which dyskerin and its cognate RNA critically regulate oral epithelial homeostasis. In particular, we will (1) determine the role and mechanism of dyskerin localization during mitosis, (2) determine the role of dyskerin in post-transcriptional gene expression and (3) determine the in vivo effects of dyskerin mutation on oral epithelium using a mouse model of X-linked DC. By elucidating novel functions for dyskerin, identifying the roles of its associated RNA, and by complementing in vitro experiments with in vivo investigations, these studies will lead to a wealth of new knowledge that may result in novel therapeutic strategies for oral leukoplakia and X-linked DC. PUBLIC HEALTH RELEVANCE: The mechanisms that regulate oral epithelial homeostasis remain incompletely understood. The studies outlined in this proposal will begin to define a novel role for dyskerin and its associated RNA in this important process. Our investigations will lead to new insight into the cellular functions of dyskerin and its bound RNA and may lead to new and novel avenues of study into the mechanisms that underlie the development of oral leukoplakia, and the pathogenesis of X-linked dyskeratosis congenita. This may eventuate in the development of new therapeutic strategies, thereby potentially increasing the quality of life and the long-term survival rate of affected individuals.