Role of dyskerin in oral epithelial homeostasis

描述(申请人提供)：应聘者致力于口腔健康相关研究，以补充他在口腔和颌面部病理学方面的培训。他正在申请K08临床科学家导师发展奖，以促进有保障的时间进行他的研究活动，以及翻译研究方法论的教学培训。在颁奖期间，他将获得正式指导，获得新技能，学习尖端方法，并直接受益于世界级调查人员的指导和专业知识。在Anil Rustgi博士和Sarah Millar博士的杰出指导下，以及多元化咨询委员会的经验和见解下，职业发展奖将确保阿拉维博士能够继续他的专业发展，并实现他成为一名独立调查员的目标。口腔鳞状上皮细胞经历明确的分化程序。为此，包括X连锁先天性角化不良(DC)在内的几种遗传性疾病会影响口腔粘膜组织。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，这些癌前病变在儿童和青春期的出现表明，正常的dyskerin功能的丧失扰乱了口腔上皮细胞的动态平衡。然而，其机制仍有待阐明。Dyskerin是核糖核蛋白生物发生所必需的，核糖核蛋白含有以H/ACA二级结构为特征的非编码小RNA分子。正是在这种作用下，dyskerin对端粒酶活性和前体rRNA的加工做出了贡献。然而，虽然这两个细胞过程在有丝分裂过程中都受到抑制，但我们发现在有丝分裂过程中Dyskerin的表达达到峰值，在有丝分裂的口腔角质形成细胞中，该蛋白定位于不同的亚细胞结构，并且Dyskerin功能的急剧丧失触发G2/M停滞，并导致具有多极纺锤体的非典型有丝分裂的积累。我们最近还证明了dyskerin的缺失会降低H/ACA小核仁RNA衍生的microRNAs(MiRNA)及其相应前体的水平。MicroRNAs通过调节转录后基因的表达，包括有丝分裂所必需的基因，在维持正常的细胞动态平衡方面发挥关键作用。在这个假设驱动的提案中，我们将使用新的形态、生化、功能和遗传学方法来确定dyskerin及其同源RNA关键调控口腔上皮内稳态的机制。特别是，我们将(1)确定有丝分裂过程中dyskerin定位的作用和机制，(2)确定dyskerin在转录后基因表达中的作用，(3)利用X连锁DC小鼠模型确定dyskerin突变对口腔上皮细胞的体内影响。通过阐明dyskerin的新功能，确定其相关RNA的作用，以及通过补充体外实验和体内研究，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.