DIFFERENTIAL FUNCTION AND REGULATION OF RUNX2 ISOFORMS

描述（由申请人提供）：复合体Cbfa 1/Runx 2基因产生两种亚型Runx 2-II和Runx 2-I，分别源自远端“骨相关”（P1）和近端（P2）启动子。除了它们的5'非翻译区（5' UTR）和N-末端的差异之外，II型和I型Cbfa 1基因产物是相同的。这些异构体赋予发育途径差异功能的分子机制仍有待确定，包括骨发生、软骨发生和牙发生，但可能源自不同的N-末端功能结构域，以及可能由其5 'UTR调节翻译效率的能力赋予的差异表达。Runx 2-II II N-末端同种型的独特功能由其在骨发育期间的后期表达提出，但是先前的缺失两种同种型的转基因研究未被设计成评估它们的单独功能。此外，Runx 2基因产生具有复杂二级结构的5 'UTR的多个mRNA，其通过蛋白质合成的内部核糖体进入位点（IRES）起始在体外调节翻译。这些研究的总体目标是建立Runx 2-II和Runx 2-I同种型的不同功能，并确认IRES依赖的翻译控制在体内调节其表达和组织特异性功能中起重要作用。在目标1中，我们将通过完成Runx 2-II同种型选择性缺陷小鼠的表征来确定“骨相关”同种型的单独功能。此外，我们将创建选择性Runx 2-I无效小鼠，其保留Runx 2-II同种型的完整表达和功能。在目标2中，我们将研究I型和Il Cbfa 1的5 'UTR通过帽和IRES依赖性机制在体外和转基因小鼠中调节翻译效率的能力。这些研究将定义N-末端结构域的独特功能，并确定翻译调控是否有助于Runx 2-I和Runx 2-II同种型的组织特异性表达。

DESCRIPTION (provided by applicant): The complex Cbfa1/Runx2 gene gives rise to two isoforms, Runx2-II and Runx2-I, respectively derived from the distal "bone-related" (P1) and proximal (P2) promoters. Except for differences in their 5' untranslated regions (5'UTRs) and N-termini, the Type II and I Cbfa1 gene products are identical. The molecular mechanisms whereby these isoforms impart differential function to developmental pathways involving osteogenesis, chondrogenesis, and odontogenesis remain to be defined, but are likely derived from distinct N-terminal functional domains and possibly differential expression imparted by the ability of their 5'UTRs to modulate translational efficiency. Distinct function of the Runx2-II II N-terminal isoform is suggested by its later expression during bone development, but prior transgenic studies that deleted both isoforms were not designed to evaluate their separate function, in addition, the Runx2 gene generates multiple mRNAs with 5'UTRs having complex secondary structures that regulate translation in vitro through internal ribosome entry site (IRES) initiation of protein synthesis. The overall goals of these investigations are to establish the distinct function of Runx2-II and Runx2-I isoforms and to confirm that IRES-dependent translation control plays an important role in regulating its expression and tissue specific functions in vivo. In aim 1, we will determine the separate function of the "bone-related" isoform by completing the characterization of mice selectively deficient in the Runx2-II isoform. In addition, we will create a selective Runx2-I null mouse that leaves the expression and function of the Runx2-II isoform intact. In aim 2, we will investigate the ability of the 5'UTRs of Type I and Il Cbfa1 to regulate translation efficiency through cap-and IRES-dependent mechanisms in vitro and in transgenic mice. These studies will define the unique functions of the N-terminal domains, and determine if translational regulation contributes to the tissue-specific expression of Runx2-I and Runx2-II isoforms.