DIFFERENTIAL FUNCTION AND REGULATION OF RUNX2 ISOFORMS

描述(申请人提供)：复杂的Cbfa1/Runx2基因产生两种亚型，Runx2-II和Runx2-I，分别来自远端的“骨相关”(P1)和近端(P2)的启动子。除了5‘非翻译区(5’UTRs)和N末端不同外，II型和I型Cbfa1基因产物是相同的。这些异构体向涉及成骨、软骨形成和牙齿形成的发育途径赋予不同功能的分子机制仍未确定，但可能来自不同的N-末端功能结构域，以及它们的5‘UTRs调节翻译效率的能力所赋予的差异表达。Runx2-II II N-末端异构体在骨骼发育过程中的后期表达提示了其独特的功能，但先前缺失这两种异构体的转基因研究并不是为了评估它们的单独功能。此外，Runx2基因产生具有复杂二级结构的多个mRNAs，这些mRNAs通过内部核糖体进入位点(IRES)启动蛋白质合成来调节体外翻译。这些研究的总体目标是建立Runx2-II和Runx2-I亚型的不同功能，并证实IRES依赖的翻译控制在调节其在体内的表达和组织特异性功能中发挥重要作用。在目标1中，我们将通过完成选择性缺乏Runx2-II亚型的小鼠的特征来确定“骨相关”亚型的单独功能。此外，我们将创造一种选择性的Runx2-I空小鼠，使Runx2-II亚型的表达和功能保持不变。在目标2中，我们将研究I型和Il Cbfa1的5‘UTRs在体外和转基因小鼠中通过依赖帽和IRES的机制调节翻译效率的能力。这些研究将确定N-末端结构域的独特功能，并确定翻译调控是否有助于Runx2-I和Runx2-II亚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.