3' end formation of human type I and II collagen mRNAs

描述(申请人提供)：我们对真核细胞的mRNA3‘末端是如何形成的一直很感兴趣。基因表达的调节可以通过调节多聚腺苷酸化来完成。我们结合了对聚腺苷酸化的兴趣和对人类I型和II型胶原的兴趣。胶原在正常的软骨发育和骨骼形态形成中起着关键作用。我们最近发现并发表了人类COL1A2多聚腺苷化受COL1A2基因3‘端非翻译区(3’UTR)顺式作用元件的调控，这些元件影响COL1A2的3‘端加工效率。我们现在发现的人类COL1A1和COL2A1也含有这些顺式作用元件。基于这些发现，本项目建议研究所有这些I型和II型胶原前体mRNAs的多聚腺苷酸化调节机制。这些研究将扩大我们的目标，更好地了解胶原蛋白的表达，以及突变和异常的基因表达调控如何引发疾病状态。我们这个项目的具体目标是：1)确定人胶原基因COL1A1、COL1A2和COL2A1中3‘端多聚腺苷化位点使用所涉及的所有顺式作用元件；2)确定什么反式作用因子(S)与这些顺式作用元件结合。首先，我们将通过缺失和定点突变来鉴定顺式作用元件，并检测它们在体外和体内影响多聚腺苷酸化效率的能力。其次，将探索和表征与这些顺式作用信号相结合的蛋白质因子。这些蛋白质因子的测定将为我们提供线索，了解顺式作用辅助元件的功能意义。了解这些人胶原基因3‘端形成调控的RNA信号和蛋白质因子将有助于我们理解胶原基因的表达调控，这将反过来导致对骨骼缺陷和其他胶原性疾病的新见解。

DESCRIPTION (provided by applicant): We have a long-standing interest in how eukaryotic mRNA 3' ends are formed. Regulation of gene expression can be accomplished by regulating polyadenylation. We have combined this interest in polyadenylation with an interest in human collagen types I and II. Collagen plays a key role in normal cartilage development and skeletal morphogenesis. We have recently discovered and published that human COL1A2 polyadenylation is regulated by cis-acting elements present in the 3' untranslated region (3' UTR) of the COL1A2 mRNA, and that these elements influence the 3' end processing efficiency. Human COL1A1 and COL2A1 we have now discovered also contain these cis-acting elements. Based upon these findings, this project proposes to investigate the mechanism of regulation of polyadenylation of all these type I and II collagen pre-mRNAs. These investigations will expand our goal of better understanding the expression of collagens, and how mutations and abnormal gene expression regulation can initiate a disease state. Our specific aims for this project are: 1) To identify all the cis-acting elements involved in 3' polyadenylation site usage in the human collagen genes COL1A1, COL1A2, and COL2A1 and 2) To determine what trans-acting factor(s) bind to these cis-acting elements. First, we will identify cis-acting elements by deletion and site-directed mutagenesis, and examine their ability to affect polyadenylation efficiency both in vitro and in vivo. Second, protein factors that bind to these cis-acting signals will be explored and characterized. Determination of these protein factors will provide us with clues as to the functional significance of cis-acting auxiliary elements. Gaining an understanding of the RNA signals and protein factors involved in regulation of 3' end formation of these human collagen genes will help us to comprehend collagen gene expression regulation, which will in turn lead to novel insights into skeletal defects and other collagen-based diseases.