PROTEOMIC ANALYSES OF PERLECAN MRNA-ASSOCIATED PROTEIN COMPLEXES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 Perlecan是基底膜上硫酸乙酰肝素蛋白多糖的主要成分，在动物体内广泛表达。它的表达在发育过程中受到动态调节，其异常表达与许多病理过程有关，如糖尿病、动脉粥样硬化、关节炎和癌症。然而，人们对Perlecan的表达如何进行适当的调控知之甚少。在这里，我们表明，通过亲和纯化，我们能够获得两个与perlecan mRNA特异相关的蛋白质复合体，可能参与其前mRNA的剪接。我们将用质谱仪检测纯化的蛋白质复合体，并分析它们在结构性剪接和选择性剪接中的功能意义。所提出的实验将为调控Perlecan Pre-mRNA的结构性和/或选择性剪接提供机制方面的见解，这将为长期研究阐明选择性剪接调控Perlecan表达的机制奠定基础。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Perlecan is a major component of heparan sulfate proteoglycans in basement membrane, which is widely expressed in animal bodies. Its expression is dynamically regulated during development and its aberrant expression is linked to many pathological processes, such as diabetes, atherosclerosis, arthritis and cancer. However, it has been poorly understood how the expression of perlecan is properly regulated. Here we show that by affinity purification we were able to obtain two protein complexes that are specifically associated with perlecan mRNA, likely involved in its pre-mRNA splicing. We will examine the purified protein complexes by mass spectrometry and analyze the proteins for their functional significances in both constitutive and alternative splicing. The proposed experiments will provide mechanistic insights into the regulation of constitutive and/or alternative splicing of perlecan pre-mRNA, which will establish a foundation for long-term studies to elucidate the mechanism of alternative splicing in regulating the expression of perlecan.