Novel Method for Isolating Actively Translated mRNAs

描述（由申请人提供）：转录调控是过去基因调控的主要焦点。然而，大量的证据表明，翻译调控在发育、细胞周期调控和急性耐药机制中起着关键作用。对主动翻译的mRNA转录物的基因表达分析提供了研究转录后调控的独特方法。以前的研究依赖于传统的蔗糖梯度超离心程序来分离多核糖体复合物，并且需要大量的细胞（高达5亿个细胞）。因此，这仍然是一个主要的瓶颈，研究转录后调控与有限数量的临床样本。因此，迫切需要开发一种新的方法来从少量细胞（10至500个细胞）中分离主动翻译的多核糖体。新方法将使我们能够系统地研究有限临床样本的潜在翻译调控。研究表明，主动翻译的mRNA与多个核糖体单位相关，新合成的多肽与分子伴侣如hsp 73密切相关。这些分子伴侣有助于新生多肽正确折叠成更高级的结构。这些分子伴侣将提供锚以将与多核糖体相关的主动翻译的mRNA与游离mRNA分离。将开发亲和抗体捕获珠以捕获与多核糖体复合物相关的hsp 73分子伴侣，使得所有多核糖体可以与单体和游离mRNA分离。分离的主动翻译的mRNA将用于高通量基因表达分析。拟议项目的具体目标是：1。开发抗体偶联的亲和捕获磁珠和条件，以从少量细胞中捕获与多核糖体复合物相关的主动翻译的mRNA。2.）通过定量RT-PCR基因表达分析，与传统的多核糖体分离方法进行比较，验证了抗体亲和捕获法用于多核糖体分离的可行性。3.）第三章从人结肠癌样本中确定5-氟尿嘧啶（5-FU）治疗期间负责确定化疗敏感性的潜在的免疫调节基因。

DESCRIPTION (provided by applicant): Transcriptional regulation has been the main focus for gene regulation in the past. However, a tremendous amount of evidence from recent studies also indicates that translational regulation plays a key role during development, cell cycle control, and mechanisms related to acute drug resistance. Gene expression analysis on actively translated mRNA transcripts provides a unique approach to study post transcriptional regulation. Previous studies have relied on a traditional sucrose gradient ultracentrifugation procedure to isolate polysome complexes and requires a large amount of cells (up to 500 million cells). As a result, this still remains a major bottleneck for the investigation of post transcriptional regulation with limited quantities of clinical samples. Therefore, there is an urgent need to develop a novel approach to isolate actively translated polysomes from a small number of cells (10 to 500 cells). The new approach will allow us to systematically study potential translational regulation with limited clinical samples. It has been shown that actively translated mRNAs are associated with multiple units of ribosomes and the newly synthesized polypeptides are closely associated with molecular chaperones such as hsp73. These molecular chaperones assist in the proper folding of nascent polypeptides into higher ordered structures. These chaperones will provide the anchor to separate actively translated mRNAs associated with polysomes from free mRNAs. Affinity antibody capture beads will be developed to capture hsp73 chaperones associated with the polysome complexes so that all polysomes can be separated from monosomes and free mRNAs. The isolated actively translated mRNAs will be used for high throughput gene expression analysis. The specific aims of the proposed project are: 1.) Develop antibody conjugated affinity capture magnetic beads and conditions to capture actively translated mRNAs associated with the polysome complex from a small number of cells. 2.) Validate the antibody affinity capture approach for polysome isolation by comparing with traditional polysome isolation protocols via quantitative RT-PCR gene expression analysis. 3.) Identify potential translationally regulated genes that are responsible for determining chemosensitivity during 5- fluorouracial (5-FU) treatment from human colon cancer samples.