Novel Method for Isolating Actively Translated mRNAs

描述（由申请人提供）：转录调控一直是过去基因调控的主要焦点。然而，最近的大量研究证据也表明，翻译调控在发育、细胞周期控制和急性耐药机制中起着关键作用。主动翻译mRNA转录本的基因表达分析为研究转录后调控提供了一种独特的方法。以前的研究依赖于传统的蔗糖梯度超离心方法分离多体复合物，并且需要大量的细胞（多达5亿个细胞）。因此，由于临床样本数量有限，这仍然是研究转录后调控的主要瓶颈。因此，迫切需要开发一种从少量细胞（10 ~ 500个细胞）中分离活性翻译多体的新方法。新的方法将允许我们系统地研究潜在的翻译调节有限的临床样本。研究表明，活性翻译mrna与多个核糖体单位相关，新合成的多肽与hsp73等分子伴侣密切相关。这些分子伴侣有助于新生多肽正确折叠成更高级的有序结构。这些伴侣将提供锚点，将与多聚体相关的主动翻译mrna与游离mrna分开。将开发亲和抗体捕获珠来捕获与多聚体复合物相关的hsp73伴侣，以便将所有多聚体与单体和游离mrna分离。分离的活性翻译mrna将用于高通量基因表达分析。拟建项目的具体目标是：1)开发抗体偶联亲和力捕获磁珠和条件，从少量细胞中捕获与多体复合物相关的主动翻译mrna。2)。通过定量RT-PCR基因表达分析，比较传统的多聚体分离方法，验证抗体亲和捕获方法对多聚体分离的有效性。3)。从人类结肠癌样本中确定在5-氟尿嘧啶（5- fu）治疗期间负责决定化学敏感性的潜在翻译调节基因。

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.