Alternate Spliced Repair Transcripts & Genome Stability

描述(申请人提供)：这个项目旨在从肺和乳腺组织的DNA修复基因中识别干扰内源性DNA修复机制的交替剪接的转录本。 已从成年鼠和人类组织中鉴定出一些DNA修复基因的交替剪接转录本。有趣的是，即使在野生型蛋白质存在的情况下，这些变异也会扰乱模型系统中的DNA修复。为了扩展这项研究，将对哺乳动物细胞中负责无错误DNA修复的基因进行信使核糖核酸剪接筛选。来自临床样本的正常组织和肿瘤(腺癌)组织将使用剪接特异性RT-PCR方案进行筛查。使用这一程序，所有来自这些组织的交替剪接的DNA修复转录本都可以被表征。通常情况下，DNA修复基因的突变对细胞是致命的。然而，令人兴奋的初步数据表明，替代剪接的转录本可以编码新的蛋白质，这改变了细胞的DNA修复活性。为了识别破坏DNA修复的剪接变体，将使用酵母模型系统进行快速功能筛选。酵母被用作真核生物的DNA修复范例，其DNA修复活动与哺乳动物的DNA修复活动在功能上是保守的。然后，将在哺乳动物细胞培养中测试主要扰乱酵母DNA修复的变种，以确保在酵母中看到的影响在人类细胞中保持不变。通过这种方式，DNA修复基因的剪接变体将被识别出来，这些基因主要扰乱DNA修复并可能导致肿瘤发生。 这个屏幕中发现的选择性剪接转录本将为肿瘤的发生提供候选的生物标记物。根据这项研究的数据，预计将进行更大规模的临床样本筛选，以建立与特定肿瘤类型的直接关联。这样的屏幕超出了本R21应用程序的范围。通过专注于剪接，一种新的生物调节可能被发现，它深刻地影响基因功能，但通常在其他临床筛查中被忽视。此外，这里概述的方法也适用于其他组织类型，预计这项研究将导致对其他人类肿瘤的进一步研究。

DESCRIPTION (provided by applicant): This project aims to identify alternately spliced transcripts from the DNA-repair genes from lung and breast tissue that perturb the endogenous DNA-repair mechanism. A number of alternately spliced transcripts of the DNA-repair genes have been identified from adult mouse and human tissue. Interestingly, these variants disrupt DNA repair in a model system, even in the presence of the wild-type protein. To extend this study, an mRNA splicing screen of the genes responsible for error free DNA repair in mammalian cells will be undertaken. Both normal and tumor (adenocarcinoma) tissue from clinical samples will be screened using a splice-specific RT-PCR protocol. Using this procedure all of the alternately spliced DNA-repair transcripts from these tissues can be characterized. Typically mutations in the DNA-repair genes are lethal to the cell. However, exciting preliminary data shows that alternatively spliced transcripts can encode novel proteins, which modify the DNA-repair activity of the cell. To identify splice variants that disrupt DNA repair, a rapid functional screen using a yeast model system will be performed. Yeast is used as the DNA-repair paradigm for eukaryotes and its DNA-repair activities are functionally conserved with mammals. Select variants that dominantly disrupt DNA-repair in yeast will then be tested in mammalian cell culture to ensure that the effects seen in yeast are conserved in human cells. In this way, splice variants of the DNA-repair genes that dominantly disrupt DNA repair and potentially lead to tumorigenesis will be identified. The alternately spliced transcripts identified in this screen will provide candidate biomarkers for tumorigenesis. Based upon the data from this study, it is anticipated that a larger screen of clinical samples will be conducted to establish a direct association with a specific tumor type. Such a screen is beyond the scope of this R21 application. By focusing upon splicing, a new class of biological regulation may be uncovered that profoundly affects gene function, but is normally overlooked in other clinical screens. In addition, the approach outlined here is applicable to other tissue types and it is anticipated that this research will lead to further study of other human tumors.