Alternate Spliced Repair Transcripts & Genome Stability

描述（由申请人提供）：本项目旨在鉴定来自肺和乳腺组织的干扰内源性DNA修复机制的DNA修复基因的交替剪接转录本。 已经从成年小鼠和人类组织中鉴定出许多DNA修复基因的交替剪接转录物。有趣的是，这些变异破坏了模型系统中的DNA修复，即使在野生型蛋白存在的情况下。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mRNA剪接筛选。将使用剪接特异性RT-PCR方案筛选来自临床样本的正常和肿瘤（腺癌）组织。使用该程序，可以表征来自这些组织的所有交替剪接的DNA修复转录物。通常，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，选择性剪接的转录本可以编码新的蛋白质，这些蛋白质可以改变细胞的DNA修复活性。为了鉴定破坏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修复活性在哺乳动物中是功能保守的。然后将在哺乳动物细胞培养物中测试主要破坏酵母中DNA修复的选择变体，以确保在酵母中观察到的效果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鉴定，这些剪接变体主要破坏DNA修复并可能导致肿瘤发生。 该筛选中鉴定的交替剪接转录本将为肿瘤发生提供候选生物标志物。根据本研究的数据，预计将对临床样本进行更大规模的筛选，以确定与特定肿瘤类型的直接相关性。这样的屏幕超出了本R21应用的范围。通过关注剪接，可以发现一类新的生物调节，它深刻地影响基因功能，但通常在其他临床筛选中被忽视。此外，本文概述的方法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.