A stable Intron from the T Cell Receptor Gene: Characterization and Functional Role

9307963威尔金森本应用的主题是一种不寻常的内含子，它在从前信使核糖核酸中切除后积累。该内含子IVS1 CB1来源于T细胞受体B(TCR-B)基因。我们发表的研究表明，IVS1CB1可能至少采用两种不同的机制来逃避其他已知哺乳动物内含子遵循的降解途径：i)IVS1CB1可能低效地去分支，从而防止核酸外切酶攻击；@)IVS1CB1可能存在于核内的保护性微环境中，如剪接体本身。我们的初步研究还表明，这个稳定的内含子可能具有一定的功能。间接证据表明，IVS1CB1的存在抑制了剪接反应后剪接体的拆解。该项目的具体目标是：1)确定IVS1CB1转录本在细胞核内的位置。这一目标很重要，因为它将为该内含子如何逃脱退化提供线索。它还可能有助于揭示IVS1 CB1如何阻止成熟的TCR-B转录本从细胞核运输出去。将使用原位杂交技术将IVS1CB1定位于细胞核内。我们将在体内和体外进行共反应实验，以探讨IVS1CB1与剪接体成分之间的关系。2)确定IVS1 CB1秘书处是否履行调节职能。特别是，将研究IVS1CB1的存在是否抑制完全剪接的转录本OUR的核运输。在这项研究中，将在哺乳动物细胞系中进行转基因实验。将评估该内含子是否影响核质运输和/或其他一些转录后事件的速率。将确定IVS1、CB1和/或邻近的外显子序列是否负责转录后调控。还将检查该内含子是否能将这一调控赋予顺式启动子中的异源转录本。大多数高等真核基因被称为内含子的非编码片段打断。这些内含子是通过RNA剪接过程从套索结构形式的前体RNA中删除的。自上世纪70年代末S首次发现内含子以来，内含子一直是一个谜。内含子从前体RNA中剔除后的命运尚不清楚。目前尚不清楚哺乳动物的内含子是否具有功能。我们打算通过研究一个不寻常的内含子来探索这些问题，该内含子在与我们的核转录本拼接后保持稳定。这个内含子IVS1 CB1来自于编码抗原的T细胞受体(TCR)的前体转录产物。我们将研究IVS1CB1是否具有控制T细胞中TCR蛋白表达的功能。在RNA剪接领域中另一个尚未解决的问题是内含子从前体转录本中剪接出来的精确机制。阻碍这一主题研究的一个障碍是缺乏系统来研究体内剪接的中间阶段。我们的研究表明，IVS1CB1的存在抑制了体内剪接后剪接的拆解。因此，IVS1CB1可能是研究完整细胞中RNA剪接中间阶段的有用工具。

9307963 Wilkinson The topic of this application is an unusual intron which accumulates following its excision from pre-mRNA. This intron, IVS1 CB1 is derived from the T cell receptor-B (TCR-B) gene. Our published investigations suggest that IVS1 CB1 may employ at least two different mechanisms to escape the degradation pathway that other known mammalian introns follow: i) IVS1 CB1 may be inefficiently debranched, thus preventing exonuclease attack; @) IVS1 CB1 may reside in a protective microenvironment in the nucleus, such as in the splicesome itself. Our preliminary studies also suggest that this stable intron may perform a function. Indirect evidence suggests that the presence of IVS1 CB1 inhibits the disassembly of the splicesome following the splicing reactions. The specific aims of this project are: 1) To determine the location of spliced IVS1 CB1 transcripts within the nucleus. This aim is important because it will provide clues as to how this intron escapes degradation. It may also help reveal how IVS1 CB1 can impede the transport of mature TCR-B transcripts out of the nucleus. In situ hybridization will be used to localize IVS1 CB1 within the nucleus. Cofraction experiments will be performed to investigate the relationship of IVS1 CB1 and splicesomal components in vivo and vitro. 2) To determine if IVS1 CB1 lariats perform a regulatory function. In particular, it will be investigated whether the presence of IVS1CB1 inhibits the transport of fully spliced transcripts our of the nucleus. For this study, transfection experiments will be performed in mammalian cell lines. It will be assessed if the rate of nuclear -to-cytoplasmic transport and/or some other post-transcriptional event is affected by this intron. It will be determined if IVS1 CB1 and/or adjacent exon sequences are responsible for the post-transcriptional regulation. It will also be examined if this intron can confer this regulation to heterologous transcripts in cis. %%% The maj ority of higher eukaryotic genes are interrupted by non- coding segments termed introns. These introns are excised from precursor RNAs in the form of lariat structures by the process of RNA splicing. Introns have been an enigma since their original discovery in the late 1970's. The fate of introns after their excision from precursor RNAs is not well understood. It is not clear if mammalian introns posses function capabilities. We intend to explore these issues by studying an unusual intron that remains stable after it is spliced our of nuclear transcripts. This intron, IVS1 CB1, is derived from precursor transcripts encoding the T cell receptor (TCR) for antigen. We will investigate whether IVS1CB1 functions to control the expression of TCR protein in T cells. Another issue that has not been resolved in the RNA splicing field is the precise mechanism by which introns are spliced out of precursor transcripts. An impediment to research on this topic is the lack of systems to study intermediate stages of the splicing in vivo. Our studies suggest that the presence of IVS1CB1 inhibits the disassembly of the splicesome following splicing in vivo. Thus, IVS1CB1 may be useful tool to study intermediate stages of RNA splicing in intact cells.