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

这个应用的主题是一个不寻常的内含子，它从pre-mRNA中切除后积累。这个内含子IVS1 CB1来源于T细胞受体- b （TCR-B）基因。我们发表的研究表明，IVS1 CB1可能采用至少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) IVS1 CB1可能无效地脱支，从而阻止外切酶攻击；IVS1 CB1可能存在于细胞核中的保护性微环境中，例如剪接体本身。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，IVS1 CB1的存在抑制剪接反应后剪接体的拆卸。该项目的具体目的是：1)确定剪接的IVS1 CB1转录本在细胞核内的位置。这一目标很重要，因为它将为内含子如何逃脱降解提供线索。这也可能有助于揭示IVS1 CB1如何阻碍成熟TCR-B转录本的转运出核。原位杂交将用于定位IVS1 CB1在细胞核内。我们将在体内和体外实验中研究IVS1 CB1与剪接体成分的关系。2)确定IVS1 CB1分支是否具有调节功能。特别是，我们将研究IVS1CB1的存在是否会抑制全剪接转录本在细胞核中的转运。在本研究中，转染实验将在哺乳动物细胞系中进行。将评估核到细胞质转运和/或其他转录后事件的速率是否受到内含子的影响。将确定IVS1 CB1和/或邻近的外显子序列是否负责转录后调控。我们还将研究这个内含子是否能顺式地将这种调控赋予异源转录本。大多数高等真核生物的基因被称为内含子的非编码片段所中断。这些内含子通过RNA剪接过程以分支结构的形式从前体RNA中切除。内含子自上世纪70年代末首次被发现以来，一直是一个谜。内含子从前体rna上切除后的命运尚不清楚。哺乳动物内含子是否具有功能能力尚不清楚。我们打算通过研究一个不寻常的内含子来探索这些问题，该内含子在剪接我们的核转录本后保持稳定。这个内含子IVS1 CB1来源于编码T细胞受体（TCR）抗原的前体细胞转录本。我们将研究IVS1CB1是否具有控制T细胞中TCR蛋白表达的功能。另一个在RNA剪接领域尚未解决的问题是内含子从前体转录物剪接出来的精确机制。研究这一课题的一个障碍是缺乏研究体内剪接中间阶段的系统。我们的研究表明，IVS1CB1的存在在体内抑制剪接后剪接体的拆卸。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.