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.
