The splicing of U12-type introns is a rate-limiting step in gene expression
This is an Open Access Thesis
Metazoan genes have recently been found to contain a novel class of introns that display non-canonical consensus sequences and are excised by a distinct splicing machinery. These introns occur very rarely, and have been called U12-type introns because recognition of their branch point sequences requires the U12 snRNP, which performs an analogous function to the U2 snRNP in splicing major-class introns. The persistence of two spliceosomes throughout virtually all of metazoan evolution suggests that the two spliceosomes play distinct and probably indispensable cellular roles. One enticing possibility is that the U12-type spliceosome functions in post-transcriptional regulation, serving as the rate-determining step in the splicing pathway of genes containing U12-type introns. To test this idea I have investigated the timing of U12-type intron removal relative to the removal of major-class introns from pre-mRNAs that contain both intron types. I have addressed this question using two different experimental approaches.One way to evaluate the order of intron removal from a transcript is to document the relative amounts of unspliced intron sequences within a steady-state population of partially-processed transcripts: if the splicing of U12-type introns is rate limiting and occurs last, then their sequences should be more abundant than those of their major-class intron neighbors. I have developed an accurate assay based on the technique of quantitative RT-PCR to measure the relative abundance of unspliced introns within several genes. Here, I present results from the analysis of three human genes showing that in all three cases splicing of the U12-type intron proceeds more slowly than splicing of the U2-type introns from the same transcript.The second experimental approach aimed to address the question of whether replacement of a naturally occurring U12-type intron with U2-type intron consensus sequences can affect the rate of production of mature mRNA and protein. Constructs were created which expressed either cyan or yellow fluorescent proteins only when completely spliced. The constructs contained either a U12-type or a U2-type intron in an arrangement which permitted correlation of color with type of splicing. By observing the relative intensities of the two fluorescent colors, it was possible to infer the relative efficiencies of the two splicing pathways within transfected Drosophila melanogaster tissue culture cells. Results of these experiments showed that replacement of a U12-type intron with canonical consensus sequences did indeed dramatically increase expression of the corresponding mRNA and protein.These results provide direct evidence that in vivo gene expression can be altered by the presence of a U12-type intron and implicate the U12-type spliceosome as a potential target in the post-transcriptional regulation of gene expression.