Human genes are split into regions that code for protein, exons, and regions that don't, introns. Upon transcription, the removal of these intervening introns is necessary if a usable mRNA molecule is to be translated. The process of intron removal and subsequent ligation of exons is called splicing and is carried out by a large complex called the spliceosome. This process is driven by sequence elements within the pre-mRNA itself and is the major contributor of diversity to the human transcriptome. Due to the ubiquitous nature of alternative splicing in almost every multi-exon gene, the regulation pathways of exon inclusion are a subject of wide study.
The different lengths of introns and exons as well as location of splice sites in a pre-mRNA molecule have been shown to have differing affects on the spliceosomes ability to recognize them. Using in vitro splicing and complex formation assays in parallel with cell transfection experiments, we determined that the distance between two splice sites across the intron or across the exon are strong predictors of splice site usage. Additionally, we found that two splice sites interact differently when placed at different lengths apart. Our findings suggest a mechanism for observed selection of specific intron/exon architectures.
Splice site recognition is also influenced by the presence of protein binding sequence elements in the pre-mRNA that alter spliceosomal recruitment. Previously, these proteins and sequence elements had been rigidly classified into splice enhancing or inhibiting categories. We show that this rigid classification is incorrect. We found that the location of these elements relative to the splice site determines their enhancing or silencing effect. That is, an enhancing element found upstream of a splice site imposes a silencing effect when relocated downstream of the splice site (and vice versa).
Spliceosomal proteins are conserved from yeast to humans. The sequence elements used in pre-mRNA sequences have been evolving over time but under pressure from multiple cellular processes, including splicing. To observe the effect of splicing on evolution, we took advantage of the synonymous mutation positions that are under the least amount of selective pressure from the genetic code. We mutated these positions and found that some caused a large decrease in exon inclusion. When we analyzed the comparative alignment data, we found that these specific nucleotide mutations were selected against across species in order to maintain exon inclusion. SNP analysis showed that this pattern of selection was broadly observable at synonymous positions throughout the human genome.
|Advisor:||Hertel, Klemens J.|
|Commitee:||Semler, Bert L., Shi, Yongsheng, Waterman, Marian L.|
|School:||University of California, Irvine|
|Department:||Biomedical Sciences - Ph.D.|
|School Location:||United States -- California|
|Source:||DAI-B 74/07(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Genetics, Bioinformatics|
|Keywords:||Alternative splicing, Mrna, Regulation, Sequence elements|
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