Immunoprecipitation of spliceosomal RNAs by antisera to galectin-1 and galectin-3

We have shown that galectin-1 and galectin-3 are functionally redundant splicing factors. Now we provide evidence that both galectins are directly associated with spliceosomes by analyzing RNAs and proteins of complexes immunoprecipitated by galectin-specific antisera. Both galectin antisera co-precipitated splicing substrate, splicing intermediates and products in active spliceosomes. Protein factors co-precipitated by the galectin antisera included the Sm core polypeptides of snRNPs, hnRNP C1/C2 and Slu7. Early spliceosomal complexes were also immunoprecipitated by these antisera. When splicing reactions were sequentially immunoprecipitated with galectin antisera, we found that galectin-1 containing spliceosomes did not contain galectin-3 and vice versa, providing an explanation for the functional redundancy of nuclear galectins in splicing. The association of galectins with spliceosomes was (i) not due to a direct interaction of galectins with the splicing substrate and (ii) easily disrupted by ionic conditions that had only a minimal effect on snRNP association. Finally, addition of excess amino terminal domain of galectin-3 inhibited incorporation of galectin-1 into splicing complexes, explaining the dominant-negative effect of the amino domain on splicing activity. We conclude that galectins are directly associated with splicing complexes throughout the splicing pathway in a mutually exclusive manner and they bind a common splicing partner through weak protein–protein interactions

Comparative and functional analysis of alternative splicing in eukaryotic genomes

Alternative splicing (AS) is a common post-transcriptional process in eukaryotic organisms, by which multiple distinct functional transcripts are produced from a single gene. Because of its potential role in expanding transcript diversity, interest in alternative splicing has been increasing over the last decade, ever since the release of the human genome draft showed it contained little more than the number of genes of a worm. Although recent studies have shown that 94% human multi-exon genes undergo AS while aberrant AS may cause disease or cancer, evolution of AS in eukaryotic genomes remains largely unexplored mainly due to the lack of comparable AS estimates. In this thesis I built a Eukaryote Comprehensive & Comparable Alternative Splicing Events Database (ECCASED) based on the analyses of over 30 million Expressed Sequence Tag (ESTs) for 114 eukaryotic genomes, including protists (22), plants (20), fungi (23), metazoan (non-vertebrates, 29) and vertebrates (20). Using this database, I addressed two main questions: 1) How does alternative splicing relate to gene duplication (GD) as an alternative mechanism to increase transcript diversity? and 2) What is the contribution of alternative splicing to eukaryote transcript diversity? I found that the previous “interchangeable model” of AS and gene duplication is a by-product of an existing relation between gene expression breadth, AS and gene family size. I also show that alternative splicing has played a key role in the expansion of transcript diversity and that this expansion is the best predictor reported to date of organisms complexity assayed as number of cell types. In addition, by comparing alternative splicing patterns in cancer and normal transcript libraries I found that cancer derived transcript libraries have increased levels of “noisy splicing”.EThOS - Electronic Theses Online ServiceGBUnited Kingdo