Abstract
Most metazoan protein coding genes contain non-coding sequence (introns) separating the protein coding sequence (exons). Removal of introns and splicing of exons from a gene mRNA transcript is catalyzed by the spliceosome, a mega-dalton ribonucleoprotein assembly. Importantly, mRNA transcripts are often spliced in alternative patterns such that a single gene can produce more than one protein variant. Despite the vital role that splicing and alternative splicing have in metazoan development, the regulatory mechanisms controlling these events are poorly understood. This is due in part to the complexity of the spliceosome and the large number of proteins often involved in regulatory events. Further, the regulatory proteins are challenging to produce in a quantity and of a quality suitable for biochemical study as they often have extended regions of non-classical structure or disorder. Despite these difficulties, I have pursued structure-function studies of the Mer1 protein (Mer1p) to gain a mechanistic understanding of how a splicing regulatory protein can influence the activity of the spliceosome. Mer1p has a critical role in regulating splicing of four mRNA transcripts in budding yeast during meiosis. Mer1p is composed of two functional ‘domains’: an N-terminal ‘activation’ domain that has no recognized structural fold, which functions to nucleate formation of the spliceosome; a C-terminal K Homology Domain (KHD), which functions to recognize and bind a specific sequence on a pre-mRNA. To examine the importance of these domains, I have produced both full-length and truncated constructs of Mer1p for heterologous expression in bacteria. I have expressed these protein variants, purified them to homogeneity, and characterized them biochemically. Mer1p is monomeric in the absence of its RNA substrate. I established a filter-binding assay to examine the affinity of Mer1p variants to its RNA substrate – finding that they bind with a Kd of ~10 µM, an affinity dependent only on its C-terminal KHD. To provide further insight into Mer1p function, I have aided efforts to build a structural model of the 0.8 MDa budding yeast U1 snRNP and have generated a hypothetical model of Mer1p in a substrate bound state and its interaction with U1 snRNP.