UC San Diego

Pre-messenger RNA (pre-mRNA) splicing is carried out by a dynamic ribonucleoprotein machine called the spliceosome. From budding yeast to mammalian cells, the majority of splicing occurs cotranscriptionally. Such spatial and temporal coupling suggest coordinated regulation. To begin to understand this coupled regulation we must first identify factors that play functional roles both in transcription and in splicing, and elucidate their mechanism of action. This motivated a directed genetic screen to identify canonical transcription factors that affect splicing in vitro. Here we describe the discovery that the essential Saccharomyces cerevisiae cyclin dependent kinase Bur1/2, which has previously been shown to regulate transcription elongation, is necessary for efficient pre-mRNA splicing in vivo. Remarkably, the complex also plays a transcription-independent role in splicing that can be observed in vitro. Using in vitro spliceosome assembly assays we demonstrate that integrity of the Bur complex is essential for early prespliceosome formation as well as catalytic spliceosome formation and activation. Our data indicates that the Bur complex associates with prespliceosomes and interacts with the pre -mRNA substrate as well as the U1 snRNA. We further show that the Bur1 kinase can phosphorylate known splicing factors both in vitro as well as in extracts. Furthermore we show that the kinase likely mediates numerous phosphorylation events leading to phosphorylated protein in prespliceosomes and spliceosomes. Finally with the use of a modular analog sensitive Bur1 kinase we uncover a role for Bur1 activation through phosphorylation in spliceosome formation. These results provide the first example in budding yeast of a cyclin dependent kinase affecting pre-mRNA splicing and spliceosomal formation through physical interactions with the spliceosome. This highlights the importance of post translational modifications, and the proteins that mediate them, in regulating critical spliceosome rearrangements. Furthermore, our data paint a picture of a model whereby a transcription factor plays a previously uncharacterized and important regulatory role in pre-mRNA splicing, and hint at an intricate coupling mechanism between multiple RNA processes