EAGER: Shifting the Spliceosome's Gears

University Of New Mexico Health Sciences Center, Albuquerque NM

Investigators

Abstract

Intellectual merit. Spliceosomal RNAs are thought to catalyze two transesterification reactions that excise the pre-mRNA's intron and ligate its two exons together during nuclear pre-mRNA splicing. Recent in vitro studies support this idea. A U6/U2 RNA complex can splice together two exons in vitro in the absence of other spliceosomal factors. Furthermore, affinity-purified yeast spliceosomes under altered conditions in vitro can act in reverse to insert an intron into spliced exons. This "reverse splicing" is consistent with the reversibility of RNA-catalyzed transesterification reactions. Reverse splicing in the affinity-purified spliceosome is achieved in vitro by altering the pH and the concentrations of mono- and divalent cations. These alterations are likely changing RNA-RNA, RNA-protein and protein-protein interactions within the spliceosome. Thus it may be possible to reverse splicing by adding protein fragments to an arrested spliceosome to alter critical interactions, and thus induce the spliceosome to reverse splice under normal pH and salt conditions. In this project, this possibility will be tested by in vitro splicing assays using affinity purified spliceosomes and various fragments of a spliceosomal protein. Although these experiments are reasonably simple to perform, there is a high risk that they will be successful. The risk is in both the hypothesis and selecting the right protein fragments to test. However, if successful, the experiments will fundamentally alter our view of the regulation of nuclear pre-mRNA splicing. Broader impact. The research will have several immediate outcomes important to science and to education and training. The results will increase our understanding of the factors that regulate splicing. The ability to drive the spliceosome either forward or backward will reveal the most fundamental interactions within the spliceosome that control its catalytic activity. The project will also provide a research opportunity for at least one undergraduate student.