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.
Intellectual Merit. The spliceosome is a large, dynamic biological machine that removes introns from the pre-messenger RNAs (pre-mRNAs) and ligates the two pre-mRNA’s exons together to form the mature mRNA in the nucleus of eukaryotic cells. Although the spliceosome is composed of nearly 200 proteins and 5 small nuclear RNAs (snRNAs), only the U2 and U6 snRNAs within the spliceosome are thought to catalyze the splicing reactions. Recent in vitro studies by the Valadkhan and Cheng groups support this idea. A U2/U6 RNA complex can splice together two exons in vitro in the absence of protein factors. Furthermore, under altered salt and pH conditions in vitro, purified yeast spliceosomes can act in reverse to insert an intron into spliced exons. This "reverse splicing" is consistent with the reversibility of RNA-catalyzed splicing reactions. This grant’s hypothesis was that spliceosomal protein fragments (peptides) added to the spliceosome might alter critical protein-protein and RNA-protein interactions to reverse splicing under normal pH and salt conditions. The specific aim of this project was to test whether or not peptides from the spliceosomal protein, Prp5, could induce reverse splicing in vitro. Prp5 is one of eight yeast DEXD/H-box proteins essential for pre-mRNA splicing in yeast and most other eukaryotic cells. These DEXD/H-box proteins share a common helicase core of 300-400 amino acids. Each DEXD/H-box protein also has unique N- and C-terminal regions flanking the helicase core. In the case of Prp5, the N- and C-terminal regions are 275 and 250 amino acids, respectively. Prp5’s N-terminal region binds to the U1 and U2 snRNPs. Additionally, Prp5 is present in the spliceosome during most of the splicing cycle -- from the early stages of spliceosome assembly to the second splicing reaction when mRNA is formed. Therefore, peptides derived from Prp5’s N-terminal region could inhibit or reverse splicing when added to in vitro splicing reactions. In this grant period, two sequences from Prp5’s N-terminal region were expressed in bacterial cells as peptides. These peptides (H-pep14 and H-pep16) were then purified by biochemical methods and tested for their ability to inhibit or reverse splicing in vitro. The results show that both peptides inhibit splicing in vitro at high micromolar concentrations and partially inhibit splicing at lower concentrations. However, H-pep14 may be a more efficient inhibitor of the two as it acts at 10-fold lower concentrations than H-pep16. The relative levels of splicing intermediates and products in partially inhibited reactions suggest that splicing inhibition, reversal or both could be occurring. Additional assays will be needed to assess what stage in the forward splicing cycle is inhibited and if the peptides can reverse splicing. Broader impact. The outcomes of this project will contribute to our understanding of the fundamental principles by which the spliceosome functions. Because pre-mRNA splicing is a basic biological process governing expression of genes in nearly all eukaryotic cells, it impacts nearly all cells, tissues, organs, and organisms. Thus increasing knowledge of the mechanism of pre-mRNA splicing enhances our understanding of biology at many levels. This project also contributed to the training and education of several people. Two undergraduate students and a technician participated in this project. All three received training in new methods and learned new concepts. The purification scheme for the peptides that the students and technician helped to develop will be important to other researchers in several fields including biochemistry and biophysics. Finally, the scientific concepts that form the basis of this grant were taught to first-year graduate students in a formal, required course.
