Abstract 9407272 A central challenge driving research on pre-mRNA splicing is to understand how splice sites are recognized with consummate specificity. We have recently identified a novel interaction between the first and last nucleotides of a pre-mRNA intron that is involved in the recognition of the 3' splice site. This /G-G/ interaction (where /G represents the 5' splice site and G/ represents the 3' splice site) is essential for the second step of splicing. This project is designed to characterize the /G-G/ interaction and place it in the context of the other RNA-RNA and RNA-protein interactions in the spliceosome. The /G-G/ interaction takes place in the mature spliceosome, which contains more than 50 proteins and at least 3 snRNAs. One of these splicing factors could participate directly in the interaction or could function indirectly to assist, stabilize, or proofread the interaction. The snRNAs are excellent candidates for such roles. Of special interest is the U5 snRNA, which base pairs with exon sequences at both splice sites, placing it in a position to facilitate the interaction. Roles of snRNAs in the /G-G/ interaction will be identified by screening banks of mutant snRNAs to isolate mutant snRNAs that increase the efficiency of this interaction. Alternatively, a protein splicing factor could be involved. Candidates for such proteins include the products of the PRP genes (pre-mRNA processing), several of which are required exclusively for the second step of splicing. These PRP genes will be mutagenized in vitro and screened for the ability to affect the /G-G/ interaction. We will also obtain biochemical evidence for the interaction using the yeast in vitro splicing system. First, we will induce random RNA-RNA crosslinks in spliceosomes formed in extracts from prp mutant strains that are defective in step ll of splicing. These spliceosomes are "frozen" in the second step of splicing, and should allow observation of crosslinks between RNAs involved in th e interaction. To test for a direct interaction, synthetic pre-mRNAs will be synthesized that contain crosslinkable nucleotide analogs at the 5' and 3' splice sites. These RNAs will be incubated in prp mutant extracts and crosslinked. The sites of crosslinking will be mapped using primer extension and RNase T1 analysis We will also use UV crosslinking to determine the timing of the /G-G/ interaction relative to other interactions in the spliceosome. Ordering the /G-G/ interaction relative to other splicing events will tell us which other splicing factors are present when the interaction occurs, allowing us to generate more detailed models for the second step of splicing. %%% Intervening sequences, also called introns, are regions of DNA that do not belong to the gene, and which must be removed in order for the gene to function properly. Introns are removed by the process of RNA splicing, which takes place after the DNA has been copied into messenger RNA. During this process, the messenger RNA is cut, the intervening sequences removed, and the RNA rejoined to produce a functional copy of the gene. Introns must be recognized with consummate specificity so that only sequences of the intron, and not of the real gene, are removed. A major unsolved challenge is to determine the means by which the beginning and end of the intron are identified. More than 50 proteins and 5 RNA molecules are required for splicing, but the precise functions of these factors in intron recognition remain unknown. These studies seek to understand the mechanism by which the ends of the introns are recognized. We have previously demonstrated that RNA sequences at the end of the intron interact with the RNA sequences at the beginning of the intron, and that this intron-intron interaction is essential for RNA splicing. In this work, we will demonstrate the exact chemical means by which this interaction takes place. We will use ultraviolet light to form chemical crosslinks between the different RNA sequences that recognize the end of the intron. This information will allow us to build precise models of the intron-intron interaction. In a complementary strategy, we will use genetic screens to identify additional RNA and protein molecules that affect this interaction. Such molecules could participate directly in the intron-intron interaction, or could function indirectly to assist, stabilize, or proofread the interaction. Identification of these molecules is essential for a complete understanding of intron recognition. Our long term goal is to understand how RNA sequences interact with other RNAs and with proteins to carry out the splicing reaction with high specificity. The studies in this grant represent an important step towards that goal by defining the means by which the ends of introns are recognized. ***
