RNA-protein crosslink mapping using TEV protease.

Characterization of novel RNA-protein interactions often demands physical mapping of the RNA binding sites in the protein. This can sometimes be accomplished using radioactively labeled RNA in covalent RNA-protein crosslinking experiments. The position of the radioactive label crosslinked to the protein can then be determined by fragmentation of the protein using a battery of sequence-specific proteolytic enzymes or chemical reagents. However, there are typically many cleavage sites in the natural protein sequence, and for large proteins, particularly when there are multiple sites of RNA-protein interaction, it may be difficult or impossible to determine the sites of crosslink formation unambiguously using this traditional physical mapping approach. We have developed an alternative method for physical mapping of RNA-protein crosslinks based on random insertion into the protein of a short peptide tag that includes the target sequence ENLYFQG (Glu-Asn-Leu-Tyr-Phe-Gln-Gly) for the highly specific TEV protease from tobacco etch virus. Covalent RNA-protein crosslinks can then be physically mapped by TEV protease digestion, fractionation of the proteolytic digestion products by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and visualization of the labeled protein fragments by phosphorimaging.

Dissection of Prp8 protein defines multiple interactions with crucial RNA sequences in the catalytic core of the spliceosome.

Current models of the core of the spliceosome include a network of RNA-RNA interactions involving the pre-mRNA and the U2, U5, and U6 snRNAs. The essential spliceosomal protein Prp8 interacts with U5 and U6 snRNAs and with specific pre-mRNA sequences that participate in catalysis. This close association with crucial RNA sequences, together with extensive genetic evidence, suggests that Prp8 could directly affect the function of the catalytic core, perhaps acting as a splicing cofactor. However, the sequence of Prp8 is almost entirely novel, and it offers few clues to the molecular basis of Prp8-RNA interactions. We have used an innovative transposon-based strategy to establish that catalytic core RNAs make multiple contacts in the central region of Prp8, underscoring the intimate relationship between this protein and the catalytic center of the spliceosome. Our analysis of RNA interactions identifies a discrete, highly conserved region of Prp8 as a prime candidate for the role of cofactor for the spliceosome's RNA core.