Bridging sulfur substitutions in the analysis of pre-mRNA splicing

Accurate excision of intervening sequences (introns) from messenger RNA precursors is accomplished by a very large and complicated ribonucleoprotein complex called the spliceosome. Elucidating the mechanisms of the two phosphotransesterification reactions that result in intron removal is important for our understanding of the molecular evolution of early genetic systems, as well as our knowledge of contemporary eukaryotic gene expression. The functional consequences of systematic alterations in the reactive groups can be invaluable for understanding catalytic mechanisms, especially for enzymes, such as the spliceosome, whose size and complexity place them beyond the reach of crystallographic and spectroscopic analysis. One type of modification that can be incorporated into a scissile phosphate linkage is the phosphorothiolate, in which a bridging phosphate oxygen is substituted with sulfur. Phosphorothiolate substitutions can be used to detect metal ion-ligand interactions by a "metal specificity switch" strategy. I review recent advances in the synthesis, incorporation, and manipulation of nucleoside phosphorothiolates (with an emphasis on 3'-S-phosphorothiolates), and describe their utility in the study of pre-mRNA splicing.