Catalytic and folding properties of protein-free spliceosomalsnRNAs

Removal of introns from eukaryotic pre-mRNAs is performed by the spliceosome, a large molecular machine made up of more than 300 proteins and five small RNA molecules (snRNAs). Splicing occurs in two consecutive transesterification steps and resembles a RNA-catalyzed reaction performed by the group II self-splicing introns. Biochemical and complementation assays both in vitro and in vivo support a critical role for the snRNAs in catalysis of the splicing reactions. Two of the snRNAs, U2 and U6, are thought to be extensively involved in formation of the active site of the spliceosome. Parts of both U2 and U6 are very conserved, sensitive to mutations, and resemble catalytically-important domains of the self-splicing group II introns. I have investigated the possibility of RNA catalysis in the spliceosome by first demonstrating that these two RNA molecules can achieve their natural, base-paired conformation in vitro without the help of proteins, and further, using a UV-crosslinking assay, I showed the presence of a tertiary interaction in this base-paired complex of U2 and U6. This interaction resembles a functionally-important interaction that has been previously observed in vivo by genetic complementation assays. By juxtaposing functionally-important regions of U6 and U2, this tertiary interaction likely helps to assemble part of the active site of the splicing reaction. I also assayed the catalytic activity of this U2/U6 base-paired complex, using a short RNA oligo that resembles one of the two substrates of the first step of splicing (the branch site). I showed that the U2/U6 base-paired complex can recognize and bind this short oligo and catalyze a reaction between a bulged adenosine in this oligo and a conserved region in U6. This reaction resembles the first step of splicing in terms of its sequence and ionic requirements, and is possibly catalyzed in a similar active site. My data provide the first direct evidence for the catalytic competence of the snRNAs and specifically their ability to perform a reaction related to the first step of splicing.