RNA caps: Formation and roles

Vaccinia virus D1-D12 is an RNA cap (guanine-N7) methyltransferase that generates a 7-methylguanosine cap (m7G) characteristic of eukaryotic and viral messenger RNAs. The active sites for all 3 enzymatic steps (triphosphatase, guanylyltransferase, methyltransferase) involved in m7G cap formation are located on D1. D12 is a stimulatory subunit for the methyltransferase component. Our collaborator Stephen Cusack has attained a 3A crystal structure of the complete D1-D12 heterodimer. This structure guided our mutational analysis of Dl-D12. The key findings derived from our functional analysis include the identification of residues at the triphosphatase-guanylyltransferase interface that are necessary for the guanylyltransferase reaction but not the triphosphatase reaction.;Yeast and human Tgs1 are orthologous RNA cap (guanine-N2) methyltransferases that convert m7G caps into the 2,2,7-trimethylguanosine (TMG) caps characteristic of spliceosomal snRNAs. TMG caps are dispensable for vegetative yeast growth, but are essential in the absence of Mud2, the putative yeast homolog of human splicing factor U2AF. We exploited the synthetic lethal interactions of tgs1Delta and mud2Delta mutations to identify essential structural features of the Tgs1 and Mud2 proteins. The structure-function data highlight a strictly essential pi-cation interaction between Trp766 and the m7G base and a network of important contacts to the cap triphosphate. We found that the Mud2 RRM domain is necessary but not sufficient for Mud2 function in the absence of Tgs1.;Saccharomyces cerevisiae Msl5(BBP) orchestrates spliceosome assembly by binding the ninon branchpoint sequence and nucleating cross intron-bridging interactions with other components of the splicing machinery. By gauging the ability of Msl5(BBP) to complement msl5Delta growth, we found that the Mud2-binding and Prp40-binding elements of the Msl5 N-terminal domain are inessential, as are the C-terminal proline-rich domain and two zinc-binding motifs. A subset of conserved branchpoint RNA-binding amino acids in the central KH-QUA2 domain was also dispensable. We used our collection of viable Msl5(BBP) mutants to interrogate synthetic genetic interactions, in cis between the inessential structural elements of the Msl5 polypeptide and in trans between Msl5 and yeast splicing factors Mud2, Nam8, and Tgs1. The results suggest a network of important but functionally buffered protein-protein and protein-RNA interactions in the Mud2-Msl5(BBP)-U1 snRNP-pre-mRNA complex.