CONDITIONAL INACTIVATION OF MESSENGER-RNA CAPPING ENZYME AFFECTS YEAST PRE-MESSENGER-RNA SPLICING IN-VIVO

Acquisition of the 5' cap is the earliest modification event during eu karyotic mRNA synthesis, The cap is thought to facilitate later proces sing steps, such as pre-mRNA splicing, If this is so, then a defect in cap synthesis should impact on splicing in vivo, We tested this hypot hesis by examining the consequences of conditional inactivation of the Saccharomyces cerevisiae CEG1 gene, which encodes mRNA guanylyltransf erase (capping enzyme), Two different ceg1-ts mutants, Y66A and C354Y, displayed a pre-mRNA processing (prp) defect, characterized by an inc rease in the amount of unspliced pre-mRNA after shift to nonpermissive temperature and a decrease in the amount of mature mRNA, The guanylyl transferase activities of the Y66A and C354Y proteins were thermolabil e, suggesting that defective capping in vivo was contributory to the p rp phenotype. Although these results provide the first genetic link be tween capping and splicing in vivo, we were unable to demonstrate a ro le for either the cap or the capping enzyme during yeast pre-mRNA spli cing in vitro.