Analysis of mutations in the yeast mRNA decapping enzyme

A major mechanism of mRNA decay in yeast is initiated by deadenylation, fol lowed by mRNA decapping, which exposes the transcript to 5' to 3' exonucleo lytic degradation. The decapping enzyme that removes the 5' cap structure i s encoded by the DCP1 gene. To understand the function of the decapping enz yme, we used alanine scanning mutagenesis to create 31 mutant versions of t he enzyme, and we examined the effects of the mutations both in vivo and in vitro. Two types of mutations that affected mRNA decapping in vivo were id entified, including a temperature-sensitive allele, First, two mutants prod uced decapping enzymes that were defective for decapping in vitro, suggesti ng that these mutated residues are required for enzymatic activity. In cont rast, several mutants that moderately affected mRNA decapping in vivo yield ed decapping enzymes that had at least the same specific activity as the wi ld-type enzyme in vitro. Combination of alleles within this group yielded d ecapping enzymes that showed a strong loss of function in vivo but that sti ll produced fully active enzymes in vitro. This suggested that interactions of the decapping enzyme with other factors may be required for efficient d ecapping in vivo, and that these particular mutations may be disrupting suc h interactions, Interestingly, partial loss of decapping activity in vivo l ed to a defect in normal deadenylation-dependent decapping, but it did not affect the rapid deadenylation-independent decapping triggered by early non sense codons. This observation suggested that these two types of mRNA decap ping differ in their requirements for the decapping enzyme.