Lariat capping as a tool to manipulate the 5' end of individual yeast mRNA species in vivo.

The 5' cap structure of eukaryotic mRNA is critical for its processing, transport, translation, and stability. The many functions of the cap and the fact that most, if not all, mRNA carries the same type of cap makes it difficult to analyze cap function in vivo at individual steps of gene expression. We have used the lariat capping ribozyme (LCrz) from the myxomycete to replace the mRNA mG cap of a single reporter mRNA species with a tiny lariat in which the first and the third nucleotide are joined by a 2', 5' phosphodiester bond. We show that the ribozyme functions in vivo in the budding yeast presumably without cofactors and that lariat capping occurs cotranscriptionally. The lariat-capped reporter mRNA is efficiently exported to the cytoplasm where it is found to be oligoadenylated and evenly distributed. Both the oligoadenylated form and a lariat-capped mRNA with a templated poly(A) tail translates poorly, underlining the critical importance of the mG cap in translation. Finally, the lariat-capped RNA exhibits a threefold longer half-life compared to its mG-capped counterpart, consistent with a key role for the mG cap in mRNA turnover. Our study emphasizes important activities of the mG cap and suggests new utilities of lariat capping as a molecular tool in vivo.