TY - JOUR
T1 - Lariat capping as a tool to manipulate the 5' end of individual yeast mRNA species in vivo
AU - Krogh, Nicolai
AU - Pietschmann, Max
AU - Schmid, Manfred
AU - Jensen, Torben Heick
AU - Nielsen, Henrik
N1 - © 2017 Krogh et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
PY - 2017
Y1 - 2017
N2 - 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 Didymium to replace the mRNA m(7)G 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 Saccharomyces cerevisiae 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 m(7)G cap in translation. Finally, the lariat-capped RNA exhibits a threefold longer half-life compared to its m(7)G-capped counterpart, consistent with a key role for the m(7)G cap in mRNA turnover. Our study emphasizes important activities of the m(7)G cap and suggests new utilities of lariat capping as a molecular tool in vivo.
AB - 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 Didymium to replace the mRNA m(7)G 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 Saccharomyces cerevisiae 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 m(7)G cap in translation. Finally, the lariat-capped RNA exhibits a threefold longer half-life compared to its m(7)G-capped counterpart, consistent with a key role for the m(7)G cap in mRNA turnover. Our study emphasizes important activities of the m(7)G cap and suggests new utilities of lariat capping as a molecular tool in vivo.
U2 - 10.1261/rna.059337.116
DO - 10.1261/rna.059337.116
M3 - Journal article
C2 - 28159804
VL - 23
SP - 683
EP - 695
JO - RNA
JF - RNA
SN - 1355-8382
IS - 5
ER -