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

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 - 2017/5

Y1 - 2017/5

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

SN - 1355-8382

VL - 23

SP - 683

EP - 695

JO - R N A

JF - R N A

IS - 5

ER -