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 myxomyceteDidymiumto replace the mRNA m7G 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 yeastSaccharomyces cerevisiaepresumably 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 m7G cap in translation. Finally, the lariat-capped RNA exhibits a threefold longer half-life compared to its m7G-capped counterpart, consistent with a key role for the m7G cap in mRNA turnover. Our study emphasizes important activities of the m7G cap and suggests new utilities of lariat capping as a molecular tool in vivo.
Department of Cellular and Molecular Medicine, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
Nicolai
N
Krogh
Department of Cellular and Molecular Medicine, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
Max
M
Pietschmann
Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark.
Manfred
M
Schmid
Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark.
Torben Heick
TH
Jensen
Department of Cellular and Molecular Medicine, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
Henrik
H
Nielsen
D002467:Cell Nucleus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D006207:Half-Life; D014176:Protein Biosynthesis; D012314:RNA Cap Analogs / Q000378:metabolism* / Q000502:physiology; D034443:RNA Transport; D016337:RNA, Catalytic / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
D012314:RNA Cap Analogs; D016337:RNA, Catalytic; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; C017516:7-methylguanosine triphosphate
lariat capping ribozyme; m7G; mRNA processing; mRNA turnover; polyadenylation