title: A general method for rapid and cost-efficient large-scale production of 5' capped RNA.
abstract: The eukaryotic mRNA 5' cap structure is indispensible for pre-mRNA processing, mRNA export, translation initiation, and mRNA stability. Despite this importance, structural and biophysical studies that involve capped RNA are challenging and rare due to the lack of a general method to prepare mRNA in sufficient quantities. Here, we show that the vaccinia capping enzyme can be used to produce capped RNA in the amounts that are required for large-scale structural studies. We have therefore designed an efficient expression and purification protocol for the vaccinia capping enzyme. Using this approach, the reaction scale can be increased in a cost-efficient manner, where the yields of the capped RNA solely depend on the amount of available uncapped RNA target. Using a large number of RNA substrates, we show that the efficiency of the capping reaction is largely independent of the sequence, length, and secondary structure of the RNA, which makes our approach generally applicable. We demonstrate that the capped RNA can be directly used for quantitative biophysical studies, including fluorescence anisotropy and high-resolution NMR spectroscopy. In combination with (13)C-methyl-labeled S-adenosyl methionine, the methyl groups in the RNA can be labeled for methyl TROSY NMR spectroscopy. Finally, we show that our approach can produce both cap-0 and cap-1 RNA in high amounts. In summary, we here introduce a general and straightforward method that opens new means for structural and functional studies of proteins and enzymes in complex with capped RNA.
authors.affiliation: Max Planck Institute for Developmental Biology, 72076 Tübingen, Germany.
authors.forename: Anna-Lisa
authors.initials: AL
authors.lastname: Fuchs
authors.affiliation: Max Planck Institute for Developmental Biology, 72076 Tübingen, Germany.
authors.forename: Ancilla
authors.initials: A
authors.lastname: Neu
authors.affiliation: Max Planck Institute for Developmental Biology, 72076 Tübingen, Germany.
authors.forename: Remco
authors.initials: R
authors.lastname: Sprangers
mesh_terms: D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism; D006801:Humans; D008780:Methyltransferases / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D012315:RNA Caps / Q000096:biosynthesis* / Q000737:chemistry; D012323:RNA Processing, Post-Transcriptional; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism
chemical_list: C527678:Dcp1 protein, S pombe; C508496:Dcp2 protein, S pombe; D039561:Eukaryotic Initiation Factor-4E; D009097:Multienzyme Complexes; D012315:RNA Caps; D029702:Schizosaccharomyces pombe Proteins; D014764:Viral Proteins; D008780:Methyltransferases; D009713:Nucleotidyltransferases; D010744:Phosphoric Monoester Hydrolases; C027731:capping enzyme, vaccinia virus
keywords: 5′ cap; 7-methylguanylate cap structure; mRNA capping; mRNA decapping; vaccinia virus capping enzyme