A cell-free yellow lupin extract containing activities of pseudouridine 35 and 55 synthases

Plant cytoplasmic tyrosine tRNA was pseudouridylated at three different positions: 35, 39 and 55. These pseudouridines were introduced by three different enzymes - pseudouridine synthases. Variants of the Arabidopsis thaliana pre-tRNATyr were constructed that allow to monitor specifically pseudouridylation at different nucleotide positions. Using such RNAs to assay pseudouridine synthesis we have prepared an extract from Lupinus luteus cv. Ventus seeds containing activities of at least Ψ35 and Ψ55 synthases. This is the first report describing the preparation of the lupin seed extract that specifically modifies plant pre-tRNATyr transcribed by T7 RNA polymerase. U35 is converted to Ψ35 only in an intron-dependent manner, while pseudouridylation of U55 is insensitive to the presence or absence of an intron.