Pleiotropic effects of intron removal on base modification pattern of yeast tRNA(Phe): An in vitro study

Cell-free yeast extract has been successfully used to catalyze the enzymatic formation of II out of the 14 naturally occurring modified nucleotides in yeast tRNA(Phe) (anticodon GAA). They are m(2)G(10), D-17, m(2)(2)G(26), Cm-32, Gm(34), psi(39), m(5)C(40), m(7)G(46), m(5)C(49), T-54 and psi(55). Only D-16, Y-37 and m(1)A(58) were not formed under in vitro conditions, However, m(1)G(37) was quantitatively produced instead of Y-37. The naturally occurring intron was absolutely required for m(5)C(40) formation while it hindered completely the enzymatic formation of Cm-32, Gm(34) and m(1)G(37). Enzymatic formation of m(2)(2)G(26), psi(39), m(7)G(46), m(5)C(49), T-54 and psi(55) Were not or only slightly affected by the presence of the intron, These results allow us to classify the different tRNA modification enzymes into three groups: intron insensitive, intron dependent, and those requiring the absence of the intron. The fact that truncated tRNA(Phe) consisting of the anticodon stem and loop prolonged with the 19 nucleotide long intron is a substrate for tRNA: cytosine-40 methylase demonstrates that the enzyme is not only strictly intron dependent, but also does not require fully structured tRNA.