Fig. 1: tRNATyr is queuosinylated at precursor level in mESCs and tissues.
From: Queuosine is incorporated into precursor tRNA before splicing
a G34 of tRNATyr is modified with queuosine (Q) and galactosyl-queuosine (galQ) by the sequential action of the QTRT1/2 complex (TGT) and QTGAL enzyme. Red rectangle indicates the cis-diol group reacting in APB Northern blot. b Schematic representation of the probes used in the Northern blots mapped onto the standard cloverleaf structure of tRNAs. The empty circles represent G34, the red triangles indicate the splicing sites. Pre-tRNA probes are designed complementary to the intron and detect only precursor tRNAs containing introns. tRNA probes are designed complementary to the 5’end of specific tRNAs and can detect both pre- and mature tRNAs. c GalQ on mature tRNATyr cannot be resolved in APB Northern blot, which shows only residual Q modification in mESCs. d Expression of pre-tRNATyr is quantified by Northern blot (Supplementary Fig. 1b) in mESCs and in the mouse brain. e APB Northern blot showing queuosinylation of tRNATyr precursors in wild type mESCs with a pool of probes targeting pre-tRNATyr 1-5, 2-1, 3-2. Qtrt1(Q1) and Qtrt2 (Q2) knockout cell lines are shown as negative control. f Specific probes against mouse pre-tRNATyr 1-4 (left panel) and pre-tRNATyr 2-1 (middle panel) show that these specific precursors are modified already at precursor level in wild type mESC. g Specific probes against mouse tRNATyr precursors show that pre-tRNATyr 1-4 is modified with Q already before the splicing in mouse brain; galQ on mature tRNATyr cannot be resolved in APB northern blot (left panel). Red arrows indicate Q, black arrows indicate unmodified or unresolved galQ, wt: wild type, Q1: Qtrt1−/−, Q2: Qtrt2−/−.
