Fig 3.
tRNA[Ser]Sec is partially modified with i6A37. (A) Schematic depictions of the aminoacyl stems of the two tRNA[Ser]Sec tRNAs predicted from the human genome. (B to D) Targeted RNase H-mediated identification of cellular tRNA[Ser]Sec isodecoder 2. Oligonucleotide DNAs complementary to the 3′ end of the acceptor stem of either potential isodecoder 1 (Sec-1) or isodecoder 2 (Sec-2) were incubated with RNase H, as indicated above the lanes, and their presence and/or elimination was monitored by Northern blotting using either the Sec-1 aminoacyl acceptor stem (AAS) probe or the Sec-2 AAS probe, as indicated. Odd- and even-numbered lanes contain RNA from control (c) and TRIT1 siRNA (si)-treated cells, respectively, as indicated above the lanes. (D) As a control, hybridization with the tRNASerCGA body probe was carried out. (E and F) The same blot used in Fig. 2 was probed for tRNA[Ser]Sec using ACL and body probes. Bands were quantified from three independent experiments and applied to the following formula: percent modification = {1 − [(amount of ACL control/amount of BP control)/(amount of ACL TRIT1 siRNA/amount of BP TRIT1 siRNA)]} × 100, where BP is body probe. The results are reported in panel L. (G to K) In vitro modification of tRNA[Ser]Sec was measured using unlabeled DMAPP and recombinant His-TRIT1 and assessed by a two-part assay. First, control or TRIT1 siRNA-treated cellular RNA was subjected to in vitro modification, followed by the PHA6 assay. (G) Ethidium bromide-stained gel, including control RNA or TRIT1 siRNA with or without the addition of DMAPP to the TRIT1 reaction mixture, as indicated above the lanes. (H and I) Sec ACL and Sec body probings. (J and K) As a control, the blot was hybridized with tRNASerUGA ACL and body probes. The bands were quantified, and the results are presented in panel L. (L) Comparison of i6A37 levels from in vivo and in vitro assays. The absence of detection of hybridization with the ACL probe in the control samples was considered 100% i6A37 modification. The error bars were determined from four independent experiments for the in vivo assays and two independent experiments for the in vitro assays. The data for tRNA[Ser]Sec were derived from experiments whose results are shown in panels E and F and panels H and I; the data for tRNASerUGA were derived from experiments whose results are shown in Fig. 2D and E and panels J and K. (M) The ratios of the steady-state levels of tRNA[Ser]Sec and tRNASerCGA in control and TRIT1 siRNA-treated samples from three independent experiments, including the probings whose results are shown in panels B and D, were compared. The ratio obtained for the control samples was set to 100%, and the percent change due to TRIT1 depletion was compared to this.