https://orcid.org/0000-0003-3886-2311
Figures & data
Figure 1. Flowchart of endogenous tiRNA purification in this study.
Figure 2. Efficiency and specificity of oligo probe-mediated pulldown. Small RNA fraction from total RNA was used for pulldown. (A–B) Pulldown of 5ʹ-tiRNA-Gly-GCC (5ʹ-Gly) using purified small RNA fraction. (A) SYBR Gold staining and (B) Northern blotting for 5ʹ-tiRNA-Gly-GCC and 5ʹ-tiRNA-iMet-CAT. (C–D) Pulldown of 5ʹ-tiRNA-Ala-AGC (5ʹ-Ala) using purified small RNA fraction. (C) SYBR Gold staining and (D) Northern blotting for 5ʹ-tiRNA-Ala-AGC and 5ʹ-tiRNA-iMet-CAT. Sup: supernatant fraction, PD: pulldown fraction.
Figure 3. Endogenous tiRNA pulldown using gel-purified tiRNA fraction. (A) tiRNA fraction was gel-excised and purified from total RNA derived from angiogenin (ANG)-treated U2OS cells. (B–C) Pulldown of 5ʹ-tiRNA-Gly-GCC (5ʹ-Gly). (B) SYBR Gold staining and (C) Northern blotting for 5ʹ-tiRNA-Gly-GCC and 5ʹ-tiRNA-iMet-CAT. Red arrowhead indicates purified 5ʹ-Gly. (D–E) Pulldown of 5ʹ-tiRNA-Ala-AGC (5ʹ-Ala). (D) SYBR Gold staining and (E) Northern blotting for 5ʹ-tiRNA-Ala-AGC and 5ʹ-tiRNA-iMet-CAT. Red arrowheads indicate purified 5ʹ-Ala. (F–H) Purified endogenous tiRNA has post-transcriptional modification. Endogeno3 USDʹ-tiRNA-Gly-GCC (3ʹ-Gly) was pulled down and compared with synthetic 3ʹ-Gly (Synth. 3ʹ-Gly). (F) SYBR Gold staining, (G) Northern blotting for 3ʹ-tiRNA-Gly-GCC and (H) Immuno-Northern Blotting (INB) for 1-methyladenosine (m1A) modification. Red arrowhead indicates the band for 3ʹ-Gly. The position of m1A in 3ʹ-Gly is indicated as green circle. Sup: supernatant fraction, PD: pulldown fraction, Synth: synthetic.
Figure 4. Endogenous mature tRNAs can be also purified. Mature tRNA-Ala-AGC (tRNA-Ala) and tRNA-Gly-GCC (tRNA-Gly) were pulled down using gel-purified mature tRNA fraction. (A) SYBR Gold staining and (B) Northern blotting for tRNA-Ala-AGC, tRNA-Gly-GCC, tRNA-His-GTG and tRNA-iMet-CAT. (C) INB for 7-methylguanosine (m7G) and pseudouridine modifications. In pulldown (PD) fraction, bands indicated by red arrowheads show the purified specific tRNAs. m7G and pseudouridine modifications are indicated as red circle and blue circle, respectively. Sup: supernatant fraction, PD: pulldown fraction.
Figure 5. Structural and functional analysis using endogenous tiRNAs. (A, B) Endogenous 5ʹ-tiRNA-Ala (5ʹ-Ala) can form G-quadruplex (G4). (A) Synthetic and endogenous 5ʹ-Ala were equilibrated in 100 mM KCl or LiCl salts. RNAs were analysed on native gels and post-stained with SYBR Gold. (B) After SYBR Gold staining, RNAs were subjected to Northern blotting for tRNA-Ala. (C) Endogenous 5ʹ-tiRNA-Gly (5ʹ-Gly) inhibits translation of mRNA reporters in vitro. Means and standard deviation were obtained from three independent experiments. *p < 0.05 vs control RNA (Ctrl RNA) and **p < 0.01 vs Ctrl RNA.