Isolation and initial structure-functional characterization of endogenous tRNA-derived stress-induced RNAs

Abstract

Recent transcriptome-wide studies have identified a diverse pool of transfer RNA (tRNA)-derived RNAs or tRNA-derived fragments (tRFs). Some of these RNAs have been demonstrated to be functional and involved in multiple biological processes ranging from the regulation of gene expression to transgenerational epigenetic inheritance. Post-transcriptional maturation of tRNAs includes various processing events including extensive decoration by various RNA modifications, which are required for correct tRNA folding and stability. Moreover, tRNA modifications determine the pattern and specificity of tRNA cleavage. The major drawbacks of many studies in the field of tRFs are that most of them used synthetic RNAs that closely mimic endogenous tRFs in their sequence, yet lack RNA modification that is found in vivo. Here, we developed a simple method to isolate tRNA-derived stress-induced RNAs (tiRNAs), a specific subset of tRFs. Our approach is scalable, cost-effective and relies on the purification of individual tiRNAs based on a sequence-specific RNA/DNA isolation technique using DNA probes. Our method facilitates functional studies of tiRNAs by addressing how physiological RNA modifications within tRNA fragments affect their biological activities. Here, we report pilot functional studies on selected endogenous tiRNAs, namely tiRNA^Ala and tiRNA^Gly. We show that natural 5'-tiRNA^Ala molecules assemble into G-quadruplex structures, and endogenous 5'-tiRNA^Gly possesses translation inhibition activity.

Keywords: angiogenin; ribonuclease; tRNA; tRNA fragments; tRNA-derived stress-induced RNAs.

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.