In lysate RNA digestion provides insights into the angiogenin's specificity towards transfer RNAs

Abstract

Under adverse conditions, tRNAs are processed into fragments called tRNA-derived stress-induced RNAs (tiRNAs) by stress-responsive ribonucleases (RNases) such as angiogenin (ANG). Recent studies have reported several biological functions of synthetic tiRNAs lacking post-transcriptional modifications found on endogenous tiRNAs. Here we describe a simple and reproducible method to efficiently isolate ANG-cleaved tiRNAs from endogenous tRNAs. Using this in vitro method, more than 50% of mature tRNAs are cleaved into tiRNAs which can be enriched using complementary oligonucleotides. Using this method, the yield of isolated endogenous 5'-tiRNA^Gly-GCC was increased about fivefold compared to when tiRNAs were obtained by cellular treatment of ANG. Although the non-specific ribonuclease activity of ANG is much lower than that of RNase A, we show that ANG cleaves physiologically folded tRNAs as efficiently as bovine RNase A. These results suggest that ANG is highly specialized to cleave physiologically folded tRNAs. Our method will greatly facilitate the analysis of endogenous tiRNAs to elucidate the physiological functions of ANG.

Keywords: stress; tRNA; tRNA-derived fragments; tiRNAs.

Figure 1.

Flowchart of in lysate ANG digestion

Figure 2.

Dose-dependency and time course of in lysate digestion method. (a-b) Dose-dependency of in lysate ANG digestion. (a) SYBR Gold staining and (b) Northern blotting for 5ʹ-tiRNA^Gly-GCC. (c-d) Time/response of in lysate ANG digestion. (c) SYBR Gold staining and (d) Northern blotting for 5ʹ-tiRNA^Gly-GCC. (d–e) Dose-dependency of in lysate digestion with bovine RNase A (bRNase A). (d) SYBR Gold staining and (e) Northern blotting for 5ʹ-tiRNA^Gly-GCC and 28S rRNA are shown

Figure 3.

Comparison of efficiency and specificity among in vivo ANG treatment, in vitro ANG digestion and in lysate ANG digestion. (a) SYBR Gold staining. (b) Northern blotting for 5ʹ-tiRNA^Gly-GCC, 5ʹ-tiRNA^Ala-AGC, 7SK RNA and 28S rRNA

Figure 4.

Effect of RNA conformation on ANG-mediated cleavage of tRNA and 28S rRNA. (a-b) EDTA pre-treatment (20 mM) of the lysate abolishes ANG-mediated tRNA cleavage and slightly promotes 28S rRNA cleavage. (a) SYBR Gold staining and (b) Northern blotting for 5ʹ-tiRNA^Gly-GCC and 28S rRNA. (c-d) bRNase A-mediated digestion (10 nM) is not affected by EDTA pre-treatment. (c) SYBR Gold staining and (d) Northern blotting for 5ʹ-tiRNA^Gly-GCC and 28S rRNA. (e-f) Calcium chelation with EGTA (10 mM) does not inhibit ANG-mediated tRNA cleavage. (e) SYBR Gold staining and (f) Northern blotting for 5ʹ-tiRNA^Gly-GCC and 5ʹ-tiRNA^Ala-AGC

Figure 5.

Comparison of yield of 5ʹ-tiRNA^Gly-GCC pulldown between in vivo ANG treatment and in lysate ANG digestion. (a) SYBR Gold staining of purified RNA obtained by 5ʹ-tiRNA^Gly-GCC pulldown method. (b) Northern blotting of purified RNA against tRNA^Gly-GCC. Densitometry analysis of 5ʹ-tiRNA^Gly-GCC signals is also shown. Data are presented as the mean ± SD (n = 4)