期刊
RNA BIOLOGY
卷 18, 期 12, 页码 2546-2555出版社
TAYLOR & FRANCIS INC
DOI: 10.1080/15476286.2021.1930758
关键词
tRNA; tiRNAs; tRNA-derived fragments; stress
资金
- National Institutes of Health [R35 GM126901, RO1 GM126150]
- Japan Society for the Promotion of Science [26860094]
- Grants-in-Aid for Scientific Research [26860094] Funding Source: KAKEN
This study introduces a simple and reproducible method to isolate ANG-cleaved tiRNAs from endogenous tRNAs, significantly increasing the yield of isolated tiRNAs and facilitating the analysis of endogenous tiRNAs to elucidate the physiological functions of ANG.
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 5MODIFIER LETTER PRIME-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.
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