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. 2022 Aug 24;50(16):9382–9396. doi: 10.1093/nar/gkac699

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© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 2. — Steady-state level, aminoacylation, and processing of tRNA^Leu(UUR) in mito-mice tRNA^Leu(UUR)2748. (A) Schematic illustration of the secondary structural of tRNA^Leu(UUR) with and without the A2748G mutation. Note that the A2748-to-G mutation in the acceptor stem will impair Watson–Crick base-pairing. (B) Northern blotting of mature tRNA levels in total RNA isolated from livers of 10-month-old mice. mt-tRNA^Leu(UUR) levels were normalized to mt-tRNA^Ile and nuclear DNA-encoded 5.8S rRNA. Quantification of tRNA^Leu(UUR) levels (C) showed no statistically significant difference between the three groups. Data are presented as the mean ± SD. n = 3 for each group. (D) Representative aminoacylated (upper band) and nonaminoacylated (lower band) tRNA^Leu(UUR) levels in total RNA from livers of 10-month-old mice were examined by northern blotting under acidic conditions. (E) Quantification of aminoacylated tRNA^Leu(UUR) levels based on Supplementary Figure S3A. Levels of aminoacylated tRNA^Leu(UUR) were normalized by the sum of aminoacylated and nonaminoacylated tRNA. Data are presented as the mean ± SD. n = 4 for each group. (F) Schematic diagram of the primary and secondary structure of precursor pre-mRNA that contains 16S rRNA, tRNA^Leu(UUR), and ND1. RNase P and RNase Z are responsible for cleaving 16S rRNA-tRNA^Leu(UUR), and tRNA^Leu(UUR)-ND1, respectively. (G) Representative northern blotting of precursor transcripts in total RNA obtained from the livers of 10-month-old mice. Hybridization of three different probes designed to recognize tRNA^Leu(UUR), ND1 and 16S rRNA revealed expression of the 2.6 kb precursor transcript RNA19, the precursor 16S rRNA-tRNA^Leu(UUR) (1.6 kb), and the precursor tRNA^Leu(UUR)-ND1 mRNA (1.0 kb). (H) Quantification of precursor transcript RNA19 levels detected with each probe based on Supplementary Figure S3B. Precursor RNA19 levels were normalized by the 18S rRNA transcripts. Data are presented as the mean ± SD; *P < 0.05, **P < 0.01 by Tukey–Kramer test. n = 4 for each group. (I) Quantification of precursor transcript levels of tRNA^Leu(UUR)-ND1 (blue arrowheads in Supplementary Figure S3B) and 16S rRNA-tRNA^Leu(UUR) (red arrowheads in Supplementary Figure S3B) detected with tRNA^Leu(UUR) probe based on the low exposure time membrane in Supplementary Figure S3B. Each precursor transcript levels were normalized by the 18S rRNA transcripts. *P < 0.05, **P < 0.01 by Tukey–Kramer test. Data are presented as the mean ± SD. n = 4 for each group.