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. 2023 Dec 11;14:8212. doi: 10.1038/s41467-023-43633-0

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — A Heatmap of a 3-nucleotide mismatch running from 5’ to 3’ throughout the double-stranded RNA. Rows represent structurally disrupted constructs at specific positions, columns represent adenosine positions, and Δ editing is color-coded after Z-score transformation (mNG series). Vertical black lines mark the 3 nt mismatch location, and dashed lines highlight ADAR2-mediated editing increase upstream. B ADAR1- and ADAR2-mediated editing offsets based on the subset of 3-nucleotide mismatch running throughout the mNG and B2 sequences. Mismatches differentially located in each construct get centered at 0 on the x-axis. The Δ editing level on the y-axis represents the change of the editing level of an adenosine, normalized to the perfect double-stranded construct. Fitted curves depict the LOESS fit (blue-colored) of Δ editing with a span of 0.05 and the gray-shaded region spans the 25th percentile and 75th percentile values of Δ editing per distance. Only adenosine positions, which have greater than 1% in editing on the perfect double-stranded construct, were considered. Vertical dashed lines are placed at −26 and −35. C Subset of TTCTTCT bulges running throughout the mNG and B2 sequences. Data is shown as the LOESS fit curve (blue-colored) of Δ editing with a span of 0.11 and the gray-shaded region spans the 25th percentile and 75th percentile. D The mismatch size affects ADAR1- and ADAR2-mediated editing on adenosines located at −35 and −26 downstream from the mismatch, respectively. Data is visualized via box-and-whisker plots, with the central line denoting the median, box edges representing the interquartile range (from the 25th to the 75th percentile), and whiskers indicating the 1.5 times interquartile range. E Library preparation: RNA was extracted, the B2 variable lower arm and barcode were reverse transcribed, and subsequently PCR amplification and sequencing using Novaseq 6000 platform with a 300 bp kit were performed. F Depiction of the subset of 3-nucleotide mismatch running throughout the stem (B2) in ADAR1-knockout HEK293T cells overexpressing ADAR2. Constant and variable arms are illustrated under each other, and nucleotide locations are aligned. Data is shown as Fig. 2B.