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. 2022 Jul 29;50(15):8674–8689. doi: 10.1093/nar/gkac626

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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 License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — polyR RAN initiation contributes to polyG translation. (A) Schematic of CGG₁₀₀-polyG-NL3xFLAG reporter, contains full 5’UTR of FMR1 (white) with CGG₁₀₀ repeat (gray) fused to nanoluciferase (yellow) and 3xFLAG (red) tags in polyG (+1) reading frame. polyR-frame ACG was mutated to AAA (red) to block polyR translation. (B) CGG₁₀₀-polyG-NL3xFLAG reporter nanoluciferase activity with native polyR-frame ACG or AAA mutation in HEK293T cells (ACG versus AAA: P< 0.0001, n = 12) (C) CGG₁₀₀-polyG-NL3xFLAG reporter 3xFLAG representative immunoblot with native polyR-frame ACG or AAA mutation in HEK293T cells, quantified at right as FLAG signal from all bands normalized to Tubulin (ACG versus AAA: P = 0.0094, n = 3). (D) CGG₁₀₀-polyG-NL3xFLAG reporter mRNA expression in HEK293T cells by RT-qPCR (P = 0.1637, n = 3). b,c,d. two-sided unpaired student's t-test. Graphs are mean ± stdev. **P< 0.01, ****P< 0.0001.