Fig. 4: m1A attenuates gene silencing by tRF-3s.
a m1A is located within the seed region of tRF-3b, which represents the guide RNA in RISC (RNA-induced silencing complex). m1A may interfere with base pairing between tRF-3b and the target RNA, thus alleviate target repression. b Scheme of the dual luciferase reporter assay to measure tRF-3 gene-silencing activity. tRF-3b complementary target sequence is inserted in the 3′ UTR of Renilla luciferase (Rluc) gene. Firefly luciferase (Fluc) signal is used for normalization. Synthetic tRF-3b with unmodified A4 or m1A-modified A4 is co-transfected with the reporter plasmid to measure relative effect of tRF-3b on the reporter activity. c–f Relative tRF-3 gene-silencing activity is measured by the dual luciferase reporter assay in HEK293T. Relative activity is calculated from Rluc signal divided by Fluc signal, and normalized to the empty site reporter; NT1 (non-targeting control1) is set as 1 in each replicate. g–j Relative tRF-3 gene-silencing activity is de-repressed after tRF-3 knock-down by LNAs (lock nucleic acids). Relative activity is calculated from Rluc signal divided by Fluc signal and normalized to the empty site reporter. Non-targeting LNA was used as control (Ctrl). All data are represented as mean ± SD; p value based on two-tailed paired student’s t test from three (four for d, e) independent experiments. Source data are provided as a Source Data file.
