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. Author manuscript; available in PMC: 2018 Jun 29.
Published in final edited form as: Cell. 2017 Jun 29;170(1):61–71.e11. doi: 10.1016/j.cell.2017.06.013

Figure 5. 18 nt 3′ tRFs do not interfere with primary transcript or protein levels but inhibit reverse transcription.

Figure 5

(A) MusD and ETn RNA transcript levels are not affected in cells which showed decreased MusD/ETn activity after transfection of ETn tRFs. Mean transcript levels ± SD were determined by quantitative Taqman RT-PCR. (B) MusD Gag protein levels are not affected by co-transfection of 18 nt, targeting tRFs. (C) Uncapped 5′-P RNA ends were sequenced by high-throughput, modified RACE. Each track represents one replicate. The position of all 5′ RNA ends including RT-RNaseH products (boxed red) and potential RNA cleavage products within ~40 bp surrounding the PBS and 5′ UTR are shown. The decrease of RNaseH products indicates that 18 nt targeting tRFs specifically inhibit accumulation of retroviral RNA intermediates. Note that for visibility, y-axis RPM maxima differ between MusD, ETn, and tRF concentrations. Welch two sample t-test, p-value ** < 0.05, * < 0.1, ns = not significant (D) Downstream retroviral DNA intermediates are decreased by ETn tRFs. TaqMan primers and probe detect extrachromosomal, retroviral DNA only (position indicated in E). Data represented as mean ± SD, normalized to total transfected plasmid DNA. (E) Outline of ETn-neo retroviral intermediates and model of retrotransposon silencing by 18 nt tRFs. See also Figure S4.