Skip to main content
. Author manuscript; available in PMC: 2023 Aug 4.
Published in final edited form as: Mol Cell. 2022 Jun 8;82(15):2797–2814.e11. doi: 10.1016/j.molcel.2022.05.016

Figure 5. Position-specific influence of ac4C on translation initiation in mRNA reporters.

Figure 5.

(A) ac4C was incorporated into specific 5’UTR locations in NanoLuc mRNA through in vitro transcription and splint ligation as in Fig. 1D. NanoLuc translation was achieved in RRL or in transfected wildtype and NAT10−/− HeLa cells.

(B) NanoLuc mRNA containing ac4C or C within a structured uORF upstream of a consensus AUG start codon was generated through splint ligation and confirmed through Northern blot/phosphorimagery (left). Uncapped mRNAs were in vitro translated in RRL. NanoLuc activity was normalized by mRNAs levels (middle), Mean±SEM, n=3. p=Two-way ANOVA (middle). Capped and polyadenylated NanoLuc mRNAs were transfected into wildtype and NAT10−/− HeLa cells along with unmodified Firefly luciferase mRNA for in vivo translation. NanoLuc activity was normalized by Firefly luciferase (right), Mean±SEM, n=3 for HeLa WT, n=8 for NAT10−/−. p=t-test.

(C) As in (B), but with NanoLuc mRNA containing C or ac4C in positions −1 and −2 of the Kozak sequence surrounding a consensus AUG start codon. Mean±SEM, n=5 for in vitro translation, n=5 for HeLa WT, n=4 for NAT10−/− p=Two-way ANOVA, in vitro results. p=t-test, in vivo results.

(D) As in (B), but with NanoLuc mRNA containing C or ac4C directly over three tandem CUG start codons. Mean±SEM, n=3 for in vitro translation, n=4 for HeLa WT. n=4 for NAT10−/−. p=Two-way ANOVA, in vitro results. p=t-test, in vivo results.