Skip to main content
. Author manuscript; available in PMC: 2024 Sep 21.
Published in final edited form as: Mol Cell. 2023 Sep 7;83(18):3303–3313.e6. doi: 10.1016/j.molcel.2023.08.008

Figure 3. Stress-dependent translational control of ALKBH5 requires a cis-acting 5′ UTR stem loop.

Figure 3.

(A) eIF3 PAR-CLIP cluster in the 5′ UTR of the ALKBH5 mRNA. (B) Luciferase activity in cells transfected with ALKBH5 5′ UTR mutant. Results are normalized to untreated cells. Δpar, deletion of eIF3 PAR-CLIP peak. (C) SHAPE-based secondary structure of the ALKBH5 WT 5′ UTR (nt 79-345) or with the eIF3 binding site (Δsl3) deleted. Nucleotides are color coded by SHAPE reactivity, with higher reactivity correlating with single-stranded probability. The results are representative of two independent experiments. Black line, main eIF3 PAR-CLIP peak; gray line, extended peak. (D) Schematic of ALKBH5 5′ UTR luciferase (Luc) reporter constructs. sl3, eIF3-binding stem loop identified by PAR-CLIP analysis. (E) eIF3 RNA-binding to ALKBH55′ UTR mutants during ISR induction. RNA-binding is normalized to eIF3 binding to WT ALKBH 5′ UTR. Δ, deletion of sl3. (F) Luciferase activity in vitro mediated by ALKBH5 5′ UTR mutants. Results are normalized to WT ALKBH5 5′ UTR luciferase activity in in vitro translation extracts made from untreated HEK293T cells. Results in (B), (E) and (F) are represented as the mean ± s.d. of three independent experiments. See also Figure S4.