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. 2020 Dec 9;296:100154. doi: 10.1074/jbc.RA120.014894

Figure 3.

Figure 3

La binds to a site proximal to the miR-21 binding site in PDCD4 3ʹUTR and reverses miR-21-mediated repression of PDCD4 translation. A, schematic diagram of full-length (FL) PDCD4 3ʹUTR and various deletion constructs from the 5ʹ- and 3ʹ-ends. The miR-21 target site (nt 228–249) as well as the La-binding sites, as determined from the RNA–protein interaction studies (below), are indicated. B, 32P-UTP labeled full-length and various deletion mutants of PDCD4 3′-UTR RNA were incubated with purified La protein (20 pmole), UV-crosslinked, digested with RNase A, and the RNA-protein complexes resolved on 10% SDS–PAGE. C, luciferase assay of MCF7 cells transfected with reporter gene constructs containing full-length and different deletion mutants of PDCD4 3ʹUTR, co-transfected with pSUPER–miR-21 (250 ng) and pCDNA3–La (250 ng) or both. Fluc values are normalized to Rluc values as transfection control. ∗ represents significant difference (p-value ≤ 0.05) from respective mock-transfected controls, # and ## represents significant difference (p-value ≤ 0.05 and ≤0.01, respectively) from miR-21 transfected sets. D, schematic diagram of deletion constructs of the 5ʹΔ2–3ʹΔ3 fragment of the PDCD4 3ʹUTR RNA having miR-21 target site (nt 228–249) and two deletions from the 5ʹ- and 3ʹ-ends. 32P-UTP labeled RNA of 5ʹΔ2–3ʹΔ3 RNA, and its deletion mutants were incubated with two concentrations (10 and 20 pmole) of purified La protein, UV-crosslinked, digested with RNase A, and resolved on 10% SDS–PAGE. E, luciferase assay of MCF7 cells transfected with reporter gene constructs containing 5ʹΔ2–3ʹΔ3 fragment of PDCD4 3ʹUTR and its two deletion mutants, co-transfected with pSUP-miR-21 (250 ng) and pCDNA3-La (250 ng) or both. Fluc values are normalized to Rluc values as transfection control. ∗ and ∗∗Represent significant difference (p-value ≤ 0.05 and ≤0.01 respectively) from respective mock-transfected controls. # and ## represent significant difference (p-value ≤ 0.05 and ≤0.01 respectively) from miR-21 transfected sets. La, lupus antigen; PDCD4, programmed cell death 4; 3ʹUTR, 3ʹ-untranslated region.