Fig. 3: GNG motif-selective clamping of eIF4A causes the repression of translation initiation. | Nature Communications

Fig. 3: GNG motif-selective clamping of eIF4A causes the repression of translation initiation.

From: DMDA-PatA mediates RNA sequence-selective translation repression by anchoring eIF4A and DDX3 to GNG motifs

Fig. 3

a Rank plot for motif prediction by ribosome profiling under 0.1 μM DMDA-PatA treatment. Spearman’s correlation coefficients (ρ, two-tailed) between the number of 4-mer motifs found in the 5′ UTRs and translation changes of the mRNAs were calculated. Motifs containing GNG are highlighted. b Rank plot for motif prediction by RNA pulldown-Seq under 0.01 μM DMDA-PatA treatment. Spearman’s correlation coefficients (ρ, two-tailed) between the number of 4-mer motifs found in 5′ UTRs and mRNA changes on SBP-tagged eIF4A1 were calculated. Motifs containing GNG are highlighted. c Schematic of reporter mRNAs with 7× NGNGNG motifs and the control CAA repeats (left). These mRNAs were subjected to in vitro translation with RRL and titration with DMDA-PatA (right). The data are presented as the mean (point) and s.d. (error) for replicates (n = 3). d, f Toeprinting assay to probe the 48S ribosomes assembled on the start codons in the indicated reporter mRNAs with or without 10 μM DMDA-PatA. cDNA synthesized with FAM-labeled reverse transcription primers was analyzed by capillary electrophoresis. A magnified view of the results for reporter mRNA with 7× AGAGAG motifs (the area defined by the dashed line in d) is shown in f. AU, arbitrary unit. e Relationships between translational repression observed during in vitro translation (at 3 μM DMDA-PatA) (c) and the reduction in 48S formation (Supplementary Fig. 3g) for the indicated reporter mRNAs. The data are presented as the mean (point) and s.d. (error) for replicates (n = 3). The regression line (dashed line) is shown. g Toeprinting assay with the recombinant eIF4A1 protein on the reporter mRNA with 7× AGAGAG motifs with or without 10 μM DMDA-PatA. cDNA synthesized with FAM-labeled reverse transcription primers was analyzed by capillary electrophoresis. A magnified view of the results is shown at the bottom. h In vitro translation of reporter mRNAs (with 7× AGAGAG motifs or CAA repeats, both at 90.9 nM) preincubated with recombinant eIF4A1 and DMDA-PatA. A size exclusion column was used to eliminate free DMDA-PatA. The data are presented as the mean (bar) and s.d. (error) for replicates (point, n = 3). The significance was calculated by Student’s t test (two-tailed). Source data are provided as a Source Data file.

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