Fig. 3

Polysome and ribosome profiling defines the role of DHX36 and DHX9 on translation. a Polysome profiling of HeLa cytoplasmic extract coupled with mass spectrometry allowed estimating the enrichment of different helicases in monosome or polysome fractions. Duplicates were used to quantitatively estimate the presence of a given helicase in each fraction. A hierarchical cluster analysis reporting the distribution of human helicases within mono/polysomes is shown. b Immunoblots of a polysome profile probing for members of the DEAH-box/RHA helicase family (left blot) confirmed the presence of DHX36 and DHX9 in both light and heavy polysomes. On the right is reported the result of affinity purifications using indicated biotinylated nucleic acids probes showing the presence of DHX9, DHX36, and DHX57 in rG4/ribonucleoprotein complexes. Lysate: 30 μg of total protein, Neg: empty beads, mut G4: mutated rG4-forming sequences, rG4: rG4-forming sequences, SL: stem-loop forming sequence. c Immunoblots of siRNA-treated cells probing DHX36 and DHX9 show that siRNA depletion of both helicases is reproducible and selective. Rep: replicate. Ribosome profiling allowed assessing change in TE upon depletion of d DHX36 and e DHX9. Are reported the frequency distributions of the ratio of TE in DHX36 (top) and DHX9 (bottom) depleted cells over TE in control (n = 3 replicates). f Comparison of fold changes of TE upon DHX36 and DHX9 depletion. Q values were calculated by combining P values using Fisher’s method. g Anticorrelation between fold changes of TE and fold changes of RPFdist upon depletion of DHX36 (red) or DHX9 (blue) for transcripts showing significant change in TE (Q value < 0.05)