Fig. 4. DDX ATPase activity controls RNA partitioning between phase-separated compartments in vivo and in vitro.

(A) Pab1-yEGFP labeled SGs are induced in an untagged Dhh1 WT or Dhh1^DQAD or dhh1Δ background. 3 biological replicates, at least 383 (WT), 93 (dhh1Δ) or 657 (Dhh1^DQAD) cells per replicate. (B) Quantification of (A), mean and SD. Unpaired t-test (two-tailed), * p = 0.0224. Dots represent mean of individual replicates. (C) Schematic representation of in vitro RNA transfer experiment (D-F) Droplets were assembled from Dhh1-mCherry (mCh) with Cy5-labelled RNA (red circles), and from Ded1-yEGFP (green circles). Cy5-RNA, Dhh1-mCh and Ded1-GFP were monitored upon addition of buffer (D) or Not1^MIF4G (F). Representative images of > 3 independent experiments. (E) Quantification of Cy5 intensity inside Ded1 areas, normalized to the intensity value at t=0 sec. Mean (line) and SD (shaded area) of 17 (“Not1 addition”) and 12 (“buffer addition”) large droplets per movie. At t = 2 min, 17.4 +/- 3.3% of the Cy5-RNA is enriched in Ded1-GFP droplets upon Not1 addition (n = 3 movies). Scale bars 5 μm. (G) Concept how DDXs could regulate multivalency, phase separation and compartment formation (I), ATPase-controlled compartment turnover and RNA release (II), and RNA transfer (III).