FIGURE 5.

Ectopic expression of human DDX5 or DDX5ΔCTE rescues the growth and cryptic transcription defects of dbp2Δ in S. cerevisiae. (A) Ectopic expression of DDX5-GFP or DDX5ΔCTE-GFP fully rescues dbp2Δ cold sensitivity and slow growth defects. Wild-type (WT) cells transformed with empty vector or dbp2Δ cells transformed with vector only, CEN plasmids expressing DBP2, DDX5-GFP, or DDX5ΔCTE-GFP, or a 2µ HAS1 plasmid were spotted in fivefold serial dilutions on selective media and grown at respective temperatures. HAS1 was isolated from a saturating suppressor screen of dbp2Δ cold sensitivity and was the sole isolate of the screen. HAS1 is a DEAD-box protein gene whose product functions in ribosome biogenesis (Emery et al. 2004). (B) The enzymatic activities of DDX5 are required for growth complementation of dbp2Δ. Serial dilution assays were conducted as above with dbp2Δ cells transformed with vector and CEN-plasmid expressing DDX5-GFP, DDX5(D248N)-GFP, and DDX5(K144N)-GFP. DDX5(K144N) and DDX5(D248N) have mutations in the Walker A and Walker B motifs, respectively, which affect ATP binding and hydrolysis (Cordin et al. 2006), and are shown to be required for the ATPase activities of DDX5 (Zhang et al. 2016). (C) RT-qPCR of DBP2, DDX5, or HAS1 genes in strains above shows similar expression of pDBP2, pDDX5-GFP, and pDDX5ΔCTE-GFP and overexpression of HAS1. Transcript levels were normalized to ACT1 with DBP2 expression in the wild type + vector strain set to one. Data show the mean ± SD of three independent biological replicates. (D) Ectopic expression of DDX5-GFP or DDX5ΔCTE-GFP rescues the cryptic transcription initiation defect in dbp2Δ cells. Northern blotting of endogenous FLO8 transcripts was performed as previously described (Cloutier et al. 2012). Full-length (2.4 kb) and the short, cryptic (0.8 kb) FLO8 transcripts were visualized by autoradiography and quantified by densitometry. (% Short) Percentage of short transcript versus total FLO8 transcripts (Full length + Short).