Fig 2: DDX6 regulates proliferation and morphology of ESCs.(A) Western blot of DDX6 in two Ddx6 knockout (KO) lines. GAPDH and ACTIN were used as loading controls. (B) Ddx6 counts per million (CPM) in nascent mRNA (4sU) or mRNA in wild-type (WT) and Ddx6 KO cells. n = 3 for wild-type, n = 4 for Ddx6 KO (2 replicates of each Ddx6 KO line) (C) Expression of pluripotency genes in Ddx6 KO ESCs based on RNA-Seq. Error bars represent 95% confidence interval. (D) Brightfield images of wild-type and Ddx6 KO ESCs. Images taken at 20X. (E) DDX6 staining in wild-type ESCs. Images taken at 20X. (F) P-body staining against DCP1a in wild-type and Ddx6 KO ESCs. Images taken at 20X. (G) Growth curves of wild-type and Ddx6 KO ESCs in ESC maintenance conditions (LIF/2i). n = 6 for wild-type cells, n = 12 for Ddx6 KO (six replicates of each Ddx6 KO line). * indicates p<0.05 using a t-test, error bars are standard deviation. See also Figure 3—figure supplement 1.

Fig 3: Translational repression alone underlies many of the downstream molecular changes associated with miRNA loss.(A) Comparison between mRNA stability changes in Dgcr8 KO versus Ddx6 KO cells. n = 3 for wild-type, n = 4 for Ddx6 KO (2 replicates of each Ddx6 KO line), n = 3 for Dgcr8 KO. (B) Comparison between translation level changes in Dgcr8 KO versus Ddx6 KO cells. n = 3 for each genotype. (C) Comparison between mRNA changes in Dgcr8 KO versus Ddx6 KO cells. The p value was calculated with correlation significance test. (D) Summary schematic comparing Dgcr8 KO cells to Ddx6 KO cells. Dgcr8 KO leads to the loss of both translational repression and mRNA destabilization of miRNA targets, while Ddx6 KO only leads to the loss of translational repression of miRNA targets. mRNA stability is measured as the ratio of mRNA/4sU reads, changes in translation level are measured as the ratio of polysome/monosome reads, protein level changes are not directly measured but are predicted based on mRNA stability and translation level changes. Changes in translation level alone in Ddx6 KO cells produce similar phenotypes and global molecular changes to Dgcr8 KO cells. See also Figure 5—figure supplement 1.