DDX6 functions as a sensor of rare codon-triggered mRNA decay in human cells.

A. Schematic representation of the reporters used in panels (B, D).

B. Wild-type (WT) and DDX6 KO HEK293T cells were transfected with indicated reporter plasmids. After 48 hr, cells were treated with actinomycin D (ActD) and harvested at the indicated time points. Reporter mRNA levels were analyzed by northern blotting. 18S rRNA ethidium bromide staining shows equal loading.

C. Relative reporter mRNA levels from panel B at time point zero (before ActD addition) were defined as 100%. Relative reporter mRNA levels were plotted as a function of time. Circles represent the mean value and error bars the standard deviation (SD) (n = 3). The decay curves were fitted to an exponential decay with a single component (dotted lines). R2 values are indicated for each curve. The half-life of each mRNA in WT and DDX6 KO cells is represented as the mean ± SD.

D. HEK293T cells were transfected with MBP or POP2 dominant negative mutant (POP2 DE-AA) and indicated reporter plasmids. After 48 hr, cells were treated with ActD and harvested at the indicated time points. Reporter mRNA levels were analyzed by northern blotting. 18S rRNA ethidium bromide staining shows equal loading.

E. Relative reporter mRNA levels from panel D at time point zero (before ActD addition) were defined as 100%. Relative reporter mRNA levels were plotted as a function of time. Circles represent the mean value and error bars the standard deviation (SD) (n = 3). The decay curves were fitted to an exponential decay with a single component (dotted lines). R2 values are indicated for each curve. The half-life of each reporter mRNA in WT and POP2 DE-AA overexpressing cells is represented as the mean ± SD.

DDX6 interacts with ribosomal proteins in human cells.

A. The interaction between the recombinant NusA-Strep-DDX6 and purified human ribosomal proteins was analyzed by SDS-PAGE and stained with Coomassie blue. Input lysate (1%) and bound fractions (20%) were loaded.

B. Western blot showing the interaction between GFP-tagged DDX6 full-length/N-ter/C-ter with HA-tagged RPL22 and endogenous RPS3A in human HEK293T cells. GFP-tagged MBP served as a negative control. For the GFP-tagged proteins, the HA-tagged RPL22 and the endogenous RPS3A, 1% of the input and 20% of the immunoprecipitate were loaded. N-ter: N-terminus; C-ter: C-terminus.

C. Immunoprecipitation assay showing the interaction of GFP-tagged DDX6 (wild-type or the indicated mutants) with HA-tagged RPL22 or endogenous CNOT1 in HEK293T cells. Samples were analyzed as described in B.

D. DDX6 KO HEK293T cells were transfected with the control R-LUC reporter or a reporter containing 30x rare codons and GFP-tagged DDX6 wild-type or mutants. After treating cells with ActD for 8 hr R-LUC mRNA levels were analyzed by northern blotting. 18S rRNA ethidium bromide staining shows equal loading.

E. Relative control reporter mRNA levels from panel D were defined as 100%. Relative 30x rare codon reporter mRNA levels were plotted. Bars represent the mean value and error bars the standard deviation (n = 3).

F. Immunoblot illustrating the expression of proteins used in the assay shown in panel D.

DDX6 controls mRNA abundance and translational efficiency in human cells.

A. Comparative analysis of translational efficiency (TE) in wild-type (WT) HEK293T and DDX6 KO cells. Genes with significantly (FDR<0.005) increased (n=1707 genes) and decreased (n=1484 genes) mRNA abundance are colored in red and blue, respectively.

B. Comparative analysis of translational efficiency (TE) in WT HEK293T and DDX6 KO cells.

Genes with significantly (FDR<0.005) increased (n=260 genes) and decreased (n=38 genes) TE are highlighted in salmon and cyan, respectively. The top 20 (total 89) of translationally upregulated zinc finger transcription factors in DDX6 KO cells are highlighted.

C. Pie charts indicating the fractions and absolute numbers of significantly (FDR<0.005) differentially expressed mRNAs in HEK293T WT and DDX6 KO cells as determined by RNA-seq.

D. Pie charts indicating the fractions and absolute numbers of significantly (FDR<0.005) differentially translated mRNAs in HEK293T WT and DDX6 KO cells as determined by Ribo-seq/RNA-seq.

E. Gene ontology of the biological processes associated with upregulated transcripts in DDX6 KO cells. Bar graph shows log10 q-values for each overrepresented category. Values and circles indicate the % of genes within each category.

F. Gene ontology of the biological processes associated with translationally upregulated transcripts in DDX6 KO cells. Bar graph shows log10 q-values for each overrepresented category. Values and circles indicate the % of genes within each category.

G. Ridgeline plots of predicted mRNA stability (Diez et al., 2022) of translationally upregulated unchanged/downregulated transcripts in DDX6 KO cells. Statistical significance was calculated with the one-sided Wilcoxon rank sum test.

H. qPCR analysis of AR and BMP2 mRNA levels in HEK293T WT and DDX6 KO and rescued with GFP-tagged DDX6 (wild-type or the indicated mutants). log2FC values for each transcript as determined by the RNA-seq experiments are indicated.

I. Immunoblot depicting the expression of proteins used in the assay shown in panel G.

DDX6 is required for ribosome-stalling mRNA degradation.

A. Schematic representation of the reporters used in panels (B, C).

B. Representative northern blots showing the decay of AR reporter mRNAs in HEK293T wild-type (WT) or DDX6 KO cells. Cells were transfected with indicated reporter plasmids and monitored after the inhibition of transcription using actinomycin D (ActD) for the indicated time. 18S rRNA ethidium bromide staining shows equal loading.

C. Relative reporter mRNA levels from panel B at time point zero (before ActD addition) were defined as 100%. Relative reporter mRNA levels were plotted as a function of time. Circles represent the mean value and error bars the standard deviation (SD) (n = 3). The decay curves were fitted to an exponential decay with a single component (dotted lines). R2 values are indicated for each curve. The half-life of each mRNA in WT and DDX6 KO cells is represented as the mean ± SD.

D. Representative northern blots showing the decay of BMP2 reporter mRNAs in HEK293T WT or DDX6 KO cells. Cells were transfected with indicated reporter plasmids and monitored after the inhibition of transcription using actinomycin D (ActD) for the indicated time. 18S rRNA ethidium bromide staining shows equal loading.

E. Relative reporter mRNA levels from panel D at time point zero (before ActD addition) were defined as 100%. Relative reporter mRNA levels were plotted as a function of time. Circles represent the mean value and error bars the standard deviation (SD) (n = 3). The decay curves were fitted to an exponential decay with a single component (dotted lines). R2 values are indicated for each curve. The half-life of each mRNA in WT and DDX6 KO cells is represented as the mean ± SD.

F. HEK293T cells were transfected with indicated R-LUC reporters containing 6×MS2 binding sites, HA-tagged RPL22, and SBP-tagged MBP-MS2 plasmids. RNA bound to V5-SBP-MBP-MS2 was immunoprecipitated with Streptavidin beads. The presence of HA-tagged RPL22 in the immunoprecipitates was determined by western blotting. V5-SBP-MBP-MS2 protein level and RT-PCR of R-LUC reporter RNA levels served as a loading control.