Summary of generated DDX6 mutants, their published phenotypes and our observations
| Mutation . | Published effects on DDX6 interactions . | Published impacts on PB formation . | Observed impact on PBs or SGs in this study . |
|---|---|---|---|
| Mut1 (Q320A, H323A, T327A, R331A) | Impairs binding to EDC3-DHH1 (Sharif et al., 2013), 4E-T-DDX6 (Ozgur et al., 2015), probably LSM14A (Brandmann et al., 2018) and weakens PAT1B-DDX6 (Sharif et al., 2013) binding | Two times less potent than DDX6wt at assembling P-bodies (Ayache et al., 2015) | Rescues PBs (increased number and docking), weak limitation of SGs |
| Mut2 (R443A, F444A, K447A, E450A, E451A) | Impairs EDC3-DHH1 (Sharif et al., 2013), then probably LSM14A (Brandmann et al., 2018) but can still bind PAT1-DHH1 (Sharif et al., 2013) | - | Rescues PBs (increased number and docking), increased SGs |
| Mut3 (S343D, Q345D, R346D) | Impairs DDX6-PAT1B binding (Sharif et al., 2013), 4E-T (Ozgur et al., 2015), and most likely LSM14A (Q345 and R346 form three side specific H bonds with LSM14A) thus mutation is expected to abolish or decrease binding to LSM14A (Brandmann et al., 2018), can still bind DDX6-EDC3 (Sharif et al., 2013). Mutation of the same helix with one overlapping residue (R346A, K352A, K353A) impairs PAT1B, DCP2, AGO2 and TNRC6A binding (Ozgur and Stoecklin, 2013). Potentially reduced/abolished RNA bindinga | No PBs with R346A, K352A, K353A mutant in HeLa cells (Ozgur and Stoecklin, 2013) | No PBs, increased SGs |
| Mut4 (R373A,T391A R421A) | Impairs RNA binding (Dutta et al., 2011), and ATP hydrolysis for DHH1 (Dutta et al., 2011), reduce binding of DDX6 to 4E-T and PAT1B and abolishes binding for LSM14A (Balak et al., 2019) | R322A, S340A, R370A in DHH1: Reduction of PBs (Mugler et al., 2016), R322A/S340A in DHH1: Smaller PBs (Dutta et al., 2011), R373Q or T391I missense mutations in DDX6 abolish PBs in patient cells (Balak et al., 2019) | No PBs, increased SGs |
| E247A | Impairs ATPase activity but is capable of RNA and ATP binding (Dutta et al., 2011) | D195A/E196A in DHH1: Increase in size/number of PBs (Dutta et al., 2011), E195Q in DHH1: Constitutive PBs (Mugler et al., 2016) | No PBs, increased SGs |
| R386E | Impairs CNOT1 binding (Mathys et al., 2014). As CNOT1 can stimulate the RNA-dependent ATPase activity (Mathys et al., 2014), we expect this mutant to behave similarly to the ATPase inactive E247A mutant | R55E, F62E, Q282E, N284E, R355E in DHH1: Constitutive PBs (Mugler et al., 2016) | No PBs, increased SGs |
| Mutation . | Published effects on DDX6 interactions . | Published impacts on PB formation . | Observed impact on PBs or SGs in this study . |
|---|---|---|---|
| Mut1 (Q320A, H323A, T327A, R331A) | Impairs binding to EDC3-DHH1 (Sharif et al., 2013), 4E-T-DDX6 (Ozgur et al., 2015), probably LSM14A (Brandmann et al., 2018) and weakens PAT1B-DDX6 (Sharif et al., 2013) binding | Two times less potent than DDX6wt at assembling P-bodies (Ayache et al., 2015) | Rescues PBs (increased number and docking), weak limitation of SGs |
| Mut2 (R443A, F444A, K447A, E450A, E451A) | Impairs EDC3-DHH1 (Sharif et al., 2013), then probably LSM14A (Brandmann et al., 2018) but can still bind PAT1-DHH1 (Sharif et al., 2013) | - | Rescues PBs (increased number and docking), increased SGs |
| Mut3 (S343D, Q345D, R346D) | Impairs DDX6-PAT1B binding (Sharif et al., 2013), 4E-T (Ozgur et al., 2015), and most likely LSM14A (Q345 and R346 form three side specific H bonds with LSM14A) thus mutation is expected to abolish or decrease binding to LSM14A (Brandmann et al., 2018), can still bind DDX6-EDC3 (Sharif et al., 2013). Mutation of the same helix with one overlapping residue (R346A, K352A, K353A) impairs PAT1B, DCP2, AGO2 and TNRC6A binding (Ozgur and Stoecklin, 2013). Potentially reduced/abolished RNA bindinga | No PBs with R346A, K352A, K353A mutant in HeLa cells (Ozgur and Stoecklin, 2013) | No PBs, increased SGs |
| Mut4 (R373A,T391A R421A) | Impairs RNA binding (Dutta et al., 2011), and ATP hydrolysis for DHH1 (Dutta et al., 2011), reduce binding of DDX6 to 4E-T and PAT1B and abolishes binding for LSM14A (Balak et al., 2019) | R322A, S340A, R370A in DHH1: Reduction of PBs (Mugler et al., 2016), R322A/S340A in DHH1: Smaller PBs (Dutta et al., 2011), R373Q or T391I missense mutations in DDX6 abolish PBs in patient cells (Balak et al., 2019) | No PBs, increased SGs |
| E247A | Impairs ATPase activity but is capable of RNA and ATP binding (Dutta et al., 2011) | D195A/E196A in DHH1: Increase in size/number of PBs (Dutta et al., 2011), E195Q in DHH1: Constitutive PBs (Mugler et al., 2016) | No PBs, increased SGs |
| R386E | Impairs CNOT1 binding (Mathys et al., 2014). As CNOT1 can stimulate the RNA-dependent ATPase activity (Mathys et al., 2014), we expect this mutant to behave similarly to the ATPase inactive E247A mutant | R55E, F62E, Q282E, N284E, R355E in DHH1: Constitutive PBs (Mugler et al., 2016) | No PBs, increased SGs |
Like previous observations in DHH1 (Sharif et al., 2013), we noticed a positively charged patch within this mutation containing helix (Fig. S5 B), suggesting partial RNA binding within this area. Our mutations to negatively charged residues could therefore significantly impair RNA binding. Moreover, all our tested parameters resemble the RNA binding incapable Mut4.