Protomer alignment modulates specificity of RNA substrate recognition by Ire1
- Department of Biochemistry and Biophysics, University of California San Francisco, United States
- Howard Hughes Medical Institute, United States
- Department of Pharmaceutical Chemistry, University of California at San Francisco, United States
- Department of Microbiology and Immunology, University of California at San Francisco, United States
- Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, United States
- Department of Cellular and Molecular Pharmacology, University of California at San Francisco, United States
Figures
Figure 1 with 1 supplement
The promiscuous RNase activity of S. pombe Ire1 causes toxicity to bacterial cells.
(A) Growth curves of bacterial cells expressing various Ire1 kinase-RNase (KR) domains. Optical densities at 600 nm (OD600) were measured every 15 min for 5 hr. Bacterial cells expressing S. …
Figure 1—figure supplement 1
4µ8C inhibits the RNase activity of S. pombe and S. cerevisiae Ire1.
(A–B) Results from the in vitro cleavage assays in Figure 1B & C were quantified using ImageJ. Cleaved portion was calculated as: cleaved RNA / (cleaved RNA +uncleaved RNA). For cleaved portion ≥0.1 …
Figure 2 with 1 supplement
S. cerevisiae Ire1-KR-mut17 has a promiscuous RNase activity.
(A) Sequence alignment of the RNase domains of Ire1 orthologs from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Schizosaccharomyces octosporus, Schizosaccharomyces cryophilus. A total of 17 …
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Figure 2—source data 1
A list of the 17 candidate residues on S. cerevisiae and S. pombe Ire1.
The oligomer interface is referred as the interface IF2C and the back-to-back dimer interface is referred as the interface IF1C in Korennykh et al., 2009.
- https://cdn.elifesciences.org/articles/67425/elife-67425-fig2-data1-v3.xlsx
Figure 2—figure supplement 1
Quantification of in vitro cleavage assays.
Results from the in vitro cleavage assays in Figure 2C–F were quantified and analyzed using the same methods as in Figure 1—figure supplement 1A.
Figure 3 with 1 supplement
Two residues at Ire1’s RNase-RNase dimer interface regulate Ire1’s RNase promiscuity.
(A) Bacterial growth assay for Sc Ire1-KR revertants. Conditions are the same as in Figure 1A. OD600 at 5 hr time-point was measured. Experiments were performed in duplicates. Dashed line marks the …
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Figure 3—source data 1
The detailed sequence information of S. cerevisiae Ire1-KR constructs used in this study.
‘Y’ indicates the mutation is present in the corresponding Ire1 construct. ‘N’ indicates the mutation is absent in the corresponding Ire1 construct.
- https://cdn.elifesciences.org/articles/67425/elife-67425-fig3-data1-v3.xlsx
Figure 3—figure supplement 1
Quantification of in vitro cleavage assays.
Results from the in vitro cleavage assays in Figure 3B–E were quantified and analyzed using the same methods as in Figure 1—figure supplement 1A.
Figure 4
S. cerevisiae Ire1-KR(K992D,Y1059R) has a promiscuous RNase activity.
(A–B) A series of twenty-four (A) and twenty-seven (B) stem-loop RNA substrates, which are derived from the S. cerevisiae HAC1 mRNA 3’ splice site (A) or the S. pombe BIP1 mRNA cleavage site (B), …
Figure 5 with 5 supplements
Structural re-arrangement at Ire1 dimer interface regulates the RNase promiscuity.
(A) Back-to-back dimer structure of WT Sc Ire1 cytosolic domain (PDB: 3FBV) with kinase domain in yellow and RNase domain in purple. K992 and Y1059 are colored in green while E988 is colored in …
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Figure 5—source data 1
In this table, 230 Ire1 orthologs were compared.
Their residues at three positions, which correspond to the S. cerevisiae Ire1 E988, K992 and Y1059, are listed.
- https://cdn.elifesciences.org/articles/67425/elife-67425-fig5-data1-v3.xlsx
Figure 5—figure supplement 1
The root-mean-square deviation (RMSD) of atomic positions of Ire1 RNase domain during the simulation.
Result of WT Sc Ire1-KR is in yellow and Sc Ire1-KR(K992D,Y1059R) is in gray.
Figure 5—figure supplement 2
Time fraction of the MD simulation during which the indicated salt bridges are present.
A 20-ns MD simulation was performed on the dimer structure of WT Sc Ire1 KR or Sc Ire1-KR(K992D,Y1059R). At a given time point during the simulation, if the distance between the two indicated …
Figure 5—figure supplement 3
Sedimentation equilibrium analytical ultracentrifugation (SE-AUC) analysis of Sc Ire1-KR and Sc Ire1-KR(K992D,Y1059R).
Each protein was examined under nine conditions: at three protein concentrations (2.5, 5, and 10 µM) and three centrifugal speeds (7,000, 10,000, and 14,000 rpm). All nine sets of data for each …
Figure 5—figure supplement 4
RNA cleavage efficiencies of Sc Ire1-KR mutants bearing mutations at back-to-back dimer interface.
kobs of the indicated Sc Ire1-KR constructs cleaving stem-loop RNA substrates derived from Sc HAC1 mRNA 3’ splice site or Sp BIP1 mRNA cleavage site. ‘BD’ indicates cleavage activity below detection …
Figure 5—figure supplement 5
Evolutionary comparison of Ire1 orthologs.
We generated a deep alignment of 230 Ire1 orthologs from fungi (211), representative plants (9) and animals (10). We compared residues at three positions, which correspond to the S. cerevisiae Ire1 …
Figure 6 with 1 supplement
Interface mutations change the protomer alignment in Ire1 dimer.
(A) Structure alignment of WT Sc Ire1-KR and Sc Ire1-KR(K992D,Y1059R). 20-ns MD simulations were performed on both WT Sc Ire1-KR and Sc Ire1-KR(K992D,Y1059R). The last simulation frame was used for …
Figure 6—figure supplement 1
Protomer alignment of RNase L.
The dimer structure of WT Sc Ire1-KR (as used in Figure 6A) and human RNase L (PDB: 4OAV) were compared. The protomer A of the Sc Ire1-KR and human RNase L dimers were aligned with minimal root mean …
Tables
Table 1
Plasmids used in this study.
In all of the plasmids, a GST and an HRV 3C protease site are N-terminally fused to Ire1-KR.
| Plasmid number | Description | Source |
|---|---|---|
| pPW1477 | Sc Ire1-KR on pGEX6P-2 | Korennykh et al., 2009 |
| pPW3205 | Sp Ire1-KR on pGEX6P-2 | Li et al., 2018 |
| pPW3244 | Sc Ire1-KR-mut17 on pGEX6P-2 | This study |
| pPW3262 | Sc Ire1-KR(K992D,Y1059R) on pGEX6P-2 | This study |
| pPW3263 | Sc Ire1-KR(K992D,H1044D,Y1059R) on pGEX6P-2 | This study |
| pPW3256 | revertant 1 (K992) on pGEX6P-2 | This study |
| pPW3245 | revertant 2 (N1001) on pGEX6P-2 | This study |
| pPW3246 | revertant 3 (M1010) on pGEX6P-2 | This study |
| pPW3247 | revertant 4 (T1032) on pGEX6P-2 | This study |
| pPW3248 | revertant 5 (F1033) on pGEX6P-2 | This study |
| pPW3257 | revertant 6 (E1038) on pGEX6P-2 | This study |
| pPW3258 | revertant 7 (R1039) on pGEX6P-2 | This study |
| pPW3259 | revertant 8 (H1044) on pGEX6P-2 | This study |
| pPW3260 | revertant 9 (S1045) on pGEX6P-2 | This study |
| pPW3249 | revertant 10 (M1049) on pGEX6P-2 | This study |
| pPW3250 | revertant 11 (Y1059) on pGEX6P-2 | This study |
| pPW3261 | revertant 12 (F1062) on pGEX6P-2 | This study |
| pPW3251 | revertant 13 (M1063) on pGEX6P-2 | This study |
| pPW3252 | revertant 14 (I1069) on pGEX6P-2 | This study |
| pPW3253 | revertant 15 (A1070) on pGEX6P-2 | This study |
| pPW3254 | revertant 16 (E1071) on pGEX6P-2 | This study |
| pPW3255 | revertant 17 (L1109) on pGEX6P-2 | This study |
| pPW3441 | Sc Ire1-KR(K992D,Y1059A) on pGEX6P-2 | This study |
| pPW3442 | Sc Ire1-KR(K992A,Y1059R) on pGEX6P-2 | This study |
| pPW3443 | Sc Ire1-KR(K992R,Y1059D) on pGEX6P-2 | This study |
| pPW3275 | Sc Ire1-KR(H1018A) on pGEX6P-2 | This study |
