Engineering a conserved RNA regulatory protein repurposes its biological function in vivo
- University of Texas Dallas, United States
- National Institute of Environmental Health Sciences, National Institutes of Health, United States
- Ithaca College, United States
- University of Wisconsin-Madison, United States
- National Institutes of Health, United States
Figures
Figure 1
Evolutionary and structural divergence among the C.elegans PUF protein family.
(A) Dendrogram of C. elegans PUF proteins based on alignment of primary sequences. The four clades are indicated: FBF, containing FBF-2 (blue and maroon); PUF-8/9, containing PUF-8 (blue and yellow);…
Figure 2
RNA recognition by PUF-8.
(A) Recognition of the conserved 5´-UGUR sequence by PUF proteins. (B) Recognition of the conserved AUA-3´ sequence by PUF proteins. Superpositions of crystal structures of PUF-8:PBE RNA (orange), …
Figure 3 with 3 supplements
Substitution of TRM residues in FBF-2 repeat R5 to SS/Y switches specificity from a 9-nt FBE to an 8-nt PBE.
(A) PUM1 binds preferentially to an 8-nt PBE by intercalating R1008 between bases A4 and A5 (left, PDB ID 3Q0L). This is distinct from FBF-2 bound to a 9-nt FBE where R364 projects away from base C5 …
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Figure 3—source data 1
Raw data for Beta-Glo assay of FBF-2 R5 SS/Y variant with different binding elements.
- https://doi.org/10.7554/eLife.43788.011
Figure 3—figure supplement 1
Modified yeast three-hybrid system to detect RNA-protein interactions.
(A) Schematic representation of RNA selection using the yeast three-hybrid system. RNA is presented to RNA-binding proteins by virtue of a LexA MS2 coat protein chimera. Hybrid RNAs containing MS2 …
Figure 3—figure supplement 2
Electrophoretic mobility shift assays confirm preferential recognition of the PBE by SS/Y.
The FBF-2 R5 SS/Y variant shifts specificity toward an 8-nt PBE over a 9-nt FBE. FBF-2 binds preferentially to a 9-nt FBE versus an 8-nt PBE (A, B), whereas FBF-2 SS/Y binds preferentially to an …
Figure 3—figure supplement 3
The FBF-2 R5 SS/Y variant binds to an 8-nt PBE without changing the overall curvature.
(A) The SS/Y substitutions do not change the overall curvature of FBF-2. Superimposed crystal structures of an FBF-2:FBE complex and the FBF-2 SS/Y:PBE complex are shown as backbone traces. The …
Figure 4 with 2 supplements
Y364 in the FBF-2 SS/Y variant is critical for 8-nt PBE selectivity.
(A) Interaction of FBF-2 TRM variants with 8-nt PBE and 9-nt FBE RNAs. Yeast 3-hybrid analyses of binding by FBF-2 WT and the FBF-2 SS/Y variant to an MS2 hairpin (None, grey) or an MS2 hairpin …
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Figure 4—source data 1
Raw data for Beta-Glo assay FBF-2 R5 variants with different binding elements.
- https://doi.org/10.7554/eLife.43788.015
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Figure 4—source data 2
Raw data for Beta-Glo assay FBF-2 R5 variants with different binding elements.
- https://doi.org/10.7554/eLife.43788.016
Figure 4—figure supplement 1
Electrophoretic mobility shift assays.
Representative binding curves and corresponding electrophoretic mobility shift assays are shown. (A, B) FBF-2 AS/Y and AQ/Y bind preferentially to an 8-nt PBE versus a 9-nt FBE. (C) FBF-2 SS/R binds …
Figure 4—figure supplement 2
The FBF-2 R5 CQ/Y mutant is expressed in yeast.
Wild-type FBF-2 is expressed at comparable levels to the CQ/Y variant. The pGADT7 vector contains an HA tag which is used to detect activation domain fusions. GAPDH is shown as a load control.
Figure 5
FBF-2 variants retain base recognition specificity at flanking positions.
Yeast 3-hybrid analyses of binding by PUF-8, FBF-2 SS/Y, FBF-2 AS/Y, and FBF-2 AQ/Y to an MS2 hairpin fused to 8-nt PBE RNAs bearing nucleotide substitutions at positions 3–5. Binding activity is …
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Figure 5—source data 1
Raw data for Beta-Glo assay of FBF-2, FBF-2 R5 variants and PUF-8 that carry R2 SE/H mutations with different length binding elements.
- https://doi.org/10.7554/eLife.43788.018
Figure 6
The FBF-2 R5 AQ/Y variant partially rescues the tumorous phenotype in the C.elegans germ line caused by loss of PUF-8 in a sensitized genetic background.
Extruded germlines were stained for nuclei (DAPI, blue) or mitotic cells (α-PHH3, red). (A) Fluorescence microscopic image of an extruded germline from an animal with wild-type fbf-2 and a …
Tables
Table 1
Data collection and refinement statistics
https://doi.org/10.7554/eLife.43788.003| Protein:RNA | PUF-8: PBE | FBF-2 SS/Y: PBE | FBF-2 AS/Y: PBE | FBF-2 AQ/Y: PBE | |
|---|---|---|---|---|---|
| Data collection | |||||
| Space group | C2 | P61 | P61 | P61 | |
| Unit Cell | a, b, c (Å) | 109.2, 189.0, 63.2 | 96.4, 96.4, 99.9 | 96.5, 96.5, 101.1 | 95.9, 95.9, 100.4 |
| α, β, γ (°) | 90, 103.6, 90 | 90, 90, 120 | 90, 90, 120 | 90, 90, 120 | |
| Resolution (Å) | 50–2.55 (2.59–2.55)* | 50–2.25 (2.29–2.25)* | 50–2.25 (2.33–2.25)* | 50–2.85 (2.9–2.85)* | |
| Rsym or Rmerge | 0.191 (0.692) | 0.101 (0.704) | 0.104 (0.772) | 0.191 (0.957) | |
| I /σI | 9.4 (1.9) | 19.2 (3.42) | 17.1 (2.98) | 12.8 (2.34) | |
| Completeness (%) | 98.9 (98.0) | 99.9 (100) | 99.9 (100) | 99.6 (99.2) | |
| Redundancy | 6.9 (3.6) | 5.7 (5.7) | 5.7 (5.7) | 10.7 (8.8) | |
| Refinement | |||||
| Resolution (Å) | 33.8–2.6 | 32.0–2.3 | 31.6–2.3 | 27.5–2.9 | |
| No. reflections | 37,625 | 25,089 | 25,386 | 12,185 | |
| Rwork/Rfree | 0.229/ 0.285 | 0.158/ 0.204 | 0.167/ 0.223 | 0.219/ 0.272 | |
| No. atoms | |||||
| Protein | 8415 | 3197 | 3194 | 3189 | |
| RNA | 507 | 150 | 168 | 168 | |
| Solvent | 229 | 169 | 109 | 21 | |
| B-factors (Å2) | |||||
| Wilson B | 29.7 | 36.6 | 35.5 | 48.4 | |
| Protein | 32.8 | 45.3 | 45.1 | 50.2 | |
| RNA | 43.6 | 51.4 | 58.8 | 64.8 | |
| Solvent | 32.7 | 49.6 | 44.3 | 23.1 | |
| R.m.s deviations | |||||
| Bond lengths (Å) | 0.002 | 0.007 | 0.007 | 0.002 | |
| Bond angles (°) | 0.45 | 0.77 | 0.78 | 0.38 | |
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*Values in parentheses are for the highest-resolution shell.
Table 2
RNA-binding analyses of PUF-8 and FBF-2 proteins
https://doi.org/10.7554/eLife.43788.005| Protein | RNA | RNA sequence | Kd (nM) | Krel* |
|---|---|---|---|---|
| PUF-8 | PBE | UGUA UAUA | 28.8 ± 0.7 | 1 |
| PBE-A5 | UGUA AAUA | 25.9 ± 3.1 | 0.9 | |
| PBE-C5 | UGUA CAUA | 44.8 ± 2.4 | 1.6 | |
| PBE-G5 | UGUA GAUA | 45.6 ± 3.1 | 1.6 | |
| FBE | UGUGCCAUA | 3110 ± 656 | 108 | |
| FBF-2 WT | PBE | ACAUGUAA AUAC | 74.6 ± 7.7 | 1 |
| FBE | ACAUGUGCCAUAC | 19.3 ± 0.6 | 0.3 | |
| FBF-2 SS/Y | PBE | ACAUGUAA AUAC | 22.7 ± 0.4 | 1 |
| FBE | ACAUGUGCCAUAC | 50.9 ± 2.3 | 2.2 | |
| FBF-2 AS/Y | PBE | ACAUGUAA AUAC | 16.3 ± 0.8 | 1 |
| FBE | ACAUGUGCCAUAC | 76.7 ± 4.3 | 4.7 | |
| FBF-2 SS/R | PBE | ACAUGUAA AUAC | 51.1 ± 1.1 | 1 |
| FBE | ACAUGUGCCAUAC | 26.9 ± 2.3 | 0.5 | |
| FBF-2 AQ/Y | PBE | ACAUGUAA AUAC | 20.2 ± 1.2 | 1 |
| FBE | ACAUGUGCCAUAC | 79.9 ± 2.0 | 4.0 |
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*Krel values are calculated for each protein with binding to the PBE RNA set to 1.
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Table 2—source data 1
Data for Table 2.
- https://doi.org/10.7554/eLife.43788.006
Table 3
Phenotypic analysis of mutant strains
https://doi.org/10.7554/eLife.43788.020| Genotype | RNAi | Wild-type | Complete tumorous | N |
|---|---|---|---|---|
| Germline, % | Germline, % | |||
| glp-1(ar202) | puf-8 | 2 | 98 | 61 |
| glp-1(ar202) | Scramble | 100 | 0 | 95 |
| fbf-2(lot14) glp-1(ar202) | puf-8 | 64 | 36 | 89 |
| fbf-2(lot14) glp-1(ar202) | Scramble | 100 | 0 | 107 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.43788.021
