Ancestral protein reconstruction reveals evolutionary events governing variation in Dicer helicase function
- Department of Biochemistry, University of Utah, United States
- Department of Microbiology and Cell Science, University of Florida, United States
Figures
Figure 1 with 4 supplements
Phylogenetic analysis of Helicase domains and DUF283 of metazoan Dicer proteins.
(A) Domain organization of Drosophila melanogaster Dicer-2 (dmDcr2) and Homo sapiens Dicer (hsDcr), with colored rectangles showing conserved domain boundaries indicated by amino acid number. Domain …
Figure 1—figure supplement 1
Maximum likelihood phylogenetic tree constructed from metazoan Dicer helicase domains and DUF283.
Dicer HEL-DUF phylogenetic tree visualized and annotated with FigTree. Resurrected ancestral nodes are indicated by black circles, with transfer bootstrap values indicated. Width of cartoon triangle …
Figure 1—figure supplement 2
Alternative reconstructions of phylogenetic tree depicting Dicer HEL-DUF evolution.
(A) Summarized phylogenetic tree showing species-accurate relationships among bilaterian phyla. Gene duplication occurs early in animal evolution. Transfer bootstrap values at select nodes are …
Figure 1—figure supplement 3
Constraining the phylogenetic tree to species-accurate relationships does not significantly impact ancestral protein reconstruction.
(A) Multiple sequence alignment illustrated with ESPript, depicting amino acid sequences for reconstructed AncD1DEUT node using either the gene tree or the species tree (Robert and Gouet, 2014). …
Figure 1—figure supplement 4
Reconstructed HEL-DUF constructs are predicted with high confidence and expressed recombinantly.
(A) Reconstructed HEL-DUFs at nodes of interest are predicted with posterior probabilities for each amino acid. Posterior probabilities for each amino acid are plotted and binned by 0.1. AncD1VERT …
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Figure 1—figure supplement 4—source data 1
Original digital image of SDS-PAGE gel used in B.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig1-figsupp4-data1-v2.zip
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Figure 1—figure supplement 4—source data 2
Posterior probabilities for ancestral states for all ancestrally reconstructed nodes in the maximum likelihood phylogeny.
Posterior probabilities represent each state in the ancestrally reconstructed protein prior to removal of low-probability insertions using an absence-presence alignment and the BIN model in RAXML-NG. Used in Figure 1—figure supplement 4.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig1-figsupp4-data2-v2.zip
Figure 2
ATP hydrolysis capability is present in ancestral metazoan Dicer but lost at the common ancestor of vertebrates.
(A–D) PhosphorImages of representative thin-layer chromatography (TLC) plates showing hydrolysis of 100 µM ATP (spiked with α-32P-ATP) by 200 nM ancestral HEL-DUFs for various times as indicated, at …
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Figure 2—source data 1
Raw digital images of thin-layer chromatography plate used in 2A.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig2-data1-v2.zip
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Figure 2—source data 2
Raw digital image of thin-layer chromatography plate used in 2A.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig2-data2-v2.zip
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Figure 2—source data 3
Raw digital image of thin-layer chromatography plate used in 2B.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig2-data3-v2.zip
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Figure 2—source data 4
Raw digital image of thin-layer chromatography plate used in 2B.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig2-data4-v2.zip
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Figure 2—source data 5
Raw digital image of thin-layer chromatography plate used in 2C.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig2-data5-v2.zip
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Figure 2—source data 6
Raw digital image of thin-layer chromatography plate used in 2C.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig2-data6-v2.zip
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Figure 2—source data 7
Raw digital image of thin-layer chromatography plate used in 2D.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig2-data7-v2.zip
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Figure 2—source data 8
Raw digital image of thin-layer chromatography plate used in 2D.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig2-data8-v2.zip
Figure 3
Binding affinity of ancestral HEL-DUF proteins to blunt (BLT) and 3’ovr dsRNA in the presence and absence of ATP.
(A) Cartoon of dsRNAs used in (B–G) showing position of 5’ 32P (*) on top, sense strand. (B–G) Representative PhosphorImages showing gel mobility shift assays using select ancestral HEL-DUF …
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Figure 3—source data 1
Raw digital image of Gel Shift phosphoimager plate used in 3B.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig3-data1-v2.zip
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Figure 3—source data 2
Raw digital image of Gel Shift phosphoimager plate used in 3C.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig3-data2-v2.zip
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Figure 3—source data 3
Raw digital image of Gel Shift phosphoimager plate used in 3C.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig3-data3-v2.zip
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Figure 3—source data 4
Raw digital image of Gel Shift phosphoimager plate used in 3D.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig3-data4-v2.zip
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Figure 3—source data 5
Raw digital image of Gel Shift phosphoimager plate used in 3E.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig3-data5-v2.zip
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Figure 3—source data 6
Raw digital image of Gel Shift phosphoimager plate used in 3F.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig3-data6-v2.zip
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Figure 3—source data 7
Raw digital image of Gel Shift phosphoimager plate used in 3G.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig3-data7-v2.zip
Figure 4 with 4 supplements
Blunt (BLT) dsRNA improves efficiency of ATP hydrolysis by improving affinity of ATP to ancient HEL-DUF enzymes.
(A) Michaelis-Menten plots for basal and dsRNA-stimulated ATP hydrolysis by AncD1D2. Basal ATP hydrolysis measured at 500 nM AncD1D2, while dsRNA-stimulated hydrolysis is measured at 100 nM. …
Figure 4—figure supplement 1
Plots of ADP production over time for ancestral HEL-DUF constructs.
(A) Basal ATP hydrolysis by 500 nM AncD1D2, measured by linear ADP production over time for indicated ATP concentrations. Velocity of each reaction is the slope of the line. (B) dsRNA-stimulated ATP …
Figure 4—figure supplement 2
Multiple sequence alignment of ancestral HEL-DUF constructs and AncD1VERT rescue constructs.
Multiple sequence alignment for ancestral HEL-DUF constructs and vertebrate HEL-DUF rescue constructs, carried out with PRANK and illustrated with ESPript. Red shading/white text indicates identity, …
Figure 4—figure supplement 3
ATP hydrolysis of ancestral HEL-DUF proteins reconstructed from incongruent nodes.
(A–B) PhosphorImages of representative thin-layer chromatography (TLC) plates showing hydrolysis of 100 µM ATP (spiked with α-32P-ATP) by 200 nM AncD1ARTH/LOPH/DEUT (A) or AncD1LOPH/DEUT (B) in the …
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Figure 4—figure supplement 3—source data 1
Raw digital image of thin-layer chromatography plate used in Figure 4—figure supplement 3A, left panel.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig4-figsupp3-data1-v2.zip
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Figure 4—figure supplement 3—source data 2
Raw digital image of thin-layer chromatography plate used in Figure 4—figure supplement 3A, right panel.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig4-figsupp3-data2-v2.zip
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Figure 4—figure supplement 3—source data 3
Raw digital image of thin-layer chromatography plate used in Figure 4—figure supplement 3B.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig4-figsupp3-data3-v2.zip
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Figure 4—figure supplement 3—source data 4
Raw digital image of thin-layer chromatography plate used in Figure 4—figure supplement 3B.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig4-figsupp3-data4-v2.zip
Figure 4—figure supplement 4
Affinity of AncD1ARTH/LOPH/DEUT and AncD1LOPH/DEUT for binding blunt (BLT) and 3’ovr dsRNA in the absence and presence of ATP.
(A–D) Representative PhosphorImages showing gel mobility shift assays using select ancestral HEL-DUF constructs as indicated, and 42 base-pair BLT or 3’ovr dsRNA in the absence or presence of 5 mM …
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Figure 4—figure supplement 4—source data 1
Raw digital image of Gel Shift phosphoimager plate used in Figure 4—figure supplement 4A.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig4-figsupp4-data1-v2.zip
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Figure 4—figure supplement 4—source data 2
Raw digital image of Gel Shift phosphoimager plate used in Figure 4—figure supplement 4B.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig4-figsupp4-data2-v2.zip
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Figure 4—figure supplement 4—source data 3
Raw digital image of Gel Shift phosphoimager plate used in Figure 4—figure supplement 4C.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig4-figsupp4-data3-v2.zip
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Figure 4—figure supplement 4—source data 4
Raw digital image of Gel Shift phosphoimager plate used in Figure 4—figure supplement 4D.
- https://cdn.elifesciences.org/articles/85120/elife-85120-fig4-figsupp4-data4-v2.zip
Figure 5
dsRNA binding triggers conformational changes in the HEL-DUF domains of Dicer.
(A) Bottom-up view of the structure of Homo sapiens Dicer (hsDcr) in the apo state (PDB: 5ZAK). Helicase subdomains and DUF283 are colored. Rest of enzyme is transparent. (B) Bottom-up view of the …
Figure 6
Model of metazoan Dicer evolution showing transition from a two-site dsRNA binding in ancestral Dicer to a 1-site dsRNA binding state in extant vertebrate and arthropod Dicers.
Early animals possessed one promiscuous Dicer enzyme capable of using both platform/PAZ and helicase domains for dsRNA recognition. After gene duplication, arthropod Dicer-2’s helicase domain …
Tables
Table 1
Summary of kinetic data for ATP hydrolysis with 100 µM ATP.
| Construct | kburst (µM/min), NO RNA kobs (min–1) | kburst (µM/min), BLT dsRNA kobs (min–1) | kburst (µM/min), 3’ovr dsRNA kobs (min–1) |
|---|---|---|---|
| AncD1D2 | - 0.06±0.01 | 14.3±1.7 0.11±0.03 | 13.9±0.5 0.11±0.02 |
| AncD2ARTH | 6.47±0.8 0.05±0.01 | 19.3±0.9 0.04±0.02 | 14.6±2.3 0.08±0.02 |
| AncD1ARTH/LOPH/DEUT | - 0.01±0.01 | 25.1±0.7 0.41±0.02 | 16.0±3.8 0.21±0.04 |
| AncD1LOPH/DEUT | - 0.07±0.02 | 7.4±0.3 0.04±0.01 | 3.8±0.6 0.03±0.01 |
| AncD1DEUT | - 0.09±0.02 | 6.0±0.7 0.06±0.03 | 1.4±0.03 0.06±0.02 |
| AncD1VERT | - | - | - |
Table 2
Dissociation constants for dsRNA binding to ancestral HEL-DUFs.
| Construct | Kd (nM) BLT, NO ATP Hill coefficient | Kd (nM) 3’ovr, NO ATP Hill coefficient | Kd (nM) BLT, 5 mM ATPHill coefficient | Kd (nM) 3’ovr, 5 mM ATP Hill coefficient |
|---|---|---|---|---|
| AncD1D2 | 3.4±0.4 1.6±0.2 | 6.5±0.8 1.4±0.2 | 6.4±0.7 1.4±0.2 | 15.9±2.4 1.3±0.2 |
| AncD2ARTH | n.d. | n.d. | n.d. | n.d. |
| AncD1ARTH/LOPH/DEUT | 23.8±2.2 2.0±0.4 | 40.1±3.7 1.7±0.3 | 17.5±2.1 1.6±0.3 | 17.3±1.5 1.7±0.2 |
| AncD1LOPH/DEUT | 60.9±5.9 1.9±0.3 | 90.8±8.8 1.4±0.2 | 38.0±3.5 2.3±0.5 | 49.0±5.2 1.4±0.2 |
| AncD1DEUT | 145.1±9.1 2.3±0.3 | 140.0±8.9 2.3±0.3 | 131.8±8.7 2.2±0.3 | 149.8±8.3 2.4±0.3 |
| AncD1VERT | 502.4±40.5 2.6±0.5 | 537.8±47.5 2.8±0.6 | 592±48.6 2.2±0.4 | 500.3±58.0 1.9±0.4 |
Table 3
Michaelis-Menten parameters for steady state ATP hydrolysis reactions.
| Construct | kcat (min–1) | KM (µM) | kcat/KM (µM–1 min–1) |
|---|---|---|---|
| AncD1D2, no dsRNA | 1117±94.5 | 35812±6,367 | 0.031 |
| AncD1D2, BLT dsRNA | 147.8±6.3 | 256±67.5 | 0.577 |
| AncD1DEUT, no dsRNA | 144.1±14.9 | 2550±855 | 0.055 |
| AncD1DEUT, BLT dsRNA | 40.31±3.78 | 336.4±142 | 0.12 |
| AncD1VERT, no dsRNA | - | - | - |
| AncD1VERT.7, no dsRNA | 257.7±28.7 | 61739±12,886 | 0.004 |
| AncD1VERT.7, BLT dsRNA | 24.87±3.52 | 5173±1929 | 0.005 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Recombinant DNA reagent | pFastBac-OSF | Thermo Fisher Scientific | Cat# 10360014 | Modified in-house to add OSF tag |
| Cell line (Spodoptera frugiperda) | Sf9 | Expression Systems | Cat# 94–001 S | Suspension insect cells |
| Strain and strain background (Escherichia coli) | DH10Bac | Thermo Fisher Scientific | Cat #10361012 | Max Efficiency Competent Cells |
| Antibody | Anti-gp64-PE (mouse, monoclonal) | Expression Systems | Cat# 97–101 | Baculovirus Titering Kit |
| Chemical compound and drug | Cellfectin II | Thermo Fisher Scientific | Cat# 10362100 | Transfection reagent |
| Software and algorithm | RAXML-NG | RAXML-NG | RRID:SCR_022066 | |
| Sequence-based reagent | 42-nucleotide sense RNA | Integrated DNA Technologies (IDT) | Single-stranded RNA | GGGAAGCUCAGAAUA UUGCACAAGUAGAGC UUCUCGAUCCCC |
| Sequence-based reagent | 42-nucleotide BLUNT antisense RNA | IDT | Single-stranded RNA | GGGGAUCGAGAAGCU CUACUUGUGCAAUAU UCUGAGCUUCCC |
| Sequence-based reagent | 42-nucleotide 3’overhang antisense RNA | IDT | Single-stranded RNA | GGAUCGAGAAGCUCUA CUUGUGCAAUAUUCUG AGCUUCCCGG |
Additional files
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Supplementary file 1
Fasta file containing amino acid sequences of ancestrally reconstructed proteins and engineered protein constructs.
- https://cdn.elifesciences.org/articles/85120/elife-85120-supp1-v2.txt
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Supplementary file 2
Fasta file containing multiple sequence alignment file used as input for phylogeny construction and ancestral protein reconstruction.
Protein accession numbers from NCBI.
- https://cdn.elifesciences.org/articles/85120/elife-85120-supp2-v2.txt
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Supplementary file 3
Text file containing the reconstructed ancestral states for all ancestral nodes in the maximum likelihood phylogenetic tree.
Reconstructions represent primary amino acid sequences prior to removal of low-probability insertions using binary states generated from the BIN model in RAXML-NG.
- https://cdn.elifesciences.org/articles/85120/elife-85120-supp3-v2.txt
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Supplementary file 4
Text file containing the reconstructed ancestral states for all ancestral nodes in the maximum likelihood phylogenetic tree, using the multiple sequence alignment in the form of an absence-presence matrix and the BIN model in RAXML-NG for ancestral protein reconstruction.
One (1) represent the presence of amino acid residues, and zero (0) represents the absence. Protein accession numbers from NCBI.
- https://cdn.elifesciences.org/articles/85120/elife-85120-supp4-v2.txt
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Supplementary file 5
Newick file of maximum likelihood metazoan Dicer HEL-DUF phylogeny used for ancestral reconstruction (shown in Figure 1 and Figure 1—figure supplement 1).
- https://cdn.elifesciences.org/articles/85120/elife-85120-supp5-v2.txt
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Supplementary file 6
Newick file of maximum likelihood phylogeny with bilaterian species constrained (shown in Figure 1—figure supplement 2A).
Protein accession numbers from NCBI.
- https://cdn.elifesciences.org/articles/85120/elife-85120-supp6-v2.txt
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Supplementary file 7
Text files containing python scripts used to convert the amino acid multiple sequence alignment to binary alignment, overlay both alignments, and remove amino acids that correspond with absence (0) in the binary alignment.
- https://cdn.elifesciences.org/articles/85120/elife-85120-supp7-v2.txt
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MDAR checklist
- https://cdn.elifesciences.org/articles/85120/elife-85120-mdarchecklist1-v2.docx
