A DCL3 dicing code within Pol IV-RDR2 transcripts diversifies the siRNA pool guiding RNA-directed DNA methylation
- Department of Biology and Department of Molecular and Cellular Biochemistry, Indiana University Bloomington, United States
- Howard Hughes Medical Institute, Indiana University, United States
Figures
Figure 1 with 1 supplement
DCL3 preferentially dices double-stranded RNAs with 3′ overhangs.
(A) A simplified model of RNA-directed DNA methylation (RdDM) highlighting the roles of Pol IV, Pol V, RDR2, DCL3, and AGO4. (B) Model depicting the hypothesis that DCL3 dicing of dsRNA precursors …
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Figure 1—source data 1
Gel images for Figure 1C.
Raw gel images are provided next to annotated gel images showing the portions of the images used in the various panels of Figure 1C. The asterisk denotes a lanes containing size markers. Numbers indicate the lanes of Figure 1C. The images show SYBR Gold-stained RNAs resolved by denaturing polyacrylamide gel electrophoresis (PAGE) and imaged using a Bio-Rad Laboratories ChemiDoc MP Imaging System.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig1-data1-v2.pdf
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Figure 1—source data 2
Gel images for Figure 1D.
Raw gel images are provided next to annotated gel images showing the portions of the images used in the various panels of Figure 1D. The asterisk denotes a lane containing size markers. The images were obtained by phosphorimaging of dried polyacrylamide gels on which 32P-labeled RNA species were resolved by denaturing PAGE.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig1-data2-v2.pdf
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Figure 1—source data 3
Gel images for Figure 1E.
Raw gel images are provided next to annotated gel images showing the portions of the images used in the various panels of Figure 1E. Asterisks denote lanes containing size markers. The images were obtained by phosphorimaging of dried polyacrylamide gels on which 32P-labeled RNA species were resolved by denaturing PAGE.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig1-data3-v2.pdf
Figure 1—figure supplement 1
Affinity purification of recombinant DCL3.
(A) Recombinant wild-type (WT) DCL3 bearing a C-terminal FLAG tag was produced in insect cells and affinity purified using anti-FLAG resin. The purified protein was analyzed by SDS-PAGE and …
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Figure 1—figure supplement 1—source data 1
Source data for Figure 1—figure supplement 1A.
Duplicate digital images of a 4%–15% gradient SDS-PAGE gel stained with Coomassie Brilliant Blue. The red rectangle in the image on the right shows the portion of the raw image used in Figure 1—figure supplement 1A. The text above the right image indicates the fractions obtained during affinity purification of DCL3. The lane marked by an asterisk includes protein standards, with masses indicated in kilodaltons (kDa).
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig1-figsupp1-data1-v2.pdf
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Figure 1—figure supplement 1—source data 2
Source data for Figure 1—figure supplement 1B.
Duplicate images obtained by phosphorimaging of a dried denaturing PAGE gel on which 32P-labeled RNA species were resolved. The red rectangle in the image on the right shows the portion of the raw image used in Figure 1—figure supplement 1B. The lane marked by an asterisk contains RNA size standards whose lengths are shown in nucleotides (nt).
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig1-figsupp1-data2-v2.pdf
Figure 2
DCL3 measures 24 nt from the recessed 5′ end of a dsRNA with a 3′ overhang.
In the experiments shown in each panel of the figure, RNA strands ranging in size from 22 to 25 nt were annealed in various permutations to form dsRNA substrates that were then tested for DCL3 …
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Figure 2—source data 1
Source data for Figure 2A and C.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig2-data1-v2.pdf
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Figure 2—source data 2
Source data for Figure 2B and D.
Duplicate digital images of a denaturing PAGE gel showing RNA species stained with SYBR Gold. The red rectangles in the image on the right show the portions of the raw image used in lanes of Figure 2 panels B or D, as indicated. Lane 1 of Figure 2B is also lane 1 of Figure 2D. The lane marked by an asterisk contains RNA size standards, with lengths shown in nucleotides (nt).
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig2-data2-v2.pdf
Figure 3 with 1 supplement
Strand cutting specificities of DCL3’s RNase III domains.
(A) Relative positions of helicase, PAZ, RNase III, and dsRNA binding domains within the 1580 amino acid sequence of DCL3. Positions of catalytic glutamate residues of RNase III domain A (E1146) and …
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Figure 3—source data 1
Source data for Figure 3B.
Duplicate digital images of a denaturing PAGE gel with RNA species stained with SYBR Gold. The red rectangle in the image on the right shows the portions of the raw image used in Figure 3B, with corresponding lane numbers. The lane marked by an asterisk contains RNA size standards whose length is shown in nucleotides (nt).
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig3-data1-v2.pdf
Figure 3—figure supplement 1
Strand cutting specificities of DCL3’s two RNase III domains tested using a dsRNA substrate with a 2 nt 3′ overhang.
This experiment was performed as in Figure 3 except that the dsRNA substrate has a 2 nt overhang rather than a 1 nt overhang. In this case, a dsRNA formed by annealing 26 and 28 nt RNAs was …
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Figure 3—figure supplement 1—source data 1
Source data for Figure 3—figure supplement 1.
Duplicate digital images of a denaturing PAGE gel showing RNA species stained with SYBR Gold. The red rectangles in the image on the right show the portions of the raw image used in Figure 3—figure supplement 1, with the corresponding lane numbers. The lane marked by an asterisk contains RNA size standards whose length is shown in nucleotides (nt).
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig3-figsupp1-data1-v2.pdf
Figure 4
DCL3 substrate recognition is influenced by 5′ terminal nucleotide and phosphorylation status.
(A) Test of top strand 5′ nucleotide preference on dicing efficiency. Top strands of 37 nt that differ by having either A, U, C, or G at their 5′ termini were 5′ end-labeled with 32P and annealed to …
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Figure 4—source data 1
Gel image used in Figure 4A.
Duplicate digital images obtained by phosphorimaging of 32P-labeled RNAs resolved by denaturing PAGE are shown, with red rectangles showing the portion of the raw image used in Figure 4A.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig4-data1-v2.pdf
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Figure 4—source data 2
Gel image of replicate experiment providing quantitative data for Figure 4A.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig4-data2-v2.pdf
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Figure 4—source data 3
Gel image of replicate experiment providing quantitative data for Figure 4A.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig4-data3-v2.pdf
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Figure 4—source data 4
Source quantitative data for triplicate experiments of Figure 4A.
Data was obtained using Image Lab version 6.0 software. The 38 nt substrate and 24 nt diced RNA bands were boxed and % dsRNA substrate cleavage was calculated as the percentage of total signal (substrate + product) represented by the 24 nt product RNA band. Means for the triplicate reactions were calculated as well as the standard error of the mean.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig4-data4-v2.xlsx
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Figure 4—source data 5
Gel image used in Figure 4B.
Duplicate digital images obtained by phosphorimaging of 32P-labeled RNA resolved by denaturing PAGE are shown, with red rectangles showing the portion of the raw image used in Figure 4B.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig4-data5-v2.pdf
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Figure 4—source data 6
Gel image of replicate experiment providing quantitative data for Figure 4B.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig4-data6-v2.pdf
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Figure 4—source data 7
Gel image of replicate experiment providing quantitative data for Figure 4B.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig4-data7-v2.pdf
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Figure 4—source data 8
Source quantitative data for triplicate experiments of Figure 4B.
Data was obtained using Image Lab version 6.0 software. The 38 nt substrate and 24 nt diced RNA bands were boxed and % dsRNA substrate cleavage was calculated as the percentage of total signal (substrate + product) represented by the 24 nt product RNA band. Means for the triplicate reactions were calculated as well as the standard error of the mean.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig4-data8-v2.xlsx
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Figure 4—source data 9
Source data for Figure 4C.
Duplicate gel images obtained by phosphorimaging of 32P-labeled RNA resolved by denaturing PAGE. The red rectangle in the image on the right shows the portion of the raw image used in the figure.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig4-data9-v2.pdf
Figure 5 with 2 supplements
DCL3 dicing does not require ATP.
(A) 37 nt top strands end-labeled with 32P were annealed to 37 nt bottom strands to generate dsRNAs with blunt ends. Resulting dsRNAs at a concentration of 25 nM were then incubated with 5 nM of …
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Figure 5—source data 1
Raw gel image for Figure 5A.
Gel images were obtained by phosphorimaging of 32P-labeled RNAs resolved by denaturing PAGE.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig5-data1-v2.pdf
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Figure 5—source data 2
Raw gel image for Figure 5B.
Gel images were obtained by phosphorimaging of 32P-labeled RNAs resolved by denaturing PAGE.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig5-data2-v2.pdf
Figure 5—figure supplement 1
Additional evidence that ATP is not required for DCL3 dicing.
Assays like those of Figure 5 were conducted but included an additional dsRNA substrate that has a 3′ overhang of 2 nt. Reactions were incubated for 30 min.
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Figure 5—figure supplement 1—source data 1
Raw gel image and the portion of the image used for Figure 5—figure supplement 1.
The gel image was obtained by phosphorimaging of 32P-labeled RNAs resolved by denaturing PAGE.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig5-figsupp1-data1-v2.pdf
Figure 5—figure supplement 2
Controls demonstrating ATP destruction by Apyrase.
(A) Apyrase was incubated with gamma-32P-ATP then heat-inactivated. Aliquots of the apyrase reaction (lane 2) and the mock apyrase reaction (lane 1) were then subjected to thin-layer chromatography …
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Figure 5—figure supplement 2—source data 1
Raw image of a thin-layer chromatogram on which 32P-ATP is resolved from 32P phosphate (Pi) following apyrase treatment, and the rectangle denotes the portion of the raw image used for Figure 5—figure supplement 2A.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig5-figsupp2-data1-v2.pdf
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Figure 5—figure supplement 2—source data 2
Gel image and portion used for Figure 5—figure supplement 2B.
The image was obtained by phosphorimaging of a 32P-end-labeling reaction using a 37 nt RNA, followed by denaturing PAGE.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig5-figsupp2-data2-v2.pdf
Figure 6
Overhangs at both ends of DCL3 substrates explain 24 and 23 nt siRNA biogenesis from both strands.
(A) Hypotheses to account for 24 and 23 nt siRNAs derived from both the Pol IV and RDR2-transcribed strands of diced dsRNAs. Scenario 1 is the hypothesis of Figure 1B and Singh et al., 2019, …
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Figure 6—source data 1
Raw gel image and the portion used for Figure 6B.
The image is that of a sheet of X-ray filmdeveloped after exposure to a dried denaturing PAGE gel on which 32P- labelledRNA species were resolved.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig6-data1-v2.pdf
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Figure 6—source data 2
Raw gel image and the portion used for Figure 6C.
Duplicate images were obtained by phosphorimaging of a dried polyacrylamide gel on which 32P- labelled RNA species were resolved by denaturing gel electrophoresis. The red rectangle in the image on the right shows the portion of the raw image used in the figure.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig6-data2-v2.pdf
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Figure 6—source data 3
Raw gel images and the portions used for Figure 6D.
In the top and bottom rows are duplicated images of two X-ray films developed after exposure to dried polyacrylamide gels on which 32P- labelled RNA species were resolved by denaturing gel electrophoresis. In the top and bottom rows are duplicated images of two X-ray films developed after exposure to dried polyacrylamide gels on which 32P- labelled RNA species were resolved by denaturing gel electrophoresis. The red rectangles in the bottom row show the groups of lanes used in the left, central and right portions of Figure 6D.
- https://cdn.elifesciences.org/articles/73260/elife-73260-fig6-data3-v2.pdf
Figure 7
Summary model for a DCL3 dicing code that diversifies the siRNA pool guiding RNA-directed DNA methylation.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Arabidopsis thaliana) | FLAG-DCL3 | Singh et al., 2019 | Synthetic gene codon-optimized for insect cells | |
| Cell line (Trichoplusia ni) | High Five Cells in Express Five Medium | Thermo Fisher Scientific | Cat # B85502 | For baculovirus replication and expression |
| Transfected construct (Escherichia coli) | pUC57-DCL3 (synthetic DCL3, codon optimized for insect cells) | Singh et al., 2019 | N/A | Strain carrying cloned FLAG-DCL3 gene |
| Transfected construct (T. ni) | pFastBacHT B-DCL3 | Singh et al., 2019 | N/A | Construct for baculovirus expression |
| Transfected construct (T. ni) | pFastBacHT B-DCL3_E1146Q_E1329Q | This paper | N/A | RNase III domain A/B double mutant |
| Transfected construct (T. ni) | pFastBacHT B-DCL3_E1146Q | This paper | N/A | RNase III domain mutant |
| Transfected construct (T. ni) | pFastBacHT B-DCL3_E1329Q | This paper | N/A | RNase III domain mutant |
| Chemical compound, drug | PMSF | Sigma-Aldrich | Cat # P7626 | Protease inhibitor |
| Chemical compound, drug | GlycoBlue | Thermo Fisher Scientific | Cat # AM9515 | |
| Chemical compound, drug | Ribolock RNase Inhibitor | Thermo Fisher Scientific | Cat # EO0384 | |
| Chemical compound, drug | RNase Inhibitor (Murine) | NEB | Cat # M0314 | |
| Chemical compound, drug | S1 nuclease | Promega | Cat # M5761 | |
| Chemical compound, drug | Proteinase K, RNA grade | Invitrogen | Cat # 25530049 | |
| Chemical compound, drug | T4 Polynucleotide Kinase | NEB | Cat # M0201S | Enzyme for end-labeling RNA |
| Chemical compound, drug | Adenosine 5′-triphosphate magnesium salt | Sigma-Aldrich | Cat # A9187 | |
| Chemical compound, drug | [γ32P]-ATP, 6000 Ci/mmol | PerkinElmer | Cat #BLU002Z250UC | Used for end-labeling RNA |
| Chemical compound, drug | Apyrase | NEB | Cat # M0398L | Hydrolyzes ATP; used in Figure 5 |
| Chemical compound, drug | SYBR Gold Nucleic Acid Gel Stain (10,000×) | Invitrogen | Cat # S11494 | |
| Chemical compound, drug | RNA Loading Dye (2×) | NEB | Cat # B0363S | |
| Chemical compound, drug | 2× TBE-Urea Sample Buffer | Invitrogen | Cat # LC6876 | |
| Chemical compound, drug | IPEGAL CA-630 | Sigma-Aldrich | Cat # I8896 | Dete |
| Chemical compound, drug | Set of rATP, rUTP, rCTP, and rGTP | Sigma-Aldrich | Cat # A1388 | Used for Pol IV-RDR2 transcription |
| Chemical compound, drug | Anti-FLAG M2 affinity gel | Sigma-Aldrich | Cat # A2220 | |
| Commercial assay or kit | Vaccinia capping system | NEB | Cat # M2080S | 5′ end-capping in Figure 6 |
| Software, algorithm | Image Lab 6.0.1 | Bio-Rad | Cat # 12012931 | |
| Other | Express FiveSFM | Thermo Fisher Scientific | Cat # 10486025 | Serum-free medium for High Five cells |
| Other | Macherey-Nagel Polygram CEL 300 PEI/UV254 Polyester Sheets | Thermo Fisher Scientific | Cat # 10013021 | For TLC assay of ATP hydrolysis |
Additional files
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Supplementary file 1
Oligonucleotides used in the study.
(a) RNA oligonucleotides used for DCL3 dicing assays. (b) Oligonucleotides used for Pol IV and RDR2 transcription assays.
- https://cdn.elifesciences.org/articles/73260/elife-73260-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/73260/elife-73260-transrepform1-v2.docx
