Skip to main content
. 2022 Jan 31;11:e73260. doi: 10.7554/eLife.73260

Figure 1. 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 can yield a 24 nt siRNA from the 5′ end of the Pol IV transcript paired to a 23 nt siRNA from the RDR2 3′ end. Pol IV transcripts tend to begin with A or G and RDR2 transcripts often have an untemplated nucleotide (N) at their 3′ termini. Green shading depicts DCL3 and its interaction with the left side of the dsRNA precursor. (C) A test of the model shown in (B). A 37 nt top strand was annealed to 37, 38, or 39 nt bottom strand to form dsRNA substrates with two blunt ends or a left-side 1 or 2 nt 3′ overhang on the bottom strand (see Supplementary file 1 for RNA strand sequences). Resulting dsRNAs (50 nM) were then incubated with 25 nM of affinity purified recombinant DCL3 (see Figure 1—figure supplement 1 – (A)). RNAs were then resolved by denaturing polyacrylamide gel electrophoresis (PAGE) and visualized using SYBR Gold staining. Lane 4 is a control that includes DCL3 but no RNA. (D) DCL3 prefers 3′ overhangs. Dicing reactions were conducted as in (C), but with either the top strand (37 nt) or bottom strand (38 or 39 nt) 5′ end-labeled with 32P and the final concentration of dsRNAs being 25 nM. In each case, a non-radioactive monophosphate is also present at the 5′ end of the complementary strand. Following incubation with (lanes 3, 4, 7, and 8) or without (lanes 1, 2, 5, and 6) DCL3, RNAs were resolved by denaturing PAGE and visualized by phosphorimaging. A related experiment comparing time courses of DCL3 cleavage for substrates with 1 or 2 nt overhangs is shown in Figure 1—figure supplement 1 – (B). (E) DCL3 cuts from both ends of precursors that have two blunt ends. Dicing reactions of 5′ end-labeled dsRNAs were conducted as in (E) but with precursors that lack a 3′ overhang at one end.

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.
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.
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.

Figure 1.

Figure 1—figure supplement 1. Affinity purification of recombinant DCL3.

Figure 1—figure supplement 1.

(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 Coomassie Blue staining. (B) Time course comparison of DCL3 dicing of substrates with either 1 or 2 nt 3′ overhangs. An end-labeled 37 nt top strand was annealed to 38 or 39 nt bottom strands to form dsRNA substrates with either 1 or 2 nt 3′ overhangs. Resulting dsRNAs (50 nM) were then incubated with 25 nM of affinity purified recombinant DCL3 for 1, 5, 10, or 15 min. RNAs were then resolved by denaturing polyacrylamide gel electrophoresis (PAGE) and visualized using SYBR Gold staining. Lanes 1 and 2 are controls that contain the different dsRNAs but no protein.
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).
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).