Figure 1. Mechanisms of long dsRNA digestion by RNase III enzymes

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Figure 1. Mechanisms of long dsRNA digestion by RNase III enzymes Figure 1. Mechanisms of long dsRNA digestion by RNase III enzymes. (A) A bacterial RNase III dimer recognizes the dsRNA ... Figure 1. Mechanisms of long dsRNA digestion by RNase III enzymes. (A) A bacterial RNase III dimer recognizes the dsRNA termini, especially those featuring a 2-nt 3′ overhang, cleaves both strands and produces a short RNA duplex of 11 nt in each strand. Under special conditions, two RNase III dimers bind to and cleave dsRNA in a cooperative manner, which produces an RNA duplex of 22 nt in each strand. (B) Human Dicer recognizes the dsRNA termini with a 2-nt 3′ overhang, cleaves both strands and produces an RNA duplex of 22 nt in each strand. (C) Two dimers of yeast Dcr1 cooperatively bind to and cleave a long dsRNA, producing RNA duplexes containing ∼22 nt in each strand. Unless provided in the caption above, the following copyright applies to the content of this slide: Published by Oxford University Press on behalf of Nucleic Acids Research 2019.This work is written by (a) US Government employee(s) and is in the public domain in the US. Nucleic Acids Res, gkz208, https://doi.org/10.1093/nar/gkz208 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 2. Bacterial RNase III cleaves long dsRNA at preferred sites Figure 2. Bacterial RNase III cleaves long dsRNA at preferred sites. (A) Schematic representation of the in vitro ... Figure 2. Bacterial RNase III cleaves long dsRNA at preferred sites. (A) Schematic representation of the in vitro cleavage experiment. (B) dsRNA of FF-luc sequence was cleaved by either Aquifex aeolicus or Escherichia coli RNase III. The cleavage products were separated on 20% polyacrylamide non-denaturing (native) gels and detected by staining. Synthetic single-stranded RNAs with certain length were used as markers. After next-generation sequencing, reads of cleavage products were mapped back to (C) FF-luc or (D) MBP, respectively. Coverage plots are presented. Unless provided in the caption above, the following copyright applies to the content of this slide: Published by Oxford University Press on behalf of Nucleic Acids Research 2019.This work is written by (a) US Government employee(s) and is in the public domain in the US. Nucleic Acids Res, gkz208, https://doi.org/10.1093/nar/gkz208 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 3. RNase III recognizes +3G in cleavage site selection Figure 3. RNase III recognizes +3G in cleavage site selection. (A) Cleavage sites were inferred by the ends of cleavage ... Figure 3. RNase III recognizes +3G in cleavage site selection. (A) Cleavage sites were inferred by the ends of cleavage products. Cleavage sites along FF-luc substrate were sorted by the counts of cleavage products’ ends and plotted. Cleavage sites supported by a count higher than 2,907, which is 5× the average (581.3), were defined as preferred cleavage sites (hotspots) and indicated by the dashed lines. (B) Sequence logos surrounding preferred AaRNase III cleavage sites on FF-luc and MBP are presented. Red arrowheads indicate the cleavage site (between positions 0 and -1), which is inferred from the end of cleavage products. Cleavage product upstream of cleavage site is in black, whereas cleavage products downstream of cleavage site are in red and boxed. Two green dashed-line boxes indicate two consensus nucleotides +3G and -6C. (C) Percentage of cleavage sites that contain +3G and/or -6C is higher than that expected by chance. (D) Schematic illustration of the RNase III:dsRNA complex. The +3G and -6C are symmetric relative to the cleavage sites that are indicated by red arrowheads. Cleavage products on one side of cleavage site are in black, whereas cleavage products on the other side of cleavage site is in red. Unless provided in the caption above, the following copyright applies to the content of this slide: Published by Oxford University Press on behalf of Nucleic Acids Research 2019.This work is written by (a) US Government employee(s) and is in the public domain in the US. Nucleic Acids Res, gkz208, https://doi.org/10.1093/nar/gkz208 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 4. Residue Q161 of AaRNase III recognizes the +3G near the cleavage site. (A) On the left: schematic ... Figure 4. Residue Q161 of AaRNase III recognizes the +3G near the cleavage site. (A) On the left: schematic illustration of the crystal structure of AaRNase III in complex with dsRNA (PDB entry: 2EZ6); On the right: residue Q161 recognizes the +3G by forming two base-specific hydrogen bonds and one hydrogen bond with the 2′-hydroxyl group. (B) The consensus sequence of cleavage site is abolished when residue 161 was mutated from Q to A. Sequence logos were created as illustrated in Figure 3. (C) Percentage of highly preferable cleavage sites of the AaRNase III Q161A mutant, with or without +3G and/or -6C, is similar to that expected by chance. Unless provided in the caption above, the following copyright applies to the content of this slide: Published by Oxford University Press on behalf of Nucleic Acids Research 2019.This work is written by (a) US Government employee(s) and is in the public domain in the US. Nucleic Acids Res, gkz208, https://doi.org/10.1093/nar/gkz208 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 5. Bacterial RNase III cleaves dsRNA into small RNAs with distinct patterns of length distribution. (A) The ... Figure 5. Bacterial RNase III cleaves dsRNA into small RNAs with distinct patterns of length distribution. (A) The length of RNase III cleavage products was measured by the next-generation sequencing analysis. The percentage of reads with certain length to total number of reads was plotted. (B) Cleavage products of FF-luc dsRNA were mapped back to the reference. Coverage plots of reads with certain length are presented. (C) A detailed look of one hotspot. The height of the bar represents the number of cleavage products covering each position. The reference sequence is listed below. Two potential cleavages are indicated: red arrows point to the cleavage sites; +3G (yellow) and -6C (blue) corresponding to each cleavage site are labeled. Unless provided in the caption above, the following copyright applies to the content of this slide: Published by Oxford University Press on behalf of Nucleic Acids Research 2019.This work is written by (a) US Government employee(s) and is in the public domain in the US. Nucleic Acids Res, gkz208, https://doi.org/10.1093/nar/gkz208 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 6. Length distribution of cleavage products is affected by the rate of product release. (A) On the left: cartoon ... Figure 6. Length distribution of cleavage products is affected by the rate of product release. (A) On the left: cartoon illustration of the crystal structure of the AaRNase III dimer in complex with dsRNA (PDB entry 2EZ6); on the right: zoom-in window shows relative positioning of side chains E37, E64 and Q161, of which the counterparts in EcRNase III are E38, E65 and Q165, respectively. (B–G) FF-luc and MBP dsRNAs were cleaved by various RNase III mutants. The length of products was compared to that of the wild-type. Fold changes in log scale are plotted against product lengths for EcE38A (B), EcE65A (C), EcQ165A (D), AaE37A (E), AaE64A (F) and AaQ161A (G). Unless provided in the caption above, the following copyright applies to the content of this slide: Published by Oxford University Press on behalf of Nucleic Acids Research 2019.This work is written by (a) US Government employee(s) and is in the public domain in the US. Nucleic Acids Res, gkz208, https://doi.org/10.1093/nar/gkz208 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 7. Engineered EcRNase III cleaves in vitro transcribed dsRNA into a heterogeneous mixture of siRNAs with a ... Figure 7. Engineered EcRNase III cleaves in vitro transcribed dsRNA into a heterogeneous mixture of siRNAs with a narrow size distribution centered at 22 nt. (A) The percentage of 22-nt products to the total number of reads was plotted for EcRNase III and its single mutants. (B) The percentage of 22-nt products to the total number of reads was plotted for EcRNase III and its double and triple mutants. (C) No consensus sequence of 22-nt cleavage products were detected in the triple mutant (EEQ) of EcRNase III. Unless provided in the caption above, the following copyright applies to the content of this slide: Published by Oxford University Press on behalf of Nucleic Acids Research 2019.This work is written by (a) US Government employee(s) and is in the public domain in the US. Nucleic Acids Res, gkz208, https://doi.org/10.1093/nar/gkz208 The content of this slide may be subject to copyright: please see the slide notes for details.