Binding and cleavage specificities of human Argonaute2

Abstract

The endonuclease Argonaute2 (Ago2) mediates the degradation of the target mRNA within the RNA-induced silencing complex. We determined the binding and cleavage properties of recombinant human Ago2. Human Ago2 was unable to cleave preformed RNA duplexes and exhibited weaker binding affinity for RNA duplexes compared with the single strand RNA. The enzyme exhibited greater RNase H activity in the presence of Mn2+ compared with Mg2+. Human Ago2 exhibited weaker binding affinities and reduced cleavage activities for antisense RNAs with either a 5'-terminal hydroxyl or abasic nucleotide. Binding kinetics suggest that the 5'-terminal heterocycle base nucleates the interaction between the enzyme and the antisense RNA, and the 5'-phosphate stabilizes the interaction. Mn2+ ameliorated the effects of the 5'-terminal hydroxyl or abasic nucleotide on Ago2 cleavage activity and binding affinity. Nucleotide substitutions at the 3' terminus of the antisense RNA had no effect on human Ago2 cleavage activity, whereas 2'-methoxyethyl substitutions at position 2 reduced binding and cleavage activity and 12-14 reduced the cleavage activity. RNase protection assays indicated that human Ago2 interacts with the first 14 nucleotides at the 5'-pole of the antisense RNA. Human Ago2 preloaded with the antisense RNA exhibited greater binding affinities for longer sense RNAs suggesting that the enzyme interacts with regions in the sense RNA outside the site for antisense hybridization. Finally, transiently expressed human Ago2 immunoprecipitated from HeLa cells contained the double strand RNA-binding protein human immunodeficiency virus, type 1, trans-activating response RNA-binding protein, and deletion mutants of Ago2 showed that trans-activating response RNA-binding protein interacts with the PIWI domain of the enzyme.

FIGURE 1.

Cleavage activity of human Ago2 for preformed RNA duplexes and single strand RNA. A, denaturing PAGE of 40-nucleotide sense RNA digested with human Ago2. Lanes 1 and 2, 19-nucleotide antisense RNA and 40-nucleotide sense RNA were annealed for 1 h prior to incubation with, respectively, GST-Ago2 or HA-Ago2 for 1 h. Lanes 3 and 4, 19-nucleotide antisense RNA was incubated with, respectively, GST-Ago2 or HA-Ago2 for 1 h prior to incubation with the 40-nucleotide sense RNA for an additional 1 h. T1, 40-nucleotide sense RNA digested with RNase T1 for 10 min at 24 °C. The positions of the T1 digestions adjacent to guanidine residues are shown next to the ladder. Arrow indicates the position of the human Ago2 cleavage site. The sequences of the 19-nucleotide antisense RNA and 5′-³²P-labeled (p) 40-nucleotide sense RNA are shown below the gel. B, denaturing polyacrylamide gel of 19-nucleotide sense RNA digested with GST-Ago2. Lane 1, 19-nucleotide antisense RNA was incubated with GST-Ago2 for 1 h prior to incubation with the 19-nucleotide sense RNA for an additional 1 h. Lane 2, 19-nucleotide antisense RNA and 19-nucleotide sense RNAs were annealed for 1 h prior to incubation with GST-Ago2 for 1 h. Arrow indicates the position of the human Ago2 cleavage site. The sequences of the 19-nucleotide antisense RNA (top sequence) and 5′-³²P-labeled (p) 19-nucleotide sense RNA (bottom sequence) are shown below the gel.

FIGURE 2.

Cleavage activities for human Ago2 containing various length antisense RNAs. A, denaturing PAGE of 40-nucleotide sense RNA digested with GST-Ago2 containing the antisense RNAs shown in C, ranging in length from 17 to 25 nucleotides. B, denaturing PAGE of 40-nucleotide sense RNA digested with HA-Ago2 containing the antisense RNAs shown in C. Antisense RNA was incubated with Ago2 for 1 h prior to incubation with the 40-nucleotide sense RNA for an additional 1 h. T1, 40-nucleotide sense RNA digested with RNase T1 for 10 min at 24 °C. The positions of the T1 digestions adjacent to guanidine residues are shown next to the ladder. Arrows indicate the position of the human Ago2 cleavage site. C, sequences of the antisense RNAs with the corresponding lane designations. The 19-nucleotide antisense RNA contained either a 5′-phosphate (p) or 5′-hydroxyl (OH). The 19-nucleotide antisense RNA containing a 5′-phosphate corresponds to the antisense RNA in Fig. 1. D, percent sense RNA cleaved with GST-Ago2 (solid bar) or HA-Ago2 (hatched bar). The standard errors reported for the Ago2 cleavage activities are based on three experiments. The antisense RNA sequences tested and corresponding lane designations are described in C.

FIGURE 3.

Influence of divalent cations and composition of the 3′ and 5′ terminus in the antisense RNA on human Ago2 cleavage activity. A, percent cleaved of the 40-nucleotide sense RNA by GST-Ago2 containing antisense sRNAs with various 5′-terminal substitutions (shown in B) in the presence of Mg²⁺ (solid bar) or Mn²⁺ (hatched bar). B, sequences of the unmodified 19-nucleotide antisense RNA (19-as) and antisense RNAs containing the following 5′-terminal substitutions: 5′-phosphorylated guanidine (p/G), 5′-phosphorylated abasic substitutions (p/a and p/aa), 5′-dephosphorylated uridine (OH/U), or 5′-dephosphorylated abasic substitutions (OH/a and OH/aa). C, percent cleaved of the 40-nucleotide sense RNA by GST-Ago2 containing antisense sRNAs with various 3′-terminal substitutions (shown in D) in the presence of Mg²⁺ (solid bar) or Mn²⁺ (hatched bar). D, sequences of the unmodified 19-nucleotide antisense RNA (19-as) and 21 (/21) nucleotide antisense RNAs containing the following 3′-terminal substitutions: riboadenine (AA), abasic residues (aa), deoxyriboadenine (_dAA), or deoxyribothymidine (_dTT). The errors reported for the Ago2 cleavage activities are based on three trials.

FIGURE 4.

Influence of divalent cations on the cleavage activities of human Ago2 containing antisense RNA with deoxyribonucleotide substitutions. A, denaturing PAGE of 40-nucleotide sense RNA digested with GST-Ago2 containing antisense RNAs with various deoxyribonucleotide substitutions in the presence of Mg²⁺. B, denaturing PAGE of 40-nucleotide sense RNA digested with GST-Ago2 containing antisense RNAs with various deoxyribonucleotide substitutions in the presence of Mn²⁺. Antisense RNA was incubated with Ago2 for 1 h prior to incubation with the 40-nucleotide sense RNA for an additional 1 h. Arrows indicate the position of the human Ago2 cleavage site. C, sequences of the antisense oligonucleotides with the corresponding lane designations. The antisense oligonucleotides contained either a 5′-phosphate (p) or 5′-hydroxyl (OH) and DNA substitutions (underlined). D, percent cleaved of the 40-nucleotide sense RNA by GST-Ago2 containing the antisense sequences shown in C in the presence of either Mg²⁺ (solid bar) or Mn²⁺ (hatched bar). The errors reported for the Ago2 cleavage activities are based on three trials.

FIGURE 5.

Effect of 2′-methoxyethyl substitutions in the antisense RNA on human Ago2 cleavage activity. A, denaturing PAGE of 40-nucleotide sense RNA digested with GST-Ago2 in the absence of antisense strand (none) and containing the antisense RNAs with 2′-methoxyethyl substitutions at various positions, counting from the 5′ terminus of the antisense RNA. The unmodified antisense RNA (0) corresponds to the 19-as RNA shown in Figs. 1–3. Arrow indicates the position of the human Ago2 cleavage site. B, sequences of the modified antisense RNAs containing the 2′-methoxyethyl substitutions at various positions (underlined).

FIGURE 6.

Characterization of the interaction between human Ago2 and TRBP. A, human Ago2 deletion mutants. Amino acid residues are numbered from the amino terminus of the enzyme. Human Ago2 deletion mutant 1 (D1) consists of amino acids 233–859 and contains the PAZ, Mid, and PIWI domains of the enzyme. Human Ago2 deletion mutant 2 (D2) consists of amino acids 371–859 and contains the Mid and PIWI domains. Human Ago2 deletion mutant 3 (D3) consists of amino acids 517–859 and contains the PAZ domain. B, Western blot analysis of HA-Ago2 expressed and immunoprecipitated from HeLa cells using HA antibody and probed with human Ago2 antibody. Lane 1, HeLa cell lysate; lane 2, immunoprecipitate from cells not transfected with HA-Ago2 plasmid; lanes 3–6, immunoprecipitates from cells transfected with, respectively, HA-Ago2, HA-D1, HA-D2, and HA-D3 plasmids. C, Western blot analysis of HA-Ago2 expressed and immunoprecipitated from HeLa cells using HA antibody and probed with human TRBP antibody. Lane 1, molecular weight ladder; lane 2, immunoprecipitate from cells not transfected with HA-Ago2 plasmid; lane 3, purified recombinant TRBP; lanes 4–7, immunoprecipitates from cells transfected with, respectively, Ha-Ago2, HA-D1, HA-D2, and HA-D3 plasmids.