Similarities between Argonautes and the alpha-sarcin-like ribotoxins: Implications for microRNA action

Abstract

We report structural, functional, and biochemical similarities between Argonautes, the effector proteins of RNA-induced silencing complexes (RISCs), and alpha-sarcin-like ribotoxins. At the structural level, regions of similarity in the amino acid sequence are located in protein loops both in the ribotoxins and in the Argonautes. In ribotoxins, these protein loops confer specificity for a highly conserved segment of ribosomal RNA, the Sarcin-Ricin-Loop (SRL) that undergoes cleavage by the ribotoxin ribonuclease. This leads to suppression of translation. In addition to the structural similarity with ribotoxins, the Argonaute proteins (Ago) show both functional and biochemical parallels. Like the ribotoxins, the Agos exhibit ribonuclease activity and like the ribotoxins, translational suppression mediated by miRISC-resident Ago is accompanied by intact polysomes. Furthermore, in both translationally suppressed systems, the puromycin reaction, reflecting correct translocation and peptidyl-transferase activities, is unharmed. These findings support a mechanism for Ago-miRISCs whereby regulated cleavage of ribosomal RNA leads to translational suppression.

Figure 1

Homology of Argonaute (Ago2) to ribotoxin (restrictocin). A: Argonaute 2 (bovine, accession number NP_991363) and the ribotoxin protein restrictocin (accession number AAA32707) were manually aligned. Location of the ribotoxin loops and beta-strands are indicated above the restrictocin sequence. Dashes in the restrictocin sequence indicate amino acid segments present in the Argonaute protein (the number of aa is indicated in square brackets in the Argonaute protein) and absent from restrictocin. B: Conservation of lysine residues in Argonaute and restrictocin.

Figure 2

Location of Ago sentinel loops homologous to the ribotoxin-restrictocin SRL-recognition loops on the Pyrococcus furiosus Argonaute protein. Left panel—Sentinel loops of the Ago protein (PDB: 1Z25) showing homology to ribotoxin loops are highlighted in dark blue and superimposed on the ribbon representation of the P. furiosus Ago protein [color scheme as in Ref. , N-terminal domain (blue), the “stalk” (light blue), the PAZ domain (red), the Mid domain (green), the PIWI domain (purple), and the interdomain connector (yellow)]. Homologous segments are indicated in the insets: identical amino acids and conserved substitutions are indicated by bold and regular red fonts, respectively. Right panel—Recognition of the SRL region of ribosomal RNA by the loop structures (indicated by dotted ovals) present on the ribotoxin protein (PDB: 1JBS). Note, the loop-recognition elements in the ribotoxin and the corresponding loops located in the Ago proteins (left panel), are indicated by red dashed ovals.

Figure 3

Model for mechanism of action of miRNA-mediated translational suppression. (A) The miRISC composed of the Argonaute protein (green) the miRNA (red) and ancillary proteins such as GW182 forms incompletely base-paired duplexes at 3′ UTR target sequences on discrete mRNAs (the curved blue line); (B and C) The Ago protein (green) in conjunction with the miRNA component (red line) recognizes and binds to a critical target site designated the SRL (enclosed by the dotted rectangle and enlarged in the inset) on the rRNA component of the large ribosome subunit. This is illustrated by the black “squiggly” line appearing on the large ribosomal subunit (pink oval). (D) The Ago protein associated with miRISC, after verging on its rRNA target site, cleaves the ribosomal RNA (cleavage indicated within the dotted red oval), thereby leading to disruption of protein synthesis: the miRISC translational suppression.