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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.

SUBMITTER: Pichinuk E

PROVIDER: S-EPMC2895252 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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

Pichinuk Edward E Wreschner Daniel H DH

Protein science : a publication of the Protein Society 20100601 6

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 riboto ...[more]

PMID: 20512980

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Project description:alpha-Sarcin is a single polypeptide chain protein which exhibits antitumour activity by degrading the larger ribosomal RNA of tumour cells. We describe the interaction of a alpha-sarcin with lipid model systems. The protein specifically interacts with negatively-charged phospholipid vesicles, resulting in protein-lipid complexes which can be isolated by ultracentrifugation in a sucrose gradient. alpha-Sarcin causes aggregation of such vesicles. The extent of this interaction progressively decreases when the molar ratio of phosphatidylcholine increases in acidic vesicles. The kinetics of the vesicle aggregation induced by the protein have been measured. This process is dependent on the ratio of alpha-sarcin present in the protein-lipid system. A saturation plot is observed from phospholipid vesicles-protein titrations. The saturating protein/lipid molar ratio is 1:50. The effect produced by the antitumour protein on the lipid vesicles is dependent on neither the length nor the degree of unsaturation of the phospholipid acyl chain. However, the aggregation is dependent on temperature, being many times higher above the phase transition temperature of the corresponding phospholipid than below it. The effects of pH and ionic strength have also been considered. An increase in the ionic strength does not abolish the protein-lipid interaction. The effect of pH may be related to conformational changes of the protein. Binding experiments reveal a strong interaction between alpha-sarcin and acidic vesicles, with Kd = 0.06 microM. The peptide bonds of the protein are protected against trypsin hydrolysis upon binding to acidic vesicles. The interaction of the protein with phosphatidylglycerol vesicles does not modify the phase transition temperature of the lipid, although it decreases the amplitude of the change of fluorescence anisotropy associated to the co-operative melting of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labelled vesicles. The results are interpreted in terms of the existence of both electrostatic and hydrophobic components for the interaction between phospholipid vesicles and the antitumour protein.

Dataset Information

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

Publications

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

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