Activation of 2′-5′ Oligoadenylate Synthetase by Single-stranded and Double-stranded RNA Aptamers

Rune Hartmann,Peder L Nørby,Pia M Martensen,Poul Jørgensen,Marion C James,Christian Jacobsen,Soren K Moestrup,Michael J Clemens,Just Justesen

doi:10.1074/jbc.273.6.3236

Rune Hartmann, Peder L Nørby + Show 7 more

Open Access

https://doi.org/10.1074/jbc.273.6.3236

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Abstract

A number of small RNA molecules that are high affinity ligands for the 46-kDa form of human 2'-5' oligoadenylate synthetase have been identified by the SELEX method. Surface plasmon resonance analysis indicates that these RNAs bind to the enzyme with dissociation constants in the nanomolar range. Competition experiments indicate that the binding site for the small RNAs on the 2'-5' oligoadenylate synthetase molecule at least partially overlaps that for the synthetic double-stranded RNA, poly(I).poly(C). Several of the RNAs function as potent activators of 2'-5' oligoadenylate synthetase in vitro, although there is no correlation between binding affinity and ability to activate. The RNA aptamers having the strongest activation potential appear to have few base-paired regions. This suggests that 2'-5' oligoadenylate synthetase, which has previously been believed to be activated only by double-stranded RNA, can also be activated by RNA ligands with little secondary structure. Since 2'-5' oligoadenylate synthetase possesses no homology to other known RNA-binding proteins, the development of small specific ligands by SELEX should facilitate studies of RNA-protein interactions and may reveal novel features of the structure-function relationships involving this enzyme.

Highlights

A number of small RNA molecules that are high affinity ligands for the 46-kDa form of human 2؅-5؅ oligoadenylate synthetase have been identified by the SELEX method
We have obtained a series of RNA aptamers using the SELEX method to select for binding to the 46-kDa form of human 2–5A synthetase
We examined 20 of these aptamers for in vitro activation of 2–5A synthetase using a simple assay previously developed in our laboratory [27] and have thereby identified a group of highly potent small RNA agonists of this enzyme

Summary

Introduction

A number of small RNA molecules that are high affinity ligands for the 46-kDa form of human 2؅-5؅ oligoadenylate synthetase have been identified by the SELEX method. The RNA aptamers having the strongest activation potential appear to have few base-paired regions This suggests that 2؅-5؅ oligoadenylate synthetase, which has previously been believed to be activated only by double-stranded RNA, can be activated by RNA ligands with little secondary structure. Double-stranded RNA (dsRNA) binds to and activates a number of interferon-induced proteins, namely the family of 2Ј-5Ј oligoadenylate synthetases (2–5A synthetase) and the protein kinase PKR The binding of TAR to PKR involves interaction of the minor groove of the doublestranded helix with a dsRNA binding motif (dsRBM) [15] This motif is not present in any of the known 2–5A synthetase sequences, and no other structural similarity has been found between PKR and 2–5A synthetase. No obvious homology is found between these proteins and 2–5A synthetase, but deletion mapping has suggested that the RNA binding domain of 2–5A synthetase is situated in the N-terminal region of the protein [17]

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