A Bipartite Model of 2-5A-dependent RNase L

Beihua Dong,Robert H Silverman

doi:10.1074/jbc.272.35.22236

Abstract

The 2-5A-dependent RNase (RNase L) is a tightly regulated endoribonuclease of higher vertebrates that is catalytically active only after engaging unusual effector molecules consisting of the 2',5'-linked oligoadenylates, p1-3A(2'p5'A)>/=2 (2-5A). Progressive truncations from either terminus have provided insight into the structure, function, and regulation of RNase L. We determined that deletion of the N-terminal 335 amino acids of RNase L, about 45% of the enzyme, produced a constitutively active endoribonuclease, thus effectively eliminating the requirement for 2-5A. The truncated nuclease had 6-fold lower catalytic activity against an oligo(rU) substrate than wild type RNase L. However, the two enzymes showed identical RNA cleavage site preferences with an mRNA as substrate. The repressor function required only the last three of a series of nine ankyrin-like repeats present in the N-terminal part of RNase L. In contrast, the entire ankyrin repeat region was necessary and sufficient for 2-5A binding activity. Deletion of a 10-amino acid sequence near the C terminus of RNase L, between residues 710 and 720, eliminated both the catalytic and RNA substrate binding functions of the enzyme. The ability to bind native RNase L in response to 2-5A required amino acid sequences near both termini of the protein. A bipartite model for the structure of RNase L emerged in which the regulatory functions of the molecule are located in the N-terminal half, while the catalytic domain is present in the C-terminal half.

Highlights

RNase L provides a unique paradigm of enzyme regulation because it is the only enzyme known to require the unusual 2Ј,5Ј-oligoadenylate(s), p1–3A(2Јp5ЈA)Ն2 (2-5A),1 for its catalytic activity [1, 2]
In murine RNase L, substitutions of both of the conserved P-loop lysine residues with asparagines resulted in a defect in 2-5A binding activity, while deletion of 89 C-terminal amino acids caused a loss of ribonuclease activity [2, 6]
We showed that the 2-5A binding domain of the murine RNase L was expressed in a polypeptide consisting of residues 1–342 [2]

Summary

A Bipartite Model of 2-5A-dependent RNase L*

We determined that deletion of the N-terminal 335 amino acids of RNase L, about 45% of the enzyme, produced a constitutively active endoribonuclease, effectively eliminating the requirement for 2-5A. The repressor function required only the last three of a series of nine ankyrin-like repeats present in the N-terminal part of RNase L. Deletion of a 10-amino acid sequence near the C terminus of RNase L, between residues 710 and 720, eliminated both the catalytic and RNA substrate binding functions of the enzyme. The ability to bind native RNase L in response to 2-5A required amino acid sequences near both termini of the protein. RNase L provides a unique paradigm of enzyme regulation because it is the only enzyme known to require the unusual 2Ј,5Ј-oligoadenylate(s), p1–3A(2Јp5ЈA)Ն2 (2-5A), for its catalytic activity [1, 2].

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION