Structural basis of lariat RNA recognition by the intron debranching enzyme Dbr1.

Eric J Montemayor,Jonathan P Schuermann,Richard Johnsson,Alexander B Taylor,Nathaniel E Clark,Stephen P Holloway,Scott W Stevens,D Joshua Combs,Masad J Damha,P John Hart,Adam Katolik

doi:10.1093/nar/gku725

Abstract

The enzymatic processing of cellular RNA molecules requires selective recognition of unique chemical and topological features. The unusual 2′,5′-phosphodiester linkages in RNA lariats produced by the spliceosome must be hydrolyzed by the intron debranching enzyme (Dbr1) before they can be metabolized or processed into essential cellular factors, such as snoRNA and miRNA. Dbr1 is also involved in the propagation of retrotransposons and retroviruses, although the precise role played by the enzyme in these processes is poorly understood. Here, we report the first structures of Dbr1 alone and in complex with several synthetic RNA compounds that mimic the branchpoint in lariat RNA. The structures, together with functional data on Dbr1 variants, reveal the molecular basis for 2′,5′-phosphodiester recognition and explain why the enzyme lacks activity toward 3′,5′-phosphodiester linkages. The findings illuminate structure/function relationships in a unique enzyme that is central to eukaryotic RNA metabolism and set the stage for the rational design of inhibitors that may represent novel therapeutic agents to treat retroviral infections and neurodegenerative disease.

Highlights

The spliceosome excises introns from nascent messenger RNA [1] in the form of a lariat containing an unusual 2,5 phosphodiester linkage [2] (Figure 1). Hydrolysis of this linkage is rate-limiting in lariat degradation after splicing [3], and is required for efficient maturation of many small nucleolar RNAs and micro RNAs that are derived from intronic RNA [4,5]
When the protein components of the 5GMPDbr1 and AK65Dbr1 complex structures are superimposed, the 5 phosphate of 5GMP and the 2 -phosphate of AK65 superimpose, providing a view of the intact lariat RNA branchpoint bound to the enzyme (Figure 5a and b)
The 2 -nucleotide in the lariat branchpoint is derived from the 5GMP component, while the branchpoint nucleotide and flanking 5 and 3 nucleotides are derived from the AK65 component (Figures 1, 4a and b, 5b and 7b)

Summary

Introduction

The spliceosome excises introns from nascent messenger RNA [1] in the form of a lariat containing an unusual 2 ,5 phosphodiester linkage [2] (Figure 1). Hydrolysis of this linkage is rate-limiting in lariat degradation after splicing [3], and is required for efficient maturation of many small nucleolar RNAs (snoRNA) and micro RNAs (mirtrons) that are derived from intronic RNA [4,5]. Montemayor, Departments of Biochemistry and Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA

Methods

Results

Conclusion