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

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.

Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1

“…In the MESPEUS database of metal sites in proteins (mined from the PDB), there are 35 examples of Cys coordinating Mn ions, 6,023 examples of Cys coordinating Fe ions, and 8,069 examples of Cys coordinating Zn ions (41). The inability of the α-pocket of Dbr1 to bind Mn(II) was evident in previous work in which EDTA-treated EhDbr1 crystallized in the presence of 1 mM MnSO 4 with Mn(II) only in the β-pocket (24). Even when the concentration of MnSO 4 in the crystallization buffer was increased to 10 mM, there was no Mn in the α-pocket.…”

“…The resulting wild type and inactive C14S EhDbr1 structures were unexpectedly mononuclear, with Mn(II) in the β-pocket and a water molecule coordinated to the Mn(II) ion occupying the position expected for the putative α-site metal (26). Alignment of the backbones of structures of EhDbr1 in complex with product analog 5′-GMP and with a synthetic RNA possessing a 2′-phosphate monoester at the branchpoint adenosine (AK65), superimposed their 5′-and 2′-phosphate moieties, respectively, suggesting a model of an intact lariat branchpoint with flanking nucleotides bound at the active site (24). The conserved 28-residue insertion loop interacts extensively with nucleotides in the putative 3′-arm of the branchpoint and is thus termed the "lariat recognition loop."…”

“…The first Dbr1 structures determined were from Entamoeba histolytica (EhDbr1) (24). EhDbr1 is shorter than Dbr1 from higher eukaryotes (354 vs. ∼550 residues) that thus far have been recalcitrant to crystallization.…”

Design, Synthesis, and Properties of Phosphoramidate 2′,5′-Linked Branched RNA: Toward the Rational Design of Inhibitors of the RNA Lariat Debranching Enzyme

“…13 Loss of Dbr1 activity results in accumulation of RNA lariats, which are believed to sequester pathogenic forms of TAR DNA binding protein 43 (TDP-43), 15 an RNA-binding protein implicated in neurodegenerative diseases associated with aging. 16 In spinal cord neurons affected by amyotrophic lateral sclerosis (ALS), inclusions enriched in TDP-43 are hallmarks of the disease.…”

“…13 The crystal structure of the 2′,5′-PS-bRNA • Eh Dbr1 complex suggests that the sulfur atom of the phosphorothioate, which has a larger van der Waals radius than that of oxygen (Δ r w ~0.3 Å), sterically hinders the accomodation of the phosphorus linkage within the active site. This prompted us to move toward bRNA analogues containing a phosphoramidate (2′N-PO 2 -O5′) bridge instead, as the size of the nitrogen atom more closely resembles that of the natural oxygen atom in the phosphodiester bond.…”