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

Abstract

Intron lariats are circular, branched RNAs (bRNAs) produced during pre-mRNA splicing. Their unusual chemical and topological properties arise from branch-point nucleotides harboring vicinal 2′,5′-and 3′,5′-phosphodiester linkages. The 2′,5′-bonds must be hydrolyzed by the RNA debranching enzyme Dbr1 before spliced introns can be degraded or processed into small nucleolar RNA and microRNA derived from intronic RNA. Here, we measure the activity of Dbr1 from Entamoeba histolytica by using a synthetic, dark-quenched bRNA substrate that fluoresces upon hydrolysis. Purified enzyme contains nearly stoichiometric equivalents of Fe and Zn per polypeptide and demonstrates turnover rates of ∼3 s −1. Similar rates are observed when apo-Dbr1 is reconstituted with Fe(II)+Zn(II) under aerobic conditions. Under anaerobic conditions, a rate of ∼4.0 s −1 is observed when apoenzyme is reconstituted with Fe(II). In contrast, apo-Dbr1 reconstituted with Mn(II) or Fe(II) under aerobic conditions is inactive. Diffraction data from crystals of purified enzyme using X-rays tuned to the Fe absorption edge show Fe partitions primarily to the β-pocket and Zn to the α-pocket. Structures of the catalytic mutant H91A in complex with 7-mer and 16-mer synthetic bRNAs reveal bona fide RNA branchpoints in the Dbr1 active site. A bridging hydroxide is in optimal position for nucleophilic attack of the scissile phosphate. The results clarify uncertainties regarding structure/function relationships in Dbr1 enzymes, and the fluorogenic probe permits high-throughput screening for inhibitors that may hold promise as treatments for retroviral infections and neurodegenerative disease.RNA debranching | intron lariat | enzyme kinetics | X-ray crystallography | Dbr1 T he enzymatic processing of diverse RNA molecules requires selective recognition of their unique physicochemical properties. The sequential trans-esterification reactions catalyzed by the spliceosome yield mature messenger RNA (mRNA) and excised intron lariats (1, 2), the latter of which contain internal branchpoint adenosine nucleotides harboring vicinal 2′,5′-and 3′,5′-phosphodiester linkages (3). Mature mRNA transcripts are exported to the cytosol for protein synthesis, but lariat introns must be linearized before they can be turned over or processed into the subset of small nucleolar RNAs and microRNAs that are derived from intronic RNA (4, 5). The lariat forms when the 2′-hydroxyl group of an adenosine nucleotide near the 3′-end of the intron acts as the nucleophile to attack the 5′-splice site, producing 5′-exon-3′-OH and intron lariat/ 3′-exon intermediates. The 3′-hydroxyl group of the 5′-exon-3′-OH intermediate subsequently acts as the nucleophile to attack the 3′-splice site, resulting in intron excision and exon ligation (6, 7) (Fig. 1A). The resulting vicinal 2′,5′-and 3′,5′-phosphodiester linkages confer unique topological and chemical features to the branchpoint and flanking nucleotides, and these lariats persist in yeast cells lacking active Dbr1 (RNA lariat debranching enzyme) ...

Crystal structure of the Entamoeba histolytica RNA lariat debranching enzyme EhDbr1 reveals a catalytic Zn2+/Mn2+ heterobinucleation

“…Our structure reveals active site features that are consistent with the previously determined structures . Namely, residues His16, Asp45, Asn90, His180, His230 and His232 directly coordinate two metal ion‐binding sites and have conserved counterparts with Mre11 proteins.…”

“…Namely, residues His16, Asp45, Asn90, His180, His230 and His232 directly coordinate two metal ion-binding sites and have conserved counterparts with Mre11 proteins. Superposition of the active site of our structure model with the active site of the Clark et al [19] model, demonstrates the similar coordinations of both metal-binding sites (Fig. 1B).…”

“…Superposition of the active site of our structure model with the active site of the Clark et al . model, demonstrates the similar coordinations of both metal‐binding sites (Fig. B).…”

“…Additionally, the metal enrichment that we observe may be catalytically suboptimal to the Fe 2+ /Zn 2+ that is observed in the EhDbr1 sample from Clark et al . .…”

Debranchase-resistant labeling of RNA using the 10DM24 deoxyribozyme and fluorescent modified nucleotides

“…16, 17 Therefore, we reasoned that if 10DM24 could incorporate fluorescently modified GMP moieties with the proper phosphorothioate stereochemistry, then the resulting labeled RNAs should be stable to cellular debranching activities. We synthesized α-S- Sp - and α-S- Rp -diastereomers of GTP (also knonw as Sp -GTP S and Rp -GTP S , respectively) and resolved the two diastereomers by RP-HPLC according to known procedures.…”