Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.

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.

The stability of 2'-deoxy-2'-fluoroarabinonucleic acid (2'F-ANA) to hydrolysis under acidic and basic conditions was compared to that of DNA, RNA and 2'F-RNA. In enzyme-free simulated gastric fluid (pH approximately 1.2), 2'F-ANA was found to have dramatically increased stability (virtually no cleavage observed after 2 days) with respect to both DNA (t(1/2) approximately 2 min) and RNA (t(1/2) approximately 3 h (PO) or 3 days (PS)). These results were observed for both phosphodiester and phosphorothioate backbones and with multiple mixed-base sequences. Under basic conditions, 2'F-ANA also showed good stability. In 1 M NaOH at 65 degrees C, 2'F-ANA had a t(1/2) of approximately 20 h, while RNA was entirely degraded in a few minutes. Furthermore, the nuclease cleavage of phosphorothioate 2'F-ANA and DNA by snake venom phosphodiesterase was studied in detail. One diastereomer of the PS-2'F-ANA linkage was found to be much more vulnerable to enzymatic cleavage than the other, which is parallel to the properties observed for PS-DNA. Additional studies of 2'F-ANA-containing oligonucleotides are warranted based on the excellent stability properties described here.

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) ...

Turnover of the branched RNA intermediates and products of pre-mRNA splicing is mediated by the lariat-debranching enzyme Dbr1. We characterized a homolog of Dbr1 from Saccharomyces cerevisiae, Drn1/Ygr093w, that has a pseudometallophosphodiesterase domain with primary sequence homology to Dbr1 but lacks essential active site residues found in Dbr1. Whereas loss of Dbr1 results in lariat-introns failing broadly to turnover, loss of Drn1 causes low levels of lariat-intron accumulation. Conserved residues in the Drn1 C-terminal CwfJ domains, which are not present in Dbr1, are required for efficient intron turnover. Drn1 interacts with Dbr1, components of the Nineteen Complex, U2 snRNA, branched intermediates, and products of splicing. Drn1 enhances debranching catalyzed by Dbr1 in vitro, but does so without significantly improving the affinity of Dbr1 for branched RNA. Splicing carried out in in vitro extracts in the absence of Drn1 results in an accumulation of branched splicing intermediates and products released from the spliceosome, likely due to less active debranching, as well as the promiscuous release of cleaved 5 ′ -exon. Drn1 enhances Dbr1-mediated turnover of lariat-intermediates and lariat-intron products, indicating that branched RNA turnover is regulated at multiple steps during splicing.