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

Tago, Nobuhiro; Katolik, Adam; Clark, Nathaniel E.; Eric, Montemayor,; Seio, Kohji; Sekine, Mitsuo; Hart, P. John; Damha, Masad J.

doi:10.1021/acs.joc.5b01719

Cited by 7 publications

(11 citation statements)

References 28 publications

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“…1B). The sequence integrity of the synthetic products was cross-validated by using recombinant Dbr1-mediated debranching assays, HPLC, and electrospray ionization mass spectrometry (25,30). AK88 is designed as a reporter of Dbr1 activity.…”

Section: Resultsmentioning

confidence: 99%

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

Clark

Katolik

Roberts

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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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) ...

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Section: Resultsmentioning

confidence: 99%

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

Clark

Katolik

Roberts

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…One view is that the reported EhiDbr1 structure reflects an incomplete active site. Alternatively, as speculated by Montemayor et al (2014) and Tago et al (2015), Dbr1 might diverge mechanistically from other structurally homologous metallophosphoesterase enzymes to catalyze phosphodiester hydrolysis via a single metal cofactor. In the hypothetical single-metal mechanism, the Dbr1 active site Cys is a cysteine thiolate anion that coordinates the water nucleophile together with the single manganese.…”

Section: Discussionmentioning

confidence: 99%

“…In the hypothetical single-metal mechanism, the Dbr1 active site Cys is a cysteine thiolate anion that coordinates the water nucleophile together with the single manganese. The cysteine thiolate would thereby act as a general base to abstract a proton from the water (Tago et al 2015).…”

Section: Discussionmentioning

confidence: 99%

“…To that end, Damha and colleagues have pursued synthesis of chemically modified branched RNAs as potential Dbr1 inhibitors. They reported that branched RNAs with a 2 ′ -phosphoramidate linkage are effective Dbr1 inhibitors in vitro and are themselves debranched slowly by Dbr1 (Tago et al 2015). Thus, the phosphoramidate branch analogs represent pseudosubstrate inhibitors.…”

Section: Introductionmentioning

confidence: 99%

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Mechanistic insights into the manganese-dependent phosphodiesterase activity of yeast Dbr1 with bis-p-nitrophenylphosphate and branched RNA substrates

Schwer

Khalid

Shuman

2016

RNA

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Saccharomyces cerevisiae Dbr1 is a manganese-dependent RNA debranching enzyme that cleaves the 2'-5' phosphodiester bond of the lariat introns formed during pre-mRNA splicing. Dbr1 is a member of the binuclear metallophosphoesterase enzyme superfamily. We showed previously via alanine scanning that RNA debranching in vivo and in vitro depends on conserved active site residues His13, Asp40, Asn85, His86, His179, His231, and His233. Here, by extending the alanine scan, we added Cys11 to the ensemble of essential active site components. We report that Dbr1 has a vigorous manganese-dependent phosphodiesterase activity with the non-RNA substrate bis-p-nitrophenylphosphate. Whereas RNA debranching requires His86, bis-p-nitrophenylphosphatase activity does not. We interpret these and other structure-activity relations reported here in light of the crystal structures of Entamoeba Dbr1 and other homologous binuclear metallophosphodiesterases. Our results suggest that (i) Dbr1 adheres to the two-metal mechanism of the enzyme superfamily, but is distinguished by its reliance on a Cys11-Xaa-His13 motif to engage one of the catalytic metals instead of the Asp-Xaa-His element typical of other clades within the superfamily; (ii) His86 is a general acid catalyst that protonates the O2' leaving group of the RNA 2'-5' phosphodiester; and (iii) the favorable pK of p-nitrophenol elides the strict need for a general acid during hydrolysis of bis-p-nitrophenylphosphate. The Dbr1 bis-p-nitrophenylphosphatase activity is well suited for high-throughput screening for inhibitors of debranching.

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“…These structures have been subsequently applied as signal amplifiers for the quantification of nucleic acids 17, 18 or to monitor lariat debranching enzyme activity. 19–21 However, synthesis of these nucleoside-based branching units having orthogonal protecting groups involves complex and lengthy synthetic procedures, hindering their widespread use.…”

Section: Introductionmentioning

confidence: 99%

Synthesis of comb-shaped DNA using a non-nucleosidic branching phosphoramidite

2018

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Branched DNAs (bDNAs) having comb-like structures have found wide utility in molecular diagnostics and DNA nanotechnology. bDNAs can be generated either by designing and assembling linear DNA molecules into rigid non-covalent structures or by using an orthogonally protected branching unit to synthesize covalently linked structures. Despite the advantages of the covalently linked structures, use of this motif has been hampered by the challenging synthesis of appropriately protected branching monomers. We report the facile synthesis of a branching monomer having orthogonal DMT and Lev protecting groups using readily available δ-velarolactone and 1,3-diaminopropan-2-ol. Using this branching monomer, a comb-shaped bDNA was synthesized having three different DNA arms. The synthesis and hybridization capability of the bDNA was assessed by fluorescence microscopy using fluorescently labeled complementary and mismatched DNA probes. Convenient access to an orthogonally protected branching monomer is anticipated to accelerate applications of bDNAs in applications including diagnostics, biosensing, gene-profiling, DNA computing, multicolor imaging, and nanotechnology.

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

Cited by 7 publications

References 28 publications

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

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

Mechanistic insights into the manganese-dependent phosphodiesterase activity of yeast Dbr1 with bis-p-nitrophenylphosphate and branched RNA substrates

Synthesis of comb-shaped DNA using a non-nucleosidic branching phosphoramidite

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