Title: 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 formore » 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.« less

Authors:

Clark, Nathaniel E.; Katolik, Adam; Roberts, Kenneth M.; Taylor, Alexander B.; Holloway, Stephen P.; Schuermann, Jonathan P.; Montemayor, Eric J. ;

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Stevens, Scott W.; Fitzpatrick, Paul F.; Damha, Masad J.; Hart, P. John

Publication Date:

Tue Dec 06 00:00:00 EST 2016

Research Org.:

Argonne National Laboratory (ANL), Argonne, IL (United States)

Sponsoring Org.:

USDOE; US Department of Veterans Affairs; Biomedical Laboratory Research and Development Service; S. Texas Veterans Health Care System; Welch Foundation; National Institutes of Health (NIH); National Science Foundation (NSF); San Antonio Cancer Inst.

OSTI Identifier:

1334675

Alternate Identifier(s):

OSTI ID: 1342237

Grant/Contract Number:  

AC02–06CH11357; BX0025801; AQ-1399; GM084246; T-32 AG021890; DBI-0905865; P41 GM103403; P30 CA054174; UL1 TR001120

Resource Type:

Published Article

Journal Name:

Proceedings of the National Academy of Sciences of the United States of America

Additional Journal Information:

Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 113 Journal Issue: 51; Journal ID: ISSN 0027-8424

Publisher:

National Academy of Sciences

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; RNA debranching; intron lariat; enzyme kinetics; X-ray crystallography; Dbr1