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doi: 10.1261/rna.079159.122. Epub 2022 Apr 22.

Metal content and kinetic properties of yeast RNA lariat debranching enzyme Dbr1

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Metal content and kinetic properties of yeast RNA lariat debranching enzyme Dbr1

Nathaniel E Clark et al. RNA.2022 Jul.

Abstract

In eukaryotic cells, intron lariats produced by the spliceosome contain a 2'5' phosphodiester linkage. The RNA lariat debranching enzyme, Dbr1, is the only enzyme known to hydrolyze this bond. Dbr1 is a member of the metallophosphoesterase (MPE) family of enzymes, and recent X-ray crystal structures and biochemistry data demonstrate that Dbr1 fromEntamoeba histolytica uses combinations of Mn2+, Zn2+, and Fe2+ as enzymatic cofactors. Here, we examine the kinetic properties and metal dependence of the Dbr1 homolog fromSaccharomyces cerevisiae (yDbr1). Elemental analysis measured stoichiometric quantities of Fe and Zn in yDbr1 purified following heterologous expressionE. coli We analyzed the ability of Fe2+, Zn2+, and Mn2+ to reconstitute activity in metal-free apoenzyme. Purified yDbr1 was highly active, turning over substrate at 5.6 sec-1, and apo-yDbr1 reconstituted with Fe2+ was the most active species, turning over at 9.2 sec-1 We treated human lymphoblastoid cells with the iron-chelator deferoxamine and measured a twofold increase in cellular lariats. These data suggest that Fe is an important biological cofactor for Dbr1 enzymes.

Keywords: RNA; debranching enzyme; introns; lariats; metalloenzymes; metallophosphoesterase.

© 2022 Clark et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

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Figures

FIGURE 1.
FIGURE 1.
Homology model of yeast Dbr1. (A) The yDbr1 homology model was calculated from EhDbr1 PDB 5k73. The α-pocket contains a Zn2+ ion, and the β-pocket contains an Fe2+ ion. All seven metal ligands are identical between EhDbr1(gray backbone) and yDbr1 (tan backbone). The OH− nucleophile is the red sphere. (B) Summary of the metal content of all EhDbr1 structures in the PDB. The metals found in the α and β pockets are indicated, along with PDB ID, and a description of the unique ligand if present.
FIGURE 2.
FIGURE 2.
Metal content and steady state kinetics of recombinant yDbr1. (A) Molar equivalents of metals ions for recombinant and apo yDbr1 measured with ICP-MS. Zn and Fe were present at 2.7 and 0.7 molar equivalents, while Ni, Cu, Mn, and Co are <0.02 equivalents. Apo-yDbr1 has <0.02 equivalents of all metals. (B) Schematic of the fluorescent AK88 bRNA substrate. When intact, the “black-hole” quencher DABCYL prevents the fluorescein (FAM) from emitting light at 488 nm. Following Dbr1 cleavage, the quencher is no longer proximal to the FAM which becomes highly fluorescent. (C) Steady-state kinetic analysis of recombinant yDbr1. The AK88 bRNA was prepared at concentrations from 0.08–5 µM and initial rates of product formation were measured. The resulting data were fit with the Michaelis–Menten equation to obtain estimates ofkcat = 5.6 sec−1,Km = 0.7 µM. No metal ions were added to any component of these reactions; the only metal ions present are those that copurified with the enzyme or those present as trace metal contaminants.
FIGURE 3.
FIGURE 3.
Reconstitution of apo-yDbr1 with Fe2+, Zn2+, and Mn2+. (A) Apo-yDbr1 (1 µM) was reconstituted with 0–100 µM of the indicated metals under anaerobic conditions. Apo-yDbr1, black diamonds at metal = 0, had no detectable activity without exogenous metals. (B) Results of curve fitting to obtain estimates ofkcat and Kmetal.
FIGURE 4.
FIGURE 4.
The iron-chelator DFO increases lariat levels in lymphoblastoid cells. (A) Genome browser screen capture showing raw read counts for the SUDS3 intron 6, an example of a lariat that is elevated in DFO-treated cells. The read density fall-off at the 3′ and 5′ splice sites is characteristic of intron lariats. (B) Lariat enrichment, calculated from branchpoint-traversing reads, is shown for duplicate samples of DFO-treated lymphoblastoid cells and controls. DFO treatment elevates lariats approximately twofold. (C) Plot of log2 fold change in lariat abundance of DFO-treated versus untreated controls cells. (D) DFO treatment does not significantly changeDBR1 expression level, FDR-adjustedP-value = 0.18. (E) DFO and EDTA inhibit Dbr1 in a time-dependent manner, with 67% and 38% inhibition, respectively, after 3 h.
Nathaniel Clark
Nathaniel Clark
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