Endonuclease V cleaves at inosines in RNA

Abstract

Endonuclease V orthologues are highly conserved proteins found in all kingdoms of life. While the prokaryotic enzymes are DNA repair proteins for removal of deaminated adenosine (inosine) from the genome, no clear role for the eukaryotic counterparts has hitherto been described. Here we report that human endonuclease V (ENDOV) and also Escherichia coli endonuclease V are highly active ribonucleases specific for inosine in RNA. Inosines are normal residues in certain RNAs introduced by specific deaminases. Adenosine-to-inosine editing is essential for proper function of these transcripts and defects are linked to various human disease. Here we show that human ENDOV cleaves an RNA substrate containing inosine in a position corresponding to a biologically important site for deamination in the Gabra-3 transcript of the GABA(A) neurotransmitter. Further, human ENDOV specifically incises transfer RNAs with inosine in the wobble position. This previously unknown RNA incision activity may suggest a role for endonuclease V in normal RNA metabolism.

Figure 1. Various incision activities for E. coli and human endonuclease V at inosines in DNA and RNA.

The substrates (a) single-stranded (ss) RNA rI, (b) double-stranded (ds) RNA rI:rU, (c) ss RNA rA, (d) ssDNA dI, (e) ds DNA dI:dT were incubated with the wild-type enzymes (1–25 nM, as indicated) or the two site-specific mutants E. coli D35A and human D52A (25 nM) and reaction products analyzed by PAGE. RNA sizes (in nt) are indicated, –=no enzyme added, r=ribonucleotide and d=deoxynucleotide. (f) Single-turnover kinetic analysis with enzyme and substrates as indicated.

Figure 2. Incision activities for human ENDOV on RNA depends on a ribonucleotide 3′ to inosine.

The substrates (a) ds DNA:RNA dI:rU, (b) ds RNA:DNA rI:dT, (c) ss DNA dI rG were incubated with the wild-type enzymes (0.1–25 nM, as indicated) or the two site-specific mutants E. coli D35A and human D52A (25 nM) and reaction products analyzed by PAGE. (d) Structural model of the active site of human ENDOV with a ribonucleotide. The 2′ OH group is close to the conserved glutamate (E100), as well as the active site water molecule (Wat) coordinating the Mg²⁺ cofactor, which bridges the 3′- and 5′-ends in the incised product. (e) The endonuclease V enzymes were tested for activity against the ds DNA dI:dT rG:dC substrate as in a–c). RNA sizes (in nt) are indicated, –=no enzyme added, r=ribonucleotide and d=deoxynucleotide.

Figure 3. Human ENDOV cleaves at inosines in in vivo-like RNA substrates.

The substrates (a) mGabra3 rI, (b) mGabra3 rA, (c) tRNA^Arg rI, (d) tRNA^Arg rA were incubated with the endonuclease V enzymes (1–25 nM, as indicated) and reaction products analyzed by PAGE. RNA sizes (in nt) are indicated, –=no enzyme added, r=ribonucleotide and glyphs illustrate the different substrates.

Figure 4. Human ENDOV cleaves at inosines in tRNA.

Northern blots of tRNA isolated from human U373 cells incubated with human wild-type (0.3 and 0.6 μM) or mutant D52A (0.6 μM) ENDOV enzymes, hybridized with DNA probes complementary to the 5′-termini of (a) tRNA^Ser(AGA), (b) tRNA^Leu(AAG), (c) tRNA^Arg(ACG), (d) tRNA^Asp(GTC), (e) tRNA^Glu(CTC) and (f) tRNA^Lys(CTT). Size markers (80, 38 and 25 nt) are shown and glyphs indicate full-length and fragment tRNA species.