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 GABAA 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.
Endonuclease V (EndoV) is an enzyme with specificity for inosines in nucleic acids. Whereas the bacterial homologs are active on both DNA and RNA, the mammalian variants only cleave RNA, at least when assayed with recombinant proteins. Here we show that ectopically expressed, as well as endogenously expressed human (h)EndoV, share the same enzymatic properties as the recombinant protein and cleaves RNA with inosine but not DNA. In search for proteins interacting with hEndoV, polyadenylate-binding protein C1 (PABPC1) was identified. The association between PABPC1 and hEndoV is RNA dependent and furthermore, PABPC1 stimulates hEndoV activity and affinity for inosine-containing RNA. Upon cellular stress, PABPC1 relocates to cytoplasmic stress granules that are multimolecular aggregates of stalled translation initiation complexes formed to aid cell recovery. Arsenite, as well as other agents, triggered relocalization also of hEndoV to cytoplasmic stress granules. As inosines in RNA are highly abundant, hEndoV activity is likely regulated in cells to avoid aberrant cleavage of inosine-containing transcripts. Indeed, we find that hEndoV cleavage is inhibited by normal intracellular ATP concentrations. The ATP stores inside a cell do not overlay stress granules and we suggest that hEndoV is redistributed to stress granules as a strategy to create a local environment low in ATP to permit hEndoV activity.Deamination of adenosine (A) to inosine (I) is a reaction that occurs spontaneously in cells and is enhanced by exposure to nitrosative agents formed as a response to inflammation, infection, or from the environment (1, 2). Both DNA and RNA as well as unincorporated (deoxy)ribonucleotides are subjected to deamination (3). Inosine is read as guanosine (G) by cellular proteins, and in DNA this event is thus considered mutagenic (3, 4). Consequences of adenosine deamination in RNA depend on which type and part of a transcript is deaminated and may result in altered coding, splicing, stability, and structure of an RNA or in intracellular relocalization (5).In addition to arbitrary deamination events, inosine is also introduced in RNA by specific enzymes in a highly regulated manner to increase transcriptomic diversity (6). Responsible enzymes are the adenosine deaminases acting on RNA (ADARs) 2 that catalyze A to I editing on mRNA and non-coding (nc)RNA including long ncRNA, micro (mi)RNA and small interfering (si)RNA. A to I editing is abundant in higher eukaryotes and edited sites amount to more than 100 million and are spread over the majority of human genes (7). Defective editing is linked to various human diseases including neurological disorders, infections, and cancer (8 -11). Also some tRNAs undergo A to I editing at the anticodon wobble adenosine A 34 , a reaction that is catalyzed by adenosine deaminases acting on tRNA (ADATs) (12). ADATs are homologs to the ADARs and their editing is essential for protein synthesis (12, 13).Until recently, no enzymatic activity specific for inosine in RNA was known. However, endon...
Endonuclease V (EndoV) is an enzyme with specificity for deaminated adenosine (inosine) in nucleic acids. EndoV from Escherichia coli (EcEndoV) acts both on inosines in DNA and RNA, whereas the human homolog cleaves only at inosines in RNA. Inosines in DNA are mutagenic and the role of EndoV in DNA repair is well established. In contrast, the biological function of EndoV in RNA processing is largely unexplored. Here we have characterized a second mammalian EndoV homolog, mouse EndoV (mEndoV), and show that mEndoV shares the same RNA selectivity as human EndoV (hEndoV). Mouse EndoV cleaves the same inosine-containing substrates as hEndoV, but with reduced efficiencies. The crystal structure of mEndoV reveals a conformation different from the hEndoV and prokaryotic EndoV structures, particularly for the conserved tyrosine in the wedge motif, suggesting that this strand separating element has some flexibility. Molecular dynamics simulations of mouse and human EndoV reveal alternative conformations for the invariant tyrosine. The configuration of the active site, on the other hand, is very similar between the prokaryotic and mammalian versions of EndoV.
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