Abstract
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.
Highlights
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]
Human endonuclease V (EndoV) Has Inosine-specific Ribonuclease Activity When Ectopically Expressed in Human Cells—Human EndoV has been identified as a ribonuclease with specificity for inosine-containing RNA [16, 17]. As this result was obtained with recombinant enzyme purified from E. coli, we tested whether hEndoV showed the same catalytic activity when expressed in human cells
In FLAG-hEndoVwt extracts, a robust cleavage was observed both with the single- and double-stranded IIUI substrates (Fig. 1A, upper and middle panels) demonstrating that hEndoV is active at inosines in RNA when expressed in human cells
Summary
Human EndoV Has Inosine-specific Ribonuclease Activity When Ectopically Expressed in Human Cells—Human EndoV has been identified as a ribonuclease with specificity for inosine-containing RNA [16, 17]. In FLAG-hEndoVwt extracts, a robust cleavage was observed both with the single- and double-stranded IIUI substrates (Fig. 1A, upper and middle panels) demonstrating that hEndoV is active at inosines in RNA when expressed in human cells. Cleavage products of similar size as those generated by FLAGhEndoV and recombinant hEndoV were found in ResS fractions 10 –14 when using the single-stranded IIUI substrate, demonstrating the endogenous hEndoV has affinity for inosines in RNA (Fig. 2B). The gel revealed that for both endogenous and recombinant hEndoV, the preferred cleavage position was 3Ј to the middle insoine (I10) with some nicking next to the furthermost 5Ј inosine (I9) especially for the double-stranded substrate (Fig. 2D) It appears that expression both in human and E. coli cells give active and comparable hEndoV enzymes suggesting that there are no extensive post-translational modifications of hEndoV that are critical for enzymatic activity.
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