Abstract
Inosine may arise in DNA as a result of oxidative deamination of adenine or misincorporation of deoxyinosine triphosphate during replication. On the other hand, the occurrence of inosine in RNA is considered a normal and essential modification induced by specific adenosine deaminases acting on mRNA and tRNA. In prokaryotes, endonuclease V (EndoV) can recognize and cleave inosine-containing DNA. In contrast, mammalian EndoVs preferentially cleave inosine-containing RNA, suggesting a role in RNA metabolism for the eukaryotic members of this protein family. We have performed a biochemical characterization of EndoV from the protozoan parasite Trypanosoma brucei. In vitro, TbEndoV efficiently processes single-stranded RNA oligonucleotides with inosine, including A to I-edited tRNA-like substrates but exhibits weak activity over DNA, except when a ribonucleotide is placed 3′ to the inosine. Immunolocalization studies performed in procyclic forms indicate that TbEndoV is mainly cytosolic yet upon nutritional stress it redistributes and accumulates in stress granules colocalizing with the DEAD-box helicase TbDhh1. RNAi-mediated depletion of TbEndoV results in moderate growth defects in procyclic cells while the two EndoV alleles could be readily knocked out in bloodstream forms. Taken together, these observations suggest an important role of TbEndoV in RNA metabolism in procyclic forms of the parasite.
Highlights
Nucleic acids are continuously exposed to endogenous and environmental damaging agents
E. coli endonuclease V (EndoV) has been described as a DNA repair enzyme for many years, it can act on inosine at RNA with
Homology searching of the Trypanosoma brucei brucei genome database, using the human endonuclease V ortholog as the query sequence, identified a protein annotated as a putative endonuclease V (Tb927.10.6860) composed of 316 amino acids and a calculated mass of 34.9 kDa
Summary
Nucleic acids are continuously exposed to endogenous and environmental damaging agents. In spite of its broad substrate specificity, biochemical and genetic evidences led to conclude a preferential role in the repair of deoxyinosine (the deoxynucleoside containing Hx) in double- and single-stranded DNA8, 15, 16. In both cases, EndoV catalyzes the cleavage of the second phosphodiester bond 3′ to the lesion without releasing the erroneous nucleotide[14]. Mouse and human EndoVs exhibit very weak activity over deoxyinosine and DNA substrates in general[21, 22] whereas they incise inosine-containing RNA very efficiently, suggesting a role in RNA metabolism instead of DNA repair for the mammalian enzymes[19, 23, 24]. Protein depletion in procyclic forms (insect-stage parasites) by RNA interference led to impaired growth and defects in cell cycle progression, suggesting a specific and vital role for TbEndoV at this life stage
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