title: Intracellular localization and induction of a dynamic RNA-editing event of macro-algal V-ATPase subunit A (VHA-A) in response to copper.
abstract: A V-ATPase subunit A protein (VHA-A) transcript together with a variant (C793 to U), which introduces a stop codon truncating the subunit immediately downstream of its ATP binding site, was identified within a Fucus vesiculosus cDNA from a heavy metal contaminated site. This is intriguing because the VHA-A subunit is the crucial catalytic subunit responsible for the hydrolysis of ATP that drives ion transport underlying heavy metal detoxification pathways. We employed a chemiluminescent hybridization protection assay to quantify the proportion of both variants directly from mRNA while performing quantification of total transcript using Q-PCR. Polyclonal antisera raised against recombinant VHA-A facilitated simultaneous detection of parent and truncated VHA-A and revealed its cellular and subcellular localization. By exploiting laboratory exposures and samples from an environmental copper gradient, we showed that total VHA-A transcript and protein, together with levels of the truncated variant, were induced by copper. The absence of a genomic sequence representing the truncated variant suggests a RNA editing event causing the production of the truncated VHA-A. Based on these observations, we propose RNA editing as a novel molecular process underpinning VHA trafficking and intracellular sequestration of heavy metals under stress.
authors.affiliation: School of Biosciences, Cardiff University, Cardiff, CF10 3AT, Wales, UK.
authors.forename: C A
authors.initials: CA
authors.lastname: Morris
authors.forename: J R
authors.initials: JR
authors.lastname: Owen
authors.forename: M C
authors.initials: MC
authors.lastname: Thomas
authors.forename: G A
authors.initials: GA
authors.lastname: El-Hiti
authors.forename: J L
authors.initials: JL
authors.lastname: Harwood
authors.forename: P
authors.initials: P
authors.lastname: Kille
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D020418:Algal Proteins / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D003300:Copper / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D044645:Fucus / Q000187:drug effects / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism; D017393:RNA Editing; D011994:Recombinant Proteins; D016415:Sequence Alignment
chemical_list: D020418:Algal Proteins; D018076:DNA, Complementary; D021122:Protein Subunits; D011994:Recombinant Proteins; D012313:RNA; D003300:Copper; D000251:Adenosine Triphosphatases
keywords: Phaeophyta; gene expression; macro-algae; protein location; toxicity.