Intracellular localization and induction of a dynamic RNA‐editing event of macro‐algal V‐ATPase subunit A (VHA‐A) in response to copper

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. Our research aims to identify key molecular mechanisms used by organisms to maintain heavy metal homeostasis particularly when challenged by environmental contamination. This study compares the macroalga Fucus vesiculosus growing in a heavy metal contaminated estuary (Fal, Cornwall) displaying an environmental copper gradient to that from the uncontaminated sites as well as utilising a suite of laboratory based copper exposures. These studies revealed copper induced the total transcript and protein levels of subunit A of the V‐ATPase (VHA‐A), the critical catalytic subunit of this proton pump, as well as inducing a single nucleotide change in the RNA transcript sufficient to stimulate the accumulation of a truncated protein. This suggests that RNA editing can be a dynamic adaptive change and this has widespread connotations for the regulation of a key enzyme which is the critical factor for a number of essential processes for life, including setting up membrane potentials that allow transport and detoxification mechanisms.