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.

Comparative proteomic analysis of two tobacco (Nicotiana tabacum) genotypes differing in Cd tolerance

“…antiport activity in plant cell vacuoles (Migocka et al 2011). And it has been previously unraveled that the abundance of V-ATPase subunit A increased with the presence of copper (Morris et al 2014). The upregulation of V-ATPases A2 subunit isoform may strengthen Cd 2?…”

“…V-ATPase A2 subunit isoform is a subunit of V-ATPases possessing ATP hydrolytic activity. It can also create endomembrane systems proton gradient (Morris et al 2014). The proton motive force is an impetus for Cd deposition via the form of Cd 2?…”

Quantitative analysis of gene expression changes in response to genotoxic compounds

“…Oligonucleotides were labelled with acridinium-ester (NHS-AE) through an internal amine linker and purified using high-performance liquid chromatography (HPLC) based on the procedures described in Nelson et al, (1992) and Morris et al (2014). HPLC purification was conducted using an instrument from Thermo…”

“…Assays based on the use of acridinium-ester (AE) as a chemiluminescent reporter molecule are well described in the literature (Arnold et al, 1989;Nelson et al, 1996 a,b) and are applied to the measurement of highly abundant ribosomal RNA of infectious agents. However, this technology has wider application as it is capable of measuring less abundant messenger RNA transcripts in a sensitive and quantitative fashion (Thomas et al, 2003a,b) with exquisite specificity (Morris et al, 2014). Here we aimed to extend the technology further by developing more sensitive chemiluminescent methods capable of measuring gene expression changes for toxicological application in conjunction with in vitro models.…”

“…In this study, the gene expression of RAD51C, TP53 and CSTA in HEPG2 cells exposed to a range of genotoxic compounds was measured by Q-PCR amplification. In parallel, we developed an alternative assay for the same 3 gene signature based on chemiluminescence (Arnold et al, 1989;Nelson et al, 1996a,b) which we have used for gene expression analysis of abundant genes (Thomas-Jones et al, 2003a, 2003b and the identification of single nucleotide differences in RNA transcripts (Morris et al, 2014). The chemiluminescence method allows the direct quantitation of RNA transcripts by the use of acridinium-ester (AE)-labelled complementary probes and is capable of analytical sensitivity of 10-50 attomol of target with a routine coefficient of variance of less than 15% (Morris et al, 2012).…”