LDHBx and MDH1x are controlled by physiological translational readthrough in Homo sapiens

Abstract

A study of translational readthrough (TR) and functional translational readthrough (FTR) in Homo sapiens. Translational readthrough involves ribosomes that translate into mRNA sequences downstream of a stop codon at a higher ratio than the normal error rate. Functional translational readthrough is defined as translational readthrough that appends extensions to parental proteins that alter its function. Translational readthrough is employed by viruses to expand the coding potential of their limited genome, however, also in more complex organisms examples of readthrough and even functional readthrough have been found. The study contains a description of the in silico regression model that was used to derive a consensus for nucleotide context based high readthrough in mammals. In a genome wide scan in a human transcriptome 57 candidates for translational readthrough were detected. The results of this genome wide scan for TR are discussed against a background of eight recent studies that applied systems biology approaches (phylogenetic approaches, ribosome profiling, and the in silico regression model) to mammalian genomes/transcriptomes. A dual reporter assay was used to test and verify the stop codon context of some transcripts. The results of 15 constructs were used to expand the database used to train the model. The database of 42.000 unique stop codon contexts was sorted according to their readthrough potential and a score was computed to estimate the probability for hidden peroxisomal targeting signals (PTS1). The subunit LDHB of lactate dehydrogenase was detected at the first position, exceeding rank two by far. MDH1, the gene coding for cytosolic malate dehydrogenase was detected at rank 175. Both enzymes are evolutionary related and were analysed in detail in terms of their translational readthrough. The proteins showed high readthrough rates and responded to treatment with aminoglycosides with elevated readthrough rates. Analysis of readthrough in multiple cell types suggests that readthrough forms of LDHB (LDHBx) and MDH1 (MDH1x) are expressed in probably all human tissues at varying levels. Their extensions are conserved in mammals, but show no conserved secondary structure. Next to immediate stop codon contexts also elaborate mRNA secondary structures can influence and mediate translational readthrough. The influence of the stop codon and of the stop codon context on TR was tested as well as codon usage frequencies. The hidden PTS1 found in the extensions of LDHBx was tested. It was found that endogenous LDHBx is imported to peroxisomes by means of translational readthrough and the hidden PTS1. Translational readthrough was quantified and analysed using immunofluorescence imaging and image analysis tools (ImageJ). LDHBx was found to co-import the other subunit LDHA into peroxisomes allowing theoretically nine new isoforms to occur in the peroxisomal matrix. The potential of the findings lies in the used methods themselves e.g. the in silico regression model, and clinical application in form of drug treatment of genetic disorders caused by premature stop codons. An outline for experiments that could allow the design of a predictor model for the response of stop codon contexts to drug treatment (aminoglycosides and their derivatives) is given.