Defining the high-translational readthrough stop codon context

Translational termination is not entirely efficient and competes with elongation, which might result in translational readthrough (TR). TR occurs when a near-cognate tRNA binds to a stop codon, (mis)interpreting it as a sense codon and producing a C-terminal extension of the protein. This process is influenced by the stop codon itself and the surrounding nucleotide sequence, known as the stop codon context (SCC). To investigate the role of these cis-acting elements beyond the high-TR motif UGA CUA G, this study examines specific positions within the SCC, both upstream and downstream of the motif, that contribute to variations in basal and aminoglycoside-induced TR. In particular, we identified a surprisingly large influence of the upstream nucleotide positions -9 and -8 (relative to the stop codon) and positions +11 and +12 on readthrough levels, revealing a complex interplay between nucleotides in the expanded SCC with effects turning out to be non-linear and, furthermore, not transferable to evolutionarily non-adapted SCCs. These findings support our understanding of translational termination and may benefit the development of pharmacological therapy for diseases caused by premature stop codon mutations.Author summary: The termination of the production of any protein in the cell is controlled by stop signals (stop codons) on the messenger-RNA (mRNA). A decade ago, we and others discovered a short stretch immediately following the stop codon that renders the termination process leaky so that the stop codon is not read as a stop signal and proteins are extended, creating new proteins (isoforms) with potential additional functions or different locations within the cell. This process is named functional translational readthrough, and Lactate and Malate dehydrogenases and the neuronal water channel Aquaporin 4 are known to have isoforms that derive from translational readthrough. The present study examined how specific base positions in the mRNA, before and after the stop codon, influence readthrough. We discovered that specific mRNA bases, both upstream and downstream of the stop codon, play an important and complex role. We also show that the individual elements of these high-readthrough sequences cannot linearly be transferred to disease-related sequences containing pathological premature stop signals. This distinction is important for efforts to develop therapies that aim to overcome such premature stop signals. Our results highlight that strategies promoting therapeutic readthrough must consider the unique sequence features of each stop signal context, rather than relying on general rules derived from high-readthrough signals.