Kinetics of Translating Ribosomes Determine the Efficiency of Programmed Stop Codon Readthrough.

During translation, a stop codon on the mRNA signals the ribosomes to terminate the process. In certain mRNAs, the termination fails due to the recoding of the canonical stop codon, and ribosomes continue translation to generate C-terminally extended protein. This process, termed stop codon readthrough (SCR), regulates several cellular functions. SCR is driven by elements/factors that act immediately downstream of the stop codon. Here, we have analysed the process of SCR using a simple mathematical model to investigate how the kinetics of translating ribosomes influences the efficiency of SCR. Surprisingly, the analysis revealed that the rate of translation inversely regulates the efficiency of SCR. We tested this prediction experimentally in mammalian AGO1 and MTCH2 mRNAs. Reduction in translation either globally by harringtonine or locally by rare codons caused an increase in the efficiency of SCR. Thus, our study has revealed a hitherto unknown mode of regulation of SCR.

Mammalian proteome expansion by stop codon readthrough.

Recognition of a stop codon by translation machinery as a sense codon results in translational readthrough instead of termination. This recoding process, termed stop codon readthrough (SCR) or translational readthrough, is found in all domains of life including mammals. The context of the stop codon, local mRNA topology, and molecules that interact with the mRNA region downstream of the stop codon determine SCR. The products of SCR can have localization, stability, and function different from those of the canonical isoforms. In this review, we discuss how recent technological and computational advances have increased our understanding of the SCR process in the mammalian system. Based on the known molecular events that occur during SCR of multiple mRNAs, we propose transient molecular roadblocks on an mRNA downstream of the stop codon as a possible mechanism for the induction of SCR. We argue, with examples, that the insights gained from the natural SCR events can guide us to develop novel strategies for the treatment of diseases caused by premature stop codons. This article is categorized under: Translation > Regulation.

Identification and functional characterization of mRNAs that exhibit stop codon readthrough in Arabidopsis thaliana.

Stop codon readthrough (SCR) is the process of continuation of translation beyond the stop codon, generating protein isoforms with C-terminal extensions. SCR has been observed in viruses, fungi, and multicellular organisms, including mammals. However, SCR is largely unexplored in plants. In this study, we have analyzed ribosome profiling datasets to identify mRNAs that exhibit SCR in Arabidopsis thaliana. Analyses of the ribosome density, ribosome coverage, and three-nucleotide periodicity of the ribosome profiling reads in the mRNA region downstream of the stop codon provided strong evidence for SCR in mRNAs of 144 genes. We show that SCR generated putative evolutionarily conserved nuclear localization signals, transmembrane helices, and intrinsically disordered regions in the C-terminal extensions of several of these proteins. Furthermore, gene ontology functional enrichment analysis revealed that these 144 genes belong to three major functional groups-translation, photosynthesis, and abiotic stress tolerance. Using a luminescence-based readthrough assay, we experimentally demonstrated SCR in representative mRNAs belonging to each of these functional classes. Finally, using microscopy, we show that the SCR product of one gene that contains a nuclear localization signal at the C-terminal extension, CURT1B, localizes to the nucleus as predicted. Based on these observations, we propose that SCR plays an important role in plant physiology by regulating protein localization and function.