N-terminal alanine-rich (NTAR) sequences drive precise start codon selection resulting in elevated translation of multiple proteins including ERK1/2

Abstract We report the discovery of N-terminal alanine-rich sequences, which we term NTARs, that act in concert with their native 5′-untranslated regions to promote selection of the proper start codon. NTARs also facilitate efficient translation initiation while limiting the production of non-functional polypeptides through leaky scanning. We first identified NTARs in the ERK1/2 kinases, which are among the most important signaling molecules in mammals. Analysis of the human proteome reveals that hundreds of proteins possess NTARs, with housekeeping proteins showing a particularly high prevalence. Our data indicate that several of these NTARs act in a manner similar to those found in the ERKs and suggest a mechanism involving some or all of the following features: alanine richness, codon rarity, a repeated amino acid stretch and a nearby second AUG. These features may help slow down the leading ribosome, causing trailing pre-initiation complexes (PICs) to pause near the native AUG, thereby facilitating accurate translation initiation. Amplification of erk genes is frequently observed in cancer, and we show that NTAR-dependent ERK protein levels are a rate-limiting step for signal output. Thus, NTAR-mediated control of translation may reflect a cellular need to precisely control translation of key transcripts such as potential oncogenes. By preventing translation in alternative reading frames, NTAR sequences may be useful in synthetic biology applications, e.g. translation from RNA vaccines. Lay Summary Initiation of translation is essential for protein synthesis. A crucial step is the correct choice of the start AUG, which leads to the production of the fully functional polypeptide. To date, nucleotide composition next to the AUG has been considered the only determinant of start codon selection. Our work identifies a large family of proteins whose start codon choice is determined by an N-terminal alanine-rich sequence (NTAR) that enables efficient protein translation. Many of these proteins are encoded by housekeeping genes. Among them, the NTARs of the pivotal kinases ERK1 and ERK2 are highly optimized in humans, shaping ERK signal transduction by increasing the kinase quantity. Our findings could be useful for applied biology, especially for mRNA-based therapeutics. Graphical Abstract Graphical Abstract