N-terminal Proteomics and Ribosome Profiling Provide a Comprehensive View of the Alternative Translation Initiation Landscape in Mice and Men

Abstract

Usage of presumed 5UTR or downstream in-frame AUG codons, next to non-AUG codons as translation start codons contributes to the diversity of a proteome as protein isoforms harboring different N-terminal extensions or truncations can serve different functions. Recent ribosome profiling data revealed a highly underestimated occurrence of database nonannotated, and thus alternative translation initiation sites (aTIS), at the mRNA level. Nterminomics data in addition showed that in higher eukaryotes around 20% of all identified protein N termini point to such aTIS, to incorrect assignments of the translation start codon, translation initiation at near-cognate start codons, or to alternative splicing. We here report on more than 1700 unique alternative protein N termini identified at the proteome level in human and murine cellular proteomes. Customized databases, created using the translation initiation mapping obtained from ribosome profiling data, additionally demonstrate the use of initiator methionine decoded near-cognate start codons besides the existence of N-terminal extended protein variants at the level of the proteome. Various newly identified aTIS were confirmed by mutagenesis, and meta-analyses demonstrated that aTIS reside in strong Kozak-like motifs and are conserved among eukaryotes, hinting to a possible biological impact. Finally, TargetP analysis predicted that the usage of aTIS often results in altered subcellular localization patterns, providing a mechanism for functional diversification. Eukaryotic protein-coding genes can give rise to multiple translation products of which the expression is regulated at multiple levels. In contrast to transcriptional regulation, protein translational regulation permits for more immediate effects to take place. Initiation, elongation, termination, and ribosome recycling constitute the different phases of the eukaryotic translation process, with translation initiation acting as the gate-keeping step by the successive steps of ternary complex recruitment, scanning, AUG codon selection, and ribosomal subunit joining. Overall, this process requires over 30 different proteins including the eukaryotic initiation factors (eIFs) 1 (1). In eukaryotes, the translation start codon is typically found by ribosome scanning, referred to as the canonical mechanism of translation initiation. Here, the 43S pre-initiation complex (PIC) composed of the initiator Met-tRNA (MettRNAi) pre-loaded onto the small (40S) ribosomal subunit, binds near the 5Ј end of the mRNA molecule in a m 7 G-cap structure/eukaryotic initiation factors 4 (i.e. eIF4E, 4G, and 4A; jointly referred to as the eIF4F complex) mediated fashion. This complex then starts to scan successive triplets of the 5Ј untranslated region (5ЈUTR) in the 3Ј direction until an AUG start codon or, alternatively, a near-cognate start codon entered the P (peptidyl) decoding site of the ribosome. Start codon recognition requires base-pairing with the anticodon loop of Met-tRNAi and triggers a scanning arrest and GTP hydrolysis of th...

GWIPS‐viz as a tool for exploring ribosome profiling evidence supporting the synthesis of alternative proteoforms

“…This is the case with the human PRKAA1 gene for which the synthesis of a truncated proteoform has been validated previously at the protein level 15, 22. Figure 2A and B shows ribo‐seq data and mRNA‐seq data aligned to the first exon of the human PRKAA1 gene and the mouse Prkaa1 gene, respectively.…”

“…While useful, these techniques suffer from high levels of signal noise, for example, elongating ribosomes pausing at particular codons 19, 20. Therefore, the use of ribo‐seq data is particularly powerful in combination with other techniques, for example, phylogenetic approaches 10 or MS 21, 22, 23.…”

Two N-Terminal Acetyltransferases Antagonistically Regulate the Stability of a Nod-Like Receptor in Arabidopsis

“…From wholeproteome or acetylome analysis, they are hardly ever found (Lee et al, 2012;Menschaert et al, 2013;Vanderperre et al, 2013; M.S. Van Damme et al, 2014;Wilhelm et al, 2014). However, when all 27,168 Arabidopsis proteins were analyzed, 620 were found to start with MM, representing over 2% of all encoded proteins.…”

“…Recent global mapping of ribosome binding sites in mammals revealed that translation initiation is more versatile and alternative translation is more prevalent than previously imagined (Lee et al, 2012;Menschaert et al, 2013;Vanderperre et al, 2013; M.S. Van Damme et al, 2014;Wilhelm et al, 2014). Such mechanisms can vastly expand the diversity of the proteome and provide greater versatility for the encoded proteins.…”

Limitless translation limits translation

“…For example, numerous small upstream ORFs (uORFs) have been identified whose translation might regulate expression of the main ORF. Ribosome profiling has also revealed a wealth of events in which translation is initiated at alternative start codons 3 (triplets of nucleotides other than the triplets at which translation is normally assumed to initiate), and read-through events 4 in which translation continues beyond the stop codon (the nucleotide triplet at the end of the ORF). Not only do these two types of event increase the overall diversity of proteoforms (molecular forms of proteins produced from genes) 5 , but they have also emerged as regulatory mechanisms for hundreds of genes in eukaryotic genomes.…”