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...

An increasing amount of studies integrate mRNA sequencing data into MS-based proteomics to complement the translation product search space. However, several factors, including extensive regulation of mRNA translation and the need for three- or six-frame-translation, impede the use of mRNA-seq data for the construction of a protein sequence search database. With that in mind, we developed the PROTEOFORMER tool that automatically processes data of the recently developed ribosome profiling method (sequencing of ribosome-protected mRNA fragments), resulting in genome-wide visualization of ribosome occupancy. Our tool also includes a translation initiation site calling algorithm allowing the delineation of the open reading frames (ORFs) of all translation products. A complete protein synthesis-based sequence database can thus be compiled for mass spectrometry-based identification. This approach increases the overall protein identification rates with 3% and 11% (improved and new identifications) for human and mouse, respectively, and enables proteome-wide detection of 5′-extended proteoforms, upstream ORF translation and near-cognate translation start sites. The PROTEOFORMER tool is available as a stand-alone pipeline and has been implemented in the galaxy framework for ease of use.

To understand the impact of alternative translation initiation on a proteome, we performed a proteome‐wide study on protein turnover using positional proteomics and ribosome profiling to distinguish between N‐terminal proteoforms of individual genes. By combining pulsed SILAC with N‐terminal COFRADIC, we monitored the stability of 1,941 human N‐terminal proteoforms, including 147 N‐terminal proteoform pairs that originate from alternative translation initiation, alternative splicing or incomplete processing of the initiator methionine. N‐terminally truncated proteoforms were less abundant than canonical proteoforms and often displayed altered stabilities, likely attributed to individual protein characteristics, including intrinsic disorder, but independent of N‐terminal amino acid identity or truncation length. We discovered that the removal of initiator methionine by methionine aminopeptidases reduced the stability of processed proteoforms, while susceptibility for N‐terminal acetylation did not seem to influence protein turnover rates. Taken together, our findings reveal differences in protein stability between N‐terminal proteoforms and point to a role for alternative translation initiation and co‐translational initiator methionine removal, next to alternative splicing, in the overall regulation of proteome homeostasis.