To New Beginnings: Riboproteogenomics Discovery of N-Terminal Proteoforms in Arabidopsis Thaliana

Alternative translation initiation is a widespread event in biology that can shape multiple protein forms or proteoforms from a single gene. However, the respective contribution of alternative translation to protein complexity remains largely enigmatic. By complementary ribosome profiling and N-terminal proteomics (i.e., riboproteogenomics), we provide clear-cut evidence for ~90 N-terminal proteoform pairs shaped by (alternative) translation initiation in Arabidopsis thaliana. Next to several cases additionally confirmed by directed mutagenesis, identified alternative protein N-termini follow the enzymatic rules of co-translational N-terminal protein acetylation and initiator methionine removal. In contrast to other eukaryotic models, N-terminal acetylation in plants cannot generally be considered as a proxy of translation initiation because of its posttranslational occurrence on mature proteolytic neo-termini (N-termini) localized in the chloroplast stroma. Quantification of N-terminal acetylation revealed differing co- vs. posttranslational N-terminal acetylation patterns. Intriguingly, our data additionally hints to alternative translation initiation serving as a common mechanism to supply protein copies in multiple cellular compartments, as alternative translation sites are often in close proximity to cleavage sites of N-terminal transit sequences of nuclear-encoded chloroplastic and mitochondrial proteins. Overall, riboproteogenomics screening enables the identification of (differential localized) N-terminal proteoforms raised upon alternative translation.

Mammalian Alternative Translation Initiation Is Mostly Nonadaptive

Alternative translation initiation (ATLI) refers to the existence of multiple translation initiation sites per gene and is a widespread phenomenon in eukaryotes. ATLI is commonly assumed to be advantageous through creating proteome diversity or regulating protein synthesis. We here propose an alternative hypothesis that ATLI arises primarily from nonadaptive initiation errors presumably due to the limited ability of ribosomes to distinguish sequence motifs truly signaling translation initiation from similar sequences. Our hypothesis, but not the adaptive hypothesis, predicts a series of global patterns of ATLI, all of which are confirmed at the genomic scale by quantitative translation initiation sequencing in multiple human and mouse cell lines and tissues. Similarly, although many codons differing from AUG by one nucleotide can serve as start codons, our analysis suggests that using non-AUG start codons is mostly disadvantageous. These and other findings strongly suggest that ATLI predominantly results from molecular error, requiring a major revision of our understanding of the precision and regulation of translation initiation.