Discovery of noncanonical translation initiation sites through mass spectrometric analysis of protein N termini

Abstract

Translation initiation generally occurs at AUG codons in eukaryotes, although it has been shown that non-AUG or noncanonical translation initiation can also occur. However, the evidence for noncanonical translation initiation sites (TISs) is largely indirect and based on ribosome profiling (Ribo-seq) studies. Here, using a strategy specifically designed to enrich N termini of proteins, we demonstrate that many human proteins are translated at noncanonical TISs. The large majority of TISs that mapped to 5' untranslated regions were noncanonical and led to N-terminal extension of annotated proteins or translation of upstream small open reading frames (uORF). It has been controversial whether the amino acid corresponding to the start codon is incorporated at the TIS or methionine is still incorporated. We found that methionine was incorporated at almost all noncanonical TISs identified in this study. Comparison of the TISs determined through mass spectrometry with ribosome profiling data revealed that about two-thirds of the novel annotations were indeed supported by the available ribosome profiling data. Sequence conservation across species and a higher abundance of noncanonical TISs than canonical ones in some cases suggests that the noncanonical TISs can have biological functions. Overall, this study provides evidence of protein translation initiation at noncanonical TISs and argues that further studies are required for elucidation of functional implications of such noncanonical translation initiation.

Chromatin-sensitive cryptic promoters encode alternative protein isoforms in yeast

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A deep proteome and transcriptome abundance atlas of 29 healthy human tissues

“…Recent research showed that there is more heterogeneity in gene models than previously anticipated, as a result of e. g. alternative translation initiation sites (aTIS) (Na et al 2018) and there is an on-going debate in the community whether or not long non-coding RNAs (lncRNAs) can be translated into proteins (Chen et al 2017). Ribosomal profiling showed that thousands of potential aTIS may exist and that ~40% of all lncRNAs can at least engage the ribosome (Kearse and Wilusz 2017).…”

Translation of upstream open reading frames in a model of neuronal differentiation

“…A straight forward approach would be to use mass spectrometry to detect uORF derived peptides; however this has previously been proven difficult, likely due to a range of complications from sample preparation, bias in annotation algorithms, as well as intrinsic factors that make these peptides difficult to detect (Bazzini, Johnstone et al 2014, Chugunova, Navalayeu et al 2018. Several methods have been developed to enhance the detection of small peptides, each with variable yields (Slavoff, Mitchell et al 2013, Bazzini, Johnstone et al 2014, Na, Barbhuiya et al 2018.…”