Combinatorial analysis of translation dynamics reveals eIF2 dependence of translation initiation at near-cognate codons

Abstract Although ribosome-profiling and translation initiation sequencing (TI-seq) analyses have identified many noncanonical initiation codons, the precise detection of translation initiation sites (TISs) remains a challenge, mainly because of experimental artifacts of such analyses. Here, we describe a new method, TISCA (TIS detection by translation Complex Analysis), for the accurate identification of TISs. TISCA proved to be more reliable for TIS detection compared with existing tools, and it identified a substantial number of near-cognate codons in Kozak-like sequence contexts. Analysis of proteomics data revealed the presence of methionine at the NH2-terminus of most proteins derived from near-cognate initiation codons. Although eukaryotic initiation factor 2 (eIF2), eIF2A and eIF2D have previously been shown to contribute to translation initiation at near-cognate codons, we found that most noncanonical initiation events are most probably dependent on eIF2, consistent with the initial amino acid being methionine. Comprehensive identification of TISs by TISCA should facilitate characterization of the mechanism of noncanonical initiation. Graphical Abstract Graphical Abstract TISCA reliably identifies translation initiation sites (TISs) and provides insight into the mechanism of translation initiation at near-cognate codons. TISCA efficiently reduces the background noise in GTI-seq analysis. The absence and nonrelease of eIF3-seq footprints are mostly eliminated as noise. Red and blue circles show the 5′ end of a GTI-seq peak and an eIF3 release point, respectively. TIS candidates are located at +11 to +13 nt from the GTI-seq peak and at −22 to −26 nt from the eIF3 release point (top). TISCA identifies a substantial number of near-cognate initiation codons, most of which are translated in a manner dependent on eIF2–Met-tRNAi (bottom).