Global mapping of translation initiation sites in mammalian cells at single-nucleotide resolution

Abstract

Understanding translational control in gene expression relies on precise and comprehensive determination of translation initiation sites (TIS) across the entire transcriptome. The recently developed ribosome-profiling technique enables global translation analysis, providing a wealth of information about both the position and the density of ribosomes on mRNAs. Here we present an approach, global translation initiation sequencing, applying in parallel the ribosome E-site translation inhibitors lactimidomycin and cycloheximide to achieve simultaneous detection of both initiation and elongation events on a genome-wide scale. This approach provides a view of alternative translation initiation in mammalian cells with single-nucleotide resolution. Systemic analysis of TIS positions supports the ribosome linear-scanning mechanism in TIS selection. The alternative TIS positions and the associated ORFs identified by global translation initiation sequencing are conserved between human and mouse cells, implying physiological significance of alternative translation. Our study establishes a practical platform for uncovering the hidden coding potential of the transcriptome and offers a greater understanding of the complexity of translation initiation.genome wide | high throughput | leaky scanning | start codon P rotein synthesis is the final step in the flow of genetic information and lies at the heart of cellular metabolism. Translation is regulated principally at the initiation stage, and during the last decade significant progress has been made in dissecting the role of initiation factors (eIFs) in the assembly of elongationcompetent 80S ribosomes (1-3). However, mechanisms underlying start codon recognition are not fully understood. Proper selection of the translation initiation site (TIS) on mRNAs is crucial for the production of desired protein products. A fundamental and longsought goal in understanding translational regulation is the precise determination of TIS codons across the entire transcriptome.In eukaryotes, ribosomal scanning is a well-accepted model for start codon selection (4). During cap-dependent translation initiation, the small ribosome subunit (40S) is recruited to the 5′ end of mRNA (the m 7 G cap) in the form of a 43S preinitiation complex (PIC). The PIC is thought to scan along the message in search of the start codon. It is commonly assumed that the first AUG codon that the scanning PIC encounters serves as the start site for translation. However, many factors influence the start codon selection. For instance, the initiator AUG triplet usually is in an optimal context, with a purine at position −3 and a guanine at position +4 (5). The presence of an mRNA secondary structure at or near the TIS position also influences the efficiency of recognition (6). In addition to these cis sequence elements, the stringency of TIS selection also is subject to regulation by trans-acting factors such as eIF1 and eIF1A (7,8). Inefficient recognition of an initiator codon results in a portion of 43S PIC continuing to ...

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.…”

“…Also displayed is the short match detected location of the GUG start codon and part of its 3′ flanking nucleotide sequence. The initiating ribosome profile was generated using the Global aggregate track in GWIPS‐viz (http://gwips.ucc.ie) using data from four studies 15, 17, 18, 29. The elongating ribosomes profile was generated using data aggregated from 13 studies 15, 17, 18, 29, 38.…”

“…(B) Initiating ribosomes profile and elongating ribosomes profile for the second exon of the mouse Stard10 gene. The description is the same as for panel A except that the initiating ribosomes profile was generated from two studies 15, 18 and the elongating ribosomes profile from five studies 15, 16, 37, 39, 40. (C) Elongating ribosomes profile for the first exon of the human SCAMP3 gene.…”

Structural basis for the inhibition of the eukaryotic ribosome

“…Lactimidomycin preferentially arrests ribosomes at the first peptide bond, whereas cycloheximide stalls ribosomes during ongoing translation [18][19][20] .…”

Ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation

“…We applied a modified Ribo-seq to HEK293 cells and examined the average pattern of ribosome density across the entire transcriptome [8,19]. As expected, the mapped RPF reads start an abrupt accumulation at the annotated start codon [11] (Figure 1A).…”