Efficient and Accurate Translation Initiation Directed by TISU Involves RPS3 and RPS10e Binding and Differential Eukaryotic Initiation Factor 1A Regulation

Abstract

Canonical translation initiation involves ribosomal scanning, but short 5= untranslated region (5=UTR) mRNAs are translated in a scanning-independent manner. The extent and mechanism of scanning-independent translation are not fully understood. Here we report that short 5=UTR mRNAs constitute a substantial fraction of the translatome. Short 5=UTR mRNAs are enriched with TISU (translation initiator of short 5=UTR), a 12-nucleotide element directing efficient scanning-independent translation. Comprehensive mutagenesis revealed that each AUG codon-flanking nucleotide of TISU contributes to translational strength, but only a few are important for accuracy. Using site-specific UV cross-linking of ribosomal complexes assembled on TISU mRNA, we demonstrate specific binding of TISU to ribosomal proteins at the E and A sites. We identified RPS3 as the major TISU binding protein in the 48S complex A site. Upon 80S complex formation, RPS3 interaction is weakened and switched to RPS10e (formerly called RPS10). We further demonstrate that TISU is particularly dependent on eukaryotic initiation factor 1A (eIF1A) which interacts with both RPS3 and RPS10e. Our findings suggest that the cap-recruited ribosome specifically binds the TISU nucleotides at the A and E sites in cooperation with eIF1A to promote scanning arrest.KEYWORDS translation initiation, TISU, RPS3, RPS10e, eIF1A, short 5=UTR, RPS10 C anonical translation initiation in eukaryotes starts with binding of eukaryotic initiation factor 4F (eIF4F) to the m 7 G-capped 5= end of the mRNA, which is then followed by the recruitment of the 43S preinitiation complex (PIC) consisting of the 40S small ribosomal subunit, initiator Met-tRNA, and a subset of initiation factors (eIF2, eIF1, eIF1A, eIF3, and eIF5). The 43S PIC then scans the mRNA 5= untranslated region (5=UTR) and inspects it for an AUG start codon. During scanning, the 43S PIC is in an "open" state, and following AUG recognition, the 48S initiation complex is switched to a "closed" conformation that arrests scanning and promotes joining of the 60S large subunit to create the 80S elongation-competent complex (reviewed in references 1, 2, 3, 4, and 5). Translation usually initiates at the first 5=-proximal AUG codon, but in certain cases, translation initiates at a downstream AUG (DS-AUG) codon, a phenomenon known as leaky scanning. The extent of leaky scanning depends on the 5=UTR length and on the AUG nucleotide context (6,7,8,9). For mRNA with a 5=UTR that is more than 30 nucleotides (nt) long, the optimal AUG context is the Kozak element, RCCAUGG, in which the most significant nucleotides (shown in boldface type) are the

Cancer-Associated Eukaryotic Translation Initiation Factor 1A Mutants Impair Rps3 and Rps10 Binding and Enhance Scanning of Cell Cycle Genes

“…In the PIC, eIF1A is located near A-site ribosomal proteins Rps3 and Rps10 and the mRNA channel (20). Both ribosomal proteins contact AUG downstream nucleotides and are implicated in scanning arrest of short 5= UTR mRNAs bearing the translation initiator of short 5' UTR (TISU) element (5). In addition, Rps3 and Rps10 were shown to interact with eIF1A in a cell-based split-Renilla assay (5).…”

“…Thus, the two tails of eIF1A play opposing roles in scanning and AUG selection. We have recently shown that the interaction of eIF1A with the 43S subunit is partly mediated by Rps3 and Rps10, which are ribosomal proteins located at the A site and which undergo a conformational change during the transition of the 43S to the 80S subunit (5). How these interactions contribute to the initiation process is presently unknown.…”

Upstream translation initiation expands the coding capacity of segmented negative-strand RNA viruses

“…This ‘start-snatching’ would require functional start codons extremely close to the 5′ end of cellular mRNAs. Start codons of this sort, referred to as Translation Initiator of Short 5′ UTR (TISU) motifs, have been reported in approximately 4 % of mammalian mRNAs, being particularly common in ‘housekeeping’ genes [16–18]. We note that this figure is comparable to the ribosomal density we observed on the 5′ UTRs of viral mRNAs (Fig 1E) and is compatible with the 8 – 10 % of viral cap-snatched leaders which we found to contain AUGs (Fig 2D).…”

“…We note that this figure is comparable to the ribosomal density we observed on the 5′ UTRs of viral mRNAs (Fig 1E) and is compatible with the 8 – 10 % of viral cap-snatched leaders which we found to contain AUGs (Fig 2D). TISU motifs are functional within 30 nt of the 5′ cap, including at sites at the extreme end of mRNA with 5’ UTRs as short as 5 nt [16–18]. As with other upstream translation sites, TISUs have been proposed to regulate the expression of downstream genes, in response to factors including energy deprivation and the circadian rhythm [47–49].…”

“…In eukaryotes, ribosomes recognise mRNAs with a terminal 5′ cap structure followed by an untranslated region (UTR), which can be tens to hundreds of nucleotides in length [9–11]. A growing body of work has shown that translation initiates at low levels in the 5′ UTRs of a large proportion of eukaryotic mRNAs, sometimes extremely close to the 5′ cap, and that translation of the resulting upstream open reading frames (uORFs) can regulate cellular gene expression [10,12–18]. As a result, the need to mimic their host’s mRNA structure could provide viruses with the potential to exploit upstream translation initiation, something which could allow them to expand the coding potential of their own genomes.…”