Translation Initiator of Short 5′ UTR (TISU) is a unique regulatory element of both transcription and translation initiation. It is present in a sizable number of genes with basic cellular functions and a very short untranslated region (5′ UTR). Here, we investigated translation initiation from short 5′ UTR mRNAs with AUG in various contexts. Reducing 5′ UTR length to the minimal functional size increases leaky scanning from weak and strong initiators but hardly affects translation initiation and ribosomal binding directed by TISU. Ribosome interaction with TISU mRNA is cap dependent and involves AUG downstream nucleotides that compensate for the absent 5′ UTR contacts. Interestingly, eIF1 inhibits cap-proximal AUG selection within weak or strong contexts but not within TISU. Furthermore, TISU-directed translation is unaffected by inhibition of the RNA helicase eIF4A. Thus, TISU directs efficient cap-dependent translation initiation without scanning, a mechanism that would be advantageous when intracellular levels of eIF1 and eIF4A fluctuate.

Protein synthesis is a major energy-consuming process, which is rapidly repressed upon energy stress by AMPK. How energy deficiency affects translation of mRNAs that cope with the stress response is poorly understood. We found that mitochondrial genes remain translationally active upon energy deprivation. Surprisingly, inhibition of translation is partially retained in AMPKα1/AMPKα2 knockout cells. Mitochondrial mRNAs are enriched with TISU, a translation initiator of short 5' UTR, which confers resistance specifically to energy stress. Purified 48S preinitiation complex is sufficient for initiation via TISU AUG, when preceded by a short 5' UTR. eIF1 stimulates TISU but inhibits non-TISU-directed initiation. Remarkably, eIF4GI shares this activity and also interacts with eIF1. Furthermore, eIF4F is released upon 48S formation on TISU. These findings describe a specialized translation tolerance mechanism enabling continuous translation of TISU genes under energy stress and reveal that a key step in start codon selection of short 5' UTR is eIF4F release.

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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