Inhibitors of eIF4G1-eIF1 uncover its regulatory role of ER/UPR stress-response genes independent of eIF2α-phosphorylation.

During translation initiation, eIF4G1 dynamically interacts with eIF4E and eIF1. While the role of eIF4E-eIF4G1 is well established, the regulatory functions of eIF4G1-eIF1 are poorly understood. Here, we report the identification of the eIF4G1-eIF1 inhibitors i14G1-10 and i14G1-12. i14G1s directly bind eIF4G1 and inhibit translation in vitro and in the cell, and their effects on translation are dependent on eIF4G1 levels. Translatome analyses revealed that i14G1s mimic eIF1 and eIF4G1 perturbations on the stringency of start codon selection and the opposing roles of eIF1-eIF4G1 in scanning-dependent and scanning-independent short 5' untranslated region (UTR) translation. Remarkably, i14G1s activate ER/unfolded protein response (UPR) stress-response genes via enhanced ribosome loading, elevated 5'UTR translation at near-cognate AUGs, and unexpected concomitant up-regulation of coding-region translation. These effects are, at least in part, independent of eIF2α-phosphorylation. Interestingly, eIF4G1-eIF1 interaction itself is negatively regulated by ER stress and mTOR inhibition. Thus, i14G1s uncover an unknown mechanism of ER/UPR translational stress response and are valuable research tools and potential drugs against diseases exhibiting dysregulated translation.

Mutant allele knockout with novel CRISPR nuclease promotes myelopoiesis in ELANE neutropenia.

Severe congenital neutropenia (SCN) is a life-threatening marrow failure disorder, usually caused by heterozygous mutations in ELANE. Potential genetic treatment strategies include biallelic knockout or gene correction via homology-directed repair (HDR). Such strategies, however, involve the potential loss of the essential function of the normal allele product or limited coverage of diverse monogenic mutations within the patient population, respectively. As an alternative, we have developed a novel CRISPR-based monoallelic knockout strategy that precisely targets the heterozygous sites of single-nucleotide polymorphisms (SNPs) associated with most ELANE mutated alleles. In vitro studies demonstrate that patients' unedited hematopoietic CD34+ cells have significant abnormalities in differentiation and maturation, consistent with the hematopoietic defect in SCN patients. Selective knockout of the mutant ELANE allele alleviated these cellular abnormalities and resulted in about 50%-70% increase in normally functioning neutrophils (p < 0.0001). Genomic analysis confirmed that ELANE knockout was specific to the mutant allele and involved no off-targets. These results demonstrate the therapeutic potential of selective allele editing that may be applicable to SCN and other autosomal dominant disorders.

Dynamic Interaction of Eukaryotic Initiation Factor 4G1 (eIF4G1) with eIF4E and eIF1 Underlies Scanning-Dependent and -Independent Translation.

Translation initiation of most mRNAs involves m7G-cap binding, ribosomal scanning, and AUG selection. Initiation from an m7G-cap-proximal AUG can be bypassed resulting in leaky scanning, except for mRNAs bearing the translation initiator of short 5' untranslated region (TISU) element. m7G-cap binding is mediated by the eukaryotic initiation factor 4E (eIF4E)-eIF4G1 complex. eIF4G1 also associates with eIF1, and both promote scanning and AUG selection. Understanding of the dynamics and significance of these interactions is lacking. We report that eIF4G1 exists in two complexes, either with eIF4E or with eIF1. Using an eIF1 mutant impaired in eIF4G1 binding, we demonstrate that eIF1-eIF4G1 interaction is important for leaky scanning and for avoiding m7G-cap-proximal initiation. Intriguingly, eIF4E-eIF4G1 antagonizes the scanning promoted by eIF1-eIF4G1 and is required for TISU. In mapping the eIF1-binding site on eIF4G1, we unexpectedly found that eIF4E also binds it indirectly. These findings uncover the RNA features underlying regulation by eIF4E-eIF4G1 and eIF1-eIF4G1 and suggest that 43S ribosome transition from the m7G-cap to scanning involves relocation of eIF4G1 from eIF4E to eIF1.

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

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.

DTIE, a novel core promoter element that directs start site selection in TATA-less genes.

The transcription start site (TSS) determines the length and composition of the 5' UTR and therefore can have a profound effect on translation. Yet, little is known about the mechanism underlying start site selection, particularly from promoters lacking conventional core elements such as TATA-box and Initiator. Here we report a novel mechanism of start site selection in the TATA- and Initiator-less promoter of miR-22, through a strictly localized downstream element termed DTIE and an upstream distal element. Changing the distance between them reduced promoter strength, altered TSS selection and diminished Pol II recruitment. Biochemical assays suggest that DTIE does not serve as a docking site for TFIID, the major core promoter-binding factor. TFIID is recruited to the promoter through DTIE but is dispensable for TSS selection. We determined DTIE consensus and found it to be remarkably prevalent, present at the same TSS downstream location in ≈20.8% of human promoters, the vast majority of which are TATA-less. Analysis of DTIE in the tumor suppressor p53 confirmed a similar function. Our findings reveal a novel mechanism of transcription initiation from TATA-less promoters.

Cap-dependent, scanning-free translation initiation mechanisms.

Eukaryotic translation initiation is an intricate and multi-step process that includes 43S Pre-Initiation Complex (PIC) assembly, attachment of the PIC to the mRNA, scanning, start codon selection and 60S subunit joining. Translation initiation of most mRNAs involves recognition of a 5'end m7G cap and ribosomal scanning in which the 5' UTR is checked for complementarity with the AUG. There is however an increasing number of mRNAs directing translation initiation that deviate from the predominant mechanism. In this review we summarize the canonical translation initiation process and describe non-canonical mechanisms that are cap-dependent but operate without scanning. In particular we focus on several examples of translation initiation driven either by mRNAs with extremely short 5' leaders or by highly complex 5' UTRs that promote ribosome shunting.

Translational tolerance of mitochondrial genes to metabolic energy stress involves TISU and eIF1-eIF4GI cooperation in start codon selection.

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.

Transcriptional activity of ATF3 in the stromal compartment of tumors promotes cancer progression.

Compelling evidences have rendered the tumor microenvironment a crucial determinant in cancer outcome. Activating transcription factor 3 (ATF3), a stress response transcription factor, is known to have a dichotomous role in tumor cells, acting either as a tumor suppressor or an oncogene in a context-dependent manner. However, its expression and possible role in the tumor microenvironment are hitherto unknown. Here we show that ATF3 is upregulated in the stromal compartment of several types of cancer. Accordingly, Cancer-associated fibroblasts (CAFs) ectopically expressing ATF3 proliferated faster as indicated by increased colony-forming capacity and promoted the growth of adjacent tumor cells when co-injected into nude mice. Utilizing a genome-wide profiling approach, we unraveled a robust gene expression program induced by ATF3 in CAFs. Focusing on a specific subset of genes, we found that the ability of stromal ATF3 to promote cancer progression is mediated by transcriptional repression of CLDN1 and induction of CXCL12 and RGS4. In addition, regulation of LIF, CLDN1, SERPINE2, HSD17B2, ITGA7 and PODXL by ATF3 mediated the increased proliferation capacity of CAFs. In sum, our findings implicate ATF3 as a novel stromal tumor promoter and suggest that targeting ATF3 pathway might be beneficial for anticancer therapy.

Unique translation initiation of mRNAs-containing TISU element.

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.