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.

A Simple Combined Use of CRISPR-Cas9 and Cre-LoxP Technologies for Generating Conditional Gene Knockouts in Mammalian Cells.

Gene knockout technologies have contributed fundamentally to our understanding of the cellular functions of various genes. Two prevalent systems used for the efficient elimination of the expression of specific genes are the Cre-LoxP system and the CRISPR-Cas9 system. Here, we present a simple method that combines the use of CRISPR-Cas9 and Cre-LoxP for the conditional deletion of essential genes in mammalian cells. First, an inducible Cre recombinase is stably expressed in the cells. Next, CRISPR-Cas9 is used to knock out an essential gene, whose function is complemented by stable expression of a FLAG-tagged version of the same protein encoded from a floxed transcription unit containing silent mutations, making it refractory to the CRISPR-Cas9 guide. This FLAG-tagged protein can be deleted by activating the expressed Cre protein, enabling evaluation of the cellular consequences of its deletion. We have further used this system to evaluate the ability of phylogenic homologues and of potential mutants to cover functionally for the deleted gene.

Identification of human nephron progenitors capable of generation of kidney structures and functional repair of chronic renal disease.

Identification of tissue-specific renal stem/progenitor cells with nephrogenic potential is a critical step in developing cell-based therapies for renal disease. In the human kidney, stem/progenitor cells are induced into the nephrogenic pathway to form nephrons until the 34 week of gestation, and no equivalent cell types can be traced in the adult kidney. Human nephron progenitor cells (hNPCs) have yet to be isolated. Here we show that growth of human foetal kidneys in serum-free defined conditions and prospective isolation of NCAM1(+) cells selects for nephron lineage that includes the SIX2-positive cap mesenchyme cells identifying a mitotically active population with in vitro clonogenic and stem/progenitor properties. After transplantation in the chick embryo, these cells-but not differentiated counterparts-efficiently formed various nephron tubule types. hNPCs engrafted and integrated in diseased murine kidneys and treatment of renal failure in the 5/6 nephrectomy kidney injury model had beneficial effects on renal function halting disease progression. These findings constitute the first definition of an intrinsic nephron precursor population, with major potential for cell-based therapeutic strategies and modelling of kidney disease.