Translation elongation factor 2 depletion by siRNA in mouse liver leads to mTOR-independent translational upregulation of ribosomal protein genes.

Due to breakthroughs in RNAi and genome editing methods in the past decade, it is now easier than ever to study fine details of protein synthesis in animal models. However, most of our understanding of translation comes from unicellular organisms and cultured mammalian cells. In this study, we demonstrate the feasibility of perturbing protein synthesis in a mouse liver by targeting translation elongation factor 2 (eEF2) with RNAi. We were able to achieve over 90% knockdown efficacy and maintain it for 2 weeks effectively slowing down the rate of translation elongation. As the total protein yield declined, both proteomics and ribosome profiling assays showed robust translational upregulation of ribosomal proteins relative to other proteins. Although all these genes bear the TOP regulatory motif, the branch of the mTOR pathway responsible for translation regulation was not activated. Paradoxically, coordinated translational upregulation of ribosomal proteins only occurred in the liver but not in murine cell culture. Thus, the upregulation of ribosomal transcripts likely occurred via passive mTOR-independent mechanisms. Impaired elongation sequesters ribosomes on mRNA and creates a shortage of free ribosomes. This leads to preferential translation of transcripts with high initiation rates such as ribosomal proteins. Furthermore, severe eEF2 shortage reduces the negative impact of positively charged amino acids frequent in ribosomal proteins on ribosome progression.

A novel uORF-based regulatory mechanism controls translation of the human MDM2 and eIF2D mRNAs during stress.

Short upstream open reading frames (uORFs) are the most prevalent cis-acting regulatory elements in the mammalian transcriptome which can orchestrate mRNA translation. Apart from being "passive roadblocks" that decrease expression of the main coding regions, particular uORFs can serve as specific sensors for changing conditions, thus regulating translation in response to cell stress. Here we report a novel uORF-based regulatory mechanism that is employed under conditions of hyperosmotic stress by at least two human mRNAs, coding for translation reinitiation/recycling factor eIF2D and E3 ubiquitin ligase MDM2. This novel mode of translational control selectively downregulates their expression and requires as few as one uORF. Using a set of reporter mRNAs and fleeting mRNA transfection (FLERT) technique, we provide evidence that the phenomenon does not rely on delayed reinitiation, altered AUG recognition, ribosome stalling, mRNA destabilization or other known mechanisms. Instead, it is based on events taking place at uORF stop codon or immediately downstream. Functional aspects and implications of the novel regulatory mechanism to cell physiology are discussed.

Late Diagnosis of POMC Deficiency and In Vitro Evidence of Residual Translation From Allele With c.-11C>A Mutation.

Context: Loss-of-function mutations in the POMC gene are associated with a syndrome with the characteristics of adrenal insufficiency, obesity, and red hair. We describe here a case of pro-opiomelanocortin (POMC) deficiency in which adrenal insufficiency was not treated until the fourth year of life. One of the disease-causative POMC mutations was characterized in vitro using a unique approach. Case Description: A boy presented in the first year of life with red hair, growth acceleration, moderate obesity, and recurrent cholestasis, which was followed by 2 episodes of hypoglycemia at the ages of 1.5 and 3 years. The diagnosis was suspected at the age of 3.6 years after documentation of undetectable levels of plasma adrenocorticotropic hormone and serum cortisol, after which replacement with hydrocortisone was initiated. Sequencing of the POMC gene revealed compound heterozygosity for c.-11C>A/p.W84X mutations. The p.W84X mutation is predicted to result in a marked truncation of preprohormone. Using a messenger RNA transfection approach followed by an in vitro translation assay, we could directly demonstrate that the transcript with c.-11C>A substitution is predominantly translated within a new open reading frame; however, translation of the POMC main reading frame is preserved, with translation efficiency being ∼17% of the wild-type transcript. Conclusions: The current report provides important information on the natural course of POMC deficiency. In vitro translation studies demonstrated residual translation of the main coding region from an allele with the c.-11C>A mutation, which at least partially explains a relatively late presentation of adrenal insufficiency in the patient.

Four translation initiation pathways employed by the leaderless mRNA in eukaryotes.

mRNAs lacking 5' untranslated regions (leaderless mRNAs) are molecular relics of an ancient translation initiation pathway. Nevertheless, they still represent a significant portion of transcriptome in some taxons, including a number of eukaryotic species. In bacteria and archaea, the leaderless mRNAs can bind non-dissociated 70 S ribosomes and initiate translation without protein initiation factors involved. Here we use the Fleeting mRNA Transfection technique (FLERT) to show that translation of a leaderless reporter mRNA is resistant to conditions when eIF2 and eIF4F, two key eukaryotic translation initiation factors, are inactivated in mammalian cells. We report an unconventional translation initiation pathway utilized by the leaderless mRNA in vitro, in addition to the previously described 80S-, eIF2-, or eIF2D-mediated modes. This mechanism is a bacterial-like eIF5B/IF2-assisted initiation that has only been reported for hepatitis C virus-like internal ribosome entry sites (IRESs). Therefore, the leaderless mRNA is able to take any of four different translation initiation pathways in eukaryotes.

Early B-cell factor 1 (EBF1) is critical for transcriptional control of SLAMF1 gene in human B cells.

Signaling lymphocytic activation molecule family member 1 (SLAMF1)/CD150 is a co-stimulatory receptor expressed on a variety of hematopoietic cells, in particular on mature lymphocytes activated by specific antigen, costimulation and cytokines. Changes in CD150 expression level have been reported in association with autoimmunity and with B-cell chronic lymphocytic leukemia. We characterized the core promoter for SLAMF1 gene in human B-cell lines and explored binding sites for a number of transcription factors involved in B cell differentiation and activation. Mutations of SP1, STAT6, IRF4, NF-kB, ELF1, TCF3, and SPI1/PU.1 sites resulted in significantly decreased promoter activity of varying magnitude, depending on the cell line tested. The most profound effect on the promoter strength was observed upon mutation of the binding site for Early B-cell factor 1 (EBF1). This mutation produced a 10-20 fold drop in promoter activity and pinpointed EBF1 as the master regulator of human SLAMF1 gene in B cells. We also identified three potent transcriptional enhancers in human SLAMF1 locus, each containing functional EBF1 binding sites. Thus, EBF1 interacts with specific binding sites located both in the promoter and in the enhancer regions of the SLAMF1 gene and is critical for its expression in human B cells.

Amicoumacin A induces cancer cell death by targeting the eukaryotic ribosome.

Amicoumacin A is an antibiotic that was recently shown to target bacterial ribosomes. It affects translocation and provides an additional contact interface between the ribosomal RNA and mRNA. The binding site of amicoumacin A is formed by universally conserved nucleotides of rRNA. In this work, we showed that amicoumacin A inhibits translation in yeast and mammalian systems by affecting translation elongation. We determined the structure of the amicoumacin A complex with yeast ribosomes at a resolution of 3.1 Å. Toxicity measurement demonstrated that human cancer cell lines are more susceptible to the inhibition by this compound as compared to non-cancerous ones. This might be used as a starting point to develop amicoumacin A derivatives with clinical value.

Sliding of a 43S ribosomal complex from the recognized AUG codon triggered by a delay in eIF2-bound GTP hydrolysis.

During eukaryotic translation initiation, 43S ribosomal complex scans mRNA leader unless an AUG codon in an appropriate context is found. Establishing the stable codon-anticodon base-pairing traps the ribosome on the initiator codon and triggers structural rearrangements, which lead to Pi release from the eIF2-bound GTP. It is generally accepted that AUG recognition by the scanning 43S complex sets the final point in the process of start codon selection, while latter stages do not contribute to this process. Here we use translation reconstitution approach and kinetic toe-printing assay to show that after the 48S complex is formed on an AUG codon, in case GTP hydrolysis is impaired, the ribosomal subunit is capable to resume scanning and slides downstream to the next AUG. In contrast to leaky scanning, this sliding is not limited to AUGs in poor nucleotide contexts and occurs after a relatively long pause at the recognized AUG. Thus, recognition of an AUG per se does not inevitably lead to this codon being selected for initiation of protein synthesis. Instead, it is eIF5-induced GTP hydrolysis and Pi release that irreversibly trap the 48S complex, and this complex is further stabilized by eIF5B and 60S joining.