eIF4F complex dynamics are important for the activation of the integrated stress response.

In response to stress, eukaryotes activate the integrated stress response (ISR) via phosphorylation of eIF2α to promote the translation of pro-survival effector genes, such as GCN4 in yeast. Complementing the ISR is the target of rapamycin (TOR) pathway, which regulates eIF4E function. Here, we probe translational control in the absence of eIF4E in Saccharomyces cerevisiae. Intriguingly, we find that loss of eIF4E leads to de-repression of GCN4 translation. In addition, we find that de-repression of GCN4 translation is accompanied by neither eIF2α phosphorylation nor reduction in initiator ternary complex (TC). Our data suggest that when eIF4E levels are depleted, GCN4 translation is de-repressed via a unique mechanism that may involve faster scanning by the small ribosome subunit due to increased local concentration of eIF4A. Overall, our findings suggest that relative levels of eIF4F components are key to ribosome dynamics and may play important roles in translational control of gene expression.

Modulation of sulfur assimilation metabolic toxicity overcomes anemia and hemochromatosis in mice.

Sulfur assimilation is an essential metabolic pathway that regulates sulfation, amino acid metabolism, nucleotide hydrolysis, and organismal homeostasis. We recently reported that mice lacking bisphosphate 3'-nucleotidase (BPNT1), a key regulator of sulfur assimilation, develop iron-deficiency anemia (IDA) and anasarca. Here we demonstrate two approaches that successfully reduce metabolic toxicity caused by loss of BPNT1: 1) dietary methionine restriction and 2) overproduction of a key transcriptional regulator hypoxia inducible factor 2α (Hif-2a). Reduction of methionine in the diet reverses IDA in mice lacking BPNT1, through a mechanism of downregulation of sulfur assimilation metabolic toxicity. Gaining Hif-2a acts through a different mechanism by restoring iron homeostatic gene expression in BPNT1 deficient mouse intestinal organoids. Finally, as loss of BPNT1 impairs expression of known genetic modifiers of iron-overload, we demonstrate that intestinal-epithelium specific loss of BPNT1 attenuates hepatic iron accumulation in mice with homozygous C282Y mutations in homeostatic iron regulator (HFEC282Y), the most common cause of hemochromatosis in humans. Overall, our study uncovers genetic and dietary strategies to overcome anemia caused by defects in sulfur assimilation and identifies BPNT1 as a potential target for the treatment of hemochromatosis.

Modulation of intestinal sulfur assimilation metabolism regulates iron homeostasis.

Sulfur assimilation is an evolutionarily conserved pathway that plays an essential role in cellular and metabolic processes, including sulfation, amino acid biosynthesis, and organismal development. We report that loss of a key enzymatic component of the pathway, bisphosphate 3'-nucleotidase (Bpnt1), in mice, both whole animal and intestine-specific, leads to iron-deficiency anemia. Analysis of mutant enterocytes demonstrates that modulation of their substrate 3'-phosphoadenosine 5'-phosphate (PAP) influences levels of key iron homeostasis factors involved in dietary iron reduction, import and transport, that in part mimic those reported for the loss of hypoxic-induced transcription factor, HIF-2α. Our studies define a genetic basis for iron-deficiency anemia, a molecular approach for rescuing loss of nucleotidase function, and an unanticipated link between nucleotide hydrolysis in the sulfur assimilation pathway and iron homeostasis.

O6-Methylguanosine leads to position-dependent effects on ribosome speed and fidelity.

Nucleic acids are under constant assault from endogenous and environmental agents that alter their physical and chemical properties. O6-methylation of guanosine (m(6)G) is particularly notable for its high mutagenicity, pairing with T, during DNA replication. Yet, while m(6)G accumulates in both DNA and RNA, little is known about its effects on RNA. Here, we investigate the effects of m(6)G on the decoding process, using a reconstituted bacterial translation system. m(6)G at the first and third position of the codon decreases the accuracy of tRNA selection. The ribosome readily incorporates near-cognate aminoacyl-tRNAs (aa-tRNAs) by forming m(6)G-uridine codon-anticodon pairs. Surprisingly, the introduction of m(6)G to the second position of the codon does not promote miscoding, but instead slows the observed rates of peptide-bond formation by >1000-fold for cognate aa-tRNAs without altering the rates for near-cognate aa-tRNAs. These in vitro observations were recapitulated in eukaryotic extracts and HEK293 cells. Interestingly, the analogous modification N6-methyladenosine (m(6)A) at the second position has only a minimal effect on tRNA selection, suggesting that the effects on tRNA selection seen with m(6)G are due to altered geometry of the base pair. Given that the m6G:U base pair is predicted to be nearly indistinguishable from a Watson-Crick base pair, our data suggest that the decoding center of the ribosome is extremely sensitive to changes at the second position. Our data, apart from highlighting the deleterious effects that these adducts pose to cellular fitness, shed new insight into decoding and the process by which the ribosome recognizes codon-anticodon pairs.

Ribosomes left in the dust: diverse strategies for Peptide-mediated translation stalling.

In two recent papers, Arenz et al. (2014a) and Bischoff et al. (2014) provide structural insights into drug-induced, peptide-mediated stalling of the ribosome.

An active role for the ribosome in determining the fate of oxidized mRNA.

Chemical damage to RNA affects its functional properties and thus may pose a significant hurdle to the translational apparatus; however, the effects of damaged mRNA on the speed and accuracy of the decoding process and their interplay with quality-control processes are not known. Here, we systematically explore the effects of oxidative damage on the decoding process using a well-defined bacterial in vitro translation system. We find that the oxidative lesion 8-oxoguanosine (8-oxoG) reduces the rate of peptide-bond formation by more than three orders of magnitude independent of its position within the codon. Interestingly, 8-oxoG had little effect on the fidelity of the selection process, suggesting that the modification stalls the translational machinery. Consistent with these findings, 8-oxoG mRNAs were observed to accumulate and associate with polyribosomes in yeast strains in which no-go decay is compromised. Our data provide compelling evidence that mRNA-surveillance mechanisms have evolved to cope with damaged mRNA.

Tissue-specific regulation of 3'-nucleotide hydrolysis and nucleolar architecture.

Sulfur is an essential micronutrient involved in diverse cellular functions ranging from the control of intracellular redox states to electron transport. Eukaryotes incorporate sulfur by metabolizing inorganic sulfate into the universal sulfur donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Sulfotransferases then catalyze the donation of the activated sulfur from PAPS to a broad range of acceptors including xenobiotic small molecules and extracellular proteoglycans while also generating the byproduct 3'-phosphoadenosine 5'-phosphate (PAP). In mammals, PAP is regulated by two related 3'-nucleotidases, Golgi-resident PAP phosphatase (gPAPP) and cytoplasmic bisphosphate 3'-nucleotidase 1 (Bpnt1), which hydrolyze PAP to 5'-AMP and whose inactivation results in severe physiological defects. Loss of Bpnt1 in mice leads to the accumulation of PAP in the liver, aberrant nucleolar architecture, and liver failure, all of which can be rescued by genetically repressing PAPS synthesis. Yet interestingly, Bpnt1 protein is expressed at high levels in a majority of tissues, suggesting that additional tissues might also be affected. To investigate this possibility, we closely examined the expression of Bpnt1 protein, accumulation of PAP, and appearance of dysmorphic nucleoli in wild-type and Bpnt1(-/-) mice. Surprisingly, we found that while Bpnt1 protein is widely expressed, only the liver, duodenum, and kidneys contain high levels of PAP and nucleolar reorganization. We hypothesize that these tissues share commonalities such as being highly polarized and situated at the interfaces of fluid reservoirs that might enhance their susceptibility to loss of Bpnt1. These studies highlight the importance of PAP metabolism in extrahepatic tissues and provide a framework for future investigations into the function of Bpnt1 in the kidney and small intestine.

Role for cytoplasmic nucleotide hydrolysis in hepatic function and protein synthesis.

Nucleotide hydrolysis is essential for many aspects of cellular function. In the case of 3',5'-bisphosphorylated nucleotides, mammals possess two related 3'-nucleotidases, Golgi-resident 3'-phosphoadenosine 5'-phosphate (PAP) phosphatase (gPAPP) and Bisphosphate 3'-nucleotidase 1 (Bpnt1). gPAPP and Bpnt1 localize to distinct subcellular compartments and are members of a conserved family of metal-dependent lithium-sensitive enzymes. Although recent studies have demonstrated the importance of gPAPP for proper skeletal development in mice and humans, the role of Bpnt1 in mammals remains largely unknown. Here we report that mice deficient for Bpnt1 do not exhibit skeletal defects but instead develop severe liver pathologies, including hypoproteinemia, hepatocellular damage, and in severe cases, frank whole-body edema and death. Accompanying these phenotypes, we observed tissue-specific elevations of the substrate PAP, up to 50-fold in liver, repressed translation, and aberrant nucleolar architecture. Remarkably, the phenotypes of the Bpnt1 knockout are rescued by generating a double mutant mouse deficient for both PAP synthesis and hydrolysis, consistent with a mechanism in which PAP accumulation is toxic to tissue function independent of sulfation. Overall, our study defines a role for Bpnt1 in mammalian physiology and provides mechanistic insights into the importance of sulfur assimilation and cytoplasmic PAP hydrolysis to normal liver function.