Molecular Determinants of the Regulation of Development and Metabolism by Neuronal eIF2α Phosphorylation in

Cell-nonautonomous effects of signaling in the nervous system of animals can influence diverse aspects of organismal physiology. We previously showed that phosphorylation of Ser49 of the α-subunit of eukaryotic translation initiation factor 2 (eIF2α) in two chemosensory neurons by PEK-1/PERK promotes entry of Caenorhabditis elegans into dauer diapause. Here, we identified and characterized the molecular determinants that confer sensitivity to effects of neuronal eIF2α phosphorylation on development and physiology of C. elegans. Isolation and characterization of mutations in eif-2Ba encoding the α-subunit of eIF2B support a conserved role, previously established by studies in yeast, for eIF2Bα in providing a binding site for phosphorylated eIF2α to inhibit the exchange factor eIF2B catalytic activity that is required for translation initiation. We also identified a mutation in eif-2c, encoding the γ-subunit of eIF2, which confers insensitivity to the effects of phosphorylated eIF2α while also altering the requirement for eIF2Bγ. In addition, we show that constitutive expression of eIF2α carrying a phosphomimetic S49D mutation in the ASI pair of sensory neurons confers dramatic effects on growth, metabolism, and reproduction in adult transgenic animals, phenocopying systemic responses to starvation. Furthermore, we show that constitutive expression of eIF2α carrying a phosphomimetic S49D mutation in the ASI neurons enhances dauer entry through bypassing the requirement for nutritionally deficient conditions. Our data suggest that the state of eIF2α phosphorylation in the ASI sensory neuron pair may modulate internal nutrient sensing and signaling pathways, with corresponding organismal effects on development and metabolism. Keywords: Caenorhabditis elegans; Dauer; EIF2α; phosphorylation; sensory neurons; translational contro

Riboswitches as Versatile Scaffolds for Ligand Recognition and as Tools for Drug Discovery

Riboswitches are highly structured, noncoding RNAs that bind specific ligands to regulate gene expression. To date, over fifty riboswitch classes have been validated to control fundamental biological processes and recognize various coenzymes, nucleotides, signaling molecules, ions, and other important ligands. As described in Chapter 1, careful study of these various riboswitches reveals an incredible diversity in their structural and biochemical characteristics as well as their function. Herein is detailed the efforts to validate long-standing orphan riboswitch classes involved in essential cellular functions such as maintaining nucleotide pools, managing ion toxicity, osmoregulation, and ATP production.The ykkC family of riboswitches are a set of at least five variant classes that share a conserved sequence and structure, but have accrued specific mutations in their ligand binding pockets to diverge and sense distinct ligands. Previous efforts to ascertain the ligand identities of these classes has resulted in the validation of the guanidine-I, ppGpp and PRPP riboswitches. These studies have revealed previously unknown biological activities, signaling networks, and complex gene regulatory systems. Chapter 2 describes the validation of the fourth variant class of the ykkC riboswitches, which promiscuously binds (d)ADP and (d)CDP to regulate hydrolase enzymes which presumably function to balance cellular nucleotide pools. The complexity of the molecular recognition of these molecules by the RNA serves to demonstrate the incredible adaptability of riboswitch sensors. Also reported in this chapter, are the efforts towards validating the last known ykkC riboswitch class, and the challenges faced in this endeavor. In Chapters 3 and 4, the first four riboswitch classes that specifically recognize the monovalent cations Na+ or Li+ are described. Many of the genes regulated by these riboswitches encode metal ion transporters that could help bacteria address ion toxicity. Other proteins whose expression are regulated by Na+-sensing riboswitches mitigate osmotic stress or harness Na+ gradients for ATP production. Some bacteria use a Na+ riboswitch in tandem with a riboswitch that recognizes the bacterial second messenger c-di-AMP to form a Boolean logic gate. This sophisticated device integrates information regarding Na+ concentration with c-di-AMP signaling to adapt to changing osmotic conditions. To date, only one protein factor has been experimentally validated to sense Na+ and regulate gene expression. Furthermore, the mechanisms of Li+ toxicity are poorly understood in general. Thus, the study of these riboswitches substantially advances our understanding of how cells sense and respond to these ions. Riboswitches are widely utilized by cells to perform sensory and regulatory tasks, but they also have great utility for various other applications. Chapter 5 describes the use of the S-adenosylhomocysteine (SAH) riboswitch as a biosensor for cellular levels of SAH in a high-throughput screen for small molecules that inhibit SAH nucleosidase, an enzyme responsible for recycling this toxic molecule. This example serves as another reminder of the amazing versatility of riboswitches as biological sensors

Mutant phenotypes for thousands of bacterial genes of unknown function.

One-third of all protein-coding genes from bacterial genomes cannot be annotated with a function. Here, to investigate the functions of these genes, we present genome-wide mutant fitness data from 32 diverse bacteria across dozens of growth conditions. We identified mutant phenotypes for 11,779 protein-coding genes that had not been annotated with a specific function. Many genes could be associated with a specific condition because the gene affected fitness only in that condition, or with another gene in the same bacterium because they had similar mutant phenotypes. Of the poorly annotated genes, 2,316 had associations that have high confidence because they are conserved in other bacteria. By combining these conserved associations with comparative genomics, we identified putative DNA repair proteins; in addition, we propose specific functions for poorly annotated enzymes and transporters and for uncharacterized protein families. Our study demonstrates the scalability of microbial genetics and its utility for improving gene annotations