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Regulation of Dauer formation by O-GlcNAcylation in Caenorhabditis elegans.

ABSTRACT: Modification of proteins at serine or threonine residues with N-acetylglucosamine, termed O-GlcNAcylation, plays an important role in most eukaryotic cells. To understand the molecular mechanism by which O-GlcNAcylation regulates the entry of Caenorhabditis elegans into the non-aging dauer state, we performed proteomic studies using two mutant strains: the O-GlcNAc transferase-deficient ogt-1(ok430) strain and the O-GlcNAcase-defective oga-1(ok1207) strain. In the presence of the dauer pheromone daumone, ogt-1 showed suppression of dauer formation, whereas oga-1 exhibited enhancement of dauer formation. Consistent with these findings, treatment of wild-type N2 worms with low concentrations of daumone and the O-GlcNAcase inhibitor O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) enhanced dauer formation, which was dependent on intact O-GlcNAcylation metabolism. We also found that the treatment of daumone enhanced O-GlcNAcylation in vivo. Seven proteins, identified by coupled two-dimensional electrophoresis/liquid chromatography-mass spectroscopy (LC-MS) analysis, were differentially expressed in oga-1(ok1207) worms compared with wild-type N2 worms. The identities of these proteins suggest that O- GlcNAcylation influences stress resistance, protein folding, and mitochondrial function. Using O-GlcNAc labeling with fluorescent dye combined with two-dimensional electrophoresis/LC-MS analysis, we also identified five proteins that were differentially O-GlcNAcylated during dauer formation. Analysis of these candidate O-GlcNAcylated proteins suggests that O-GlcNAcylation may regulate cytoskeleton modifications and protein turnover during dauer formation.

SUBMITTER: Lee J

PROVIDER: S-EPMC2823417 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Regulation of Dauer formation by O-GlcNAcylation in Caenorhabditis elegans.

Lee Jeeyong J Kim Kwang-Youl KY Lee Jihyun J Paik Young-Ki YK

The Journal of biological chemistry 20091123 5

Modification of proteins at serine or threonine residues with N-acetylglucosamine, termed O-GlcNAcylation, plays an important role in most eukaryotic cells. To understand the molecular mechanism by which O-GlcNAcylation regulates the entry of Caenorhabditis elegans into the non-aging dauer state, we performed proteomic studies using two mutant strains: the O-GlcNAc transferase-deficient ogt-1(ok430) strain and the O-GlcNAcase-defective oga-1(ok1207) strain. In the presence of the dauer pheromone ...[more]

PMID: 19940149

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Project description:Identifying the physiological functions of microRNAs (miRNAs) is often challenging because miRNAs commonly impact gene expression under specific physiological conditions through complex miRNA::mRNA interaction networks and in coordination with other means of gene regulation, such as transcriptional regulation and protein degradation. Such complexity creates difficulties in dissecting miRNA functions through traditional genetic methods using individual miRNA mutations. To investigate the physiological functions of miRNAs in neurons, we combined a genetic "enhancer" approach complemented by biochemical analysis of neuronal miRNA-induced silencing complexes (miRISCs) in C. elegans. Total miRNA function can be compromised by mutating one of the two GW182 proteins (AIN-1), an important component of miRISC. We found that combining an ain-1 mutation with a mutation in unc-3, a neuronal transcription factor, resulted in an inappropriate entrance into the stress-induced, alternative larval stage known as dauer, indicating a role of miRNAs in preventing aberrant dauer formation. Analysis of this genetic interaction suggests that neuronal miRNAs perform such a role partly by regulating endogenous cyclic guanosine monophosphate (cGMP) signaling, potentially influencing two other dauer-regulating pathways. Through tissue-specific immunoprecipitations of miRISC, we identified miRNAs and their likely target mRNAs within neuronal tissue. We verified the biological relevance of several of these miRNAs and found that many miRNAs likely regulate dauer formation through multiple dauer-related targets. Further analysis of target mRNAs suggests potential miRNA involvement in various neuronal processes, but the importance of these miRNA::mRNA interactions remains unclear. Finally, we found that neuronal genes may be more highly regulated by miRNAs than intestinal genes. Overall, our study identifies miRNAs and their targets, and a physiological function of these miRNAs in neurons. It also suggests that compromising other aspects of gene expression, along with miRISC, can be an effective approach to reveal miRNA functions in specific tissues under specific physiological conditions.

Dataset Information

Regulation of Dauer formation by O-GlcNAcylation in Caenorhabditis elegans.

Publications

Regulation of Dauer formation by O-GlcNAcylation in Caenorhabditis elegans.

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