The mammalian Nrf/CNC proteins (Nrf1, Nrf2, Nrf3, p45 NF-E2) perform a wide range of cellular protective and maintenance functions. The most thoroughly described of these proteins, Nrf2, is best known as a regulator of antioxidant and xenobiotic defense, but more recently has been implicated in additional functions that include proteostasis and metabolic regulation. In the nematode Caenorhabditis elegans, which offers many advantages for genetic analyses, the Nrf/CNC proteins are represented by their ortholog SKN-1. Although SKN-1 has diverged in aspects of how it binds DNA, it exhibits remarkable functional conservation with Nrf/CNC proteins in other species and regulates many of the same target gene families. C. elegans may therefore have considerable predictive value as a discovery model for understanding how mammalian Nrf/CNC proteins function and are regulated in vivo. Work in C. elegans indicates that SKN-1 regulation is surprisingly complex and is influenced by numerous growth, nutrient, and metabolic signals. SKN-1 is also involved in a wide range of homeostatic functions that extend well beyond the canonical Nrf2 function in responses to acute stress. Importantly, SKN-1 plays a central role in diverse genetic and pharmacologic interventions that promote C. elegans longevity, suggesting that mechanisms regulated by SKN-1 may be of conserved importance in aging. These C. elegans studies predict that mammalian Nrf/CNC protein functions and regulation may be similarly complex and that the proteins and processes that they regulate are likely to have a major influence on mammalian life- and healthspan.

Summary mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70k to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.

Highlights d Dietary restriction longevity requires modulation of nutrientregulated immunity d Nutrients activate the p38-ATF-7 immunometabolic pathway independently of mTORC1 d Insulin/IGF-1 signaling affects immunity and aging in part by curtailing food intake d DAF-16/FOXO lowers food consumption, linking feeding and immunity to growth signals

BackgroundMicroRNAs (miRNA) are an abundant and ubiquitous class of small RNAs that play prominent roles in gene regulation. A significant fraction of miRNA genes reside in the introns of the host genes in the same orientation and are thought to be co-processed from the host gene mRNAs and thus depend on the host gene promoter for their expression. However, several lines of evidence for independent expression of intronic miRNAs exist in the literature but the extent of this independence remains unclear.ResultsWe performed a systematic analysis of genomic regions surrounding intronic miRNAs in the nematode Caenorhabditis elegans and found that, in many cases, there are extended intronic sequences immediately upstream of the miRNAs that are well-conserved between the nematodes. We have generated transcriptional green fluorescent protein reporter fusions in transgenic C. elegans lines and demonstrated that, in all seven investigated cases, the conserved sequences show promoter properties and produce specific expression patterns that are different from the host gene expression patterns. The observed expression patterns are corroborated by the published small RNA sequencing data.ConclusionsOur analysis reveals that the number of intronic miRNAs that do not rely on their host genes for expression is substantially higher than previously appreciated. At least one-third of the same-strand intronic miRNAs in C. elegans posses their own promoters and, thus, could be transcribed independently from their host genes. These findings provide a new insight into the regulation of miRNA genes and will be useful for the analysis of interactions between miRNAs and their host genes.

Diverse stresses and aging alter expression levels of microRNAs, suggesting a role for these posttranscriptional regulators of gene expression in stress modulation and longevity. Earlier studies demonstrated a central role for the miR-34 family in promoting cell cycle arrest and cell death following stress in human cells. However, the biological significance of this response was unclear. Here we show that in C. elegans mir-34 upregulation is necessary for developmental arrest, correct morphogenesis, and adaptation to a lower metabolic state to protect animals against stress-related damage. Either deletion or overexpression of mir-34 lead to an impaired stress response, which can largely be explained by perturbations in DAF-16/FOXO target gene expression. We demonstrate that mir-34 expression is regulated by the insulin signaling pathway via a negative feedback loop between miR-34 and DAF-16/FOXO. We propose that mir-34 provides robustness to stress response programs by controlling noise in the DAF-16/FOXO-regulated gene network.

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Mitochondrial defects underlie a multitude of human diseases. Genetic manipulation of mitochondrial regulatory pathways represents a potential therapeutic approach. We have carried out a highthroughput overexpression screen for genes that affect mitochondrial abundance or activity using flow-cytometry-based enrichment of a cell population expressing a high-complexity, concentrationnormalized pool of human ORFs. The screen identified 94 candidate mitochondrial regulators including the nuclear protein GLTSCR2, also known as PICT1. GLTSCR2 enhances mitochondrial function and is required for the maintenance of oxygen consumption, consistent with a pivotal role in the control of cellular respiration. RNAi inactivation of the Caenorhabditis elegans ortholog of GLTSCR2 reduces respiration in worms, indicating functional conservation across species. GLTSCR2 controls cellular proliferation and metabolism via the transcription factor Myc, and is induced by mitochondrial stress, suggesting it may constitute a significant component of the mitochondrial signaling pathway.

The ability to generate transgenic animals to study gene expression and function is a powerful and important part of the Caenorhabditis elegans genetic toolbox. Transgenic animals can be created by introducing exogenous DNA into the worm germline either by microinjection or by microparticle bombardment (biolistic transformation). In this chapter we describe a simple and robust protocol to generate transgenic C. elegans animals by biolistic transformation with gold particles using the Bio-Rad PDS-1000/He system with Hepta adapter and unc-119 selection marker. We also point out the steps that need special attention to achieve successful transformations.