Reliability analysis of the Ahringer Caenorhabditis elegans RNAi feeding library: a guide for genome-wide screens

<p>Abstract</p> <p>Background</p> <p>The Ahringer <it>C. elegans </it>RNAi feeding library prepared by cloning genomic DNA fragments has been widely used in genome-wide analysis of gene function. However, the library has not been thoroughly validated by direct sequencing, and there are potential errors, including: 1) mis-annotation (the clone with the retired gene name should be remapped to the actual target gene); 2) nonspecific PCR amplification; 3) cross-RNAi; 4) mis-operation such as sample loading error, <it>etc</it>.</p> <p>Results</p> <p>Here we performed a reliability analysis on the Ahringer <it>C. elegans </it>RNAi feeding library, which contains 16,256 bacterial strains, using a bioinformatics approach. Results demonstrated that most (98.3%) of the bacterial strains in the library are reliable. However, we also found that 2,851 (17.54%) bacterial strains need to be re-annotated even they are reliable. Most of these bacterial strains are the clones having the retired gene names. Besides, 28 strains are grouped into unreliable category and 226 strains are marginal because of probably expressing unrelated double-stranded RNAs (dsRNAs). The accuracy of the prediction was further confirmed by direct sequencing analysis of 496 bacterial strains. Finally, a freely accessible database named CelRNAi (<url>http://biocompute.bmi.ac.cn/CelRNAi/</url>) was developed as a valuable complement resource for the feeding RNAi library by providing the predicted information on all bacterial strains. Moreover, submission of the direct sequencing result or any other annotations for the bacterial strains to the database are allowed and will be integrated into the CelRNAi database to improve the accuracy of the library. In addition, we provide five candidate primer sets for each of the unreliable and marginal bacterial strains for users to construct an alternative vector for their own RNAi studies.</p> <p>Conclusions</p> <p>Because of the potential unreliability of the Ahringer <it>C. elegans </it>RNAi feeding library, we strongly suggest the user examine the reliability information of the bacterial strains in the CelRNAi database before performing RNAi experiments, as well as the post-RNAi experiment analysis.</p

The Homeobox Protein CEH-23 Mediates Prolonged Longevity in Response to Impaired Mitochondrial Electron Transport Chain in C. elegans

Recent findings indicate that perturbations of the mitochondrial electron transport chain (METC) can cause extended longevity in evolutionarily diverse organisms. To uncover the molecular basis of how altered METC increases lifespan in C. elegans, we performed an RNAi screen and revealed that three predicted transcription factors are specifically required for the extended longevity of mitochondrial mutants. In particular, we demonstrated that the nuclear homeobox protein CEH-23 uniquely mediates the longevity but not the slow development, reduced brood size, or resistance to oxidative stress associated with mitochondrial mutations. Furthermore, we showed that ceh-23 expression levels are responsive to altered METC, and enforced overexpression of ceh-23 is sufficient to extend lifespan in wild-type background. Our data point to mitochondria-to-nucleus communications to be key for longevity determination and highlight CEH-23 as a novel longevity factor capable of responding to mitochondrial perturbations. These findings provide a new paradigm for how mitochondria impact aging and age-dependent diseases

Relationship Between Mitochondrial Electron Transport Chain Dysfunction, Development, and Life Extension in Caenorhabditis elegans

Prior studies have shown that disruption of mitochondrial electron transport chain (ETC) function in the nematode Caenorhabditis elegans can result in life extension. Counter to these findings, many mutations that disrupt ETC function in humans are known to be pathologically life-shortening. In this study, we have undertaken the first formal investigation of the role of partial mitochondrial ETC inhibition and its contribution to the life-extension phenotype of C. elegans. We have developed a novel RNA interference (RNAi) dilution strategy to incrementally reduce the expression level of five genes encoding mitochondrial proteins in C. elegans: atp-3, nuo-2, isp-1, cco-1, and frataxin (frh-1). We observed that each RNAi treatment led to marked alterations in multiple ETC components. Using this dilution technique, we observed a consistent, three-phase lifespan response to increasingly greater inhibition by RNAi: at low levels of inhibition, there was no response, then as inhibition increased, lifespan responded by monotonically lengthening. Finally, at the highest levels of RNAi inhibition, lifespan began to shorten. Indirect measurements of whole-animal oxidative stress showed no correlation with life extension. Instead, larval development, fertility, and adult size all became coordinately affected at the same point at which lifespan began to increase. We show that a specific signal, initiated during the L3/L4 larval stage of development, is sufficient for initiating mitochondrial dysfunction–dependent life extension in C. elegans. This stage of development is characterized by the last somatic cell divisions normally undertaken by C. elegans and also by massive mitochondrial DNA expansion. The coordinate effects of mitochondrial dysfunction on several cell cycle–dependent phenotypes, coupled with recent findings directly linking cell cycle progression with mitochondrial activity in C. elegans, lead us to propose that cell cycle checkpoint control plays a key role in specifying longevity of mitochondrial mutants

Fatty Acid Desaturation Links Germ Cell Loss to Longevity Through NHR-80/HNF4 in C. elegans

Lifespan extension induced by germline ablation in C. elegans is regulated by the nuclear hormone receptor NHR-80 in a process that requires the production of oleic acid by activation of the lipid desaturase FAT-6/SCD1

Sub-Telomeric core X and Y' Elements in S.cerevisiae Suppress Extreme Variations in Gene Silencing

Telomere Position Effect (TPE) is governed by strong repression signals emitted by telomeres via the Sir2/3/4 Histone Deacetylase complex. These signals are then relayed by weak proto-silencers residing in the subtelomeric core X and Y' elements. Subtelomeres also contain Sub-Telomeric Anti-silencing Regions (STARs). In this study we have prepared telomeres built of different combinations of core X, Y' and STARs and have analyzed them in strains lacking Histone-Acetyltransferase genes as well as in cdc6-1 and Δrif1 strains. We show that core X and Y' dramatically reduce both positive and negative variations in TPE, that are caused by these mutations. We also show that the deletion of Histone-Acetyltransferase genes reduce the silencing activity of an ACS proto-silencer, but also reduce the anti-silencing activity of a STAR. We postulate that core X and Y' act as epigenetic “cushioning” cis-elements

Daf-2 Signaling Modifies Mutant SOD1 Toxicity in C. elegans

The DAF-2 Insulin/IGF-1 signaling (IIS) pathway is a strong modifier of Caenorhabditis elegans longevity and healthspan. As aging is the greatest risk factor for developing neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS), we were interested in determining if DAF-2 signaling modifies disease pathology in mutant superoxide dismutase 1 (SOD1) expressing C. elegans. Worms with pan-neuronal G85R SOD1 expression demonstrate significantly impaired locomotion as compared to WT SOD1 expressing controls and they develop insoluble SOD1 aggregates. Reductions in DAF-2 signaling, either through a hypomorphic allele or neuronally targeted RNAi, decreases the abundance of aggregated SOD1 and results in improved locomotion in a DAF-16 dependant manner. These results suggest that manipulation of the DAF-2 Insulin/IGF-1 signaling pathway may have therapeutic potential for the treatment of ALS

Role of CBP and SATB-1 in Aging, Dietary Restriction, and Insulin-Like Signaling

Increased transcriptional complex activity, or pharmacological mimics of increased complex activity, predict lifespan in mice and mediate the protective effects of dietary restriction during aging

EXD2 governs germ stem cell homeostasis and lifespan by promoting mitoribosome integrity and translation

Mitochondria are subcellular organelles critical for meeting the bioenergetic and biosynthetic needs of the cell. Mitochondrial function relies on genes and RNA species encoded both in the nucleus and mitochondria, as well as their coordinated translation, import and respiratory complex assembly. Here we describe the characterization of exonuclease domain like 2 (EXD2), a nuclear encoded gene that we show is targeted to the mitochondria and prevents the aberrant association of mRNAs with the mitochondrial ribosome. The loss of EXD2 resulted in defective mitochondrial translation, impaired respiration, reduced ATP production, increased reactive oxygen species and widespread metabolic abnormalities. Depletion of EXD2/CG6744 in D.melanogaster caused developmental delays and premature female germline stem cell attrition, reduced fecundity and a dramatic extension of lifespan that could be reversed with an anti-oxidant diet. Our results define a conserved role for EXD2 in mitochondrial translation that influences development and aging