Project description:Cellular response to redox imbalance is crucial for organismal health. microRNAs are implicated in stress responses. ALG-1, the C. elegans ortholog of human AGO2, plays an essential role in microRNA processing and function. Here we investigated the mechanisms governing ALG-1 expression in C. elegans and the players controlling lifespan and stress resistance downstream of ALG-1. We found that upregulated ALG-1 is a shared feature in conditions linked to increased longevity (e.g., germline-deficient glp-1 mutants). ALG-1 knockdown reduces lifespan and oxidative stress resistance, while overexpression enhances survival against pro-oxidant agents but not heat or reductive stress. R02D3.7 represses alg-1 expression, impacting oxidative stress resistance at least in part via ALG-1. microRNAs upregulated in glp-1 mutants (miR-87-3p, miR-230-3p, and miR-235-3p) can target genes in the protein disulfide isomerase pathway and protect against oxidative stress. This study unveils a tightly regulated network involving transcription factors and microRNAs which controls organisms’ ability to withstand oxidative stress.
Project description:Investigated the specific miRNAs that are assocaited with ALG-1 and ALG-2 in adult (D5) C. elegans. ALG-1 and ALG-2 were IP-ed from D5 collected C.elegans, RNA was isolated and used for small RNA sequencing. Two biological replicates were used.
Project description:Here, we report a new phosphorylation site on ALG-1 that modulates its ability to bind miRNAs. Mutating ALG-1 S642 into a phosphomimetic residue strongly impairs binding to miRNAs. Furthermore, this mutation causes embryonic lethality which are not observed in animals depleted of alg-1 suggesting that it may consequently impair the normal function of its homolog alg-2. Quantification of miRNAs in the phosphorylation mutants of alg-1 reveals that the miRNA passenger strands are strongly increased but not preferentially loaded into ALG-1, indicating that the defects in miRNA binding may also lead to an accumulation of miRNA duplexes.
Project description:MicroRNAs are regulators of gene expression whose functions are critical for normal development and physiology. We have previously characterized mutations in a Caenorhabditis elegans microRNA-specific Argonaute ALG-1 (Argonaute-like gene) that are antimorphic [alg-1(anti)]. alg-1(anti) mutants have dramatically stronger microRNA-related phenotypes than animals with a complete loss of ALG-1. ALG-1(anti) miRISC (microRNA induced silencing complex) fails to undergo a functional transition from microRNA processing to target repression. To better understand this transition, we characterized the small RNA population associated with ALG-1(anti) complexes in vivo. alg-1(anti) mutants dramatically overaccumulated microRNA* (passenger) strands, and immunoprecipitated ALG-1(anti) complexes contained nonstoichiometric yields of mature microRNA and microRNA* strands, with some microRNA* strands present in the ALG-1(anti) Argonaute far in excess of the corresponding mature microRNAs. We show complex and microRNA-specific defects in microRNA strand selection and microRNA* strand disposal. For certain microRNAs (for example mir-58), microRNA guide strand selection by ALG-1(anti) appeared normal, but microRNA* strand release was inefficient. For other microRNAs (such as mir-2), both the microRNA and microRNA* strands were selected as guide by ALG-1(anti), indicating a defect in normal specificity of the strand choice. Our results suggest that wild-type ALG-1 complexes recognize structural features of particular microRNAs in the context of conducting the strand selection and microRNA* ejection steps of miRISC maturation.
Project description:Caenorhabditis elegans contains twenty-five Argonautes, of which, only ALG-1 and ALG-2 are known to interact with miRNAs. ALG-5 belongs to the AGO subfamily of Argonautes that includes ALG-1 and ALG-2, but its role in small RNA pathways is unknown. We analyzed by high-throughput sequencing the small RNAs associated with ALG-5, ALG-1, and ALG-2, as well as changes in mRNA expression in alg-5, alg-1, and alg-2 mutants.
Project description:Argonaute proteins are at the core of the microRNA-mediated gene silencing pathway essential for animals. In C. elegans, the microRNA-specific Argonautes ALG-1 and ALG-2 regulate multiple processes required for proper animal developmental timing and viability. Here, we identified a new phosphorylation site, serine 642, on ALG-1 that modulates microRNA association. Mutating ALG-1 serine 642 into a phospho-mimicking residue impairs microRNA binding and causes embryonic lethality and post-embryonic phenotypes that are common with alteration of microRNA functions. Monitoring microRNA levels in alg-1 phosphorylation mutant animals reveal that miRNA passenger strands strongly increase but are not preferentially loaded into ALG-1, indicating that the miRNA binding defects could also lead to miRNA duplexes accumulation. Our genetic and biochemical experiments support the protein kinase A KIN-1 as the putative kinase that phosphorylates ALG-1 serine 642. Altogether, our data indicate that PKA triggers the ALG-1 phosphorylation to regulate its microRNAs association during C. elegans development.
Project description:As part of the ENCODE consortium project (NIH, NHGRI), we conducted genome-wide location analysis for various transcription factors in various cell types. The long-term goal of this project is to determine the function of each DNA nucleotide in the genome. Measurement of transcription factor binding events is an important part of this effort. Here we measure genome-wide binding events for a variety of important transcriptional regulators. Keywords: ChIP-chip
Project description:To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein (C. elegans strain WS4303). We crossed the TAP::ALG-1 transgene into the mir-58(n4640) mutant background to generate the strain WS5041. For simplicity, we will hereafter term the TAP::ALG-1 transgenic animals as “wild type” and the transgenic WS5041 animals as “mir-58”. In addition to immunopurifying the TAP::ALG-1 and associated RNAs from these strains, we also compared total mRNA levels between these two strains. Total RNA was isolated from the same WS4303 and WS5041 total extracts that was further used for the TAP::ALG-1 RIP. Three independent biological replicates were analyzed. Long-oligo whole-genome C. elegans arrays, produced by the Genome Sequencing Center at Washington University in St. Louis (http://genome.wustl.edu/genome/celegans/microarray/ma_gen_info.cgi), were used for these experiments. A total of 10 µg of total RNA was used for cDNA synthesis.
Project description:We aimed to characterize the cell-type specific loading patterns of miRNAs in c.elegans. We expressed cell-type specific HA-epitope tagged versions of Argonaute-like 1 (ALG-1) and ALG-2 from three major tissue types i.e. intestine, nervous system and body wall mucle. We found that most miRNAs display highly cell-type specific loading patterns. ALG-1 is more ubiquitously loaded whereas ALG-2 is eniriched for miRNA loading within the nervous system. Addtionally we show that there is flexibility in ALG loading which changes during the aging process.