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:This study analyzes miRNA association with ALG-1 and ALG-2 in different stages during larval development of C. elegans Staged animals (L1, L2, L3, L4) - the alg-2 mutant expressing tagged-ALG-2, alg-1 mutant, and wild type controls - were lysed. ALG-1 IP or ALG-2 (GFP) IP was performed using tags from all four samples at all four timepoints. Small RNAs were released from antibodies and 5' end-labelled with AlexaFluor532. Labelled RNA was hybridized to a custom microRNA microarray platform to quantify miRNA content.
Project description:In order to examine the misregulation of genes in loss of function alg-1 and alg-2 versus WT, RNA-seq was performed on D5 C.elegans in WT(N2), alg-1(gk214), alg-2(ok304)
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. Deep-sequencing was performed on cDNA libraries made from total RNA and RNA immunoprecipitated with ALG-1 from mixed-staged populations of three strains: three biological replicates from wild-type animals and two biological replicates each from alg-1(ma192) and alg-1(ma202) mutant animals. In addition, deep-sequencing was performed on cDNA libraries made from L2-staged total RNA in two biological replicates from wildtype and alg-1(ma202) animals and one biological replicate of alg-1(ma192).
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: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: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.
Project description:Comprehensive discovery of endogenous Argonaute binding sites in C. elegans Experiment Overall Design: Total RNA were extracted from whole alg-1(gk214) (alg-1(-)) and N2 (wildtype) worms and used to compare the expression profiles of genes that were targeted by ALG-1 protein.