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: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:microRNAs fine-tune the regulation of numerous biological processes by mediating gene expression post-transcriptionally. Mature miRNAs associate with argonaute (AGO) proteins to form the RNA-induced silencing complex (RISC) that binds to and blocks translation of target mRNAs. In our study, we address the function of miRNAs in regulating DNA damage responses in-vivo, by exploiting the nematode model organism Caenorhabditis elegans. Using next-generation sequencing, we obtained miRNA expression profiles of WT worms as well as mutants harboring a loss-of-function deletion of the C. elegans AGO gene alg-2 that display abnormal cell death in response to genotoxic stress. 2.5 hours after exposure to ionizing irradiation, the miRNA transcriptomes of both WT and alg-2(ok304) mutant worms were only mildly altered in comparison to their respective untreated controls. However, irrespective of treatment, alg-2(ok304) mutants exhibited noticeable dysregulation of several miRNA families with known physiological functions. Reduced levels of some of these miRNAs might explain the abnormal DNA damage response of alg-2-deficient C. elegans.
Project description:C.elegans small RNAs from HA::ALG-1, HA::ALG-2 and HA::RDE-1 IP and rde-1 mutants Small RNAs were cloned from transgenic or mutant C. elegans adults. Sequencing was performed using 454 and Illumina platforms.
Project description:MicroRNAs (miRNA) associate with Argonaute proteins and negatively regulate gene expression by base pairing with complementary sequences in the 3’ UTRs of target genes. De novo coding variants in the human Argonaute gene AGO1 were reported to cause neurodevelopmental disorder (NDD) with intellectual disability (ID). Most of the altered amino acids are conserved between the miRNA associated Argonautes in H. sapiens and C. elegans, suggesting that the human AGO1 mutations could disrupt evolutionarily conserved functions in miRNA biogenesis or target repression. We genetically modeled four human AGO1 mutations in C. elegans by introducing identical mutations into the C. elegans AGO1 homolog, ALG-1. These alg-1 NDD mutations caused phenotypes in C. elegans indicative of disrupted miRNA processing, miRISC formation, and/or target repression. We show that the alg-1 NDD mutations are antimorphic as they cause developmental and molecular phenotypes stronger than those exhibited by the alg-1 null mutants, likely by sequestrating functional miRNA silencing complex (miRISC) components into non-functional complexes that fail to confer robust gene repression. The alg-1 NDD mutations cause allele-specific disruptions in mature miRNA profiles, both in overall abundances and ALG-1 NDD association, accompanied by perturbation of downstream gene expression, including altered translational efficiency and/or mRNA abundance. The perturbed genes include those with human orthologs whose dysfunction is associated with NDD. These cross-clade genetic studies illuminate fundamental Argonaute functions and provide insights into the conservation of miRNA-mediated post-transcriptional regulatory mechanisms.
Project description:microRNAs (miRNAs) are potent regulators of gene expression that function in diverse developmental and physiological processes. Argonaute proteins loaded with miRNAs form the miRNA Induced Silencing Complexes (miRISCs) that repress gene expression at the post-transcriptional level. miRISCs target genes through partial sequence complementarity between the miRNA and the target mRNA’s 3’ UTR. In addition to being targeted by miRNAs, these mRNAs are also extensively regulated by RNA-binding proteins (RBPs) through RNA processing, transport, stability, and translation regulation. While the degree to which RBPs and miRISCs interact to regulate gene expression is likely extensive, we have only begun to unravel the mechanisms of this functional cooperation. An RNAi-based screen of putative ALG-1 Argonaute interactors has identified a role for a conserved RNA binding protein, HRPK- 1, in modulating miRNA activity during C. elegans development. Here, we report the physical and genetic interaction between HRPK-1 and ALG-1/miRNAs. Specifically, we report the genetic and molecular characterizations of hrpk-1 and its role in C. elegans development and miRNA-mediated target repression. We show that loss of hrpk-1 causes numerous developmental defects and enhances the mutant phenotypes associated with reduction of miRNA activity, including those of lsy-6, mir-35-family, and let-7-family miRNAs. In addition to hrpk-1 genetic interaction with these miRNA families, hrpk-1 is required for efficient regulation of lsy-6 target cog-1. We report that hrpk-1 plays a role in processing of some but not all miRNAs and is not required for ALG-1/AIN-1 miRISC assembly. We suggest that HRPK-1 may functionally interact with miRNAs by both affecting miRNA processing and by enhancing miRNA/miRISC gene regulatory activity and present models for its activity.
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: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.