Unknown,Transcriptomics,Genomics,Proteomics

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Identification of miRNA target genes in C. elegans by RIP-chip-SRM

ABSTRACT: RIP-chip-SRM : a New Combinatorial Large Scale Approach Identifies a Set of Translationally Regulated bantam/miR 58 Targets in C. elegans RNA binding protein immunopurification + microarray + targeted protein quantification via Selected Reaction Monitoring This SuperSeries is composed of the following subset Series: GSE32941: Purification of TAP::ALG-1 complexes and associated RNA from mixed-stage TAP::ALG-1 transgenic (WS4303) and wild-type (N2) animals GSE32942: Microarray analysis of TAP::ALG-1 associated RNAs isolated from synchronized 'wild-type' animals and 'mir-58' mutants GSE32943: Expression analysis of WS4303 (wild type) vs WS5041 (mir-58 mutants) total mRNA Refer to individual Series

ORGANISM(S): Caenorhabditis elegans

SUBMITTER: Marko Jovanovic

PROVIDER: E-GEOD-32944 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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).

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Identification of miRNA target genes in C. elegans by RIP-chip-SRM

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