Project description:Dicer is a deeply conserved endoribonuclease with key functions in small RNA biogenesis. Here we employed PAR-CLIP/iPAR-CLIP to identify direct Dicer binding sites in the transcriptomes of human cells and human. We found hundreds of novel miRNAs and non-canonical Dicer substrates with high sensitivity. Small RNA production depended on structure of the binding site and is globally biased towards the 5' arm of hairpins. Unexpectedly, in both species Dicer bound numerous hairpins inside mRNAs without observable small RNA production. Our data revealed ~100 mRNAs of protein coding genes to be targeted in both human and worm. These mRNAs significantly overlapped with the RNAi pathway. We also, unexpectedly, found that mitochondrial transcripts are Dicer targets in both species. We demonstrate functional consequences of Dicer binding by perturbation analysis. Taken together,we provide the first genome-wide catalog of direct Dicer targets. Our results suggest widespread function outside of miRNA biogenesis. PAR-CLIP basically as described previously (Hafner et al. 2010).

2014-11-20 | E-GEOD-55324 | biostudies-arrayexpress

Transcriptome wide identification of Dicer binding in human and C. elegans reveals a variety of substrates (CEL PAR-CLIP)

Project description:Dicer is a deeply conserved endoribonuclease with key functions in small RNA biogenesis. Here we employed PAR-CLIP/iPAR-CLIP to identify direct Dicer binding sites in the transcriptomes of human cells and human. We found hundreds of novel miRNAs and non-canonical Dicer substrates with high sensitivity. Small RNA production depended on structure of the binding site and is globally biased towards the 5' arm of hairpins. Unexpectedly, in both species Dicer bound numerous hairpins inside mRNAs without observable small RNA production. Our data revealed ~100 mRNAs of protein coding genes to be targeted in both human and worm. These mRNAs significantly overlapped with the RNAi pathway. We also, unexpectedly, found that mitochondrial transcripts are Dicer targets in both species. We demonstrate functional consequences of Dicer binding by perturbation analysis. Taken together,we provide the first genome-wide catalog of direct Dicer targets. Our results suggest widespread function outside of miRNA biogenesis. In vivo PAR-CLIP basically as described previously (Jungkamp et al. 2011). FLAG tagged DCR-1 expressing (rescue strain) and 4SU labeled animals were UV irradiated at young adult stage. Worm lysate (NP40) was treated with RNase T1 and RNase V1. After that, PAR-CLIP proceeded as described in Hafner et al. 2010.

2014-11-20 | E-GEOD-55325 | biostudies-arrayexpress

Homo sapiens

Project description:A variety of Dicer substrates in human and C. elegans (HEK PAR-CLIP)

| PRJNA267836 | ENA

Transcriptome wide identification of Dicer binding in human and C. elegans reveals a variety of substrates (HEK RNA-Seq)

Project description:Dicer is a deeply conserved endoribonuclease with key functions in small RNA biogenesis. Here we employed PAR-CLIP/iPAR-CLIP to identify direct Dicer binding sites in the transcriptomes of human cells and human. We found hundreds of novel miRNAs and non-canonical Dicer substrates with high sensitivity. Small RNA production depended on structure of the binding site and is globally biased towards the 5' arm of hairpins. Unexpectedly, in both species Dicer bound numerous hairpins inside mRNAs without observable small RNA production. Our data revealed ~100 mRNAs of protein coding genes to be targeted in both human and worm. These mRNAs significantly overlapped with the RNAi pathway. We also, unexpectedly, found that mitochondrial transcripts are Dicer targets in both species. We demonstrate functional consequences of Dicer binding by perturbation analysis. Taken together,we provide the first genome-wide catalog of direct Dicer targets. Our results suggest widespread function outside of miRNA biogenesis. Regulatory impact of Dicer binding was assessed by knock down experiments in human HEK293 cells. Drosha knockdown and mock transfections were used as controls. In total 3 samples.

2014-11-20 | E-GEOD-55326 | biostudies-arrayexpress

Transcriptome wide identification of Dicer binding in human and C. elegans reveals a variety of substrates (small RNA AGO-IP)

Project description:Dicer is a deeply conserved endoribonuclease with key functions in small RNA biogenesis. Here we employed PAR-CLIP/iPAR-CLIP to identify direct Dicer binding sites in the transcriptomes of human cells and human. We found hundreds of novel miRNAs and non-canonical Dicer substrates with high sensitivity. Small RNA production depended on structure of the binding site and is globally biased towards the 5' arm of hairpins. Unexpectedly, in both species Dicer bound numerous hairpins inside mRNAs without observable small RNA production. Our data revealed ~100 mRNAs of protein coding genes to be targeted in both human and worm. These mRNAs significantly overlapped with the RNAi pathway. We also, unexpectedly, found that mitochondrial transcripts are Dicer targets in both species. We demonstrate functional consequences of Dicer binding by perturbation analysis. Taken together,we provide the first genome-wide catalog of direct Dicer targets. Our results suggest widespread function outside of miRNA biogenesis. Argonaute loaded small RNAs were extracted from FLAG:AGO2 and FLAG:AGO3 expressing HEK293 cells. Small RNA was purified and length selected (see supplementary methods).

2014-11-20 | E-GEOD-55331 | biostudies-arrayexpress

Transcriptome wide identification of Dicer binding in human and C. elegans reveals a variety of substrates (CEL RNA-Seq)

Project description:Dicer is a deeply conserved endoribonuclease with key functions in small RNA biogenesis. Here we employed PAR-CLIP/iPAR-CLIP to identify direct Dicer binding sites in the transcriptomes of human cells and C.elegans. We found hundreds of novel miRNAs and non-canonical Dicer substrates with high sensitivity. Small RNA production depended on structure of the binding site and is globally biased towards the 5' arm of hairpins. Unexpectedly, in both species Dicer bound numerous hairpins inside mRNAs without observable small RNA production. Our data revealed ~100 mRNAs of protein coding genes to be targeted in both human and worm. These mRNAs significantly overlapped with the RNAi pathway. We also, unexpectedly, found that mitochondrial transcripts are Dicer targets in both species. We demonstrate functional consequences of Dicer binding by perturbation analysis. Taken together,we provide the first genome-wide catalog of direct Dicer targets. Our results suggest widespread function outside of miRNA biogenesis. Regulatory impact of Dicer binding was assessed by knock down experiments in human HEK293 cells and C.elegans. Drosha knockdown and mock transfections were used as controls. In total 6 samples (3 human, 3 nematode)

2014-11-20 | E-GEOD-55329 | biostudies-arrayexpress