Project description:RNA structure is vital for its function. Current transcriptome-wide RNA structure probing methods only capture partial structure information. Measuring RNA structure in full length is critical to the function and regulation study of small RNAs and short fragments of functional sites. Here, we present icSHAPE-MaP, an approach combining in vivo click selective 2′-hydroxyl acylation and mutational profiling to probe intact RNA structures. We further showcase the RNA structural landscape of substrates bound by human Dicer, by combining RNA immunoprecipitation pull-down and small RNA structure profiling through icSHAPE-MaP. Structural categories of Dicer substrates were unveiled with distinct patterns in correlation to their binding affinity and cleavage efficiency. And by tertiary structural modeling for pre-miRNAs, one of the major binding and cleavage substrates for Dicer, we find the spatial distance measuring as an important parameter for Dicer cleavage-site selection.
Project description:The endoribonuclease Dicer is known for its central role in the biogenesis of eukaryotic small RNAs/microRNAs. Despite its importance, Dicer target transcripts have not been directly mapped. Here, we apply biochemical methods to human cells and C. elegans and identify thousands of Dicer binding sites. We find known and hundreds of novel miRNAs with high sensitivity and specificity. We also report structural RNAs, promoter RNAs, and mitochondrial transcripts as Dicer targets. Interestingly, most Dicer binding sites reside on mRNAs/lncRNAs and are not significantly processed into small RNAs. These passive sites typically harbor small, Dicer-bound hairpins within intact transcripts and generally stabilize target expression. We show that passive sites can sequester Dicer and reduce microRNA expression. mRNAs with passive sites were in human and worm significantly associated with processing-body/granule function. Together, we provide the first transcriptome-wide map of Dicer targets and suggest conserved binding modes and functions outside the miRNA pathway. Regulatory impact of Dicer binding was assessed by knock down experiments in human HEK293 cells and Caenorhabditis elegans. Drosha knockdown and mock transfections were used as controls. In total 6 samples (3 human, 3 nematode)
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).