The first nt of piRNAs was not considered in the base pairing. (E) Capacity of embryonic piRNAs to target Aub-bound mRNAs with different complementarities within ☖0 nt of crosslink sites. (C and D) Length distribution of TE-derived small RNAs in sense (blue) and antisense (red) orientations. miRNAs were identified by their sequences. Smg Is in Complex with piRNAs, and Embryonic piRNAs Have the Potential to Target Aub-Crosslinked mRNAs with Imperfect Base Pairing (A and B) Length distribution of small RNAs that either coimmunoprecipitated with Smg in 0-to 2-hr embryos (A) or were present in 0-to 2-hr embryos (B). See also Figure S1 and Tables S1-S3 and S4. (F) Fold enrichment of reproduced crosslinks in gene regions, relative to the size of the corresponding regions in the whole genome. Pearson's correlation coefficient (r) is indicated. (E) Plots of the number of uniquely mapped reads per gene in two iCLIP biological replicates. The 1U bias is consistent with a proportion of these reads corresponding to full-length piRNAs. (D) nt distribution in 23-to 29-nt reads mapping to piRNA clusters. (C) Size distribution of reads from iCLIP1 that map to piRNA clusters. Graph of the percentages of crosslinked nt with a given cDNA count that were reproduced in at least two biological replicates. (B) Reproducibility of crosslink positions. The red squares indicate the regions of the membrane cut out for RNA extractions. The asterisks indicate the size of Aub, whose presence was validated by western blot. The gel on the right represents an independent experiment in which embryos expressing GFP-Aub and GFP-Aub AA were used in parallel. No complexes were formed in the absence of UV or when rabbit serum was used for immunoprecipitation. All rights reserved.Īub iCLIP Reveals Direct Interactions of Aub with piRNAs and Cellular mRNAs (A) 32 P-labeled Aub-RNA complexes showing decreasing size with increasing amounts of RNase I (+ to +++). These results suggest general regulation of maternal mRNAs by Aub and piRNAs, which plays a key developmental role in the embryo through decay and localization of mRNAs encoding germ cell determinants.Ĭopyright © 2015 The Authors. Base pairing between piRNAs and mRNAs can induce mRNA cleavage and decay that are essential for embryonic development. iCLIP with an Aub mutant that is unable to bind piRNAs confirms piRNA-dependent binding of Aub to mRNAs. Strikingly, Aub-dependent unstable mRNAs encode germ cell determinants. Gene expression profiling reveals that a proportion of these mRNAs undergo Aub-dependent destabilization during the maternal-to-zygotic transition. Here, we perform iCLIP with the PIWI protein Aubergine (Aub) and identify hundreds of maternal mRNAs interacting with Aub in the early Drosophila embryo. Other functions of piRNAs such as post-transcriptional regulation of mRNAs are now emerging. Other versions: HITS-CLIP, PAR-CLIP, eCLIP, irCLIP.The Piwi-interacting RNA (piRNA) pathway plays an essential role in the repression of transposons in the germline. Deep sequencing of these amplified fragments provides nucleotide resolution of the protein-binding site eCLIP is an improvement over iCLIP that avoids circularizing the cDNA to reduce artifacts. These circularized fragments are linearized and PCR-amplified. Upon RT, cDNA truncates at the binding site and is circularized. The complexes are treated with proteinase K, as the protein crosslinked at the binding site remains undigested. In iCLIP, specific crosslinked RNA-protein complexes are immunoprecipitated. This approach includes additional steps to digest the proteins after crosslinking and to map the crosslink sites with reverse transcriptase. ICLIP maps protein-RNA interactions, in a process similar to HITS-CLIP and PAR-CLIP.
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