1/13/2024 0 Comments Iclip germany![]() Target sites of miR-297 and miR-299, where hnRNP L modulates miRNA repression are highlighted by a green arrowhead below. The middle panel (miRNA sites in green) shows conserved mammalian miRNA regulatory target sites for conserved miRNA families in the 3′ UTR regions of Refseq genes, as predicted by ‘TargetScanHuman 5.1’ ( ). ( A) UCSC Genome Browser view of VEGFA exon/intron structure with crosslink-site distribution determined by iCLIP (hnRNP L-IP vs. HnRNP L binds preferentially to miRNA target sites in 3′ UTRs. PCR products derived from all three immunoprecipitations were pooled for sequencing.įigure 5. The three different size fractions resulted from prior size selection of the cDNA, following reverse transcription. PCR products for high-throughput sequencing were visualized by ethidium bromide staining on a 6% polyacrylamide-TBE gel. ( C) Library preparation from iCLIP-processed hnRNP L- (IP lanes 1–3) or control FLAG-immunoprecipitated material (FLAG-IP lanes 4–6). ( B) Western blot analysis of the same membrane as in panel ( A), using anti-hnRNP L and GAPDH antibodies and including 5% input material. Marker positions and the mobilities of hnRNP L-RNA adducts, limit-digest hnRNP L, and of the antibody heavy chain are marked. Boxed regions were cut out and subjected to RNA isolation and library preparation. In each case, different amounts of RNase I were applied (as indicated). As additional control, UV irradiation was left out (lanes 7–8). Autoradiography of iCLIP membrane with bound 32P-labeled RNA–protein complexes, comparing after UV-irradiation (+UV) hnRNP L- (lanes 1–3) with control FLAG immunoprecipitated material (IP, lanes 4–6). ( A) A representative experiment is shown: Briefly, protein–RNA interactions were crosslinked by UV-irradiation of HeLa cells, followed by lysate preparation, limited RNase digestion, immunoprecipitation of hnRNP L-RNA adducts, 3′-RNA-linker addition to the RNA tags, 5′-terminal 32P-labeling, and gel separation of the covalent RNA–protein complexes under denaturing conditions. iCLIP-Seq analysis for transcriptome-wide mapping of hnRNP L RNA-binding sites. Translational regulation by hnRNP L was validated for a subset of predicted target 3'UTRs.ĬLIP hnRNP L microRNA splicing regulation.įigure 1. Finally, regarding 3' UTR binding, hnRNP L binding preferentially overlaps with predicted microRNA target sites, indicating global competition between hnRNP L and microRNA binding. These findings shed light on the longstanding question of differential hnRNP L-mediated splicing regulation. Additionally, position-dependent splicing regulation by hnRNP L was demonstrated: The protein represses splicing when bound to intronic regions upstream of alternative exons, and in contrast, activates splicing when bound to the downstream intron. Genome-wide mapping of hnRNP L binding revealed that the protein preferably binds to introns and 3' UTR. Sequence analysis of the iCLIP crosslink sites showed significant enrichment of C/A motifs, which perfectly agrees with the in vitro binding consensus obtained earlier by a SELEX approach, indicating that in vivo hnRNP L binding targets are mainly determined by the RNA-binding activity of the protein. ![]() To analyze activities of hnRNP L on a genome-wide level, we performed individual-nucleotide resolution crosslinking-immunoprecipitation in combination with deep-sequencing (iCLIP-Seq). Interestingly, hnRNP L can both activate and repress splicing of alternative exons, but the precise mechanism of hnRNP L-mediated splicing regulation remained unclear. We have previously characterized hnRNP L as a global regulator of alternative splicing, binding to CA-repeat, and CA-rich RNA elements. All rights reserved.Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is a multifunctional RNA-binding protein that is involved in many different processes, such as regulation of transcription, translation, and RNA stability. Overall, the methodological advances in iCLIP2 allow efficient library generation and thereby promote the versatile and flexible application of this important technology.ĬLIP High-throughput sequencing Protein-RNA interaction RNA-binding protein UV crosslinking iCLIP.Ĭopyright © 2019 The Authors. ![]() Our advances significantly increase the complexity of the iCLIP2 libraries, resulting in a more comprehensive representation of RBP binding sites. The full procedure can be completed within four days. The new protocol comprises separate adapter ligations, two cDNA amplification steps and bead-based size selection. Here, we present the new iCLIP2 protocol that allows to obtain high-quality iCLIP libraries in a fast and efficient manner. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) is a state-of-the-art technology to map the RNA interaction sites of an RNA-binding protein (RBP) across the transcriptome.
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