Dataset Information

Low-Dose Ionizing Radiation-Crosslinking Immunoprecipitation (LDIR-CLIP) Identified Irradiation-Sensitive RNAs for RNA-Binding Protein HuR-Mediated Decay.

ABSTRACT: Although ionizing radiation (IR) is widely used for therapeutic and research purposes, studies on low-dose ionizing radiation (LDIR) are limited compared with those on other IR approaches, such as high-dose gamma irradiation and ultraviolet irradiation. High-dose IR affects DNA damage response and nucleotide-protein crosslinking, among other processes; however, the molecular consequences of LDIR have been poorly investigated. Here, we developed a method to profile RNA species crosslinked to an RNA-binding protein, namely, human antigen R (HuR), using LDIR and high-throughput RNA sequencing. The RNA fragments isolated via LDIR-crosslinking and immunoprecipitation sequencing were crosslinked to HuR and protected from RNase-mediated digestion. Upon crosslinking HuR to target mRNAs such as PAX6, ZFP91, NR2F6, and CAND2, the transcripts degraded rapidly in human cell lines. Additionally, PAX6 and NR2F6 downregulation mediated the beneficial effects of LDIR on cell viability. Thus, our approach provides a method for investigating post-transcriptional gene regulation using LDIR.

SUBMITTER: Lee JW

PROVIDER: S-EPMC10740889 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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Low-Dose Ionizing Radiation-Crosslinking Immunoprecipitation (LDIR-CLIP) Identified Irradiation-Sensitive RNAs for RNA-Binding Protein HuR-Mediated Decay.

Lee Ji Won JW Mun Hyejin H Kim Jeong-Hyun JH Ko Seungbeom S Kim Young-Kook YK Shim Min Ji MJ Kim Kyungmin K Ho Chul Woong CW Park Hyun Bong HB Kim Meesun M Lee Chaeyoung C Choi Si Ho SH Kim Jung-Woong JW Jeong Ji-Hoon JH Yoon Je-Hyun JH Min Kyung-Won KW Son Tae Gen TG

Biology 20231215 12

Although ionizing radiation (IR) is widely used for therapeutic and research purposes, studies on low-dose ionizing radiation (LDIR) are limited compared with those on other IR approaches, such as high-dose gamma irradiation and ultraviolet irradiation. High-dose IR affects DNA damage response and nucleotide-protein crosslinking, among other processes; however, the molecular consequences of LDIR have been poorly investigated. Here, we developed a method to profile RNA species crosslinked to an R ...[more]

PMID: 38132359

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Project description:BackgroundSchistosomiasis, caused by schistosomes, is one of the most prevalent and serious parasitic diseases in tropical and subtropical countries. This pathogen has a complex life cycle and harbors a unique repertoire of genes expressed at different life-stages. Understanding the gene regulation of schistosomes will contribute to identification of novel drug targets and vaccine candidates. Some conserved and novel microRNAs (miRNAs) have been identified in schistosomes as key transcriptional and post-transcriptional regulators in the past few years; however, little is known about their specific targets.MethodsHigh-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation (HITS-CLIP) was used to covalently crosslink native Argonaute protein-RNA complexes in Schistosoma japonicum. An antibody against S.japonicum Argonaute proteins, was generated and used for immunoprecipitation of the crosslinked SjAgo-RNA complex from soluble adult worm extract. Small RNAs, including miRNAs and their target mRNAs associated with the native SjAgo in adult parasites, were enriched and extracted for library construction.ResultsHigh-throughput sequencing produced a total of ~7.4 million high-quality reads, of which approximately 45.07 % were composed of 769 miRNAs and 35.54 % were composed of 11,854 mRNAs target sites. Further bioinformatics analysis identified 43 conserved known miRNAs and 256 novel miRNAs in the SjAgo-associated small RNA population. An average of approximately 15 target sites were predicted for each miRNA. Moreover, a positive rate of 50 % has been achieved in a small-scale verification test of the putative target sites of miRNA1.ConclusionIn this study, we isolated and identified small RNAs including miRNAs and their targets associated with the S. japonicum Argonaute proteins, by the HITS-CLIP method combined with bioinformatics and biologic experimental analysis. These data reveal a genome-wide miRNA-mRNA interaction map in S. japonicum in vivo, which will help us understand the complex gene regulatory network in this pathogen and thereby facilitate the development of novel drug approaches against schistosomiasis.

Dataset Information

Low-Dose Ionizing Radiation-Crosslinking Immunoprecipitation (LDIR-CLIP) Identified Irradiation-Sensitive RNAs for RNA-Binding Protein HuR-Mediated Decay.

Publications

Low-Dose Ionizing Radiation-Crosslinking Immunoprecipitation (LDIR-CLIP) Identified Irradiation-Sensitive RNAs for RNA-Binding Protein HuR-Mediated Decay.

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