#ICLIP TECHNIQUE CODE#

CLIP-seq.), UV crosslinking immunoprecipitation (CLIP) combined with high-throughput sequencing (HITS), is a powerful technique to identify the RNA binding sites for any RBPs at the transcriptome wide. In addition, they are major components of the subcellular architecture, mediating protein-RNA interactions through translocation from the nucleus to the cytoplasm. RNA-binding proteins (RBPs) play central roles in the regulation of multiple post-transcriptional processes such as alternative splicing, mRNA stability, translation, and mRNA transport. There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials. received funding in the form of salary from Takeda Pharmaceutical Company, Ltd. Yano is a scientific advisor of K Pharma, Inc. is a paid member of the Scientific Advisory Board of San Bio Co., Ltd. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: H.O. The specific roles of these authors are articulated in the ‘author contributions’ section.

JP17H05598 and JP19H03543), the Foundation for Dietary Scientific Research, the Ichiro Kanehara Foundation, the Takeda Science Foundation and the Serika Fund (to M. Yano and H.O.), the MEXT Grant-in-Aid (grant no.

#ICLIP TECHNIQUE CODE#

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: The data underlying the results presented in the study are available, e.g BrdU-CLIP, optimized CLIP and RNA-seq data have been deposited in the Gene Expression Omnibus under the accession code GSE138726.įunding: This work was supported by grants from the SIL Research Fund from Takeda Pharmaceutical Company, Ltd. Received: JanuAccepted: MaPublished: April 17, 2020Ĭopyright: © 2020 Yugami et al. PLoS ONE 15(4):Įditor: Thomas Preiss, John Curtin School of Medical Research, AUSTRALIA This optimized method enables us to accurately identify target genes and provides a snapshot of the protein-RNA interactions of nucleocytoplasmic shuttling RBPs.Ĭitation: Yugami M, Okano H, Nakanishi A, Yano M (2020) Analysis of the nucleocytoplasmic shuttling RNA-binding protein HNRNPU using optimized HITS-CLIP method. Importantly, this interaction was only observed in the cytoplasmic fraction, suggesting a role of cytoplasmic HNRNPU in mRNA stability control. Using this, we validated IL-6 expression by a nuclear RBP, HNRNPU, which directly binds the 3’-UTR of IL-6 mRNA in HeLa cells. Variance of the yields was also reduced, and the experimental period was shortened by 2 days. Our protocol produced a 10-fold greater yield of pre-amplified CLIP library, which resulted in a low duplicate rate of CLIP-tag reads because the number of PCR cycles required for library amplification was reduced. Here we improved a more efficient, rapid, and reproducible CLIP method by optimizing BrdU-CLIP. However, HITS-CLIP protocol is still required for some optimization due to experimental complication, low efficiency and time-consuming, whose library has to be generated from very small amounts of RNAs. Crosslinking immunoprecipitation (CLIP), coupled with high-throughput sequencing (HITS-CLIP), is a powerful technique for investigating the molecular mechanisms underlying disease pathogenesis by comprehensive identification of RBP target sequences at the transcriptome level. RNA-binding proteins (RBPs) control many types of post-transcriptional regulation, including mRNA splicing, mRNA stability, and translational efficiency, by directly binding to their target RNAs and their mutation and dysfunction are often associated with several human neurological diseases and tumorigenesis.

Iclip technique