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Expanding the map of protein-RNA interaction sites via cell fusion followed by PAR-CLIP

Item Type:Article
Title:Expanding the map of protein-RNA interaction sites via cell fusion followed by PAR-CLIP
Creators Name:Hinze, F. and Drewe-Boss, P. and Milek, M. and Ohler, U. and Landthaler, M. and Gotthardt, M.
Abstract:PAR-CLIP (photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation) facilitates the identification and mapping of protein/RNA interactions. So far, it has been limited to select cell-lines as it requires efficient 4SU uptake. To increase transcriptome complexity and thus identify additional RNA-protein interaction sites we fused HEK 293 T-Rex cells (HEK293-Y) that express the RNA binding protein YBX1 with PC12 cells expressing eGFP (PC12-eGFP). The resulting hybrids enable PAR-CLIP on a neuronally expanded transcriptome (Fusion-CLIP) and serve as a proof of principle. The fusion cells express both parental marker genes YBX1 and eGFP and the expanded transcriptome contains human and rat transcripts. PAR-CLIP of fused cells versus the parental HEK293-Y identified 768 novel RNA targets of YBX1. We were able to trace the origin of the majority of the short PAR-CLIP reads as they differentially mapped to the human and rat genome. Furthermore, Fusion-CLIP expanded the CAUC RNA binding motif of YBX1 to UCUUUNNCAUC. The fusion of HEK293-Y and PC12-eGFP cells resulted in cells with a diverse genome expressing human and rat transcripts that enabled the identification of novel YBX1 substrates. The technique allows the expansion of the HEK 293 transcriptome and makes PAR-CLIP available to fusion cells of diverse origin.
Keywords:PAR-CLIP, Cell Fusion, YBX1, RNAseq, Transcriptomics, RNA Processing, RNA-Binding Protein, Animals, Rats
Source:RNA Biology
Publisher:Taylor & Francis
Page Range:359-368
Date:4 March 2018
Official Publication:https://doi.org/10.1080/15476286.2017.1384120
External Fulltext:View full text on PubMed Central
PubMed:View item in PubMed

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