Expanding the map of protein-RNA interaction sites via cell fusion followed by PAR-CLIP. Issue 3 (4th March 2018)

• Record Type: Journal Article
• Authors: Hinze, Florian; Drewe-Boss, Philipp; Milek, Miha; Ohler, Uwe; Landthaler, Markus; Gotthardt, Michael
• Is Part Of: RNA biology. Volume 15:Issue 3(2018)
• Page Start: 359
• Page End: 368
• Publication Date: 2018-03-04
• Subjects: Cell fusion -- PAR-CLIP -- RNAseq -- RNA-binding protein -- RNA processing -- transcriptomics -- YBX1; RNA -- Periodicals; Molecular biology -- Periodicals; Molecular biology; RNA; Periodicals; 572.8805
• DOI: 10.1080/15476286.2017.1384120
• Languages: English
• ISSNs: 1547-6286

Abstract

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.