Journal
RNA BIOLOGY
Volume 15, Issue 3, Pages 359-368Publisher
TAYLOR & FRANCIS INC
DOI: 10.1080/15476286.2017.1384120
Keywords
Cell fusion; PAR-CLIP; RNAseq; RNA-binding protein; RNA processing; transcriptomics; YBX1
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Funding
- Deutsche Forschungsgemeinschaft, Bonn, Germany [Go865/11-1]
- European Research Council [StG282078]
- Bundesministerium fur Bildung und Forschung
- German Center for Cardiovascular Research (DZHK), Berlin
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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 RNAprotein 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.
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