Expanding the map of protein-RNA interaction sites via cell fusion followed by PAR-CLIP
| Autor: | Philipp Drewe-Boss, Uwe Ohler, Florian Hinze, Markus Landthaler, Miha Milek, Michael Gotthardt |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Immunoprecipitation Amino Acid Motifs Green Fluorescent Proteins RNA-binding protein PAR-CLIP Biology PC12 Cells Transcriptome Cell Fusion 03 medical and health sciences 0302 clinical medicine Animals Humans RNA Messenger Molecular Biology Gene Cell fusion Binding Sites Technical Paper Gene Expression Profiling HEK 293 cells RNA Cell Biology Molecular biology Cell biology Rats 030104 developmental biology Cross-Linking Reagents HEK293 Cells 030220 oncology & carcinogenesis Y-Box-Binding Protein 1 Protein Binding |
| Zdroj: | RNA biology. 15(3) |
| ISSN: | 1555-8584 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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