Isolation and identification of Oct4 RNA-binding proteins / 上海交通大学学报（医学版）

ABSTRACT

Objective: To identify the RNA binding proteins of Oct4, a key factor of embryonic stem cells. Methods: Total RNA was extracted from mouse embryonic stem cells (R1), which was reverse-transcribed into cDNA. Then PCR product of Oct4 mRNA were transcribed into Oct4 mRNA. Finally the candidate RNA-binding proteins were eluted applying the streptomycin affinity chromatography, and submitted for high-performance liquid chromatography combined with the mass spectrometry. The identified RNA binding proteins were further confirmed by RNA immunoprecipitation (RIP). Results: 121 RNA binding proteins of Oct4 3' untranslated region (UTR) and Oct4 5' UTR were identified with liquid chromatography / mass spectrometry. There were 11 proteins with the number of peptide spectrum matches more or equal to 2, and 7 of them were selected for additional confirmation using RIP method. RIP results showed that Oct4 interacted with DSP, SOD1, LMNA, NPM1, PSIP1, NCL and HDGF. Among them, HDGF had the strongest binding ability to Oct4 mRNA. Conclusion: Identification of Oct4 RNA binding proteins will provide a theoretical basis for the regulation mechanism of Oct4, and will be a basis for the further study of its function.