亲和沉降及质谱法分离和鉴定与EGFR 3'UTR结合的蛋白质

Isolation and Identification of Proteins Bonded to EGFR 3'UTR by Affinity Pull-down and Mass Spectrometry

随着后基因组学研究的逐步深入,非编码RNA成为生物学研究的热点之一,其中对mRNA非翻译区的功能研究也逐渐广泛。mRNA非翻译区(UTR)的调控序列与一些蛋白质特异结合,对其自身mRNA命运进行调控。表皮生长因子受体(EGFR)是细胞信号转导的关键因子,其特异酪氨酸残基发生磷酸化,可招募诸如Shc、Grb2、磷脂酶Cγ和Src等信号蛋白,进而激活下游信号通路,研究证实EGFR与一些癌症的发生相关,因此探讨EGFR非翻译区对其mRNA的调控机制就显得尤为重要。运用亲和沉降及质谱,从HeLa细胞中分离到3种蛋白质与EGFR 3'UTR相结合:Ilf3、hnRNP A1和hnRNP A2/B1。这些蛋白质均为RNA结合蛋白并参与mRNA代谢。这为研究EGFR 3'UTR的调控机制提供了依据。

With the development of post-genomics, non-coding RNA has become one of the biological research hotspots. Researches about the function of mRNA untranslated region （UTR） are increasingly widespread. The regulatory sequence of mRNA UTR is bonded to some proteins to regulate mRNA fate. Epidermal growth factor receptor （EGFR） is the key factor in cell signal transduction. Phosphorylation of EGFR specific tyrosine residues can recruit specific signal proteins, such as She, Grb2,Phospholipase C gamma and Src,and then activate the downstream signal channel. The previous research confirmed that EGFR is associated with the occurrence of some cancers,so it is very important to investigate the regulatory mechanism of EGFR UTR on its mRNA regulation. Affinity pull-down and mass spectrometry technology were used to isolate and identify three kinds of proteins from HeLa cell lysate to interact with EGFR 3’UTR： Ilf3,hnRNP Al and hnRNP A2/B1. These are all RNA binding proteins and participate in the metabolism of mRNA. The findings in our study may provide references to further investigate the regulatory mechanism of EGFR 3’UTR.