水稻RNA结合蛋白C3H12与RNA结合位点的鉴定

Identification of Binding Center Site of RNA-binding Protein C3H12 in Rice

CCCH型锌指蛋白质C3H12是进化上保守的RNA结合蛋白质,它含有5个串联的CCCH锌指结构域ZnF1-5,形成2个紧密的锌指簇ZnF1-3和ZnF4-5。早期的研究发现,C3H12可能通过与mRNA结合的方式在转录后水平调控基因的表达。然而,与C3H12结合的mRNA类型和他们的结合模式,并未通过实验得到证明。本文表达纯化了一系列C3H12截短及全长蛋白质,并合成了一些潜在RNA底物ARE9、ARE19及对照Random21。通过等温滴定量热法(isothermal titration calorimetry,ITC)确定了C3H12与富含腺嘌呤尿嘧啶单元(AU-rich element,ARE)mRNA底物的结合,并揭示了互作核心区域和热力学性质。通过荧光光谱分析和微型热泳动(microscale thermophoresis,MST)技术对ITC的结果进一步佐证。结果表明:(1)C3H12与ARE底物的相互作用是焓驱动的能量有利的(ΔG<0)特异性结合,结合比为1:1。(2)C3H12与ARE19的亲和力较ARE9更高(约2倍)。(3)C3H12中ZnF1-3在与ARE类底物的结合活性中发挥主导作用。(4)C3H12结构中的141个氨基酸残基的接头不直接参与和ARE底物的相互作用。本研究揭示的CCCH型锌指蛋白质C3H12与ARE底物结合模式,将为进一步在分子结构水平阐明C3H12与ARE底物结合的机制奠定基础。

The CCCH zinc finger protein C3H12 is an evolutionary conserved RNA-binding protein that contains five tandem CCCH zinc finger motifs(ZnF1-5) forming two compact clusters, ZnF1-3 and ZnF4-5. A tentative inference on previous study is that C3H12 may regulate the target gene expression at post-transcriptional level by binding its mRNA. However, the substrate mRNA type and the binding pattern are not well defined. We produced a series of truncated C3H12 as well as the full-length protein, and synthesized potential mRNA substrates ARE9, ARE19 and Random 21 as a control. The interaction and thermodynamic properties between C3H12 and AU-rich element(ARE) mRNA substrates were characterized for the first time by isothermal titration calorimetry(ITC). The ITC results were further verified by spectrofluorimetry and microscale thermophoresis(MST). All results showed that(1) C3H12 specially binds to ARE substrates in a ratio of 1∶1, and the binding is enthalpy-driven and energy-favorable(ΔG<0).(2) The binding affinity between C3H12 and ARE19 is as twice higher as that between C3H12 and ARE9.(3) ZnF1-3 domain in C3H12 plays a dominant role in the binding activity between C3H12 and ARE substrates.(4) The linker with 141 amino acid residues in C3H12 is not directly involved in the interaction with ARE substrates. The binding pattern between C3H12 and ARE substrate revealed in our research can provide useful information for further illuminating the structural mechanism of C3H12-RNA interaction.