Ferric, not ferrous, heme activates RNA-binding protein DGCR8 for primary microRNA processing
Ferric, not ferrous, heme activates RNA-binding protein DGCR8 for primary microRNA processing
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DOI：
10.1073/pnas.1114514109
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发表时间：
2012-02-07
影响因子：
11.1
通讯作者：
Guo, Feng
中科院分区：
文献类型：
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作者：
Barr, Ian;Smith, Aaron T.;Guo, Feng
RNA结合蛋白DiGeorge关键区8(Dgcr8)及其伴侣核酸酶DROSHA是动物microRNA(MiRNA)初级转录本(pri-miRNAs)加工所必需的。以前的工作表明，Dgcr8以两个内源性半胱氨酸为轴向配体，与铁[Fe(III)]血红素形成高度稳定和活性的络合物。在这里，我们报告说，在DgCr8中，血红素铁还原到亚铁[Fe(II)]状态，取消了pri-miRNA的加工活性。这种减少导致血红素从Dgcr8解离的速度急剧增加，使络合物变得不稳定。电子吸收、磁圆二色谱和共振拉曼光谱表明，血红素铁的还原伴随着作为轴向配体的半胱氨酸的丢失。ApoDGCR8二聚体是通过还原和去除血红素而产生的，在体外的pri-miRNA加工中显示出低水平的活性。重要的是，亚铁，但不是亚铁，血红素将apoDGCR8的活性恢复到天然铁复合体的水平。本研究证明了Dgcr8与铁血红素的结合特异性，为铁血红素作为miRNA成熟的激活剂提供了直接的生化证据，并表明细胞内环境增加铁血红素的利用率可能会提高pri-miRNA的加工效率。
The RNA-binding protein DiGeorge Critical Region 8 (DGCR8) and its partner nuclease Drosha are essential for processing of microRNA (miRNA) primary transcripts (pri-miRNAs) in animals. Previous work showed that DGCR8 forms a highly stable and active complex with ferric [Fe(III)] heme using two endogenous cysteines as axial ligands. Here we report that reduction of the heme iron to the ferrous [Fe(II)] state in DGCR8 abolishes the pri-miRNA processing activity. The reduction causes a dramatic increase in the rate of heme dissociation from DGCR8, rendering the complex labile. Electronic absorption, magnetic circular dichroism, and resonance Raman spectroscopies indicate that reduction of the heme iron is accompanied by loss of the cysteines as axial ligands. ApoDGCR8 dimers, generated through reduction and removal of the heme, show low levels of activity in pri-miRNA processing in vitro. Importantly, ferric, but not ferrous, heme restores the activity of apoDGCR8 to the level of the native ferric complex. This study demonstrates binding specificity of DGCR8 for ferric heme, provides direct biochemical evidence for ferric heme serving as an activator for miRNA maturation, and suggests that an intracellular environment increasing the availability of ferric heme may enhance the efficiency of pri-miRNA processing.