Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA.
Protein Sci. 2012 Jun;21(6):797-808. doi: 10.1002/pro.2062. Epub 2012 Apr 23.
DGCR8 (DiGeorge Critical Region 8) is an essential microRNA (miRNA) processing protein that recognizes primary transcripts of miRNAs (pri-miRNAs) and triggers their cleavage by the Drosha nuclease. We previously found that Fe(III) heme binds and activates DGCR8. Here we report that in HeLa cells, DGCR8 undergoes two proteolytic events that produce two C-terminal fragments called DGCR8(C1) and DGCR8(C2) , respectively. DGCR8(C2) accumulates during apoptosis and is generated through cleavage by a caspase. The caspase cleavage site is located in the central loop of the heme-binding domain. Cleavage of DGCR8 by caspase-3 in vitro results in loss of the otherwise tightly bound Fe(III) heme cofactor, dissociation of the N- and C-terminal proteolytic fragments, and inhibition of the pri-miRNA processing activity. These results reveal an intrinsic mechanism in the DGCR8 protein that seems to have evolved for regulating miRNA processing via association with Fe(III) heme and proteolytic cleavage by caspases. Decreased expression of miRNAs has been observed in apoptotic cells, and this change was attributed to caspase-mediated cleavage of a down-stream miRNA processing nuclease Dicer. We suggest that both the Drosha and Dicer cleavage steps of the miRNA maturation pathway may be inhibited in apoptosis and other biological processes where caspases are activated.
DGCR8（DiGeorge 关键区 8）是一种重要的 microRNA（miRNA）加工蛋白，它能识别 miRNA 的初级转录本（pri-miRNAs），并触发 Drosha 核酸酶对其进行切割。我们之前发现，Fe（III）血红素结合并激活 DGCR8。在这里，我们报告说在 HeLa 细胞中，DGCR8 经历了两个蛋白水解事件，分别产生两个 C 末端片段，称为 DGCR8（C1）和 DGCR8（C2）。DGCR8（C2）在细胞凋亡过程中积累，并且通过半胱天冬酶的切割产生。半胱天冬酶的切割位点位于血红素结合域的中心环中。体外用 caspase-3 切割 DGCR8 会导致原本紧密结合的 Fe（III）血红素辅因子丢失、N 端和 C 端蛋白水解片段的解离，以及 pri-miRNA 加工活性的抑制。这些结果揭示了 DGCR8 蛋白中的一种内在机制，该机制似乎通过与 Fe（III）血红素结合和半胱天冬酶的蛋白水解切割来调节 miRNA 加工而进化而来。在凋亡细胞中观察到 miRNA 的表达减少，并且这种变化归因于 caspase 介导的下游 miRNA 加工核酸酶 Dicer 的切割。我们认为，miRNA 成熟途径的 Drosha 和 Dicer 切割步骤都可能在细胞凋亡和其他激活半胱天冬酶的生物学过程中受到抑制。
