裂殖酵母RNA选择性消除机器的结构生物学研究

During vegetative growth, the transcription of S. pombe meiotic genes is not completely repressed. In mitotic cells, to avoid impairments caused by the presence of unnecessary meiotic gene transcripts, S. pombe utilizes elimination machinery to remove these mRNA. Mmi1, a YTH-family RNA-binding protein, plays an indispensable role in this process , together with nuclear poly(A)-binding protein Iss10, Red1 and Red5. In the RNA elimination process, Mmi1 binds the DSR motif (UNAAAC) specific for meiotic transcripts and directs them to the exosome for degradation. However, the mechanisms of specific targeting of DSR by Mmi1 and the assembly of the RNA elimination machinery remain unknown. To understand the molecular mechanism of this process, knowing the structure of the Mmi1 YTH domain in complex with a DSR motif at atomic resolution is essential. Here, we present the crystal structure of the Mmi1 YTH domain in the apo state and in complex with a DSR motif-containing RNA. The structure reveal how Mmi1 YTH domain specifically readout the UUAAAC motif. Furthermore, this complex structure reveals a unique RNA-binding mode distinct from the m6A RNA-binding mode utilized by other YTH domains, in which the RNA is bound in a long groove opposite the putative m6A-binding pocket of Mmi1. Subsequent experiments are ongoing.

在裂殖酵母的营养生长期，减数分裂基因的转录并没有被完全抑制。为了避免这些转录产物对细胞带来的损害，裂殖酵母使用RNA选择性消除器来降解这些转录产物，其分子机理是：减数分裂基因mRNA的富含UNAAAC序列（N是可变的碱基）被蛋白Mmi1识别，然后Mmi1伙同Red1, Red5 和Iss10把减数分裂基因mRNA招募到外切体进行降解。Mmi1特异识别UNAAAC序列的结构机理以及RNA消除器组装的结构机理还是未知的，我们对这两个问题开展结构研究。我们证明了Mmi1的YTH 结构域负责结合UNAAAC序列并解析了Mmi1-YTH结构域与RNA的复合物结构，发现Mmi1-YTH结构域不结合UUAAA序列+2位点而结合其他位点，从结构上解释Mmi1识别UNAAAC序列的机制。Mmi1结合RNA的模式与之YTH结构域识别RNA的模式不同，是全新的RNA识别模式。后续的相关实验正在进行。

本研究通过生化和结构生物学手段研究裂殖酵母RNA消除器的组装和工作机理，首次发现：（1）通过解析Mmi1-YTH结构域与DSR-RNA复合物结构，发现Mmi1包含一个全新的RNA结合模式，结合界面与之前报道的其它YTH结构域完全不同；（2）与之前报的YTH结构域不同，Mmi1-YTH结构域并不能结合m6A修饰的RNA，是因为Mmi1-YTH结构域并不存在结合m6A-RNA的疏水口袋和正电表面；（3）首次鉴定出Mmi1-ERH1复合物最短结合区域；（4）位于Mmi1 N-端一段小肽结合在ERH1二聚界面，并且以2：2的模式结合。