Release date: 2018-05-03
On April 26th, Professor Wang Hongwei, research group of Tsinghua University School of Life, Tsinghua-Peking University Life Science Joint Center and Beijing Structural Biology Innovation Center, published a book titled Cryo-EM structure of human in Cell. The research paper on Dicer and its complexes with a pre-miRNA substrate first reported the full-length high-resolution structure of the human endonuclease Dicer protein. It also reported that the human endonuclease Dicer protein binds to a small RNA. Two different structural states of the precursor pre-let-7 substrate.
This article was reprinted from the "College of Life Sciences at Tsinghua University". The original title: Wang Hongwei's research group published a paper in "Cell" magazine to report the cryo-electron structure of human Dicer and Dicer-pre-miRNA complexes.
RNA interference (RNAi) is the most commonly used means of knocking down a gene expression. The small RNA that causes endogenous RNA interference is mainly microRNA (miRNA). So far, as many as 1,800 kinds of microRNAs have been found in human bodies. More and more reports in the literature suggest that the occurrence, development and metastasis of many tumors are closely related to the abnormal expression of microRNAs.
The vast majority of microRNAs in the human body mature and form an endonuclease, which we call Dicer protein. Interestingly, there is only one copy of the endonuclease Dicer gene in the human body, which expresses the only human endonuclease Dicer protein, but is responsible for the formation of most microRNAs in the human body. Therefore, the importance of the endonuclease Dicer protein in human cells is self-evident. Endonuclease Dicer is an endonuclease that cleaves only double-stranded RNA substrates. It has a molecular weight of approximately 220 kDa and is composed of multiple domains. Some of these domains are responsible for binding RNA substrates, some are responsible for cleavage of RNA substrates, and some domains are like a ruler to accurately measure where RNA needs to be cleaved. The human endonuclease Dicer protein recognizes a number of different microRNA precursor substrates in the cell and then processes to generate mature small RNAs of approximately 22 bases in length and two free bases at the 3' end.
Unfortunately, the overall three-dimensional structure of the human endonuclease Dicer protein has not been resolved, and the mechanism by which the human endonuclease Dicer protein accurately processes these microRNA precursors remains unclear. There are several reasons why the human endonuclease Dicer protein has not obtained high-resolution three-dimensional structure: 1. The human endonuclease Dicer protein is about 220 kDa, and the molecular weight is obtained by using crystallography to obtain three-dimensional structure. Larger, difficult to crystallize; 2, the use of single particle reconstruction method to obtain high-resolution three-dimensional structure, its molecular weight is relatively small; 3, endonuclease Dicer protein three-dimensional structure is L-shaped, no symmetry Sexuality, the distribution in the ice is mostly strip-shaped, the contrast is low, the distribution is uneven, and the dominant orientation has formed a great technical obstacle to the three-dimensional reconstruction of single particles.
Over the past decade, Wang Hongwei and other research groups have attempted to use the single-particle electron microscopy method to resolve the three-dimensional structure of the human endonuclease Dicer protein. After continuous exploration, the research team solved the technical problems of protein sample preparation, frozen sample preparation, data collection and processing. Finally, the protein complex was co-expressed from mammalian 293F cell line, and the protein was separated and purified by affinity chromatography. The frozen electron microscopy samples with relatively uniform distribution and relatively few dominant orientations were prepared by pure gold or gold-plated grids, and the high-resolution three-dimensional structure (4.4 angstroms) of human Dicer and its cofactor protein TRBP complex was obtained. Figure 1), the first time the high-resolution overall structure of the human endonuclease Dicer protein was analyzed, and the precise three-dimensional distribution of the domains in the endonuclease Dicer protein and the spatial relationship between the domains were observed.
Figure 1. High-resolution structure and domain distribution of the human Dicer-TRBP complex (TRBP is an RNA-binding protein) obtained by cryo-electron microscopy analysis
In order to further understand how the human endonuclease Dicer protein processes microRNA precursors, Wang Hongwei obtained a human precursor Dicer-TRBP complex and a microRNA precursor pre-let by in vitro recombination. The ternary complex formed by -7, and the two three-dimensional structural states of the composite are analyzed. One is that the stem of pre-let-7 is completely complementary (hDicer-TRBP-pre-let-7 complex class I), and the other is that the stem of pre-let-7 is partially dissociated (hDicer -TRBP-pre-let-7 complex class II) (Figure 2). Wang Hongwei's research team collaborated with Zhang Qianfeng's research group of Tsinghua University. Through chemical modification sequencing (icSHAPE), ribonuclease digestion, sequencing gel electrophoresis and other techniques, it was found that pre-let-7 has a dynamic conformational equilibrium in solution, and part of pre- The stem of let-7 is a perfectly paired double-stranded helix, and some stems of pre-let-7 are partially dissociated. In-depth studies have shown that the combination of human Dicer protein and TRBP complex with pre-let-7 can promote the double-stranded helical structure conversion of the RNA to the stem, ensuring the conformation of the stem before being digested by Dicer protein. Sex, thus ensuring the exact length of the microRNA product. It is this mechanism that makes it possible to ensure that human Dicer proteins accurately cleave numerous microRNA precursors with different structural features into products with a common 22 nt length. This work laid the foundation for further analysis of the maturation mechanism of microRNAs.
Figure 2. Refrigerated electron microscopy structure of two hDicer-TRBP-pre-let-7 complexes
In this work, Wang Hongwei's research team members, Tsinghua University Life College postdoctoral Liu Zhongmin, Wang Jia, 2015 PhD student Cheng Hang, 2015 PhD student Ke Xin as the co-first author, Professor Wang Hongwei is the author of this article. In addition, Professor Zhang Qiangfeng from the Life College of Tsinghua University and Dr. Sun Lei from 2013 conducted the experiment of icSHAPE, which provided important experimental evidence for this work. The research was supported by Tsinghua University's cryo-electron microscope platform, high-performance computing platform and protein separation and purification and identification platform. The data processing was carried out on the National Protein Science (Beijing) facility Tsinghua University high-performance computing platform.
This work has won strong support from the National Natural Science Foundation of China, the Ministry of Science and Technology, the Beijing Municipal Science and Technology Commission, the Tsinghua-Peking University Life Science Joint Center and the Beijing Structural Biology Innovation Center.
Paper link:
Https://doi.org/10.1016/j.cell.2018.03.080
Source: School of Life Sciences, Tsinghua University
Mackerel
Mackerel,King Mackerel,Jack Mackerel,Saba Mackerel
ZHEJIANG EVERNEW SEAFOOD CO.,LTD , https://www.evernewseafood.com