The central rule of life activity is that messenger RNA is generated from DNA transcription of genetic material, and then translated into protein by messenger RNA, so as to complete various physiological functions such as metabolism, growth and development. However, there are still a large number of untranslated RNA in cells (especially in higher biological cells), which is called noncoding RNA. They play an important role in key life processes such as gene expression regulation, and are closely related to cell differentiation, individual development and disease occurrence and development. One of the most important small non coding RNAs is microRNA (miRNA), which has about 21-24 bases and is widely found in higher organisms from nematodes to human beings, and many of the biological functions of miRNA are conserved in the evolutionary process. Since its first discovery in 1993, tens of thousands of miRNAs have been identified, some of which have been used as diagnostic markers and drug development targets for cancer and other diseases. Therefore, the function of miRNA and its own generation and regulation mechanism have always been the focus of biomedical research.
The functional miRNA is produced by a two-step cleavage reaction of a longer transcript (also known as PRI miRNA) containing a neck ring structure. The first step of the cleavage reaction is carried out in the nucleus, which is catalyzed by Drosha / dgcr8 complex. Drosha is a type III RNA cleavage enzyme, the core catalytic component, and dgcr8 is a double stranded RNA binding protein, responsible for the recruitment of PRI miRNA substrate. In the nucleus, about 60-70 bases of precursor miRNA are produced by cutting, and then the precursor miRNA is out of the nucleus. In the cytoplasm, Dicer RNA enzyme completes the second step of cutting. The first step is the cutting reaction in the nucleus, which is particularly important. On the one hand, to remove the lengthy irrelevant sequence, from the PRI miRNA of thousands of base length to produce the precursor miRNA of only 60-70 bases; on the other hand, the 3 'end produced by the cutting is the end of the final mature miRNA, which is very important for the function of miRNA, so the cutting site is required to be very accurate.
Drosha / dgcr8 complex, as the only PRI miRNA cleavage enzyme in the nucleus, was found in 2003-2004. Although a lot of research has been done in the past decade, including the identification of key sequences on PRI miRNA and the analysis of important functional domains of proteins, how to accurately identify PRI miRNA and how to define the cleavage site by Drosha / dgcr8 have not been clear Answer. On March 27, 2020, "structural basis for PRI miRNA" was published online by molecular cell magazine, which was composed by Xu Ruiming group of Institute of Biophysics and Wang Hongwei group of Tsinghua University In this paper, the structure of the complex of Drosha / dgcr8 and PRI miRNA was analyzed by means of single freezing electron microscope, and the molecular mechanism of PRI miRNA processing in the nucleus was revealed. Their results confirmed the decisive role of Drosha in the definition of cleavage sites, and found that the Paz, MB helix and dsRBD domains of Drosha play an important role in pri miRNA recognition and collaborative completion of cleavage site location. Among them, Paz domain has obvious conformational changes before and after RNA binding. It combines with MB helix on both sides of the single and double strand junction of PRI miRNA, forming a unique pattern of key characteristics recognition of PRI miRNA. The binding mode of Paz domain and RNA is totally different from that of other protein Paz domain which only recognizes the 3 'end of RNA. In summary, this study first revealed the molecular mechanism of Drosha specific recognition of PRI miRNA key sequence and structural characteristics, found a novel Paz domain conformation and its new pattern of binding RNA, and clarified the key molecular mechanism that has puzzled the research field for many years.
In addition, this study also proposed that Drosha / dgcr8 complex has different active states. When there is no substrate RNA, the Paz hairpin like double helix of Drosha occupies the cutting active center area, which hinders the binding of Drosha to RNA; while when the PRI miRNA substrate is recognized, the helix changes its conformation, which promotes and stabilizes the binding of substrate. According to the researchers, this is a transition from self inhibition state to activation state, indicating that Drosha has an active self-regulation mechanism, which is beneficial to identify the correct substrate for cutting in vivo environment.
Researcher Suo xuruiming of Chinese Academy of Sciences and Professor Wang Hongwei of Life Science College of Tsinghua University are the co correspondents of the paper. Associate researcher Jin Wenxing of Institute of Biophysics and Doctor Wang Jia of Tsinghua University are the co first authors. The main participants of the work are also associate researcher Liu chaopei of Institute of Biophysics. Zhang Xinzheng, a researcher at the Institute of Biophysics, and Dr. Cao duanfang provided valuable suggestions for the research work. The research was supported by the Institute of Biophysics, the center for biological imaging, Tsinghua University's frozen electron microscopy and high-performance computing platform, and supported by the National Natural Science Foundation, the national key R & D program of the Ministry of science and technology, the Beijing Natural Science Foundation and the youth innovation promotion association of the Chinese Academy of Sciences.
