The transposon was first discovered in the maize genome by American geneticist Barbara McClintock, and it is widely present in the genomes of higher organisms.
The images in this article are provided by the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences
Transposons can "copy and paste" their own DNA in the host genome for the purpose of self-"reproduction", which poses a serious threat to the stability of the genome, and higher organisms maintain the stability of the genome by branding the transposon DNA with a methyl chemical brand to inhibit the activity of the transposon.
In response, plants evolved an RNA-guided DNA methylation pathway (plant RdDM pathway) to complete dna methylation. In this pathway, two protein machines, Pol IV and RDR2, perform different functions, collaborate to produce a double-stranded guide RNA molecule that then helps plant cells precisely methylate transposon DNA. Although Pol IV and RDR2 have been discovered for years, the internal makeup of these two transcription machines and how they work together to synthesize double-stranded RNA has not been elucidated.
3D structure GIF of the complex of Pol IV-RDR2
Pol IV is the fourth RNA polymerase encoded by plant nuclei, and compared with eukaryotes Pol I, Pol II, and Pol III, its genomic transcriptional regions, interacting transcription factors, working patterns, and physiological functions are significantly different. In the past 30 years, the three-dimensional structure of Pol I, Pol II and Pol III has been analyzed, but the three-dimensional structure and working mode of Pol IV have still not been answered.
Researchers are working
On December 24, 2021, Zhang Yu's research team and Wang Jiawei's research team from the Center for Excellence in Molecular Plant Sciences of the Chinese Academy of Sciences and Feng Yu's team from Zhejiang University collaborated to publish a research paper entitled "Pol IV and RDR2: A two-RNA-polymerase machine that produces double-stranded RNA" in the form of Research Article in Science. The study resolved the three-dimensional structure of the Pol IV-RDR2 protein complex and proposed a unique molecular mechanism for the synthesis of double-stranded RNA by Pol IV-RDR2 using double-stranded DNA as a template.
What is the difficulty of cracking the working mechanism of RNA polymerase? Zhang Yu revealed that on the one hand, it is difficult to obtain high-quality protein samples, because there are 20,000 proteins in the plant, and the abundance of Pol IV is extremely low, on the other hand, it is necessary to resolve its three-dimensional structure.
Zhang Yu was troubled by this, and a discussion with researcher Wang Jiawei in the canteen brought a turning point to solve this problem. Through interdisciplinary collaboration, they achieved a methodological innovation, namely "constructing a plant suspension cell line to purify proteins", which stably integrates one of the subunits of the Pol IV complex in the plant cell genome, which has a protein purification tag that can be assembled into a complex with other subunits, and then purified to obtain a protein sample of Pol IV by large-scale culture of suspended cells. "And the plant culture solution is quite cheap, and it is also feasible in terms of research cost input." Wang Jiawei revealed.
Through this method innovation, the research team obtained a high-quality protein sample of the Pol IV-RDR2 complex and resolved the three-dimensional structure of the complex, which showed that Pol IV and RDR2 resembled two separate workshops for the synthesis of different RNAs, which cleverly merged together and built an internal channel between the two otherwise separate workshops.
The work of the Pol IV synthesis workshop is to synthesize single-stranded RNA with double-stranded DNA as a template, and the synthesized single-stranded RNA products can be directly transmitted to the synthesis workshop of RDR2 through internal channels, so that RDR2 can directly synthesize double-stranded RNA with single-stranded RNA as a template.
Based on this, the researchers proposed that the protein machine Pol IV-RDR2 complex is like a factory that efficiently synthesizes double-stranded RNA, using double-stranded DNA as a template to deliver single-stranded RNA intermediates inside the protein machine, continuously and efficiently synthesizing double-stranded RNA.
The study successfully resolved the structure of the fourth eukaryote RNA polymerase, reported the unique structure and synergistic mechanism of the dual RNA polymerase complex, and proposed a novel working mode of the transcriptional protein machine. The research results are an important breakthrough in the frontier field of molecular biology and plant science.