Rikkyo University

In eukaryotes, small RNAs of about 20-30 bases combine with proteins called argonautes (AGOs) form RNA-induced silencing complexes (RISCs), which bind to complementary RNAs to regulate the expression of target genes. This mechanism is called RNA silencing and plays an important role in various biological processes, such as the development and differentiation of organisms and antiviral responses. Plants encode multiple AGOs, which are divided into three groups based on the homology of their amino acid sequences. The functions of each group’s RISCs have been studied, but which nucleic acid sequences they bind strongly with has not been demonstrated.

In his research, Rikkyo University Associate Professor Hiro-oki Iwakawa revealed that the 3 groups of RISCs found in plants each have different nucleic acid binding properties. He showed that the 2 groups that function in the cytoplasm bind strongly to RNA, while the group that acts in the nucleus binds to DNA with equal or greater strength than it does to RNA. These findings suggest the existence of a previously unknown mechanism—that of plant RISCs directly binding to DNA—and could be of great help in developing new techniques for regulating gene expression.

This research was conducted using a grant from the Japan Science and Technology Agency's FOREST program (Fusion Oriented REsearch for disruptive Science and Technology) "Understanding and Application of Plant RNAi: Toward Flexible Artificial Genome Expression" (Principal Investigator: Hiro-oki Iwakawa).

Argonautes (AGOs) are proteins that are widely found in organisms from prokaryotes to eukaryotes, which have the function of cleaving nucleic acids (a general term for RNA and DNA). It is known that prokaryote AGO forms complexes with small DNA or RNA, then binds to and cleaves the DNA of complementary plasmids^*2 and phages^*3. In eukaryotes such as humans and plants, AGO forms functional complexes called RNA-induced silencing complexes (RISCs) with small RNA, which bind to complementary RNA. RISCs play an important role in various eukaryotic biological processes, such as growth, morphogenesis, and antiviral responses, through sequence-specific RNA cleavage, protein synthesis inhibition, and transcription inhibition.

The model plant Arabidopsis thaliana encodes 10 AGOs, which can be separated into 3 groups based on amino acid sequence homology. RISCs belonging to groups 1 and 2 mainly act in the cytoplasm by cleaving target RNA and inhibiting protein synthesis. AGO1-RISC, which belongs to group 1, plays an important role in plant development, differentiation, and stress responses, while AGO2-RISC, part of group 2, is involved in antiviral responses. Group 3 RISCs are known to promote methylation of nearby DNA by binding to RNA being synthesized in the nucleus. They play a role in silencing non-self genes and transposons, which are forms of DNA that possess transfer abilities (Figure 1). Although our understanding of how plant RISCs function has progressed, much remains unknown about the nucleic acid sequences that each RISC binds to strongly.

In his research, Rikkyo University Associate Professor Hiro-oki Iwakawa described the nucleic acid binding properties of 3 groups of RISCs in Arabidopsis thaliana. He started by synthesizing AGO proteins using a plant cell-free translation system^*4, to which small RNA was added to form AGO1-RISC from group 1, AGO2-RISC from group 2, and AGO4-RISC, AGO6-RISC, and AGO9-RISC from group 3. Purified RISC was mixed with single-stranded RNA or DNA that is either completely complementary (base-paired) or partially mismatched (not base-paired) with the small RNA, and their binding affinity was quantified using a biochemical technique called filter binding assay^*5 (Figure 2). This showed that compared to groups 1 and 2, group 3 RISC was capable of binding (tolerating mismatches) even in the presence of low complementarity of the 3' supplementary region^*6 (Figure 3). Of even more interest, he found that group 1 and 2 RISCs, which function in the cytoplasm, bind strongly to RNA, while group 3 RISCs, which act in the nucleus, binds more strongly to DNA than to RNA (Figure 3). These findings suggest that the RISCs in each group have developed different target binding properties for executing their unique functions.

This research not only deepens our understanding of how RNA silencing works in plants, it also suggests the existence of a previously unknown mechanism, in which eukaryotic RISCs act by directly binding to DNA. These findings could provide a gateway for figuring out how to use plant RISC to regulate gene expression.

Journal name: Nucleic Acids Research (online version)
Article title: The clade-specific target recognition mechanisms of plant RISCs
Author: Hiro-oki Iwakawa
DOI number: 10.1093/nar/gkae257

This study was supported by the Japan Science and Technology Agency's FOREST program (Principal Investigator: Hiro-oki Iwakawa, Project Number: JPMJFR204O) and Strategic Basic Research Program PRESTO (Principal Investigator: Hiro-oki Iwakawa, Project Number: JPMJPR18K2), and Ministry of Education, Culture, Sports, Science and Technology Grants-in-Aid for Young Scientists A (Principal Investigator: Hiro-oki Iwakawa, Project Number: 16H06159) and for Basic Research B (Principal Investigator: Hiro-oki Iwakawa, Project Number: 23H02412).