Research
About half of the genome—the blueprint of life—is made up of transposons, sequences that copy themselves much like viruses. When transposons move around the genome they can break genes and prevent correct genetic information from being passed on. To keep them in check, animals evolved a system of small RNAs called PIWI-interacting RNAs (piRNAs). piRNAs carry sequences that match transposons and, together with PIWI proteins, recognize and silence “non-self” in the genome. They are especially important in germ cells, which pass genetic information to the next generation, and in many animals the loss of piRNAs causes infertility.
We see piRNAs not merely as defensive factors but as a molecular memory that is rewritten as non-self keeps changing. Transposons invade anew and alter their sequences to escape silencing, so the defense cannot stay fixed either. How do piRNAs tell non-self apart, and how do they keep and update this memory? We approach these questions through very different kinds of non-self—transposons, symbionts, and viruses—and aim to turn the principles we find into new technologies for controlling RNA.
Responding to changing transposons
Self-organization and “fluctuation” of piRNAs
Transposons mutate to escape silencing. piRNA sequence patterns are not fixed but fluctuate, and we think this lets piRNAs re-target transposons from new positions—a fast response to that change.
piRNA sequence patterns are not produced in a fixed way from fixed positions. We have found that they show “fluctuation,” shifting little by little over time and across conditions. piRNAs are flexibly chosen from multiple candidates, and we think this sparse, fluctuating nature is what allows silencing positions to be rearranged when transposon sequences change.
We regard this as self-organization of piRNAs: through local interactions, individual piRNAs autonomously settle into an arrangement that silences transposons more efficiently as a whole. If this idea is correct, even when a transposon mutates to escape silencing, piRNAs should be able to restore it by using different positions. We are working to reveal the molecular mechanisms behind this self-organization and its biological meaning.
How piRNA clusters form
Defense against newly arising transposons is written as memory into piRNA clusters, the genomic regions that supply piRNAs, and keeps being updated. We look for the rules of this slow, evolutionary-scale renewal.
piRNA clusters are genomic regions that serve as the main source of piRNAs. Because they contain many transposon fragments, they are thought to store the memory of “non-self” encountered in the past. At the same time, these regions are poorly conserved between species and are highly dynamic, arising and disappearing during evolution.
We see piRNA clusters not just as sources of piRNAs but as the genomic basis for holding and updating defensive memory. When a new transposon appears, how is it written into this memory? Under what conditions, and what actually happens? No one knows yet. Looking ahead, we aim to understand how short-term sequence fluctuations over a few generations connect to long-term cluster reorganization thought to occur over thousands to millions of years.
Non-self beyond transposons
piRNAs and intracellular symbionts
How does germline RNA control deal with symbionts such as Wolbachia that pass from mother to offspring? We explore the line between partners to remove and partners to carry on.
Intracellular symbionts, exemplified by Wolbachia, infect a wide range of insects and are passed from mother to offspring through the egg. In other words, symbionts are “non-self” that stay inside the germ cells—where piRNAs act—into the next generation.
Symbionts intervene heavily in host reproduction, for example by manipulating host sex. Through collaborations, we have contributed to revealing how Wolbachia kills males by targeting the masculinizing gene and to identifying the factor responsible.
What we want to know now is whether piRNAs recognize or control such symbionts, and conversely whether symbionts manipulate the host piRNA pathway. How does germline RNA control tell apart partners to be eliminated, such as pathogens, from partners that coexist and are passed on? We think the answer will offer new clues to host defense, evolution, and the continuity of species.
piRNAs and viruses
Can piRNAs also fight viruses that come from outside? Using lepidopteran insects and their viruses, we study how piRNAs act against viruses and how this ability evolved.
Viruses are also “non-self”: they enter cells and try to multiply their own RNA. Antiviral defense in insects has been thought to rely mainly on the siRNA pathway, and how far piRNAs are involved remains unclear.
Using silkworms and other lepidopteran insects together with baculoviruses and RNA viruses that persistently infect cells, we study the conditions under which virus-derived piRNAs are produced and how they suppress viral replication. We are also interested in the possibility that viral fragments integrated into the genome act as piRNA “memory,” just as transposon fragments do.
Approach
We combine bioinformatics—analyzing small RNA and mRNA data from high-throughput sequencers on computers—with molecular biology and biochemistry that test phenomena at the molecular level. Forming hypotheses from data, testing them by experiment, and feeding the results back into the analysis: this back-and-forth is at the core of our research.
Main research materials
- The silkworm (Bombyx mori) and its cultured cells, one of the few cell culture systems with an active piRNA pathway
- Cultured cells from other lepidopteran insects
- A wide range of organisms, including Drosophila, through collaborations
Main methods
- Analysis of large-scale data such as small RNA-seq and RNA-seq
- Predicting piRNA production with computer simulations and machine learning
- Genome editing and gene transfer in cultured cells
- Biochemical analysis
Beyond describing what we observe, we aim to predict from data where piRNAs tend to arise, under what conditions their sequence fluctuation grows, and which transposon changes they can respond to. In the future, we hope to build a foundation for applications in viral responses, disease, and RNA design.