Exploring retrotransposon regulation mechanisms
via piRNA

SAITO, Kuniaki Professor

Invertebrate Genetics Laboratory,
Department of Chromosome Science.

Dr. Saito graduated from the Department of Material and Biological Chemistry, Faculty of Science, Yamagata University and completed his Ph.D. (Science) course at
the Graduate School of Natural Sciences, Kobe University. He worked as a postdoctoral fellow at the Institute for Genome Research, Tokushima University, andAssistant Professor and Associate Professor at the Department of Molecular Biology, Keio University School of Medicine. In 2017, he was appointed as a Professor
at the National Institute of Genetics.

Saito took a new position at NIG in 2017 and has launched many projects, such as the production mechanisms of smallRNAs, formation and maintenance of germ cells, regulatory mechanism of transposons that move within the genome,
and the structure of Drosophila chromosomes. Not that he had a long-standing interest in all of these topics; he statedthat these subjects began to interest him as he conducted a careful analysis of one particular topic. Saito told us about
the trajectory of his research and his future visions.

It all started with research on theproduction mechanisms of small RNAThe original research that led to my current studies goes back to a
research on small RNAs. During the period between the 1990s andthe beginning of 2000, genome projects were conducted wherelarge-scale genomic analyses were performed, covering everythingfrom nematodes and Drosophila to mice and humans. As a result, thegene number of each species was precisely counted and the functionof each gene began to become clear. At the same time, it was foundthat there was a large number of RNAs (non-coding RNAs, ncRNAs)
that do not code for proteins after being transcribed from DNA (aprocess called transcription). Although it was considered at the timethat these ncRNAs were junk and waste with no function, they werenumerous. Therefore, researchers suspected that they must havesome function, and so functional analyses were initiated. While thefunctional analyses were ongoing, it was revealed that small RNAs
with lengths of 20–30 bases tended to suppress the expression ofgenes. For example, a single-stranded small RNA (called amicroRNA or miRNA), with a length of approximately 20 bases,
anneals with its complementary sequence in messenger RNA(mRNA) to regulate the expression of genes involved indevelopmental timing and cell growth. Abnormalities in such
regulations have a connection with various diseases, such as cancer.
In 2004 when I obtained Ph.D. and started working as a postdoc,
microRNAs were known to be synthesized from a long precursor witha hairpin shape, and subsequently processed to form a short, mature
microRNA. I was interested in the mechanism of microRNA
synthesis, so I decided to study the molecular mechanism of theprecise systematic processing of long precursor RNA. For example, it
was known at the time that a precursor of microRNA is cleaved by aspecific protein called “Dicer”. However, I discovered that Dicer does
not function alone, but functions via interactions with a different
protein called “Loquacious”. Later, it was found that double-stranded
RNA created by Dicer/Loquacious is denatured to produce asingle-stranded RNA, and single-stranded RNA binds to a protein
called “Argonaute” to become a functional microRNA, which canregulate the expression of genes.

Progression to the piwi protein, piRNA,and transposons
While conducting my research, I noticed that five proteins with anamino acid sequence and structure (called a domain) similar to the
Argonaute protein had been reported in Drosophila, and furthermore
seven proteins had been reported in mammals. Among these
proteins, there was an interesting protein called Piwi (P-elementinduced wimpy testis). piwi gene had been identified in Drosophila in1997. Mutations in this gene were reported to cause abnormalities in
the formation and maintenance of reproductive cells in both males
and females, resulting in an inability to produce offspring (i.e.,sterility).

Later, Aubergine (Aub) and AGO3, which were similar to piwi, wereidentified; proteins coded by these three genes are grouped together
and are now called the PIWI subfamily group. Based on findings thatthese three proteins possess a structure similar to that of theargonaute protein and that AGO1, one of argonaute family proteins,binds to microRNA, I hypothesized that the PIWI subfamily group canalso bind to small RNA. At the time, I was working at the Institute for
Genome Research at Tokushima University where I reported that thePIWI protein binds to small RNA in Drosophila ovary.
However, contrary to my expectation, the small RNA was found tobe anywhere between 25 and 30 bases, which is a little longer thanmicroRNA. After careful consideration, I began to think that this small
RNA was different from microRNA, and so I decided to study its
sequence and genomic origin. As a result, I discovered that thePIWI-associated small RNAs possessed a sequence complementary
to a part of a retrotransposon. Later, Aub and AGO3 proteins werealso found to bind to the retrotransposon-derived small RNA. As aresult, these small RNAs are now being called piRNA(PIWI-interacting RNA) as the small RNA group that binds to the PIWI
subfamily proteins. This means a new player to my research:
retrotransposon biology.

Studies examining piRNA brought bigchanges
At the time, we had only biochemically identified piRNA as a factor
that bound to the PIWI subfamily group; there was no extensive
information regarding the biological functions of piRNAs, how they
were distinguished from other small RNAs and specifically loaded

onto PIWI proteins, and how piwi mutations caused sterility.
Therefore, we decided to move away from microRNA research and[begin a new field]. At the time, research on small RNA, including
microRNA, was extremely competitive worldwide. Thus, I wanted tomove toward a more challenging field, one with great mystery.

Retrotransposons are transposable elements that are integrated
throughout the genome, and some retrotransposons are long,
approximately 10,000 bp. A characteristic of retrotransposons is thatthey first transcribe their own DNA into RNA, and then, while
replicating DNA from the RNA through reverse transcription, they
integrate (i.e., move around) within the genome. It is easy tounderstand if you imagine copying and pasting words using a word
processor. Retrotransposons became integrated into the genome
during the development of organism following a retroviral infection,and retrotransposons contribute to the increasing size and genome
diversity. For example, in cases where a retrotransposon integrated
into a gene, a new function may be added to the gene or, inversely,
the existing function of the gene may be deleted. According to
genomic analysis, 45% of the human genome and 20% of theDrosophila genome is occupied by retrotransposons.
Going back to my experiments, we had already discovered that theargonaute protein suppress the expression of target genes by binding
to small RNAs. Therefore, we thought that piRNA also suppressed
the function of retrotransposons by binding to the piwi protein. Thatis, we predicted that the copy and paste function in variants withabnormal piwi will be increased. Therefore, we examined suchvariants using individual or cultured Drosophila cells. As predicted,
the expression of retrotransposons was increased in piwi mutant.

We are beginning to understand therelationship between the piwi protein,piRNA, and retrotransposons
After further analyses, it was discovered that piwi is localized in thenucleus of germline stem cells and in the ovarian somatic cells
surrounding the germ cells in the testis and ovaries of Drosophila. In
contrast, Aub and AGO3 were localized in the cytoplasm of germ
cells of both the testis and ovaries. Therefore, we conducted anexperiment to suppress the expression of piwi and found thatsuppression caused the overexpression of retrotransposons andcaused abnormalities in the development of germ cells. Phenomena
such as overexpressed retrotransposons damaging genes important

for the production and maintenance of germ cells are suspected, but
additional studies are required to elucidate the details.
Meanwhile, I discovered that the PIWI protein interacts with histone
H1, a protein that regulates the tightness of the chromatin structure. I
believe that interactions between target retrotransposon locus andhistone H1 are increased by the PIWI protein, causing a condensed
chromatin structure, and that the retrotransposon activity issuppressed as a result. Thus, we believe that piRNAs are also
required and that the interaction between the PIWI protein and piRNA
suppresses the expression of retrotransposons. However, thepresence of other proteins that interact with the PIWI protein has
been discovered, so we need to more closely examine these proteinsfor better understanding.
A system wherein RNAs derived from retrotransposons suppress
the activity of retrotransposons is a conflict system. Whereas, theamplification of retrotransposons can be a driving force for genomic
diversity, integration of retrotransposons can cause the destruction ofgenes. Therefore, we believe that this system was acquired during
the earliest stages of genomic diversification on Earth.

NIG is a privileged environment
It has only been approximately 6 months since I have started
working at NIG, so I am currently constructing various Drosophila
constructs in which specific genes are tagged with epitopes, with thehelp of an assistant professor, Dr. Shu Kondo, as well as preparing
the experimental environment. I have consistently used Drosophila
for my research because it is easy to handle because of its small
size, has a short cycle of maturation and reproduction (i.e.,alternation of generations), introducing and inactivating its genes isrelatively easy, and results obtained may be easily applied to higherorganisms, such as mice and humans. NIG is equipped with awonderful environment, where studies using Drosophila can reach
their full potential. In our laboratory, various Drosophila strains are
separately stored, and there is an RNAi library that can artificiallysuppress more than 9,000 of the total 14,000 Drosophila genes.

devotion that scientists here have for their research.
I also like how naturally English education is being conducted here.
For example, NIG has its own textbook with audio for English
education, called the NIG Method, by Dr. Tatsumi Hirata at the Brain
Function Research Lab, Dr. Yasushi Hiromi who is head of office forresearch development at NIG and professor emeritus at the Graduate
University for Advanced Studies, SOKENDAI, and others together
with an assistant professor in English. This text book is also availableto the public. It is said that the NIG Method was developed based on
the idea that researchers need to be able to think logically andconduct discussions in English. The method offers excellent tips,
such as describing unusable English that researchers and students
tend to use, as well as describing how to prepare effective slides andpresentations.
Furthermore, a journal club run without a barrier between labs isvery meaningful in terms of improving both research and English.
There are currently 22 journal clubs, each with roughly 15
researchers and addressing a different research area and theme; I
belong to a journal club for germ cells and developmental biology. Atmeetings of the journal club, the speaker of the day introduces apublished paper written in English that is suitable to their field. Afterthe introduction of the paper by the speaker, everybody discusses thepaper. Speakers are rotated, with each individual speaking about
once every 6 months. Staff and students all take turns in speaking.

I want students to know that research
is funI do not have any students in my lab at the moment; however, I
plan to have one student in April 2018. If anyone is interested in myresearch as I have described it, I invite them to join my lab. I thinkthat anybody who is interested in performing experiments who cancome up with their own hypothesis and plan experiments to support
or deny the hypothesis can join us, regardless of their background. In
the first place, the SOKENDAI entrance exam, despite being
considered an entrance exam, is not a typical exam but tests thequality of individuals. Therefore, I would encourage students toconsider joining us.
At the beginning of a doctoral program in my lab, students will
notice that there are many occasions where they will learn fromobservation. However, I think that they will gradually be able todesign their own experiments. One more assistant professor isscheduled to join us in January 2018, so I am anticipating that we will
have enough researchers to conduct various experiments.

More opportunities lie ahead as westeadily move forward, step by step

In addition, I can tell that researchers in NIG are passionate about
their research; there are several inquisitive people here. It has only
been 6 months since I moved here, and I am already very pleased to
be a part of this laboratory. Before I came here, an associate at theKeio University told me a story about a researcher at NIG whocontinued conducting his experiment on a table at a faculty meetingroom during discussions. This story describes the passion andPeople think that scientific research should be practical and returna benefit to the society. However, I try to focus on the present work. I
think that results are obtained if you are focused on a proper target,
have trust in yourself, and are devoted to research. Collaborators andrivals will naturally appear, and discussions with the same will leadyour research to a more advanced stage. For example, I will not
conduct research involving humans, but I would be happy if otherresearchers were to apply our results from Drosophila to mammalsand humans.
An example of the application of good research is the developmentof the CRISPR/Cas9 system, which is now an indispensable tool ingenome editing. CRISPR/Cas9 is a system where a targeted DNA
sequence is clipped using DNA endonuclease (Cas9) guided byRNA. This system originated in bacteria and protects them fromattack by foreign materials, such as bacteriophages. The
CRISPR/Cas9 mechanism is very similar to the retrotransposon
regulation mechanism of the PIWI proteins and piRNA. I believe it islikely that the origin of both mechanisms has something in common. I
once encountered an occasion where an argonaute researcherchanged his research area to CRISPR/Cas9 research. This
happened several years ago prior to a report suggesting that theCRISPR/Cas9 system can be used as a gene manipulation tool. Boththe CRISPR/Cas9 system and argonaute protein suppress thespecific gene expressions via small RNA. Therefore, I think that theoverseas researcher thought that the CRISPR/Cas9 system is usefulas a gene manipulation tool. Thus, my research at the time was very
similar to the CRISPR/Cas9 research, which is now a possible
nominee for the Nobel Prize. I should have moved on toCRISPR/Cas9 research at that time; however, I expect that a similar
opportunity will appear if I diligently continue my research.

I would like to expand my research to
unknown fields
Competition in piRNA research is becoming intense. The group ofJulius Brennecke and his colleagues in Austria published an article in
Nature stating that they identified an important factor involved in theregulation of piRNA transcription. The research was accomplishedusing next generation sequencing, which can determine nucleotide
sequences at a high speed and accuracy, and I feel that they have
solved the issue that I was interested in. I do not want to change myinterests or publish papers without having confirmed evidence andconclusions. Research begun with the production mechanism ofsmall RNAs led to the field of developmental biology such as theformation and maintenance of germ cells and has further expanded
to the regulation of piRNA, retrotransposons, and chromosomes. I
have no idea where my research will lead in the future, but I want toadvance onward, enjoying research in unknown fields.

Interviewer:
Naoko Nishimura, Science writer
Photographer:
Mitsuhiko Kurusu, Office for Research Development
September 2017