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Ryuya Fukunaga, Ph.D.

Photo of Dr. Ryuya Fukunaga, Ph.D.

Assistant Professor of Biological Chemistry

Research Interests: Mechanism and biology of small silencing RNAs

Background

Dr. Ryuya Fukunaga is an Assistant Professor of Biological Chemistry at the Johns Hopkins University School of Medicine. Dr. Fukunaga’s research focuses on mechanism and biology of small silencing RNAs using biochemistry, Drosophila genetics, cell culture, high throughput sequencing, and X-ray crystallography.

Dr. Fukunaga received her undergraduate degree in biochemistry and biophysics from University of Tokyo. He earned his Ph.D. in biochemistry and biophysics from University of Tokyo. He completed postdoctoral training in RNA silencing mechanism at the University of California Berkeley and University of Massachusetts Medical School. Dr. Fukunaga joined the Johns Hopkins faculty in 2013.

He is a member of the RNA Society and the American Heart Association. He serves as an ad hoc peer reviewer for several journals. He received American Heart Association National Scientist Development Award for 2015-2018. He received the Research fellowship of the Japan society for the promotion of science for young scientists (2004-2007), the Research Postdoctoral Fellowship of the Japan Society for the Promotion of Science for Research Abroad (2007-2009), and the Research Fellowship of King Trust Postdoctoral Fellowship (2010-2012). 

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Titles

  • Assistant Professor of Biological Chemistry

Departments / Divisions

Education

Degrees

  • B.S., University of Tokyo (Japan) (2002)
  • Ph.D., University of Tokyo (Japan) (2007)

Additional Training

University of California, Berkeley, Berkeley, California/USA, 2009, Molecular and Cellular Biology; University of Massachusetts Medical School, Worcester, Massachusetts/USA, 2013, Biochemistry and Molecular Pharmacology

Research & Publications

Research Summary

Overview

The Fukunaga lab investigates the mechanism and biology of small silencing RNAs. We try to understand how small silencing RNAs, such as microRNAs (miRNAs), small interfering RNAs (siRNAs) and piwi-interacting RNAs (piRNAs), are produced and how they function. We use a combination of biochemistry, biophysics, fly genetics, cell culture, X-ray crystallography and next-generation sequencing, in order to understand the biogenesis and function of small silencing RNAs from the atomic to the organismal level.

1. miRNA

miRNAs are 21-24 nt long RNA. In fruit fly Drosophila, miRNAs are transcribed as long primary transcripts called pri-miRNAs (Figure 1). The pri-miRNA is cleaved into pre-miRNA in the nucleus by the RNase III enzyme Drosha, aided by the dsRNA-binding partner protein Pasha. The Exportin-5/Ran-GTP complex transports pre-miRNA from the nucleus to the cytoplasm. In cytoplasm, Dicer-1, aided by the dsRNA-binding partner protein Loqs-PA or Loqs-PB, cleaves the pre-miRNA into miRNA duplex. miRNA is then loaded to Argonaute1 and binds target mRNAs through base complementarity of the miRNA sequence at positions 2-8 (called seed sequence). miRNA-Ago1 binding to the target mRNAs causes translational repression and mRNA degradation.

Loqs-PB, but not its alternative splicing isoform Loqs-PA, changes the nucleotide positions at which Dicer-1 cleaves pre-miRNA and produces miRNA with distinct length (Figure 2). These alternatively produced miRNAs can have distinct seed sequences and therefore regulate different target mRNAs. The mammalian Dicer partner protein TRBP, but not its paralogue PACT, changes the length and the seed sequence of miRNAs produced by Dicer in mammals. The Fukunaga lab investigates how Dicer partner proteins (Loqs-PB in fly and TRBP in mammals) change the miRNA length generated by the Dicer enzymes. We also try to uncover biological significance of the alternative miRNA production. Our hypothesis is that the alternative splicing of Loqs-PA/Loqs-PB in fly and the gene expression of TRBP/PACT in mammals are finely regulated in each tissue and developmental stage, leading to regulated production of distinct miRNA isoforms, and that such fine regulation is important for biology. In addition, we investigate the functions of Drosha-Pasha complex focusing whether Pasha changes the pri-miRNA cleavage site choice of Drosha. Furthermore, we are trying to discover novel factors and mechanisms regulating the miRNA production and function.

In another project, as collaboration with a physician scientist, Dr. Roselle Abraham at the Cardiology Division of Department of Medicine, we are studying functional effects of a miRNA SNP mutation found from Hypertrophic cardiomyopathy (HCM) patients. This project may lead to development of novel diagnosis and therapeutics for cardiovascular diseases including HCM in the future.

2. siRNA 

Drosophila Dicer-2 associates with the dsRNA-binding partner proteins Loqs-PD and R2D2 and produces 21 nt long siRNAs from long dsRNA (Figure 1). siRNA is loaded to Argonaute2 and silences highly complementary target RNAs by cleaving them—a process typically called RNAi. One of the biological functions of the siRNA pathway is to fight against exogenously derived viral infection and against genome encoded transposon invasion. In addition, Dicer-2 produces endogenous siRNAs (endo-siRNAs) derived from genome encoded long hairpin RNA or overlapping mRNAs. The biological functions of these classes of endo-siRNAs are not well understood. We are interested in how viral and endogenously derived RNAs are recognized and cleaved into siRNAs by Dicer-2 and how the produced siRNAs function in biology. We also try to identify and characterize novel factors involved in or regulating the siRNA pathways. We are also interested in understanding how the two Dicer enzymes achieve their respective substrate specificities (pre-miRNA for Dicer-1 and long dsRNA for Dicer-2). Physiological concentration of inorganic phosphate, a small molecule found in all the cells, restricts the substrate specificity of Dicer-2 to long dsRNA by inhibiting Dicer-2 from cleaving pre-miRNA, without affecting cleavage of long dsRNA. We investigate the underlying molecular mechanisms.

3. piRNA

piRNAs (26-31 nt) are mostly produced in gonads (ovaries and testes). Unlike miRNAs and siRNAs, Dicer enzymes are not involved in the piRNA production. piRNAs are produced in the primary processing pathway and the ping-pong pathway, which are not yet fully understood (Figure 1). piRNAs are loaded onto PIWI proteins and function in epigenetic and post-transcriptional gene silencing of transposons and other genetic elements in order to maintain genome integrity of germline cells. Interestingly, piRNAs are recently implicated also in sex determination, neuronal functions in brain, and tumorigenesis in cancer cells. We are interested in the mechanisms for biogenesis and function of piRNAs. 

4. RNA helicase

RNA helicases are involved in allmost all the aspects in the RNA biology: RNA transcription, transport, translation, silencing, localization, structural rearangement, decay, and so on. The Dicer enzymes also have a N-terminal 'helicase' domain. We are studying molecular and physiological roles of DEAD-box RNA helicases.

Summary

Our lab uses multi-disciplinary approaches to understand the biogenesis and function of small silencing RNAs from the atomic to the organismal level. Small silencing RNAs play crucial roles in various aspects in biology. In fact, mutations in the small RNA genes or in the genes involved in the pathways cause many diseases in human including cancers. Our research projects will answer fundamental biological questions and also potentially lead to therapeutic application to human disease. 

Student and postdoc positions are available. Please contact the PI if interested.

Lab Website: Ryuya Fukunaga Lab

Selected Publications

View all on Pubmed

Fukunaga, R., Doudna, J.A. (2009). dsRNA with 5' overhangs contributes to endogenous and antiviral RNA silencing pathways in plants. EMBO J. 28(5):545-55. PMCID: PMC2657584

Cenik, E.S., Fukunaga, R., Lu, G., Dutcher, R., Wang, Y., Tanaka Hall, T.M., Zamore, P.D. (2011). Phosphate and R2D2 restrict the substrate specificity of Dicer-2, an ATP-driven ribonuclease. Mol Cell. 42(2):172-84. PMCID: PMC3115569

Fukunaga, R., Han, B.W., Hung, J.H., Xu, J., Weng, Z., Zamore, P.D. (2012). Dicer partner proteins tune the length of mature miRNAs in flies and mammals. Cell. 151(3):533-46. PMCID: PMC3609031


Fukunaga, R., Colpan, C., Han, B.W., Zamore, P.D. (2014). Inorganic phosphate blocks binding of pre-miRNA to Dicer-2 via its PAZ domain. EMBO J. 18;33(4):371-84. PMCID: PMC3989643


Fukunaga, R., Zamore, P.D. (2014). A universal small molecule, inorganic phosphate, restricts the substrate specificity of Dicer-2 in small RNA biogenesis. Cell Cycle. 13(11):1671-6. PMCID: PMC4111713

Patents

Modified tRna Containing Unnatural Base And Use Thereof
Patent # US patent 8,715,984 | 05/06/2014

Mutant SepRS, and method for site-specific introduction of phosphoserine into protein using the same
Patent # US patent 8,178,301 | 05/15/2012

Academic Affiliations & Courses

Graduate Program Affiliation

Biochemistry, Cellular and Molecular Biology (BCMB) Graduate Program

Biological Chemistry (BC) Graduate Program

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