Assistant Professor of Biological Chemistry
Johns Hopkins University School of Medicine
725 N. Wolfe Street
521A Physiology Bldg
Office Phone: 410-955-3790
Lab Phone: 410-955-3458
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Mechanism and biology of small silencing RNAs
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.
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 will investigate how Dicer partner proteins (Loqs-PB in fly and TRBP in mammals) change the miRNA length generated by the Dicer enzymes. We will 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 will investigate the functions of Drosha-Pasha complex focusing whether Pasha changes the pri-miRNA cleavage site choice of Drosha. Furthermore, we will try to find novel factors and mechanisms regulating the miRNA production and function.
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 will 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 will investigate the underlying molecular mechanisms.
piRNAs (26-31 nt) are longer than miRNAs and siRNAs. 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. Interestingly, piRNAs are recently implicated in neuronal functions in brain. We are interested in the mechanisms for biogenesis and function of piRNAs.
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.
Fukunaga R, Han BW, Hung JH, Xu J, Weng Z, Zamore PD, "Dicer Partner Proteins Tune the Length of Mature miRNAs in Flies and Mammals" Cell, 151, 533-46, (2012) PubMed Reference
Cenik ES, Fukunaga R, Lu G, Dutcher R, Wang Y, Tanaka Hall TM, Zamore PD, “Phosphate and R2D2 Restrict the Substrate Specificity of Dicer-2, an ATP-Driven Ribonuclease” Mol. Cell, 42, 172-84, (2011) PubMed Reference
Fukunaga R, Doudna JA, “dsRNA with 5¢ overhangs contributes to endogenous and antiviral RNA silencing pathways in plants” EMBO J., 28, 545-55, (2009) PubMed Reference
Fukunaga R, Harada Y, Hirao I, Yokoyama S, “Phosphoserine aminoacylation of tRNA bearing an unnatural base anticodon” Biochem Biophys Res Commun., 1, 372, 480-5, (2008) PubMed Reference
Fukunaga R, Yokoyama S, “Structural insights into the second step of RNA-dependent cysteine biosynthesis in archaea: crystal structure of Sep-tRNA:Cys-tRNA synthase from Archaeoglobus fulgidus” J. Mol. Biol., 29, 370, 128-41, (2007) PubMed Reference
Fukunaga R, Yokoyama S, “The C-terminal domain of the archaeal leucyl-tRNA synthetase prevents misediting of isoleucyl-tRNAIle” Biochemistry, 1, 46, 4985-96, (2007) PubMed Reference
Fukunaga R, Yokoyama S, “Structural insights into the first step of RNA-dependent cysteine biosynthesis in archaea. Structural basis of phosphoserine ligation to tRNA for genetic code evolution” Nat. Struct. Mol. Biol., 14, 272-9, (2007) PubMed Reference
Fukunaga R, Yokoyama S, “Structure of the AlaX-M trans-editing enzyme from Pyrococcus horikoshii” Acta Crystallogr. D, 63, 390-400, (2007) PubMed Reference
Fukunaga R, Yokoyama S, “Structural basis for substrate recognition by the editing domain of isoleucyl-tRNA synthetase” J. Mol. Biol. 359, 901-12, (2006) PubMed Reference
Fukunaga R, Yokoyama S, “Aminoacylation complex structures of leucyl-tRNA synthetase and tRNALeu reveal two modes of discriminator base recognition for 3¢-end relocation toward the editing domain” Nat. Struct. Mol. Biol. 12, 915-922, (2005) PubMed Reference
Fukunaga R, Yokoyama S, “Structural basis for non-cognate amino acid discrimination by the valyl-tRNA synthetase editing domain” J. Biol. Chem. 280, 29937-29945, (2005) PubMed Reference
Fukunaga R, Yokoyama S, “Crystal Structure of Leucyl-tRNA Synthetase from the Archaeon Pyrococcus horikoshii Reveals a novel editing domain orientation” J. Mol. Biol. 346, 57-71, (2005). PubMed Reference
Fukunaga R, Fukai S, Ishitani R, Nureki O, Yokoyama S, “Crystal Structures of the CP1 Domain from Thermus thermophilus Isoleucyl-tRNA synthetase and Its Complex with L-Valine” J. Biol. Chem. 279, 8396-8402, (2004) PubMed Reference
Fukunaga R, Colpan C, Han BW, Zamore PD, "Inorganic phosphate blocks binding of pre-miRNA to Dicer-2 via its PAZ domain" EMBO Journal, 18, 371-84, (2014)