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Mechanism and biology of small silencing RNAs
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. For this end, we are trying to make miR-307a knockout flies and plan to analyze the molecular phenotypes. 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 2). 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). Recently, we found that 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 (Figure 4). We propose that inorganic phosphate occupies the phosphate-binding pocket in Dicer-2 and thereby block access of pre-miRNA. Currently we are investigation the function of the phosphate-binding pocket. 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 5). 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. We are trying to identify new factors involved in the piRNA pathway, using a fly reporter system. 4. RNA helicase RNA helicases are involved in almost all the aspects in the RNA biology: RNA transcription, transport, translation, silencing, localization, structural rearrangement, 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. Particularly, we are currently focusing on Drosophila belle, a DEAD-box RNA helicase that essential for fly viability and fertility and is conserved from yeast to human (Figure 6). We are making various mutant Belle and analyzing them genetically and biochemically. 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. Positions available Postdoc and student positions are available. Please contact the PI if interested.
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)
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)
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)
Fukunaga R, Doudna JA, “dsRNA with 5ў overhangs contributes to endogenous and antiviral RNA silencing pathways in plants” EMBO J., 28, 545-55, (2009)
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)
Fukunaga R, Yokoyama S, “Structural insights into the first 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)
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)
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)
Fukunaga R, Yokoyama S, “Structure of the AlaX-M trans-editing enzyme from Pyrococcus horikoshii” Acta Crystallogr. D, 63, 390-400, (2007)
Fukunaga R, Yokoyama S, “Structural basis for substrate recognition by the editing domain of isoleucyl-tRNA synthetase” J. Mol. Biol. 359, 901-12, (2006)
Fukunaga R, Yokoyama S, “Aminoacylation complex structures of leucyl-tRNA synthetase and tRNALeu reveal two modes of discriminator base recognition for 3ў-end relocation toward the editing domain” Nat. Struct. Mol. Biol. 12, 915-922, (2005)
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).
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).
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)