We are studying RNA biology, focusing on post-transcriptional gene regulation by RNA-binding proteins and small silencing RNAs such as miRNAs and siRNAs. We are particularly interested in elucidating the mechanism in small RNA biogenesis and finding novel RNA-binding proteins that have crucial functions in post-transcriptional gene regulation. We use multi-disciplinary approaches including biochemistry, Drosophila genetics, tissue culture system, and high-throughput sequencing.
For the small silencing RNA projects, we are particularly interested in the mechanisms by which the small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) are produced by Dicer enzyme and by which the Dicer enzyme is regulated by Dicer-partner RNA-binding proteins in this small RNA biogenesis functions. Specifically, we aim to understand the molecular mechanism by which the length of small silencing RNAs produced by Dicer is defined and regulated, which is a biologically significant question.
For the RNA-binding proteins projects, we are interested in novel post-transcriptional gene regulation mechanism performed by poorly characterized RNA-binding proteins. We use Drosophila oogenesis as one of the model systems since post-transcriptional gene regulation is particularly important during oogenesis.
We use a combination of biochemistry, biophysics, Drosophila genetics, cell culture, and next-generation sequencing, in order to understand RNA biology from the atomic to the organismal level.
1. Biogenesis of miRNAs by Dicer enzyme and regulation by Dicer-partner RNA-binding proteins
miRNAs are 21-24 nt non-coding silencing RNA. In Drosophila, miRNAs are transcribed as long primary transcripts called pri-miRNAs. The pri-miRNA is cleaved into pre-miRNA by the RNase III enzyme Drosha, aided by the partner dsRNA-binding protein Pasha. The pre-miRNA is cleaved into miRNA duplex by the RNase III enzyme Dicer-1, aided by the partner dsRNA-binding protein Loquacious-PA or Loquacious-PB (Loqs-PA, Loqs-PB). 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-bound by Argonaute1 causes translational repression and destabilization of target mRNAs.
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. 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. However, the molecular mechanism by which Loqs-PB/TRBP changes the miRNA length is not understood at all. We are trying to understand the mechanism using biochemistry and high-throughput sequencing. We developed a novel high-throughput approach (DRAM-seq; Dice Randomized pre-miRNAs and seq) to achieve this goal. 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 another project, as collaboration with a physician scientist, Dr. Roselle Abraham, who was in the Cardiology Division of Department of Medicine at Johns Hopkins University School of Medicine until 2017 and is now at UCSF, we are studying roles of miRNAs in Hypertrophic cardiomyopathy (HCM) patients and mouse models.
Fukunaga lab belong to the Program for microRNA Biology at Johns Hopkins University.
2. Biogenesis of siRNAs by Dicer-2 enzyme
Drosophila Dicer-2 associates with the dsRNA-binding partner proteins Loqs-PD and R2D2 and produces siRNAs of precisely 21 nt long with a remarkably high length-fidelity from long dsRNA substrates derived from virus and transposon. siRNA is loaded to Argonaute2 and silences highly complementary target RNAs by cleaving them—a process typically called RNAi. Argonaute2 specifically binds 21 nt long RNA. Therefore, production of 21 nt siRNAs with high length-fidelity by Dicer-2 is crucial for efficient RNA silencing. 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. We are particularly interested to understand the molecular mechanism by which Dicer-2 produces 21 nt siRNA with a remarkably high length-fidelity.
We found that Dicer-2 makes 21 nt siRNAs with high length-fidelity in vitro only when the RNA substrates has a 5' terminal monophosphate.
We identified a unique phosphate-binding pocket in the Dicer-2 PAZ domain. When this pocket is mutated, the length-fidelity in siRNA production was lowered. The phosphate-pocket mutant Dicer-2 produced less 21 nt siRNAs and more 20 and 22 nt siRNAs compared with the control wild-type Dicer-2 in vivo and in vitro.
We propose a model that recognition and anchoring of the 5' terminal monophosphate of substrate RNA in the unique phosphate-binding pocket in the Dicer-2 PAZ domain are crucial for the high-fidelity siRNA production.
We are trying to uncover more mechanisms by which Dicer-2 produces siRNAs of precisely 21 nt length with remarkably high length-fidelity.
3. Novel mechanism of post-transcriptional gene regulation controlled by poorly characterized RNA-binding proteins.
RNA-binding proteins play crucial roles in post-transcriptional gene regulation. By characterizing the molecular and biological functions of poorly characterized RNA-binding proteins, we aim to uncover novel mechanisms in post-transcriptional gene regulation. Post-transcriptional gene regulation plays particularly important roles during oogenesis. We use Drosophila oogenesis as a model system to study biological and molecular functions of RNA-binding proteins in post-transcriptional gene regulation.
In one of such projects, we are studying RNA-binding protein MARF1 (Meiosis Regulator And mRNA Stability Factor 1), MARF1 has RNA-binding motif and six tandem LOTUS domains. While MARF1 protein and LOTUS domains are conserved from flies to human, their molecular functions and mechanisms were unknown. We created MARF1 knockout fly strains by CRISPR-Cas9 system. Interestingly, the mutant flies showed complete female sterility and impaired oocyte maturation. We also found that the LOTUS domain of MARF1 binds the CCR4-NOT deadenylase complex to shorten poly-A tail of target mRNAs and there by silence translation of the target mRNAs. We are extensively working on to identify target mRNAs of MARF1 in a transcript-wide manner. We are also studying how MARF1 is regulated.
3B. ATP-dependent RNA helicase
In another project, we are studying an ATP-dependent RNA helicase, which is thought to bind and restructure RNA or RNA-protein complex. By using Drosophila genetics, we are characterizing the RNA helicase mutants that cannot bind ATP, cannot hydrolyze ATP, or cannot release the ATP. We observe impaired oogenesis in these mutant flies. We also observe developmental phenotypes in eyes when the mutants were expressed in eyes.ene pheno We are extensively working to uncover underlying molecular phenotypes. Current data in our hands suggest that this RNA helicase regulates translation of a specific target mRNAs, in an ATPase activity dependent manner.
3C. RNA-post-transcriptional modification enzyme
We are also starting several new projects, including RNA-post-transcriptional modification enzymes and specific ribonuclease enzymes. We found that RNAi knockdown in ovaries of an RNA-post-transcriptional modification enzyme causes sterility. We are currently trying to understand underlying molecular mechanism and functions of this gene and RNA modification.
We found that knockout of a specific ribonuclease enzyme causes lethality at the late pupa stage. We are currently trying to understand underlying molecular mechanism and functions of this gene and its target RNAs.
Student and postdoc positions are available. Please contact the PI if interested.