How do small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) regulate gene expression and protect the genome? RNAi encompasses the cellular response to exogenous double-stranded RNA (dsRNA)-delivered experimentally or generated during a viral infection-and the response to parasitic genetic elements, such as transposons and highly repeated sequences. Our experimental strategy uses Drosophila melanogaster as a model system, because flies combine outstanding genetic, biochemical, and phenotypic tools.
Aim 1 will explore why Drosophila Dicer-1 and Dicer-2 use ATP. Dicer enzymes make siRNAs and miRNAs. Dicer enzymes contain protein motifs that suggest they are ATPases. The function of Dicer proteins is modified by their dsRNA-binding partner proteins-Loquacious (Loqs) and R2D2 in flies. How do R2D2 and the three different isoforms of Loqs extend or restrict the function of Dicer-1 and Dicer- 2? siRNAs associate with the protein Argonaute2 to form the RNA-induced silencing complex (RISC), which mediates RNAi.
In Aim 2, new technologies will be used to study the function of RISC, including single-molecule fluorescence techniques to study the cycle of RISC function, and deep sequencing methods that allow us to compare millions of different sequences in a single experiment to test the contribution of each siRNA nucleotide to binding and catalysis.
Aim 3 proposes to define the biological functions of endogenous siRNAs. One function of endo-siRNAs is to protect the somatic genome from transposon expression, but endo-siRNAs also map to convergently transcribed mRNAs, and at least one endo-siRNAs acts in trans to regulate expression of an highly complementary mRNA. A combination of genetics, high throughput sequencing, and bioinformatic analysis will be employed to define the target repertoire of Drosophila endogenous siRNAs.
Aim 4 seeks to identify the role of Drosophila piRNAs in epigenetic silencing. piRNAs provide the primary genomic defense in the germ line against transposons and repetitive sequence elements, yet how they are produced or how they suppress transposon or cellular gene expression remains unknown. Of particular interest is testing the idea that piRNAs acting early in development can set the epigenetic state of genes whose expression and phenotypic consequences can be observed only in the adult fly.

Public Health Relevance

The Drosophila RNAi and piRNA pathways are highly similar to their mammalian counterparts, so what is learned in these studies will advance our understanding of RNA silencing in humans. Moreover, RNAi has become an essential laboratory tool and siRNA therapeutics are currently in human trials. Thus, the proposed studies promise to contribute to the development of practical applications of RNAi as a gene discovery technology and as a human therapy.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
Project #
Application #
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Bender, Michael T
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Massachusetts Medical School Worcester
Schools of Medicine
United States
Zip Code
Fu, Yu; Yang, Yujing; Zhang, Han et al. (2018) The genome of the Hi5 germ cell line from Trichoplusia ni, an agricultural pest and novel model for small RNA biology. Elife 7:
Gainetdinov, Ildar; Colpan, Cansu; Arif, Amena et al. (2018) A Single Mechanism of Biogenesis, Initiated and Directed by PIWI Proteins, Explains piRNA Production in Most Animals. Mol Cell 71:775-790.e5
Lewis, Samuel H; Quarles, Kaycee A; Yang, Yujing et al. (2018) Pan-arthropod analysis reveals somatic piRNAs as an ancestral defence against transposable elements. Nat Ecol Evol 2:174-181
Fu, Yu; Wu, Pei-Hsuan; Beane, Timothy et al. (2018) Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers. BMC Genomics 19:531
Koh, Cha San; Madireddy, Rohini; Beane, Timothy J et al. (2017) Small methyltransferase RlmH assembles a composite active site to methylate a ribosomal pseudouridine. Sci Rep 7:969
Ouimet, Mireille; Koster, Stefan; Sakowski, Erik et al. (2016) Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism. Nat Immunol 17:677-86
Ge, Daniel Tianfang; Tipping, Cindy; Brodsky, Michael H et al. (2016) Rapid Screening for CRISPR-Directed Editing of the Drosophila Genome Using white Coconversion. G3 (Bethesda) 6:3197-3206
Han, Bo W; Wang, Wei; Li, Chengjian et al. (2015) Noncoding RNA. piRNA-guided transposon cleavage initiates Zucchini-dependent, phased piRNA production. Science 348:817-21
Roy, Christian K; Olson, Sara; Graveley, Brenton R et al. (2015) Assessing long-distance RNA sequence connectivity via RNA-templated DNA-DNA ligation. Elife 4:
Chou, Min-Te; Han, Bo W; Hsiao, Chiung-Po et al. (2015) Tailor: a computational framework for detecting non-templated tailing of small silencing RNAs. Nucleic Acids Res 43:e109

Showing the most recent 10 out of 46 publications