Shuo Gu, Ph.D.
RNA Biology Laboratory
Building 560, Room 11-6B
Frederick, MD 21702
Research in the Gu lab aims to unveil the roles of non-coding RNAs in gene regulation and to develop new therapeutic approaches for cancer treatment. MicroRNA (miRNA), as a small non-coding RNA, plays an essential role in gene regulation networks, human diseases and cancer. We are interested in understanding the mechanisms of miRNA biogenesis, post-transcriptional modifications, and biological functions in mammalian systems. This information will then be used to test novel RNA-based approaches designed to alter gene expression with improved safety, off-targeting, and potency profiles, which can then be used as tools for biological discovery and therapeutics.
Fidelity of miRNA biogenesis. Enormous efforts have focused on studying the alteration in miRNA expression levels during development and disease. However, imprecise biogenesis or post-maturation modification alters miRNA function by changing the sequence of the final product rather than its abundance. The biological importance of these changes in normal and disease states is not well defined. We are interested in unveiling the molecular mechanism controlling the fidelity of miRNA biogenesis and its effects on miRNA function. Recent advances in sequencing technology are making it possible to reveal such information in living cells. Together with applying classical biochemical approaches and reporter-based functional assays, we established a "loop-counting" rule governing the preciseness of Dicer processing, which is critical for miRNA biogenesis and function. We will extend our efforts to study other events in miRNA biogenesis to develop a more complete picture of how the ends of miRNA are generated, which can then serve as a reference point in investigating post-maturation modification of miRNAs.
In vivo study of RISC assembly. MicroRNAs and siRNAs interact with target sequences, inducing gene repression through the RNA-induced silencing complex (RISC) that consists of one of four mammalian Argonaute (Ago) proteins. Despite its importance, our understanding of RISC assembly is limited. The majority of the knowledge about this process is drawn from experiments performed within cell-free or reconstituted systems. During the past few years, we have developed a cell-based assay to specifically measure the RISC assembly process of cleaving Ago (Ago2) and non-cleaving Agos (Ago1/3/4) in vivo. Applying such a system, we are interested in studying how miRNA isoforms, which are generated by non-precise processing during biogenesis, affect RISC assembly and function in normal and disease states.
Novel design of potent DNA-directed RNAi (shRNA) with minimal off-target effects. DNA-directed RNAi (shRNA expressed from plasmids) is more desirable than traditional synthetic siRNAs in many applications. It is preferred or required in genetic screens and specific RNAi approaches in gene therapy settings. However, the application of ddRNAi is hampered due to unwanted off-target effects. Currently, attempts in reducing off-target effects are mainly based on empirical approaches with limited success.
We are interested in achieving such a goal through rational design based on the knowledge gained in the mechanistic studies outlined above. Our finding on Dicer processing has established the loop-counting rule, which laid the groundwork in designing Pol III-driven pre-miRNA-like shRNAs free of the off-target effects resulting from heterogeneous processing. We are currently working on transferring such a design into the Pol II system. In addition, based on the recent findings of how RISC interacts with its target mRNA on a molecular level, we are developing a novel strategy for designing shRNA with minimal off-target effects originating from the miRNA-like pathway.
Bofill-De Ros X, Gu S. Guidelines for the optimal design of miRNA-based shRNAs. Methods. 2016;103:157-66.
Valdmanis PN, Gu S, Chu K, Jin L, Zhang F, Munding EM, Zhang Y, Huang Y, Kutay H, Ghoshal K, Lisowski L, Kay MA. RNA interference-induced hepatotoxicity results from loss of the first synthesized isoform of microRNA-122 in mice. Nat Med. 2016;22(5):557-62.
Gu S, Jin L, Zhang Y, Huang Y, Zhang F, Valdmanis PN, Kay MA. The loop position of shRNAs and pre-miRNAs is critical for the accuracy of dicer processing in vivo. Cell. 2012;151(4):900-11.
Gu S, Jin L, Huang Y, Zhang F, Kay MA. Slicing-independent RISC activation requires the argonaute PAZ domain. Curr Biol. 2012;22(16):1536-42.
Gu S, Jin L, Zhang F, Huang Y, Grimm D, Rossi JJ, Kay MA. Thermodynamic stability of small hairpin RNAs highly influences the loading process of different mammalian Argonautes. Proc Natl Acad Sci U S A. 2011;108(22):9208-13.
Related Scientific Focus Areas
Genetics and Genomics
Molecular Biology and Biochemistry
This page was last updated on February 4th, 2019