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Mechanistic insights into the slicing specificity of Argonaute and development of a programmable RNA endonuclease

Dayeh, Daniel M, Dayeh

Abstract Details

2018, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.
The central dogma of molecular biology defines the flow of information from genes to proteins by way of RNA. In addition to messenger RNA (mRNA), which serves as the intermediate between DNA and protein, ribosomal RNA (rRNA) and transfer RNA (tRNA) provide the foundation for functional noncoding RNAs. Other gene transcripts also have cellular functions, but as regulatory noncoding RNAs. For example, ~23 nucleotide (nt)-long small interfering RNAs (siRNAs) are loaded into Argonaute (AGO) proteins. The resultant ribonucleoprotein assembly, the RNA-induced silencing complex (RISC), cleaves RNAs that are extensively base-paired with the loaded siRNA. To date, base complementarity is recognized as the major determinant of specific target cleavage (or slicing), but little is known about how AGO inspects base pairing before cleavage. A hallmark of AGO proteins is their bilobal structure, but despite the significance of this structure for curtailing slicing activity against mismatched targets, the molecular mechanism remains elusive. Here, our structural and functional studies of a bilobed yeast AGO protein and its isolated catalytic C-terminal lobe (C-lobe) revealed that the C-lobe alone retains almost all properties of bilobed AGO: siRNA-duplex loading, passenger cleavage/ejection, and siRNA-dependent RNA cleavage. Our crystal structure revealed that the catalytic C-lobe mirrors the bilobed AGO in terms of guide-RNA recognition and that all requirements for transitioning to the catalytically active conformation reside in the C-lobe. Nevertheless, we found that in the absence of the N-terminal lobe (N-lobe), target RNAs are scanned for complementarity using only a partial region of the RNA before endonucleolytic cleavage, thereby allowing for some off-target cleavage. Of note, acquisition of an N-lobe expanded the range of the guide RNA strand used for inspecting target complementarity to positions 2–23. These findings offer clues to the evolution of the bilobal structure of catalytically active AGO proteins. Our structure-function studies of the AGO C-lobe in comparison with its full-length parent construct revealed the high-specificity of yeast AGO and its meticulous investigation of targets before slicing. This property prompted us to further explore the utility of AGO as a laboratory tool. The identification and development of RNA-guided enzymes for programmable cleavage of target nucleic acids offers exciting possibilities for both therapeutic and biotechnological applications. However, critical challenges such as expensive guide RNAs and inability to predict the efficiency of target recognition, especially for highly-structured RNAs, remain to be addressed. We introduce a programmable RNA restriction enzyme, based on a budding yeast AGO, programmed with cost-effective single-stranded (ss) DNAs as guides. DNA guides offer the advantage that diverse sequences can be easily designed and purchased, enabling high-throughput screening to identify optimal recognition sites in the target RNA. Using this DNA-induced slicing complex (DISC) programmed with DNAs designed to span the sequence, sites of cleavage were identified in the 352-nt human immunodeficiency virus type 1 5'-untranslated region. Comparison between DISC cleavage and Ribonuclease H (RNase H) cleavage reveals that DISC not only cleaves solvent-exposed sites, but also sites that become more accessible upon DISC binding. This study demonstrates the advantages of the DISC system for programmable cleavage of highly-structured, functional RNAs. Collectively, the structural and functional analyses of yeast AGO provide insights into the molecular mechanism of how AGO ensures high-specificity for its targets. The knowledge gained during our study motivated us to employ AGO for new in vitro applications as an RNA restriction enzyme. The success of programmable RNA restriction would offer new and exciting opportunities for technological and therapeutic applications.
Kotaro Nakanishi (Advisor)
Charles Bell (Committee Member)
Michael Ibba (Committee Member)
Daniel Schoenberg (Committee Member)
185 p.

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Citations

  • Dayeh, Dayeh, D. M. (2018). Mechanistic insights into the slicing specificity of Argonaute and development of a programmable RNA endonuclease [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1543438392875464

    APA Style (7th edition)

  • Dayeh, Dayeh, Daniel . Mechanistic insights into the slicing specificity of Argonaute and development of a programmable RNA endonuclease. 2018. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1543438392875464.

    MLA Style (8th edition)

  • Dayeh, Dayeh, Daniel . "Mechanistic insights into the slicing specificity of Argonaute and development of a programmable RNA endonuclease." Doctoral dissertation, Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1543438392875464

    Chicago Manual of Style (17th edition)