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Principal Investigator: Kotaro Nakanishi
Organization: OHIO STATE UNIVERSITY
Fiscal Year: 2024
Award: $316,592
Funding agency: National Institute of General Medical Sciences
PROJECT SUMMARY
In eukaryotic cells, gene expression is regulated at multiple levels, including post-transcriptional gene
silencing, where microRNAs (miRNAs) bind to complementary target RNAs and cause translational repression.
Argonaute (AGO) proteins and miRNAs form RNA-induced silencing complexes (RISCs), the core players in
gene silencing. Humans have four AGO proteins, AGO1-4, which share a high sequence identity, and the
majority of miRNAs bound are common across all AGOs. Therefore, it has been thought that the four AGOs
work redundantly. Nevertheless, an increasing number of studies have found that each AGO has its unique
roles in various biological processes and diseases in addition to gene silencing. Although the interaction of all
four AGOs with miRNAs has been well characterized, little is known about how each RISC recognizes its target
RNAs. Elucidation of this recognition will provide insight into the unique roles of each AGO. Meanwhile,
characterization of RISC and target interactions will facilitate target prediction accuracy by improving prediction
algorithms, which will take account of not only the complementarity between guide and target but also the type
of AGO and target interaction. In this proposed study, we will pursue the following specific aims. In Aim 1, we
will use cryo-electron microscopy and X-ray crystallography to determine the structures of all four
homogenously purified RISCs with the same guide and target RNAs, which will provide insight into the
differences in target recognition by the four AGOs. In Aim 2, to clarify these differences, we recently developed
a novel SHAPE-based technique which allows us to visualize the conformational dynamics of target RNA bound
to RISC. The method will enable us to characterize this interaction within the RISC binding channel and its
periphery at a single-nucleotide resolution and can be expanded to understand how RISCs recognize guide-
binding sites buried within highly structured target RNAs. In Aim 3, we will first use mass spectrometry to identify
the unique protein binding partners of each AGO and their specific sites of interaction. Then, we will use tandem
immunoprecipitation, followed by RNA sequencing, to determine how the binding of these proteins influences
the target specificity of each AGO and directs their functionality towards alternative cellular events. The
outcome from this study will provide a solid foundation for fields beyond gene silencing and enable the
development of new strategies for higher accuracy guide-RNA drug design in therapeutic applications.
Terms: <5' Untranslated Regions><5'UTR><AIDS Virus><Acquired Immune Deficiency Syndrome Virus><Acquired Immunodeficiency Syndrome Virus><Acylation><Algorithms><Amino Acids><Anti-viral Response><Assay><Binding><Binding Proteins><Binding Sites><Bioassay><Biological Assay><Biological Function><Biological Process><Cancers><Cell Body><Cells><Chimera><Chimera organism><Combining Site><Complex><Cryo-electron Microscopy><Cryoelectron Microscopy><Customized Drugs><Designer Drugs><Development><Disease><Disorder><Dissociation><Drug Design><Electron Cryomicroscopy><Equipment and supply inventories><Eukaryotic Cell><Event><Foundations><Funding><Gene Expression><Gene Inactivation><Gene Silencing><Goals><Guide RNA><HIV><Human><Human Immunodeficiency Viruses><Hydroxyl><Hydroxyl Radical><IRES><Immune Precipitation><Immunoprecipitation><Individual><Internal Ribosome Entry Segment><Internal Ribosome Entry Site><Inventory><Investigation><Knowledge><LAV-HTLV-III><Ligand Binding Protein><Ligand Binding Protein Gene><Lymphadenopathy-Associated Virus><Malignant Neoplasms><Malignant Tumor><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Measures><Mediating><Messenger RNA><Methods><Micro RNA><MicroRNAs><Modern Man><Molecular><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><N-methylisatoic anhydride><Neural Development><Non-Polyadenylated RNA><Nucleotides><Outcome><Outcome Study><Post-Transcriptional Gene Silencing><Posttranscriptional Gene Silencing><Primer Extension><Property><Protein Binding><Proteins><RISC Multicomponent Nuclease><RNA><RNA Binding><RNA Gene Products><RNA Interference><RNA Seq><RNA Silencing><RNA bound><RNA purification><RNA sequencing><RNA-Induced Silencing Complex><RNAi><RNAseq><Reactive Site><Resolution><Ribonucleic Acid><Ribosome Entry Site><Role><Sequence-Specific Posttranscriptional Gene Silencing><Shapes><Single Crystal Diffraction><Site><Solid><Specificity><Structure><Techniques><Testing><Therapeutic><Translational Inhibition><Translational Repression><Translations><Virus-HIV><Visualization><Work><X Ray Crystallographies><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray/Neutron Crystallography><Xray Crystallography><aminoacid><base><bases><bound protein><chimeras><conformation><conformational><conformational state><conformationally><conformations><cryo-EM><cryoEM><cryogenic electron microscopy><design><designing><developmental><experiment><experimental research><experimental study><experiments><flexibility><flexible><gRNA><hydroxyl group><improved><insight><mRNA><mRNA Leader Sequences><malignancy><miRNA><miRNAs><mutant><neoplasm/cancer><neurodevelopment><new therapeutic approach><new therapeutic intervention><new therapeutic strategies><new therapy approaches><new treatment approach><new treatment strategy><novel><novel therapeutic approach><novel therapeutic intervention><novel therapeutic strategies><novel therapy approach><prediction algorithm><prevent><preventing><resolutions><scaffold><scaffolding><social role><transcriptional silencing><transcriptome sequencing><transcriptomic sequencing><translation>