Browsing by Author "Aoki, Scott T."

Now showing 1 - 6 of 6
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    An improved in vivo tethering assay with single molecule FISH reveals that a nematode Nanos enhances reporter expression and mRNA stability
    (RNA Society, 2021) Doenier, Jonathan; Lynch, Tina R.; Kimble, Judith; Aoki, Scott T.; Biochemistry and Molecular Biology, School of Medicine
    Robust methods are critical for testing the in vivo regulatory mechanism of RNA binding proteins. Here we report improvement of a protein–mRNA tethering assay to probe the function of an RNA binding protein in its natural context within the C. elegans adult germline. The assay relies on a dual reporter expressing two mRNAs from a single promoter and resolved by trans-splicing. The gfp reporter 3′UTR harbors functional binding elements for λN22 peptide, while the mCherry reporter 3′UTR carries mutated nonfunctional elements. This strategy enables internally controlled quantitation of reporter protein by immunofluorescence and mRNA by smFISH. To test the new system, we analyzed a C. elegans Nanos protein, NOS-3, which serves as a post-transcriptional regulator of germ cell fate. Unexpectedly, tethered NOS-3 enhanced reporter expression. We confirmed this enhancement activity with a second reporter engineered at an endogenous germline gene. NOS-3 enhancement of reporter expression was associated with its amino-terminal intrinsically disordered region, not its carboxy-terminal zinc fingers. RNA quantitation revealed that tethered NOS-3 enhances stability of the reporter mRNA. We suggest that this direct NOS-3 enhancement activity may explain a paradox: Classically Nanos proteins are expected to repress RNA, but nos-3 had been found to promote gld-1 expression, an effect that could be direct. Regardless, the new dual reporter dramatically improves in situ quantitation of reporter expression after RNA binding protein tethering to determine its molecular mechanism in a multicellular tissue.
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    Developing Novel Methods to Identify RNA-Associated Mechanisms for Inheritance
    (2020-11) Ettaki, Zacharia Nabil; Aoki, Scott T.; Georgiadis, Millie; Quilliam, Lawrence
    Animals depend on inheriting non-genetic information early in life to grow and develop naturally. This inherited, non-genetic information was previously thought to be limited to DNA modifications and DNA binding proteins. But recent studies have expanded our understanding of inheritance to include RNA and RNA binding proteins. We currently lack methods to identify and enrich for RNA binding proteins that might be involved in providing non-genetic information from mother to daughter cells. Others have developed a method using modified enzyme tags to pulse-label proteins with small molecule fluorescent ligands and follow these proteins as they are inherited by cells. Here I characterized and tested the application of a fluorescent small molecule targeting antibody to enrich for these labeled proteins. I first tested the ability of this antibody to bind to fluorescent ligand-labeled enzymes. I determined that the antibody can efficiently bind to at least one of the labeled enzymes. Second, I determined crystallization conditions for the ligand binding antibody fragment. This thesis sets the stage for structure determination and to test whether this antibody can work in vivo to enrich for RNA binding proteins involved in the delivery of non-genetic information to cells.
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    Functional and Quantitative Mass Spectrometry-Based Approaches for Mapping the Lysine Methylome
    (2024-09) Berryhill, Christine Annette; Cornett, Evan M.; Aoki, Scott T.; Georgiadis, Millie M.; Mosley, Amber L.; Turchi, John J.
    Proteins are frequently modified with small chemical tags, or modifications, that play a key role in controlling their functions within the cell. One modification, lysine methylation, is found on thousands of human proteins and is added and removed by lysine methyltransferases (KMTs) and lysine demethylases (KDMs), respectively. Recognition of methylated lysines by specific reader proteins regulates downstream processes. Lysine methylation, KMTs, KDMs, and reader proteins therefore create a signaling network. Components of lysine methylation signaling networks are frequently dysregulated in human disease, but current methods to detect lysine methylation present barriers for understanding the impact an awry signaling network has on lysine methylation. In this study, we investigated the use of mass spectrometry (MS)-based proteomics to better detect and quantify both lysine methylation sites and methyl regulators across multiple samples. We investigated the sequence bias of commercially available pan-methyllysine antibodies using both a lysine-oriented peptide library and immunoprecipitation mass spectrometry. Our results showed that most antibodies have a preference for certain sequences. Furthermore, we observed that unenriched samples obtained the same number of identified lysine methylation sites as enriched samples. Following the establishment of an efficient and quantitative MS-based proteomics approach, we applied it to profile both lysine methylation and KDMs within breast cancer cell lines. Studies have repeatedly shown that components of the lysine methylation signaling network are overexpressed within breast cancer. Indeed, we characterized distinct lysine methylation and KDM patterns across the cell lines, suggesting the existence of different lysine methylation signaling. Given the ability to quantitatively profile lysine methylation, this work also characterized the impact of a compound known to disrupt the lysine methylation signaling network, 3-deazanplanocin A. The observed transcript, protein, and lysine methylation site abundance changes highlight how dysregulation of methyl mediators impacts lysine methylation and cellular signaling. Overall, we developed a reproducible pipeline that promises to enable a deeper understanding of how a dysregulated lysine methylation landscape influences cellular signaling and associated phenotypes.
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    In silico λ-dynamics predicts protein binding specificities to modified RNAs
    (bioRxiv, 2024-01-27) Angelo, Murphy; Zhang, Wen; Vilseck, Jonah Z.; Aoki, Scott T.; Biochemistry and Molecular Biology, School of Medicine
    RNA modifications shape gene expression through a smorgasbord of chemical changes to canonical RNA bases. Although numbering in the hundreds, only a few RNA modifications are well characterized, in part due to the absence of methods to identify modification sites. Antibodies remain a common tool to identify modified RNA and infer modification sites through straightforward applications. However, specificity issues can result in off-target binding and confound conclusions. This work utilizes in silico λ-dynamics to efficiently estimate binding free energy differences of modification-targeting antibodies between a variety of naturally occurring RNA modifications. Crystal structures of inosine and N6-methyladenosine (m6A) targeting antibodies bound to their modified ribonucleosides were determined and served as structural starting points. λ-Dynamics was utilized to predict RNA modifications that permit or inhibit binding to these antibodies. In vitro RNA-antibody binding assays supported the accuracy of these in silico results. High agreement between experimental and computed binding propensities demonstrated that λ-dynamics can serve as a predictive screen for antibody specificity against libraries of RNA modifications. More importantly, this strategy is an innovative way to elucidate how hundreds of known RNA modifications interact with biological molecules without the limitations imposed by in vitro or in vivo methodologies.
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    In silico λ-dynamics predicts protein binding specificities to modified RNAs
    (Oxford University Press, 2025) Angelo, Murphy; Zhang, Wen; Vilseck, Jonah Z.; Aoki, Scott T.; Biochemistry and Molecular Biology, School of Medicine
    RNA modifications shape gene expression through a variety of chemical changes to canonical RNA bases. Although numbering in the hundreds, only a few RNA modifications are well characterized, in part due to the absence of methods to identify modification sites. Antibodies remain a common tool to identify modified RNA and infer modification sites through straightforward applications. However, specificity issues can result in off-target binding and confound conclusions. This work utilizes in silico λ-dynamics to efficiently estimate binding free energy differences of modification-targeting antibodies between a variety of naturally occurring RNA modifications. Crystal structures of inosine and N6-methyladenosine (m6A) targeting antibodies bound to their modified ribonucleosides were determined and served as structural starting points. λ-Dynamics was utilized to predict RNA modifications that permit or inhibit binding to these antibodies. In vitro RNA-antibody binding assays supported the accuracy of these in silico results. High agreement between experimental and computed binding propensities demonstrated that λ-dynamics can serve as a predictive screen for antibody specificity against libraries of RNA modifications. More importantly, this strategy is an innovative way to elucidate how hundreds of known RNA modifications interact with biological molecules without the limitations imposed by in vitro or in vivo methodologies.
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    In vivo pulse-chase in C. elegans reveals intestinal histone turnover changes upon starvation
    (bioRxiv, 2025-02-16) Borchers, Christopher; Osburn, Kara; Roh, Hyun Cheol; Aoki, Scott T.; Biochemistry and Molecular Biology, School of Medicine
    The ability to study protein dynamics and function in the authentic context of a multicellular organism is paramount to better understand biological phenomena in animal health and disease. Pulse-chase of self-labeling fusion protein tags provide the opportunity to label proteins of interest and track those proteins over time. There are currently several challenges associated with performing in vivo protein pulse-chase in animals, such as cost, reproducibility, and accurate detection methods. The C. elegans model organism has attributes that alleviate many of these challenges. This work tests the feasibility of applying the Halo modified enzyme (HaloTag) for in vivo protein pulse-chase in C. elegans. HaloTag intestinal histone reporters were created in the worm and used to demonstrate that reporter protein could be efficiently pulse-labeled by soaking animals in ligand. Labeled protein stability could be monitored over time by fluorescent confocal microscopy. Further investigation revealed reporter protein stability was dependent on the animal's nutritional state. Chromatin Immunoprecipitation and sequencing (ChIP-seq) of the reporters showed incorporation in chromatin with little change hours into starvation, implying a lack of chromatin regulation at the time point tested. Collectively, this work presents a straightforward method to label and track proteins of interest in C. elegans that can address a multitude of biological questions surrounding protein stability and dynamics in this animal model.