Browsing by Author "Aoki, Scott Takeo"

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    Accurate in silico predictions of modified RNA interactions to a prototypical RNA-binding protein with λ-dynamics
    (bioRxiv, 2024-12-11) Angelo, Murphy; Bhargava, Yash; Kierzek, Elzbieta; Kierzek, Ryszard; Hayes, Ryan L.; Zhang, Wen; Vilseck, Jonah Z.; Aoki, Scott Takeo; Biochemistry and Molecular Biology, School of Medicine
    RNA-binding proteins shape biology through their widespread functions in RNA biochemistry. Their function requires the recognition of specific RNA motifs for targeted binding. These RNA binding elements can be composed of both unmodified and chemically modified RNAs, of which over 170 chemical modifications have been identified in biology. Unmodified RNA sequence preferences for RNA-binding proteins have been widely studied, with numerous methods available to identify their preferred sequence motifs. However, only a few techniques can detect preferred RNA modifications, and no current method can comprehensively screen the vast array of hundreds of natural RNA modifications. Prior work demonstrated that λ-dynamics is an accurate in silico method to predict RNA base binding preferences of an RNA-binding antibody. This work extends that effort by using λ-dynamics to predict unmodified and modified RNA binding preferences of human Pumilio, a prototypical RNA binding protein. A library of RNA modifications was screened at eight nucleotide positions along the RNA to identify modifications predicted to affect Pumilio binding. Computed binding affinities were compared with experimental data to reveal high predictive accuracy. In silico force field accuracies were also evaluated between CHARMM and Amber RNA force fields to determine the best parameter set to use in binding calculations. This work demonstrates that λ-dynamics can predict RNA interactions to a bona fide RNA-binding protein without the requirements of chemical reagents or new methods to experimentally test binding at the bench. Advancing in silico methods like λ-dynamics will unlock new frontiers in understanding how RNA modifications shape RNA biochemistry.
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    C. elegans germ granules require both assembly and localized regulators for mRNA repression
    (Springer Nature, 2021-02-12) Aoki, Scott Takeo; Lynch, Tina R.; Crittenden, Sarah L.; Bingman, Craig A.; Wickens, Marvin; Kimble, Judith; Biochemistry and Molecular Biology, School of Medicine
    Cytoplasmic RNA–protein (RNP) granules have diverse biophysical properties, from liquid to solid, and play enigmatic roles in RNA metabolism. Nematode P granules are paradigmatic liquid droplet granules and central to germ cell development. Here we analyze a key P granule scaffolding protein, PGL-1, to investigate the functional relationship between P granule assembly and function. Using a protein–RNA tethering assay, we find that reporter mRNA expression is repressed when recruited to PGL-1. We determine the crystal structure of the PGL-1 N-terminal region to 1.5 Å, discover its dimerization, and identify key residues at the dimer interface. Mutations of those interface residues prevent P granule assembly in vivo, de-repress PGL-1 tethered mRNA, and reduce fertility. Therefore, PGL-1 dimerization lies at the heart of both P granule assembly and function. Finally, we identify the P granule-associated Argonaute WAGO-1 as crucial for repression of PGL-1 tethered mRNA. We conclude that P granule function requires both assembly and localized regulators.