Biology subjects

Vilseck, J. Z.

Publications and source records attributed to Vilseck, J. Z..

8 recordsLinked to original sources

bioRxiv2026

EXOSC3 G191 Variants Trigger System-Wide Recalibration of RNA Processing Machinery

Pathogenic variants in EXOSC3, a noncatalytic subunit of the RNA exosome, cause pontocerebellar hypoplasia type 1B (PCH1B), yet substantial variability in disease severity is observed among individuals carrying different EXOSC3 alleles. The molecular mechanisms of RNA exosome dysfunction in individuals carrying EXOSC3 p.G191 variants remains unresolved. To address this, we generated CRISPR/Cas9-engineered human cell models harboring EXOSC3 p.G191 variants and performed integrated transcriptomic, proteomic, and computational structural analyses. EXOSC3 p.G191 variants produced extensive, allele- and dosage-dependent alterations in gene expression and splicing, with heterozygous variants causing broad but attenuated disruption relative to homozygous EXOSC3 G191D/G191D cells. All EXOSC3 G191 variants promoted increased skipping of exon 3 in EXOSC3 transcripts, generating a short isoform predicted to encode an unstable proteoform. Molecular dynamics and {lambda}-dynamics simulations predicted substantial thermodynamic destabilization of all EXOSC3 G191 variant proteoforms, consistent with reduced protein abundance and thermal stability measured by global proteomics and PISA. At the protein complex level, EXOSC3 p.G191 variants were associated with coordinated decreases in all RNA exosome core subunits and the exonuclease EXOSC10, consistent with destabilization of RNA exosome assembly and orphan protein decay. In contrast, the catalytic exonuclease DIS3 and multiple independent RNA processing pathways were upregulated, indicating compensatory recalibration of RNA metabolism. Together, these findings link variant-induced alternative splicing, RNA exosome complex destabilization, and adaptive network responses to phenotypic variability in EXOSC3 p.G191-associated disease.

systems biology↗

bioRxiv2025

Quantifying the Structural and Energetic Consequences of EXOSC3 S1 Domain Variants from a Comparative Assessment of {lambda}-Dynamics with Two Charge-Changing Perturbation Strategies

Charge-changing perturbations are notoriously dificult to investigate with alchemical free energy calculations. The routine use of periodic boundary conditions and electrostatic approximations, such as particle-mesh Ewald (PME), may produce finite-size efect errors that become non-negligible as a perturbation changes a simulation cells net charge away from zero. Two prevalent strategies exist to correct for these errors: the analytic correction (AC) and co-alchemical ion (CI) methods. Both correction schemes have been found to produce comparable relative free energy results for small molecule perturbations, but these methods have not been compared using {lambda}-dynamics ({lambda}D) free energy calculations or for protein side chain mutations. Recently, we investigated relative folding and binding free energies ({Delta}{Delta}Gs) of a series of EXOSC3 variants involved in a rare neurodegenerative disorder, including D132A, G135R, and G191D charge-change perturbations, with a simplified AC scheme in {lambda}D. In this study, these perturbations are reevaluated with the CI scheme for comparison with AC to identify the best correction strategy for {lambda}D. The collected AC- and CI-corrected {Delta}{Delta}Gs show excellent agreement with a mean unsigned error of 0.4 kcal/mol. However, reduced sampling proficiency and increased dificulties of evaluating multisite perturbations with the CI method suggest that a simplified AC approach may be more generalizable for future {lambda}D calculations. Previously, the use of the CI approach with {lambda}D has been limited due to a lack of infrastructure available to users to simplify its more involved setup procedure. This study introduces an automated workflow for implementation of the CI approach with {lambda}D, laying the foundation for future comparisons between charge-change correction schemes. These studies facilitated analysis of the {lambda}D trajectories to identify structural changes within EXOSC3 and the RNA exosome complex that clearly rationalize the calculated {Delta}{Delta}Gs for the D132A, G135R, G191C, and G191D EXOSC3 variants, providing insight into potential disease-causing mechanisms of EXOSC3 modifications.

biophysics↗

bioRxiv2025

EXOSC3 S1-domain variants implicated in PCH1b alter RNA exosome cap subunit abundance and thermal stability disrupting rRNA processing and targeting of AU-rich mRNA

Missense variants in EXOSC3, an RNA exosome subunit, have been identified in patients with PCH1b. We investigated three missense variants in the S1 domain of EXOSC3, including one variant of uncertain significance (VUS) and two pathogenic variants (hence S1 variants). EXOSC3 S1 variant cell lines were generated using CRISPR-Cas9 resulting in widespread proteome changes including decreases in some RNA exosome subunits paired with increases in the catalytic subunit DIS3. Thermal stability, analyzed by PISA, revealed extensive destabilization of RNA exosome cap subunits and the cap-associated exonuclease EXOSC10. Functionally, S1 variants altered rRNA processing with corresponding protein compensation observed in rRNA processing proteins outside the RNA exosome. Exogenous overexpression of EXOSC3 rescues many molecular defects caused by S1 variants suggesting that protein destabilization and turnover strongly contribute to molecular defects. Overall, our findings define the mechanisms through which cells respond to EXOSC3 S1 variant disruption of RNA processing homeostasis.

systems biology↗

bioRxiv2024

Accurate in silico predictions of modified RNA interactions to a prototypical RNA-binding protein with {lambda}-dynamics

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 {lambda}-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 {lambda}-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 {lambda}-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 {lambda}-dynamics will unlock new frontiers in understanding how RNA modifications shape RNA biochemistry.

biochemistry↗

bioRxiv2024

A {lambda}-dynamics investigation of insulin Wakayama and other A3 variant binding affinities to the insulin receptor

Insulin Wakayama is a clinical insulin variant where a conserved valine at the third residue on insulins A chain (ValA3) is replaced with a leucine (LeuA3), impairing insulin receptor (IR) binding by 140-500 fold. This severe impact on binding from such a subtle modification has posed an intriguing problem for decades. Although experimental investigations of natural and unnatural A3 mutations have highlighted the sensitivity of insulin-IR binding to minor changes at this site, an atomistic explanation of these binding trends has remained elusive. We investigate this problem computationally using {lambda}-dynamics free energy calculations to model structural changes in response to perturbations of the ValA3 side chain and to calculate associated relative changes in binding free energy ({Delta}{Delta}Gbind). The Wakayama LeuA3 mutation and seven other A3 substitutions were studied in this work. The calculated {Delta}{Delta}Gbind results showed high agreement compared to experimental binding potencies with a Pearson correlation of 0.88 and a mean unsigned error of 0.68 kcal/mol. Extensive structural analyses of {lambda}-dynamics trajectories revealed that critical interactions were disrupted between insulin and the insulin receptor as a result of the A3 mutations. This investigation also quantifies the effect that adding an A3 C{delta} atom or losing an A3 C{gamma} atom has on insulins binding affinity to the IR. Thus, {lambda}-dynamics was able to successfully model the effects of subtle modifications to insulins A3 side chain on its protein-protein interactions with the IR and shed new light on a decades-old mystery: the exquisite sensitivity of hormone-receptor binding to a subtle modification of an invariant insulin residue. SIGNIFICANCE STATEMENTThis work addresses a decades-old question of how subtle modifications to insulins A3 side chain affects its binding affinity to the insulin receptor. {lambda}-Dynamics computed free energies of binding match experimental activity trends with high accuracy. Atomistic insights into hormone-receptor protein-protein interactions were obtained through a detailed investigation of {lambda}-dynamic trajectories. This work quantifies the effects of adding and removing atoms to insulins conserved A3 residue and identifies clear conformational preferences for insulin A3 residues when bound to the insulin receptor.

biophysics↗

bioRxiv2024

In silico {lambda}-dynamics predicts protein binding specificities to modified RNAs

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 {lambda}-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. {lambda}-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 {lambda}-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.

biochemistry↗

bioRxiv2023

Chemically induced partial unfolding of the multifunctional Apurinic/apyrimidinic endonuclease 1

Apurinic/apyrimidinic endonuclease I (APE1) acts as both an endonuclease and a redox factor to ensure cell survival. The two activities require different conformations of APE1. As an endonuclease, APE1 is fully folded. As a redox factor, APE1 must be partially unfolded to expose the buried residue Cys65, which reduces transcription factors including AP-1, NF-{kappa}B, and HIF-1 and thereby enables them to bind DNA. To determine a molecular basis for partial unfolding associated with APE1s redox activity, we characterized specific interactions of a known redox inhibitor APX3330 with APE1 through waterLOGSY and 1H-15N HSQC NMR approaches using ethanol and acetonitrile as co-solvents. We find that APX3330 binds to the endonuclease active site in both co-solvents and to a distant small pocket in acetonitrile. Prolonged exposure of APE1 with APX3330 in acetonitrile resulted in a time-dependent loss of 1H-15N HSQC chemical shifts ([~]35%), consistent with partial unfolding. Regions that are partially unfolded include adjacent N- and C-terminal beta strands within one of the two sheets comprising the core, which converge within the small binding pocket defined by the CSPs. Removal of APX3330 via dialysis resulted in a slow reappearance of the 1H-15N HSQC chemical shifts suggesting that the effect of APX3330 is reversible. APX3330 significantly decreases the melting temperature of APE1 but has no effect on endonuclease activity using a standard assay in either co-solvent. Our results provide insights on reversible partial unfolding of APE1 relevant for its redox function as well as the mechanism of redox inhibition by APX3330. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/547112v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@13f9fe2org.highwire.dtl.DTLVardef@19cbcborg.highwire.dtl.DTLVardef@1927c7aorg.highwire.dtl.DTLVardef@2a3cc0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Substrate selectivity of the PRDM9 lysine methyltransferase domain.

Lysine methylation is a dynamic, post-translational mark that regulates the function of histone and non-histone proteins. Many of the enzymes that mediate lysine methylation, known as lysine methyltransferases (KMTs), were originally identified to modify histone proteins but have also been discovered to methylate non-histone proteins. In this work, we investigate the substrate selectivity of the lysine methyltransferase PRDM9 to identify both potential histone and non-histone substrates. Though normally expressed in germ cells, PRDM9 is significantly upregulated across many cancer types. The methyltransferase activity of PRDM9 is essential for double-strand break formation during meiotic recombination. PRDM9 has been reported to methylate histone H3 at lysine residues 4 and 36; however, PRDM9 KMT activity had not previously been evaluated on non-histone proteins. Using lysine-oriented peptide (K-OPL) libraries to screen potential substrates of PRDM9, we determined that PRDM9 preferentially methylates peptide sequences not found in any histone protein. We confirmed PRDM9 selectivity through in vitro KMT reactions using peptides with substitutions at critical positions. A multisite {lambda}-dynamics computational analysis provided a structural rationale for the observed PRDM9 selectivity. The substrate selectivity profile was then used to identify putative non-histone substrates, which were tested by peptide spot array. Finally, PRDM9 methylation non-histone substrates were validated at the protein level by in vitro KMT assays on recombinant proteins. The selectivity profile of PRDM9 will be useful in identifying putative PRDM9 substrates in different cellular contexts, and future studies are required to determine whether PRDM9 methylates non-histone proteins in the context of meiotic recombination or cancer.

biochemistry↗