Biology subjects

O'Leary, S. E.

Publications and source records attributed to O'Leary, S. E..

7 recordsLinked to original sources

bioRxiv2025

Dynamics and Regulation of mRNA Cap Recognition by Human eIF4F

Efficient eukaryotic messenger RNA translation requires dynamic collaboration between the three subunits of initiation factor 4F (eIF4F, eIF4E*G*A), which recognises and activates mRNA at its 5' cap structure for ribosome recruitment. Despite its high biological and pharmacological importance, the dynamics of full human eIF4F-mRNA engagement remain largely uncharacterised, hindering mechanistic understanding of translation initiation and its regulation. Here we observed human eIF4F activity with single-molecule fluorescence assays that directly visualise mRNA cap recognition by its eIF4E subunit. Unexpectedly, we find that inherently transient eIF4E-cap binding is repressed by full-length human eIF4G, predominantly through its C-terminus, representing an unanticipated role for eIF4G as a central rate-limiting factor in the eIF4F complex. This repression is relieved by nucleotide-bound eIF4A in the eIF4F heterotrimer, placing eIF4A as a crucial determinant of efficient cap recognition for translation. Molecular dynamics simulations reveal that electrostatic modulation of eIF4E-mRNA interaction allows eIF4G to control the cap-recognition frequency. Our findings also indicate that intrinsic eIF4F- mRNA dynamics are insufficient to support cap-tethered ribosomal scanning to locate translation start sites. They illuminate fundamental design-principle differences for the overall mechanism and division of labour among eIF4F subunits during mRNA recognition in humans and yeast.

biophysics↗

bioRxiv2025

Genome-Wide Interrogation of SARS-CoV-2 RNA-Protein Interactions Uncovers Hidden Regulatory Sites

The global impact of the COVID-19 pandemic underscores the critical need for a comprehensive understanding of SARS-CoV-2 replication mechanisms. While the central roles of the RNA dependent RNA polymerase (NSP12), primase protein (NSP8), and nucleocapsid protein (N) in the virus life cycle are extensively studied, the precise nature of their interactions with the full-length viral RNA genome remain incompletely characterized. In this study, we sought to address this knowledge gap by employing enhanced crosslinking and immunoprecipitation (eCLIP) to map the binding sites of NSP8, NSP12, and N proteins across the SARS-CoV-2 genome at early stages of viral RNA and protein synthesis and late stages of virion assembly. Our findings revealed interactions of NSP8 and NSP12 to the 5 and 3 untranslated regions (UTRs) of both positive and negative sense RNA, regions known to regulate viral replication, transcription, and translation. We identified a surprising and essential NSP12 binding site within the RNA sequence encoding the conserved Y1 domain of NSP3, which regulates RNA abundance upstream of the site. Additionally, we found that N protein interacts with the 5 UTR and influences translation efficiency. Finally, we report a novel regulatory function of N protein in modulating ribosomal frameshifting proximal to the frameshift element, a crucial process for maintaining viral protein stoichiometry. Our results provide a detailed molecular map of SARS-CoV-2 protein-RNA interactions, revealing potential therapeutic targets for attenuating viral fitness and informing the development of next-generation antiviral strategies.

molecular biology↗

bioRxiv2023

Single-Molecule Tracking Reveals Dynamic Regulation of Ribosomal Scanning

To initiate protein synthesis, the eukaryotic ribosomal pre-initiation complex must survey a messenger RNA leader sequence to identify the correct start codon.1 This pre-initiation complex motion through the leader, termed scanning, is coordinated by an intricate and highly-dynamic assemblage of translation factors, mRNA, initiator tRNA, and the small ribosomal subunit.2,3,4 Fundamental aspects of scanning dynamics remain poorly understood: estimates of its rate vary widely, and mechanisms that establish and regulate the motion remain largely unknown. Here we show, at the single-molecule level, that the Saccharomyces cerevisiae pre-initiation complex scans a diverse set of mRNA leaders at a rate of 10 - 20 nt s-1. Our data quantitatively support a scanning mechanism in which the mRNA leader is inspected base by base, essentially unidirectionally, and with modest sensitivity to mRNA structure. Unexpectedly, scanning bypasses canonical start sites where the initiator tRNA is present but GTP hydrolysis in the pre-initiation complex is impaired. Conversely, binding of the S. cerevisiae poly(A)-binding protein Pab1p to its own mRNA leader hinders scanning in a concentration-dependent manner. At saturating, physiological concentrations, Pab1p prolongs scanning by more than four-fold, evoking an autoregulation mechanism for translation initiation. Our data provide a real-time mechanistic framework for scanning regulation and energetics.

biophysics↗

bioRxiv2021

mRNA- and Factor-Driven Dynamic Variability Controls eIF4F-Cap Recognition for Translation Initiation.

mRNA 5' cap recognition by eIF4F is a key step in eukaryotic translational control. While different mRNAs respond differently to eIF4F-directed regulation, the molecular basis for this variability remains unclear. We developed single-molecule fluorescence assays to directly observe eIF4F- mRNA interactions. We uncovered a complex interplay of mRNA features with factor activities that differentiates cap recognition between mRNAs. eIF4E-cap association rates are anticorrelated with mRNA length. eIF4A leverages ATP binding to differentially accelerate eIF4E- mRNA association; the extent of this acceleration correlates with translation efficiency in vivo. eIF4G lengthens eIF4E-cap binding to persist on the initiation timescale. The full eIF4F complex discriminates between mRNAs in an ATP-dependent manner. After eIF4F-mRNA binding, eIF4E is ejected from the cap by eIF4A ATP hydrolysis. Our results suggest features throughout mRNA coordinate in controlling cap recognition at the 5' end, and suggest a model for how eIF4F-mRNA dynamics establish mRNA sensitivity to translational control processes.

biophysics↗

bioRxiv2021

Single-Molecule Dynamics of SARS-CoV-2 5' Cap Recognition by Human eIF4F

Coronaviruses initiate translation through recognition of the viral RNA 5 m7GpppAm cap by translation factor eIF4F. eIF4F is a heterotrimeric protein complex with cap-binding, RNA-binding, and RNA helicase activities. Modulating eIF4F function through cellular regulation or small-molecule inhibition impacts coronavirus replication, including for SARS-CoV-2. Translation initiation involves highly coordinated dynamics of translation factors with messenger or viral RNA. However, how the eIF4F subunits coordinate on the initiation timescale to define cap-binding efficiency remains incompletely understood. Here we report that translation supported by the SARS-CoV-2 5-UTR is highly sensitive to eIF4A inhibition by rocaglamide. Through a single-molecule fluorescence approach that reports on eIF4E-cap interaction, we dissect how eIF4F subunits contribute to cap-recognition efficiency on the SARS-CoV-2 5 UTR. We find that free eIF4A enhances cap accessibility for eIF4E binding, but eIF4G alone does not change the kinetics of eIF4E-RNA interaction. Conversely, formation of the full eIF4F complex significantly alters eIF4E-cap interaction, suggesting that coordinated eIF4E and eIF4A activities establish the net eIF4F-cap recognition efficiency. Moreover, the eIF4F complex formed with phosphomimetic eIF4E(S209D) binds the viral UTR more efficiently than with wild-type eIF4E. These results highlight a dynamic interplay of eIF4F subunits and mRNA that determines cap-recognition efficiency.

biophysics↗

bioRxiv2020

Direct readout of heterochromatic H3K9me3 regulates DNMT1-mediated maintenance DNA methylation

In mammals, repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3), frequently coexist with DNA methylation, producing a more stable and silenced chromatin state. However, it remains elusive how these epigenetic modifications crosstalk. Here, through structural and biochemical characterizations, we identified the replication foci targeting sequence (RFTS) domain of maintenance DNA methyltransferase DNMT1, a module known to bind the ubiquitylated H3 (H3Ub), as a specific reader for H3K9me3/H3Ub, with the recognition mode distinct from the typical trimethyl-lysine reader. Disruption of the interaction between RFTS and the H3K9me3Ub affects the localization of DNMT1 in stem cells and profoundly impairs the global DNA methylation and genomic stability. Together, this study reveals a previously unappreciated pathway through which H3K9me3 directly reinforces DNMT1-mediated maintenance DNA methylation.

genetics↗

bioRxiv2019

TANGLED1 mediates interactions between microtubules that may promote spindle organization and phragmoplast guidance to the division site in maize

The microtubule cytoskeleton serves as a dynamic structural framework for mitosis in eukaryotic cells. TANGLED1 (TAN1) is a microtubule-binding protein that localizes to the division site and mitotic microtubules and plays a critical role in division plane orientation in plants. Here, in vitro experiments demonstrate that TAN1 directly binds microtubules, mediating microtubule zippering or end-on microtubule interactions, depending on their contact angle. Maize tan1 mutant cells improperly position the preprophase band (PPB), which predicts the future division site. However, cell-shape-based modeling indicates that PPB positioning defects are likely a consequence of abnormal cell shapes and not due to TAN1 absence. Spindle defects in the tan1 mutant suggest that TAN1-mediated microtubule zippering may contribute to metaphase spindle organization. In telophase, co-localization of growing microtubules ends from the phragmoplast with TAN1 at the division site suggests that TAN1 interacts with microtubule tips end-on. Together, our results suggest that TAN1 contributes to spindle and phragmoplast microtubule organization to ensure proper division plane orientation.

plant biology↗