Biology subjects

Ashe, M. P.

Publications and source records attributed to Ashe, M. P..

7 recordsLinked to original sources

bioRxiv2025

Genomic foundation models reveal RNA-binding proteins regulating translation and mRNA degradation during Drosophila development

BackgroundThe regulation of mRNA decay and translation is crucial for cellular function and development; however, the complex interplay of RNA-binding proteins (RBPs) regulating these processes remains incompletely understood. Recent advances in genomic foundation models present new opportunities for decoding the regulatory grammar embedded within mRNA untranslated regions (UTRs). Here, we leverage explainable artificial intelligence to systematically identify RBP motifs that influence translation and mRNA decay during Drosophila melanogaster development. ResultsWe extended the training of GENA-LM Fly, a genomic foundation model, on all 5 and 3 UTR pairs from the D. melanogaster genome. We separately fine-tuned it using ribosome density (RD) and mRNA decay (half-life) data from the embryonic maternal-to-zygotic transition (MZT). Using SHapley Additive exPlanations (SHAP) analysis, we identified the sequence regions most influential for prediction and performed motif enrichment analysis to discover associated RBP binding sites. We identified 42 unique RBPs associated with increasing (n=23; e.g., Rnp4f, Mxt) or decreasing (n=19; e.g., Aret/Bruno, Rox8, Sxl, Orb2) RD and 18 unique RBPs associated with increasing (n=6) and decreasing (n=12; e.g., Cnot4, Rbp9, Rox8) mRNA half-life. Using publicly available PAR-CLIP data, we validated our Orb2 signal in a Drosophila cell line. Furthermore, feature ablation and shuffling experiments revealed the contributions of different sequence components to model performance. Our approach significantly outperformed naive high-versus-low RD comparisons, demonstrating the power of model explainability in biological discovery. ConclusionsThis study demonstrates that genomic foundation models, when combined with explainability methods, can discover meaningful biology even without drastically improving the underlying prediction accuracy. The identified RBP motifs provide new insights into post-transcriptional regulatory elements that govern RD and decay during early development.

bioinformatics↗

bioRxiv2025

Eukaryotic translation initiation factor 3d regulates stress granule assembly via its RNA binding domain

Stress granules are cytoplasmic mRNA-protein complexes that form by liquid-liquid phase separation in response to a variety of stresses. Their assembly is contingent upon the inhibition of mRNA translation. Depending on the type of stress and severity, they can promote stress resistance or act to reduce cellular fitness. As such, stress granules are implicated in ageing and a range of related pathologies. Many translation factors are components of stress granules, but it is unclear how they contribute to granule assembly. Here we show that the eIF3d component of the eIF3 translation initiation complex is recruited to stress granules in human cells and is required for stress granule assembly in response to specific stresses. The RNA-binding domain of eIF3d mediates its recruitment to stress granules and deletion of this domain blocks granule formation and decreases cell viability. Furthermore, the exogenous expression of just the eIF3d RNA-binding domain can rescue stress granule assembly in eIF3d-depleted cells. We confirmed the importance of eIF3d for robust stress granule assembly in vivo using the nematode worm C. elegans. This study demonstrates that eIF3d is a critical evolutionary conserved stress granule assembly factor, rather than simply coalescing passively into stress granules following the inhibition of translation initiation.

cell biology↗

bioRxiv2025

Exploiting the SunTag system to study the developmental regulation of mRNA translation

The ability to quantitatively study mRNA translation using SunTag imaging is transforming our understanding of the translation process. Here, we expand the SunTag method to study new aspects of translation regulation in Drosophila. Repression of the maternal hunchback (hb) mRNA in the posterior of the Drosophila embryo is a textbook example of translational control. Using SunTag imaging to quantitate translation of maternal SunTag-hb mRNAs, we show that repression in the posterior is leaky as [~]5% of SunTag-hb mRNAs are translated. In the anterior of the embryo, the maternal and zygotic SunTag-hb mRNAs show similar translation efficiency despite having different UTRs. We demonstrate that the SunTag-hb mRNA can be used as a reporter to study ribosome pausing at single-mRNA resolution, by exploiting the conserved xbp1 mRNA and A60 pausing sequences. Finally, we adapt the detector component of the SunTag system to visualise and quantitate translation of the short gastrulation (sog) mRNA, encoding an essential secreted extracellular BMP regulator, at the endoplasmic reticulum in fixed and live embryos. Together, these tools will facilitate the future dissection of translation regulatory mechanisms during development.

developmental biology↗

bioRxiv2021

Cytosolic aspartate aminotransferase moonlights as a ribosome binding modulator of Gcn2 activity during oxidative stress

Regulation of translation is a fundamental facet of the cellular response to rapidly changing external conditions. Specific RNA-binding proteins (RBPs) co-ordinate the translational regulation of distinct mRNA cohorts during stress. To identify RBPs with previously under-appreciated roles in translational control, we used polysome profiling and mass spectrometry to identify and quantify proteins associated with translating ribosomes in unstressed yeast cells and during oxidative stress and amino acid starvation, which both induce the integrated stress response (ISR). Over 800 proteins were identified across polysome gradient fractions, including ribosomal proteins, translation factors and many others without previously described translation-related roles, including numerous metabolic enzymes. We identified variations in patterns of polysome enrichment in both unstressed and stressed cells and identified proteins enriched in heavy polysomes during stress. Genetic screening of polysome-enriched RBPs identified the cytosolic aspartate aminotransferase, Aat2, as a ribosome-associated protein whose deletion conferred growth sensitivity to oxidative stress. Loss of Aat2 caused aberrantly high activation of the ISR via enhanced eIF2 phosphorylation and GCN4 activation. Importantly, non-catalytic AAT2 mutants retained polysome association and did not show heightened stress sensitivity. Aat2 therefore has a separate ribosome-associated translational regulatory or moonlighting function that modulates the ISR independent of its aspartate aminotransferase activity.

biochemistry↗

bioRxiv2020

Integrated multi-omics reveals common properties underlying stress granule and P-body formation

Non-membrane-bound compartments such as P-bodies (PBs) and stress granules (SGs) play important roles in the regulation of gene expression following environmental stresses. We have systematically determined the protein and mRNA composition of PBs and SGs formed in response to a common stress condition imposed by glucose depletion. We find that high molecular weight (HMW) complexes exist prior to glucose depletion that may act as seeds for the further condensation of proteins forming mature PBs and SGs. Both before and after glucose depletion, these HMW complexes are enriched for proteins containing low complexity and RNA binding domains. The mRNA content of these HMW complexes is enriched for long, structured mRNAs that become more poorly translated following glucose depletion. Many proteins and mRNAs are shared between PBs and SGs including several multivalent RNA binding proteins that may promote condensate interactions during liquid-liquid phase separation. Even where the precise identity of mRNAs and proteins localizing to PBs and SGs is distinct, the mRNAs and proteins share common biophysical and chemical features that likely trigger their phase separation.

biochemistry↗

bioRxiv2019

CoFe granules focus coordinated glycolytic mRNA localization and translation to fuel glucose fermentation

Glycolysis is a fundamental metabolic pathway for glucose catabolism across biology, and glycolytic enzymes are amongst the most abundant proteins in cells. Their expression at such levels provides a particular challenge. Here we demonstrate that the glycolytic mRNAs are localized to granules in yeast and human cells. Detailed live cell and smFISH studies in yeast show that the mRNAs are actively translated in granules, and this translation appears critical for the localization. Furthermore, this arrangement is likely to facilitate the higher level organisation and control of the glycolytic pathway. Indeed, the degree of fermentation required by cells is intrinsically connected to the extent of mRNA localization to granules. On this basis, we term these granules, Core Fermentation (CoFe) granules; they appear to represent translation factories allowing high-level co-ordinated enzyme synthesis for a critical metabolic pathway.

cell biology↗

bioRxiv2018

Translation factor mRNA granules direct protein synthetic capacity to regions of polarized growth

mRNA localization serves key functions in localized protein production making it critical that the translation machinery itself is present at these locations. Here we show that translation factor mRNAs are localized to distinct granules within yeast cells. In contrast to many mRNP granules, such as P-bodies and stress granules, which contain translationally repressed mRNAs, these granules harbor translated mRNAs under active growth conditions. The granules require Pablp for their integrity and are inherited by developing daughter cells in a She2p/ She3p dependent manner. These results point to a model where roughly half the mRNA for certain translation factors are specifically directed in granules toward the tip of the developing daughter cell where protein synthesis is most heavily required, which has particular implications for filamentous forms of growth. Such a feedforward mechanism would ensure adequate provision of the translation machinery where it is to be needed most over the coming growth cycle. SummaryThis study shows that mRNAs encoding a range of translation factors are localized to granules that get transported into the yeast daughter cell using the She2p/She3p machinery. This likely supports an intensification of protein synthetic activity to facilitate apical polarized growth.

cell biology↗