Biology subjects

Hodge, K.

Publications and source records attributed to Hodge, K..

6 recordsLinked to original sources

bioRxiv2025

The differential mechanisms of eIF4A1-mediated translational activation instructed by distinct RNA features

All mRNAs require eukaryotic translation initiation factor (eIF) 4A1 for translation through its different functions: loading of the pre-initiation complex onto mRNAs and unwinding of RNA structure. eIF4A1 is the catalytic subunit of the cap- binding eIF4F complex and presumed to select the mRNAs for translation through these activities, instructed by signalling pathways driving cell fate. The mechanisms underlying activation of the distinct eIF4A1 functions and establish translational selectivity are unknown. Here, we have unravelled the complexity of mRNA selection by eIF4A1. We have mechanistically characterised the biological and atomic basis of inhibition by gain- and loss-of-function eIF4A1-inhibitors eFT226 and hippuristanol, and used machine learning to model the mRNA features associated with specific inhibition. This uncovered the eIF4A1 function - mRNA sequence relationship: 5UTRs containing C/CG-rich require efficient mRNA loading by eIF4F which is specifically targeted by hippuristanol, while 5UTRs harbouring alternatives starts sites together with AG-rich motifs utilise eIF4A1 for start site selection, specifically perturbed by eFT226. Our model is validated through a massively parallel reporter assay using 5UTRs from a distinct evolutionary origin. This prompted us to examine the conservation between mRNA sequence and eIF4A1 function, and revealed their co- development. Our findings highlight opportunities for novel therapeutic strategies targeting eIF4A1 and for improved design of mRNA-based therapeutics.

cell biology↗

bioRxiv2023

Engineered immunomodulatory extracellular vesicles derived from epithelial cells acquire capacity for positive and negative T cell co-stimulation in cancer and autoimmunity

Extracellular vesicles (EVs) are generated by all cells and systemic administration of allogenic EVs derived from epithelial and mesenchymal cells have been shown to be safe, despite carrying an array of functional molecules, including thousands of proteins. To address whether epithelial cells derived EVs can be modified to acquire the capacity to induce immune response, we engineered 293T EVs to harbor the immunomodulatory CD80, OX40L and PD-L1 molecules. We demonstrated abundant levels of these proteins on the engineered cells and EVs. Functionally, the engineered EVs efficiently elicit positive and negative co-stimulation in human and murine T cells. In the setting of cancer and auto-immune hepatitis, the engineered EVs modulate T cell functions and alter disease progression. Moreover, OX40L EVs provide additional benefit to anti-CTLA-4 treatment in melanoma-bearing mice. Our work provides evidence that epithelial cell derived EVs can be engineered to induce immune responses with translational potential to modulate T cell functions in distinct pathological settings.

cancer biology↗

bioRxiv2023

The eIF4A2 negative regulator of mRNA translation promotes extracellular matrix deposition to accelerate hepatocellular carcinoma initiation

Increased protein synthesis supports growth of established tumours. However, how mRNA translation contributes to early tumorigenesis remains unclear. Here we show that following oncogene activation, hepatocytes enter a non-proliferative/senescent-like phase characterized by 5{beta}1 integrin-dependent deposition of fibronectin-rich extracellular matrix (ECM) niches. These niches then promote exit from oncogene-induced senescence to permit progression to proliferating hepatocellular carcinoma (HCC). Removal of eIF4A2, a negative regulator of mRNA translation, boosts the synthesis of membrane/secretory proteins which drives a compensatory increase in the turnover/degradation of membrane proteins including 5{beta}1 integrin. This increased membrane protein degradation, in turn, compromises generation of ECM-rich tumour initiation niches, senescence-exit and progression to proliferating HCC. Consistently, pharmacological inhibition of mRNA translation following eIF4A2 loss restores ECM deposition and reinstates HCC progression. Thus, although inhibition of protein synthesis may be an effective way to reduce tumour biomass and the growth of established tumours, our results highlight how agents which reduce mRNA translation, if administered during early tumorigenesis, may awaken senescent cells and promote tumour progression.

cancer biology↗

bioRxiv2022

Purine-rich RNA sequences in the 5'UTR site-specifically regulate eIF4A1-unwinding through eIF4A1-multimerisation to facilitate translation

Oncogenic translational programmes underpin cancer development and are often driven by dysregulation of oncogenic signalling pathways that converge on the eukaryotic translation initiation (eIF) 4F complex. Altered eIF4F activity promotes translation of oncogene mRNAs that typically contain highly structured 5UTRs rendering their translation strongly dependent on RNA unwinding by the DEAD-box helicase eIF4A1 subunit of the eIF4F complex. While eIF4A1-dependent mRNAs have been widely investigated, it is still unclear how highly structured mRNAs recruit and activate eIF4A1 unwinding specifically to facilitate their preferential translation. Here, we show that RNA sequence motifs regulate eIF4A1 unwinding activity in cells. Our data demonstrate that eIF4A1-dependent mRNAs contain AG-rich motifs within their 5UTR which recruit and stimulate eIF4A1 unwinding of localised RNA structure to facilitate mRNA translation. This mode of eIF4A1 regulation is used by mRNAs encoding components of mTORC-signalling and cell cycle progression and renders these mRNAs particularly sensitive to eIF4A1-inhibition. Mechanistically, we show that binding of eIF4A1 to AG-rich sequences leads to multimerization of eIF4A1 with eIF4A1 subunits performing distinct enzymatic activities. Our structural data suggest that RNA-binding of multimeric eIF4A1 induces conformational changes in the RNA substrate resulting in an optimal positioning of eIF4A1 proximal to the RNA duplex region that supports efficient unwinding. Hence, we conclude a model in which mRNAs utilise AG-rich sequences to specifically recruit eIF4A1, enabling assembly of the helicase-active multimeric eIF4A1 complex, and positioning these complexes proximal to stable localised RNA structure allowing ribosomal subunit scanning.

molecular biology↗

bioRxiv2021

Differential regulation of mRNA fate by the human Ccr4-Not complex is driven by CDS composition and mRNA localisation

BackgroundRegulation of protein output at the level of translation allows for a rapid adaptation to dynamic changes to the cells requirements. This precise control of gene expression is achieved by complex and interlinked biochemical processes that modulate both the protein synthesis rate and stability of each individual mRNA. A major factor coordinating this regulation is the Ccr4-Not complex. Despite playing a role in most stages of the mRNA life cycle, no attempt has been made to take a global integrated view of how the Ccr4-Not complex affects gene expression. ResultsThis study has taken a comprehensive approach to investigate post-transcriptional regulation mediated by the Ccr4-Not complex assessing steady-state mRNA levels, ribosome position, mRNA stability and protein production transcriptome-wide. Depletion of the scaffold protein CNOT1 results in a global upregulation of mRNA stability and the preferential stabilisation of mRNAs enriched for G/C-ending codons. We also uncover that mRNAs targeted to the ER for their translation have reduced translational efficiency when CNOT1 is depleted, specifically downstream of the signal sequence cleavage site. In contrast, translationally upregulated mRNAs are normally localised in p-bodies, contain disorder-promoting amino acids and encode nuclear localised proteins. Finally, we identify ribosome pause sites that are resolved or induced by the depletion of CNOT1. ConclusionWe define the key mRNA features that determine how the human Ccr4-Not complex differentially regulates mRNA fate and protein synthesis through a mechanism linked to codon composition, amino acid usage, and mRNA localisation.

molecular biology↗

bioRxiv2019

Evolutionary relationships among Massospora spp. (Entomophthorales), obligate pathogens of cicadas

The fungal genus Massospora (Zoopagomycota: Entomophthorales) includes more than a dozen obligate, sexually transmissible pathogenic species that infect cicadas (Hemiptera) worldwide. At least two species are known to produce psychoactive compounds during infection, which has garnered considerable interest for this enigmatic genus. As with many Entomophthorales, the evolutionary relationships and host associations of Massospora spp. are not well understood. The acquisition of M. diceroproctae from Arizona, M. tettigatis from Chile, and M. platypediae from California and Colorado provided an opportunity to conduct molecular phylogenetic analyses and morphological studies to investigate if these fungi represent a monophyletic group and delimit species boundaries. In a three-locus phylogenetic analysis including the D1-D2 domains of the nuclear 28S rRNA gene (28S), elongation factor 1 alpha-like (EFL), and beta-tubulin (BTUB), Massospora was resolved in a strongly supported monophyletic group containing four well-supported genealogically exclusive lineages, based on two of three methods of phylogenetic inference. There was incongruence among the single-gene trees: two methods of phylogenetic inference recovered trees with either the same topology as the 3-gene concatenated tree (EFL), or a basal polytomy (28S, BTUB). Massospora levispora and M. platypediae isolates formed a single lineage in all analyses and are synonymized here as M. levispora. Massospora diceroproctae was sister to M. cicadina in all three single-gene trees and on an extremely long branch relative to the other Massospora, and even the outgroup taxa, which may reflect an accelerated rate of molecular evolution and/or incomplete taxa sampling. The results of the morphological study presented here indicate that spore measurements may not be phylogenetically or diagnostically informative. Despite recent advances in understanding the ecology of Massospora, much about its host range and diversity remains unexplored. The emerging phylogenetic framework can provide a foundation for exploring co-evolutionary relationships with cicada hosts and the evolution of behavior-altering compounds.

microbiology↗