Biology subjects

Willis, A. E.

Publications and source records attributed to Willis, A. E..

7 recordsLinked to original sources

bioRxiv2023

Benchmarking tRNA-Seq quantification approaches by realistic tRNA-Seq data simulation identifies two novel approaches with higher accuracy

Quantification of transfer RNA (tRNA) using illumina sequencing based tRNA-Seq is complicated due to their degree of redundancy and extensive modifications. As such, no tRNA-Seq method has become well established, while various approaches have been proposed to quantify tRNAs from sequencing reads. Here, we use realistic tRNA-Seq simulations to benchmark tRNA-Seq quantification approaches, including two novel approaches. We demonstrate that these novel approaches are consistently the most accurate, using data simulated to mimic five different tRNA-Seq methods. This simulation-based benchmarking also identifies specific shortfalls for each quantification approach and suggests that up to 13% of the variance observed between cell lines in real tRNA-Seq data could be due to systematic differences in quantification accuracy.

bioinformatics↗

bioRxiv2023

Interrogation of RNA-protein interaction dynamics in bacterial growth

Characterising RNA-protein interaction dynamics is fundamental to understand how bacteria respond to their environment. In this study, we have analysed the dynamics of 91% of the Escherichia coli expressed proteome and the RNA-interaction properties of 271 RNA-binding proteins (RBPs) at different growth phases. We find that 68% of RBPs differentially bind RNA across growth phases and characterise 17 previously unannotated proteins as bacterial RBPs including YfiF, a ncRNA-binding protein. While these new RBPs are mostly present in Proteobacteria, two of them have human orthologs in the form of mitochondrial proteins associated with rare metabolic disorders. Moreover, we reveal novel RBP functions for proteins such as the chaperone HtpG, a new stationary phase tRNA-binding protein. Altogether, we provide the first dynamic RBPome of a bacterium, showcasing how this approach can reveal the function of uncharacterised proteins, and identify critical RNA-protein interactions for cell growth which could inform new antimicrobial therapies.

microbiology↗

bioRxiv2023

HSP70 binds to specific non-coding RNA and regulates human RNA Polymerase III

Molecular chaperones are critical for protein homeostasis and are implicated in several human pathologies such as neurodegeneration and cancer. While the binding of chaperones to nascent and misfolded proteins has been studied in great detail, the direct interaction between chaperones and RNA has not been systematically investigated. Here we provide the evidence for widespread interaction between chaperones and RNA in human cells. We show that the major chaperone Heat-Shock Protein 70 (HSP70) binds to non-coding RNA transcribed by RNA Polymerase III (Pol III) such as tRNA and 5S rRNA. Global chromatin profiling revealed that HSP70 binds genomic sites of transcription by Pol III. Detailed biochemical analyses showed that HSP70 facilitates transcription of its target non-coding RNA by binding to Pol III. Thus our study uncovers an unexpected role of HSP70-RNA interaction in the biogenesis of a specific class of non-coding RNA with wider implications in neurodegeneration and cancer.

biochemistry↗

bioRxiv2023

Metabolic reprogramming provides a novel approach to overcome resistance to BH3-mimetics in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive malignancy linked to asbestos exposure and highly resistant to chemotherapy, potentially due to upregulated expression of the pro-survival proteins, BCL2/BCL-XL/MCL-1. Using clinically-relevant models of MPM we show that patient-derived primary MPM cell lines and ex-vivo 3D tumour explants are highly resistant to apoptosis induced by the BCL2/BCL-XL inhibitor, ABT-737. Importantly, we discover that 2-deoxyglucose (2DG), a glycolytic inhibitor, can sensitize MPM cells to ABT-737 and show this correlates with loss of the pro-survival protein, MCL-1. siRNA knockdown of MCL-1 (MCL-1 KD) combined with ABT-737 induced BAX/BAK-dependent, but BIM/PUMA-independent apoptosis, mimicking 2DG/ABT-737 treatment. MCL-1 KD/ABT-737 induced mitochondrial cytochrome c release and caspase-independent inhibition of mitochondrial respiration. Moreover, we observed a hitherto unreported caspase-dependent cleavage of glycolytic enzymes and subsequent inhibition of glycolysis. 2DG inhibited ERK/STAT3 activity, decreased MCL-1 mRNA and protein levels, with concurrent activation of AKT, which limited loss of MCL-1 protein. However, co-treatment with a specific AKT inhibitor, AZD5363, and 2DG/ABT-737 potently induced cell death and inhibited clonogenic cell survival, while in MPM 3D tumour explants MCL-1 protein expression decreased significantly following 2DG or 2DG/AZD5363 treatment. Notably, a similar synergy was observed in MPM cell lines and MPM 3D tumour explants using ABT-737 in combination with the recently developed MCL-1 inhibitor, S63845. Importantly, our study provides a mechanistic explanation for the chemoresistance of MPM and highlights how this can be overcome by a combination of metabolic reprogramming and/or simultaneous targeting of MCL-1 and BCL-2/BCL-XL using BH3-mimetics.

cell biology↗

bioRxiv2022

The imprinted Mir483 is a growth suppressor and metabolic regulator functioning through IGF1

Mir483 is a conserved and highly expressed microRNA in placental mammals, embedded within the Igf2 gene. Here, we uncover the control mechanisms and physiological functions of Mir483 in vivo, by generating constitutive loss-of-function and over-expressing mice. Mir483 expression is imprinted and dependent on the Igf2 promoters and Igf2/H19 imprinting control region. Over-expression of Mir483 causes severe mid-gestation fetal, but not placental, growth restriction, and late lethality. Fetal death is prevented by restoring Mir483 to endogenous levels using an inducible transgenic system. Continuous postnatal Mir483 over-expression induces growth stunting, elevated hepatic lipid content, increased adiposity, reduced local and systemic IGF1 levels and increased GH. The growth phenotypes are rescued by IGF1 infusion. Our findings provide evidence for a novel functional antagonism between a growth-suppressor microRNA and its growth-promoter host gene, and suggest that Mir483 evolved to limit excessive tissue growth through repression of IGF ligand signalling.

developmental biology↗

bioRxiv2022

A system-wide quantitative map of RNA and protein subcellular localisation dynamics

Existing methods to analyse RNA localisation are constrained to specific RNAs or subcellular niches, precluding the cell-wide mapping of RNA. We present Localisation of RNA (LoRNA), which maps, at once, RNAs to membranous (nucleus, ER and mitochondria) and membraneless compartments (cytosol, nucleolus and phase-separated granules). Simultaneous interrogation of all RNA locations allows the system-wide quantification of RNA proportional distribution and the comprehensive analysis of RNA subcellular dynamics. Moreover, we have re-engineered the LOPIT (Localisation Of Proteins by Isotope Tagging) method, enabling integration with LoRNA, to jointly map RNA and protein subcellular localisation. Applying this framework, we obtain a global re-localisation map for 31839 transcripts and 5314 proteins during the unfolded protein response, uncovering that ER-localised transcripts are more efficiently recruited to stress granules than cytosolic RNAs, and revealing eIF3d is key to sustain cytoskeletal function. Overall, we provide the most exhaustive map to date of RNA and protein subcellular dynamics.

cell biology↗

bioRxiv2021

Rpl24Bst mutation suppresses colorectal cancer by promoting eEF2 phosphorylation via eEF2K

Increased protein synthesis supports the rapid proliferation associated with cancer. The Rpl24Bst mutant mouse reduces the expression of the ribosomal protein RPL24 and has been used to suppress translation and limit tumorigenesis in multiple mouse models of cancer. Here we show that Rpl24Bst also suppresses tumorigenesis and proliferation in a model of colorectal cancer with two common patient mutations, Apc and Kras. In contrast to previous reports, Rpl24Bst mutation has no effect on ribosomal subunit abundance but suppresses translation elongation through phosphorylation of eEF2, reducing protein synthesis by 40% in tumour cells. Ablating eEF2 phosphorylation in Rpl24Bst mutant mice by inactivating its kinase, eEF2K, completely restores the rates of elongation and protein synthesis. Furthermore, eEF2K activity is required for the Rpl24Bst mutant to suppress tumorigenesis. This work demonstrates that elevation of eEF2 phosphorylation is an effective means to suppress colorectal tumorigenesis with two driver mutations. This positions translation elongation as a therapeutic target in colorectal cancer, as well as other cancers where the Rpl24Bst mutation has a tumour suppressive effect in mouse models.

cancer biology↗