ORCID Profile
0000-0002-6103-5165
Current Organisations
University of South Australia
,
University of Adelaide
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Publisher: Springer Science and Business Media LLC
Date: 03-09-2020
DOI: 10.1038/S41598-020-71323-0
Abstract: The attachment of unique molecular identifiers (UMIs) to RNA molecules prior to PCR lification and sequencing, makes it possible to lify libraries to a level that is sufficient to identify rare molecules, whilst simultaneously eliminating PCR bias through the identification of duplicated reads. Accurate de-duplication is dependent upon a sufficiently complex pool of UMIs to allow unique labelling. In applications dealing with complex libraries, such as total RNA-seq, only a limited variety of UMIs are required as the variation in molecules to be sequenced is enormous. However, when sequencing a less complex library, such as small RNAs for which there is a more limited range of possible sequences, we find increased variation in UMIs are required, even beyond that provided in a commercial kit specifically designed for the preparation of small RNA libraries for sequencing. We show that a pool of UMIs randomly varying across eight nucleotides is not of sufficient depth to uniquely tag the microRNAs to be sequenced. This results in over de-duplication of reads and the marked under-estimation of expression of the more abundant microRNAs. Whilst still arguing for the utility of UMIs, this work demonstrates the importance of their considered design to avoid errors in the estimation of gene expression in libraries derived from select regions of the transcriptome or small genomes.
Publisher: American Society for Microbiology
Date: 04-1997
DOI: 10.1128/JB.179.8.2740-2747.1997
Abstract: The sequence of part of the rfb region of Vibrio cholerae serogroup O139 and the physical map of a 35-kb region of the O139 chromosome have been determined. The O139 rfb region presented contains a number of open reading frames which show similarities to other rfb and capsular biosynthesis genes found in members of the Enterobacteriaceae family and in V. cholerae O1. The cloned and sequenced region can complement the defects in O139 antigen biosynthesis in transposon insertions within the O139 rfb cluster. Linkage is demonstrated among IS1358 of V. cholerae O139, the rfb region, and the recently reported otnA and otnB genes (E. M. Bik, A. E. Bunschoten, R. D. Gouw, and F. R. Mooi, EMBO J. 14:209-216, 1995). In addition, the whole of this region has been linked to the rfaD gene. Furthermore, determination of the sequence flanking IS1358 has revealed homology to other rfb-like genes. The exact site of insertion with respect to rfaD is defined for the novel DNAs of both the Bengal and the Argentinian O139 isolates.
Publisher: Public Library of Science (PLoS)
Date: 29-06-2011
Publisher: Public Library of Science (PLoS)
Date: 13-06-2011
Publisher: Informa UK Limited
Date: 07-2003
Publisher: Life Science Alliance, LLC
Date: 03-08-2023
Abstract: Epithelial–mesenchymal transition is essential for tissue patterning and organization. It involves both regulation of cell motility and alterations in the composition and organization of the ECM—a complex environment of proteoglycans and fibrous proteins essential for tissue homeostasis, signaling in response to chemical and biomechanical stimuli, and is often dysregulated under conditions such as cancer, fibrosis, and chronic wounds. Here, we demonstrate that basonuclin-2 (BNC2), a mesenchymal-expressed gene, that is, strongly associated with cancer and developmental defects across genome-wide association studies, is a novel regulator of ECM composition and degradation. We find that at endogenous levels, BNC2 controls the expression of specific collagens, matrix metalloproteases, and other matrisomal components in breast cancer cells, and in fibroblasts that are primarily responsible for the production and processing of the ECM within the tumour microenvironment. In so doing, BNC2 modulates the motile and invasive properties of cancers, which likely explains the association of high BNC2 expression with increasing cancer grade and poor patient prognosis.
Publisher: Cold Spring Harbor Laboratory
Date: 18-04-2020
DOI: 10.1101/2020.04.18.047571
Abstract: Translation of eukaryotic mRNAs starts with binding of the m 7 G cap to the protein eIF4E followed by recruitment of other translation initiation factors. eIF4E’s essential role in translation suggests the cellular eIF4E-mRNA interactome (or ‘eIF4E cap-ome’) may serve as a faithful proxy of cellular translational activity. Here we describe capCLIP, a novel method to systematically capture and quantify the eIF4E cap-ome. To validate capCLIP, we identified the cap-omes in human cells ± the partial mTORC1 inhibitor rapamycin. As expected, TOP (terminal oligopyrimidine) mRNA representation is systematically reduced in rapamycin-treated cells. capCLIP tag data permits refinement of a 7-nucleotide TOP motif (5′-CUYUYYC-3′). We also apply capCLIP to probe the consequences of phosphorylation of eIF4E, whose function had remained unclear. eIF4E phosphorylation drives an overall reduction in eIF4E-mRNA association strikingly, mRNAs most sensitive to phosphorylation possess short 5′-UTRs. capCLIP provides a sensitive and comprehensive measure of cellular translational activity. We foresee its application as a high-throughput way to assess translation in contexts not amenable to existing methodologies.
Publisher: Wiley
Date: 31-03-2005
Publisher: Proceedings of the National Academy of Sciences
Date: 24-10-1995
Abstract: The recent emergence of a pathogenic new non-O1 serotype (O139) of Vibrio cholerae has led to numerous studies in an attempt to identify the origins of this new strain. Our studies indicate that O139 strains have clear differences in the surface polysaccharides when compared with O1 strains: the lipopolysaccharide can be described as semi-rough. Southern hybridization with the O1 rfb region demonstrates that O139 strains no longer contain any of the rfb genes required for the synthesis of the O1 O-antigen or its modification and also lack at least 6 kb of additional contiguous DNA. However, O139 strains have retained rfaD and have a single open reading frame closely related to three small open reading frames of the O1 rfb region. This region is closely related to the H-repeat of Escherichia coli and to the transposases of a number of insertion sequence elements and has all the features of an insertion sequence element that has been designated VcIS1. Transposon insertion mutants defective in O139 O-antigen (and capsule) biosynthesis map to the same fragment as VcIS1. Preliminary sequence data of complementing clones indicate that this DNA encodes a galactosyl-transferase and other enzymes for the utilization of galactose in polysaccharide biosynthesis. We propose a mechanism by which both the Ogawa serotype of O1 strains and the O139 serotype strains may have evolved.
Publisher: Elsevier BV
Date: 02-2000
DOI: 10.1016/S0896-6273(00)80900-9
Abstract: We have combined genetic and biochemical approaches to analyze the function of the RNA-binding protein Nova-1, the paraneoplastic opsoclonus-myoclonus ataxia (POMA) antigen. Nova-1 null mice die postnatally from a motor deficit associated with apoptotic death of spinal and brainstem neurons. Nova-1 null mice show specific splicing defects in two inhibitory receptor pre-mRNAs, glycine alpha2 exon 3A (GlyRalpha2 E3A) and GABA(A) exon gamma2L. Nova protein in brain extracts specifically bound to a previously identified GlyRalpha2 intronic (UCAUY)3 Nova target sequence, and Nova-1 acted directly on this element to increase E3A splicing in cotransfection assays. We conclude that Nova-1 binds RNA in a sequence-specific manner to regulate neuronal pre-mRNA alternative splicing the defect in splicing in Nova-1 null mice provides a model for understanding the motor dysfunction in POMA.
Publisher: Springer Science and Business Media LLC
Date: 2001
DOI: 10.1038/35049061
Abstract: Splicing of pre-messenger RNA is regulated differently in the brain compared with other tissues. Recognition of aberrations in splicing events that are associated with neurological disease has contributed to our understanding of disease pathogenesis in some cases. Neuron-specific proteins involved in RNA splicing and metabolism are also affected in several neurological disorders. These findings have begun to bridge what we know about the mechanisms regulating neuron-specific splicing and our understanding of neural function and disease.
Publisher: EMBO
Date: 06-06-2018
Publisher: Oxford University Press (OUP)
Date: 13-07-2021
DOI: 10.1093/NAR/GKAB604
Abstract: Translation of eukaryotic mRNAs begins with binding of their m7G cap to eIF4E, followed by recruitment of other translation initiation factor proteins. We describe capCLIP, a novel method to comprehensively capture and quantify the eIF4E (eukaryotic initiation factor 4E) ‘cap-ome’ and apply it to examine the biological consequences of eIF4E–cap binding in distinct cellular contexts. First, we use capCLIP to identify the eIF4E cap-omes in human cells with/without the mTORC1 (mechanistic target of rapamycin, complex 1) inhibitor rapamycin, there being an emerging consensus that rapamycin inhibits translation of TOP (terminal oligopyrimidine) mRNAs by displacing eIF4E from their caps. capCLIP reveals that the representation of TOP mRNAs in the cap-ome is indeed systematically reduced by rapamycin, thus validating our new methodology. capCLIP also refines the requirements for a functional TOP sequence. Second, we apply capCLIP to probe the consequences of phosphorylation of eIF4E. We show eIF4E phosphorylation reduces overall eIF4E–mRNA association and, strikingly, causes preferential dissociation of mRNAs with short 5′-UTRs. capCLIP is a valuable new tool to probe the function of eIF4E and of other cap-binding proteins such as eIF4E2/eIF4E3.
Publisher: EMBO
Date: 28-07-2014
Abstract: The micro RNA s of the miR‐200 family maintain the central characteristics of epithelia and inhibit tumor cell motility and invasiveness. Using the Ago‐ HITS ‐ CLIP technology for transcriptome‐wide identification of direct micro RNA targets in living cells, along with extensive validation to verify the reliability of the approach, we have identified hundreds of miR‐200a and miR‐200b targets, providing insights into general features of mi RNA target site selection. Gene ontology analysis revealed a predominant effect of miR‐200 targets in widespread coordinate control of actin cytoskeleton dynamics. Functional characterization of the miR‐200 targets indicates that they constitute subnetworks that underlie the ability of cancer cells to migrate and invade, including coordinate effects on Rho‐ ROCK signaling, invadopodia formation, MMP activity, and focal adhesions. Thus, the miR‐200 family maintains the central characteristics of the epithelial phenotype by acting on numerous targets at multiple levels, encompassing both cytoskeletal effectors that control actin filament organization and dynamics, and upstream signals that locally regulate the cytoskeleton to maintain cell morphology and prevent cell migration.
Publisher: Elsevier BV
Date: 2021
DOI: 10.1016/J.CELREP.2020.108585
Abstract: Potent therapeutic inhibition of the androgen receptor (AR) in prostate adenocarcinoma can lead to the emergence of neuroendocrine prostate cancer (NEPC), a phenomenon associated with enhanced cell plasticity. Here, we show that microRNA-194 (miR-194) is a regulator of epithelial-neuroendocrine transdifferentiation. In clinical prostate cancer s les, miR-194 expression and activity were elevated in NEPC and inversely correlated with AR signaling. miR-194 facilitated the emergence of neuroendocrine features in prostate cancer cells, a process mediated by its ability to directly target a suite of genes involved in cell plasticity. One such target was FOXA1, which encodes a transcription factor with a vital role in maintaining the prostate epithelial lineage. Importantly, a miR-194 inhibitor blocked epithelial-neuroendocrine transdifferentiation and inhibited the growth of cell lines and patient-derived organoids possessing neuroendocrine features. Overall, our study reveals a post-transcriptional mechanism regulating the plasticity of prostate cancer cells and provides a rationale for targeting miR-194 in NEPC.
Publisher: American Society of Hematology
Date: 30-06-2022
Abstract: Inducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R–mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent for survival. Targeting this novel ISR pathway, in combination with the Bcl-2 inhibitor venetoclax, synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anticancer effects of ceramide and preclinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.
Publisher: Cold Spring Harbor Laboratory
Date: 02-12-2022
DOI: 10.1101/2022.12.01.518773
Abstract: Epithelial-mesenchymal transition (EMT) plays important roles in tumour progression and is orchestrated by dynamic changes in gene expression. While it is well established that post-transcriptional regulation plays a significant role in EMT, the extent of alternative polyadenylation (APA) during EMT has not yet been explored. Using 3’ end anchored RNA sequencing, we mapped the alternative polyadenylation landscape (APA) following TGF-β-mediated induction of EMT in human mammary epithelial cells and found APA generally causes 3’UTR lengthening during this cell state transition. Analysis of the RNA-binding protein Quaking (QKI), a splicing factor induced during EMT, revealed enrichment of its binding adjacent to cleavage and polyadenylation sites within 3’UTRs. Following QKI knockdown, APA of many transcripts are altered to produce predominantly shorter 3’UTRs associated with reduced gene expression. Among these, QKI binds to its own cleavage site to produce a transcript with a longer 3’UTR. These findings reveal extensive changes in APA occur during EMT and identify a novel function for QKI in this process.
Publisher: Oxford University Press (OUP)
Date: 31-07-2023
DOI: 10.1093/NAR/GKAD645
Abstract: MiRNAs post-transcriptionally repress gene expression by binding to mRNA 3′UTRs, but the extent to which they act through protein coding regions (CDS regions) is less well established. MiRNA interaction studies show a substantial proportion of binding occurs in CDS regions, however sequencing studies show much weaker effects on mRNA levels than from 3′UTR interactions, presumably due to competition from the translating ribosome. Consequently, most target prediction algorithms consider only 3′UTR interactions. However, the consequences of CDS interactions may have been underestimated, with the reporting of a novel mode of miRNA-CDS interaction requiring base pairing of the miRNA 3′ end, but not the canonical seed site, leading to repression of translation with little effect on mRNA turnover. Using extensive reporter, western blotting and bioinformatic analyses, we confirm that miRNAs can indeed suppress genes through CDS-interaction in special circumstances. However, in contrast to that previously reported, we find repression requires extensive base-pairing, including of the canonical seed, but does not strictly require base pairing of the 3′ miRNA terminus and is mediated through reducing mRNA levels. We conclude that suppression of endogenous genes can occur through miRNAs binding to CDS, but the requirement for extensive base-pairing likely limits the regulatory impacts to modest effects on a small subset of targets.
No related grants have been discovered for Kate Dredge.