ORCID Profile
0000-0002-5848-9019
Current Organisation
John Curtin School of Medical Research
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Gene Expression (incl. Microarray and other genome-wide approaches) | Genomics | Bioinformatics | Genetics
Expanding Knowledge in the Biological Sciences | Expanding Knowledge in the Medical and Health Sciences |
Publisher: Springer US
Date: 2022
DOI: 10.1007/978-1-0716-2380-0_7
Abstract: Piwi-interacting RNAs (piRNAs) are 25- to 32-nucleotide-long small RNAs that silence transposable elements (transposons) in animal gonads. piRNAs have a large sequence ersity (over one million different sequences per organism) to target a variety of transposon sequences. This is achieved by flexible and distinct biogenesis pathways that are evolutionarily conserved. In this chapter, I describe a detailed method of purifying and cloning piRNAs from freshly dissected tissue s les, such as fruit fly ovaries, for the high-throughput sequencing. I also describe how to computationally process the sequencing data and interrogate the characteristic pattern of piRNA biogenesis, including ping-pong lification and head-to-tail phasing.
Publisher: Cold Spring Harbor Laboratory
Date: 21-07-2022
DOI: 10.1101/2022.07.20.500385
Abstract: The S. pombe orthologue of the human PAXT complex, Mtl1-Red1 Core (MTREC), is an eleven-subunit complex which targets cryptic unstable transcripts (CUTs) to the nuclear RNA exosome for degradation. It encompasses the canonical poly(A) polymerase Pla1, responsible for polyadenylation of nascent RNA transcripts as part of the cleavage and polyadenylation factor (CPF/CPSF). In this study we identified and characterised the interaction between Pla1 and the MTREC complex core component Red1 and analysed the functional relevance of this interaction in vivo . Our crystal structure of the Pla1-Red1 complex showed that a 58-residue fragment in Red1 binds to the RNA recognition motif domain of Pla1 and tethers it to the MTREC complex. Structure-based Pla1-Red1 interaction mutations showed that Pla1, as part of MTREC complex, hyper-adenylates CUTs for their efficient degradation. Interestingly, the Red1-Pla1 interaction was also required for the efficient assembly of the fission yeast facultative heterochromatic islands. Together, our data suggest a complex interplay between the RNA surveillance and 3’-end processing machineries.
Publisher: Elsevier BV
Date: 2022
DOI: 10.1016/J.GIM.2021.09.001
Abstract: Genetic variants causing aberrant premessenger RNA splicing are increasingly being recognized as causal variants in genetic disorders. In this study, we devise standardized practices for polymerase chain reaction (PCR)-based RNA diagnostics using clinically accessible specimens (blood, fibroblasts, urothelia, biopsy). A total of 74 families with erse monogenic conditions (31% prenatal-congenital onset, 47% early childhood, and 22% teenage-adult onset) were triaged into PCR-based RNA testing, with comparative RNA sequencing for 19 cases. Informative RNA assay data were obtained for 96% of cases, enabling variant reclassification for 75% variants that can be used for genetic counseling (71%), to inform clinical care (32%) and prenatal counseling (41%). Variant-associated mis-splicing was highly reproducible for 28 cases with s les from ≥2 affected in iduals or heterozygotes and 10 cases with ≥2 biospecimens. PCR licons encompassing another segregated heterozygous variant was vital for clinical interpretation of 22 of 79 variants to phase RNA splicing events and discern complete from partial mis-splicing. RNA diagnostics enabled provision of a genetic diagnosis for 64% of recruited cases. PCR-based RNA diagnostics has capacity to analyze 81.3% of clinically significant genes, with long licons providing an advantage over RNA sequencing to phase RNA splicing events. The Australasian Consortium for RNA Diagnostics (SpliceACORD) provide clinically-endorsed, standardized protocols and recommendations for interpreting RNA assay data.
Publisher: Elsevier BV
Date: 05-2007
DOI: 10.1016/J.MITO.2006.10.005
Abstract: We studied the transcriptional regulation of the human mitochondrial translation initiation factor 2 (IF2mt) gene. The minimal promoter region for the human IF2mt gene contains binding sites for Nuclear Respiratory Factor 2 (NRF-2), which is often involved in the transcription of mitochondrial-related genes. Electrophoresis mobility shift assay (EMSA) analyses indicated that NRF-2alpha/beta binds to the IF2mt promoter. Reporter assays, where HEK293T cells were co-transfected with an NRF-2alpha/beta-expressing vector and/or an IF2mt promoter reporter vector, revealed that NRF-2 trans-activates the IF2mt promoter. NRF-2 sites were also found in the promoters of several other mitochondrial translation factors, which suggests NRF-2 may play a key role in the regulation of mitochondrial protein synthesis.
Publisher: Cold Spring Harbor Laboratory
Date: 16-03-2022
DOI: 10.1101/2022.03.14.484124
Abstract: The epitranscriptome embodies many new and largely unexplored functions of RNA. A major roadblock in the epitranscriptomics field is the lack of transcriptome-wide methods to detect more than a single RNA modification type at a time, identify RNA modifications in in idual molecules, and estimate modification stoichiometry accurately. We address these issues with CHEUI (CH3 (methylation) Estimation Using Ionic current), a new method that concurrently detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in in idual RNA molecules from the same s le, as well as differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals with convolutional neural networks to achieve high single-molecule accuracy and outperforms other methods in detecting m6A and m5C sites and quantifying their stoichiometry. CHEUI’s unique capability to identify two modification types in the same s le reveals a non-random co-occurrence of m6A and m5C in mRNA transcripts in cell lines and tissues. CHEUI unlocks an unprecedented potential to study RNA modification configurations and discover new epitranscriptome functions.
Publisher: Proceedings of the National Academy of Sciences
Date: 16-11-2020
Abstract: We report on structure-function studies of the conserved 3′-to-5′ exoribonuclease Nibbler (Nbr) toward understanding its role in 3′-end trimming of small regulatory RNAs. Our studies of the Nbr N-terminal domain provide insights into its role as a double-strand RNA-binding recruitment platform that is required for Nbr-directed microRNA trimming and that experimentally can be bypassed through an Argonaute-interacting peptide. Structural studies on Nbr’s exonucleolytic domain suggest a substrate selectivity of the Mn 2+ -coordinated DEDDy catalytic domain toward non–2′- O -methylated small RNAs. Our findings provide conceptual insights into how Nbr participates in small RNA biogenesis.
Publisher: Cold Spring Harbor Laboratory
Date: 24-09-2022
DOI: 10.1101/2022.09.23.509222
Abstract: Long-read sequencing enables isoform-resolved detection of functionally important RNA elements, such as RNA chemical modifications, RNA secondary structure, or RNA-protein interaction sites. Importantly, the functional impact of these elements can relate to their positions relative to isoform-specific transcript features, such as start and stop codons, open reading frames, exon-exon junctions and transcript termini. Relating transcriptomic and genomic features during sequencing data analysis is challenged by the flexibility of RNA biogenesis and the ersity of alternative mRNA transcripts. To address these challenges and streamline the mapping between transcriptome-mapped data and genome-mapped data, we developed R2Dtool. We illustrate R2Dtool’s capability to process long-read transcriptomic information and potential for interpretation in the context of transcript and genomic annotation features using epitranscriptomics data. R2Dtool embraces and expedites analysis of RNA complexity, and we anticipate it will empower multiple transcriptomic studies for interpretation and discovery. R2Dtool is freely available under the MIT license from omprna/R2Dtool .
Publisher: Springer Science and Business Media LLC
Date: 11-2016
DOI: 10.1038/NATURE20162
Publisher: Public Library of Science (PLoS)
Date: 06-06-2023
DOI: 10.1371/JOURNAL.PBIO.3002099
Abstract: Organisms require mechanisms to distinguish self and non-self-RNA. This distinction is crucial to initiate the biogenesis of Piwi-interacting RNAs (piRNAs). In Drosophila ovaries, PIWI-guided slicing and the recognition of piRNA precursor transcripts by the DEAD-box RNA helicase Yb are the 2 known mechanisms to licence an RNA for piRNA biogenesis in the germline and the soma, respectively. Both the PIWI proteins and Yb are highly conserved across most Drosophila species and are thought to be essential to the piRNA pathway and for silencing transposons. However, we find that species closely related to Drosophila melanogaster have lost the yb gene, as well as the PIWI gene Ago3 . We show that the precursor RNA is still selected in the absence of Yb to abundantly generate transposon antisense piRNAs in the soma. We further demonstrate that Drosophila eugracilis , which lacks Ago3 , is completely devoid of ping-pong piRNAs and exclusively produces phased piRNAs in the absence of slicing. Thus, core piRNA pathway genes can be lost in evolution while still maintaining efficient transposon silencing.
Publisher: Cold Spring Harbor Laboratory
Date: 15-11-2022
DOI: 10.1101/2022.11.14.516378
Abstract: Organisms require mechanisms to distinguish self and non-self RNA. This distinction is crucial to initiate the biogenesis of piRNAs. In Drosophila ovaries, PIWI-guided slicing and the recognition of piRNA precursor transcripts by the DEAD-box RNA helicase Yb are the two known mechanisms to licence an RNA for piRNA biogenesis in the germline and the soma, respectively. Both, the PIWI proteins and Yb are highly conserved across most Drosophila species and are thought to be essential to the piRNA pathway and for silencing transposons. However, we find that species closely related to D. melanogaster have lost the yb gene, as well as the PIWI gene Ago3 . We show that the precursor RNA is still selected in the absence of Yb to abundantly generate transposon antisense piRNAs in the soma. We further demonstrate that D. eugracilis , which lacks Ago3 , is completely devoid of ping-pong piRNAs and exclusively produces phased piRNAs in the absence of slicing. Thus, there are more possible routes through which the piRNA pathway can achieve specificity than previously suggested.
Publisher: Elsevier BV
Date: 09-2013
DOI: 10.1016/J.JMB.2013.07.007
Abstract: Nuclear respiratory factor 2 (NRF-2) is a mammalian transcription factor composed of two distinct and unrelated proteins: NRF-2α, which binds to DNA through its Ets domain, and NRF-2β, which contains the transcription activation domain. The activity of NRF-2 in neurons is regulated by nuclear localization however, the mechanism by which NRF-2 is imported into the nucleus remains unknown. By using in vitro nuclear import assays and immuno-cytofluorescence, we dissect the nuclear import pathways of NRF-2. We show that both NRF-2α and NRF-2β contain intrinsic nuclear localization signals (NLSs): the Ets domain within NRF-2α and the NLS within NRF-2β (amino acids 311/321: EEPPAKRQCIE) that is recognized by importin-α:β. When NRF-2α and NRF-2β form a complex, the nuclear import of NRF-2αβ becomes strictly dependent on the NLS within NRF-2β. Therefore, the nuclear import mechanism of NRF-2 is unique among Ets factors. The NRF-2β NLS contains only two lysine/arginine residues, unlike other known importin-α:β-dependent NLSs. Using ELISA-based binding assays, we show that it is bound by importin-α in almost the same manner and with similar affinity to that of the classical monopartite NLSs, such as c-myc and SV40 T-antigen NLSs. However, the part of the tryptophan array of importin-α that is essential for the recognition of classical monopartite NLSs by generating apolar pockets for the P3 and the P5 lysine/arginine side chains is not required for the recognition of the NRF-2β NLS. We conclude that the NRF-2β NLS is an unusual but is, nevertheless, a bona fide monopartite-type NLS.
Publisher: Elsevier BV
Date: 09-2017
Publisher: Cold Spring Harbor Laboratory
Date: 31-07-2014
Abstract: Splicing of pre-mRNAs results in the deposition of the exon junction complex (EJC) upstream of exon–exon boundaries. The EJC plays crucial post-splicing roles in export, translation, localization, and nonsense-mediated decay of mRNAs. It also aids faithful splicing of pre-mRNAs containing large introns, albeit via an unknown mechanism. Here, we show that the core EJC plus the accessory factors RnpS1 and Acinus aid in definition and efficient splicing of neighboring introns. This requires prior deposition of the EJC in close proximity to either an upstream or downstream splicing event. If present in isolation, EJC-dependent introns are splicing-defective also in wild-type cells. Interestingly, the most affected intron belongs to the piwi locus, which explains the reported transposon desilencing in EJC-depleted Drosophila ovaries. Based on a transcriptome-wide analysis, we propose that the dependency of splicing on the EJC is exploited as a means to control the temporal order of splicing events.
Publisher: Oxford University Press (OUP)
Date: 04-2014
Abstract: We have screened chromosome arm 3L for ethyl methanesulfonate−induced mutations that disrupt localization of fluorescently labeled gurken (grk) messenger (m)RNA, whose transport along microtubules establishes both major body axes of the developing Drosophila oocyte. Rapid identification of causative mutations by single-nucleotide polymorphism recombinational mapping and whole-genomic sequencing allowed us to define nine complementation groups affecting grk mRNA localization and other aspects of oogenesis, including alleles of elg1, scaf6, quemao, nudE, Tsc2/gigas, rasp, and Chd5/Wrb, and several null alleles of the armitage Piwi-pathway gene. Analysis of a newly induced kinesin light chain allele shows that kinesin motor activity is required for both efficient grk mRNA localization and oocyte centrosome integrity. We also show that initiation of the dorsoanterior localization of grk mRNA precedes centrosome localization, suggesting that microtubule self-organization contributes to breaking axial symmetry to generate a unique dorsoventral axis.
Publisher: Springer Science and Business Media LLC
Date: 11-02-2023
DOI: 10.1038/S41467-023-36402-6
Abstract: The S. pombe orthologue of the human PAXT connection, Mtl1-Red1 Core (MTREC), is an eleven-subunit complex that targets cryptic unstable transcripts (CUTs) to the nuclear RNA exosome for degradation. It encompasses the canonical poly(A) polymerase Pla1, responsible for polyadenylation of nascent RNA transcripts as part of the cleavage and polyadenylation factor (CPF/CPSF). In this study we identify and characterise the interaction between Pla1 and the MTREC complex core component Red1 and analyse the functional relevance of this interaction in vivo. Our crystal structure of the Pla1-Red1 complex shows that a 58-residue fragment in Red1 binds to the RNA recognition motif domain of Pla1 and tethers it to the MTREC complex. Structure-based Pla1-Red1 interaction mutations show that Pla1, as part of MTREC complex, hyper-adenylates CUTs for their efficient degradation. Interestingly, the Red1-Pla1 interaction is also required for the efficient assembly of the fission yeast facultative heterochromatic islands. Together, our data suggest a complex interplay between the RNA surveillance and 3’-end processing machineries.
Publisher: Cold Spring Harbor Laboratory
Date: 28-10-2023
Publisher: Oxford University Press (OUP)
Date: 25-05-2020
DOI: 10.1093/NAR/GKAA435
Abstract: Monocytes and macrophages are essential components of the innate immune system. Herein, we report that intron retention (IR) plays an important role in the development and function of these cells. Using Illumina mRNA sequencing, Nanopore direct cDNA sequencing and proteomics analysis, we identify IR events that affect the expression of key genes roteins involved in macrophage development and function. We demonstrate that decreased IR in nuclear-detained mRNA is coupled with increased expression of genes encoding regulators of macrophage transcription, phagocytosis and inflammatory signalling, including ID2, IRF7, ENG and LAT. We further show that this dynamic IR program persists during the polarisation of resting macrophages into activated macrophages. In the presence of proinflammatory stimuli, intron-retaining CXCL2 and NFKBIZ transcripts are rapidly spliced, enabling timely expression of these key inflammatory regulators by macrophages. Our study provides novel insights into the molecular factors controlling vital regulators of the innate immune response.
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 2005
End Date: 2008
Funder: Japan Society for the Promotion of Science
View Funded ActivityStart Date: 2021
End Date: 12-2023
Amount: $746,020.00
Funder: Australian Research Council
View Funded Activity