Publications by authors named "Charles S Bond"

71 Publications

A synthetic RNA editing factor edits its target site in chloroplasts and bacteria.

Commun Biol 2021 May 10;4(1):545. Epub 2021 May 10.

Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Crawley, WA, Australia.

Members of the pentatricopeptide repeat (PPR) protein family act as specificity factors in C-to-U RNA editing. The expansion of the PPR superfamily in plants provides the sequence variation required for design of consensus-based RNA-binding proteins. We used this approach to design a synthetic RNA editing factor to target one of the sites in the Arabidopsis chloroplast transcriptome recognised by the natural editing factor CHLOROPLAST BIOGENESIS 19 (CLB19). We show that our synthetic editing factor specifically recognises the target sequence in in vitro binding assays. The designed factor is equally specific for the target rpoA site when expressed in chloroplasts and in the bacterium E. coli. This study serves as a successful pilot into the design and application of programmable RNA editing factors based on plant PPR proteins.
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http://dx.doi.org/10.1038/s42003-021-02062-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8110955PMC
May 2021

Evolving origin-of-transfer sequences on staphylococcal conjugative and mobilizable plasmids-who's mimicking whom?

Nucleic Acids Res 2021 05;49(9):5177-5188

Curtin Health Innovation Research Institute, Curtin University, Perth, WA 6102, Australia.

In Staphylococcus aureus, most multiresistance plasmids lack conjugation or mobilization genes for horizontal transfer. However, most are mobilizable due to carriage of origin-of-transfer (oriT) sequences mimicking those of conjugative plasmids related to pWBG749. pWBG749-family plasmids have diverged to carry five distinct oriT subtypes and non-conjugative plasmids have been identified that contain mimics of each. The relaxasome accessory factor SmpO, encoded by each conjugative plasmid, determines specificity for its cognate oriT. Here we characterized the binding of SmpO proteins to each oriT. SmpO proteins predominantly formed tetramers in solution and bound 5'-GNNNNC-3' sites within each oriT. Four of the five SmpO proteins specifically bound their cognate oriT. An F7K substitution in pWBG749 SmpO switched oriT-binding specificity in vitro. In vivo, the F7K substitution reduced but did not abolish self-transfer of pWBG749. Notably, the substitution broadened the oriT subtypes that were mobilized. Thus, this substitution represents a potential evolutionary intermediate with promiscuous DNA-binding specificity that could facilitate a switch between oriT specificities. Phylogenetic analysis suggests pWBG749-family plasmids have switched oriT specificity more than once during evolution. We hypothesize the convergent evolution of oriT specificity in distinct branches of the pWBG749-family phylogeny reflects indirect selection pressure to mobilize plasmids carrying non-cognate oriT-mimics.
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http://dx.doi.org/10.1093/nar/gkab303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8136818PMC
May 2021

Caveolin-1-driven membrane remodelling regulates hnRNPK-mediated exosomal microRNA sorting in cancer.

Clin Transl Med 2021 Apr;11(4):e381

The University of Queensland Diamantina Institute, The University of Queensland, Woolloongabba, Queensland, Australia.

Background: Caveolae proteins play diverse roles in cancer development and progression. In prostate cancer, non-caveolar caveolin-1 (CAV1) promotes metastasis, while CAVIN1 attenuates CAV1-induced metastasis. Here, we unveil a novel mechanism linking CAV1 to selective loading of exosomes with metastasis-promoting microRNAs.

Results: We identify hnRNPK as a CAV1-regulated microRNA binding protein. In the absence of CAVIN1, non-caveolar CAV1 drives localisation of hnRPNK to multi-vesicular bodies (MVBs), recruiting AsUGnA motif-containing miRNAs and causing their release within exosomes. This process is dependent on the lipid environment of membranes as shown by cholesterol depletion using methyl-β-cyclodextrin or by treatment with n-3 polyunsaturated fatty acids. Consistent with a role in bone metastasis, knockdown of hnRNPK in prostate cancer PC3 cells abolished the ability of PC3 extracellular vesicles (EV) to induce osteoclastogenesis, and biofluid EV hnRNPK is elevated in metastatic prostate and colorectal cancer.

Conclusions: Taken together, these results support a novel pan-cancer mechanism for CAV1-driven exosomal release of hnRNPK and associated miRNA in metastasis, which is modulated by the membrane lipid environment.
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http://dx.doi.org/10.1002/ctm2.381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8031663PMC
April 2021

Amylin and beta amyloid proteins interact to form amorphous heterocomplexes with enhanced toxicity in neuronal cells.

Sci Rep 2020 06 25;10(1):10356. Epub 2020 Jun 25.

School of Pharmacy and Biomedical Sciences, Curtin Health and Innovation Research Institute (CHIRI), Faculty of Health Sciences, Curtin University, Bentley, WA, 6107, Australia.

Human pancreatic islet amyloid polypeptide (hIAPP) and beta amyloid (Aβ) can accumulate in Type 2 diabetes (T2D) and Alzheimer's disease (AD) brains and evidence suggests that interaction between the two amyloidogenic proteins can lead to the formation of heterocomplex aggregates. However, the structure and consequences of the formation of these complexes remains to be determined. The main objective of this study was to characterise the different types and morphology of Aβ-hIAPP heterocomplexes and determine if formation of such complexes exacerbate neurotoxicity. We demonstrate that hIAPP promotes Aβ oligomerization and formation of small oligomer and large aggregate heterocomplexes. Co-oligomerized Aβ42-hIAPP mixtures displayed distinct amorphous structures and a 3-fold increase in neuronal cell death as compared to Aβ and hIAPP alone. However, in contrast to hIAPP, non-amyloidogenic rat amylin (rIAPP) reduced oligomer Aβ-mediated neuronal cell death. rIAPP exhibited reductions in Aβ induced neuronal cell death that was independent of its ability to interact with Aβ and form heterocomplexes; suggesting mediation by other pathways. Our findings reveal distinct effects of IAPP peptides in modulating Aβ aggregation and toxicity and provide new insight into the potential pathogenic effects of Aβ-IAPP hetero-oligomerization and development of IAPP based therapies for AD and T2D.
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http://dx.doi.org/10.1038/s41598-020-66602-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316712PMC
June 2020

High resolution crystal structure of a KRAS promoter G-quadruplex reveals a dimer with extensive poly-A π-stacking interactions for small-molecule recognition.

Nucleic Acids Res 2020 06;48(10):5766-5776

School of Molecular Sciences, University of Western Australia, Crawley, WA 6009, Australia.

Aberrant KRAS signaling is a driver of many cancers and yet remains an elusive target for drug therapy. The nuclease hypersensitive element of the KRAS promoter has been reported to form secondary DNA structures called G-quadruplexes (G4s) which may play important roles in regulating KRAS expression, and has spurred interest in structural elucidation studies of the KRAS G-quadruplexes. Here, we report the first high-resolution crystal structure (1.6 Å) of a KRAS G-quadruplex as a 5'-head-to-head dimer with extensive poly-A π-stacking interactions observed across the dimer. Molecular dynamics simulations confirmed that the poly-A π-stacking interactions are also maintained in the G4 monomers. Docking and molecular dynamics simulations with two G4 ligands that display high stabilization of the KRAS G4 indicated the poly-A loop was a binding site for these ligands in addition to the 5'-G-tetrad. Given sequence and structural variability in the loop regions provide the opportunity for small-molecule targeting of specific G4s, we envisage this high-resolution crystal structure for the KRAS G-quadruplex will aid in the rational design of ligands to selectively target KRAS.
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http://dx.doi.org/10.1093/nar/gkaa262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261167PMC
June 2020

Divergent receptor proteins confer responses to different karrikins in two ephemeral weeds.

Nat Commun 2020 03 9;11(1):1264. Epub 2020 Mar 9.

School of Molecular Sciences, The University of Western Australia, 35 Stirling Hwy, Perth, WA, 6009, Australia.

Wildfires can encourage the establishment of invasive plants by releasing potent germination stimulants, such as karrikins. Seed germination of Brassica tournefortii, a noxious weed of Mediterranean climates, is strongly stimulated by KAR, the archetypal karrikin produced from burning vegetation. In contrast, the closely-related yet non-fire-associated ephemeral Arabidopsis thaliana is unusual because it responds preferentially to KAR. The α/β-hydrolase KARRIKIN INSENSITIVE 2 (KAI2) is the putative karrikin receptor identified in Arabidopsis. Here we show that B. tournefortii expresses three KAI2 homologues, and the most highly-expressed homologue is sufficient to confer enhanced responses to KAR relative to KAR when expressed in Arabidopsis. We identify two amino acid residues near the KAI2 active site that explain the ligand selectivity, and show that this combination has arisen independently multiple times within dicots. Our results suggest that duplication and diversification of KAI2 proteins could confer differential responses to chemical cues produced by environmental disturbance, including fire.
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http://dx.doi.org/10.1038/s41467-020-14991-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062792PMC
March 2020

The Expansion and Diversification of Pentatricopeptide Repeat RNA-Editing Factors in Plants.

Mol Plant 2020 02 21;13(2):215-230. Epub 2019 Nov 21.

Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, Crawley, Perth 6009, WA, Australia; School of Molecular Sciences, The University of Western Australia, Crawley, Perth 6009, WA, Australia. Electronic address:

The RNA-binding pentatricopeptide repeat (PPR) family comprises hundreds to thousands of genes in most plants, but only a few dozen in algae, indicating massive gene expansions during land plant evolution. The nature and timing of these expansions has not been well defined due to the sparse sequence data available from early-diverging land plant lineages. In this study, we exploit the comprehensive OneKP datasets of over 1000 transcriptomes from diverse plants and algae toward establishing a clear picture of the evolution of this massive gene family, focusing on the proteins typically associated with RNA editing, which show the most spectacular variation in numbers and domain composition across the plant kingdom. We characterize over 2 250 000 PPR motifs in over 400 000 proteins. In lycophytes, polypod ferns, and hornworts, nearly 10% of expressed protein-coding genes encode putative PPR editing factors, whereas they are absent from algae and complex-thalloid liverworts. We show that rather than a single expansion, most land plant lineages with high numbers of editing factors have continued to generate novel sequence diversity. We identify sequence variations that imply functional differences between PPR proteins in seed plants versus non-seed plants and variations we propose to be linked to seed-plant-specific editing co-factors. Finally, using the sequence variations across the datasets, we develop a structural model of the catalytic DYW domain associated with C-to-U editing and identify a clade of unique DYW variants that are strong candidates as U-to-C RNA-editing factors, given their phylogenetic distribution and sequence characteristics.
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http://dx.doi.org/10.1016/j.molp.2019.11.002DOI Listing
February 2020

TGF-β-induced fibrotic stress increases G-quadruplex formation in human fibroblasts.

FEBS Lett 2019 11 15;593(22):3149-3161. Epub 2019 Nov 15.

School of Molecular Sciences, The University of Western Australia, Perth, Australia.

Scar formation after wound healing is a major medical problem. A better understanding of the dynamic nuclear architecture of the genome during wound healing could provide insights into the underlying pathophysiology and enable novel therapeutic strategies. Here, we demonstrate that TGF-β-induced fibrotic stress increases formation of the dynamic secondary DNA structures called G-quadruplexes in skin fibroblasts, which is coincident with increased expression of collagen 1. This G-quadruplex formation is attenuated by a small molecule inhibitor of intracellular Ca influx and an anti-fibrotic compound. In addition, we identify G-quadruplex-forming sequences in the promoter region of COL1A1, which encodes collagen 1, and confirm their ability to form G-quadruplex structures under physiologically relevant conditions. Our findings reveal a link between G-quadruplexes and scar formation that may lead to novel therapeutic interventions.
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http://dx.doi.org/10.1002/1873-3468.13658DOI Listing
November 2019

Evolution of a 72-Kilobase Cointegrant, Conjugative Multiresistance Plasmid in Community-Associated Methicillin-Resistant Staphylococcus aureus Isolates from the Early 1990s.

Antimicrob Agents Chemother 2019 11 22;63(11). Epub 2019 Oct 22.

Curtin Health Innovation Research Institute, Curtin University, Perth, WA, Australia

Horizontal transfer of plasmids encoding antimicrobial resistance and virulence determinants has been instrumental in evolution, including the emergence of community-associated methicillin-resistant (CA-MRSA). In the early 1990s, the first CA-MRSA strain isolated in Western Australia (WA), WA-5, encoded cadmium, tetracycline, and penicillin resistance genes on plasmid pWBG753 (∼30 kb). WA-5 and pWBG753 appeared only briefly in WA; however, fusidic acid resistance plasmids related to pWBG753 were also present in the first European CA-MRSA isolates at the time. Here, we characterize a 72-kb conjugative plasmid, pWBG731, present in multiresistant WA-5-like clones from the same period. pWBG731 was a cointegrant formed from pWBG753 and a pWBG749 family conjugative plasmid. pWBG731 carried mupirocin, trimethoprim, cadmium, and penicillin resistance genes. The stepwise evolution of pWBG731 likely occurred through the combined actions of IS, IS-dependent miniature inverted-repeat transposable elements (MITEs), and the BinL resolution system of the β-lactamase transposon Tn An evolutionarily intermediate ∼42-kb nonconjugative plasmid, pWBG715, possessed the same resistance genes as pWBG731 but retained an integrated copy of the small tetracycline resistance plasmid pT181. IS likely facilitated the replacement of pT181 with conjugation genes on pWBG731, thus enabling autonomous transfer. Like conjugative plasmid pWBG749, pWBG731 also mobilized nonconjugative plasmids carrying mimics. It seems likely that pWBG731 represents the product of multiple recombination events between the WA-5 pWBG753 plasmid and other mobile genetic elements present in indigenous community-associated methicillin-sensitive (CA-MSSA) isolates. The molecular evolution of pWBG731 saliently illustrates how diverse mobile genetic elements can together facilitate rapid accrual and horizontal dissemination of multiresistance in CA-MRSA.
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http://dx.doi.org/10.1128/AAC.01560-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6811413PMC
November 2019

Bridging Crystal Engineering and Drug Discovery by Utilizing Intermolecular Interactions and Molecular Shapes in Crystals.

Angew Chem Int Ed Engl 2019 11 19;58(47):16780-16784. Epub 2019 Aug 19.

School of Molecular Sciences, University of Western Australia, Perth, WA, 6009, Australia.

Most structure-based drug discovery methods utilize crystal structures of receptor proteins. Crystal engineering, on the other hand, utilizes the wealth of chemical information inherent in small-molecule crystal structures in the Cambridge Structural Database (CSD). We show that the interaction surfaces and shapes of molecules in experimentally determined small-molecule crystal structures can serve as effective tools in drug discovery. Our description of the shape and interaction propensities of molecules in their crystal structures can be used to screen them for specific binding compatibility with protein targets, as demonstrated through the high-throughput profiling of around 138 000 small-molecule structures in the CSD and a series of drug-protein crystal structures. Electron-density-based intermolecular boundary surfaces in small-molecule crystal structures and in target-protein pockets are utilized to identify potential ligand molecules from the CSD based on 3D shape and intermolecular interaction matching.
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http://dx.doi.org/10.1002/anie.201906602DOI Listing
November 2019

Delineation of the integrase-attachment and origin-of-transfer regions of the symbiosis island ICEMlSym.

Plasmid 2019 07 9;104:102416. Epub 2019 May 9.

School of Pharmacy and Biomedical Sciences and Curtin Health Innovation Research Institute, Curtin University, Perth, WA, Australia. Electronic address:

Integrative and conjugative elements (ICEs) are chromosomally-integrated mobile genetic elements that excise from their host chromosome and transfer to other bacteria via conjugation. ICEMlSym is the prototypical member of a large family of "symbiosis ICEs" which confer upon their hosts the ability to form a nitrogen-fixing symbiosis with a variety of legume species. Mesorhizobial symbiosis ICEs carry a common core of mobilisation genes required for integration, excision and conjugative transfer. IntS of ICEMlSym enables recombination between the ICEMlSym attachment site attP and the 3' end of the phe-tRNA gene. Here we identified putative IntS attP arm (P) sites within the attP region and demonstrated that the outermost P1 and P5 sites demarcated the minimal region for efficient IntS-mediated integration. We also identified the ICEMlSym origin-of-transfer (oriT) site directly upstream of the relaxase-gene rlxS. The ICEMlSym conjugation system mobilised a plasmid carrying the cloned oriT to Escherichia coli in an rlxS-dependent manner. Surprisingly, an in-frame, markerless deletion mutation in the ICEMlSym recombination directionality factor (excisionase) gene rdfS, but not a mutation in intS, abolished mobilisation, suggesting the rdfS deletion tentatively has downstream effects on conjugation or its regulation. In summary, this work defines two critical cis-acting regions required for excision and transfer of ICEMlSym and related ICEs.
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http://dx.doi.org/10.1016/j.plasmid.2019.102416DOI Listing
July 2019

The macrocyclizing protease butelase 1 remains autocatalytic and reveals the structural basis for ligase activity.

Plant J 2019 06 28;98(6):988-999. Epub 2019 Mar 28.

School of Molecular Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, Perth, 6009, Australia.

Plant asparaginyl endopeptidases (AEPs) are expressed as inactive zymogens that perform maturation of seed storage protein upon cleavage-dependent autoactivation in the low-pH environment of storage vacuoles. The AEPs have attracted attention for their macrocyclization reactions, and have been classified as cleavage or ligation specialists. However, we have recently shown that the ability of AEPs to produce either cyclic or acyclic products can be altered by mutations to the active site region, and that several AEPs are capable of macrocyclization given favorable pH conditions. One AEP extracted from Clitoria ternatea seeds (butelase 1) is classified as a ligase rather than a protease, presenting an opportunity to test for loss of cleavage activity. Here, making recombinant butelase 1 and rescuing an Arabidopsis thaliana mutant lacking AEP, we show that butelase 1 retains cleavage functions in vitro and in vivo. The in vivo rescue was incomplete, consistent with some trade-off for butelase 1 specialization toward macrocyclization. Its crystal structure showed an active site with only subtle differences from cleaving AEPs, suggesting the many differences in its peptide-binding region are the source of its efficient macrocyclization. All considered, it seems that either butelase 1 has not fully specialized or a requirement for autocatalytic cleavage is an evolutionary constraint upon macrocyclizing AEPs.
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http://dx.doi.org/10.1111/tpj.14293DOI Listing
June 2019

Intracellular speciation of gold nanorods alters the conformational dynamics of genomic DNA.

Nat Nanotechnol 2018 12 8;13(12):1148-1153. Epub 2018 Oct 8.

School of Molecular Sciences, The University of Western Australia, Perth, Western Australia, Australia.

Gold nanorods are one of the most widely explored inorganic materials in nanomedicine for diagnostics, therapeutics and sensing. It has been shown that gold nanorods are not cytotoxic and localize within cytoplasmic vesicles following endocytosis, with no nuclear localization, but other studies have reported alterations in gene expression profiles in cells following exposure to gold nanorods, via unknown mechanisms. In this work we describe a pathway that can contribute to this phenomenon. By mapping the intracellular chemical speciation process of gold nanorods, we show that the commonly used Au-thiol conjugation, which is important for maintaining the noble (inert) properties of gold nanostructures, is altered following endocytosis, resulting in the formation of Au(I)-thiolates that localize in the nucleus. Furthermore, we show that nuclear localization of the gold species perturbs the dynamic microenvironment within the nucleus and triggers alteration of gene expression in human cells. We demonstrate this using quantitative visualization of ubiquitous DNA G-quadruplex structures, which are sensitive to ionic imbalances, as an indicator of the formation of structural alterations in genomic DNA.
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http://dx.doi.org/10.1038/s41565-018-0272-2DOI Listing
December 2018

NONO Detects the Nuclear HIV Capsid to Promote cGAS-Mediated Innate Immune Activation.

Cell 2018 10 27;175(2):488-501.e22. Epub 2018 Sep 27.

Immunity and Cancer Department, Institut Curie, PSL Research University, INSERM U932, 75005 Paris, France. Electronic address:

Detection of viruses by innate immune sensors induces protective antiviral immunity. The viral DNA sensor cyclic GMP-AMP synthase (cGAS) is necessary for detection of HIV by human dendritic cells and macrophages. However, synthesis of HIV DNA during infection is not sufficient for immune activation. The capsid protein, which associates with viral DNA, has a pivotal role in enabling cGAS-mediated immune activation. We now find that NONO is an essential sensor of the HIV capsid in the nucleus. NONO protein directly binds capsid with higher affinity for weakly pathogenic HIV-2 than highly pathogenic HIV-1. Upon infection, NONO is essential for cGAS activation by HIV and cGAS association with HIV DNA in the nucleus. NONO recognizes a conserved region in HIV capsid with limited tolerance for escape mutations. Detection of nuclear viral capsid by NONO to promote DNA sensing by cGAS reveals an innate strategy to achieve distinction of viruses from self in the nucleus.
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http://dx.doi.org/10.1016/j.cell.2018.08.062DOI Listing
October 2018

Functional Domains of NEAT1 Architectural lncRNA Induce Paraspeckle Assembly through Phase Separation.

Mol Cell 2018 06;70(6):1038-1053.e7

Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan. Electronic address:

A class of long noncoding RNAs (lncRNAs) has architectural functions in nuclear body construction; however, specific RNA domains dictating their architectural functions remain uninvestigated. Here, we identified the domains of the architectural NEAT1 lncRNA that construct paraspeckles. Systematic deletion of NEAT1 portions using CRISPR/Cas9 in haploid cells revealed modular domains of NEAT1 important for RNA stability, isoform switching, and paraspeckle assembly. The middle domain, containing functionally redundant subdomains, was responsible for paraspeckle assembly. Artificial tethering of the NONO protein to a NEAT1_2 mutant lacking the functional subdomains rescued paraspeckle assembly, and this required the NOPS dimerization domain of NONO. Paraspeckles exhibit phase-separated properties including susceptibility to 1,6-hexanediol treatment. RNA fragments of the NEAT1_2 subdomains preferentially bound NONO/SFPQ, leading to phase-separated aggregates in vitro. Thus, we demonstrate that the enrichment of NONO dimers on the redundant NEAT1_2 subdomains initiates construction of phase-separated paraspeckles, providing mechanistic insights into lncRNA-based nuclear body formation.
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http://dx.doi.org/10.1016/j.molcel.2018.05.019DOI Listing
June 2018

Crystal structure of a SFPQ/PSPC1 heterodimer provides insights into preferential heterodimerization of human DBHS family proteins.

J Biol Chem 2018 04 12;293(17):6593-6602. Epub 2018 Mar 12.

From the Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086 and

Members of the behavior human splicing (DBHS) protein family are nuclear proteins implicated in many layers of nuclear functions, including RNA biogenesis as well as DNA repair. Definitive of the DBHS protein family, the conserved DBHS domain provides a dimerization platform that is critical for the structural integrity and function of these proteins. The three human DBHS proteins, splicing factor proline- and glutamine-rich (SFPQ), paraspeckle component 1 (PSPC1), and non-POU domain-containing octamer-binding protein (NONO), form either homo- or heterodimers; however, the relative affinity and mechanistic details of preferential heterodimerization are yet to be deciphered. Here we report the crystal structure of a SFPQ/PSPC1 heterodimer to 2.3-Å resolution and analyzed the subtle structural differences between the SFPQ/PSPC1 heterodimer and the previously characterized SFPQ homodimer. Analytical ultracentrifugation to estimate the dimerization equilibrium of the SFPQ-containing dimers revealed that the SFPQ-containing dimers dissociate at low micromolar concentrations and that the heterodimers have higher affinities than the homodimer. Moreover, we observed that the apparent dissociation constant for the SFPQ/PSPC1 heterodimer was over 6-fold lower than that of the SFPQ/NONO heterodimer. We propose that these differences in dimerization affinity may represent a potential mechanism by which PSPC1 at a lower relative cellular abundance can outcompete NONO to heterodimerize with SFPQ.
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http://dx.doi.org/10.1074/jbc.RA117.001451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5925804PMC
April 2018

Structural basis of ribosomal peptide macrocyclization in plants.

Elife 2018 01 31;7. Epub 2018 Jan 31.

School of Molecular Sciences, The University of Western Australia, Perth, Australia.

Constrained, cyclic peptides encoded by plant genes represent a new generation of drug leads. Evolution has repeatedly recruited the Cys-protease asparaginyl endopeptidase (AEP) to perform their head-to-tail ligation. These macrocyclization reactions use the substrates amino terminus instead of water to deacylate, so a peptide bond is formed. How solvent-exposed plant AEPs macrocyclize is poorly understood. Here we present the crystal structure of an active plant AEP from the common sunflower, . The active site contained electron density for a tetrahedral intermediate with partial occupancy that predicted a binding mode for peptide macrocyclization. By substituting catalytic residues we could alter the ratio of cyclic to acyclic products. Moreover, we showed AEPs from other species lacking cyclic peptides can perform macrocyclization under favorable pH conditions. This structural characterization of AEP presents a logical framework for engineering superior enzymes that generate macrocyclic peptide drug leads.
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http://dx.doi.org/10.7554/eLife.32955DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834244PMC
January 2018

Paraspeckles: Where Long Noncoding RNA Meets Phase Separation.

Trends Biochem Sci 2018 02 27;43(2):124-135. Epub 2017 Dec 27.

School of Molecular Sciences, University of Western Australia, Crawley, Western Australia, 6009, Australia.

Long noncoding RNA (lncRNA) molecules are some of the newest and least understood players in gene regulation. Hence, we need good model systems with well-defined RNA and protein components. One such system is paraspeckles - protein-rich nuclear organelles built around a specific lncRNA scaffold. New discoveries show how paraspeckles are formed through multiple RNA-protein and protein-protein interactions, some of which involve extensive polymerization, and others with multivalent interactions driving phase separation. Once formed, paraspeckles influence gene regulation through sequestration of component proteins and RNAs, with subsequent depletion in other compartments. Here we focus on the dual aspects of paraspeckle structure and function, revealing an emerging role for these dynamic bodies in a multitude of cellular settings.
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http://dx.doi.org/10.1016/j.tibs.2017.12.001DOI Listing
February 2018

Non-nuclear Pool of Splicing Factor SFPQ Regulates Axonal Transcripts Required for Normal Motor Development.

Neuron 2017 Apr 6;94(2):322-336.e5. Epub 2017 Apr 6.

Centre for Developmental Neurobiology and MRC CNDD, IoPPN, Guy's Campus, King's College London, London SE1 1UL, UK. Electronic address:

Recent progress revealed the complexity of RNA processing and its association to human disorders. Here, we unveil a new facet of this complexity. Complete loss of function of the ubiquitous splicing factor SFPQ affects zebrafish motoneuron differentiation cell autonomously. In addition to its nuclear localization, the protein unexpectedly localizes to motor axons. The cytosolic version of SFPQ abolishes motor axonal defects, rescuing key transcripts, and restores motility in the paralyzed sfpq null mutants, indicating a non-nuclear processing role in motor axons. Novel variants affecting the conserved coiled-coil domain, so far exclusively found in fALS exomes, specifically affect the ability of SFPQ to localize in axons. They broadly rescue morphology and motility in the zebrafish mutant, but alter motor axon morphology, demonstrating functional requirement for axonal SFPQ. Altogether, we uncover the axonal function of the splicing factor SFPQ in motor development and highlight the importance of the coiled-coil domain in this process. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.neuron.2017.03.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5405110PMC
April 2017

Evidence for Ancient Origins of Bowman-Birk Inhibitors from .

Plant Cell 2017 Mar 14;29(3):461-473. Epub 2017 Mar 14.

School of Molecular Sciences, The University of Western Australia, Crawley, Perth 6009, Australia

Bowman-Birk Inhibitors (BBIs) are a well-known family of plant protease inhibitors first described 70 years ago. BBIs are known only in the legume (Fabaceae) and cereal (Poaceae) families, but peptides that mimic their trypsin-inhibitory loops exist in sunflowers () and frogs. The disparate biosynthetic origins and distant phylogenetic distribution implies these loops evolved independently, but their structural similarity suggests a common ancestor. Targeted bioinformatic searches for the BBI inhibitory loop discovered highly divergent BBI-like sequences in the seedless, vascular spikemoss Using de novo transcriptomics, we confirmed expression of five transcripts in whose encoded proteins share homology with BBI inhibitory loops. The most highly expressed, , encodes a protein that inhibits trypsin. We needed to mutate two lysine residues to abolish trypsin inhibition, suggesting BBI3's mechanism of double-headed inhibition is shared with BBIs from angiosperms. As belongs to the lycopod plant lineage, which diverged ∼200 to 230 million years before the common ancestor of angiosperms, its BBI-like proteins imply there was a common ancestor for legume and cereal BBIs. Indeed, we discovered sequences in six angiosperm families outside the Fabaceae and Poaceae. These findings provide the evolutionary missing links between the well-known legume and cereal gene families.
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http://dx.doi.org/10.1105/tpc.16.00831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5385957PMC
March 2017

Whaddaya Know: A Guide to Uncertainty and Subjectivity in Structural Biology.

Trends Biochem Sci 2017 02 12;42(2):155-167. Epub 2017 Jan 12.

School of Chemistry and Biochemistry, The University of Western Australia, Crawley, WA 6014, Australia.

The methods of structural biology, while powerful, are technically complex. Although the Protein Data Bank (PDB) provides a repository that allows anyone to download any structure, many users would not appreciate the caveats that should be considered when examining a structure. Here, we describe several key uncertainties associated with the application of X-ray crystallography, NMR spectroscopy, single-particle electron microscopy (SPEM), and small-angle scattering (SAS) to biological macromolecules. The take-home message is that structures are not absolute truths - they are models that fit the experimental data and therefore have uncertainty and subjectivity associated with them. These uncertainties must be appreciated - careful reading of the associated paper, and any validation report provided by the structure database, is highly recommended.
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http://dx.doi.org/10.1016/j.tibs.2016.11.002DOI Listing
February 2017

Determinants of affinity and specificity in RNA-binding proteins.

Curr Opin Struct Biol 2016 06 14;38:83-91. Epub 2016 Jun 14.

School of Life and Environmental Sciences, The University of Sydney, NSW 2006, Australia. Electronic address:

Emerging data suggest that the mechanisms by which RNA-binding proteins (RBPs) interact with RNA and the rules governing specificity might be substantially more complex than those underlying their DNA-binding counterparts. Even our knowledge of what constitutes the RNA-bound proteome is contentious; recent studies suggest that 10-30% of RBPs contain no known RNA-binding domain. Adding to this situation is a growing disconnect between the avalanche of identified interactions between proteins and long noncoding RNAs and the absence of biophysical data on these interactions. RNA-protein interactions are also at the centre of what might emerge as one of the biggest shifts in thinking about cell and molecular biology this century, following from recent reports of ribonucleoprotein complexes that drive reversible membrane-free phase separation events within the cell. Unexpectedly, low-complexity motifs are important in the formation of these structures. Here we briefly survey recent advances in our understanding of the specificity of RBPs.
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http://dx.doi.org/10.1016/j.sbi.2016.05.005DOI Listing
June 2016

A crystallographic study of human NONO (p54(nrb)): overcoming pathological problems with purification, data collection and noncrystallographic symmetry.

Acta Crystallogr D Struct Biol 2016 06 25;72(Pt 6):761-9. Epub 2016 May 25.

School of Chemistry and Biochemistry, The University of Western Australia, Crawley, WA 6009, Australia.

Non-POU domain-containing octamer-binding protein (NONO, a.k.a. p54(nrb)) is a central player in nuclear gene regulation with rapidly emerging medical significance. NONO is a member of the highly conserved Drosophila behaviour/human splicing (DBHS) protein family, a dynamic family of obligatory dimeric nuclear regulatory mediators. However, work with the NONO homodimer has been limited by rapid irreversible sample aggregation. Here, it is reported that L-proline stabilizes purified NONO homodimers, enabling good-quality solution small-angle X-ray structure determination and crystallization. NONO crystallized in the apparent space group P21 with a unique axis (b) of 408.9 Å and with evidence of twinning, as indicated by the cumulative intensity distribution L statistic, suggesting the possibility of space group P1. Structure solution by molecular replacement shows a superhelical arrangement of six NONO homodimers (or 12 in P1) oriented parallel to the long axis, resulting in extensive noncrystallographic symmetry. Further analysis revealed that the crystal was not twinned, but the collected data suffered from highly overlapping reflections that obscured the L-test. Optimized data collection on a new crystal using higher energy X-rays, a smaller beam width and an increased sample-to-detector distance produced non-overlapping reflections to 2.6 Å resolution. The steps taken to analyse and overcome this series of practical difficulties and to produce a biologically informative structure are discussed.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5038197PMC
http://dx.doi.org/10.1107/S2059798316005830DOI Listing
June 2016

The DBHS proteins SFPQ, NONO and PSPC1: a multipurpose molecular scaffold.

Nucleic Acids Res 2016 05 15;44(9):3989-4004. Epub 2016 Apr 15.

School of Anatomy, Physiology and Human Biology, The University of Western Australia, Crawley, Western Australia, WA 6009, Australia Harry Perkins Institute of Medical Research, QEII Medical Centre, Nedlands, WA 6009, Australia

Nuclear proteins are often given a concise title that captures their function, such as 'transcription factor,' 'polymerase' or 'nuclear-receptor.' However, for members of the Drosophila behavior/human splicing (DBHS) protein family, no such clean-cut title exists. DBHS proteins are frequently identified engaging in almost every step of gene regulation, including but not limited to, transcriptional regulation, RNA processing and transport, and DNA repair. Herein, we present a coherent picture of DBHS proteins, integrating recent structural insights on dimerization, nucleic acid binding modalities and oligomerization propensity with biological function. The emerging paradigm describes a family of dynamic proteins mediating a wide range of protein-protein and protein-nucleic acid interactions, on the whole acting as a multipurpose molecular scaffold. Overall, significant steps toward appreciating the role of DBHS proteins have been made, but we are only beginning to understand the complexity and broader importance of this family in cellular biology.
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http://dx.doi.org/10.1093/nar/gkw271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4872119PMC
May 2016

Redefining the structural motifs that determine RNA binding and RNA editing by pentatricopeptide repeat proteins in land plants.

Plant J 2016 Feb;85(4):532-47

Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley, 6009, Australia.

The pentatricopeptide repeat (PPR) proteins form one of the largest protein families in land plants. They are characterised by tandem 30-40 amino acid motifs that form an extended binding surface capable of sequence-specific recognition of RNA strands. Almost all of them are post-translationally targeted to plastids and mitochondria, where they play important roles in post-transcriptional processes including splicing, RNA editing and the initiation of translation. A code describing how PPR proteins recognise their RNA targets promises to accelerate research on these proteins, but making use of this code requires accurate definition and annotation of all of the various nucleotide-binding motifs in each protein. We have used a structural modelling approach to define 10 different variants of the PPR motif found in plant proteins, in addition to the putative deaminase motif that is found at the C-terminus of many RNA-editing factors. We show that the super-helical RNA-binding surface of RNA-editing factors is potentially longer than previously recognised. We used the redefined motifs to develop accurate and consistent annotations of PPR sequences from 109 genomes. We report a high error rate in PPR gene models in many public plant proteomes, due to gene fusions and insertions of spurious introns. These consistently annotated datasets across a wide range of species are valuable resources for future comparative genomics studies, and an essential pre-requisite for accurate large-scale computational predictions of PPR targets. We have created a web portal (http://www.plantppr.com) that provides open access to these resources for the community.
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http://dx.doi.org/10.1111/tpj.13121DOI Listing
February 2016

Caenorhabditis elegans NONO-1: Insights into DBHS protein structure, architecture, and function.

Protein Sci 2015 Dec 29;24(12):2033-43. Epub 2015 Oct 29.

School of Chemistry and Biochemistry, The University of Western Australia, Crawley, Western Australia, 6009, Australia.

Members of the Drosophila behavior/human splicing (DBHS) protein family have been characterized in the vertebrates Homo sapiens and Mus musculus, and the invertebrates Drosophila melanogaster and Chironomus tentans. Collectively, both vertebrate and invertebrate DBHS proteins function throughout gene regulation, largely but not always, within the nucleus. In this study, we report a structural and bioinformatic analysis of the DBHS protein family to guide future studies into DBHS protein function. To explore the structural plasticity of the family, we describe the 2.4 Å crystal structure of Caenorhabditis elegans non-POU domain-containing octamer-binding protein 1 (NONO-1). The structure is dimeric, with a domain arrangement consistent with mammalian DBHS proteins. Comparison with the DBHS structures available from H. sapiens reveals that there is inherent domain flexibility within the homologous DBHS region. Mapping amino acid similarity within the family to the NONO-1 dimer highlights the dimer interface, coiled-coil oligomerization motif, and putative RNA binding surfaces. Surprisingly, the interior surface of RNA recognition motif 2 (RRM2) that faces a large internal void is highly variable, but the external β2-β3 loops of RRM2 show remarkable preservation. Overall, the DBHS region is under strong purifying selection, whereas the sequences N- and C-terminal to the DBHS region are less constrained. The findings described in this study provide a molecular basis for further investigation into the mechanistic function of the DBHS protein family in biology.
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http://dx.doi.org/10.1002/pro.2816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4815227PMC
December 2015

The ins and outs of lncRNA structure: How, why and what comes next?

Biochim Biophys Acta 2016 Jan 29;1859(1):46-58. Epub 2015 Aug 29.

School of Chemistry and Biochemistry, The University of Western Australia, Crawley, Western Australia 6009, Australia. Electronic address:

The field of structural biology has the unique advantage of being able to provide a comprehensive picture of biological mechanisms at the molecular and atomic level. Long noncoding RNAs (lncRNAs) represent the new frontier in the molecular biology of complex organisms yet remain the least characterised of all the classes of RNA. Thousands of new lncRNAs are being reported each year yet very little structural data exists for this rapidly expanding field. The length of lncRNAs ranges from 200 nt to over 100 kb in length and they generally exhibit low cellular abundance. Therefore, obtaining sufficient quantities of lncRNA to use for structural analysis is challenging. However, as technologies develop structures of lncRNAs are starting to emerge providing important information regarding their mechanism of action. Here we review the current methods used to determine the structure of lncRNA and lncRNA:protein complexes and describe the significant contribution structural biology has and will make to the field of lncRNA research. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa.
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http://dx.doi.org/10.1016/j.bbagrm.2015.08.009DOI Listing
January 2016

Prion-like domains in RNA binding proteins are essential for building subnuclear paraspeckles.

J Cell Biol 2015 Aug;210(4):529-39

The Harry Perkins Institute of Medical Research, Queen Elizabeth II Medical Centre, Nedlands, WA 6009, Australia The Centre for Medical Research, The University of Western Australia, Crawley, WA 6009, Australia

Prion-like domains (PLDs) are low complexity sequences found in RNA binding proteins associated with the neurodegenerative disorder amyotrophic lateral sclerosis. Recently, PLDs have been implicated in mediating gene regulation via liquid-phase transitions that drive ribonucleoprotein granule assembly. In this paper, we report many PLDs in proteins associated with paraspeckles, subnuclear bodies that form around long noncoding RNA. We mapped the interactome network of paraspeckle proteins, finding enrichment of PLDs. We show that one protein, RBM14, connects key paraspeckle subcomplexes via interactions mediated by its PLD. We further show that the RBM14 PLD, as well as the PLD of another essential paraspeckle protein, FUS, is required to rescue paraspeckle formation in cells in which their endogenous counterpart has been knocked down. Similar to FUS, the RBM14 PLD also forms hydrogels with amyloid-like properties. These results suggest a role for PLD-mediated liquid-phase transitions in paraspeckle formation, highlighting this nuclear body as an excellent model system for understanding the perturbation of such processes in neurodegeneration.
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http://dx.doi.org/10.1083/jcb.201504117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539981PMC
August 2015

PLANT EVOLUTION. Convergent evolution of strigolactone perception enabled host detection in parasitic plants.

Science 2015 Jul;349(6247):540-3

Department of Genetics, University of Georgia, Athens, GA 30602, USA.

Obligate parasitic plants in the Orobanchaceae germinate after sensing plant hormones, strigolactones, exuded from host roots. In Arabidopsis thaliana, the α/β-hydrolase D14 acts as a strigolactone receptor that controls shoot branching, whereas its ancestral paralog, KAI2, mediates karrikin-specific germination responses. We observed that KAI2, but not D14, is present at higher copy numbers in parasitic species than in nonparasitic relatives. KAI2 paralogs in parasites are distributed into three phylogenetic clades. The fastest-evolving clade, KAI2d, contains the majority of KAI2 paralogs. Homology models predict that the ligand-binding pockets of KAI2d resemble D14. KAI2d transgenes confer strigolactone-specific germination responses to Arabidopsis thaliana. Thus, the KAI2 paralogs D14 and KAI2d underwent convergent evolution of strigolactone recognition, respectively enabling developmental responses to strigolactones in angiosperms and host detection in parasites.
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http://dx.doi.org/10.1126/science.aab1140DOI Listing
July 2015