Publications by authors named "Rachel Allison"

20 Publications

  • Page 1 of 1

De Novo VPS4A Mutations Cause Multisystem Disease with Abnormal Neurodevelopment.

Am J Hum Genet 2020 12 12;107(6):1129-1148. Epub 2020 Nov 12.

Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK; Department of Medical Genetics, University of Cambridge, Cambridge CB2 0QQ, UK. Electronic address:

The endosomal sorting complexes required for transport (ESCRTs) are essential for multiple membrane modeling and membrane-independent cellular processes. Here we describe six unrelated individuals with de novo missense variants affecting the ATPase domain of VPS4A, a critical enzyme regulating ESCRT function. Probands had structural brain abnormalities, severe neurodevelopmental delay, cataracts, growth impairment, and anemia. In cultured cells, overexpression of VPS4A mutants caused enlarged endosomal vacuoles resembling those induced by expression of known dominant-negative ATPase-defective forms of VPS4A. Proband-derived fibroblasts had enlarged endosomal structures with abnormal accumulation of the ESCRT protein IST1 on the limiting membrane. VPS4A function was also required for normal endosomal morphology and IST1 localization in iPSC-derived human neurons. Mutations affected other ESCRT-dependent cellular processes, including regulation of centrosome number, primary cilium morphology, nuclear membrane morphology, chromosome segregation, mitotic spindle formation, and cell cycle progression. We thus characterize a distinct multisystem disorder caused by mutations affecting VPS4A and demonstrate that its normal function is required for multiple human developmental and cellular processes.
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http://dx.doi.org/10.1016/j.ajhg.2020.10.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820634PMC
December 2020

Spastin MIT Domain Disease-Associated Mutations Disrupt Lysosomal Function.

Front Neurosci 2019 8;13:1179. Epub 2019 Nov 8.

Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.

The hereditary spastic paraplegias (HSPs) are genetic motor neuron diseases characterized by progressive degeneration of corticospinal tract axons. Mutations in SPAST, encoding the microtubule-severing ATPase spastin, are the most common causes of HSP. The broad SPAST mutational spectrum indicates a haploinsufficiency pathogenic mechanism in most cases. Most missense mutations cluster in the ATPase domain, where they disrupt the protein's ability to sever microtubules. However, several putative missense mutations in the protein's microtubule interacting and trafficking (MIT) domain have also been described, but the pathogenicity of these mutations has not been verified with functional studies. Spastin promotes endosomal tubule fission, and defects in this lead to lysosomal enzyme mistrafficking and downstream lysosomal abnormalities. We investigated the function of three disease-associated spastin MIT mutants and found that none was able to promote normal endosomal tubule fission, lysosomal enzyme receptor trafficking, or lysosomal morphology. One of the mutations affected recruitment of spastin to endosomes, a property that requires the canonical function of the MIT domain in binding endosomal sorting complex required for transport (ESCRT)-III proteins. However, the other mutants did not affect spastin's endosomal recruitment, raising the possibility of pathologically important non-canonical roles for the MIT domain. In conclusion, we demonstrate that spastin MIT mutants cause functional abnormalities related to the pathogenesis of HSP. These mutations do not directly affect spastin's microtubule-severing capacity, and so we identify a new molecular pathological mechanism by which spastin mutations may cause disease.
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http://dx.doi.org/10.3389/fnins.2019.01179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856053PMC
November 2019

ESCRT-III-associated proteins and spastin inhibit protrudin-dependent polarised membrane traffic.

Cell Mol Life Sci 2020 Jul 5;77(13):2641-2658. Epub 2019 Oct 5.

Department of Medical Genetics and Cambridge Institute for Medical Research, The Keith Peters Building, Cambridge Biomedical Campus, University of Cambridge, Cambridge, CB2 0XY, UK.

Mutations in the gene encoding the microtubule severing ATPase spastin are the most frequent cause of hereditary spastic paraplegia, a genetic condition characterised by length-dependent axonal degeneration. Here, we show that HeLa cells lacking spastin and embryonic fibroblasts from a spastin knock-in mouse model become highly polarised and develop cellular protrusions. In HeLa cells, this phenotype was rescued by wild-type spastin, but not by forms unable to sever microtubules or interact with endosomal ESCRT-III proteins. Cells lacking the spastin-interacting ESCRT-III-associated proteins IST1 or CHMP1B also developed protrusions. The protrusion phenotype required protrudin, a RAB-interacting protein that interacts with spastin and localises to ER-endosome contact sites, where it promotes KIF5-dependent endosomal motility to protrusions. Consistent with this, the protrusion phenotype in cells lacking spastin also required KIF5. Lack or mutation of spastin resulted in functional consequences for receptor traffic of a pathway implicated in HSP, as Bone Morphogenetic Protein receptor distribution became polarised. Our results, therefore, identify a novel role for ESCRT-III proteins and spastin in regulating polarised membrane traffic.
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http://dx.doi.org/10.1007/s00018-019-03313-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7320071PMC
July 2020

BMP- and neuropilin 1-mediated motor axon navigation relies on spastin alternative translation.

Development 2018 09 12;145(17). Epub 2018 Sep 12.

Sorbonne Universités, UPMC Université Paris 06, INSERM, CNRS, Neuroscience Paris Seine - Institut de Biologie Paris-Seine (NPS-IBPS), 75005 Paris, France

Functional analyses of genes responsible for neurodegenerative disorders have unveiled crucial links between neurodegenerative processes and key developmental signalling pathways. Mutations in -encoding spastin cause hereditary spastic paraplegia (HSP). Spastin is involved in diverse cellular processes that couple microtubule severing to membrane remodelling. Two main spastin isoforms are synthesised from alternative translational start sites (M1 and M87). However, their specific roles in neuronal development and homeostasis remain largely unknown. To selectively unravel their neuronal function, we blocked spastin synthesis from each initiation codon during zebrafish development and performed rescue analyses. The knockdown of each isoform led to different motor neuron and locomotion defects, which were not rescued by the selective expression of the other isoform. Notably, both morphant neuronal phenotypes were observed in a CRISPR/Cas9 mutant. We next showed that M1 spastin, together with HSP proteins atlastin 1 and NIPA1, drives motor axon targeting by repressing BMP signalling, whereas M87 spastin acts downstream of neuropilin 1 to control motor neuron migration. Our data therefore suggest that defective BMP and neuropilin 1 signalling may contribute to the motor phenotype in a vertebrate model of spastin depletion.
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http://dx.doi.org/10.1242/dev.162701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6141775PMC
September 2018

Mechanistic basis of an epistatic interaction reducing age at onset in hereditary spastic paraplegia.

Brain 2018 05;141(5):1286-1299

Department of Medical Genetics and Cambridge Institute for Medical Research, University of Cambridge, UK.

Many genetic neurological disorders exhibit variable expression within affected families, often exemplified by variations in disease age at onset. Epistatic effects (i.e. effects of modifier genes on the disease gene) may underlie this variation, but the mechanistic basis for such epistatic interactions is rarely understood. Here we report a novel epistatic interaction between SPAST and the contiguous gene DPY30, which modifies age at onset in hereditary spastic paraplegia, a genetic axonopathy. We found that patients with hereditary spastic paraplegia caused by genomic deletions of SPAST that extended into DPY30 had a significantly younger age at onset. We show that, like spastin, the protein encoded by SPAST, the DPY30 protein controls endosomal tubule fission, traffic of mannose 6-phosphate receptors from endosomes to the Golgi, and lysosomal ultrastructural morphology. We propose that additive effects on this pathway explain the reduced age at onset of hereditary spastic paraplegia in patients who are haploinsufficient for both genes.
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http://dx.doi.org/10.1093/brain/awy034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5917785PMC
May 2018

Role of Elemental Sulfur in Forming Latent Precursors of HS in Wine.

J Agric Food Chem 2017 Dec 28;65(48):10542-10549. Epub 2017 Nov 28.

Department of Food Science, Cornell University , Stocking Hall, Ithaca, New York 14853, United States.

The level of hydrogen sulfide (HS) can increase during abiotic storage of wines, and potential latent sources of HS are still under investigation. We demonstrate that elemental sulfur (S) residues on grapes not only can produce HS during fermentation but also can form precursors capable of generating additional HS after bottle storage for 3 months. HS could be released from S-derived precursors by addition of a reducing agent (TCEP), but not by addition of strong brine to induce release of HS from metal sulfide complexes. The size of the TCEP-releasable pool varied among yeast strains. Using the TCEP assay, multiple polar S-derived precursors were detected following normal-phase preparative chromatography. Using reversed-phase liquid chromatography and high-resolution mass spectrometry, we detected an increase in the levels of diglutathione trisulfane (GSSSG) and glutathione disulfide (GSSG) in S-fermented red wine and an increase in the levels of glutathione S-sulfonate (GSSO) and tetrathionate (SO) in S-fermented white wine as compared to controls. GSSSG, but not SO, was shown to evolve HS in the presence of TCEP. Pathways for the formation of GSSSG, GSSG, GSSO, and SO from S are proposed.
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http://dx.doi.org/10.1021/acs.jafc.7b04015DOI Listing
December 2017

Defects in ER-endosome contacts impact lysosome function in hereditary spastic paraplegia.

J Cell Biol 2017 05 7;216(5):1337-1355. Epub 2017 Apr 7.

Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, England, UK

Contacts between endosomes and the endoplasmic reticulum (ER) promote endosomal tubule fission, but the mechanisms involved and consequences of tubule fission failure are incompletely understood. We found that interaction between the microtubule-severing enzyme spastin and the ESCRT protein IST1 at ER-endosome contacts drives endosomal tubule fission. Failure of fission caused defective sorting of mannose 6-phosphate receptor, with consequently disrupted lysosomal enzyme trafficking and abnormal lysosomal morphology, including in mouse primary neurons and human stem cell-derived neurons. Consistent with a role for ER-mediated endosomal tubule fission in lysosome function, similar lysosomal abnormalities were seen in cellular models lacking the WASH complex component strumpellin or the ER morphogen REEP1. Mutations in , , or cause hereditary spastic paraplegia (HSP), a disease characterized by axonal degeneration. Our results implicate failure of the ER-endosome contact process in axonopathy and suggest that coupling of ER-mediated endosomal tubule fission to lysosome function links different classes of HSP proteins, previously considered functionally distinct, into a unifying pathway for axonal degeneration.
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http://dx.doi.org/10.1083/jcb.201609033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5412567PMC
May 2017

Quantitative Gait Analysis Using a Motorized Treadmill System Sensitively Detects Motor Abnormalities in Mice Expressing ATPase Defective Spastin.

PLoS One 2016 28;11(3):e0152413. Epub 2016 Mar 28.

Department of Medical Genetics and Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.

The hereditary spastic paraplegias (HSPs) are genetic conditions in which there is progressive axonal degeneration in the corticospinal tract. Autosomal dominant mutations, including nonsense, frameshift and missense changes, in the gene encoding the microtubule severing ATPase spastin are the most common cause of HSP in North America and northern Europe. In this study we report quantitative gait analysis using a motorized treadmill system, carried out on mice knocked-in for a disease-associated mutation affecting a critical residue in the Walker A motif of the spastin ATPase domain. At 4 months and at one year of age homozygous mutant mice had a number of abnormal gait parameters, including in stride length and stride duration, compared to heterozygous and wild-type littermates. Gait parameters in heterozygous animals did not differ from wild-type littermates. We conclude that quantitative gait analysis using the DigiGait system sensitively detects motor abnormalities in a hereditary spastic paraplegia model, and would be a useful method for analyzing the effects of pharmacological treatments for HSP.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0152413PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4809716PMC
August 2016

Preclinical evaluation of a miniaturized Deep Brain Stimulation electrode lead.

Annu Int Conf IEEE Eng Med Biol Soc 2015 ;2015:6908-11

The effect of miniaturizing the electrode lead for Deep Brain Stimulation (DBS) therapy was investigated in this work. A direct comparison was made between a miniature lead (0.65 mm diameter) and a lead of standard size (1.3 mm). Acute in vivo implantation in two cat brains was performed to evaluate surgical trauma and confirm capacity to target thalamic nuclei. Insertion into a homogeneous gel model of neural tissue was used to compare insertion forces while visualizing the process. The standard size cannula, used first to guide lead insertion, required substantially higher insertion force compared with the miniature version and produced a significantly larger region of tissue disruption. The characteristic hemorrhage and edema extended 119-352 μm from the implanted track surface of the miniature lead and cannula, while these extended 311-571 μm for the standard size lead and cannula. A miniature DBS implant can reduce the extent of trauma and could potentially help improve neural function preservation after functional neurosurgery.
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http://dx.doi.org/10.1109/EMBC.2015.7319981DOI Listing
September 2016

Ultrasound-guided hip injections: a comparative study with fluoroscopy-guided injections.

Arthroscopy 2014 Jan;30(1):42-6

Nashville Sports Medicine Foundation, Nashville, Tennessee, U.S.A.

Purpose: The purpose was to assess ultrasound-guided injections through patient satisfaction in a comparative internally controlled study of fluoroscopic versus ultrasound technique and to quantitate the reliability of the ultrasound method. In addition, the reliability of the ultrasound method was quantitated.

Methods: This study consisted of the first 50 consecutive patients to undergo ultrasound-guided intra-articular injection of the hip (by a nurse practitioner) and who had previously undergone fluoroscopy-guided intra-articular injections by our center's fellowship-trained musculoskeletal radiologists. The patients rated the ultrasound and fluoroscopic experiences on a scale from 1 to 10 for convenience and pain; in addition, they indicated their preference between the 2 techniques. Success of the injection was documented among a total of 206 consecutive patients who underwent ultrasound-guided injections during the period of the controlled study.

Results: For convenience, ultrasound injection had a mean rating of 9.8 whereas fluoroscopic injection had a mean rating of 3.1. For pain, ultrasound had a mean rating of 3 and fluoroscopy had a mean rating of 5.6. These differences were statistically significant (P < .01) in favor of ultrasound. For preference, 49 of 50 patients in the control study (98%) stated that they would prefer the ultrasound injection, whereas 1 was uncertain. The injection was successful in 202 of the first 206 patients (98%) to undergo ultrasound injection, whereas 4 patients required a second pass for successful injection.

Conclusions: In this study in-office ultrasound-guided injections of the hip were more convenient and less painful than fluoroscopy-guided hospital-based injections and were preferred by patients who have undergone both. Furthermore, the ultrasound-guided injections were performed by a recently trained physician extender in contrast to the fluoroscopic method, which was performed by experienced fellowship-trained musculoskeletal radiologists. The procedure is highly successful in the hands of a properly trained clinician.

Level Of Evidence: Level II, prospective comparative study.
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http://dx.doi.org/10.1016/j.arthro.2013.09.083DOI Listing
January 2014

An ESCRT-spastin interaction promotes fission of recycling tubules from the endosome.

J Cell Biol 2013 Aug 29;202(3):527-43. Epub 2013 Jul 29.

Department of Medical Genetics, University of Cambridge, Cambridge CB2 0XY, England, UK.

Mechanisms coordinating endosomal degradation and recycling are poorly understood, as are the cellular roles of microtubule (MT) severing. We show that cells lacking the MT-severing protein spastin had increased tubulation of and defective receptor sorting through endosomal tubular recycling compartments. Spastin required the ability to sever MTs and to interact with ESCRT-III (a complex controlling cargo degradation) proteins to regulate endosomal tubulation. Cells lacking IST1 (increased sodium tolerance 1), an endosomal sorting complex required for transport (ESCRT) component to which spastin binds, also had increased endosomal tubulation. Our results suggest that inclusion of IST1 into the ESCRT complex allows recruitment of spastin to promote fission of recycling tubules from the endosome. Thus, we reveal a novel cellular role for MT severing and identify a mechanism by which endosomal recycling can be coordinated with the degradative machinery. Spastin is mutated in the axonopathy hereditary spastic paraplegia. Zebrafish spinal motor axons depleted of spastin or IST1 also had abnormal endosomal tubulation, so we propose this phenotype is important for axonal degeneration.
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http://dx.doi.org/10.1083/jcb.201211045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734076PMC
August 2013

A double standard for "Hooking Up": How far have we come toward gender equality?

Soc Sci Res 2013 Sep 3;42(5):1191-206. Epub 2013 May 3.

Department of Sociology, University of Illinois at Chicago, 1007 W. Harrison St., M/C 312, Chicago, IL 60607, United States. Electronic address:

While sexual attitudes have liberalized in the past half century, research is mixed as to whether attitudes have become less gendered over time. Recent studies on college students' sexual and romantic relationships suggest that a sexual double standard continues to organize sexuality on many campuses. Data from the Online College Social Life Survey shed light on students' evaluation of casual sex, or "hooking up." In addition to exploring gendered attitudinal patterns, we use gender structure theory to explore how individual characteristics and normative expectations of campus group affiliations shape attitudes. While three quarters of students do not hold different standards for men and women's hooking up, attitudes are more conservative than liberal, with almost half of students losing respect for men and women who hook up "a lot." However, men are more likely to hold a traditional double standard, while women are more likely to espouse egalitarian conservative attitudes. Individual characteristics, including age, religion, race, social class and sexual orientation are frequently related to sexual attitudes, as are number of hook ups, fraternity/sorority affiliation and varsity athletic participation.
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http://dx.doi.org/10.1016/j.ssresearch.2013.04.006DOI Listing
September 2013

Mutations in the ER-shaping protein reticulon 2 cause the axon-degenerative disorder hereditary spastic paraplegia type 12.

J Clin Invest 2012 Feb 9;122(2):538-44. Epub 2012 Jan 9.

Department of Human Genetics, University of Miami Miller School of Medicine, Miami, Florida, USA.

Hereditary spastic paraplegias (HSPs) are a group of genetically heterogeneous neurodegenerative conditions. They are characterized by progressive spastic paralysis of the legs as a result of selective, length-dependent degeneration of the axons of the corticospinal tract. Mutations in 3 genes encoding proteins that work together to shape the ER into sheets and tubules - receptor accessory protein 1 (REEP1), atlastin-1 (ATL1), and spastin (SPAST) - have been found to underlie many cases of HSP in Northern Europe and North America. Applying Sanger and exome sequencing, we have now identified 3 mutations in reticulon 2 (RTN2), which encodes a member of the reticulon family of prototypic ER-shaping proteins, in families with spastic paraplegia 12 (SPG12). These autosomal dominant mutations included a complete deletion of RTN2 and a frameshift mutation predicted to produce a highly truncated protein. Wild-type reticulon 2, but not the truncated protein potentially encoded by the frameshift allele, localized to the ER. RTN2 interacted with spastin, and this interaction required a hydrophobic region in spastin that is involved in ER localization and that is predicted to form a curvature-inducing/sensing hairpin loop domain. Our results directly implicate a reticulon protein in axonopathy, show that this protein participates in a network of interactions among HSP proteins involved in ER shaping, and further support the hypothesis that abnormal ER morphogenesis is a pathogenic mechanism in HSP.
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http://dx.doi.org/10.1172/JCI60560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3266795PMC
February 2012

The AAA ATPase spastin links microtubule severing to membrane modelling.

Biochim Biophys Acta 2012 Jan 25;1823(1):192-7. Epub 2011 Aug 25.

Department of Medical Genetics and Cambridge Institute for Medical Research, University of Cambridge, UK.

In 1999, mutations in the gene encoding the microtubule severing AAA ATPase spastin were identified as a major cause of a genetic neurodegenerative condition termed hereditary spastic paraplegia (HSP). This finding stimulated intense study of the spastin protein and over the last decade, a combination of cell biological, in vivo, in vitro and structural studies have provided important mechanistic insights into the cellular functions of the protein, as well as elucidating cell biological pathways that might be involved in axonal maintenance and degeneration. Roles for spastin have emerged in shaping the endoplasmic reticulum and the abscission stage of cytokinesis, in which spastin appears to couple membrane modelling to microtubule regulation by severing.
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http://dx.doi.org/10.1016/j.bbamcr.2011.08.010DOI Listing
January 2012

Convenient, inexpensive quantification of elemental sulfur by simultaneous in situ reduction and colorimetric detection.

Anal Chim Acta 2011 Oct 19;703(1):52-7. Epub 2011 Jul 19.

Department of Food Science, Cornell University, Geneva, NY, USA.

Rapid, inexpensive, and convenient methods for quantifying elemental sulfur (S(0)) with low or sub-μgg(-1) limits of detection would be useful for a range of applications where S(0) can act as a precursor for noxious off-aromas, e.g., S(0) in pesticide residues on winegrapes or as a contaminant in drywall. However, existing quantification methods rely on toxic reagents, expensive and cumbersome equipment, or demonstrate poor selectivity. We have developed and optimized an inexpensive, rapid method (∼15 min per sample) for quantifying S(0) in complex matrices. Following dispersion of the sample in PEG-400 and buffering, S(0) is quantitatively reduced to H(2)S in situ by dithiothreitol and simultaneously quantified by commercially available colorimetric H(2)S detection tubes. By employing multiple tubes, the method demonstrated linearity from 0.03 to 100 μg S(0) g(-1) for a 5 g sample (R(2)=0.994, mean CV=6.4%), and the methodological detection limit was 0.01 μg S(0) g(-1). Interferences from sulfite or sulfate were not observed. Mean recovery of an S(0) containing sulfur fungicide in grape macerate was 84.7% with a mean CV of 10.4%. Mean recovery of S(0) in a colloidal sulfur preparation from a drywall matrix was 106.6% with a mean CV of 6.9%. Comparable methodological detection limits, sensitivity, and recoveries were achieved in grape juice, grape macerate and with 1g drywall samples, indicating that the methodology should be robust across a range of complex matrices.
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http://dx.doi.org/10.1016/j.aca.2011.07.010DOI Listing
October 2011

Activation of a microRNA response in trans reveals a new role for poly(A) in translational repression.

Nucleic Acids Res 2011 Jul 8;39(12):5215-31. Epub 2011 Mar 8.

Ecole Normale Supérieure de Lyon, Unité de Virologie Humaine, IFR 128, Lyon, F-69364 France.

Here, we report that the untreated rabbit reticulocyte lysate contains over 300 different endogenous microRNAs together with the major components of the RNA-induced silencing complex and thus can be used as a model in vitro system to study the effects of microRNAs on gene expression. By using this system, we were able to show that microRNA hybridization to its target resulted in a very rapid and strong inhibition of expression that was exerted exclusively at the level of translation initiation with no involvement of transcript degradation or deadenylation. Moreover, we demonstrate that the magnitude of microRNA-induced repression can only be recapitulated in the context of a competitive translating environment. By using a wide spectrum of competitor cellular and viral RNAs, we could further show that competition was not exerted at the level of general components of the translational machinery, but relied exclusively on the presence of the poly(A) tail with virtually no involvement of the cap structure.
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http://dx.doi.org/10.1093/nar/gkr086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3130266PMC
July 2011

Translational control assessed using the tethered function assay in Xenopus oocytes.

Methods 2010 May 25;51(1):165-9. Epub 2010 Feb 25.

Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, UK.

The tethered function assay is a method designed to address the role of an RNA-binding protein upon the metabolism of a reporter RNA. The basis of this assay is to artificially tether a test protein to a reporter mRNA by employing an unrelated bacteriophage MS2 or lambda N RNA-protein interaction, and to assess the effects of the test protein on the reporter RNA. In this chapter, we first discuss the principles and validity of the tethered function approach, drawing on appropriate examples from several cell types and of many proteins that regulate RNA in a variety of processes, including RNA processing (splicing, polyadenylation/deadenylation, decay), localisation and protein synthesis. Secondly, we will focus on the use of this approach to monitor translational activation and repression in Xenopus oocytes, giving a detailed protocol, and discussing possible optimizations we have explored.
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http://dx.doi.org/10.1016/j.ymeth.2010.02.018DOI Listing
May 2010

Vg1RBP phosphorylation by Erk2 MAP kinase correlates with the cortical release of Vg1 mRNA during meiotic maturation of Xenopus oocytes.

RNA 2009 Jun 17;15(6):1121-33. Epub 2009 Apr 17.

Department of Biochemistry, University of Cambridge, Cambridge CB21GA, United Kingdom.

Xenopus Vg1RBP is a member of the highly conserved IMP family of four KH-domain RNA binding proteins, with roles in RNA localization, translational control, RNA stability, and cell motility. Vg1RBP has been implicated in localizing Vg1 mRNAs to the vegetal cortex during oogenesis, in a process mediated by microtubules and microfilaments, and in migration of neural crest cells in embryos. Using c-mos morpholino, kinase inhibitors, and constitutely active recombinant kinases we show that Vg1RBP undergoes regulated phosphorylation by Erk2 MAPK during meiotic maturation, on a single residue, S402, located between the KH2 and KH3 domains. Phosphorylation temporally correlates with the release of Vg1 mRNA from its tight cortical association, assayed in lysates in physiological salt buffers, but does not affect RNA binding, nor self-association of Vg1RBP. U0126, a MAP kinase inhibitor, prevents Vg1RBP cortical release and Vg1 mRNA solubilization in meiotically maturing eggs, while injection of MKK6-DD, a constitutively activated MAP kinase kinase, promotes the release of both Vg1RBP and Vg1 mRNA from insoluble cortical structures. We propose that Erk2 MAP kinase phosphorylation of Vg1RBP regulates the protein:protein-mediated association of Vg1 mRNP with the cytoskeleton and/or ER. Since the MAP kinase site in Vg1RBP is conserved in several IMP homologs, this modification also has important implications for the regulation of IMP proteins in somatic cells.
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http://dx.doi.org/10.1261/rna.1195709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2685525PMC
June 2009

Asymmetrical beta-actin mRNA translation in growth cones mediates attractive turning to netrin-1.

Nat Neurosci 2006 Oct 17;9(10):1247-56. Epub 2006 Sep 17.

Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK.

Local protein synthesis regulates the turning of growth cones to guidance cues, yet little is known about which proteins are synthesized or how they contribute to directional steering. Here we show that beta-actin mRNA resides in Xenopus laevis retinal growth cones where it binds to the RNA-binding protein Vg1RBP. Netrin-1 induces the movement of Vg1RBP granules into filopodia, suggesting that it may direct the localization and translation of mRNAs in growth cones. Indeed, a gradient of netrin-1 activates a translation initiation regulator, eIF-4E-binding protein 1 (4EBP), asymmetrically and triggers a polarized increase in beta-actin translation on the near side of the growth cone before growth cone turning. Inhibition of beta-actin translation abolishes both the asymmetric rise in beta-actin and attractive, but not repulsive, turning. Our data suggest that newly synthesized beta-actin, concentrated near sites of signal reception, provides the directional bias for polymerizing actin in the direction of an attractive stimulus.
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http://dx.doi.org/10.1038/nn1775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1997306PMC
October 2006

Two distinct Staufen isoforms in Xenopus are vegetally localized during oogenesis.

RNA 2004 Nov;10(11):1751-63

Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom.

Localization of mRNA is an important way of generating early asymmetries in the developing embryo. In Drosophila, Staufen is intimately involved in the localization of maternally inherited mRNAs critical for cell fate determination in the embryo. We show that double-stranded RNA-binding Staufen proteins are present in the oocytes of a vertebrate, Xenopus, and are localized to the vegetal cytoplasm, a region where important mRNAs including VegT and Vg1 mRNA become localized. We identified two Staufen isoforms named XStau1 and XStau2, where XStau1 was found to be the principal Staufen protein in oocytes, eggs, and embryos, the levels of both proteins peaking during mid-oogenesis. In adults, Xenopus Staufens are principally expressed in ovary and testis. XStau1 was detectable throughout the oocyte cytoplasm by immunofluorescence and was concentrated in the vegetal cortical region from stage II onward. It showed partial codistribution with subcortical endoplasmic reticulum (ER), raising the possibility that Staufen may anchor mRNAs to specific ER-rich domains. We further showed that XStau proteins are transiently phosphorylated by the MAPK pathway during meiotic maturation, a period during which RNAs such as Vg1 RNA are released from their tight localization at the vegetal cortex. These findings provide evidence that Staufen proteins are involved in targeting and/or anchoring of maternal determinants to the vegetal cortex of the oocyte in Xenopus. The Xenopus oocyte should thus provide a valuable system to dissect the role of Staufen proteins in RNA localization and vertebrate development.
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http://dx.doi.org/10.1261/rna.7450204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1370663PMC
November 2004