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«RNA Meeting 2015 Tuesday 2nd June 2015 Room 3WN 2.1 University of Bath Bath BA2 7AY Meeting proudly sponsored by: TIME SPEAKER INSTITUTION TALK TITLE ...»

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www.southwest.rna.org.uk

RNA Meeting 2015

Tuesday 2nd June 2015

Room 3WN 2.1

University of Bath

Bath BA2 7AY

Meeting proudly sponsored by:

TIME SPEAKER INSTITUTION TALK TITLE

Welcome and Introduction: 9.15-9.20

Mark Lindsay

GENE EXPRESSION 9.20-9.35 Michael Ladomery UWE Bristol The Scd6 protein xRAPB has properties

Chair: Michael Ladomery different from RAP55 relating to early translation in Xenopus oocytes King’s College 9.35-9.50 Dipen Rajgor Mammalian microtubule P-body dynamics London are mediated by nesprin-1 9.50-10.05 Clare Pritchard Cardiff University Cytoplasmic RNA regulation and cell motility 10.05-10.20 Yuhui Doi Birmingham Screening for novel UPF1 interacting University factors in Schizosaccharomyces pombe 10.20-10.35 Subhendu Birmingham Exon Junction Complex (EJC) protein Choudhury University components associate with transcription sites independently of splicing in Drosophila melanogaster Please visit our sponsors’ stalls Tea/Coffee Pre-mRNA SPLICING 11.15-11.30 Emma Woods Cardiff University The role of CD44 variants in fibroblast Chair: Lorna Harries differentiation and monocyte binding 11.30-11.45 Adam Midgley Cardiff University Hyaluronidase-2 dependent regulation of CD44 splicing in anti-fibrotic versus profibrotic cells 11.45-12.00 Ling Li University of Bristol Control of epithelial splicing regulatory proteins (ESRPs) in epithelial

–  –  –

Michael Ladomery* and John Sommerville** **Biomedical Sciences Research Complex, Biomolecular Sciences Building, University of St Andrews, North Haugh, St Andrews, KY16 9TS, UK.

*Present address: Faculty of Health and Applied Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY, UK.

Oocytes accumulate mRNAs in the form of maternal ribonucleoprotein (RNP) particles, the protein components of which determine the location and stability of individual mRNAs prior to translation. The Scd6 family of proteins, typified by RAP55, functions in a wide range of eukaryotes in repressing translation and relocating mRNPs to processing bodies and stress granules.

Here we describe in Xenopus laevis a variant of RAP55, xRAPB, a member of the LSM14B family of proteins found in many other organisms, which also contains conserved Lsm and FDF domains but differs in containing fewer RGG repeats.. xRAPB differs from xRAPA in other respects: it is expressed at high concentration earlier in oogenesis; it interacts specifically with the RNA helicase Xp54; it is first distributed to a subcortical layer and the Balbiani body; it is a component of mRNP particles with a different size distribution; its over-expression can lead to an increase in protein synthesis, whereas knock-down can lead to a decrease. Since Xp54 is a dominant repressor of translation, activation appears to be effected by the dislocation of Xp54 from xRAPB.

MAMMALIAN MICROTUBULE P-BODY DYNAMICS ARE MEDIATED

BY NESPRIN-1 Dipen Rajgor1, Jason A. Mellad1, Daniel Soong1, Jerome B. Rattner2, Marvin J. Fritzler2, and Catherine M. Shanahan1 Cardiovascular Division, BHF Centre of Excellence, James Black Centre, King’s College London Department of Biochemistry and Molecular Biology, University of Calgary, Alberta, Canada Nesprins are a multi-isomeric family of spectrin-repeat (SR) proteins, predominantly known as nuclear envelope scaffolds. However, isoforms that function beyond the nuclear envelope remain poorly examined. Here, we characterize p50Nesp1, a 50-kD isoform that localizes to processing bodies (PBs), where it acts as a microtubule-associated protein capable of linking mRNP complexes to microtubules. Overexpression of dominant-negative p50Nesp1 caused PB displacement from microtubules, resulting in reduced PB movement and cross talk with stress granules (SGs). These cells were unable to disassemble canonical SGs, leading to cell death and revealing PB–microtubule attachment is required for SG anti-apoptotic functions.

Furthermore, p50Nesp1 was required for miRNA-mediated silencing and interacted with core miRISC silencers Ago2 and DDx6 in an RNA-dependent manner. These data identify p50Nesp1 as a multi-functional PB component and microtubule scaffold necessary for RNA granule dynamics and provides evidence for PB and SG micro-heterogeneity.

CYTOPLASMIC RNA REGULATION AND CELL MOTILITY

Clare Pritchard1, Kate Comber2, Will Wood2 and Sonia López de Quinto1

1. Cardiff University, School of Biosciences, Museum Avenue, Cardiff CF10 3AX.

2. University of Bristol, School of Cellular and Molecular Medicine, University Walk, Bristol BS8 1TD Motile cells are highly polarised. One way in which this polarity may be achieved is through the localisation and local translation of mRNA by RNA-binding proteins (RBPs). In cultured cells, β-actin mRNA is localised and translationally-repressed at the leading edge of migratory fibroblasts by the RBP, Zipcode-binding protein 1 (ZBP1). Delocalisation of β-actin mRNA results in a loss of directional cell migration, suggesting that RNA localisation is required for proper cell motility. However, it is still unclear whether this type of cytoplasmic RNA regulation is relevant in vivo, in the 3D context of a living organism.





We have established an in vivo model system, using Drosophila embryonic macrophages, to study the role of RNA regulation in cell motility within the context of a living organism. Analysis of the localisation of Imp (Drosophila ZBP1 homologue) in Drosophila macrophages revealed that, contrary to published reports of cultured cells, Imp is not localised at the leading edge of migratory cells. Conversely, Imp was found enriched at the base of small protrusions which may correspond to either microtubule or actin extensions.

Overexpression of Imp in macrophages impaired both their developmental migration and directed migration to epithelial wounds. Our in vivo characterization revealed that haemocytes over-expressing Imp phenocopied the migratory defects observed upon reduction of β-integrin levels in Drosophila macrophages. Interestingly, we found that Imp binds the 3’UTR of βintegrin mRNA, and both proteins - Imp and b-integrin – co-localise in distinct cytoplasmic granules in Drosophila cultured haemocytes. Furthermore, we observed an increase in βintegrin protein levels upon Imp-RNAi treatment, suggesting that Imp represents a novel regulator of β-integrin expression.

SCREENING FOR NOVEL UPF1 INTERACTING FACTORS IN

SCHIZOSACCHAROMYCES POMBE

Yuhui Dou*, Jianming Wang and Saverio Brogna School of Bioscience, University of Birmingham, Birmingham, UK UPF1 is an essential factor in nonsense-mediated mRNA decay (NMD), it also play key roles in other mRNA decay mechanisms. In recent years its nuclear roles are emerging. However, the role of UPF1 is still not fully understood. To have a better understanding of the role of UPF1, we used Schizosaccharomyces pombe as a model organism to screen for UPF1 interacting factors. A systematic genetic interaction screen was carried out to identify novel UPF1 interacting factors. A group of putative UPF1 interacting factors were identified in this study. And among these factors, AIR1 and PPN1 which have RNA metabolic roles and function in the nucleus were selected for further study. Through tetrad dissection and spot assay, the interaction between UPF1 and AIR1, UPF1 and PPN1 were further confirmed. And a C-terminal HA-tagged AIR1 strain was constructed for further investigations on physical interactions. This study discovered novel UPF1 interacting factors, which can improve our understanding on the role of UPF and also provided fundamental information and materials for further study.

EXON JUNCTION COMPLEX (EJC) PROTEIN COMPONENTS

ASSOCIATE WITH TRANSCRIPTION SITES INDEPENDENTLY OF

SPLICING IN DROSOPHILA MELANOGASTER

Subhendu Roy Choudhury* and Saverio Brogna School of Bioscience, University of Birmingham, Birmingham, UK The results of number of studies across organisms show that pre-mRNA splicing, a strictly nuclear process, affects NMD, a process expected to be strictly cytoplasmic. It has been proposed that this link is mediated by the exon junction complex (EJC), a multiprotein complex deposited during splicing in the nucleus, which remains associated with the mRNA during export to the cytoplasm. Some observations are not consistent with this function attributed to the EJC. Additionally, all of the proteins that constitute the EJC are well conserved in Drosophila, yet these proteins are not required for NMD in this organism. To understand better the function of the EJC, we aimed to visualize its association with nascent RNA at the polytene chromosomes of Drosophila. Surprisingly, EJC recruitment is RNA-independent at both introncontaining and intron-less genes. Additionally, we found that not all of the EJC components are always present at transcription sites, suggesting that EJC assembly is not an obligatory step in mRNP biogenesis. Similar observations were made in S2 Drosophila cells using chromatin immunoprecepitation coupled to high throughput sequencing (ChIP-seq). Our data suggest that the core EJC components Y14 and Mago regulate transcription.

THE ROLE OF CD44 VARIANTS IN FIBROBLAST DIFFERENTIATION

AND MONOCYTE BINDING

E Woods, A. Midgley, T Bowen, R Steadman Cardiff University School of Medicine, Institute of Molecular and Experimental Medicine, Section of Nephrology, Cardiff Institute of Tissue Engineering and Repair, Heath Park CF14 4XN CD44 is a transmembrane surface receptor that is expressed as multiple variants. This variability in protein expression is due to the alternative splicing of 10 exons within the CD44 gene and a high level of post-transcriptional modifications.

Chronic kidney disease (CKD) is a progressive fibrotic disease that results in the destruction of kidney tissue and the loss of renal function. Transforming Growth Factor Beta (TGF-β1) has been widely implicated in fibrosis through its promotion of fibroblast to myofibroblast differentiation. Myofibroblasts form collagen rich scars and have a contractile phenotype due to the expression of α smooth muscle actin (αSMA) intracellular stress fibres. Central to the function of TGF-β1 is the assembly of a cell-surface protein complex consisting of CD44 (receptor for hyaluronan) and the epidermal growth factor receptor (EGFR). Without the formation of this complex, myofibroblasts cannot differentiate from fibroblasts. Furthermore, the inflammatory cytokine interleukin 1 beta (IL-1) enhances monoctye binding to resident fibroblasts. The increase in monoctyes present in the surrounding tissue activates an inflammatory response, hence, contributing to the fibrotic progression. Fibroblasts stimulated with IL-1 have a re-organisation of Hyaluronan (HA) on cell membrane protrusions where CD44 co-localises with intercellular adhesion molecule 1 (ICAM-1).

This study aimed to identify which CD44 spliced variants are expressed in fibroblasts and to elucidate which of the variants are implicated in TGF-1 induced fibroblast differentiation and Il1- induced monocyte binding.

HYALURONIDASE-2 DEPENDENT REGULATION OF CD44 SPLICING

IN ANTI-FIBROTIC VERSUS PRO-FIBROTIC CELLS.

Adam Midgley1, Robert Steadman1, Vincent Hascall2, Timothy Bowen1, Aled Phillips1 & Soma Meran1 Institute of Nephrology, School of Medicine, Cardiff University, Cardiff UK.

Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, Ohio.

This work was funded by the UK Medical Research Council Progressive fibrosis resulting in loss of organ function comprises a wide variety of chronic disorders including cardiac, kidney, lung and liver failure. Fibroblasts are the principal effector cells in fibrosis, and differentiate to their active pro-fibrotic phenotype (myofibroblasts) under the influence of the cytokine Transforming Growth Factor (TGF)-1. Our recent studies indicate that the anti-fibrotic growth factor Bone Morphogenetic Protein-(BMP)-7 antagonises the effects of TGF-1 in myofibroblasts and renders these cells resistant to differentiation.

Hyal2 and a specific variant isoform of CD44, CD44v7/8 were noted to play critical roles in BMP7-driven resistance to myofibroblast differentiation. Hyal2 knockdown modulated the expression of CD44s and CD44v7/8, however the exact mechanism through which Hyal2 mediates CD44v7/8 expression and BMP7 anti-fibrotic effects were unclear. Here, we investigate the role of Hyal2 in the regulation of CD44 (standard and variant isoform) expression.

CD44 and its variant isoform expression levels differ depending on cellular treatment.

Fibroblasts were incubated with TGF-1 to induce myofibroblast differentiation, and compared to cells incubated with BMP7 or IL1 to induce the differentiation-resistant and immunoassociated phenotypes respectively. Hyal2 expression, distribution and cellular localisation were assessed using QPCR, immunocytochemistry and immunoblotting. ChIP and siRNA techniques were used to determine the functional significance of Hyal2 and splice regulators in the experimental systems. A panel of candidate splice regulators common to mesenchymal cell types which target CD44 were assessed for changes in expression: TRA2, SLM2, SRSF2 and SRSF6.

CONTROL OF EPITHELIAL SPLICING REGULATORY PROTEINS

(ESRPs) IN EPITHELIAL-MESENCHYMAL TRANSITIONS

Ling Li and 1Sebastian Oltean

School of Physiology and Pharmacology, University of Bristol, Bristol BS1 3NY



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