DOCSLIB.ORG

Retinal Pigment Epithelium Degeneration Caused by Aggregation of PRPF31 and the Role of HSP70 Family of Proteins Lourdes Valdés-Sánchez1†, Sofia M

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Retinal Pigment Epithelium Degeneration Caused by Aggregation of PRPF31 and the Role of HSP70 Family of Proteins Lourdes Valdés-Sánchez1†, Sofia M Valdés-Sánchez et al. Molecular Medicine (2020) 26:1 Molecular Medicine https://doi.org/10.1186/s10020-019-0124-z RESEARCH ARTICLE Open Access Retinal pigment epithelium degeneration caused by aggregation of PRPF31 and the role of HSP70 family of proteins Lourdes Valdés-Sánchez1†, Sofia M. Calado1,2†, Berta de la Cerda1, Ana Aramburu1,3, Ana Belén García-Delgado1, Simone Massalini1,4, Adoración Montero-Sánchez1, Vaibhav Bhatia1, Eduardo Rodríguez-Bocanegra1,5, Andrea Diez-Lloret1, Daniel Rodríguez-Martínez1, Christina Chakarova6, Shom S. Bhattacharya6* and Francisco J. Díaz-Corrales1* Abstract Background: Mutations in pre-mRNA splicing factor PRPF31 can lead to retinitis pigmentosa (RP). Although the exact disease mechanism remains unknown, it has been hypothesized that haploinsufficiency might be involved in the pathophysiology of the disease. Methods: In this study, we have analyzed a mouse model containing the p.A216P mutation in Prpf31 gene. Results: We found that mutant Prpf31 protein produces cytoplasmic aggregates in the retinal pigment epithelium and decreasing the protein levels of this splicing factor in the nucleus. Additionally, normal protein was recruited in insoluble aggregates when the mutant protein was overexpressed in vitro. In response to protein aggregation, Hspa4l is overexpressed. This member of the HSP70 family of chaperones might contribute to the correct folding and solubilization of the mutant protein, allowing its translocation to the nucleus. Conclusions: Our data suggests that a mechanism haploinsufficiency and dominant-negative is involved in retinal degeneration due to mutations in PRPF31. HSP70 over-expression might be a new therapeutic target for the treatment of retinal degeneration due to PRPF31 mutations. Keywords: HSP70, PRPF31, Retinal degeneration, Retinal pigment epithelium, Retinitis pigmentosa Background night blindness, which usually starts during adoles- Retinitis pigmentosa (RP) is one of a diverse group of ret- cence and progresses with constriction of the visual inal dystrophies and one of the commonest causes of inher- field and a marked reduction in the amplitude of ited blindness in adults, affecting around 1:4000 individuals electroretinogram (ERG) waves. So far, mutations in worldwide (Verbakel et al., 2018). RP initially presents a more than 80 genes have been implicated in non- progressive impairment and cell death of rod photorecep- syndromic RP (Verbakel et al., 2018). Many of these tors, followed by loss of cones and retinal pigment genes encode for retinal-specific proteins; however, epithelium (RPE). Clinically, RP is characterized by some are ubiquitously expressed, such as splicing fac- tors PRPF3, PRPF4, PRPF6, PRPF8 and PRPF31 (Liu &Zack,2013; Ruzickova & Stanek, 2017). * Correspondence: [email protected]; [email protected] †Lourdes Valdés-Sánchez and Sofia M. Calado contributed equally to this Pre-mRNA splicing is a general cellular function cru- work. cial for the expression of eukaryotic transcripts. It is cat- 6Institute of Ophthalmology, University College London, 11-43 Bath Street, alyzed by the spliceosome, a large ribonucleoprotein London EC1V 9EL, UK 1Regeneration and Cell Therapy Department, Andalusian Molecular Biology complex composed of five small nuclear ribonucleopro- and Regenerative Medicine Centre-CABIMER (Junta de Andalucía), CSIC, tein complexes (Ruzickova & Stanek, 2017). In humans, Universidad de Sevilla, Universidad Pablo de Olavide, Avda. Americo PRPF31 encodes the homolog of S. cerevisiae pre- Vespucio 24, 41092 Seville, Spain Full list of author information is available at the end of the article mRNA processing factor 31, also known as PRPF31 © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Valdés-Sánchez et al. Molecular Medicine (2020) 26:1 Page 2 of 22 protein (Vithana et al., 2001). PRPF31 is required for modulation as a new therapeutic target for the treatment the U4/U6-U5 tri-snRNP formation and spliceosome of RP due to PRPF31 mutations. activity (Makarova et al., 2002; Schaffert et al., 2004). Mutations in PRPF31 have been described as the sec- Methods ond most common cause of autosomal dominant RP Animal handling and eye samples (adRP) known as RP11 (Vithana et al., 2001;Al- Eight to sixteen-month old C57BL/6 J Prpf31+/+ (WT) Maghtheh et al., 1998;Roseetal.,2016)and,al- and C57BL/6 J Prpf31A216P/+ (KI) mice were housed in though PRPF31 is necessary for pre-mRNA splicing in the Biological Resources Unit of CABIMER and kept in every cell, adRP is the only clinical entity associated a temperature-controlled environment (21 ± 1 °C), with a with these mutations. relative humidity of 55 ± 5%, a light/dark cycle 08:00–20: Curiously, within the PRPF31-affected families, it is 00 and given standard mouse chow and water ad libi- common to find asymptomatic carriers due to overex- tum. Mouse genotyping was performed as previously de- pression of the WT allele inherited from the normal par- scribed (Bujakowska et al., 2009). Due to homozygous ent. Therefore, the differential expression of the WT Prpf31A216P/A216P mice are not viable, we use allele explains the incomplete penetrance associated with Prpf31A216P/+ and Prpf31+/+ mice to obtain a similar pro- this RP locus (Rose et al., 2016; Vithana et al., 2003). It portion of KI and WT in each litter. The WT mice used has recently been described that the expression level of as controls in each experiment belonged to the same lit- PRPF31 is regulated by the number of copies of a minis- ter of the Prpf31A216P/+ mice. The rd8 mutants were dis- atellite repeat element-MSR1 located 200 bp upstream of carded in these mice using the specific primers: forward the promoter. High-expressing WT alleles are found in 5′-GCC CCT GTT TGC ATG GAG GAA ACT TGG asymptomatic carriers and low-expressing alleles are as- AAG ACA GTC ACA GTT CTT CTG-3′ and reverse sociated with the disease, where the amount of WT 5′-GCC CCA TTT GCA CAC TGA TGA C-3’ (Matta- PRPF31 protein produced is beneath its threshold for pallil et al., 2012). A group of WT CD-1 mice were also normal function (Rose et al., 2016). used for the immunohistochemistry experiments. Although haploinsufficiency contributes to the physio- All experiments described in this work were per- pathology of the disease, it is still not clear how retinal formed in compliance with the Spanish and European degeneration occurs in patients carrying PRPF31 muta- Laboratory Animal Science Association-FELASA Guide tions. To explore disease mechanisms, two animal for the Care and Use of Laboratory Animals, the Euro- models were previously generated (Bujakowska et al., pean Union Council Directive 2010/63/EU for the use of 2009). One was a heterozygous knockout (KO) mouse animals and the Association for Research in Vision and − (Prpf31+/ ) and the second a knock-in (KI) mouse carry- Ophthalmology-ARVO for the use of animals in oph- ing the point mutation p.A216P (Prpf31A216P/+). This thalmic and vision research. Animal manipulation and mutation was previously identified in RP11 patients with experimental methods have been approved by the Ethics a severe retinal phenotype (Vithana et al., 2001). How- Committee for Animal Experimentation of CABIMER, ever, both heterozygous mouse models did not show any Seville, Spain. All efforts were made to minimize the sign of photoreceptor degeneration and, as expected, the number of animals used and their suffering. Pig and cow homozygous mutant mice were found to be embryonic eye samples were obtained from a local slaughterhouse. lethal (Bujakowska et al., 2009). Based on these results, it Human eye sample for Western blotting was obtained was speculated that Prpf31 is essential for survival and from a deceased healthy donor, in a procedure approved the presence of one WT Prpf31 allele is enough to main- by the Ethics Committee of University Hospital Virgen tain retinal function with no dominant-negative effect of Macarena, Seville, Spain. the p.A216P mutation in mice. More recently, it has been published that three Immunohistochemistry and immunofluorescence splicing-factor mouse models (Prpf3T494M/+, experiments − Prpf8H2309P/+ and Prpf31+/ ) develop late-onset morpho- Immunohistochemistry was performed to evaluate distri- logical changes and dysfunction in the RPE rather than bution of Prpf31 protein in retinal sections of WT CD-1 photoreceptor degeneration (Farkas et al., 2014;Graziotto mice. The animals were euthanized by cervical disloca- et al., 2011). Therefore, in this work, we decided to study tion and the eyes excised quickly and fixed in ice-cold the effect of the p.A216P mutation on RPE. We found 4% paraformaldehyde (PFA) in PBS, overnight, at 4 °C. mislocalization and aggregation of the mutant Prpf31 pro- The fixed eyes were then cryoprotected in 30% sucrose tein with concomitant depletion of normal protein. These in PBS, and embedded in optimal cutting temperature results indicate mixed haploinsufficiency and dominant- compound for cryotome sections. Serial sections of negative mechanisms involved in retinal degeneration due 18 μm thick were mounted in five parallel series and to mutations in PRPF31. Also, this work postulates HSP70 processed for immunohistochemistry. Briefly, the retinal Valdés-Sánchez et al. Molecular Medicine (2020) 26:1 Page 3 of 22 sections were kept in 3% H2O2 in PBS for 30 min. Sam- Incubation with primary antibodies: goat anti-PRPF31 ples were then washed in 0.2% Triton X-100/PBS (PBS- (1:100; OriGene Technologies Inc., TA302582) and T) and blocked in 1% BSA/PBS-T at room temperature mouse anti-HSP70 (1:100; Santa Cruz Biotechnology, for 1 h.
Load more

Recommended publications
  • Premature Termination Codons in PRPF31 Cause Retinitis Pigmentosa Via Haploinsufficiency Due to Nonsense-Mediated Mrna Decay
    Premature termination codons in PRPF31 cause retinitis pigmentosa via haploinsufficiency due to nonsense-mediated mRNA decay Thomas Rio Frio, … , Jacques S. Beckmann, Carlo Rivolta J Clin Invest. 2008;118(4):1519-1531. https://doi.org/10.1172/JCI34211. Research Article Genetics Dominant mutations in the gene encoding the mRNA splicing factor PRPF31 cause retinitis pigmentosa, a hereditary form of retinal degeneration. Most of these mutations are characterized by DNA changes that lead to premature termination codons. We investigated 6 different PRPF31 mutations, represented by single-base substitutions or microdeletions, in cell lines derived from 9 patients with dominant retinitis pigmentosa. Five of these mutations lead to premature termination codons, and 1 leads to the skipping of exon 2. Allele-specific measurement of PRPF31 transcripts revealed a strong reduction in the expression of mutant alleles. As a consequence, total PRPF31 protein abundance was decreased, and no truncated proteins were detected. Subnuclear localization of the full-length PRPF31 that was present remained unaffected. Blocking nonsense-mediated mRNA decay significantly restored the amount of mutant PRPF31 mRNA but did not restore the synthesis of mutant proteins, even in conjunction with inhibitors of protein degradation pathways. Our results indicate that most PRPF31 mutations ultimately result in null alleles through the activation of surveillance mechanisms that inactivate mutant mRNA and, possibly, proteins. Furthermore, these data provide compelling evidence that the pathogenic effect of PRPF31 mutations is likely due to haploinsufficiency rather than to gain of function. Find the latest version: https://jci.me/34211/pdf Research article Premature termination codons in PRPF31 cause retinitis pigmentosa via haploinsufficiency due to nonsense-mediated mRNA decay Thomas Rio Frio,1 Nicholas M.
    [Show full text]
  • Organ Level Protein Networks As a Reference for the Host Effects of the Microbiome
    Downloaded from genome.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press 1 Organ level protein networks as a reference for the host effects of the microbiome 2 3 Robert H. Millsa,b,c,d, Jacob M. Wozniaka,b, Alison Vrbanacc, Anaamika Campeaua,b, Benoit 4 Chassainge,f,g,h, Andrew Gewirtze, Rob Knightc,d, and David J. Gonzaleza,b,d,# 5 6 a Department of Pharmacology, University of California, San Diego, California, USA 7 b Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 8 California, USA 9 c Department of Pediatrics, and Department of Computer Science and Engineering, University of 10 California, San Diego California, USA 11 d Center for Microbiome Innovation, University of California, San Diego, California, USA 12 e Center for Inflammation, Immunity and Infection, Institute for Biomedical Sciences, Georgia State 13 University, Atlanta, GA, USA 14 f Neuroscience Institute, Georgia State University, Atlanta, GA, USA 15 g INSERM, U1016, Paris, France. 16 h Université de Paris, Paris, France. 17 18 Key words: Microbiota, Tandem Mass Tags, Organ Proteomics, Gnotobiotic Mice, Germ-free Mice, 19 Protein Networks, Proteomics 20 21 # Address Correspondence to: 22 David J. Gonzalez, PhD 23 Department of Pharmacology and Pharmacy 24 University of California, San Diego 25 La Jolla, CA 92093 26 E-mail: [email protected] 27 Phone: 858-822-1218 28 1 Downloaded from genome.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press 29 Abstract 30 Connections between the microbiome and health are rapidly emerging in a wide range of 31 diseases.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Molecular Signatures in IASLC/ATS/ERS Classified Growth Patterns of Lung Adenocarcinoma
    RESEARCH ARTICLE Molecular signatures in IASLC/ATS/ERS classified growth patterns of lung adenocarcinoma 1 1 1,2 1¤a Heike ZabeckID *, Hendrik Dienemann , Hans Hoffmann , Joachim Pfannschmidt , Arne Warth2,3, Philipp A. Schnabel3¤b, Thomas Muley2,4, Michael Meister2,4, Holger SuÈ ltmann2,5, Holger FroÈ hlich6, Ruprecht Kuner2,5¤c, Felix Lasitschka3 1 Department of Thoracic Surgery, Thoraxklinik, University Hospital Heidelberg, Heidelberg, Germany, 2 Translational Lung Research Centre Heidelberg (TLRC-H), German Centre for Lung Research (DZL), a1111111111 Heidelberg, Germany, 3 Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany, a1111111111 4 Translational Research Unit (STF), Thoraxklinik, University of Heidelberg, Heidelberg, Germany, 5 Cancer a1111111111 Genome Research (B063), German Cancer Research Center (DKFZ) and German Cancer Consortium a1111111111 (DKTK), Heidelberg, Germany, 6 Institute for Computer Science, c/o Bonn-Aachen International Center for a1111111111 IT, Algorithmic Bioinformatics, University of Bonn, Bonn, Germany ¤a Current address: Department of Thoracic Surgery, Lung Clinic Heckeshorn at HELIOS Hospital Emil von Behring, Berlin, Germany ¤b Current address: Institute of Pathology, Saarland University, Homburg/Saar, Germany ¤c Current address: TRONÐTranslational Oncology at the University Medical Center of Johannes OPEN ACCESS Gutenberg University, Mainz, Germany * [email protected] Citation: Zabeck H, Dienemann H, Hoffmann H, Pfannschmidt J, Warth A, Schnabel PA, et al. (2018) Molecular signatures in IASLC/ATS/ERS classified growth patterns of lung adenocarcinoma. Abstract PLoS ONE 13(10): e0206132. https://doi.org/ 10.1371/journal.pone.0206132 Editor: Stefania Crispi, Institute for Bioscience and Background Biotechnology Research, ITALY The current classification of human lung adenocarcinoma defines five different histological Received: April 4, 2018 growth patterns within the group of conventional invasive adenocarcinomas.
    [Show full text]
  • Transcriptome Sequencing and Genome-Wide Association Analyses Reveal Lysosomal Function and Actin Cytoskeleton Remodeling in Schizophrenia and Bipolar Disorder
    Molecular Psychiatry (2015) 20, 563–572 © 2015 Macmillan Publishers Limited All rights reserved 1359-4184/15 www.nature.com/mp ORIGINAL ARTICLE Transcriptome sequencing and genome-wide association analyses reveal lysosomal function and actin cytoskeleton remodeling in schizophrenia and bipolar disorder Z Zhao1,6,JXu2,6, J Chen3,6, S Kim4, M Reimers3, S-A Bacanu3,HYu1, C Liu5, J Sun1, Q Wang1, P Jia1,FXu2, Y Zhang2, KS Kendler3, Z Peng2 and X Chen3 Schizophrenia (SCZ) and bipolar disorder (BPD) are severe mental disorders with high heritability. Clinicians have long noticed the similarities of clinic symptoms between these disorders. In recent years, accumulating evidence indicates some shared genetic liabilities. However, what is shared remains elusive. In this study, we conducted whole transcriptome analysis of post-mortem brain tissues (cingulate cortex) from SCZ, BPD and control subjects, and identified differentially expressed genes in these disorders. We found 105 and 153 genes differentially expressed in SCZ and BPD, respectively. By comparing the t-test scores, we found that many of the genes differentially expressed in SCZ and BPD are concordant in their expression level (q ⩽ 0.01, 53 genes; q ⩽ 0.05, 213 genes; q ⩽ 0.1, 885 genes). Using genome-wide association data from the Psychiatric Genomics Consortium, we found that these differentially and concordantly expressed genes were enriched in association signals for both SCZ (Po10 − 7) and BPD (P = 0.029). To our knowledge, this is the first time that a substantially large number of genes show concordant expression and association for both SCZ and BPD. Pathway analyses of these genes indicated that they are involved in the lysosome, Fc gamma receptor-mediated phagocytosis, regulation of actin cytoskeleton pathways, along with several cancer pathways.
    [Show full text]
  • Genes with 5' Terminal Oligopyrimidine Tracts Preferentially Escape Global Suppression of Translation by the SARS-Cov-2 NSP1 Protein
    Downloaded from rnajournal.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Genes with 5′ terminal oligopyrimidine tracts preferentially escape global suppression of translation by the SARS-CoV-2 Nsp1 protein Shilpa Raoa, Ian Hoskinsa, Tori Tonna, P. Daniela Garciaa, Hakan Ozadama, Elif Sarinay Cenika, Can Cenika,1 a Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, USA 1Corresponding author: [email protected] Key words: SARS-CoV-2, Nsp1, MeTAFlow, translation, ribosome profiling, RNA-Seq, 5′ TOP, Ribo-Seq, gene expression 1 Downloaded from rnajournal.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Abstract Viruses rely on the host translation machinery to synthesize their own proteins. Consequently, they have evolved varied mechanisms to co-opt host translation for their survival. SARS-CoV-2 relies on a non-structural protein, Nsp1, for shutting down host translation. However, it is currently unknown how viral proteins and host factors critical for viral replication can escape a global shutdown of host translation. Here, using a novel FACS-based assay called MeTAFlow, we report a dose-dependent reduction in both nascent protein synthesis and mRNA abundance in cells expressing Nsp1. We perform RNA-Seq and matched ribosome profiling experiments to identify gene-specific changes both at the mRNA expression and translation level. We discover that a functionally-coherent subset of human genes are preferentially translated in the context of Nsp1 expression. These genes include the translation machinery components, RNA binding proteins, and others important for viral pathogenicity. Importantly, we uncovered a remarkable enrichment of 5′ terminal oligo-pyrimidine (TOP) tracts among preferentially translated genes.
    [Show full text]
  • Nuclear PTEN Safeguards Pre-Mrna Splicing to Link Golgi Apparatus for Its Tumor Suppressive Role
    ARTICLE DOI: 10.1038/s41467-018-04760-1 OPEN Nuclear PTEN safeguards pre-mRNA splicing to link Golgi apparatus for its tumor suppressive role Shao-Ming Shen1, Yan Ji2, Cheng Zhang1, Shuang-Shu Dong2, Shuo Yang1, Zhong Xiong1, Meng-Kai Ge1, Yun Yu1, Li Xia1, Meng Guo1, Jin-Ke Cheng3, Jun-Ling Liu1,3, Jian-Xiu Yu1,3 & Guo-Qiang Chen1 Dysregulation of pre-mRNA alternative splicing (AS) is closely associated with cancers. However, the relationships between the AS and classic oncogenes/tumor suppressors are 1234567890():,; largely unknown. Here we show that the deletion of tumor suppressor PTEN alters pre-mRNA splicing in a phosphatase-independent manner, and identify 262 PTEN-regulated AS events in 293T cells by RNA sequencing, which are associated with significant worse outcome of cancer patients. Based on these findings, we report that nuclear PTEN interacts with the splicing machinery, spliceosome, to regulate its assembly and pre-mRNA splicing. We also identify a new exon 2b in GOLGA2 transcript and the exon exclusion contributes to PTEN knockdown-induced tumorigenesis by promoting dramatic Golgi extension and secretion, and PTEN depletion significantly sensitizes cancer cells to secretion inhibitors brefeldin A and golgicide A. Our results suggest that Golgi secretion inhibitors alone or in combination with PI3K/Akt kinase inhibitors may be therapeutically useful for PTEN-deficient cancers. 1 Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025, China. 2 Institute of Health Sciences, Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences and SJTU-SM, Shanghai 200025, China.
    [Show full text]
  • Three Gene-Targeted Mouse Models of RNA Splicing Factor RP Show Late-Onset RPE and Retinal Degeneration John J
    Three Gene-Targeted Mouse Models of RNA Splicing Factor RP Show Late-Onset RPE and Retinal Degeneration John J. Graziotto, Michael Farkas, Kinga Bujakowska, Bertrand M. Deramaudt, Qi Zhang, Emeline F. Nandrot, Chris F. Inglehearn, Shomi S. Bhattacharya, Eric A. Pierce To cite this version: John J. Graziotto, Michael Farkas, Kinga Bujakowska, Bertrand M. Deramaudt, Qi Zhang, et al.. Three Gene-Targeted Mouse Models of RNA Splicing Factor RP Show Late-Onset RPE and Retinal Degeneration. Investigative Ophthalmology & Visual Science, Association for Research in Vision and Ophthalmology, 2011. hal-03086760 HAL Id: hal-03086760 https://hal.archives-ouvertes.fr/hal-03086760 Submitted on 22 Dec 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Biochemistry and Molecular Biology Three Gene-Targeted Mouse Models of RNA Splicing Factor RP Show Late-Onset RPE and Retinal Degeneration John J. Graziotto,1,2 Michael H. Farkas,1 Kinga Bujakowska,3 Bertrand M. Deramaudt,1 Qi Zhang,1 Emeline F. Nandrot,3 Chris F. Inglehearn,4 Shomi S. Bhattacharya,3,5 and Eric A. Pierce1 PURPOSE. Mutations in genes that produce proteins involved in mice, and were associated with decreased rod function in the mRNA splicing, including pre-mRNA processing factors 3, 8, Prpf3-T494M mice.
    [Show full text]
  • Interaction Profiling of RNA-Binding Ubiquitin Ligases Reveals A
    www.nature.com/scientificreports OPEN Interaction profling of RNA- binding ubiquitin ligases reveals a link between posttranscriptional Received: 7 March 2017 Accepted: 14 September 2017 regulation and the ubiquitin system Published: xx xx xxxx Andrea Hildebrandt1, Gregorio Alanis-Lobato1,2, Andrea Voigt1, Kathi Zarnack3, Miguel A. Andrade-Navarro1,2, Petra Beli1 & Julian König1 RNA-binding ubiquitin ligases (RBULs) have the potential to link RNA-mediated mechanisms to protein ubiquitylation. Despite this, the cellular functions, substrates and interaction partners of most RBULs remain poorly characterized. Afnity purifcation (AP) combined with quantitative mass spectrometry (MS)-based proteomics is a powerful approach for analyzing protein functions. Mapping the physiological interaction partners of RNA-binding proteins has been hampered by their intrinsic properties, in particular the existence of low-complexity regions, which are prone to engage in non- physiological interactions. Here, we used an adapted AP approach to identify the interaction partners of human RBULs harboring diferent RNA-binding domains. To increase the likelihood of recovering physiological interactions, we combined control and bait-expressing cells prior to lysis. In this setup, only stable interactions that were originally present in the cell will be identifed. We exploit gene function similarity between the bait proteins and their interactors to benchmark our approach in its ability to recover physiological interactions. We reveal that RBULs engage in stable interactions with RNA-binding proteins involved in diferent steps of RNA metabolism as well as with components of the ubiquitin conjugation machinery and ubiquitin-binding proteins. Our results thus demonstrate their capacity to link posttranscriptional regulation with the ubiquitin system.
    [Show full text]
  • Targeted Exome Capture and Sequencing Identifies Novel
    Open Access Research BMJ Open: first published as 10.1136/bmjopen-2013-004030 on 7 November 2013. Downloaded from Targeted exome capture and sequencing identifies novel PRPF31 mutations in autosomal dominant retinitis pigmentosa in Chinese families Liping Yang,1 Xiaobei Yin,2 Lemeng Wu,1 Ningning Chen,1 Huirong Zhang,1 Genlin Li,2 Zhizhong Ma1 To cite: Yang L, Yin X, Wu L, ABSTRACT et al Strengths and limitations of this study . Targeted exome capture Objectives: To identify disease-causing mutations in and sequencing identifies two Chinese families with autosomal dominant retinitis ▪ The HEDEP based on targeted exome capture novel PRPF31 mutations in pigmentosa (adRP). autosomal dominant retinitis technology is an efficient method for molecular pigmentosa in Chinese Design: Prospective analysis. diagnosis in adRP patients. families. BMJ Open 2013;3: Patients: Two Chinese adRP families underwent ▪ Both mutations result in premature termination e004030. doi:10.1136/ genetic diagnosis. A specific hereditary eye disease codons before the last exon, thus insufficient bmjopen-2013-004030 enrichment panel (HEDEP) based on targeted exome functioning due to haploinsufficiency instead of capture technology was used to collect the protein aberrant function of the mutated proteins seems ▸ Prepublication history and coding regions of targeted 371 hereditary eye disease to be the most probably reason in these two additional material for this genes; high throughput sequencing was done with the families. However no experiment was done to paper is available online. To Illumina HiSeq 2000 platform. The identified variants prove it in this study. view these files please visit were confirmed with Sanger sequencing. the journal online Setting: All experiments were performed in a large (http://dx.doi.org/10.1136/ laboratory specialising in genetic studies in the bmjopen-2013-004030).
    [Show full text]
  • The Alter Retina: Alternative Splicing of Retinal Genes in Health and Disease
    International Journal of Molecular Sciences Review The Alter Retina: Alternative Splicing of Retinal Genes in Health and Disease Izarbe Aísa-Marín 1,2 , Rocío García-Arroyo 1,3 , Serena Mirra 1,2 and Gemma Marfany 1,2,3,* 1 Departament of Genetics, Microbiology and Statistics, Avda. Diagonal 643, Universitat de Barcelona, 08028 Barcelona, Spain; [email protected] (I.A.-M.); [email protected] (R.G.-A.); [email protected] (S.M.) 2 Centro de Investigación Biomédica en Red Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), Universitat de Barcelona, 08028 Barcelona, Spain 3 Institute of Biomedicine (IBUB, IBUB-IRSJD), Universitat de Barcelona, 08028 Barcelona, Spain * Correspondence: [email protected] Abstract: Alternative splicing of mRNA is an essential mechanism to regulate and increase the diversity of the transcriptome and proteome. Alternative splicing frequently occurs in a tissue- or time-specific manner, contributing to differential gene expression between cell types during development. Neural tissues present extremely complex splicing programs and display the highest number of alternative splicing events. As an extension of the central nervous system, the retina constitutes an excellent system to illustrate the high diversity of neural transcripts. The retina expresses retinal specific splicing factors and produces a large number of alternative transcripts, including exclusive tissue-specific exons, which require an exquisite regulation. In fact, a current challenge in the genetic diagnosis of inherited retinal diseases stems from the lack of information regarding alternative splicing of retinal genes, as a considerable percentage of mutations alter splicing Citation: Aísa-Marín, I.; or the relative production of alternative transcripts. Modulation of alternative splicing in the retina García-Arroyo, R.; Mirra, S.; Marfany, is also instrumental in the design of novel therapeutic approaches for retinal dystrophies, since it G.
    [Show full text]
  • Mrna Editing, Processing and Quality Control in Caenorhabditis Elegans
    | WORMBOOK mRNA Editing, Processing and Quality Control in Caenorhabditis elegans Joshua A. Arribere,*,1 Hidehito Kuroyanagi,†,1 and Heather A. Hundley‡,1 *Department of MCD Biology, UC Santa Cruz, California 95064, †Laboratory of Gene Expression, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan, and ‡Medical Sciences Program, Indiana University School of Medicine-Bloomington, Indiana 47405 ABSTRACT While DNA serves as the blueprint of life, the distinct functions of each cell are determined by the dynamic expression of genes from the static genome. The amount and specific sequences of RNAs expressed in a given cell involves a number of regulated processes including RNA synthesis (transcription), processing, splicing, modification, polyadenylation, stability, translation, and degradation. As errors during mRNA production can create gene products that are deleterious to the organism, quality control mechanisms exist to survey and remove errors in mRNA expression and processing. Here, we will provide an overview of mRNA processing and quality control mechanisms that occur in Caenorhabditis elegans, with a focus on those that occur on protein-coding genes after transcription initiation. In addition, we will describe the genetic and technical approaches that have allowed studies in C. elegans to reveal important mechanistic insight into these processes. KEYWORDS Caenorhabditis elegans; splicing; RNA editing; RNA modification; polyadenylation; quality control; WormBook TABLE OF CONTENTS Abstract 531 RNA Editing and Modification 533 Adenosine-to-inosine RNA editing 533 The C. elegans A-to-I editing machinery 534 RNA editing in space and time 535 ADARs regulate the levels and fates of endogenous dsRNA 537 Are other modifications present in C.
    [Show full text]