DOCSLIB.ORG

SR Proteins in Microrna/Mrna Biogenesis by Han Wu Department

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
SR Proteins in Microrna/Mrna Biogenesis by Han Wu Department SR proteins in microRNA/mRNA biogenesis by Han Wu Department of Cell Biology Duke University Date:_______________________ Approved: ___________________________ Jun Zhu, Supervisor ___________________________ Brigid L.M. Hogan, Chair ___________________________ Christopher B. Newgard ___________________________ Uwe Ohler ___________________________ Kenneth D. Poss ___________________________ Yuan Zhuang Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell Biology in the Graduate School of Duke University 2011 ABSTRACT SR proteins in microRNA/mRNA biogenesis by Han Wu Department of Cell Biology Duke University Date:_______________________ Approved: ___________________________ Jun Zhu, Supervisor ___________________________ Brigid L.M. Hogan, Chair ___________________________ Christopher B. Newgard ___________________________ Uwe Ohler ___________________________ Kenneth D. Poss ___________________________ Yuan Zhuang An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell Biology in the Graduate School of Duke University 2011 Copyright by Han Wu 2011 Abstract Serine/arginine‐rich proteins or SR proteins are well‐known protein factors involved in splicing regulation. Despite the tremendous efforts that have been made in characterizing their functions, several fundamental questions still remain: how are the expression levels of SR proteins regulated; what are the molecular mechanisms underlying SR protein‐mediated gene regulation; and what are the physiological targets of SR proteins in vivo. In my dissertation study, I employed a number of genomic and molecular approaches to study the functional involvement of two SR proteins, SF2/ASF and SRp20, in regulating microRNA/mRNA biogenesis. Negative feedback regulation has been shown as a common mechanism to maintain SR protein homeostasis (i.e. SC35 and SRp20). In the first part of my thesis, I set out to examine the potential involvement of miRNA‐mediated feedback regulation in maintaining the steady‐state level of SF2/ASF. MicroRNA deep sequencing was employed to identify differentially expressed miRNAs by elevating SF2/ASF level in an inducible cell line system. The sequencing data were further integrated with miRNA target predictions, which allowed me to identify a putative SF2/ASF‐miR‐7 negative feedback loop. A series of molecular and cellular techniques were then employed to validate the circuit structure. To our knowledge, this is the first negative feedback circuit iv reported between an SR protein (SF2/ASF) and a miRNA (miR‐7), and the paradigm may be broadly applicable in regulating the homeostasis of other SR proteins. This initial study was then extended to characterize the mechanism underlying SF2/ASF‐enhanced miR‐7 expression. Notably, one of the miR‐7 primary transcripts, miR‐7‐1, is embedded in an alternatively spliced intron of the hnRNPK gene. It provides a unique system to simultaneously study the involvement of SF2/ASF in alternative splicing and miRNA biogenesis. Through a series of mutagenesis assays in a minigene reporter system, I have uncovered a novel splicing‐independent function of SF2/ASF in regulating miRNA biogenesis. Direct interaction between SF2/ASF and primary‐miR‐7 (pri‐miR‐7) was demonstrated by cross‐linking and immunoprecipitation assay (CLIP) as well as by RNA affinity purification assay. Furthermore, I showed that this interaction is required for efficient miR‐7 processing in vivo. Finally, an in vitro pri‐miRNA processing assay was employed, and the results showed that SF2/ASF promotes the Drosha cleavage step of pri‐miR‐7 depending on the predicted binding site. Taken together, this was the first study showing the direct involvement of an SR protein in miRNA biogenesis. In order to study the global involvement of SR proteins in RNA biogenesis, one important stepping stone is to identify their targets in vivo. To this end, I focused on SRp20, another classic SR protein. PAR‐CLIP (Photoactivatable‐Ribonucleoside‐ Enhanced Cross‐linking and immunoprecipitation assay) combined with Illumina v sequencing was employed to systematically identify potential SRp20 binding targets, a subset of which were randomly selected and validated. As expected, our results showed that SRp20 primarily targets exonic regions for splicing regulation, and such interactions are likely to be mediated by a CWWCW motif. Surprisingly, extensive interactions between SRp20 and the 3’ UTRs of mRNAs were also observed. Such interactions presumably affect the choices between alternative polyadenylation sites. Thus, SRp20 may be one of the master regulators of both splicing and 3’ end processing. In summary, my thesis study was centered on functional characterization of two SR proteins, SF2/ASF and SRp20, in post‐transcriptional gene regulation. I have identified a negative feedback circuit between SF2/ASF and miR‐7: SF2/ASF directly binds to pri‐miR‐7 and promotes its maturation in a splicing‐independent manner; while mature miR‐7 negatively regulates SF2/ASF at the translational level. Furthermore, PAR‐ CLIP assay identified genome‐wide interactions between SRp20 and its potential targets, which may be linked to splicing regulation as well as alternative 3’ end processing. Altogether, this study has provided novel insights into how SR protein homeostasis can be achieved through negative feedback regulation, and the regulatory role of SR proteins in miRNA/mRNA biogenesis. vi Contents Abstract ......................................................................................................................................... iv List of Tables ................................................................................................................................. xi List of Figures .............................................................................................................................. xii List of Abbreviations .................................................................................................................xiv Acknowledgements ................................................................................................................... xix 1. Introduction ............................................................................................................................... 1 1.1 Pre‐mRNA splicing .......................................................................................................... 2 1.2 The SR family of splicing factors .................................................................................... 4 1.3 SR proteins in splicing regulation .................................................................................. 6 1.4 Other regulatory functions of SR proteins in gene expression .................................. 8 1.5 SR protein‐mediated gene regulation: functional significance ................................ 10 1.6 The substrate specificities of SR proteins .................................................................... 13 1.7 SR protein homeostasis .................................................................................................. 16 1.8 MicroRNA biogenesis .................................................................................................... 18 1.9 MicroRNA functions ...................................................................................................... 22 1.10 MicroRNA‐mediated gene regulatory networks ..................................................... 26 2. Methods .................................................................................................................................... 29 2.1 Cell culture and transfection ......................................................................................... 29 2.2 Plasmids ........................................................................................................................... 29 2.3 Profiling miRNA expression by deep sequencing ..................................................... 30 vii 2.3.1 Construction of miRNA sequencing library .......................................................... 30 2.3.2 Analysis of miRNA deep sequencing data ............................................................ 31 2.4 Dual‐Luciferase reporter assay ..................................................................................... 32 2.5 Semi‐quantitative PCR ................................................................................................... 33 2.6 Cross‐linking and Immunoprecipitation (CLIP) ........................................................ 34 2.6.1 CLIP‐PCR .................................................................................................................... 34 2.6.2 PAR‐CLIP .................................................................................................................... 35 2.6.3 Analysis of PAR‐CLIP sequencing data ................................................................. 36 2.7 Northern blotting ............................................................................................................ 37 2.8 Western blotting ............................................................................................................. 38 2.9 RNA affinity purification .............................................................................................. 39 2.10 RNA interference .........................................................................................................
Load more

Recommended publications
  • Cooperativity of Imprinted Genes Inactivated by Acquired Chromosome 20Q Deletions
    Cooperativity of imprinted genes inactivated by acquired chromosome 20q deletions Athar Aziz, … , Anne C. Ferguson-Smith, Anthony R. Green J Clin Invest. 2013;123(5):2169-2182. https://doi.org/10.1172/JCI66113. Research Article Oncology Large regions of recurrent genomic loss are common in cancers; however, with a few well-characterized exceptions, how they contribute to tumor pathogenesis remains largely obscure. Here we identified primate-restricted imprinting of a gene cluster on chromosome 20 in the region commonly deleted in chronic myeloid malignancies. We showed that a single heterozygous 20q deletion consistently resulted in the complete loss of expression of the imprinted genes L3MBTL1 and SGK2, indicative of a pathogenetic role for loss of the active paternally inherited locus. Concomitant loss of both L3MBTL1 and SGK2 dysregulated erythropoiesis and megakaryopoiesis, 2 lineages commonly affected in chronic myeloid malignancies, with distinct consequences in each lineage. We demonstrated that L3MBTL1 and SGK2 collaborated in the transcriptional regulation of MYC by influencing different aspects of chromatin structure. L3MBTL1 is known to regulate nucleosomal compaction, and we here showed that SGK2 inactivated BRG1, a key ATP-dependent helicase within the SWI/SNF complex that regulates nucleosomal positioning. These results demonstrate a link between an imprinted gene cluster and malignancy, reveal a new pathogenetic mechanism associated with acquired regions of genomic loss, and underline the complex molecular and cellular consequences of “simple” cancer-associated chromosome deletions. Find the latest version: https://jci.me/66113/pdf Research article Cooperativity of imprinted genes inactivated by acquired chromosome 20q deletions Athar Aziz,1,2 E. Joanna Baxter,1,2,3 Carol Edwards,4 Clara Yujing Cheong,5 Mitsuteru Ito,4 Anthony Bench,3 Rebecca Kelley,1,2 Yvonne Silber,1,2 Philip A.
    [Show full text]
  • Lupus-Associated Endogenous Retroviral LTR Polymorphism and Epigenetic Imprinting Promote HRES-1/Rab4 Expression and Mtor Activation
    Lupus-associated endogenous retroviral LTR polymorphism and epigenetic imprinting promote HRES-1/Rab4 expression and mTOR activation Aparna Godavarthy, … , Katalin Banki, Andras Perl JCI Insight. 2019. https://doi.org/10.1172/jci.insight.134010. Research In-Press Preview Immunology Graphical abstract Find the latest version: https://jci.me/134010/pdf LUPUS-ASSOCIATED ENDOGENOUS RETROVIRAL LTR POLYMORPHISM AND EPIGENETIC IMPRINTING PROMOTE HRES-1/RAB4 EXPRESSION AND MTOR ACTIVATION Aparna Godavarthy*1, Ryan Kelly*1, John Jimah1, Miguel Beckford1, Tiffany Caza1,2, David Fernandez1,2, Nick Huang1,3, Manuel Duarte1,2, Joshua Lewis1,2, Hind J. Fadel4, Eric M. Poeschla4, Katalin Banki5, and Andras Perl1,2,3 * These authors contributed equally to the study. 1, Division of Rheumatology, Department of Medicine; 2, Department of Microbiology and Immunology; 3, Department of Biochemistry and Molecular Biology, and 5, Department of Pathology, State University of New York, Upstate Medical University, College of Medicine, 750 East Adams Street, Syracuse, New York 13210; 4, Department of Molecular Medicine; Mayo Clinic College of Medicine, 200 First Street SW, Rochester 55905, USA; Correspondence: Andras Perl, M.D., Ph.D. , State University of New York, College of Medicine, 750 East Adams Street, Syracuse, New York 13210; Phone: (315) 464-4194; Fax: (315) 464- 4176; E-mail: [email protected] Key Words: Systemic Lupus Erythematosus, T Cells, HRES-1/Rab4. mTOR, Autoimmunity The authors have declared that no conflict of interest exists. Supplementary Materials include Supplemental Methods and Supplementary Figures S1-S26. 1 ABSTRACT Overexpression and long terminal repeat (LTR) polymorphism of the HRES-1/Rab4 human endogenous retrovirus locus have been associated with T-cell activation and disease manifestations in systemic lupus erythematosus (SLE).
    [Show full text]
  • Serine Arginine-Rich Protein-Dependent Suppression Of
    Serine͞arginine-rich protein-dependent suppression of exon skipping by exonic splicing enhancers El Che´ rif Ibrahim*, Thomas D. Schaal†, Klemens J. Hertel‡, Robin Reed*, and Tom Maniatis†§ *Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115; †Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138; and ‡Department of Microbiology and Molecular Genetics, University of California, Irvine, CA 92697-4025 Contributed by Tom Maniatis, January 28, 2005 The 5؅ and 3؅ splice sites within an intron can, in principle, be joined mechanisms by which exon skipping is prevented. Our data to those within any other intron during pre-mRNA splicing. How- reveal that splicing to the distal 3Ј splice site is suppressed by ever, exons are joined in a strict 5؅ to 3؅ linear order in constitu- proximal exonic sequences and that SR proteins are required for tively spliced pre-mRNAs. Thus, specific mechanisms must exist to this suppression. Thus, SR protein͞exonic enhancer complexes prevent the random joining of exons. Here we report that insertion not only function in exon and splice-site recognition but also play of exon sequences into an intron can inhibit splicing to the a role in ensuring that 5Ј and 3Ј splice sites within the same intron .downstream 3؅ splice site and that this inhibition is independent of are used, thus suppressing exon skipping intron size. The exon sequences required for splicing inhibition were found to be exonic enhancer elements, and their inhibitory Materials and Methods activity requires the binding of serine͞arginine-rich splicing fac- Construction of Plasmids.
    [Show full text]
  • 1 Supporting Information for a Microrna Network Regulates
    Supporting Information for A microRNA Network Regulates Expression and Biosynthesis of CFTR and CFTR-ΔF508 Shyam Ramachandrana,b, Philip H. Karpc, Peng Jiangc, Lynda S. Ostedgaardc, Amy E. Walza, John T. Fishere, Shaf Keshavjeeh, Kim A. Lennoxi, Ashley M. Jacobii, Scott D. Rosei, Mark A. Behlkei, Michael J. Welshb,c,d,g, Yi Xingb,c,f, Paul B. McCray Jr.a,b,c Author Affiliations: Department of Pediatricsa, Interdisciplinary Program in Geneticsb, Departments of Internal Medicinec, Molecular Physiology and Biophysicsd, Anatomy and Cell Biologye, Biomedical Engineeringf, Howard Hughes Medical Instituteg, Carver College of Medicine, University of Iowa, Iowa City, IA-52242 Division of Thoracic Surgeryh, Toronto General Hospital, University Health Network, University of Toronto, Toronto, Canada-M5G 2C4 Integrated DNA Technologiesi, Coralville, IA-52241 To whom correspondence should be addressed: Email: [email protected] (M.J.W.); yi- [email protected] (Y.X.); Email: [email protected] (P.B.M.) This PDF file includes: Materials and Methods References Fig. S1. miR-138 regulates SIN3A in a dose-dependent and site-specific manner. Fig. S2. miR-138 regulates endogenous SIN3A protein expression. Fig. S3. miR-138 regulates endogenous CFTR protein expression in Calu-3 cells. Fig. S4. miR-138 regulates endogenous CFTR protein expression in primary human airway epithelia. Fig. S5. miR-138 regulates CFTR expression in HeLa cells. Fig. S6. miR-138 regulates CFTR expression in HEK293T cells. Fig. S7. HeLa cells exhibit CFTR channel activity. Fig. S8. miR-138 improves CFTR processing. Fig. S9. miR-138 improves CFTR-ΔF508 processing. Fig. S10. SIN3A inhibition yields partial rescue of Cl- transport in CF epithelia.
    [Show full text]
  • 1 Inventory of Supplemental Information Presented in Relation To
    Inventory of Supplemental Information presented in relation to each of the main figures in the manuscript 1. Figure S1. Related to Figure 1. 2. Figure S2. Related to Figure 2. 3. Figure S3. Related to Figure 3. 4. Figure S4. Related to Figure 4. 5. Figure S5. Related to Figure 5. 6. Figure S6. Related to Figure 6. 7. Figure S7. Related to Figure 7. 8. Table S1. Related to Figures 1 and S1. 9. Table S2. Related to Figures 3, 4, 6 and 7. 10. Table S3. Related to Figure 3. Supplemental Experimental Procedures 1. Patients and samples 2. Cell culture 3. Long term culture-initiating cells (LTC-IC) assay 4. Differentiation of CD34+ cord blood cells 5. Bi-phasic erythroid differentiation assay 6. Clonal assay 7. Mutation and sequencing analysis 8. Bisulphite sequencing and quantitative pyrosequencing 9. Human SNP genotyping 10. Strand-specific cDNA synthesis and PCR 11. Chromatin Immuno-precipitation (ChIP) 12. Immunoprecipitation and western blot 13. Colony genotyping 14. shRNA generation and viral infection 15. Retrovirus generation and transduction 16. Study approval 17. Statistical Methods 18. Primers list Supplementary References (19) 1 Additional SGK2 families 2 3 4 ♂ ♀ ♂ ♀ ♂ ♀ C/G C/C C/C C/G C/C C/G ♀ ♀ ♂ ♀ C/G C/G C/G C/G T T C T G C T A G A T T C T C C T A G A T T C T C C T A G A T T C T C C T A G A T-Cells T-Cells T-Cells T-Cells Additional GDAP1L1 familiy 2 ♂ ♀ G/G A/A ♀ G/A A C A C G G T G Erythroblasts Figure S1 - .
    [Show full text]
  • Aneuploidy: Using Genetic Instability to Preserve a Haploid Genome?
    Health Science Campus FINAL APPROVAL OF DISSERTATION Doctor of Philosophy in Biomedical Science (Cancer Biology) Aneuploidy: Using genetic instability to preserve a haploid genome? Submitted by: Ramona Ramdath In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Science Examination Committee Signature/Date Major Advisor: David Allison, M.D., Ph.D. Academic James Trempe, Ph.D. Advisory Committee: David Giovanucci, Ph.D. Randall Ruch, Ph.D. Ronald Mellgren, Ph.D. Senior Associate Dean College of Graduate Studies Michael S. Bisesi, Ph.D. Date of Defense: April 10, 2009 Aneuploidy: Using genetic instability to preserve a haploid genome? Ramona Ramdath University of Toledo, Health Science Campus 2009 Dedication I dedicate this dissertation to my grandfather who died of lung cancer two years ago, but who always instilled in us the value and importance of education. And to my mom and sister, both of whom have been pillars of support and stimulating conversations. To my sister, Rehanna, especially- I hope this inspires you to achieve all that you want to in life, academically and otherwise. ii Acknowledgements As we go through these academic journeys, there are so many along the way that make an impact not only on our work, but on our lives as well, and I would like to say a heartfelt thank you to all of those people: My Committee members- Dr. James Trempe, Dr. David Giovanucchi, Dr. Ronald Mellgren and Dr. Randall Ruch for their guidance, suggestions, support and confidence in me. My major advisor- Dr. David Allison, for his constructive criticism and positive reinforcement.
    [Show full text]
  • A Novel Resveratrol Analog: Its Cell Cycle Inhibitory, Pro-Apoptotic and Anti-Inflammatory Activities on Human Tumor Cells
    A NOVEL RESVERATROL ANALOG : ITS CELL CYCLE INHIBITORY, PRO-APOPTOTIC AND ANTI-INFLAMMATORY ACTIVITIES ON HUMAN TUMOR CELLS A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Boren Lin May 2006 Dissertation written by Boren Lin B.S., Tunghai University, 1996 M.S., Kent State University, 2003 Ph. D., Kent State University, 2006 Approved by Dr. Chun-che Tsai , Chair, Doctoral Dissertation Committee Dr. Bryan R. G. Williams , Co-chair, Doctoral Dissertation Committee Dr. Johnnie W. Baker , Members, Doctoral Dissertation Committee Dr. James L. Blank , Dr. Bansidhar Datta , Dr. Gail C. Fraizer , Accepted by Dr. Robert V. Dorman , Director, School of Biomedical Sciences Dr. John R. Stalvey , Dean, College of Arts and Sciences ii TABLE OF CONTENTS LIST OF FIGURES……………………………………………………………….………v LIST OF TABLES……………………………………………………………………….vii ACKNOWLEDGEMENTS….………………………………………………………….viii I INTRODUCTION….………………………………………………….1 Background and Significance……………………………………………………..1 Specific Aims………………………………………………………………………12 II MATERIALS AND METHODS.…………………………………………….16 Cell Culture and Compounds…….……………….…………………………….….16 MTT Cell Viability Assay………………………………………………………….16 Trypan Blue Exclusive Assay……………………………………………………...18 Flow Cytometry for Cell Cycle Analysis……………..……………....……………19 DNA Fragmentation Assay……………………………………………...…………23 Caspase-3 Activity Assay………………………………...……….….…….………24 Annexin V-FITC Staining Assay…………………………………..…...….………28 NF-kappa B p65 Activity Assay……………………………………..………….…29
    [Show full text]
  • (P -Value<0.05, Fold Change≥1.4), 4 Vs. 0 Gy Irradiation
    Table S1: Significant differentially expressed genes (P -Value<0.05, Fold Change≥1.4), 4 vs. 0 Gy irradiation Genbank Fold Change P -Value Gene Symbol Description Accession Q9F8M7_CARHY (Q9F8M7) DTDP-glucose 4,6-dehydratase (Fragment), partial (9%) 6.70 0.017399678 THC2699065 [THC2719287] 5.53 0.003379195 BC013657 BC013657 Homo sapiens cDNA clone IMAGE:4152983, partial cds. [BC013657] 5.10 0.024641735 THC2750781 Ciliary dynein heavy chain 5 (Axonemal beta dynein heavy chain 5) (HL1). 4.07 0.04353262 DNAH5 [Source:Uniprot/SWISSPROT;Acc:Q8TE73] [ENST00000382416] 3.81 0.002855909 NM_145263 SPATA18 Homo sapiens spermatogenesis associated 18 homolog (rat) (SPATA18), mRNA [NM_145263] AA418814 zw01a02.s1 Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE:767978 3', 3.69 0.03203913 AA418814 AA418814 mRNA sequence [AA418814] AL356953 leucine-rich repeat-containing G protein-coupled receptor 6 {Homo sapiens} (exp=0; 3.63 0.0277936 THC2705989 wgp=1; cg=0), partial (4%) [THC2752981] AA484677 ne64a07.s1 NCI_CGAP_Alv1 Homo sapiens cDNA clone IMAGE:909012, mRNA 3.63 0.027098073 AA484677 AA484677 sequence [AA484677] oe06h09.s1 NCI_CGAP_Ov2 Homo sapiens cDNA clone IMAGE:1385153, mRNA sequence 3.48 0.04468495 AA837799 AA837799 [AA837799] Homo sapiens hypothetical protein LOC340109, mRNA (cDNA clone IMAGE:5578073), partial 3.27 0.031178378 BC039509 LOC643401 cds. [BC039509] Homo sapiens Fas (TNF receptor superfamily, member 6) (FAS), transcript variant 1, mRNA 3.24 0.022156298 NM_000043 FAS [NM_000043] 3.20 0.021043295 A_32_P125056 BF803942 CM2-CI0135-021100-477-g08 CI0135 Homo sapiens cDNA, mRNA sequence 3.04 0.043389246 BF803942 BF803942 [BF803942] 3.03 0.002430239 NM_015920 RPS27L Homo sapiens ribosomal protein S27-like (RPS27L), mRNA [NM_015920] Homo sapiens tumor necrosis factor receptor superfamily, member 10c, decoy without an 2.98 0.021202829 NM_003841 TNFRSF10C intracellular domain (TNFRSF10C), mRNA [NM_003841] 2.97 0.03243901 AB002384 C6orf32 Homo sapiens mRNA for KIAA0386 gene, partial cds.
    [Show full text]
  • Sorting out the Complexity of SR Protein Functions
    Downloaded from rnajournal.cshlp.org on February 6, 2009 - Published by Cold Spring Harbor Laboratory Press RNA (2000), 6:1197–1211+ Cambridge University Press+ Printed in the USA+ Copyright © 2000 RNA Society+ REVIEW Sorting out the complexity of SR protein functions BRENTON R. GRAVELEY Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA INTRODUCTION been clear whether all of these activities occur during the removal of each intron+ Recent studies now sug- Members of the serine/arginine-rich (SR) protein fam- gest that all of the proposed SR protein functions are ily have multiple functions in the pre-mRNA splicing carried out during each round of splicing, and at least reaction+ In addition to being required for the removal some of these functions are performed by independent of constitutively spliced introns, SR proteins can func- SR protein molecules+ This review discusses recent tion to regulate alternative splicing both in vitro and in advances in understanding the diverse functions of SR vivo (Ge & Manley, 1990; Krainer et al+, 1990a; Fu proteins in metazoan pre-mRNA splicing and presents et al+, 1992; Zahler et al+, 1993a; Caceres et al+, 1994; a model that takes these new findings into account+ Wang & Manley, 1995)+ In the cell, SR proteins migrate Although the reader should keep in mind that the ac- from speckles—subnuclear domains that may function tivity of SR proteins in vivo can be influenced by mod- as storage sites for certain splicing factors—to
    [Show full text]
  • Pirfenidone Is Renoprotective in Diabetic Kidney Disease
    BASIC RESEARCH www.jasn.org Pirfenidone Is Renoprotective in Diabetic Kidney Disease ʈ Satish P. RamachandraRao,*†‡ Yanqing Zhu,‡ Timothy Ravasi,§ Tracy A. McGowan,‡ Irene Toh,‡ Stephen R. Dunn,‡¶ Shinichi Okada,*† Michael A. Shaw,** and Kumar Sharma*†‡ *Center for Renal Translational Medicine, Division of Nephrology-Hypertension, Department of Medicine, and ʈ §Department of Bioengineering, Jacobs School of Engineering, University of California, San Diego, Scripps NeuroAIDS Preclinical Studies Centre, and †Veterans Administration San Diego Healthcare System, La Jolla, California, ‡Center for Novel Therapies in Kidney Disease, Department of Medicine, ¶Cancer Genomics Facility, Kimmel Cancer Center, and **Proteomics and Mass Spectrometry Core Facility, Department of Cancer Biology, Thomas Jefferson University, Philadelphia, Pennsylvania ABSTRACT Although several interventions slow the progression of diabetic nephropathy, current therapies do not halt progression completely. Recent preclinical studies suggested that pirfenidone (PFD) prevents fibrosis in various diseases, but the mechanisms underlying its antifibrotic action are incompletely understood. Here, we evaluated the role of PFD in regulation of the extracellular matrix. In mouse mesangial cells, PFD decreased TGF-␤ promoter activity, reduced TGF-␤ protein secretion, and inhibited TGF-␤–induced Smad2-phosphor- ylation, 3TP-lux promoter activity, and generation of reactive oxygen species. To explore the therapeutic potential of PFD, we administered PFD to 17-wk-old db/db mice for 4 wk. PFD treatment significantly reduced mesangial matrix expansion and expression of renal matrix genes but did not affect albuminuria. Using liquid chromatography with subsequent electrospray ionization tandem mass spectrometry, we iden- tified 21 proteins unique to PFD-treated diabetic kidneys. Analysis of gene ontology and protein–protein interactions of these proteins suggested that PFD may regulate RNA processing.
    [Show full text]
  • Expression Analysis of Genes Located Within the Common Deleted Region
    Leukemia Research 84 (2019) 106175 Contents lists available at ScienceDirect Leukemia Research journal homepage: www.elsevier.com/locate/leukres Research paper Expression analysis of genes located within the common deleted region of del(20q) in patients with myelodysplastic syndromes T ⁎ Masayuki Shiseki , Mayuko Ishii, Michiko Okada, Mari Ohwashi, Yan-Hua Wang, Satoko Osanai, Kentaro Yoshinaga, Naoki Mori, Toshiko Motoji, Junji Tanaka Department of Hematology, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan ARTICLE INFO ABSTRACT Keywords: Deletion of the long arm of chromosome 20 (del(20q)) is observed in 5–10% of patients with myelodysplastic Deletion 20q syndromes (MDS). We examined the expression of 28 genes within the common deleted region (CDR) of del Common deleted region (20q), which we previously determined by a CGH array using clinical samples, in 48 MDS patients with (n = 28) Myelodysplastic syndromes or without (n = 20) chromosome 20 abnormalities and control subjects (n = 10). The expression level of 8 of 28 genes was significantly reduced in MDS patients with chromosome 20 abnormalities compared to that of control subjects. In addition, the expression of BCAS4, ADA, and YWHAB genes was significantly reduced in MDS pa- tients without chromosome 20 abnormalities, which suggests that these three genes were commonly involved in the molecular pathogenesis of MDS. To evaluate the clinical significance, we analyzed the impact of the ex- pression level of each gene on overall survival (OS). According to the Cox proportional hazard model, multi- variate analysis indicated that reduced BCAS4 expression was associated with inferior OS, but the difference was not significant (HR, 3.77; 95% CI, 0.995-17.17; P = 0.0509).
    [Show full text]
  • Functional Control of HIV-1 Post-Transcriptional Gene Expression by Host Cell Factors
    Functional control of HIV-1 post-transcriptional gene expression by host cell factors DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Amit Sharma, B.Tech. Graduate Program in Molecular Genetics The Ohio State University 2012 Dissertation Committee Dr. Kathleen Boris-Lawrie, Advisor Dr. Anita Hopper Dr. Karin Musier-Forsyth Dr. Stephen Osmani Copyright by Amit Sharma 2012 Abstract Retroviruses are etiological agents of several human and animal immunosuppressive disorders. They are associated with certain types of cancer and are useful tools for gene transfer applications. All retroviruses encode a single primary transcript that encodes a complex proteome. The RNA genome is reverse transcribed into DNA, integrated into the host genome, and uses host cell factors to transcribe, process and traffic transcripts that encode viral proteins and act as virion precursor RNA, which is packaged into the progeny virions. The functionality of retroviral RNA is governed by ribonucleoprotein (RNP) complexes formed by host RNA helicases and other RNA- binding proteins. The 5’ leader of retroviral RNA undergoes alternative inter- and intra- molecular RNA-RNA and RNA-protein interactions to complete multiple steps of the viral life cycle. Retroviruses do not encode any RNA helicases and are dependent on host enzymes and RNA chaperones. Several members of the host RNA helicase superfamily are necessary for progressive steps during the retroviral replication. RNA helicase A (RHA) interacts with the redundant structural elements in the 5’ untranslated region (UTR) of retroviral and selected cellular mRNAs and this interaction is necessary to facilitate polyribosome formation and productive protein synthesis.
    [Show full text]