DOCSLIB.ORG

DCP1 Forms Asymmetric Trimers to Assemble Into Active Mrna Decapping Complexes in Metazoa

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
DCP1 Forms Asymmetric Trimers to Assemble Into Active Mrna Decapping Complexes in Metazoa DCP1 forms asymmetric trimers to assemble into active mRNA decapping complexes in metazoa Felix Tritschler1, Joerg E. Braun1, Carina Motz, Catia Igreja, Gabrielle Haas, Vincent Truffault, Elisa Izaurralde2, and Oliver Weichenrieder2 Department of Biochemistry, Max Planck Institute for Developmental Biology, Spemannstrasse 35, D-72076 Tu¨bingen, Germany Edited by James E. Dahlberg, University of Wisconsin Medical School, Madison, WI, and approved October 29, 2009 (received for review August 28, 2009) DCP1 stimulates the decapping enzyme DCP2, which removes the DCP1a in mRNA decapping we expressed the domain in E. coli -mRNA 5؅ cap structure committing mRNAs to degradation. In (DCP1a residues S539 to L582). Using static light scattering mea multicellular eukaryotes, DCP1-DCP2 interaction is stabilized by surements coupled with size exclusion chromatography, we found additional proteins, including EDC4. However, most information unexpectedly that the purified domain forms stable trimers in on DCP2 activation stems from studies in S. cerevisiae, which lacks solution (Table S1). We have termed this domain the DCP1- EDC4. Furthermore, DCP1 orthologs from multicellular eukaryotes trimerization domain (DCP1-TD). Furthermore, although the re- have a C-terminal extension, absent in fungi. Here, we show that in combinant polypeptide contains 51 residues per monomer (i.e., 44 metazoa, a conserved DCP1 C-terminal domain drives DCP1 trim- from DCP1a-TD and seven from the expression vector), NMR erization. Crystal structures of the DCP1-trimerization domain re- spectroscopy yields Ͼ115 peaks in the 15N-HSQC spectrum (Fig. veal an antiparallel assembly comprised of three kinked ␣-helices. S2), suggesting that in solution the trimers are asymmetric (assum- Trimerization is required for DCP1 to be incorporated into active ing a single trimeric assembly). decapping complexes and for efficient mRNA decapping in vivo. We determined the crystal structure of this unusual assembly Our results reveal an unexpected connectivity and complexity of to a resolution of 2.3 Å (Rwork ϭ 20.8, Rfree ϭ 25.2, Table S2). the mRNA decapping network in multicellular eukaryotes, which The structure reveals an unprecedented, antiparallel bundle of likely enhances opportunities for regulating mRNA degradation. three kinked ␣-helices (two up, one down), in which the struc- tural environment for a given side-chain differs for each of the ͉ ͉ ͉ ͉ BIOCHEMISTRY DCP2 miRNAs P-bodies EDC4 Ge-1 three molecules (Fig. 1 B–F). The central sequence (K544-L571) of each molecule is ␣-helical, with a strong kink at D558 that n eukaryotes, removal of the mRNA 5Ј cap structure is separates helix ␣1 from helix ␣2 with an elbow angle of Ϸ90° Icatalyzed by the decapping enzyme DCP2 (1, 2); to be fully (Fig. 1F). At the kink, residue L554 from helix ␣1 makes van der active and/or stable, DCP2 requires additional proteins (1, 2). Waals contacts with F561 from helix ␣2, while D558 caps the N Yeast DCP2 interacts directly with DCP1 and this interaction is terminus of helix ␣-2 by hydrogen-bonding with the peptide required for decapping in vivo and in vitro (3–7). In humans, the NH-groups of residues S560 and F561 (Fig. 1F). Alignment of DCP2-DCP1 interaction requires additional proteins, which DCP1 sequences from various species shows only these three together assemble into multimeric decapping complexes that residues are invariant (Fig. 2A, asterisks), indicating the elbow is also include the enhancers of decapping 3 and 4 (EDC3 and a conserved structural feature of DCP1 trimerization domains ECD4), and the DEAD-box protein DDX6/RCK (8, 9). from multicellular eukaryotes. DCP2 is highly conserved and most information on DCP2 The three polypeptide chains (termed A, B, and C in Fig. 1 activation stems mainly from studies in S. cerevisiae and S. pombe B–F) superimpose over the central sequence, with a maximal C␣ (3–7). Fungi, however, lack EDC4 as well as many extensions and r.m.s.d. of 1.15 Å (chain A and B, residues 544–571). To additional domains present in decapping activators of metazoan assemble the trimer, chain B interacts with chains A and C in an orthologs (8–10). For example, all eukaryotic DCP1 proteins antiparallel fashion, causing helices ␣1 of chain A and ␣2 of chain contain an N-terminal EVH1 domain (3, 5, 6); however, DCP1 C to interact in parallel. Chains A and B are thus related by a orthologs from metazoa and plants also have a proline-rich pseudotwofold axis close to F561 (Fig. 1D), while chains C and C-terminal extension (9, 10). The sequence of this extension is B are related by a pseudotwofold axis close to I552 (Fig. 1E). not conserved except for a 14-residue short motif (motif I, MI) This arrangement places most hydrophobic side chains into a C. elegans A conserved in metazoa with the exception of (Fig. 1 densely packed core (Figs. 1B and 2A, residues shaded in blue), and Fig. S1) and a C-terminal domain conserved in plants and explaining the stability of the trimer. The resulting DCP1a metazoa (Fig. 1A, referred to as TD). trimerization domain could be characterized as a novel fold if the The DCP1 C-terminal domain is predicted to adopt an chains were connected in cis. ␣-helical conformation. In this work, we show that this domain Using the structural information, we constructed three mu- trimerizes in an asymmetric fashion. We solved the crystal structure of the trimerized domain for both human and D. melanogaster DCP1 and show that the trimer adopts an unprec- Author contributions: E.I. designed research; F.T., J.E.B., C.M., C.I., G.H., V.T., and O.W. edented fold, with no current similarities in the protein database. performed research; F.T., J.E.B., C.M., C.I., G.H., V.T., E.I., and O.W. analyzed data; and F.T., We further show that DCP1 trimerization is required for the J.E.B., E.I., and O.W. wrote the paper. assembly of active decapping complexes and for mRNA decap- The authors declare no conflict of interest. ping in vivo. The conservation of structurally critical residues This article is a PNAS Direct Submission. indicates that this domain adopts a similar fold in DCP1 or- Freely available online through the PNAS open access option. thologs of other multicellular eukaryotes. Consequently, within Data deposition: Coordinates of the human DCP1a and the D. melanogaster DCP1 trimer- mRNA decapping complexes in these organisms, the stoichiom- ization domains have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code etry of the protein components is likely more complex than 2WX3 and 2WX4). previously thought. 1F.T. and J.E.B. contributed equally to this work. 2To whom correspondence may be addressed. E-mail: [email protected] Results and Discussion or [email protected]. Crystal Structure of the Human DCP1a Trimerization Domain. To This article contains supporting information online at www.pnas.org/cgi/content/full/ investigate the role of the conserved C-terminal domain of human 0909871106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0909871106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 30, 2021 Crystal Structure of the Drosophila melanogaster DCP1 Trimerization Domain. Despite the apparent stability of this unusual asymmet- ric homotrimeric assembly, we could not formally rule out the possibility that this particular protein sequence causes a unique artifact. Therefore, we crystallized the trimerization domain of D. melanogaster DCP1 (residues L328 to D366), which is only 36% identical to the human DCP1a trimerization domain (iden- tity calculated over the central sequence of the TDs: HsDCP1a 544–571, DmDCP1 331–358; Fig. 2A). We found it also trim- erizes with the same topology but in a different crystal packing environment and with two independent copies in the asymmetric unit (Fig. 1C, Fig. S3, and Table S2). Moreover, when D. melanogaster DCP1 is mutated at structural positions equivalent to those in human DCP1a mutants (Mut-1, Mut-2, and Mut-3), trimerization is again disrupted (Table S1). We therefore con- clude that the C terminus of DCP1 contains a trimerization domain, which is conserved in metazoa but absent in fungi. Functional Analysis of the Human and D. melanogaster DCP1 Trimer- ization Domain. Further analysis showed DCP1 also oligomerizes in vivo. In human HEK293 cells, we coexpressed hemagglutinin (HA)-tagged DCP1a with green fluorescent protein (GFP)- tagged wild-type or mutant DCP1a, and then performed coim- munoprecipitations with anti-GFP antibodies. HA-DCP1a co- immunoprecipitated with GFP-DCP1a, but not with the negative control protein GFP-MBP (Fig. 2B, lane 9 vs. 8). As expected, the trimerization domain (GFP-DCP1a-TD) is sufficient for oligomerization (Fig. 2C, lane 6); while for DCP1a mutants 1, 2 and 3 oligomerization is strongly impaired (Fig. 2B, lanes 10–12). Accordingly, deleting the trimerization domain prevents DCP1a oligomerization (Fig. 2B, lane 13). Similarly, we confirmed that the trimerization domain of D. melanogaster DCP1 is also sufficient for oligomerization in vivo (Fig. 2D). Because we were able to disrupt oligomerization in vivo using information from the crystal structure, we infer DCP1 in human and D. melanogaster cells exist as asymmetric trimers. Accord- ingly, the possibility that the observed DCP1a self-association is indirect and results from the incorporation of several monomeric DCP1a copies into larger decapping complexes can be ruled out, as it is inconsistent with the crystal structures and with the findings described below. We next showed human DCP1a trimerization is required for assembly of active decapping complexes. We immunopurified GFP-DCP1a (wild-type and mutants) from HEK293 cells and, using an m7G-capped RNA substrate, tested for decapping activity in vitro. Decapping activity coimmunopurified with Fig. 1. Structure of the DCP1-trimerization domain.
Load more

Recommended publications
  • IP6K1 Upregulates the Formation of Processing Bodies by Promoting Proteome Remodeling on the Mrna Cap
    bioRxiv preprint doi: https://doi.org/10.1101/2020.07.13.199828; this version posted July 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. IP6K1 upregulates the formation of processing bodies by promoting proteome remodeling on the mRNA cap Akruti Shah1,2 and Rashna Bhandari1* 1Laboratory of Cell Signalling, Centre for DNA Fingerprinting and Diagnostics (CDFD), Inner Ring Road, Uppal, Hyderabad 500039, India. 2Graduate studies, Manipal Academy of Higher Education, Manipal 576104, India. *Correspondence to Rashna Bhandari; Email: [email protected] Running title: IP6K1 promotes mRNA turnover to induce P-bodies ORCID IDs Akruti Shah - 0000-0001-9557-4952 Rashna Bhandari - 0000-0003-3101-0204 This PDF file includes: Main Text Figures 1 to 6 Keywords mRNA decay/mRNA metabolism/P-bodies/translation suppression 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.13.199828; this version posted July 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract Inositol hexakisphosphate kinases (IP6Ks) are ubiquitously expressed small molecule kinases that catalyze the conversion of the inositol phosphate IP6 to 5-IP7. IP6Ks have been reported to influence cellular functions by protein-protein interactions independent of their enzymatic activity.
    [Show full text]
  • Mutations in DCPS and EDC3 in Autosomal Recessive Intellectual
    Human Molecular Genetics, 2015, Vol. 24, No. 11 3172–3180 doi: 10.1093/hmg/ddv069 Advance Access Publication Date: 20 February 2015 Original Article Downloaded from ORIGINAL ARTICLE Mutations in DCPS and EDC3 in autosomal recessive intellectual disability indicate a crucial role for mRNA http://hmg.oxfordjournals.org/ decapping in neurodevelopment Iltaf Ahmed1,2,†, Rebecca Buchert3,†, Mi Zhou5,†, Xinfu Jiao5,†, Kirti Mittal1, Taimoor I. Sheikh1, Ute Scheller3, Nasim Vasli1, Muhammad Arshad Rafiq1, 6 1 7 2 M. Qasim Brohi , Anna Mikhailov , Muhammad Ayaz , Attya Bhatti , at Universitaet Erlangen-Nuernberg, Wirtschafts- und Sozialwissenschaftliche Z on August 15, 2016 Heinrich Sticht4, Tanveer Nasr8,9, Melissa T. Carter10, Steffen Uebe3, André Reis3, Muhammad Ayub7,11, Peter John2, Megerditch Kiledjian5,*, John B. Vincent1,12,13,* and Rami Abou Jamra3,* 1Molecular Neuropsychiatry and Development Lab, Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, 250 College Street, Toronto, Ontario, Canada M5T 1R8, 2Atta-ur-Rehman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Islamabad 44000, Pakistan, 3Institute of Human Genetics and 4Bioinformatics, Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen 91054, Germany, 5Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854, USA, 6Sir Cowasji Jehangir Institute of Psychiatry, Hyderabad, Sindh 71000, Pakistan, 7Lahore Institute of Research and Development,
    [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]
  • 1 AGING Supplementary Table 2
    SUPPLEMENTARY TABLES Supplementary Table 1. Details of the eight domain chains of KIAA0101. Serial IDENTITY MAX IN COMP- INTERFACE ID POSITION RESOLUTION EXPERIMENT TYPE number START STOP SCORE IDENTITY LEX WITH CAVITY A 4D2G_D 52 - 69 52 69 100 100 2.65 Å PCNA X-RAY DIFFRACTION √ B 4D2G_E 52 - 69 52 69 100 100 2.65 Å PCNA X-RAY DIFFRACTION √ C 6EHT_D 52 - 71 52 71 100 100 3.2Å PCNA X-RAY DIFFRACTION √ D 6EHT_E 52 - 71 52 71 100 100 3.2Å PCNA X-RAY DIFFRACTION √ E 6GWS_D 41-72 41 72 100 100 3.2Å PCNA X-RAY DIFFRACTION √ F 6GWS_E 41-72 41 72 100 100 2.9Å PCNA X-RAY DIFFRACTION √ G 6GWS_F 41-72 41 72 100 100 2.9Å PCNA X-RAY DIFFRACTION √ H 6IIW_B 2-11 2 11 100 100 1.699Å UHRF1 X-RAY DIFFRACTION √ www.aging-us.com 1 AGING Supplementary Table 2. Significantly enriched gene ontology (GO) annotations (cellular components) of KIAA0101 in lung adenocarcinoma (LinkedOmics). Leading Description FDR Leading Edge Gene EdgeNum RAD51, SPC25, CCNB1, BIRC5, NCAPG, ZWINT, MAD2L1, SKA3, NUF2, BUB1B, CENPA, SKA1, AURKB, NEK2, CENPW, HJURP, NDC80, CDCA5, NCAPH, BUB1, ZWILCH, CENPK, KIF2C, AURKA, CENPN, TOP2A, CENPM, PLK1, ERCC6L, CDT1, CHEK1, SPAG5, CENPH, condensed 66 0 SPC24, NUP37, BLM, CENPE, BUB3, CDK2, FANCD2, CENPO, CENPF, BRCA1, DSN1, chromosome MKI67, NCAPG2, H2AFX, HMGB2, SUV39H1, CBX3, TUBG1, KNTC1, PPP1CC, SMC2, BANF1, NCAPD2, SKA2, NUP107, BRCA2, NUP85, ITGB3BP, SYCE2, TOPBP1, DMC1, SMC4, INCENP. RAD51, OIP5, CDK1, SPC25, CCNB1, BIRC5, NCAPG, ZWINT, MAD2L1, SKA3, NUF2, BUB1B, CENPA, SKA1, AURKB, NEK2, ESCO2, CENPW, HJURP, TTK, NDC80, CDCA5, BUB1, ZWILCH, CENPK, KIF2C, AURKA, DSCC1, CENPN, CDCA8, CENPM, PLK1, MCM6, ERCC6L, CDT1, HELLS, CHEK1, SPAG5, CENPH, PCNA, SPC24, CENPI, NUP37, FEN1, chromosomal 94 0 CENPL, BLM, KIF18A, CENPE, MCM4, BUB3, SUV39H2, MCM2, CDK2, PIF1, DNA2, region CENPO, CENPF, CHEK2, DSN1, H2AFX, MCM7, SUV39H1, MTBP, CBX3, RECQL4, KNTC1, PPP1CC, CENPP, CENPQ, PTGES3, NCAPD2, DYNLL1, SKA2, HAT1, NUP107, MCM5, MCM3, MSH2, BRCA2, NUP85, SSB, ITGB3BP, DMC1, INCENP, THOC3, XPO1, APEX1, XRCC5, KIF22, DCLRE1A, SEH1L, XRCC3, NSMCE2, RAD21.
    [Show full text]
  • DEAD-Box RNA Helicases in Cell Cycle Control and Clinical Therapy
    cells Review DEAD-Box RNA Helicases in Cell Cycle Control and Clinical Therapy Lu Zhang 1,2 and Xiaogang Li 2,3,* 1 Department of Nephrology, Renmin Hospital of Wuhan University, Wuhan 430060, China; [email protected] 2 Department of Internal Medicine, Mayo Clinic, 200 1st Street, SW, Rochester, MN 55905, USA 3 Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 1st Street, SW, Rochester, MN 55905, USA * Correspondence: [email protected]; Tel.: +1-507-266-0110 Abstract: Cell cycle is regulated through numerous signaling pathways that determine whether cells will proliferate, remain quiescent, arrest, or undergo apoptosis. Abnormal cell cycle regula- tion has been linked to many diseases. Thus, there is an urgent need to understand the diverse molecular mechanisms of how the cell cycle is controlled. RNA helicases constitute a large family of proteins with functions in all aspects of RNA metabolism, including unwinding or annealing of RNA molecules to regulate pre-mRNA, rRNA and miRNA processing, clamping protein complexes on RNA, or remodeling ribonucleoprotein complexes, to regulate gene expression. RNA helicases also regulate the activity of specific proteins through direct interaction. Abnormal expression of RNA helicases has been associated with different diseases, including cancer, neurological disorders, aging, and autosomal dominant polycystic kidney disease (ADPKD) via regulation of a diverse range of cellular processes such as cell proliferation, cell cycle arrest, and apoptosis. Recent studies showed that RNA helicases participate in the regulation of the cell cycle progression at each cell cycle phase, including G1-S transition, S phase, G2-M transition, mitosis, and cytokinesis.
    [Show full text]
  • Supplemental Information
    Supplemental information Dissection of the genomic structure of the miR-183/96/182 gene. Previously, we showed that the miR-183/96/182 cluster is an intergenic miRNA cluster, located in a ~60-kb interval between the genes encoding nuclear respiratory factor-1 (Nrf1) and ubiquitin-conjugating enzyme E2H (Ube2h) on mouse chr6qA3.3 (1). To start to uncover the genomic structure of the miR- 183/96/182 gene, we first studied genomic features around miR-183/96/182 in the UCSC genome browser (http://genome.UCSC.edu/), and identified two CpG islands 3.4-6.5 kb 5’ of pre-miR-183, the most 5’ miRNA of the cluster (Fig. 1A; Fig. S1 and Seq. S1). A cDNA clone, AK044220, located at 3.2-4.6 kb 5’ to pre-miR-183, encompasses the second CpG island (Fig. 1A; Fig. S1). We hypothesized that this cDNA clone was derived from 5’ exon(s) of the primary transcript of the miR-183/96/182 gene, as CpG islands are often associated with promoters (2). Supporting this hypothesis, multiple expressed sequences detected by gene-trap clones, including clone D016D06 (3, 4), were co-localized with the cDNA clone AK044220 (Fig. 1A; Fig. S1). Clone D016D06, deposited by the German GeneTrap Consortium (GGTC) (http://tikus.gsf.de) (3, 4), was derived from insertion of a retroviral construct, rFlpROSAβgeo in 129S2 ES cells (Fig. 1A and C). The rFlpROSAβgeo construct carries a promoterless reporter gene, the β−geo cassette - an in-frame fusion of the β-galactosidase and neomycin resistance (Neor) gene (5), with a splicing acceptor (SA) immediately upstream, and a polyA signal downstream of the β−geo cassette (Fig.
    [Show full text]
  • Comprehensive Protein Interactome Analysis of a Key RNA Helicase: Detection of Novel Stress Granule Proteins
    Biomolecules 2015, 5, 1441-1466; doi:10.3390/biom5031441 OPEN ACCESS biomolecules ISSN 2218-273X www.mdpi.com/journal/biomolecules/ Article Comprehensive Protein Interactome Analysis of a Key RNA Helicase: Detection of Novel Stress Granule Proteins Rebecca Bish 1,†, Nerea Cuevas-Polo 1,†, Zhe Cheng 1, Dolores Hambardzumyan 2, Mathias Munschauer 3, Markus Landthaler 3 and Christine Vogel 1,* 1 Center for Genomics and Systems Biology, Department of Biology, New York University, 12 Waverly Place, New York, NY 10003, USA; E-Mails: [email protected] (R.B.); [email protected] (N.C.-P.); [email protected] (Z.C.) 2 The Cleveland Clinic, Department of Neurosciences, Lerner Research Institute, 9500 Euclid Avenue, Cleveland, OH 44195, USA; E-Mail: [email protected] 3 RNA Biology and Post-Transcriptional Regulation, Max-Delbrück-Center for Molecular Medicine, Berlin-Buch, Robert-Rössle-Str. 10, Berlin 13092, Germany; E-Mails: [email protected] (M.M.); [email protected] (M.L.) † These authors contributed equally to this work. * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +1-212-998-3976; Fax: +1-212-995-4015. Academic Editor: André P. Gerber Received: 10 May 2015 / Accepted: 15 June 2015 / Published: 15 July 2015 Abstract: DDX6 (p54/RCK) is a human RNA helicase with central roles in mRNA decay and translation repression. To help our understanding of how DDX6 performs these multiple functions, we conducted the first unbiased, large-scale study to map the DDX6-centric protein-protein interactome using immunoprecipitation and mass spectrometry. Using DDX6 as bait, we identify a high-confidence and high-quality set of protein interaction partners which are enriched for functions in RNA metabolism and ribosomal proteins.
    [Show full text]
  • Gene Expression During Normal and FSHD Myogenesis Tsumagari Et Al
    Gene expression during normal and FSHD myogenesis Tsumagari et al. Tsumagari et al. BMC Medical Genomics 2011, 4:67 http://www.biomedcentral.com/1755-8794/4/67 (27 September 2011) Tsumagari et al. BMC Medical Genomics 2011, 4:67 http://www.biomedcentral.com/1755-8794/4/67 RESEARCHARTICLE Open Access Gene expression during normal and FSHD myogenesis Koji Tsumagari1, Shao-Chi Chang1, Michelle Lacey2,3, Carl Baribault2,3, Sridar V Chittur4, Janet Sowden5, Rabi Tawil5, Gregory E Crawford6 and Melanie Ehrlich1,3* Abstract Background: Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disease linked to contraction of an array of tandem 3.3-kb repeats (D4Z4) at 4q35. Within each repeat unit is a gene, DUX4, that can encode a protein containing two homeodomains. A DUX4 transcript derived from the last repeat unit in a contracted array is associated with pathogenesis but it is unclear how. Methods: Using exon-based microarrays, the expression profiles of myogenic precursor cells were determined. Both undifferentiated myoblasts and myoblasts differentiated to myotubes derived from FSHD patients and controls were studied after immunocytochemical verification of the quality of the cultures. To further our understanding of FSHD and normal myogenesis, the expression profiles obtained were compared to those of 19 non-muscle cell types analyzed by identical methods. Results: Many of the ~17,000 examined genes were differentially expressed (> 2-fold, p < 0.01) in control myoblasts or myotubes vs. non-muscle cells (2185 and 3006, respectively) or in FSHD vs. control myoblasts or myotubes (295 and 797, respectively). Surprisingly, despite the morphologically normal differentiation of FSHD myoblasts to myotubes, most of the disease-related dysregulation was seen as dampening of normal myogenesis- specific expression changes, including in genes for muscle structure, mitochondrial function, stress responses, and signal transduction.
    [Show full text]
  • Lorena Novoa-Aponte
    A Strain: fl/fl ∆hep Strain: fl/fl ∆hep AnalysisAAV: ofLuc the ironLuc chaperonePCBP1-WT PCBP1PCBP1-∆Fe interactome:PCBP1-∆RNA PCBP1 variant intersection of DNA repair and iron traffickingAAV: Luc Luc WT ∆Fe ∆RNA PCBP1 −40 Lorena Novoa-Aponte a*, Sarju J. Patel a, Olga Protchenko a, James Wohlschlegel b, Caroline C. Philpott a −40 PCBP1, IHC PCBP1, GAPDH - a Genetics and Metabolism Section,a NIDDK, NIH, Bethesda, MD, USA. b Department of Biological Chemistry, UCLA, Los Angeles, CA, USA C.C. Philpott, et al. *[email protected] 80 B P = 0.0262 60 40 1. Iron is essential but toxic 2. Increased DNA damage in cells lacking PCBP1 ns H&E ? + 20 C.C. Philpott, et al. Iron is used as an essential cofactor by several enzymes involved in DNA ⦿ Increased TUNEL in cells and tissue livers from mice lacking PCBP1. ? replication and repair. However, unchaperoned iron promotes redox ⦿ PCBP1 binds both, iron and single-stranded nucleicTG, nmol/mg protein 0 acids. stress that may affect DNA stability. Strain: Δhep Δhep Δhep ? ⦿ The iron binding activity of PCBP1 controls suppressionAAV: of DNA damage C WT ∆Fe ∆RNA PCBP1 variant ? Iron storage HEK293 cells Mouse Liver ? A 8 siNT+P1var 100 ? A 8 var siPCBP1 2 ? Mammals use the iron siNT+P1 ns var Fe-S cluster assembly 6 siPCBP1+P1siPCBP1 80 ADI1 TUNEL chaperone PCBP1 to var ? 6 siPCBP1+P1 metalate several iron- 60 = 0.0468 = 0.0354 = 0.0465 cell / mm population P P P population + dependent enzymes + 4 + 4 ? 40 Degradation of HIF1α Fig. 3. Iron chaperone-mediated handling of cytosolic labile iron pool.
    [Show full text]
  • Investigation of RNA Quality Control Pathways in RNP Hypo-Assembly
    Investigation of RNA quality control pathways in RNP hypo-assembly diseases by Siddharth Shukla Integrated M.Sc., Indian Institute of Technology Bombay, 2011 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirement for the degree of Doctor of Philosophy Department of Chemistry and Biochemistry 2016 i This thesis entitled: Investigation of RNA quality control pathways in RNP hypo-assembly diseases written by Siddharth Shukla has been approved for the Department of Chemistry and Biochemistry ________________________________ Roy Parker ________________________________ James Goodrich Date ___________ The final copy of this thesis has been examined by the signatories, and we find that both the content and the form meet acceptable presentation standards of scholarly work in the above mentioned discipline. ii Shukla, Siddharth (Ph.D., Biochemistry) Investigation of RNA quality control pathways in RNP hypo-assembly diseases Thesis directed by Professor Roy Parker A key aspect of cellular function is the proper assembly and utilization of ribonucleoproteins (RNPs). Defects in the formation of RNPs lead to "RNP hypo- assembly diseases", which can be caused by RNA degradation out-competing RNP assembly. Examples of such human diseases include Dyskeratosis Congenita (DC) and Spinal Muscular Atrophy (SMA). In order to test the hypothesis that specific RNA quality control pathways were responsible for degradation of RNAs in these diseases, I used yeast and mammalian cell lines as model systems to investigate two different diseases, SMA and DC. In SMA, Sm-site mutations in yeast U1 snRNA led to their rapid degradation by two different RNA decay pathways: 3’ to 5’ decay in the nucleus by Trf4/Rrp6, and 5’ to 3’ decay in the cytoplasm by Dcp2/Xrn1.
    [Show full text]
  • A Non-Canonical Role for the EDC4 Decapping Factor in Regulating
    RESEARCH ARTICLE A non-canonical role for the EDC4 decapping factor in regulating MARF1- mediated mRNA decay William R Brothers1, Steven Hebert1, Claudia L Kleinman1,2, Marc R Fabian1,3,4* 1Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Canada; 2Department of Human Genetics, McGill University, Montreal, Canada; 3Department of Biochemistry, McGill University, Montreal, Canada; 4Department of Oncology, McGill University, Montreal, Canada Abstract EDC4 is a core component of processing (P)-bodies that binds the DCP2 decapping enzyme and stimulates mRNA decay. EDC4 also interacts with mammalian MARF1, a recently identified endoribonuclease that promotes oogenesis and contains a number of RNA binding domains, including two RRMs and multiple LOTUS domains. How EDC4 regulates MARF1 action and the identity of MARF1 target mRNAs is not known. Our transcriptome-wide analysis identifies bona fide MARF1 target mRNAs and indicates that MARF1 predominantly binds their 3’ UTRs via its LOTUS domains to promote their decay. We also show that a MARF1 RRM plays an essential role in enhancing its endonuclease activity. Importantly, we establish that EDC4 impairs MARF1 activity by preventing its LOTUS domains from binding target mRNAs. Thus, EDC4 not only serves as an enhancer of mRNA turnover that binds DCP2, but also as a repressor that binds MARF1 to prevent the decay of MARF1 target mRNAs. Introduction *For correspondence: The regulated decay of eukaryotic mRNA populations plays an important role in the post-transcrip- [email protected] tional control (PTC) of gene expression. These PTC programs are, in turn, critical for regulating a number of biological processes, including during early development, cell proliferation and immune Competing interests: The response.
    [Show full text]
  • Identification of Edc3p As an Enhancer of Mrna Decapping In
    Copyright 2004 by the Genetics Society of America Identification of Edc3p as an Enhancer of mRNA Decapping in Saccharomyces cerevisiae Meenakshi Kshirsagar and Roy Parker1 Department of Molecular and Cellular Biology and Howard Hughes Medical Institute, University of Arizona, Tucson, Arizona 85721-0106 Manuscript received July 8, 2003 Accepted for publication October 27, 2003 ABSTRACT The major pathway of mRNA decay in yeast initiates with deadenylation, followed by mRNA decapping and 5Ј–3Ј exonuclease digestion. An in silico approach was used to identify new proteins involved in the mRNA decay pathway. One such protein, Edc3p, was identified as a conserved protein of unknown function having extensive two-hybrid interactions with several proteins involved in mRNA decapping and 5Ј–3Ј degradation including Dcp1p, Dcp2p, Dhh1p, Lsm1p, and the 5Ј–3Ј exonuclease, Xrn1p. We show that Edc3p can stimulate mRNA decapping of both unstable and stable mRNAs in yeast when the decapping enzyme is compromised by temperature-sensitive alleles of either the DCP1 or the DCP2 genes. In these cases, deletion of EDC3 caused a synergistic mRNA-decapping defect at the permissive temperatures. The edc3⌬ had no effect when combined with the lsm1⌬, dhh1⌬,orpat1⌬ mutations, which appear to affect an early step in the decapping pathway. This suggests that Edc3p specifically affects the function of the decapping enzyme per se. Consistent with a functional role in decapping, GFP-tagged Edc3p localizes to cytoplasmic foci involved in mRNA decapping referred to as P-bodies. These results identify Edc3p as a new protein involved in the decapping reaction. N eukaryotic cells, mRNA turnover and its regulation al.
    [Show full text]