The Encyclopaedia of DNA Elements (ENCODE) is an NIH-backed multi-million dollar project, that brings together an international consortium of scientists in industry and academia with the aim of identifying all the functional elements in the ENCODE human genome. The e fg orts of the ENCODE consortium are focused entirely on DNA aspects of the project, such as transcription sites, TRANSFRAGS, non–protein-coding genes and sequences that mediate chromosome structure and dynamics. The pilot project is studying 30MB from 44 regions comprising 1% of the genome. 15MB come from regions chosen for their scientific interest, the rest are chosen via a stratified random sampling method. Tress et al., PNAS, in press.
ENCODE r112 r221 r121 r231 r113 m002 r212 ENCODE 5 4 r331 3 r131 1 2 m011 m010 m009 r334 r223 r123 r332 r114 m013 r323 m012 m001 m003 r222 m014 r321 r312 r232 11 8 9 7 10 6 12 m008 r111 r211 r213 r233 r311 r313 r122 r322 16 17 18 r132 15 13 14 r333 m004 m005 m007 r133 r324 20 21 22 Y 19 m006 X Tress et al., PNAS, in press.
ENCODE ENCODE Tress et al., PNAS, in press.
Mutually Exclusive Exons This is the famous form of alternative splicing – where the gene has a choice between two mutually exclusive alternative internal exons, eg. DSCAM in drosophila. Alt. Exon 1 Alt. Exon 2 ENCODE It was very rare - just 2 of the 250 genes with splice AMP variants displayed this form of splicing. Gene ACSL6 (Long-chain-fatty- acid--CoA ligase 6). It a fg ects binding a ffj nities of AMP. Isoform 008 SQWAPTCADVHISYLPLAHMFERMVQ . : .:: ::.:::::::::..: Isoform 004 KVIFPRQDDVLISFLPLAHMFERVIQ Tress et al., PNAS, in press.
Database Searches for Constitutive 36 gene products have known structure and models can be built for a high number (245) of the 434 genes. ENCODE PDB Structural Database Growth Tress et al., PNAS, in press.
ENCODE ENCODE ? Tress et al., PNAS, in press.
ENCODE ENCODE Tress et al., PNAS, in press.
Alternatively Spliced Isoforms and The insertion or deletion of complete functional domains through alternative splicing was rare. The e fg ect was most marked with the immunoglobulin domain ENCODE Functional domains that were cut in two by splicing were much more common - splicing events occurred inside of Pfam-A hand-curated functional domains in 46.5% of sequence-distinct isoforms. Tress et al., PNAS, in press.
Do Alternative Isoforms Have a Role in the Cell? Alternatively spliced transcripts have been implicated in a number of cellular processes, but how often do we see evidence of this in this set? Many variants are transcribed. Individual RT-PCR experiments have been carried out for a number of the genes (eg TAZ, ITSN1). ENCODE Some variants are translated. Individual cases known from experiment. Need for HT experimental MTCP-2 confirmation. Occasionally variants have known structure (see left). But we only found one gene with isoforms that had recorded di fg erent functions (ACSL6). MTCP-1 There are alternative isoforms implicated in disease states: - isoform 011 from locus TNNT3 in facioscapulohumeral muscular dystrophy
90% Main chain of the “core” very similar, The methods extended common “core” 70% Homologous proteins Main chain of the 50% “core” somehwat similar, limited common “core” 30% Same topology 10% Sequence identity
Drug design? 90% Main chain of the “core” very similar, The methods extended common “core” 70% Homologous proteins Biochemistry? Main chain of the 50% “core” somehwat similar, limited common “core” 30% Molecular biology? Same topology 10% Sequence identity
HIV Accessory proteins Regulatory proteins gag Nucleocapsid core proteins The examples pol env Reverse transcriptase, Viral coat proteins protease and integrase
HIV HIV CD4/coreceptor regulation The examples tat, ref, nef HIV-RNA RT transcription gag, pol, env Integration assembly Reverse transcription “budding” + maturation
HIV protease The examples ! D D Pearl and Taylor Nature, 1987
HIV protease The examples
HCV The examples RNA 5’ untranslated Structural proteins Non structural proteins 3’ untranslated C E1 E2 p7 NS2 NS3 NS4A NS4B NS5A NS5B ? core Protease RNA cofactor polymerase ? ? Envelope Glycoprotein 1 ? Envelope Protease/helicase Glycoprotein 2
HCV protease The examples Pizzi et al PNAS, 1994
HCV protease The examples Pizzi et al PNAS, 1994
leptin AVGIFRAAVCTRGVAKAVD FVPVESMETTMRSPVFTDN SSPPAVPQSFQVAHLHAPT GSGKSTKVPAAYAAQGYKV LVLNPSVAATLGFGAYMSK The examples AHGIDPNIRTGVRTITTGA PVTYS Madej et al FEBS Lett, 1995
Claudia Bertonati Michele Ceriani Giuliana Brunetti Domenico Cozzetto Enrico Capobianco Emanuela Giombini Simone Carcangiu Alejandro Giorgetti Alberto de la Fuente Paolo Marcatili Matteo Floris Veronica Morea Elisabetta Marras Romina Oliva Joël Masciocchi Domenico Raimondo Elisabetta Muscas Alessandra Godi Massimiliano Orsini Stefania Bosi Enrico Pieroni Frédéric Reinier Claudia Bonaccini Patricia Rodriguez Tome’ Marialuisa Pellegrini Alphonse Thanaraj Thangavel Simonetta Soro Maria Valentini Tiziana Castrignanò P. D’Onorio De Meo Danilo Carrabino EU BioSapiens Network of Excellence Human Frontiers Program Regione Sardegna Istituto Pasteur Universita' di Roma
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