SLIDE 1 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 human genome. The efgorts 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.
ENCODE
Tress et al., PNAS, in press.
SLIDE 2 m001 m002 m003 m004 m005 m007 m008 m009 m010 m011 m012 m013 m014 r111 r112 r113 r114 r121 r122 r123 r131 r132 r133 r211 r212 r213 r221 r222 r223 r231 r232 r233 r311 r312 r313 r321 r322 r323 r334 r324 m006 r331 r332 r333
1 2 3 4 5 6 9 8 7 10 12 11 13 15 14 20 19 16 22 21 Y X 17 18
ENCODE
Tress et al., PNAS, in press.
ENCODE
SLIDE 3
ENCODE
ENCODE
Tress et al., PNAS, in press.
SLIDE 4 Isoform 008 SQWAPTCADVHISYLPLAHMFERMVQ . : .:: ::.:::::::::..: Isoform 004 KVIFPRQDDVLISFLPLAHMFERVIQ
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.
It was very rare - just 2 of the 250 genes with splice variants displayed this form of splicing.
Gene ACSL6 (Long-chain-fatty- acid--CoA ligase 6). It afgects binding affjnities of AMP.
AMP
Mutually Exclusive Exons
Tress et al., PNAS, in press.
ENCODE
SLIDE 5 PDB Structural Database Growth
Database Searches for Constitutive
36 gene products have known structure and models can be built for a high number (245) of the 434 genes. Tress et al., PNAS, in press.
ENCODE
SLIDE 6
?
ENCODE
Tress et al., PNAS, in press.
ENCODE
SLIDE 7
ENCODE
Tress et al., PNAS, in press.
ENCODE
SLIDE 8
The insertion or deletion of complete functional domains through alternative splicing was rare. The efgect was most marked with the immunoglobulin domain 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.
Alternatively Spliced Isoforms and
Tress et al., PNAS, in press.
ENCODE
SLIDE 9 Many variants are transcribed. Individual RT-PCR experiments have been carried out for a number of the genes (eg TAZ, ITSN1). Some variants are translated. Individual cases known from experiment. Need for HT experimental confirmation. Occasionally variants have known structure (see left). But we only found one gene with isoforms that had recorded difgerent functions (ACSL6). There are alternative isoforms implicated in disease states:
- isoform 011 from locus TNNT3 in
facioscapulohumeral muscular dystrophy
Do Alternative Isoforms Have a Role in the Cell?
Alternatively spliced transcripts have been implicated in a number
- f cellular processes, but how often do we see evidence of this in
this set?
MTCP-2 MTCP-1
ENCODE
SLIDE 10 Sequence identity
10% 30% 50% 70% 90% Homologous proteins
Same topology Main chain of the “core” very similar, extended common “core” Main chain of the “core” somehwat similar, limited common “core”
The methods
SLIDE 11 Sequence identity
10% 30% 50% 70% 90% Homologous proteins
Same topology Main chain of the “core” very similar, extended common “core” Main chain of the “core” somehwat similar, limited common “core”
Drug design? Molecular biology? Biochemistry?
The methods
SLIDE 12
gag Nucleocapsid core proteins pol Reverse transcriptase, protease and integrase env Viral coat proteins Regulatory proteins Accessory proteins
The examples
HIV
SLIDE 13
HIV CD4/coreceptor HIV-RNA Integration transcription Reverse transcription “budding” + maturation regulation tat, ref, nef gag, pol, env assembly RT
The examples
HIV
SLIDE 14
!
D D
The examples
HIV protease
Pearl and Taylor Nature, 1987
SLIDE 15
The examples
HIV protease
SLIDE 16 C E1 E2 p7 NS2 NS3 NS4A NS4B NS5A NS5B
5’ untranslated 3’ untranslated Structural proteins Non structural proteins
core Envelope Glycoprotein 1 Envelope Glycoprotein 2
?
Protease/helicase Protease cofactor RNA polymerase
? ? ?
RNA
The examples
HCV
SLIDE 17
The examples
HCV protease
Pizzi et al PNAS, 1994
SLIDE 18
The examples
HCV protease
Pizzi et al PNAS, 1994
SLIDE 19 AVGIFRAAVCTRGVAKAVD FVPVESMETTMRSPVFTDN SSPPAVPQSFQVAHLHAPT GSGKSTKVPAAYAAQGYKV LVLNPSVAATLGFGAYMSK AHGIDPNIRTGVRTITTGA PVTYS
The examples
leptin
Madej et al FEBS Lett, 1995
SLIDE 20 Giuliana Brunetti Enrico Capobianco Simone Carcangiu Alberto de la Fuente Matteo Floris Elisabetta Marras Joël Masciocchi Elisabetta Muscas Massimiliano Orsini Enrico Pieroni Frédéric Reinier Patricia Rodriguez Tome’ Alphonse Thanaraj Thangavel Maria Valentini Claudia Bertonati Michele Ceriani Domenico Cozzetto Emanuela Giombini Alejandro Giorgetti Paolo Marcatili Veronica Morea Romina Oliva Domenico Raimondo Alessandra Godi Stefania Bosi Claudia Bonaccini Marialuisa Pellegrini Simonetta Soro Tiziana Castrignanò
Danilo Carrabino EU BioSapiens Network of Excellence Human Frontiers Program Regione Sardegna Istituto Pasteur Universita' di Roma
