Searching for functional regions (coding Searching for functional regions (coding or non-coding) in mammalian genomes or non-coding) in mammalian genomes Organization of the human genome Human genome project: present status Human sequence data in GenBank/EMBL Prediction of functional elements by computer analysis of genomic sequences State of the art Success and pitfalls of different approaches Prediction of function by homology Orthology/paralogy
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Searching for functional regions (coding or non-coding) in mammalian genomes Organization of the human genome Human genome project: present status Human.
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Searching for functional regions (coding or non-coding) Searching for functional regions (coding or non-coding) in mammalian genomes in mammalian genomes
Organization of the human genome Human genome project: present status Human sequence data in GenBank/EMBL Prediction of functional elements by computer analysis of
genomic sequences State of the art Success and pitfalls of different approaches
Prediction of function by homology Orthology/paralogy
Functional elements in the human genomeFunctional elements in the human genome
3.4 109 nt 50,000-100,000 protein-coding genes
81% no known function43%38%introns4%12%protein-coding regions
centromeres, telomeres,
RNA2%intergenic
Untranslated RNAs: Xist, H19, His-1, bic, etc.
Regulatory elements: promoters, enhancers, etc.
Repeated sequences (SINES, LINES, HERV, etc.) : 40% of the human genome
Repeat SequencesRepeat Sequences
Tandem repeats
motif bloc size % human genome satellite: 2-2000 nt up to 10 Mb 10% minisatellite: 2-64 nt 100-20,000 bp ? microsatellite: 1-6 nt 10-100 bp 2%
Interspersed repeats
SINE (non-autonomous retroelement) LINE (retrotransposon) Endogenous Retrovirus (HERV, LTR- retrotransposon) DNA transposons
Fréquence des éléments transposables Fréquence des éléments transposables dans le génome humaindans le génome humain
Total = 42% (Smit 1999)
0%4%8%12%AluLINE1MIRLINE2LTR elementsDNAtranposon
RetropseudogènesRetropseudogènes
23,000 à 33,000 retropseudogènes dans le génome humain (6-10 copies / Mb)
Les gènes qui génèrent des retropseudogènes sont généralement de type housekeeping
Gonçalves et al. 2000
Structure of human protein genesStructure of human protein genes
1396 complete human genes (exons + introns) from GenBank Average size (25%, 75%)
Gene 15 kb ± 23 kb (4, 16) (10% > 35 kb) CDS 1300 nt ± 1200 (600, 1500) Exon (coding) 200 nt ± 180 (110, 200) Intron 1800 nt ± 3000 (500, 2000) 5'UTR 210 nt (Pesole et al. 1999) 3'UTR 740 nt (Pesole et al. 1999)
Intron/exon Number of introns: 6 ±3 introns / kb CDS Introns / (introns + CDS): 80% 5' introns in 15% of genes (more ?), 3 ’introns very rare
Alternative splicing in more than 30% of human genes (Hanke et al. 1999)
Structure of human protein genesStructure of human protein genes GenBank: bias towards short genes 1396 complete human genes (exons + introns)
Structure of human protein genesStructure of human protein genes GenBank: bias towards short genes 1396 complete human genes (exons + introns) 9268 complete human mRNA
Sequence:cDNA
complete gene (exons+introns)
400800120016002000889092949698Average CDS size (nt)Publication date
Isochore organization of the human genomeIsochore organization of the human genome
Insertion of repeated sequences (A. Smit 1996) Recombination frequency (Eyre-Walker 1993) Chromosome banding (Saccone, 1993) Replication timing (Bernardi, 1998) Gene density (Mouchiroud, 1991) Gene expression ?? -> No Gene structure (Duret, 1995)
isochore %C+G % total genomic DNA
L1+L2 : 33%-44% 62 %
H1+H2 : 44%-51% 31%
H3 : 51%-60% 3-5%
H1+H2L1+L2H3H1+H2L1+L2L1+L2>300 kbBernardi et al. 1985
Isochores and insertion of repeat sequencesIsochores and insertion of repeat sequences
4%8%12%16%20%AluLINE-1LTR-
elements
Density in repeat sequencesG+C content of genomic sequence:G+C < 39%G+C > 47%G+C 39%-47%
4419 human genomic sequences > 50 kb4419 human genomic sequences > 50 kb
Isochores and gene densityIsochores and gene density
MHC locus (3.6 Mb) MHC locus (3.6 Mb) (The MHC sequencing consortium 1999)(The MHC sequencing consortium 1999)
Class I, class II (H1-H2 isochores): 20 genes/Mb, many pseudogenesClass I, class II (H1-H2 isochores): 20 genes/Mb, many pseudogenesClass III (H3 isochore): 84 genes/Mb, no pseudogeneClass III (H3 isochore): 84 genes/Mb, no pseudogene
Class II boundaries correlate with switching of replication timingClass II boundaries correlate with switching of replication timing
isochore % total genomic DNA %total genes
L1+L2 : 62 % 31%
H1+H2 : 31% 39%
H3 : 3-5% 30%
2060100140Number of genes / MbL1+L2H1+H2H3Mouchiroud et al. 1991
Isochores and introns lengthIsochores and introns length
Inventory of all mRNAs expressed by an organism, in different tissues, development stages, pathologies, …
Single pass sequences: high error rate (>1%), partial mRNA sequences Usually derived from poly-dT-primed cDNA -> bad coverage of 5' regions of long mRNAs 60-80% of human genes represented in public EST database, but only 25-50% of the total
Number of ESTs (Sep. 2000)Number of ESTs (Sep. 2000)
large insert DNA library (BAC): 150-250 kbgenomesmall insert library (M13)sequencingcontig assemblyfinished sequencecloningsub-cloningfinishing (filling gaps)Phase 0 single-few pass reads of a single clone (not contigs).Phase 1 Unfinished, may be unordered, unoriented contigs, with gaps.Phase 2 Unfinished, ordered, oriented contigs, with or without gaps. Phase 3 Finished, no gaps (with or without annotations)
GenBank/EMBL divisionPhase 0
Phase 1
Phase 2
Phase 3
HTG PRI (nr)GenBank/EMBL HTG division : High Troughput Genome sequences
Genomic SequencesGenomic Sequences
(draft)(draft)
Exponential growth of sequence dataExponential growth of sequence data
Doubling time: 13 mounths
-500
0
500
1000
1500
2000
2500
3000
3500
82 86 90 94 98Date
0.1
1
10
100
1000
10000
82 86 90 94 98Date
Publicly available sequences (Mb)
Human Genome Sequence DataHuman Genome Sequence Data Traditional sequences: correspond to biologically
characterized genes, annotated by reearchers or database curators, usually relatively short (<20,000).
Finished genome sequences: long contiguous sequences, correspond to clones (cosmid, BAC, PAC); partly automatically generated annotations covers repetitive elements, kown and predicted genes, EST matches
Unfinished genome sequences (draft): large sequence entries consisting of unordered pieces separated by runs of N's, correspond to clones, contain minimal annotation.
Genome survey sequences: low-quality, single pass sequences from a variety of different projects (BAC end sequencing, polymorphism studies, CpG islands, etc.), minimal annotation.
Different types of nucleotide sequences in current databasesDifferent types of nucleotide sequences in current databases
StandardHigh throughput genome (HTG)
Genome survey sequence (GSS)
Expressed sequence tags (EST)
Contents
biologically characterized genes and RNAs, finished clones from genome projects
The human genome sequencing projectThe human genome sequencing projectWhere are we today (July 17 2000) ?Where are we today (July 17 2000) ?
According to Phillip Bucher (SIB, Lausanne) statistics and genome coverage estimates (see also EBI's statistics: http://www.ebi.ac.uk/~sterk/ genome-MOT)
Prédiction de gènes protéiques completsPrédiction de gènes protéiques complets C. elegans: la plupart des ‘ gènes ’ annotés sont seulement des prédictions Peut-on utiliser ces méthodes pour annoter les séquences génomique humaines ?
+ les faux positifs !
00.20.40.60.8113579111315Sensibilité par exon:90%80%
Probabilité de détecter tous les exons d’un gènesNombre d’exons du gène
Un peu d ’optimismeUn peu d ’optimisme Fraction de la longueur des gènes correctement prédits:
70-80%
Probabilité que deux exons potentiels consécutifs soient réels (et donc positifs en RT-PCR)
0.5
Prediction of functional elements (2)Prediction of functional elements (2)
Large scale transcriptome projects: ESTs, full-length cDNA Identification of transcribed genes (protein or non-coding RNA) Information on alternative splicing, polyadenylation (Hanke et al.
1999, Gautheret et al. 1998), expression pattern SIM4: align a cDNA to genomic DNA Very useful but ...
Problems with genes expressed at low level, narrow tissue distribution, stage-specific expression, …
Limited tissue sampling Artifacts in ESTs (introns, partially matured RNA, …) Limited to polyadenylated RNA
Prediction of functional elements (3)Prediction of functional elements (3) Comparative sequence analysis (phylogenetic footprinting)
Function => selective pressure
Corollary Sequence conservation = selective pressure = function
provided the number of aligned homologous sequences represents enough evolutionary time for the accumulation of mutations at the less constrained (presumably selectively neutral)
base positions.
Evolutionary rate in non-functional DNA: ~ 0.3% / My (± 0.069)
Man/Mouse: ~ 80 Myrs 46-58% identity
Mammals/Birds: ~ 300 Myr 26-28% identity
Random sequences 25% identity
Analyse comparative des gènes de Analyse comparative des gènes de -actine de l'homme et de la carpe-actine de l'homme et de la carpe
CarpeHomme5’UTR 3’UTR site polyA échelle de similarité: pas de similarité significative70 - 80% identité80 - 90% identitérégions codantes: éléments régulateurs:introns:ATGcodon stop
Works for all kinds of functional elements (transcribed or not, coding or not) as far as the information is in the primary sequence
Does not require any a priori knowledge of the functional elements
Limits Absence of evolutionary conservation does not mean absence of function No efficient method to detect unknown conserved secondary structure in RNA Function, but what function ? Depends on the sequencing status of other genomes
Human, mouse, fugu, C. elegans, drosophila, yeast, A. thaliana Number of sequences to compare : > 200 Myrs of evolution
Prédiction de gènes eucaryotes (suite)Prédiction de gènes eucaryotes (suite)
Approche comparative Comparaison d ’une séquence génomique avec des gènes déjà caractérisés
dans d ’autres espèces (WISE2: alignement ADN/protéine avec épissage) Comparaison de séquences génomiques (non-annotées) homologues
– Locus mnd2 (homme souris) (Jang et al. 1999): >80 kb– Prédiction d ’exons internes basée sur la conservation de séquence
ORF ≥ 80 nt
Séquence protéique ≥ 70% similarité
Séquence ADN ≥50% identité
GT AG conservés
=> détection de tous les exons internes du gène D6Mm5e
– Généralisation de la méthode (Guigo 2000). Sensibilité ? Spécificité ?
Next steps in genome projectsNext steps in genome projects
Identify genes and other functional elements within genomic sequence (where are the genes ?)
Determine the function of genes (what do they do ?)
Prédiction de fonction par homologie ?Prédiction de fonction par homologie ? Similarité entre séquences homologie Homologie structure conservée Structure conservée fonction conservée
Oui, mais … Fonction: concept flou
– activité biochimique identique ? e.g. même ligand pour un récepteur, même substrat pour une enzyme, même gènes cibles pour un facteur de transcription.
– distribution tissulaire ? (isoformes tissu-spécifiques).– compartimentalisation cellulaire: cytoplasme, mitochondrie, etc.
Protéines homologues de fonction différentes – Protéines homologues ligands (activateur/répresseur) d ’un même récepteur– Recrutement pour une fonction totalement différente: -cristalline / -énolase
Orthologie/paralogie
Évolution modulaire
Prédiction de fonction par homologie ?Prédiction de fonction par homologie ?
326 Highly Conserved 326 Highly Conserved Regions (HCRs)Regions (HCRs)
• > 70% identity over 50 to 2000 nt after more than 300 Myrs
• Unique sequences
• Generally specific of only one gene
• Longest HCR:
84% identity over 1930 nt after 300 Myrs
3’UTR deltaEF1 transcription factor
• Oldest HCRs: 500 to 600 Myrs
• No HCR between vertebrates and insects or nematode
Oldest HCRsOldest HCRsMillion yearsPorifera (sponge)Nematodes (C. elegans)Arthropods (Drosophila)EchinodermsUrochordataCephalochordata (amphioxus)Jawless fisheschondrichthyes (ray, shark)actinopterygii (bony fishes)amphibians mammals birds reptiles600400200800Sequencing effort: 9 to 100 Mb 0.8 to 2.4 Mb less than 0.2 MbHistone 3’UTR- actin3’UTR
3 5’HOX UTRVertebrates
Conservation pattern in Conservation pattern in 3’UTRs3’UTRs
position relative to the stop codon (nt)10005000150020002400c-fosTransferrin receptorbirdmammalEndoplasmic-reticulum Ca2+ ATPase birdmammalbirdmammalsimilarity: <60% ≥60% ≥70% ≥80%
Distribution of HCRs within Distribution of HCRs within genesgenes3'-non-coding5'-non-codingintrons0%10%20%30%40%mammals / birdsmammals / amphibiansmammals / bony fishes2841917296512563812 Frequency of orthologous
genes containing HCRs
HCRs and multigenic familiesHCRs and multigenic familiesHistone replacement variant H3.3A0400600100014001800AAAAAAAAUGStopAUGStopAAAAAAAHistone replacement variant H3.3BHistone replacement variant H3.3A and H3.3B, Calmodulinsnt• several genes coding for a same protein
• non-coding sequences are distinct, and conserved
Function of 3’HCRs: Function of 3’HCRs: mRNA stability, translationmRNA stability, translationA+U-rich element: stability, translationposition relative to the stop codon (nt)10005000150020002400c-fosTransferrin receptorbirdmammalbirdmammalsimilarity: <60% ≥60% ≥70% ≥80%IRE : Iron Responsive Element
IRP : Iron Regulatory Protein
CCAGUGN5'3'
Function of 3’HCRs:Function of 3’HCRs:mRNA subcellular localizationmRNA subcellular localization
Myosin heavy chain, c-myc, vimentin, -actin
chickencarp (bony fish)site poly(A)site poly(A)0200400600800position relative to the stop codon (nt)localization signalssimilarity: <60% ≥60% ≥70% ≥80%- 3’actin UTR