1. Monday, October 18, 1:43:47 PM
Outline for today
Lec 06
Gene Prediction: What are genes? Where are genes? Why do we care about a definition?
Prokaryotic vs. eukaryotic gene models
Introns/exons
Splicing
Alternative splicing
Genes-in-genes, genes-ad-genes
Multi-subunit proteins
Gene identification
Homology-based gene prediction
Similarity Searches (e.g. BLAST, BLAT)
Genome Browsers
RNA evidence (ESTs)
Ab initio gene prediction
Gene prediction programs: prokaryotes, eukaryotes
Promoter prediction
PolyA-signal prediction
Splice site, start/stop-codon predictions
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2. Monday, October 18, 1:43:47 PM
Lec 06
Alternative splicing
Alternative splicing can be either constitutive or regulated
Constitutive alternative splicing: more than one product is always made from a pre-mRNA.
Regulative alternative splicing: different forms of mRNA are produced at different time, under different conditions, or in different cell or tissue types.
Alternative splicing is regulated by activators and repressors.
The regulating sequences : exonic or intronic; splicing enhancers (ESE or ISE) or silencers (ESS and ISS). The former enhance and the latter repress splicing.
Proteins that regulate splicing bind to these specific sites for their action.
Mo Chen & James L. Manley (2009): Nature Reviews Molecular Cell Biology 10,
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3. Monday, October 18, 1:43:47 PM
Lec 06
Alternative splicing
Alternative splicing can generate tens of thousands of mRNAs from a single primary transcript. Alternative splicing generates segments of mRNA variability that can insert or remove amino acids, shift the reading frame, or introduce a termination codon.
The typical human gene contains an average of 8 exons.
Up to 59% of human genes generate multiple mRNAs by alternative splicing and ∼80% of alternative splicing results in changes in the encoded protein.
A large fraction of alternative splicing undergoes cell specific regulation in which splicing pathways are modulated according to cell type, developmental stage, gender, or in response to external stimuli.
Heart muscle mRNA 1 2 3 5
Pre-mRNA 1 2 3 4 5
Uterine muscle mRNA 1 3 4 5
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4. Monday, October 18, 1:43:47 PM
Lec 06
Alternative splicing
Alternative splicing is the process where one gene produces more than one type of mRNA.
Environment, Development
DNA
mRNA
80%
20%
Cell type 1
10%
90%
Cell type 2
absent
100%
Cell type 3
The phenotype is determined by the proteome & transcriptome.
Selection acts on the phenotype, and is blind to the genotype.
Therefore: two species/individuals that have different forms of a protein will be selected differently - even if the genes DNA sequence is identical.
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5. Monday, October 18, 1:43:47 PM
Alternative splicing
Lec 06
Alternative splicing can generate mRNAs encoding proteins with different, even opposite functions.
Alternative splicing of the fas apoptosis receptor
Fas pre-mRNA
Fas
Fas ligand
(membrane-associated) 7 6 5
APOPTOSIS
Soluble Fas
(membrane)
(+)
(-)
Intron 1
Intron 2
Therefore, understanding the mechanism of RNA splicing in normal cells and how it is regulated in different tissues and at different stages of development of an organism is essential in order to develop strategies to correct aberrant splicing in human pathologies.
Slide 136

6. Pathologies resulting from aberrant splicing
Monday, October 18, 1:43:47 PM
Pathologies resulting from aberrant splicing
can be grouped in two major categories
Lec 06
Mutations affecting a specific messenger RNA and disturbing its normal splicing pattern.
Examples:
ß-Thalassemia
Duchenne Muscular Dystrophy
Cystic Fibrosis
Frasier Syndrome
Frontotemporal Dementia and Parkinsonism
Mutations affecting proteins that are involved in splicing.
Spinal Muscular Atrophy
Retinitis Pigmentosa
Myotonic Dystrophy
Slide 137

7. Splice variant detection
Monday, October 18, 1:43:47 PM
Splice variant detection
Lec 06
PCR method: simple, sensitive, with std curve enough accurate, however, only internal changes are detectable and can’t scaled up.
Capture probe: very sensitive and accurate, complicated probe design, expensive.
Microarray method: can be scaled up to an entire genome (high throughput), so any types of splice variants are detectable, but not very accurate, complex and expensive. 1 2A 3 2B
Slide 138

8. Monday, October 18, 1:43:47 PM
Outline for today
Lec 06
Gene Prediction: What are genes? Where are genes? Why do we care about a definition?
Prokaryotic vs. eukaryotic gene models
Introns/exons
Splicing
Alternative splicing
Genes-in-genes, genes-ad-genes
Multi-subunit proteins
Gene identification
Homology-based gene prediction
Similarity Searches (e.g. BLAST, BLAT)
Genome Browsers
RNA evidence (ESTs)
Ab initio gene prediction
Gene prediction programs: prokaryotes, eukaryotes
Promoter prediction
PolyA-signal prediction
Splice site, start/stop-codon predictions
Slide 139

9. Bidirectional and partially overlapping genes
Monday, October 18, 1:43:47 PM
Bidirectional and partially overlapping genes
Lec 06
Not very common in human genome.
Provides possibility for common regulation of a gene pair.
Partially overlapping genes are usually encoded by opposite DNA strands.
Found in dense gene areas, as HLA class III complex on 6p21.3.
Could represent sense-antisense pair with one gene is coding mRNA, another is non-coding.
Slide 140

10. Genes within genes Monday, October 18, 1:43:47 PM
Lec 06
Nested intronic genes
Neurofibromatosis gene (NF1)
OGMP-Oligodendrocyte myelin glycoprotein
EVI2A and EVO2B homologues of ecotropic viral intergration sites in mouse.
Two overlapping genes encoded by same strand of mt DNA (unique example).
Two independent AUG located in frame-shift to each other, second stop codon is derived from TA + A from poly-A.
Slide 141

11. Gene prediction Comparative Genomics
Monday, October 18, 1:43:47 PM
Gene prediction
Lec 06
Comparative Genomics
When we BLAST a sequence is that comparative genomics?
Entire genome compared to other entire genomes.
Use information from many genomes to learn more about the individual genes.
What are some questions that comparative genomics can address?
How has the organism evolved?
What differentiates species?
Which genes are required for organisms to survive in a certain environment?
Which non-coding regions are important?
Slide 142

12. Gene prediction through comparative genomics
Monday, October 18, 1:43:47 PM
Gene prediction through comparative genomics
Lec 06
Different questions require different comparisons
Highly similar (conserved) regions between two genomes are useful or else they would have diverged.
If genomes are too closely related all regions are similar, not just genes.
If genomes are too far apart, analogous regions may be too dissimilar to be found.
Slide 143

13. Prokaryotes gene prediction
Monday, October 18, 1:43:47 PM
Prokaryotes gene prediction
Lec 06
NCBI ORF finder
ORF Finder - identifies all possible ORFs in a DNA sequence by locating the standard and alternative stop and start codons.
The deduced amino acid sequences can then be used to BLAST against GenBank. ORF finder is also packaged in the sequence submission software Sequin.
Based on NCBI ORF finder 90 ORFs were identified in the Contig3 (28715 bp).
This method is still not proper way for gene identification!
Slide 144

14. Prokaryotes gene prediction
Monday, October 18, 1:43:47 PM
Prokaryotes gene prediction
Lec 06
Gene calling anomalies
Short genes: a gene is called 'short' when it has been truncated significantly at the 5'-end. Such genes are significantly shorter than their homologs in other species. Often this truncation causes the loss of important functional domains, resulting in theoretical loss of function of the gene.
Slide 145

15. Prokaryotes gene prediction
Monday, October 18, 1:43:47 PM
Prokaryotes gene prediction
Lec 06
Gene calling anomalies
Long genes: a gene is called 'long' when it has been extended at the 5'-end. Such genes are significantly longer than their homolog's in other species. A long gene can create overlaps with neighbouring features, with the result being that neighbouring genes are called short or features in the flanking intergenic regions are missed.
Slide 146

16. Prokaryotes gene prediction
Monday, October 18, 1:43:47 PM
Prokaryotes gene prediction
Lec 06
Gene calling anomalies
Unique gene: a gene is called 'unique' when it has no known homolog's in other species. For such genes, Blast comparisons at the amino acid level with genes in other organisms return no hits. Often, such a gene call is an anomaly which, in turn, causes other anomalies, e.g. neighbouring genes called short.
DdesDRAFT_0263 is a unique gene. If DdesDRAFT_0264 were detected as a short gene, DdesDRAFT_0263 would actually be responsible for this short call.
Dubious (uncertain) gene: a gene called as unique that is too short to be a functional gene is classified as 'dubious.' In actual practice, very few (1-10) dubious genes are found in the gene calls. When present, both unique and dubious genes are included when searching intergenic regions for missed genes.
Slide 147

17. Prokaryotes gene prediction
Monday, October 18, 1:43:47 PM
Prokaryotes gene prediction
Lec 06
Gene calling anomalies
Split genes interrupted by frame shifts and stop codons: a reported split gene could be a good gene that is interrupted by frame-shifts or stop codons. Such a gene is called as a series of consecutive smaller genes, all of which have many blast hits in common.
Split genes DdesDRAFT_1032 and DdesDRAFT_1033 interrupted by a frame-shift.
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18. Prokaryotes gene prediction
Monday, October 18, 1:43:47 PM
Prokaryotes gene prediction
Lec 06
Gene calling anomalies
Missed genes: gene prediction programs often miss genes however, an alignment of this region indicates the presence of a perfectly good gene.
No genes had been predicted in the region between DdesDRAFt_0231 and DdesDRAFT_0232.
However, an alignment of this region indicates the presence of a perfectly good gene.
Slide 149

19. Free gene prediction software
Monday, October 18, 1:43:47 PM
Free gene prediction software
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GeneMark: Georgia Institute of Technology, Atlanta, Georgia, USA.
Based on GeneMark gene prediction software 14 genes were predicted in the Contig3 (28715 bp).
Slide 150

20. Free gene prediction software
Monday, October 18, 1:43:47 PM
Free gene prediction software
Lec 06
Softberry: (FGENESB) Bacterial Operon and Gene Prediction.
Based on Softberry gene prediction software 6 genes were predicted in the Contig3 (28715 bp).
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21. Free gene prediction software
Monday, October 18, 1:43:47 PM
Free gene prediction software
Lec 06
EasyGene: gene finding in prokaryotes (1.2b Server).
Based on EasyGene 1.2b Server 6 genes were predicted in the Contig3 (28715 bp).
Slide 152

22. Free gene prediction software
Monday, October 18, 1:43:47 PM
Free gene prediction software
Lec 06
Glimmer: NCBI Microbial Genome Annotation Tools.
Based on Glimmer 81 genes were predicted in the Contig3 (28715 bp).
Slide 153