Transcript Comparative genomics analysis

Towards a new gene
definition: the
hindrance of
Alternative Splicing
Laura Fontrodona Montals
Mòdul 4: Genòmica i Proteòmica
Màster Oficial de Genètica-UAB
Background
High–throughput methods used for AS analysis
A. Transcript/genomic alignments B. Splicing-sensitive microarrays
Ben-Dov et al., 2008
Molecular basis of Splicing Mechanism
Kim et al., 2007
Splice signals that delineate the proper exon-intron boundaries.
• 5’ splice site (5’ss)
• 3’ splice site (3’ss)
• Branch site (BS)
• Polypyrimidine tract
Molecular basis of Splicing Mechanism
Wang et al., 2007
Regulation of Splicing (both constitutive and alternative).
• Cis-acting factors: ESEs, ISEs, ESSs, ISSs
• Trans-acting factors: the spliceosome ( 5 diferent RNPs and
more than 150 proteins)
Origin and evolution of
Alternative Splicing:
Comparative genomics
Types of Alternative Splicing
A. Exon skipping
B. Alternative 3’ss selection
C. Alternative 5’ss selection
D. Intron retention
Kim et al., 2007
AS is believed to be a major source for the phenotypic complexity in higher
eukaryotes.
Exon skipping exhibits a gradual increase in its relative prevalence along the
eukaryotic tree, suggesting it is the more important event in shaping phenotypic
complexity.
Do the same evolutionary forces apply to
Alternative and Constitutive Splice Exons?
Comparative genomics analysis
Kim et al., 2007
Exons that are alternatively spliced in both human and mouse are more conserved
than constitutive exons.
Do the same evolutionary forces apply to
Alternative and Constitutive Splice Exons?
Comparative genomics analysis
Kim et al., 2007
The sequence conservation extends into the flanking introns of conserved alternative
cassette exons for 80 to 100 nucleotides compared to constitutive ones.
Do the same evolutionary forces apply to
Alternative and Constitutive Splice Exons?
Comparative genomics analysis
Kim et al., 2007
The type of the 5’ss dictates constitutive or alternative splicing and also the level of
inclusion/skipping in alternative splicing.
Do the same evolutionary forces apply to
Alternative and Constitutive Splice Exons?
Comparative genomics analysis
Kim et al., 2007
Alternative exons are shorter and are flanked by longer introns than constitutively
spliced ones.
Do the same evolutionary forces apply to
Alternative and Constitutive Splice Exons?
Comparative genomics analysis
Kim et al., 2007
A maximum of 60% of identity
A maximum of 80% of identity
Exonic Splicing Regulatory sequences (ESR) are significantly more conserved in
alternatively spliced exons.
Do the same evolutionary forces apply to
Alternative and Constitutive Splice Exons?
Comparative genomics analysis
Kim et al., 2007
Exclusion or inclusion of symmetrical exons would mantain the reading frame of the
downstream exons.
Origin of alternative cassette exons
Kim et al., 2007
Origin of alternative cassette exons
• Alternative cassette exons with low inclusion levels probably originated from
de novo exonization of intronic sequences.
• Alternative cassette exons with high inclusion levels probably originated from
exons that were previously constitutively spliced.
The inclusion level may serve as an indicator of the age of
alternatively spliced exons and, consequently, of their origin and
evolution
What is a gene?
From the abstract elements of
heredity of Mendel and Morgan to
the present-day ORFs
“One thing only I know, and that is that I
know nothing”
Socrates
“The more I know, the less I understand”
A. Einstein
Problems with the current definition of a gene
o Gene regulation
o Overlapping and spliced genes
o Overlapping
o Splicing
o Trans-splicing/tandem chimerism
o Parasitic and mobile genes
o The large amount of “junk DNA” under selection
The ENCODE view: dispersed genome activity
o Unannotated transcription
TARs (Transcriptionally Active Regions)
o Unannotated and alternative TSSs
TSSs (Transcription Start Sites)
Unannotated and alternative TSSs
Gerstein et al., 2007
Many known protein genes have alternative TSSs that are sometimes
>100 kb upstream of the annotated transcription start site.
The ENCODE view: dispersed genome activity
o Unannotated transcription
TARs (Transcriptionally Active Regions)
o Unannotated and alternative TSSs
TSSs (Transcription Start Sites)
o More alternative splicing
o Dispersed regulation
Dispersed regulation
Gerstein et al., 2007
Many regulatory elements actually reside within the first exon, introns, or
the entire body of a gene.
Toward an updated definition of a gene
Gerstein et al., 2007
Take-home
messages
Take-home messages
A.
Why do cells tolerate noise instead of turning down the
volume?
o Some truncated mRNA molecules, which are generated as a result of AS, are
translated into proteins.
o There are little evidences to indicate whether this alternative splicing isoforms
have a role as functional proteins or whether they are merely “noise” of the
splicing machinery.
B.
Sequencing of genomes and transcripts from more
organisms from a broad range of lineages will further our
understanding of the alternative splicing mechanism.
Take-home messages
C.
The classical view of a gene as a unit of hereditary
information aligned along a chromosome, each coding for one
protein, has changed dramatically over the past century. And
now the ENCODE project has increased the complexity still
further.
Bibliography
-International Human Genome Sequencing Cosortium. Initial sequencing and analysis of the human
genome. Nature, Vol 409. February 2001
-Mouse Genome Sequencing Consortium. Initial sequencing and comparative analysis of the mouse
genome. Nature, Vol. 420. December 2002
-Douglas L. Black. Mechanisms of Alternative pre-messenger RNA Splicing. Annual Reviews
Biochemistry, 2003
-Rotem Sorek, Ron Shamir, and Gil Ast. How prevalent is functional alternative splicing in the human
genome?. Trends in Genetics, Vol. 20. February 2004
-Manuel Irimia, David Penny, and Scott W. Roy. Coevolution of genomic intron number and splice
sites. Trends in Genetics, Vol 23. April 2007
-Rotem Sorek. The birth of new exons: Mechanisms and evolutionary consequences. RNA, Vol 13.
2007
-Eddo Kim, Amir Goren, and Gil Ast. Alternative splicing: current perspectives. BioEssays, 2007
Bibliography
-M. Gerstein, C. Bruce, J. Rozowsky, D. Zheng, J. Du, J. Korbel, O. Emanuelsson, Z. Zhang, S.
Weissman, and M. Snyder. What is a gene, post-ENCODE? History and updated definition. Genome
Research, 2007
-Martha J. Fedor. Alternative Splicing Minireview Series: Combinatorial Control Facilitates Splicing
Regulation of Gene Expression and Enhances Genome Diversity. Journal of Biological Chemistry, Vol.
283. January 2008
-Claudia Ben-Dov, Britta Hartmann, Josefin Lundgren, and Juan Valcárcel. Genome-wide Analysis of
Alternative Pre-mRNA Splicing. Journal of Biological Chemistry, Vol 283. January 2008
Thank you for your attention!!