On the biological significance of alternative splicing: a bioinformatics approach

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On the biological significance of alternative
splicing a bioinformatics approach
Sandro J. de Souza TDR, 07/05/2004
RNA 10757-765, 2004
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Genomics
Bioinformatics
Large-scale Biology
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The Real Revolution
Early 20th century Mendel and the inheritance
laws Mid 20th century DNA as the genetic
element (Avery) Mid 20th century Watson and
Crick and the structure of DNA. 70s and 80s
Molecular biology/biotechnology 90s and 21th
century Genomics and Bioinformatics
Paradigm in Biology Evolution by means of
natural selection (Darwin and Wallace, mid 19th
century)
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Bioinformatics

• Development of tools
• Gateway to explore new datasets
• Processing of data derived from large-scale
  projects
• A new way to do hypothesis-driven science

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Splicing (1977)Roberts and Sharp (Nobel 1993)
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Exons
Introns
mRNA
Coding Non-coding
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Splicing
Splicing depends on recognition of exon-intron
boundaries
Splice sites are generic and consist solely
of 5 boundary 3 boundary Acceptor
site Polypyrimidine tract
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.....if they occur at the boundaries of the
regions to be spliced out, can change the
splicing pattern, resulting in the deletion or
addition of whole sequences of amino
acids. Walter Gilbert. Why genes in pieces.
Nature 271501, 1978.
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At least half of all human genes undergo
alternative splicing
Biological significance or spurious events?
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Alternative splicing

• 1. Chromosomal ratio activates txn of Sxl in
  females only
• 2. SXL controls splicing of tra-2 mRNA
• 3. Females exon 2 (which has a stop codon) is
  removed via SXL
• Males exon 2 is not removed.
• Males no active TRA
• Females TRA is made.
• 5. TRA directs splicing of dsx mRNA in specific
  manner in males default splicing occurs.

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Alternative Splicing Auditory Hair Cells
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Types of alternative splicing
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3
mRNA
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Large-scale analysis of intron retention in the
human transcriptome
Pedro F.A. Galante, Noboru Jo Sakabe, Natanja
Slager, Sandro J. de Souza
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Examples of intron retention events with
biological significance

• Msl2 in Drosophila
• P element in Drosophila
• retroviruses

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Intron retention and cancer
CD44 several tumors Gastrin receptor pancreas
Ret tyrosine kinase pheochromocytomas Fas
receptor T-cell lymphoma
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Known mRNAs
EST data
SAGE data
Transcriptome Database
Genome Data
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Genome-based cDNA clustering

Exon 1
Exon 2
Exon 3
DNA
RNAm
cluster
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Transcript Mapping
P53
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Types of Data
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Dataset
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Experimental validation
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14 of all human genes show evidence of intron
retention
Kan, States Gish (2002) 36 of RefSeq
database! After sample statistics 5
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Distribution of events along transcripts.
p ltlt 0.005
This bias can be a product of
Underreporting of sequences
p ltlt 0.005
Nonsense-mediated decay (NMD)
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• 2563 out of 3195 (80) sequences with a retained
  intron had an exon/exon boundary downstream of
  the retention event.

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Retained introns are shorter
Pltltltlt0.001
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Domains encoded by retained introns
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Number of domains entirely encoded by Retained
introns only 02 Exon-intron-exon 31 Number
of domains partially encoded by Retained introns
only 25 Exon-intron-exon 10
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Retained introns have a higher GC content
Pltltltlt0.001
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Did retained introns encode protein domains?

• Only retained introns in the CDS were used.
• Only retained introns defined by full-length
  mRNAs were used.
• Protein sequences were searched against PFAM
  database.

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Codon Usage
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Conservation of intron retention in mouse cDNA
sequences
40-57 of all retained introns present a mouse
hit
Identity of orthologous retained introns is 84
Non-retained introns is 60 Exons 87
Mouse cDNA also corresponds to an retention
variant
26 - 10 out of 46
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Frequency of stop codon
retained intron
exon
exon
TACTTGTGCGTAGTCCCCGCGATCTAACGCCACGATGGATGACACTGTGA
TACTTGTGCGTAGTCCCCGCGATCTAACGCCACGATGGATGACAC
Stop codons TAG, TGA, TAA
Found 651 stop codons
Expected 1064
p-value ltlt 0.005
88 cases where the retention generates a putative
truncated protein
mRNA
cds
stop
mRNA
cds
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GC content for sequences upstream and downstream
the premature stop codon 88 cases
exon
exon
retained intron
5
3
stop
GC 58
GC 49
Are under selective pressure for coding potential
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Why the argument of selection is important?

• As noted originally by Gilbert (1978), mutations
  that affect splicing can allow the production of
  new proteins without the loss of the original one

• Therefore, there should not be any negative
  selection on this variant.

• If, however, the new variant has some biological
  significance, selection will act to maintain the
  function of this variant.

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Intron Retention in Tumors
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Towards a reliable set of intron retention events
full-length vs full-length set and retained
intron entirely in the CDS
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Second International Conference on
Bioinformatics and Computational
Biology www.icobicobi.com.br
25-28/10/2004 Angra dos Reis
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Group of Computational Biology
Sandro J. de Souza tennis player Helena
Samaia Research Assistant Ana C. Pereira Admin.
Assistant Maarten Leerkes Ph.D student Noboru
Sakabe Ph.D student Maria Vibranovski Ph.D
student Elza Helena Ph.D student Natanja
Slater Ph.D student Pedro Galante Ph.D
student Elisson C. Osorio programmer Jorge E. de
Souza Ph.D student Rodrigo Soares programmer Andr
e Zaiats system admin.
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