Alternative Splicing: Combinatorial Output from the Genome

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Alternative Splicing Combinatorial Output from
the Genome

• Gavin C. Roberts and Christopher W. J. Smith,
  June 2002

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Introduction

• Focus of the Review
• Explain recent findings concerning alternative
  pre-mRNA splicing as a generator of protein
  diversity
• Explore the conservation of alternate splicing
  between different multicellular organisms
• Explore alternative splicing as a mechanism for
  combinatorial gene output

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Key Terms

• Transcriptomics-the comprehension of the entire
  complement of mRNAs for any given organism or
  cell
• Proteome-the complete set of proteins in a cell
  or organism
• Spliceosome-a 60s ribonucleoprotein complex
  within which splicing of pre-mRNA occurs
• Alternate Splicing-different arrangements of
  exons resulting in isoformeric proteins
• Isoform-proteins created through alternate
  splicing of exons

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Examples of Alternate Splicing

• Human Immunogenes can be spliced to create great
  diversity

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Examples of Alternate Splicing

• The human genome has a 10 to 100 fold greater
  complexity in its expressed proteome as compared
  to its relatively limited number of genes

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Splicing

• Pre-mRNA splicing is essential for expression of
  many eukaryotic genes
• Genes contain non-coding introns
• Introns are copied into initial pre-mRNA
• Once introns are removed, exons spliced together
• Occurs within the spliceosome

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Spliceosomes

• 60s ribonucleoprotein complex
• Contains 50 to 100 protein subunits
• Active core is primarily composed of RNA
• Believed to be a Ribozyme
• Core itself is capable of performing the first
  step of splicing

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Types of Alternate Splicing
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Alternate Splicing

• Typical genes can contain multiple introns, and
  often exons can be joined in several ways to
  generate multiple mRNAs
• This allows for optional inclusions or
  substitutions of exons
• Results in protein Isoforms, with specific
  functional domains while sharing common functions

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Alternate Splicing

• Alternate Splicing can also lead to
• Premature termination of ORFs
• Use of the same mRNA sequence in two different
  reading frames

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Alternate Splicing

• Products themselves are important regulators and
  can influence critical developmental paths
• Sexuality in Drosophilia where male-specific
  splicing leads to inclusion premature stop codons
• Apoptosis is influenced by alternate splicing
  events within genes for cell surface receptors
• Bcl proteins and caspases produce antagonistic
  isoforms that promote either cell death or
  survival

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Alternate Splicing can lead to Disease

• Can be caused by cases in which the ratios of
  authentic protein isoforms vary
• Insulin resistance in skeletal muscle of myotonic
  dystrophy patients
• Frasier syndrome

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Functional Consequences

• Can cause significant functional changes for
  proteins expressed by individual genes
• Even when the changes in amino acid sequences
  appear to be very minor
• This is due to two factors its prevalence and
  the extreme combinatorial output of some genes

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Historical Look

• Phil Sharp, in his 1993 Nobel lecture, speculated
  that up to 5 of genes were alternatively spliced
• Ensuing expressed-sequence tag (EST) and
  genome-sequencing projects have given rise to
  estimates which are uniformly higher
• In fact, recent evidence has shown alternate
  splicing in at least one-third of all human genes
• Combinatorial splicing was first described in the
  rat troponin-T gene, where a pair of mutually
  exclusive exons and an array of five cassette
  exons yields up to 64 isoforms

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Historical Look

• The most modern comprehensive studies which
  aligned full-length mRNAs alongside the entire
  genome and identified splicing events in 42 of
  genes
• The true figure may be much higher
• A similar analysis limited to the transcripts
  from chromosome 22 estimated 59 of the genes to
  be alternatively spliced, with an average of 2.6
  distinct transcripts per gene

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Combinatorial output exampleCD44

• Combinatorial Output Examples
• CD44 is a structural transmembrane glycoprotein
  involved in intercellular and cell-matrix
  contacts and signal transduction
• Gene contains 21 exons, at least 10 of which can
  be alternatively spliced
• With just 10 variant exons there are 1024
  isoforms possible (210)
• However some combinations are more frequent, as
  only 30 isoforms have been detected

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Combinatorial output exampleDrosophila

• Dscam is an Ig superfamily member involved in
  axon guidance in the developing brain
• It contains 115 exons, 95 of which can be
  alternatively spliced
• This has the potential to generate 38,016
  isoforms which is 2 fold more the the total of
  all Drosophila genes
• At least a fraction of the potential diversity is
  harnessed for the purposes of correct neural
  wiring

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Genomic Biology

• Shifts emphasis to global, hypothesis-free
  observations
• Leads to new reason for analyzing alternative
  splicing comprehension of the entire complement
  of mRNAs for any given cell or organism, known as
  transcriptomics
• Helps further our understanding of the functions
  of all genes of an organism and how they work
  together to produce life

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Working to Define Transcriptomes

• Combination of experimental observations and
  bioinformatic tools are exploited
• Utilizes
• Existing mRNA/cDNA databases as a staring point
• Genome-scanning software
• Tilting arrays

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Obstacles to overcome

• Properties of the encoded proteins
• Where and when they are expressed
• Prediction of structure and function from
  sequences

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Possible Solutions

• Clues to function may be achieved using software
  that identifies know domains and motifs
• Laboratory-based assays carried out on massively
  parallel scale
• cDNA microarrays and oligonucleotide chips

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Conclusions

• Alternative pre-mRNA splicing is emerging as a
  widespread mechanism for modulating the function
  of the genome
• Appears likely that most human genes produce
  multiple, different proteins with different
  properties
• More technologies must be developed to fully
  understand the function of all possible mRNAs and
  monitor their expression

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