07 - Alternative Splicing

Process of Splicing (two steps):

• 5 critical bases: 5’ donor SS (GU), branch point (A), 3’ acceptor SS (AG)

  

• first step:

  • cleavage at the 5’ SS

  • joining of the 5′ end of the intron to an A within the intron (the branch point)

  • —> lariat-like (lasso) intermediate —> intron forms a loop

• second step:

  • cleavage at the 3′ splice site and ligation of the exons

  • —> result: excision of the intron as a lariat-like structure

Effects of AS:

1. multiple isoforms of a gene —> Protein diversity

2. Tissue-specific regulation of gene expression

   
   

Larger product:

• Change protein stability

• Change enzymatic/ signaling activity

• Negative control

  • repressor protein binds to the primary RNA in tissue 2

  • prevents intron removal by the splicing machinery

• Positive control

  • splicing machinery needs activator protein to remove intron sequence efficiently

• ~ 25.000 genes

• > 35% undergo AS

• constitutive:

  • always > 1 isoform per pre-mRNA

• regulated:

  • splicing forms depends on tissue/condition/time

• add protein parts: 75% of AS in CDS —> change structure

  • stability (e.g., inclusion of cleavage sites)

  • alter enzymatic or signaling activities

  • alter protein binding (e.g., receptor/ligand)

• alter 5’ / 3’ UTR regions:

  • RNA either degraded or not

  • RNA in cytoplasm or exported

• regulation of biological events:

  • Cell differentiation

  • Neuron development

  • Apoptosis

• Expressed sequence tag

• cDNA -> RNA that start from either

  • 5’ -> 5’ EST

  • 3’ -> 3’ EST

• Insertion and deletion in Expressed Sequence Tags (ESTs) relative to mRNA

• two splice sites are mutually exclusive —> check donor/acceptor site (are highly conserved, 99.24% of introns have a GT-AG at their 5′ and 3′ ends)

—> heavily depends on ESTs

—> analyzing RNA isoforms

• using RT-PCR with primers that flank the alternatively spliced region → different lengths of PCR product

• using microarrays (high-throughput approach) with exon-exon junction probes

Computation of splice junction difference ratio (SJD)

—> 0 = the same, 1 = completely different