CSB349H1 Lecture Notes - Lecture 9: Fibroblast Growth Factor Receptor 2, Reading Frame, Exon
14 May 2018
School
Department
Course
Professor
Lecture 9(c): RNA Splicing
PTB Proteins – Key Splicing Repressors:
• Polyprymidine Tract Binding (PTB) proteins are a class of proteins related to hnRNPs
o PTB proteins associate with pre-mRNA and functions to control RNA processing
(especially during splicing)
§ Also involved in RNA transport, 3’ end processing, localization & stability
ð PTB proteins will compete with the spliceosome to access the 3’ splice site
o High levels of PTB proteins will prevent splicing at those regions
Two-Color Splicing Reports:
• Unc-16 gene: found in C. Elegans, expressed in neurons & has 2 alternatively spliced variants
o One of the splice variants has the “A” exon while the other one does not
• Used the gene to create a reporter gene to visualize the alternative splicing:
o Initial part was fused with:
§ GFP (at reading frame #1) – followed by a stop codon
§ RFP (at reading frame #2)
• If “A” exon is not included, reading frame #1
is active and we can detect GFP
• If “A” exon is included, reading frame #2
is active and we can detect RFP
ð Reveals diversity of alternative splicing & understand the concept of a reading frame
Regulation of Alternative Splicing:
1. Histone Modifications
o FGFR2 can be alternatively spliced (mutually exclusive splicing variants)
depending on cell type: epithelial vs mesenchymal
§ Epithelial – exon 3b is found | Mesenchymal – exon 3c is found
Experiments:
a) qPCR Analysis:
o Was used to exclusively identify how different cells had different exon regions (b&c)
b) ChIP Analysis:
o Antibodies used to identify histone marks on both cell types to identify differences:
§ Epithelial cells – more H3K27 marks found on exon 3b
§ Mesenchymal cells – more H3K36 marks found on exon 3c
c) Modifying Histone Marks:
o SET2 (methyl transferase) was added to epithelial cells to increase H3K36 marks:
§ mRNA transcripts with 3b exon decreased | no effect on transcripts with 3c
o SET2 added to mesenchymal cells has no effect
ð MRG15, chromatin binding protein binds to H3K36 marks and overexpressing it will
generate the same phenotype in mesenchymal cells, but see an increase of 3c in epithelial cells
o If we knock-out the gene (MRG15) we get an increase of 3b in mesenchymal cells
Document Summary
Lecture 9(c): rna splicing: polyprymidine tract binding (ptb) proteins are a class of proteins related to hnrnps, ptb proteins associate with pre-mrna and functions to control rna processing (especially during splicing) Also involved in rna transport, 3" end processing, localization & stability. Ptb proteins will compete with the spliceosome to access the 3" splice site: high levels of ptb proteins will prevent splicing at those regions. Gfp (at reading frame #1) followed by a stop codon. If a exon is not included, reading frame #1 is active and we can detect gfp. If a exon is included, reading frame #2 is active and we can detect rfp. Reveals diversity of alternative splicing & understand the concept of a reading frame. Regulation of alternative splicing: histone modifications, fgfr2 can be alternatively spliced (mutually exclusive splicing variants) depending on cell type: epithelial vs mesenchymal. Epithelial exon 3b is found | mesenchymal exon 3c is found.
