discovery of alternative splicing
DESCRIPTION
Discovery of Alternative Splicing. First discovered with an Immunoglobulin heavy chain gene (D. Baltimore et al.) Alternative splicing gives two forms of the protein with different C-termini 1 form is shorter and secreted Other stays anchored in the plasma membrane via C-terminus - PowerPoint PPT PresentationTRANSCRIPT
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Discovery of Alternative Splicing
First discovered with an Immunoglobulin heavychain gene (D. Baltimore et al.)
• Alternative splicing gives two forms of the protein with different C-termini
– 1 form is shorter and secreted– Other stays anchored in the plasma membrane
via C-terminus~40% of human genes produce alternatively spliced
transcripts!
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Alternative splicing of the mouse immunoglobulin μ heavy chain gene
Fig. 14.38
S-signal peptide C - constant regionV- variable region green – membrane anchorRed- untranslated reg. yellow – end of coding reg. for secreted form
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Regulation of Alternative splicing• Sex determination in Drosophila involves 3 regulatory
genes that are differentially spliced in females versus males; 2 of them affect alternative splicing
1. Sxl (sex-lethal) - promotes alternative splicing of tra (exon 2 is skipped) and of its own (exon 3 is skipped) pre-mRNA
2. Tra – promotes alternative splicing of dsx (last 2 exons are excluded)
3. Dsx (double-sex) - Alternatively spliced form of dsx needed to maintain female state
Fig. 14.38
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Alternative splicing
Sxl and Tra are SR proteins!Tra and Tra-2 bind a repeated element in exon 4 of dsx mRNA, causing it to be retained in mature mRNA.
Alternative splicing in Drosophila maintains the female state.
Fig. 14.39
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Trans-Splicing (Ch. 16.3)
• Intermolecular splicing of pre-mRNAs• First discovered in African trypanosomes, a
disease(African Sleeping Sickness)-causing parasitic protozoan.
• The mRNAs had 35 nt not encoded in the main gene – called the spliced leader sequence.
• Spliced leader (SL) is encoded separately, and there about 200 copies in the genome .
• SL primary transcript contains ~100 nt that resemble the 5’ end of a NmRNA intron.
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Organisms that trans-splice nuclear genes.
Trypanosome Schistosoma Ascaris Euglena
from Fig. 16.8
Trans-splicing also occurs in plant chloroplast and mitochondrial genes!
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2 possible models to explain the joining of the SL to the coding region of a mRNA
1. Primed transcription by SL
2. Trans-splicing model
Fig. 16.9
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Trans-splicing in Trypanosomes
Fig. 16.10
SL
Trans-splicing should yield some unique “Y –shaped” intron-exon intermediates containing the SL half-intron.
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Release of the SL half-intron from larger RNAs by a debranching enzyme.
This result is consistent with a trans-splicing model rather than a cis-splicing mechanism.
Figs. 16.11, 16.12
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Some of these organisms (Trypanosomes and Euglena) also have polycistronic genes.
Trypanosome Schistosoma Ascaris Euglena
Parasitic Worms
Fig. 16.8
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Cap stimulates splicing of the first intron in a multi-intron pre-mRNA
May have been methylation of Cap in extract.
Splicing of 1st intron very poor with uncapped pre-mRNA.
32P-labeled substrate RNAs were incubated in a Hela nuclear extract.
Fig. 15.30
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CAP Binding Complex (CBP)
• Contains 2 proteins of 80 (CBP80) and 20 (CBP20) kiloDaltons
• Depletion of CBP from a splicing extract using antibody against CBP80 inhibited splicing of the first intron in a model pre-mRNA– Further analysis showed an inhibition of
spliceosome formation• CBP may be important for spliceosome
formation in vivo on first intron
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Poly A-Dependent Splicing of the Last Intron in a 2-intron pre-RNA
Splicing of the 2nd intron in this pre-mRNA is reduced by a mutation in the polyadenylation signal (wild-type hexamer=AAUAAA).Splicing of the 1st intron is normal.
Fig. 15.31
Double-spliced mRNA
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RNA Splicing and Disease
• ~ 15 % of the mutations that cause genetic diseases affect pre-mRNA splicing.
• Many are cis-acting mutations at the splice-sites, the branch point, or sequences that promote (enhancers) or inhibit (silencers) splicing of certain exons.
• OMIM (Online Mendelian Inheritance in Man) - database of human genetic mutations and disorders at NCBI, a.k.a. the National Center for Biotechnology Information) (link on Blackboard)