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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)