Discovery of Alternative Splicing

1
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
• 40 of human genes produce alternatively spliced
  transcripts!

2
Alternative splicing of the mouse immunoglobulin
µ heavy chain gene
S-signal peptide C - constant
region V- variable region green
membrane anchor Red- untranslated reg. yellow
end of coding reg. for secreted form
Fig. 14.38
3
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
• Sxl (sex-lethal) - promotes alternative splicing
  of tra (exon 2 is skipped) and of its own (exon
  3 is skipped) pre-mRNA
• Tra promotes alternative splicing of dsx (last
  2 exons are excluded)
• Dsx (double-sex) - Alternatively spliced form of
  dsx needed to maintain female state

Fig. 14.38
4
Alternative splicing in Drosophila maintains the
female state.
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.
Fig. 14.39
5
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.

6
Organisms that trans-splice nuclear genes.
from Fig. 16.8
Trypanosome Schistosoma Ascaris
Euglena
Trans-splicing also occurs in plant chloroplast
and mitochondrial genes!
7
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
8
Trans-splicing in Trypanosomes
SL
Trans-splicing should yield some unique Y
shaped intron-exon intermediates containing the
SL half-intron.
Fig. 16.10
9
SL half-intron is attached to polyA RNA (mRNA)
Reverse transcriptase stopped
Primer anneals to SL half intron, which is
extended with reverse transcriptase (RT) in the
presence of a limiting ddNTP. PolyA RNA
(mRNA) was used as the RNA template. This would
detect the intron-exon splicing intermediate, and
the nt that becomes part of the branch (the RT
falls off).
Fig. 16.14, 2ed
10
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
11
Some of these organisms (Trypanosomes and
Euglena) also have polycistronic genes.
Trypanosome Schistosoma Ascaris Euglena
Parasitic Worms
Fig. 16.8
12
Cap stimulates splicing of the first intron in a
multi-intron pre-mRNA
32P-labeled substrate RNAs were incubated in a
Hela nuclear extract.
Splicing of 1st intron very poor with uncapped
pre-mRNA.
May have been methylation of Cap in extract.
Fig. 15.30
13
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

14
Poly A-Dependent Splicing of the Last Intron in a
2-intron pre-RNA
Double-spliced mRNA
Splicing of the 2nd intron in this pre-mRNA is
reduced by a mutation in the polyadenylation
signal (wild-type hexamerAAUAAA). Splicing of
the 1st intron is normal.
Fig. 15.31
15
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.