Proteins that promote formation of the Commitment Complex

1
Proteins that promote formation of the Commitment
Complex

• In humans the SR proteins SC35 and SF2 commit
  splicing on globin pre-mRNA
• SR proteins have domains rich in serine and
  arginine
• In yeast the branch-point bridging protein (BBP)
  binds to the U1 snurp at the 5 end of the intron
  and near the 3 end of the intron
• Helps define the intron prior to splicing

2
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

3
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. 14.37
4
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
5
Alternative splicing in Drosophila maintains the
female state.
Alternative splicing
Sxl and Tra are SR proteins! Tra binds exon 4 in
dsx mRNA causing it to be retained in mature
mRNA.
6
Trans-Splicing (Chapter 16 505-509)

• 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 transcript also contains 100 nt that
  resembles the 5 end of a NmRNA intron.

7
Organisms that Trans-splice nuclear genes.
Trypanosome Schistosoma Ascaris Euglena
Parasitic Worms
Fig. 16.11
8
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
9
Trans-splicing in Trypanosomes
SL
Trans-splicing should yield some unique Y
shaped intron-exon intermediates containing the
SL half-intron.
Fig. 16.14
10
SL half-intron is attached to polyA RNA (mRNA)
Reverse transcriptase stopped
Primer anneals to SL half intron, which is
extended with reverse transcriptase 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 site.
Fig. 16.14
11
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. 16.15, 16.16
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. 15.40
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
Splicing of the 2nd intron in this pre-mRNA is
reduced by a mutation in the polyadenylation
signal (WT hexamerAAUAAA). Splicing of the 1st
intron was normal.
Fig. 15.43
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