NmRNA Splicing occurs on Spliceosomes

1
NmRNA Splicing occurs on Spliceosomes!
Strategy An Adenovirus pre-mRNA (32P-labeled)
was incubated in a HeLa cell nuclear extract to
allow splicing to begin. Then the extract was
centrifuged down a glycerol gradient to size the
complexes that formed.
Result The intron-exon 2 intermediate sedimented
at 60S on a glycerol gradient much bigger than
expected for naked RNA.
Question What else is in the 60S spliceosome
complex?
Fig. 14.13, 2ed.
2
Spliceosomes contain Snurps (a.k.a., snRNPs or
small nuclear ribonucleoproteins)

• A snurp contains a small, nuclear, U-rich RNA
  (snRNAs U1, U2, U4, U5 or U6), and gt 7
  proteins, 5 (Sm) are common.
• The snRNAs base-pair with the pre-mRNA (U1, U2,
  U5, U6), and some with each other (U4-U6 pair in
  snurps, and U2-U6 pair in spliceosome).
• Lupus patients have antibodies to snurps mainly
  the Sm proteins.

3
Roles of snRNAs/Snurps

• U1 base-pairs with the 5 splice-site
• U2 binds/pairs with the branch point also pairs
  with U6 in the assembled spliceosome
• U4 pairs with U6 in SnRNPs, but unpairs during
  spliceosome assembly
• U5 interacts with both exons (only 1-2 nt
  adjacent to intron) helps bring exons together
• U6 displaces U1 at the 5 splice-site (pairs with
  nt in the intron) it also pairs with U2 in the
  catalytic center of the spliceosome

4
U1 and U2 paired with pre-mRNA in yeast
5
Similar active sites (catalytic center) in
Spliceosomal and Group II introns?
(both models after first step)
Fig. 14.23
6
The yeast Spliceosome cycle of assembly, Rxn, and
disassembly
Fig. 14.28
7
Intermediate complexes in the Spliceosome cycle

• CC is the commitment complex (contains U1 on
  the pre-mRNA)
• A also contains U2
• B1 also contains U6-U4/U5
• B2 lacks U1 and U4, activated spliceosome
• C1 contains 5-exon intron-exon
• C2 contains intron-lariat and ligated exons

8
Some Unique Features of the Spliceosome

• Transient complex that forms on pre-mRNA.
  Contrast with ribosomal subunits, which are
  completely stable.
• Ribonucleoprotein components of the spliceosome,
  snurps, are stable structures.
• In yeast, the spliceosome sediments at 40S
  whereas in humans it is 60S (ribosomal subunits
  from these species are similar in size).

9
Proteins that promote formation of the Commitment
Complex

• In humans the SR proteins SC35 and SF2 commit
  splicing on globin HIV Tat 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 RNA and Mud2p near the 3 end of the
  intron
• Helps define the intron prior to splicing

10
Figure 14.36
Fig. 14.36
SS - splice site BP - branch point

11
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!

12
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. 14.40
13
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
14
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.
Fig. 14.41
15
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.

16
Organisms that Trans-splice nuclear genes.
from Fig. 16.10
Trypanosome Schistosoma Ascaris
Euglena
Trans-splicing also occurs in plant chloroplast
and mitochondrial genes!
17
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. 16.11
18
Trans-splicing in Trypanosomes
SL
Trans-splicing should yield some unique Y
shaped intron-exon intermediates containing the
SL half-intron.
Fig. 16.12
19
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
20
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. 16.13, 16.14
21
Some of these organisms (Trypanosomes and
Euglena) also have polycistronic genes.
Trypanosome Schistosoma Ascaris Euglena
Parasitic Worms
Fig. 16.10
22
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. 15.32
23
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

24
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. 15.34a
25
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.