Consensus Group II intron Structure
6 domains,
“Helical Wheel”
Domain I
contains binding
sites for the 5’
exon (keeps the
5’ exon from
floating away
after the first
splicing step)
Group II Splicing
Pathway
(1) The 2’ OH of a special
internal A attacks the 5’ splice
site creating a branched intron
structure.
(2) The 3’ OH of the 5’ exon
attacks the 3’ splice site,
ligating the exons and
releasing the intron as a lariat
structure.
Structure of NmRNA Introns
1. Most begin with GU and end with AG.
2. Most of the internal sequences are not
conserved.
3. However, there are other important
consensus sequences near the ends
(in addition to GU and AG).
Consensus Mammalian NmRNA
Splice Signals
5’ ag/GUAAGU -------YNCURAC---YnNAG/g 3’
Y - pyrimidine (U or C)
Yn - string of ~ 9 pyrimidines
R - purine (A or G)
N - any base
Branch site
sequence
Nm RNA Splicing Mechanism:
How it has been studied.
•
Elucidating the overall mechanism, cis
elements, and trans factors depended on:
1. Site-directed mutagenesis of genes in vitro, and
subsequent expression in vivo (yeast, Hela
cells, and others).
2. Development of accurate splicing extracts
(HeLa cells and yeast).
3. Isolation of temperature-sensitive yeast mutants
defective in NmRNA splicing.
In yeast, the branch-point sequence determines
which downstream AG is used.
Exon 1
branch
Exon 2
Inserted sequence
Branch sequence
Branch site moved into exon 2.
RNAs that were tested for splicing in vivo.
Fig. 14.8
Exon 2
Kinetics of In vitro
Splicing in a Hela
cell nuclear extract
Pre - radioactively labeled
precursor RNA
- The splicing reactions were separated by
gel electrophoresis. Notice that the intron
and intron-exon RNAs have an unusually
reduced mobility in these polyacrylamideurea gels.
There is also some cleavage at the Exon 2
- Intron 2 splice-site, producing the Spliced
Exons molecule.
Spliced exons
Fig.
14.5
Plot of changes in amounts of products and intermediates
during the splicing reaction in the previous slide.
Fig. 14.5
NmRNA Splicing occurs on
Spliceosomes!
Strategy: An Adenovirus pre-mRNA (32Plabeled) 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, 2nd ed.
Spliceosomes contain Snurps
(snRNPs, small nuclear ribonucleoproteins)
1. A snurp contains a small, nuclear, U-rich
RNA (snRNAs = U1, U2, U4, U5 or U6), and
> 7 proteins, 7 (Sm) are common.
2. The snRNAs base-pair with the pre-mRNA
(U1, U2, U5, U6) and/or with each other
(U4-U6 and U2-U6).
3. Lupus patients have antibodies to snurps;
mainly the Sm proteins.
Fig. 14.28
Structure (in stereo) of the U1 SnRNP
Proteins 70K, A, and C are specific to U1 snurp
U1 and U2 paired with pre-mRNA in yeast
Roles of snRNAs/Snurps
1. U1 pairs with the 5’ splice-site.
2. U2 pairs with the branch point; also pairs with U6 in
the assembled spliceosome.
3. U4 pairs with U6 in SnRNPs, but unpairs during
spliceosome assembly.
4. U5 interacts with both exons (only 1-2 nt adjacent to
intron); helps bring exons together.
5. 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.
Similar active sites (catalytic center) in Spliceosomal
and Group II introns?
(both models after first step)
Fig. 14.22
The Spliceosome Cycle of Assembly, Rxn, and Disassembly
Fig. 14.27
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
Some Unique Features of the
Spliceosome
1. Transient complex that forms on pre-mRNA.
Contrast with ribosomal subunits, which are
completely stable.
2. Ribonucleoprotein components of the
spliceosome, snurps, are stable structures.
3. In yeast, the spliceosome sediments at ~40S
whereas in humans it is ~60S (ribosomal
subunits from these species are similar in size).
Proteins that promote formation
of the Commitment Complex
• In humans: the SR proteins SC35 and SF2
commit splicing on globin & HIV Tat premRNA
– 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 the RNA and Mud2p protein
near the 3’ end of the intron
– Helps define the intron prior to splicing
Fig. 14.34a
Figure 14.36
SS - splice site
BP - branch point
Branch-point Bridging Protein (BBP) binds RNA
(near the 3’ end of intron) and 2 proteins (U1 SnRNP
& Mud2p). Helps define the intron portion.