Transcript Lec. 18

RNA Splicing
• RNA splicing is the
removal of
intervening
sequences (IVS)
that interrupt the
coding region of a
gene
• Excision of the IVS
(intron) is
accompanied by the
precise ligation of
the coding regions
(exons)
Discovery of Split Genes (1977)
• P. Sharp and R. Roberts - 1993 Nobel Prize in
Physiology & Medicine
• Discovered using R-loop Analysis
– Cloned genomic DNAs of a few highly expressed
nuclear genes (e.g., hemoglobin, ovalbumin), and
certain Adenoviral genes were hybridized to RNA
fractions and visualized by EM
– Loops form from RNA annealing to the template
strand and displacing coding strand of DNA
Genomic DNA fragment containing a Globin gene was annealed to
large heterogenous nuclear RNA (hnRNA), which contained globin
mRNA precursors.
Template strand
coding
Dotted line is
RNA
DNA
Fig. 14.3a
template strand
Coding strand
When genomic globin gene was annealed to cytoplasmic
mRNA (which contained mature globin mRNA) got an
internal loop of single-stranded DNA (= spliced out intron).
Fig. 14.3b
Intron Classes & Distribution
1. Group I - common in organelles, nuclear
rRNA genes of lower eukaryotes, a few
prokaryotes
2. Group II - common in organelles, also in
some prokaryotes and archaea
3. Nuclear mRNA (NmRNA) - ubiquitous in
eucaryotes
4. Nuclear tRNA- some eucaryotes
Relationships of the 4 Intron Classes
1. Each has a distinctive structure.
2. The chemistry of splicing of Groups I, II and
NmRNA is similar – i.e, transesterification
reactions
3. The splicing pathway for Group II and nuclear
mRNA introns is similar.
4. Splicing of Groups I, II and possibly NmRNA
introns are RNA-catalyzed
Self-Splicing Introns
1. Some Group I & II introns self-splice in vitro in
the absence of proteins - are “ribozymes.
2. Conserved secondary structure but not primary
sequence.
3. Require Mg2+ to fold into a catalytically active
ribozyme.
4. Group I introns also require a guanosine
nucleotide in the first step.
Tetrahymena rRNA Group I Intron
• First self-splicing intron discovered by T. Cech’s lab
in 1981
• In the 26S rRNA gene in Tetrahymena
• First example of a catalytic RNA!
• Nobel Prize in Chemistry to T. Cech and S. Altman
(showed that RNase P was a true “turnover”
riboenzyme in vivo), 1989
Group I splicing mechanism
GOH –
guanosine
nucleotide,
guanosine
will work
because the
phosphates
don’t
participate in
the reaction.
The 3’
terminal G
of the intron
is nearly
100%
conserved.
In vivo, GTP
probably
used.
Fig. 14.47
Cr.LSU intron: 2ndary structure of a group I intron
Old style drawing
Newer representation
Exon seq. in lower case and boxed
Shows how splice sites can
be brought close together by
“internal guide sequence”.
Conserved core
5’ splice site
RNA structures seen in Group I
introns
1. G  U pairs
2. Stacked helices
3. Long-range base pairings (P3 and P7,
also form last)
4. Triple helices (3 strands); (P4-P6
junction area)
3-D Model of
Tetrahymena
rRNA Intron
Catalytic core consists of two
stacked helices domains:
1. P5 – P4 – P6 –P6a (in green)
2. P9 – P7 – P3 – P8 (in purple)
The “substrate is the P1 – P10
domain (in red and black), it
contains both the 5’ and 3’ splice
sites.
A two-metal ion mechanism for group I intron splicing
(Second Step)
M. R. Stahley et al., Science 309, 1587 -1590 (2005)
Guanosine binding site of Group I
Introns
1. Mainly the G of a G-C pair in the P7 helix of
the conserved core.
2. Highly specific for Guanosine (Km ~20
μM).
3. Also binds the 3-terminal G of the intron in
the second splicing step.
Splicing Factors for Self-Splicing
Introns
• Some Group I and many Group II introns can’t
self-splice in vitro (need protein factors)
• Even self-splicing introns get help from
proteins in vivo
– Based on fungal (yeast and Neurospora)
mutants deficient in splicing of
mitochondrial introns (respiratory-deficient)
Protein Splicing Factors for
Group I (and Group II) Introns
•
2 types:
1. Intron-encoded
- promote splicing of only the intron that
encodes it
2. Nuclear-encoded
- Splice organellar introns
Split Genes of Yeast Mitochondria
From Phil Perlman
Proteins encoded within these introns:
Mat – maturase (promotes splicing)
Endo – DNA endonuclease, promotes intron invasion
•
Nuclear-encoded splicing factors function by:
1. Promoting correct folding of the intron (cbp2)
- CBP2 promotes folding of a cytochrome b intron (bI5)
2. Stabilizing the correctly folded structure (cyt-18)
a) Cyt-18 promotes splicing of a number of Mt Group I
introns in Neurospora.
b) Cyt-18 is also the Mt tyrosyl-tRNA synthetase, dualfunction protein.
c) Evolved from the tyrosyl-tRNA synthetase
by acquiring a new RNA-binding
surface.
Alan Lambowitz
Figure 5. Models of CYT-18/ΔC424-669 with Bound RNA Substrates(A) Dimeric CYT-18/ΔC424-669 with the T. thermophilus tRNATyr (orange) docked as in the T. thermophilus TyrRS/tRNATyr
cocrystal structure (Yaremchuk et al. 2002; PDB ID: 1H3E). Subunits A (Sub. A; magenta) and B (Sub. B; blue) are defined as those that bind the tRNA acceptor and anticodon arms, respectively.
Side chains at positions that did or did not give specific EPD-Fe-induced cleavages in the ND1 intron are shown in space-filling representations colored yellow and black, respectively.(B) Stereoview
of dimeric CYT-18/ΔC424-669 with docked ND1 intron RNA. The model is based on optimized fit to directed hydroxyl radical cleavage data summarized in Figure 4B. The ND1 intron RNA (residues
27–182) is shown as a green ribbon, with purple balls indicating phosphate-backbone protections from full-length CYT-18 protein (Caprara et al., 1996a), and red ribbon segments indicating EPDFe cleavage sites. The C-terminal domain of T. thermophilus TyrRS (yellow) is shown positioned on subunit B as in the T. thermophilus/tRNATyr cocrystal structure (Yaremchuk et al., 2002).