Splicing RNA: Mechanisms
Splicing of Group I and II introns
• Introns in fungal mitochondria, plastids,
Tetrahymena pre-rRNA
• Group I
– Self-splicing
– Initiate splicing with a G nucleotide
– Uses a phosphoester transfer mechanism
– Does not require ATP hydrolysis.
• Group II
– self-splicing
– Initiate splicing with an internal A
– Uses a phosphoester transfer mechanism
– Does not require ATP hydrolysis
Self-splicing in pre-rRNA in Tetrahymena :
T. Cech et al. 1981
+
Exon 1
Intron 1 Exon 2
Exon 1 Exon 2
Intron 1
•Products of splicing were resolved by gel electrophoresis:
+
+
+
pre-rRNA +
Nuclear extract Additional proteins
+
+
GTP +
+
are NOT needed for
pre-rRNA
Spliced exon
Intron circle
Intron linear
splicing of this prerRNA!
Do need a G
nucleotide (GMP,
GDP, GTP or
Guanosine).
Self-splicing by a phosphoester transfer
mechanism
G
OH
U
P P
Exon 1
U U
Intron 1
P P
A
Exon 1
G U
P P
+
Exon 2
G A
P
Exon 2
Intron 1
N15 N16
P
G
P OH
G A
N15
OH
P
+
Circular intron
A catalytic activity in Group I intron
• Self-splicing uses the intron in a
stoichiometric fashion.
• But the excised intron can catalyze
cleavage and addition of C’s to CCCCC
Group I intron catalyzes cleavage
and nucleotide addition
2 pCCCCC-OH
pCCCC-OH + pCCCCCC-OH
3'G
OH
5'pCCCCC-OH
GGGAGG
3'G
C-OH
5'
G
3'C-OH
5'pCCCC-OH
GGGAGG
5'pCCCCC-OH
GGGAGG
5'
3'G
OH
5'
GGGAGG
5'
+ 5'pCCCCCC-OH
The intron folds into a particular 3-D
structure
• Has active site for phosphoester transfer
• Has G-nucleotide binding site
Active sites in Group I intron self-splicing
ex2
3'
3'G414
OH
414
G
G-binding site
G-OH
5' G
ex1
5'
Substrate
binding
site
3'
CUCUCU
GGGAGG
2nd transfer
IGS
UUUACCU
GGGAGG
ex1
+
ex2
1st transfer
ex2
3rd transfer
G414
ex1
5'
G 414
CUCUCU OH
GGGAGG
G 5'
GGGAGG
+
5' G
UUUACCU
Domains of the Group I intron ribozyme
http://www.tulane.edu/~biochem/nolan/lectures/rna/grz.htm
RNAs that function as enzymes
•
•
•
•
•
•
RNase P
Group I introns
Group II introns
rRNA: peptide bond formation
Hammerhead ribozymes: cleavage
snRNAs involved in splicing
Hammerhead ribozymes
• A 58 nt structure is used in self-cleavage
• The sequence CUGA adjacent to stemloops is sufficient for cleavage
5'
3'
AA
A
GGCC
CCGG A
CG
U A
C G
AUC
U
G
GU A
Bond that is cleaved.
ACCAC
C UGGUG
CUGA is required for catalysis
Design hammerhead ribozymes to
cleave target RNAs
AA
A
substrate strand 5' GGCC
enzyme strand 3' CCGG A
UA
G A
CG
U A
C G
AUC
GU
A
GU
Bond that is cleaved.
ACCAC
C UGGUG
Potential therapy for genetic disease.
Mechanism of hammerhead ribozyme
• The folded RNA forms an active site for
binding a metal hydroxide
• Abstracts a proton from the 2’ OH of the
nucleotide at the cleavage site.
• This is now a nucleophile for attack on the 3’
phosphate and cleavage of the
phosphodiester bond.
Phosphotransfers for Group I vs. Group II
& pre-mRNA
Exon 1
3’ G
HO
2’
Exon 1
Exon 2
HO
Exon 2
2’ A
G
OH
OH
Exon 1+2
A
Exon 1+2
+
+ G
2’ A
OH
Group I
Group II and pre-mRNA
Splicing of pre-mRNA
• The introns begin and end with almost
invariant sequences: 5’ GU…AG 3’
• Use ATP to assemble a large spliceosome
• Mechanism is similar to that of the Group II
fungal introns:
– Initiate splicing with an internal A
– Uses a phosphoester transfer mechanism
for splicing
Initiation of phosphoester transfers in pre-mRNA
• Uses 2’ OH of an A internal to the intron
• Forms a branch point by attacking the 5’
phosphate on the first nucleotide of the
intron
• Forms a lariat structure in the intron
• Exons are joined and intron is excised as a
lariat
• A debranching enzyme cleaves the lariat at
the branch to generate a linear intron
• Linear intron is degraded
Splicing of pre-mRNA, step 1
Splicing of pre-mRNA, step 2
Investigation of splicing intermediates
In vitro splicing reaction:
nuclear extracts + ATP+ labeled pre-mRNA
Resolve reaction intermediates and products on gels.
Some intermediates move slower than pre-mRNA.
Suggest they are not linear.
Use RNase H to investigate structure of intermediate.
RNase H cuts RNA in duplex with RNA or DNA.
RNase H
RNA
5'
3'
|||||
5'
oligodeoxyribonucleotide
+
RNase H + oligonucleotides complementary to
different regions give very different products
precursor RNA
exon 1
5'
intron
exon 2
3'
splicing reaction
exons joined in a linear molecule+ excised intron, non-linear molecule
1
5'
2
3'
5'
3
Map of positions of
oligodeoxyribonucleotides
that annealed to different
regions of the excised
intron. This is not the
structure of the excised
intron.
4
RNase H
3'
3'
5'
3'
+
3'
5'
Ans wer:
intron
exon 2
3'
s plicing reaction
exons joined in a linear m olecule + excis ed intron, non-linear m olecule
1
2
3
Map of pos itions of
oligodeoxyribonucleotides
that annealed to different
regions of the excis ed
intron.
4
2
GU
Analysis
reveals a
lariate
structure
in intermediate
precurs or RNA
exon 1
5'
A
3
1
AG
4
oligo 1
oligo 2
oligo 4
3'
5'
oligo 3
3'
After annealing with
the oligo, the
heteroduplexes were
treated with RNas e H
3'
5'
3'
+
3'
5'
Involvement of snRNAs and snRNPs
• snRNAs = small nuclear RNAs
• snRNPs = small nuclear ribonucleoprotein
particles
• Antibodies from patients with the autoimmune
disease systemic lupus erythematosus (SLE) can
react with proteins in snRNPs
– Sm proteins
• Addition of these antibodies to an in vitro premRNA splicing reaction blocked splicing.
• Thus the snRNPs were implicated in splicing
snRNPs
• U1, U2, U4/U6, and U5 snRNPs
– Have snRNA in each: U1, U2, U4/U6, U5
– Conserved from yeast to human
– Assemble into spliceosome
– Catalyze splicing
• Sm proteins bind “Sm RNA motif” in snRNAs
– 7 Sm proteins: B/B’, D1, D2, D3, E, F, G
– Each has similar 3-D structure: alpha helix
followed by 5 beta strands
– Sm proteins interact via beta strands, may form
circle around RNA
Sm proteins may form ring around snRNAs
ANGUS I. LAMOND
Nature 397, 655 - 656
(1999)
RNA splicing:
Running rings around
RNA
Predicted structure of assembled Sm
proteins
4th beta strand
of one Sm protein
interacts with
5th beta strand
of next.
Channel for single
strand of RNA
ANGUS I. LAMOND
Nature 397, 655 - 656
(1999)
RNA splicing: Running
rings around RNA
Assembly of spliceosome
• The spliceosome is a large protein-RNA complex
in which splicing of pre-mRNAs occurs.
• snRNPs are assembled progressively into the
spliceosome.
–
–
–
–
U1 snRNP binds (and base pairs) to the 5’ splice site
U2 snRNP binds (and base pairs) to the branch point
U4-U6 snRNP binds, and U4 snRNP dissociates
U5 snRNP binds
• Assembly requires ATP hydrolysis
• Assembly is aided by various auxiliary factors and
splicing factors.
Spliceosome assembly and catalysis
U2 snRNP
2’
A
HO
Exon 1
U1
snRNP
2’
A
2’
HO
Exon 2
A
HO
Sm proteins
U6
U5
snRNP
Other proteins
U4/U6
snRNP
snRNAs
U
G
O
H
U4
A
U6
U2
Exons 1+2
U1
U
2’ A
G O
H
U6
U4?
2’ A
U5
Spliceosome
Catalysis by U6/U2 on branch oligonucleotide in vitro
Figure 1 Base-pairing interactions in the in vitro-assembled complex of
U2–U6 and the branch oligonucleotide (Br). Shaded boxes mark the invariant
regions in U6 and previously established base-paired regions are indicated.
Dashed lines connect psoralen-crosslinkable nucleotides (S.V. and J.L.M.,
unpublished data). The circled residues connected by a zigzag can be
crosslinked by ultraviolet light. The underlined residues in Br constitute the
yeast branch consensus sequence. Asterisks denote the residues involved in
the covalent link between Br and U6 in RNA X (see text). Arrowheads point
to residues involved in a genetically proven interaction in yeast22. Numbers
indicate nucleotide positions from the 5' ends of full-length human U2 and U6.
Nature 413, 701 - 707 (2001)
Splicing-related catalysis by protein-free
snRNAs
SABA VALADKHAN &
JAMES L. MANLEY
RNA editing
• RNA editing is the process of changing the
sequence of RNA after transcription.
• In some RNAs, as much as 55% of the
nucleotide sequence is not encoded in the
(primary) gene, but is added after
transcription.
• Examples: mitochondrial genes in
trypanosomes and Leishmania.
• Can add, delete or change nucleotides by
editing
Addition of nucleotides by editing
• Uses a guide RNA that is encoded elsewhere in
the genome
• Part of the guide RNA is complementary to the
mRNA in vicinity of editing
• U nt at the the 3’ end of the guide RNA initiates a
series of phosphoester transfers that result in
insertion of that U at the correct place.
• More U’s are added sequentially at positions
directed by the guide RNA
• Similar mechanism to that used in splicing
What is a gene?
• Making a correctly edited mRNA requires one
segment of DNA to encode the initial transcript
and a different segment of DNA to encode each
guide RNA.
• Thus making one mRNA that uses 2 guide RNAs
requires 3 segments of DNA - is this 3 genes or 1
gene?
• Loss-of-function mutations in any of those 3 DNA
segments result in an nonfunctional product
(enzyme), but they will complement in trans in a
diploid analysis!
• This is an exception to the powerful cis-trans
complementation analysis to define genes.
Mammalian example of editing
• Apolipoprotein B in the intestine is much
shorter than apolipoprotein B in the liver.
• They are encoded by the same gene.
• The difference results from a single nt
change in codon 2153:
• CAA for Gln in liver, but UAA for termination
of translation in intestine
• The C is converted to U in intestine by a
specific deaminating enzyme, not by a guide
RNA.