Study Guide/OutlineRNA Processing

RNA Processing: rRNA genes, tRNA genes, self-splicing, spliceosomal

Structural genes
Pre-RNA Processing
What kind of processing must occur to pre-mRNA in eukaryotes? Where
does this take place?
What happens to the RNA molecule if it does not undergo processing?
Purpose of each RNA processing step
What is a methyl guanine cap?
How is a polyA tail added? How does this tail contribute to the stability of the
mRNA?
How are nucleotides numbered in genes with exons and introns?

Spliceosomes
What is a spliceosome and what class of genes use spliceosomes?
What consensus sequences are needed in introns in order for correct
splicing to occur? What would happen if there was a mutation in a splice
site consensus sequence?
What is the significance of the lariat structure in splicing out introns?
mRNA and protein synthesis are
coupled in bacteria
In eukaryotes mRNA must be processed
and transported out of nucleus for
translation
iGenetics, 1
st
ed. Russell
Prokaryotes vs. Eukaryotes
Prokaryotic
Polycistronic (one promoter,
multiple genes)
Introns thought to be non-
existent in prokaryotes until
very recently
Transcription and
translation can occur
concordantly
Exceptions: archaebacteria,
bacteriaphage (virus),
mitochondria, chloroplasts
Eukaryotic
Monocistronic (one
promoter, one gene)
Introns are common
High amounts of junk
DNA in genome.
RNA requires significant
processing
Size of introns is roughly
correlated with
complexity of the
organism.

Structure of the
methylguanine
cap
1
2 3
4
5
O
O
O
1
2 3
4
5
1
2 3
4
5
14 - 47
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RNA polymerase II transcribes a gene
past the polyA signal sequence.
The RNA is cleaved just past the
polyA signal sequence. RNA
polymerase continues transcribing
the DNA.
PolyA signal sequence
3
5
5
3
5
3
Allosteric model: After passing the polyA signal sequence,
RNA polymerase II is destabilized due to the release of
elongation factors or the binding of termination factors (not
shown). Termination occurs.
Torpedo model: An exonuclease
binds to the 5 end of the RNA
that is still being transcribed and
degrades it in a 5 to 3 direction.
3
3
3
5 3
5
Exonuclease catches
up to RNA polymerase II
and causes termination.
Exonuclease
3
5
5
Figure 14.15
5
3
5
5
3
Endonuclease cleavage occurs
about 20 nucleotides downstream
from the AAUAAA sequence.
PolyA-polymerase adds
adenine nucleotides
to the 3 end.
Polyadenylation signal sequence
AAUAAA
AAUAAA
AAUAAA
PolyA tail
AAAAAAAAAAAA....
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Brooker Figure 14.22
Consensus sequence in higher
eukaryotes
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
Termination of RNA synthesis in (eukaryotic) RNA Pol II
Regular
transcript
3 UTR
Site of
cleavage and
addition of
polyA tail
Animation of cap addition and poly-A tail addition
http://vcell.ndsu.nodak.edu/animations/

http://vcell.ndsu.nodak.edu/animations/
Length of poly-A Tail and Stability (half-life of
mRNA)
Prokaryotic
Degradation at 5 end begins immediately (before 3
transcription is completed)
Eukaryotic
cFOS mRNA (cell
cycle gene)

Half-life: 10-30
min
Short poly-A tail
Hemoglobin Half-life: 24 hours Long poly-A tail
Major types of introns
Type of intron Gene type Splicing
Mechanism
tRNAs and
rRNAs
tRNA genes
Enzymatic
Nuclear (pre-
mRNA)
Protein-encoding
genes in nuclear
chromosomes
Spliceosomal
Group I
Some rRNA genes
Self-splicing
Group II
Protein-encoding
genes in
mitochondria
Self-splicing
Promoter
Transcription
Cleavage
(the light pink regions
are degraded)
45S rRNA
transcript
18S 5.8S 28S
5 3
18S 5.8S 28S
18S 5.8S 28S
rRNA rRNA rRNA
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Functional RNAs that are key in
ribosome structure
This processing
occurs in the
nucleolus
Brooker Figure 14.16
Processing of ribosomal RNA
3 5
Endonuclease
Exonuclease
(RNaseD)
P = Pseudouridine
T = 4-Thiouridine
IP = 2-Isopentenyladenosine
Endonuclease
(RNaseP)
T
T
A
P
P
IP
C
C
Anticodon
m
G = Methylguanosine
m
G
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Brooker Figure 14.17
Covalently
modified bases
Processing of
tRNAs
RNA-DNA hybrid reveals intron sequences as
they loop out
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Brooker Figure 14.19
3
5
3 splice site Branch site
Intron Exon Exon
UACUUAUCC
Py
12
N Py AGG A
/
C
GGU Pu AGUA
5 splice site
Splice site consensus sequences
Rough overview of splicing mechanism
(formation of lariat structure)
Mechanism of
Spliceosome
U1
3 5
5 splice site 3 splice site
Branch site
A
GU
Exon 1 Exon 2
U1 binds to 5 splice site.
U2 binds to branch site.
AG
3 5
A
U4/U6 and U5 trimer binds. Intron loops out
and exons are brought closer together.
U1 snRNP U2 snRNP
3
5
A
U5 snRNP
U4/U6 snRNP
U2
Intron loops out
and exons brought
closer together
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Brooker, Fig 14.20
Mechanism of Spliceosome (Brooker)
U1
U4
3
5
3 5
5 splice site is cut.
5 end of intron is connected to the
A in the branch site to form a lariat.
U1 and U4 are released.
3 splice site is cut.
Exon 1 is connected to exon 2.
The intron (in the form of a lariat) is released along with
U2, U5, and U6 (intron will be degraded).
A
A
U5
U6
U5 U6
U2
Intron plus U2,
U5, and U6
Two connected
exons
Exon 1
Exon 2
U2
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Brooker, Fig 14.20
Intron will be degraded
and the snRNPs used
again
Mechanism of Spliceosome (Brooker), cont.
Rare mutations in intron sequences can cause
a phenotype (e.g. in the large gene, Dystrophin)
Duchennes Muscular
Dystrophy (X-linked)
Caused by mutations in
Dystrophin gene:
2+ million nt gene (many
exons)
Relatively high incidence
(1/3500 males) due to large
size of gene and
hemizygosity in males

Advanced
Duchennes
Muscular
Dystrophy
Fig from Medical Genetics, Jorde et al., 3
rd
ed.
Major types of introns
Type of intron Gene type Splicing
Mechanism
tRNAs and
rRNAs
tRNA genes
Enzymatic
Nuclear (pre-
mRNA)
Protein-encoding
genes in nuclear
chromosomes
Spliceosomal
Group I
Some rRNA genes
Self-splicing
Group II
Protein-encoding
genes in
mitochondria
Self-splicing
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Brooker Fig 14.18a and b
H H
OH OH
H
G
G
G
O
CH
2
OH
Guanosine
H H
O
OH
H
CH
2
O
O
P
P
A
Self-splicing introns
(relatively uncommon)
Exon 1
Intron
Exon 2 Exon 1
Intron
Exon 2
Guanosine
binding site
G
G
P
H H
O
O
H
CH
2
O
O
P
P
P
A
3
OH
3
3
3
3
3
3 5 3 5
5
5
OH
G
G
H H
O
O
H
CH
2
O
O
P
P
A
RNA RNA
5 5
5 5
(a) Group I (b) Group II
3
2
2
2
Self splicing Introns
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