Transcript RNA Processing

Study Guide/Outline—RNA 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, 1st ed. Russell
Prokaryotes vs. Eukaryotes
Prokaryotic
• Polycistronic (one promoter,
multiple genes)
• Introns thought to be nonexistent 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
5
O
4
1
2
3
5
O
4
1
3
2
5
O
4
1
3
2
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RNA polymerase II transcribes a gene
past the polyA signal sequence.
5′
3′
PolyA signal sequence
The RNA is cleaved just past the
polyA signal sequence. RNA
polymerase continues transcribing
the DNA.
5′
3′
5′
3′
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.
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.
5′
3′
5′
3′
5′
3′
Exonuclease catches
Exonuclease
up to RNA polymerase II
and causes termination.
3′
5′
3′
Figure 14.15
14 - 47
Polyadenylation signal sequence
5′
3′
AAUAAA
Consensus sequence in higher
eukaryotes
5′
Endonuclease cleavage occurs
about 20 nucleotides downstream
from the AAUAAA sequence.
AAUAAA
PolyA-polymerase adds
adenine nucleotides
to the 3′ end.
5′
AAUAAA
AAAAAAAAAAAA.... 3′
PolyA tail
Brooker Figure 14.22
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Termination of RNA synthesis in (eukaryotic) RNA Pol II
3’ UTR
Regular
transcript
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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 Half-life: 10-30
cycle gene)
min
Hemoglobin
Short poly-A tail
Half-life: 24 hours Long poly-A tail
Major types of introns
Type of intron
Gene type
Splicing
Mechanism
Enzymatic
tRNAs and
rRNAs
Nuclear (premRNA)
tRNA genes
Protein-encoding
genes in nuclear
chromosomes
Spliceosomal
Group I
Some rRNA genes
Self-splicing
Group II
Protein-encoding
genes in
mitochondria
Self-splicing
Processing of ribosomal RNA
Promoter
18S
5.8S
28S
Transcription
5′
45S rRNA
transcript
18S
5.8S
28S
Cleavage
(the light pink regions
are degraded)
This processing
occurs in the
nucleolus
18S
rRNA
5.8S
rRNA
28S
rRNA
Functional RNAs that are key in
ribosome structure
Brooker Figure 14.16
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3′
Processing of
tRNAs
Endonuclease
5′
Endonuclease
(RNaseP)
mG
3′
A
C
C Exonuclease
(RNaseD)
T
T
P
Covalently
modified bases
P
mG
IP
= Methylguanosine
P = Pseudouridine
T = 4-Thiouridine
Brooker Figure 14.17
Anticodon
IP = 2-Isopentenyladenosine
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RNA-DNA hybrid reveals intron sequences as
they “loop out”
Splice site consensus sequences
Exon
5′
Intron
A/ GGU
C
Pu AGUA
5′ splice site
Exon
UACUUAUCC
Py12N Py AGG
Branch site
3′ splice site
Brooker Figure 14.19
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3′
Rough overview of splicing mechanism
(formation of lariat structure)
Mechanism of
Spliceosome
Mechanism of Spliceosome (Brooker)
Exon 1
Exon 2
A
GU
5′
AG
5′ splice site Branch site
3′
3′ splice site
U1 binds to 5′ splice site.
U2 binds to branch site.
U1 snRNP
U2 snRNP
A
5′
Intron loops out
and exons brought
closer together
3′
U4/U6 and U5 trimer binds. Intron loops out
and exons are brought closer together.
A
U2
U4/U6 snRNP
U1
5′
U5 snRNP
3′
Brooker, Fig 14.20
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Mechanism of Spliceosome (Brooker), cont.
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.
U1
U4
U2
A
U6 U5
5′
3′
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).
Intron will be degraded
and the snRNPs used
again
U2
A
Intron plus U2,
U5, and U6
U6
5′
Exon 1
U5
Exon 2
3′
Two connected
exons
Brooker, Fig 14.20
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Rare mutations in intron sequences can cause
a phenotype (e.g. in the large gene, Dystrophin)
Duchenne’s 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
Duchenne’s
Muscular
Dystrophy
Fig from Medical Genetics, Jorde et al., 3rd ed.
Major types of introns
Type of intron
Gene type
Splicing
Mechanism
Enzymatic
tRNAs and
rRNAs
Nuclear (premRNA)
tRNA genes
Protein-encoding
genes in nuclear
chromosomes
Spliceosomal
Group I
Some rRNA genes
Self-splicing
Group II
Protein-encoding
genes in
mitochondria
Self-splicing
Self splicing Introns
Self-splicing introns
(relatively uncommon)
CH2OH
O
H 3′
G
H
Intron
Guanosine
binding site
G
Intron
H
OH
OH
Guanosine 5′
A
Exon 1
G
Exon 2
G
3′ 5′
Exon 1
HH
O
O
(a) Group I
P
O
CH2
O
HH
O
O
5′
RNA
3′
3′
P
H 2′
P
3′
Exon 2
P
O
CH2
O
A
5′
P
3′
OH
G
5′G
O
H 2′
5′
3′
OH
HH
OH
P
5′
O
H 2′
A
3′
5′
G
P
O
CH2
P
3′
RNA
(b) Group II
Brooker Fig 14.18a and b
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