generalised mRNA Decay

1
(generalised) mRNA Decay
2
mRNA Decay
AAAAAAAAAAAAAAAA
m7Gppp
poly A shortening
Deadenylase complex
AAA
m7Gppp
Decapitation
Decapping enzyme (Dcp1 complex)
AAA
5-3 exonucleolytic cleavage
Xrn1 complex
rAMP, rUMP, rCMP rGMP
3
Translating mRNA
eIF4G
PABP
PABP
AAAAAAAAAAAAAAAA
m7Gppp
dissociation
Transient dissociation
PABP
PABP
AAAAAAAAAAA
m7Gppp
A
A
A
A
A
A
De-adenylation
A
4
eIF4G
De-adenylated mRNA
Mrt1p
A
m7Gppp
Recruitment of Lsm by Mrt1P
m7Gppp
LSM complex
m7Gppp
decapping
exosome
Xrn1p
A
m7Gppp
5 ? 3
3 ? 5
progressive degradation
progressive degradation
5
5- Degradation in Yeast
Following deadenylation, 5 to 3 degradation
requires access to the cap by a decapping
enzyme.
eIF4G
PABP
PABP
AAAAAAAAAAAAAAAAA
m7Gppp
Competition between Dcp1 and eIF4E Kd of eIF4E
cap gtgt Kd of Dcp cap However,
interaction of eIF4G strengthens eIF4E
interaction with cap and eIF4G interaction with
PAB may help stabilize mRNP Mutations in PABP,
eIF4G, eIF4E or eIF3 lead to increased decapping
by the Dcp complex.
Dcp Complex
6
AU-rich elements (AREs)
7
Messenger RNA structure 3 NCR
A / U rich Elements (AREs) - binding
sites for stabilising / destabilisng proteins
stop
orf
AAAAAAAAAAAAA
The turnover of an mRNA is mostly regulated by
cis-acting elements located in the 3' UTR, such
as the AU-rich elements (AREs), which promote
mRNA decay in response to a variety of specific
intra- and extra-cellular signals.
8
AREs have been experimentally grouped into three
classes class I and II AREs are characterized
by the presence of multiple copies of the
pentanucleotide AUUUA, which is absent from class
III AREs. Class I AREs control the
cytoplasmic deadenylation of mRNAs by the
degradation of all parts of the poly(A) tail at
the same rate, generating intermediates with
poly(A) tails of 30-60 nucleotides, which are
then completely degraded. These elements are
found mainly in mRNAs encoding nuclear
transcription factors such as c-Fos and c-Myc
(the products of 'fast response' genes) and also
in mRNAs for some cytokines, such as interleukins
4 and 6. The presence of one or more copies of
the pentanucleotide AUUUA next to a U-rich region
is the structural characteristic of class I AREs.
9
Class II AREs mediate asynchronous cytoplasmic
deadenylylation, in other words the poly(A) tail
is degraded at different rates in different
transcripts, generating mRNAs without poly(A)
tails. Among mRNAs containing this signal are
those encoding the cytokines GM-CSF, interleukin
2, tumor necrosis factor a (TNF-a) and
interferon-a. Class II AREs are characterized
by tandem reiterations of the AUUUA pentamer, and
an AU-rich region is usually found upstream of
these repeats. Class III AREs The mRNAs
containing class III AREs, such as those encoding
c-Jun, do not contain the pentanucleotide AUUUA
but have only a U-rich segment they show
degradation kinetics similar to those of mRNAs
containing class I AREs.
10
translation in the nucleus
11
Coupled Transcription and Translation within
Nuclei of Mammalian Cells
Iborra, F.I., Jackson, D.A. and Cooke, P.R.
(2000) Science 293,
1139-1142.
cells permeabilised and incubated with (a) 3H
- lysine
(b) biotinylated-lysyl-tRN
A
(c) BIODIPY-lysyl-tRNA
distribution of label 90 cytoplasmic
10 nucleus
12
nonsense-mediated decay (NMD)
13
mRNA surveillance
The removal of introns is error-prone. Even very
small mistakes (1 nt !) result in open reading
frame-shifts and the introduction of premature
stop codons. Failure to remove an intron
introduces a (formerly) non-coding sequence
into an open reading frame with similar
disastrous results. To prevent the potentially
deleterious effects of producing truncated
translation products, the mRNA is
quality-controlled to ensure only correctly
processed mRNAs, with no premature stop codons
are exported to the cytoplasm. The two
mechanisms used are (i) mRNA export and (ii)
nonsense-mediated decay
14
correct stop codon
premature stop codon
UAA
UAG
AUG
m7Gppp
AAAAAAAAAAA
Translation
C-terminally truncated translation product
15
Exon
Intron
correct stop codon
premature stop codon
introns removed by splicing
a complex of (at least 5) proteins the EJC
(exon-exon junction complex) is deposited
at each splice site
16
The factors involved were first identified in the
yeast Saccarhomyces cerevisiae
Upf1, Upf2 / NMD2, Upf3 and
Hrp1 Human orthologues have also been
identified
hUpf1, hUpf2, hUpf3a and hUpf3b. Upf1
(cytoplasmic RNA helicase) interacts with Upf2,
which in turn binds to Upf3. Although these
proteins can exist as a complex, Upf3 is part of
the exon-exon junction complex (EJC) and
interacts with Y14 a protein which marks an
mRNA for export.
17
During transcription the RNA becomes coated with
hnRNPs. Splicing commitment factors direct the
spliceosome to exon / intron junctions. The
pre-mRNA 5 and 3 ends are bound by nuclear
cap-binding protein complex(CBC) and polyA
binding protein II (PABPII), respectively. After
intron excision, a post-splicing complex is
deposited upstream (20nts) of the exon / exon
boundary boundary. A protein in the post-splicing
complex (REF/Aly) interacts with the nuclear mRNA
export factor TAP, and the mRNA is exported
through the nuclear pore to the
cytoplasm. During the first round of
translation, the ribosome displaces most of the
post-splicing complexes. If a premature stop
codon is encountered upstream of a post-splicing
complex, then mRNA is decapped and degraded
. The hUpf proteins form a dynamic bridge
between the translation release factor and the
post-splicing complex. hUpf3p (3) may associate
with the post-splicing complex before the mRNA
leaves the nucleus.
18
NMD occurs when translation terminates 50-55nts
upstream of the 3 most exon/exon junction.
NMD
UAA
gt 50- 55nts
3 most exon / exon junction
An mRNA is immune to NMD if translation
terminates less than 50-55nts upstream of the 3
most exon/exon junction, or, if translation
terminates downstream of the 3 most exon/exon
junction.
NMD
UAA
UAA
lt 50- 55nts
3 most exon / exon junction
19
caps of mRNAs subject to NMD in mammalian cells
are bound to nuclear proteins CBP80 and
CBP20
- Pioneer translation
?
eIF4G
?
eIF4A
?
?
Upf2
PABPII
PABPII
CBP80
43S
Upf3
AAAAAAAAAAAAAAAA
m7Gppp
CBP20
20
prokaryotic messenger RNA
21
Attenuation of Translation The Tryptophan Operon
trpE
trpD
trpC
trpB
trpA
Promoter
Operator
Leader
Attenuator
trpE
AUG AAA GCA AUU UUC GUA CUG AAA GGU UGG UGG CGC
ACU UCC UGA (N43)
AUUUUUUUUGAACAAAA
G-C
M K A I F V L K G W W R
T S
C-G
C-G
C-G
G-C
C-G
The "hairpin" RNA structure preceding the U8
A
sequence forms the intrinsic transcription
C-G
termination feature.
U U
AA
22
Sequence 1 is complementary to 2
Sequence 3 is complementary to 2 4
UGCGUAAA
CGUAAA
U-A
G
G G
G-C
C
U C
G-C
A
A A
U-A
A
C A
G-C
U
C U
U
C
U A C
G-U
A
G C A
C A
G
G U G
G-U
A
U U-A
C-G
U
1 2
G A-U
A-U
A
G U-A
C-G
C

C G-C
U-A
C
G U C
U C
C
3 4
C G-C
C-G
A-U UUUUUU
A A A
C-G
G-C
C C-G U UUUUUUU
U-G
C-G
U G-C C
G C
C-G
U G-C G
A A
C-G
C C C G
A
G-C
C U G AAA G G
C-G
C C
A
C UAAU GAG
C-G
U U
AA
Sequence 2 3 base-pair
terminator hairpin destroyed.
Sequence 3 4 base-pair
form terminator hairpin.
23
(No Transcript)
24
(No Transcript)
25
Translational Attenuation   Ribosomal Protein
Operon Leader Sequences     Met Lys Arg Ile Ser
Thr Thr Ile Thr Thr Thr Ile Thr Ile Thr Thr - AUG
AAA CGC AUU AGC ACC ACC AUU ACC ACC ACC AUC ACC
AUU ACC ACA -   Met Lys His Ile Pro Phe Phe Phe
Ala Phe Phe Phe Thr Phe Pro AUG AAA CAC AUA
CCG UUU UUC UUC GCA UUC UUU UUU ACC UCC CCC
UGA   Met Thr Arg Val Gln Phe Lys His His His His
His His His His Pro Asp - AUG ACA CGC GUU CAA UUU
AAA CAC CAC CAC CAC CAC CAC CAC CAC CCU GAC -
26
 Translational Repression Ribosomal
Proteins     1. There are more than 50 kinds of
ribosomal proteins.   2. Their genes are located
in more than 20 operons.   3. Their synthesis is
balanced.   4. Control of synthesis is at the
level of translation - not transcription.   5. At
least one protein encoded by each such operon
serves as a translational repressor.   6. It
binds to mRNA near its own initiation site and
blocks the synthesis of several proteins in the
polycistronic message.   7. Ribosomal proteins
bind more strongly to rRNA than mRNA   8. RNA
'pseudoknot' structure is present at the 5'
regions of translationally repressed mRNAs  
 
27
Ribosomal protein S8 binds to 16S RNA (ribosome
biogenesis) .. but it can also bind to a
specific site of the polycistronic mRNA encoding
the entire operon. Excess S8 (has not bound to
rRNA) will, therefore, repress the translation of
itself, and other ribosomal proteins encoded in
that operon.
28
In prokaryotes, translation can be inhibited by
anti-sense RNA
29
eukaryotic messenger RNA
30

• Where is the mRNA ?
• estimated that only 5-10 of RNA transcribed
  in the
• nucleus is ever exported to the cytoplasm
• RNA undergoes extensive processing incompletely
  / incorrectly
• processed RNA is degraded in the nucleus -
  exosome

mRNA

• How good is the mRNA ?
• what is the yield of protein from a specific
  steady state
• level of mRNA?
• what other factors can affect the level of
  translation ?

• What is the half-life of the mRNA ?
• once exported to the cytoplasm, what is the
  longevity of an mRNA?
• what factors can modify the half-life of an
  mRNA?

31
mRNA exported from the nucleus
generalised mRNA degradation local
protection at specific sites
specific mRNAs transported to specific cellular
sites by the cytoskeleton
random mRNA diffusion trapping at specific
sites
32
Messenger RNA structure 3 NCR
A / U rich Elements (AREs) - binding
sites for stabilising
/ destabilising proteins
stop
orf
AAAAAAAAAAAAA
mRNA localisation elements (usually located in
the 3NCR) - binding sites for proteins
which bind to the cytoskeleton - binding
sites for proteins (located at specific cellular
sites) which anchor the mRNA in that
location
33
repression of translation
34
Iron response element-binding protein
(IRE-BP)
IRE-BP
IRE-BP
Iron starvation
ferritin mRNA
transferrin receptor mRNA
m7Gppp
An
m7Gppp
An
translation blocked
mRNA translated
no ferritin made
transferrin receptor made
Excess Iron
IRE-BP
Fe
Fe
IRE-BP
ferritin mRNA
transferrin receptor mRNA
m7Gppp
An
m7Gppp
An
mRNA translated
mRNA degraded
no transferrin receptor made
ferritin made
35
upstream ORFs (uORFs)
36
Eukaryotic ribosomes, in general, do not
reinitiate downstream of a stop codon. The
presence of a short leader uORF or upstream ORF
on the 5' side of the main structural ORF can act
to divert translational initiation from the major
polypeptide translation to a minor leader
oligopeptide.
scanning
AUG
AUG
UAG
43S
main ORF
uORF
Expression of the major ORF depends on
either (i) leaky scanning (sub-optimal
context for the uORF AUG causing it to be
skipped) (ii) (low-level) reinitiation of
translation, (iii) entry of a new initiation
complex at an IRES (iv) RNA secondary structure
37
Internal Ribosome Entry Sequence (IRES)
FMDV IRES
Cellular IRES
AUG
m7Gppp
uORF
main ORF
38
Yeast GCN4 is a gene regulatory protein that is
required for the activation of many genes (50)
involved in amino acid metabolism. amino acid
starvation protein synthesis is inhibited, but
translation of GCN4 is markedly enhanced GCN4
mRNA contains 4 uORFS upstream of the main coding
sequence (containing only 2 or 3 codons each)
AUG
main ORF
after translating uORF1, ribosomes can
re-initiate at uORF2 (50) downstream uORFs
show much higher inhibitory effects after
translating uORF4, only 1 of ribosomes
re-initiate translation at the main ORF length
of uORFs and the spacing
39
after translating a uORF, ribosomes must regain
the ability to initiate if this happens before
they encounter the next uORF, then that uORF is
translated if this does not happen before they
encounter the next uORF, then the main ORF is
translated the rate at which the ability to
re-initiate is acquired is controlled by
phosphorylation at Ser 51 of eIF2a, the factor
that activates the initiator tRNA when the cell
is starved of amino acids, the high level of
uncharged tRNAs activates the phosphorylation of
eIF2? (mediated by the GCN2 kinase) The
transcriptional activator protein GCN4 is only
translated when eIF2a is highly
phosphorylated. This mechanism is involved in a
global mechanism for response to amino acid
starvation in yeast. A similar mechanism is
involved in the control of the heme synthesis
pathway in reticulocytes a heme-controlled
repressor (HCR) phosphorylates eIF2a only in the
presence of heme