mRNA Turnover: Why Should We Care

1
(No Transcript)
2
mRNA Turnover Why Should We Care?

• Control of Gene Expression
• Quality Control of mRNA Biogenesis

3
The Biological Issue Differential mRNA Turnover
t1/2
Time After Transcriptional Repression
0 3 6 10 15 20 30
40 50 60
Stable (PGK1)
35'
Tell me in detail how this experiment was done!
4
Two General Pathways of Eukaryotic mRNA Turnover
AUG
UAA
m7Gppp
AAAAAAA70
DEADENYLATION
Aoligo
AUG
UAA
m7Gppp
3' -gt 5' EXONUCLEOLYTIC DECAY
DECAPPING
m7Gppp
m7Gppp
AUG
UAA
Aoligo
5' -gt 3' EXONUCLEOLYTIC DECAY
m7Gpp
AUG
UAA
p
Aoligo
5
Decapping is an Important Control Point in mRNA
Decay
Dcp1p/Dcp2p
AUG
m7Gppp
PGK1 Stability Element
PAB1
PAB1
AAU
AAAAAAAAAAAAA
MFA2 3' UTR Instability Element
6
MFA2 instability element
t1/23'
MFA1
t1/215'
Describe how the experiment was done for these
two RNAs. Also, design experiments to test that
the sequence shown in MFA2 is indeed an
instability element.
7
What is the Relationship Between Decapping and
the Complex of Translation Factors on the 5' Cap
Structure?
Dcp1p Dcp2p
AUG
4B
eIF-4E
m7Gppp
eIF-4A
eIF-4G
PAB1
PAB1
AAU
AAAAAAAAAAAA
Absence of eIf-4E leads to faster degradation of
messages. Describe and draw the results showing
the rate of degradation for a typical mRNA in a
wild type cell and in one which has a "bad" form
of eIF-4E that is unable to associate efficiently
with the 5' CAP.
8
(No Transcript)
9
mRNA Turnover Why Should We Care?

• Control of Gene Expression
• Quality Control of mRNA Biogenesis

10
mRNAs that lack a termination codon are unstable
(nonstop decay)
t1/2
Time after transcription repression
PGK1
0 2 4 6 8 10 15
30 45 60
m7Gppp-
AAAAA
UAA
30'
NONSTOP-PGK1
lt2'
m7Gppp-
AAAAAA
11
Premature Polyadenylation Generates Nonstop
mRNAs
Polyadenylation
Premature polyadenylation
m7Gppp
m7Gppp
AAAAAAA
AAAAAAA
nonstop mRNA
normal mRNA
truncated protein
deleterious effect
12
Premature Polyadenylation Occurs Frequently at
Many Different Sites
- At steady state 1 of yeast transcripts are
prematurely polyadenlyated within the coding
region (Graber et al. 1999).
- Nonstop mRNAs are ten-fold less stable than
normal mRNAs.
13
Nonsense-mediated Decayan Example of Quality
Control
Upf1
Upf2
AUG
UAA
UAA
AAAA
m7Gppp
Upf3
Recognition by UPF Proteins Triggers Degradation
Degrades mRNAs with premature stop
codons mRNAs with extended 3' UTRs
unspliced pre-mRNAs
14
5' Nonsense Codons Show Greater Effects than 3'
Nonsense Codons
AUG
UAA
UAA
UAA
AAAAAAAA
m7Gppp
very unstable
unstable
normal
Draw a gel showing the degradation rates for
these three messages.
15
Nonsense-mediated Decay Triggers Deadenylation
Independent Decapping
m7Gppp
AUG
UAA
UAA
AAAA
AUG
UAA
m7Gppp
AAAA
16
Almost 20 of mRNAs in humans have a premature
stop codon. All of these are degraded by NMD.
Where do you think all these mistakes are coming
from? That is, which process in the biogenesis
of an mRNA molecule is the most prone to errors?
17
Transcription Splicing Polyadenylation
AAA
m7Gpp
AAA
m7Gpp
18
Where is mRNA degradation occuring?
19
Yeast Dhh1p is found in discrete cytoplasmic foci
GFP alone control
Dhh1p-GFP
20
Other decapping factors are found in cytoplasmic
foci
Dcp1p-GFP
Pat1p-GFP
Dcp2p-GFP
Lsm1p-GFP
Dhh1p-GFP
Decapping enzyme
Activators of decapping
21
Yin Yang Model of mRNA Physiology
Non-Translating Pool (Stored mRNAs)
Translating Pool
Reinitiate and return to translating pool
AAAAAAA
m7Gppp
Rounds of Initiation, elongation termination
m7Gppp
A10
Localization in P-Bodies
40S
eIF3
4G
4e
A10
m7Gppp
ribosome free mRNA