Advisor: Li-Kwan Chang, Ph.D. Date: 2012/10/08. Cell 147(7): 1473-1483, 2011
.... Rap1p can bind to Rap1p-binding site (RapBS) found in ~90% of ribosomal.
Promoter elements regulate cytoplasmic mRNA decay Cell 147(7): 1473-1483, 2011
Speaker: Ting-Yu Lin Advisor: Li-Kwan Chang, Ph.D. Date: 2012/10/08
Central dogma
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DNA
Pre-mRNA mature mRNA
RNA mRNA stability regulation
Protein 3
Control of mRNA levels mRNA synthesis
mRNA decay
Promoter
Exosome 3’-to-5’
Preinitiation complex (PIC)
mRNA 4
Xrn1 5’-to-3’
Promoter DNA cis-acting elements that enable transcription. Core promoter yeast promoter transcription start site
Upstream activating sequence similar to enhancers, repress or enhance assembly of basal transcription apparatus
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recruit RNA polymerase II and the basal transcription apparatus.
mRNA synthesis Preinitiation complex (PIC)
Two major cytoplasmic decay pathways of mRNA in yeast
mature mRNA
5’ to 3’ decay
3’-polyA tail removal
3’ to 5’ decay
Exosome
XRN1
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Nature Review molecular cell biology
The half-lives of mRNAs in yeast vary wildly.
Ranging from 3 min to more than 90 min The natural ACT1 and RPL30 mRNAs are degraded with different kinetics.
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Q. What determines the half-life of a specific mRNA? mRNAs carry all of the necessary information, both in their sequence and structure After releasing from RNA Pol II, mRNA in the cytoplasm is unaffected by the promoter.
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RNA Polymerase II can control the fate of its transcripts in the cytoplasm.
Pol II impacts mRNA decay (2008)
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Specific aim Q What determines the half-life of a specific mRNA? RNA Pol II
Cis-acting elements on the promoter 10
?
mRNA decay
Flow chart mRNA mapping (5’ end, 3’ end)
Reporter genes encode identical mRNA.
RPL30 ACT1
• mRNA decay rate: RPL30 > ACT1 UAS can affect the stability of the resulting mRNA.
UAS can regulate the Xrn1p-dependent pathway. Rap1p-binding sites are necessary to confer enhanced mRNA decay.
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Constructs Upstream activating sequence
G18 tract
ACT1 UAS
RPL30pG RPL30 UAS
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3’ end mapping of mRNAs
3’ end mapping
probe
RNaseH cleavage
RNaseH digestion
probe RPL30pG hybridization
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Reporter genes encode identical mRNA.
RPL30 deletion
Northern blot analysis
3’ end mapping
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5’ end mapping
Flow chart Reporter genes encode identical mRNA.
UAS can affect the stability of the resulting mRNA.
UAS can regulate the Xrn1p-dependent pathway. Rap1p-binding sites are necessary to confer enhanced mRNA decay.
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Upstream activating sequence can affect the stability of the resulting mRNA. transcription arrest by 1,10-phenanthroline
5’-to-3’ decay
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Fragment
Flow chart Reporter genes encode identical mRNA.
UAS can affect the stability of the resulting mRNA.
UAS can regulate the Xrn1p-dependent pathway. Rap1p-binding sites are necessary to confer enhanced mRNA decay.
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Xrn1p or Exosome?
Two major cytoplasmic decay pathways of mRNA in yeast
3’-polyA tail removal
5’ to 3’ decay
3’ to 5’ decay
Exosome
XRN1
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Nature Review molecular cell biology
UAS can regulate the Xrn1p-dependent pathway. block Xrn1p-mediated 5’-to-3’ mRNA degradation
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Deletion of SKI7 did not stabilize mRNA A or mRNA B. block 3’-to-5’ exosome-mediated mRNA degradation
SKI7: • An adaptor that links the SKI complex with the exosome. • required for exosome activity
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Flow chart Reporter genes encode identical mRNA.
UAS can affect the stability of the resulting mRNA.
UAS can regulate the Xrn1p-dependent pathway. Rap1p-binding sites are necessary to confer enhanced mRNA decay.
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Rap1p-binding sites are necessary to confer enhanced mRNA decay. Rap1p-binding site (RapBS)
found in ~90% of ribosomal protein promoter
• Rap1p: repressor activator protein 1 • Rap1p can bind to Rap1p-binding site (RapBS)
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Rap1p-binding sites are necessary to confer enhanced mRNA decay. insertion of RapBS
decay rate: B>F
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removal of RapBS
decay rate: E>A
Depletion of Rap1p increased mRNA B stability.
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Rap1p depletion affected the decay of the endogenous RPL30 mRNA. depletion of Rap1p
Rap1p stimulated transcription of RPL30. steady state mRNA
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Summary mRNA mapping (5’ end, 3’ end)
Reporter genes encode identical mRNA.
UAS can affect the stability of the resulting mRNA.
UAS can regulate the Xrn1p-dependent pathway. Rap1p and Rap1p-binding site (RapBS) are both required for the regulation of mRNA synthesis and decay.
