Promoter elements regulate cytoplasmic mRNA decay

Speaker: Ting-Yu LinAdvisor: Li-Kwan Chang, Ph.D.

Date: 2012/10/08

Cell 147(7): 1473-1483, 2011

Central dogma

2

DNA

RNA

Protein

3

mRNA stability regulation

Pre-mRNA

mature mRNA

Control of mRNA levels

mRNA synthesis mRNA decay

4

Preinitiation complex (PIC)

Xrn15’-to-3’

Exosome3’-to-5’

Promoter

mRNA

Promoter

5

DNA cis-acting elements that enable transcription.

yeast promoter

transcription start site

Core promoter

recruit RNA polymerase II and the basal transcription apparatus.

Upstream activating sequence

similar to enhancers,repress or enhance assembly of basal transcription apparatus

Preinitiation complex (PIC)

mRNA synthesis

Two major cytoplasmic decay pathways of mRNA in yeast

6

3’-polyA tail removal

5’ to 3’ decay 3’ to 5’ decay

Exosome

XRN1Nature Review molecular cell biology

mature mRNA

The half-lives of mRNAs in yeast vary wildly.

7

Ranging from 3 min to more than 90 min

The natural ACT1 and RPL30 mRNAs are degraded with different kinetics.

8

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.

RNA Polymerase II can control the fate of its transcripts in the cytoplasm.

Pol II impacts mRNA decay (2008)

9

Specific aim

10

Q What determines the half-life of a specific mRNA?

RNA Pol II

mRNA decayCis-acting elements on the promoter ?

Flow chart

11

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.

RPL30ACT1

mRNA mapping(5’ end, 3’ end)

• mRNA decay rate: RPL30 > ACT1

Constructs

G18 tract

ACT1 UAS

RPL30 UASRPL30pG

Upstream activating sequence

12

3’ end mapping of mRNAs

13

RNaseH cleavage

probe3’ end mapping

probe RPL30pG hybridization

RNaseHdigestion

Reporter genes encode identical mRNA.

14

Northern blot analysis

3’ end mapping 5’ end mapping

RPL30 deletion

Flow chart

15

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.

Upstream activating sequence can affect the stability of the resulting mRNA.

16

transcription arrestby 1,10-phenanthroline

Fragment5’-to-3’ decay

Flow chart

17

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.

Xrn1p or Exosome?

Two major cytoplasmic decay pathways of mRNA in yeast

18

3’-polyA tail removal

5’ to 3’ decay 3’ to 5’ decay

Exosome

XRN1Nature Review molecular cell biology

UAS can regulate the Xrn1p-dependent pathway.

19

block Xrn1p-mediated 5’-to-3’ mRNA degradation

Deletion of SKI7 did not stabilize mRNA A or mRNA B.

20

SKI7: • An adaptor that links the SKI complex with the exosome.• required for exosome activity

block 3’-to-5’ exosome-mediated mRNA degradation

Flow chart

21

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.

Rap1p-binding sites are necessary to confer enhanced mRNA decay.

22

Rap1p-binding site (RapBS)

• Rap1p: repressor activator protein 1• Rap1p can bind to Rap1p-binding site (RapBS)

found in ~90% of ribosomal protein promoter

Rap1p-binding sites are necessary to confer enhanced mRNA decay.

23

removal of RapBSinsertion of RapBS

decay rate: B>F decay rate: E>A

Depletion of Rap1p increased mRNA B stability.

24

Rap1p depletion affected the decay of the endogenous RPL30 mRNA.

25

depletion of Rap1p

steady state mRNA

Rap1p stimulated transcription of RPL30.

Summary

26

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.

RPL30ACT1

mRNA mapping(5’ end, 3’ end)

Proposed model

27

mRNA imprinting (choder,2011)

RapBSTATA

nucleus

Rap1p unknown

Pol II

unknown

cytoplasm Xrn1p-mediated mRNA decay

28

Thanks for your attention!