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Dégradation des ARN et régulation de l'expression génique chez Saccharomyces cerevisiae. Introduction. Dégradation nucléaire des ARN et mécanisme de surveillance. Identification exhaustive des snoRNAs H/ACA chez Saccharomyces cerevisiae.

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Dégradation des ARN et régulation de l'expression génique chez Saccharomyces cerevisiae

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Slide1 l.jpg

Dégradation des ARN et régulation de l'expression génique chez Saccharomyces cerevisiae

Introduction

Dégradation nucléaire des ARN et mécanisme de surveillance

Identification exhaustive des snoRNAs H/ACA chez Saccharomyces cerevisiae

Régulation de la dégradation des ARNm: exemple de l'ANRm RPS28B

AReNA Toulouse 2005


Slide2 l.jpg

CCR4

POP2

NOT

CCR4

POP2

NOT

LSM 1-7

PAB1

XRN1

( 5 ’->3 ’)

XRN1

( 5 ’->3 ’)

DCP1

DCP1

DCP2

DCP2

m7GpppN

AA

Exo

3’>5 ’

pN

A

Cytoplasmic mRNA degradation

Stabilisation

PAB1

Cap binding complex

PAB1

PAB1

m7GpppN

AAAAAAAAAAAA

From Tharun et al. 2001 & Thucker et al. 2002


Slide3 l.jpg

7

Gppp

m

Mtr4p

Nuclear

Exosome

AAAAAA

A…AAAAA

A

7

m

Xrn1p

Gppp

Ski2p

Rat1p

Homologous degradation pathways in the nucleus and cytoplasm

Cytoplasmic mRNA degradation

Dcp2p

Cytoplasmic

Exosome

AAAA…AA

A

Nuclear mRNA degradation

D. Tollerey


Slide4 l.jpg

Ski3

Ski8

Rrp41

Rrp4

Rrp45

Rrp41

Rrp4

Rrp45

Rrp47

Rrp6

Ski2

Mtr4

Exosome

3’->5’ exonucleases (complex)

cytoplasm

nucleus


Slide5 l.jpg

Ski3

degradation of mRNAs with premature STOP codons (NMD)

Ski8

Rrp41

Rrp4

Rrp45

mRNA degradation

Rrp41

Rrp4

Rrp45

Rrp47

Rrp6

Ski2

Mtr4

degradation of mRNAs without STOP codon

cytoplasm

nucleus


Slide6 l.jpg

Ski3

Ski8

Rrp41

Rrp4

Rrp45

Rrp41

Rrp4

Rrp45

Rrp47

Rrp6

Ski2

Mtr4

maturation/degradation of rRNAs, snRNAs, snoRNAs and tRNAs

cytoplasm

nucleus

Degradation

of pre-mRNAs


Slide7 l.jpg

Ski3

Ski8

Rrp41

Rrp4

Rrp45

Rrp41

Rrp4

Rrp45

Rrp47

Rrp6

Ski2

Mtr4

Other roles

in a WT cell ?

cytoplasm

nucleus


Slide8 l.jpg

∆rrp6

snRNAs

snoRNAs

WT


Snrnas rrna and snornas get poly a tails and become visible for the dna chip l.jpg

snRNAs, rRNA and snoRNAs get poly(A) tails and become "visible" for the DNA chip

WT

TTTTTT

rrp6∆

AAAAAAAAA

van Hoof, 2000; Kuai, 2004


Slide10 l.jpg

∆rrp6

intergenic SAGE = short sequence known to be transcribed

snRNAs

snoRNAs

SAGE

Serial Analysis

of Gene Expression

WT


Intergenic regions are expressed in rrp6 l.jpg

Intergenic regions are expressed in ∆rrp6

∆rrp6

∆rrp6

∆rrp6

∆rrp6

WT

WT

WT

WT

NEL025c

NBL001c

NGR060w

NPL040w


Quantitative rt pcr confirms transcription from intergenic regions l.jpg

Quantitative RT-PCR confirms transcription from intergenic regions

Fold increase in ∆rrp6


Quantitative rt pcr confirms transcription from intergenic regions13 l.jpg

Quantitative RT-PCR confirms transcription from intergenic regions

Fold increase in ∆rrp6

1000 nt


Nel025c transcripts have a defined capped 5 end l.jpg

NEL025c transcripts have a defined capped 5' end

wt

∆rrp6

-

+

-

+

RNAse H

5'

3'

AAAAAA…

568 nt

5'

3'

AAAAAA…

~300 nt

oligonucleotide anti-sens

5'

3'

186 nt

AAAAAA…

RNAse H cleavage

112 nt (U6)

Northern Blot

Libri lab


Nel025c transcripts are polyadenylated in rrp6 l.jpg

NEL025c transcripts are polyadenylated in ∆rrp6

Oligo-dT selected

Total

∆rrp6

∆rrp6

WT

MW

WT

622 nt.

TTTTTT

AAAAA

527 nt.

404 nt.

NEL025c

307 nt.

242 nt.

238 nt.

217 nt.

201 nt.

190 nt.

180 nt.

Oligo-dT chromatography to select poly(A) RNAs

RPS28A


Nel025c transcripts have heterogenous 3 ends in rrp6 l.jpg

NEL025c transcripts have heterogenous 3' ends in ∆rrp6

wt

∆rrp6

-

+

-

+

RNAse H

5'

3'

AAAAAA…

5'

3'

NEL025C

AAAAAA…

oligo-dT

5'

3'

RNAse H cleavage

RPS28A

Northern Blot

Libri lab


Nel025 is transcribed by rna polymerase ii and its turnover is slower in rrp6 l.jpg

Pol II inactivation

RNA analysis (NEL025C)

time

% of

time 0

time (min)

NEL025 is transcribed by RNA polymerase II and its turnover is slower in ∆rrp6

∆rrp6

wt

Libri lab


Intergenic transcripts are rna polymerase ii products l.jpg

Intergenic transcripts are RNA polymerase II products

Capped and polyadenylated

Unstable in a wild type strain (but detectable)

Degraded by the nuclear exosome

CUTs: Criptic Unstable Transcripts

Who's responsible for CUTs polyadenylation?


Pap1 is the major nuclear poly a polymerase but not for cuts l.jpg

pap1-1

RRP6

Pap1 is the major nuclear poly(A) polymerase - but not for CUTs

∆rrp6 + pap1-1

pap1-1

∆rrp6

wt

37°C 1h

NEL025C

Shatkin & Manley, 2000

poly(A)+

RPS28A

5S RNA


Trf4 a good candidate for a novel poly a polymerase linked to rna turnover l.jpg

Trf4 - a good candidate for a novel poly(A)-polymeraselinked to RNA turnover

Trf4

Looks like a poly(A)-polymerase - BLAST p=0.06, best hit of Pap1

Is nuclear (Huh et al., 2003)

Trf4 interacts with Mtr4 - a nuclear helicase involved in exosome function (RNOMics)


Purified trf4 shows a poly a polymerase activity in vitro l.jpg

Purified Trf4 shows a poly(A) polymerase activity in vitro

TAP - Tandem Affinity Purification

Trf4-TAP

0

AAAAAA

AAAAA

RNA (labeled)

ATP

Trf4-TAP

AAAAAA

AAA

AAAAAAAAAAAAAAA

Séraphin lab


Slide22 l.jpg

Trf4, Air2 and Mtr4 form a complex ("TRAMP")

Séraphin lab


Tr f4 a ir2 and m tr4 form a complex tramp l.jpg

Trf4, Air2 and Mtr4 form a complex ("TRAMP")

no

tag

Air2

TAP

Trf4

TAP

Mtr4

TAP

Is Trf4/TRAMP required for the polyadenylation and the degradation of CUTs?

*

Mtr4

*

Trf4

*

Air2

TAP - Tandem Affinity Purification

tagged protein

*


Trf4 is essential for cuts polyadenylation and degradation l.jpg

Trf4 is essential for CUTs polyadenylation and degradation

∆rrp6 + ∆trf4

∆rrp6

∆trf4

wt

total RNA

NEL025C

The absence of Trf4 leads to accumulation of unpolyadenylated NEL025C transcripts

poly(A)+

total RNA

RPS28A

poly(A)+


Slide25 l.jpg

?

exosome

TRAMP complex

(Trf4, Air2, Mtr4)

Pap1

AAAAAAA

AAAAAAA

AAAAAAA

AAAAAAA


Slide26 l.jpg

?

exosome

TRAMP complex

Pap1

AAAAAAA

How widespread are CUTs in yeast ?

AAAAAAA

AAAAAAA

AAAAAAA


Non polyadenylated cuts accumulation in rrp6 trf4 l.jpg

Non-polyadenylated CUTs accumulation in ∆rrp6 + ∆trf4

∆rrp6 + ∆trf4

∆rrp6

∆trf4

wt

total RNA

NEL025C

poly(A)+

total RNA

RPS28A

poly(A)+


Slide29 l.jpg

∆rrp6

intergenic SAGE = short sequence known to be transcribed

snRNAs

snoRNAs

SAGE

Serial Analysis

of Gene Expression

WT


Shift in the distribution of transcript ratios mutant wt for intergenic sages in rrp6 l.jpg

Shift in the distribution of transcript ratios mutant/wt for intergenic SAGEs in ∆rrp6

wt vs wt

∆rrp6 vs. wt


Shift in the distribution of transcript ratios mutant wt for intergenic sages in rrp631 l.jpg

Shift in the distribution of transcript ratios mutant/wt for intergenic SAGEs in ∆rrp6

wt vs wt

∆rrp6 vs. wt

1,5-3,5 x induction


3 to 200 fold induction for new cuts in the double mutant rrp6 trf4 l.jpg

3 to 200 fold induction for new CUTs in the double mutant: ∆rrp6 + ∆trf4


Further questions for widespread cuts l.jpg

AAAAAA

Further questions for widespread CUTs

  • More than 10% of the intergenic regions are transcribed in yeast to detectable levels (SAGE, Velculescu et al., 1997)

  • The exosome and the TRAMP complex degrade CUTs in the nucleus very efficiently

  • What targets a CUT to degradation:

    • promoter region ?

    • terminator ?

    • chromatin ?

  • The mechanics of exosome activation…

  • Possible functions for the CUTs ?


Slide36 l.jpg

Domenico Libri

Mathieu Rougemaille

Jocelyne Boulay

Alain Jacquier

Gwenael Breard

Cosmin Saveanu

GIM

Bertrand Séraphin

Françoise Wyers

Edda Kastenhuber

Abdelkader Namane

Jean-Claude Rousselle

PF3

University of Edinburgh

Béatrice Régnault

PF2

John LaCava

Jonathan Houseley

David Tollervey

EEC grant: RNOMICS

Wyers et al., Cell, 2005

La Cava et al., Cell, 2005


Slide37 l.jpg

new snoRNA


Slide38 l.jpg

The snoARNs (small nucleolar ARN)

  • 2 snoARN families:

  • Box C/D snoARNs

  • Box H/ACAsnoARNs

  • 2 main classes of RNA modifications in the ribosome:

  • Methylations

  • Pseudouridylations


Slide39 l.jpg

- Almost no consensus sequences

- guides consist of two short sequences

- four specifically associated proteins


Slide40 l.jpg

Il existe 44 sites  sur l’ARNr chez la levure

15 sites « orphelins » : sans snoARN guide associé


Slide41 l.jpg

Strategy used

Yeast total extract

Nhp2p

or Gar1

TAP-tag

RNA extraction

ARN

Direct labeling

Fluorochrome 546

First affinity column

IgG sepharose beads

TEV protease cleavage

Genomic DNA microarrays :

genes and intergenic regions

Second affinity column

Calmoduline beads

Direct labeling

Fluorochrome 647

Elution

RNA extraction


Slide42 l.jpg

102pb

U18 : known box C/D snoRNA

WT

Nop1p-TAP

Nhp2p-TAP

Tot

1

2

Tot

1

2

Tot

1

2

274pb

snR191 : known H/ACA snoRNA


Slide43 l.jpg

Distribution des gènes en fonction du log2(Ratio R/V)

pas ou très peu immunoprécipité par Nhp2p

Transposons et ORF (dont beaucoup d’ARN fortement exprimés (glycolyse, etc…)). Immunoprécipités de façon non spécifique par Nhp2p

SNR37, SNR10, RUF1, iYML103c, SNR32, SNR11, SNR35, SNR44, SNR42, RUF3, SNR43, SNR3, SNR191, SNR161, SNR30, SNR31/5, SNR36, iYBR044c, SNR189, SNR34, SNR33, SNR8, SNR44, ZEO1, FAR1, SNR9, SNR49, SNR189, iYMR246w, RPS28A, iYEL055c


Slide44 l.jpg

102pb

U18 : known box C/D snoRNA

170pb

iYML103c : new snoRNA (snR85)

1000pb

iYMR246w : new sno RNA (snR86)

175pb

iYEL055c : new snoRNA (snR80)

WT

Nop1p-TAP

Nhp2p-TAP

Tot

1

2

Tot

1

2

Tot

1

2

274pb

snR191 : known H/ACA snoRNA

300pb

iYBR044c

275pb

260pb


Slide45 l.jpg

Are all H/ACA snoRNAs now identified ?

22 snoARN H/ACA already known +7 new ones.

Hypothesis :all rRNA s are guided by these snoRNAs

(there are no more missing snoRNAs)

-> Verify the 8 predicted guide snoARNs

-> Determine the guiding snoARN for the 16 remaining sites


Slide46 l.jpg

Are all H/ACA snoRNAs now identified ?

- Construction de 23 souches D snoARN

- Chacune des souches a été testé pour l’ensemble des positions modifiées


Slide48 l.jpg

oligo CT36

oligo GB295

oligo GB178

oligo GB289

+

M

- +

M

- +

M

M

- +

1 2

1 2

1 2

1 2

1 2

217

Y

106

Y

1289

90

242

Y

120

201

Y

632

Y

466

76

217

190

67

201

190

90

90

180

Y

759

Y

1414

160

Y

211

Y

766

147

76

76

67

Y

302

217

201

190


Slide49 l.jpg

7 predictions verified (one was wrong)

15 entirely new guides identified

Only one position (LSU 1051) with no guide identified


Slide50 l.jpg

P. Schattner et al. (2004) Nucl. Acids Res. vol.32, 4281-4296

Use of a bioinformatic approach

snR81 (new)


Slide51 l.jpg

Main conclusions

29 H/ACA snoRNAs are responsible for guiding all 44 rRNA s

In yeast, all or nearly all H/ACA snoRNAs are involved in rRNA modification


Slide52 l.jpg

3 snoRNAs guide more than two positions.

snR49

RUF2

snR3


Slide54 l.jpg

ARC

FRM

ENS Paris

Frédéric Devaux

Institut Pasteur

Claire Torchet

Gwénaël Badis

Ginny Costanzo

Alain Jacquier

CEA Saclay

Michel Werner

Torchet et al., RNA, 2005


Slide55 l.jpg

CCR4

POP2

NOT

CCR4

POP2

NOT

LSM 1-7

PAB1

XRN1

( 5 ’->3 ’)

XRN1

( 5 ’->3 ’)

DCP1

DCP1

DCP2

DCP2

m7GpppN

AA

Exo

3’>5 ’

pN

A

Cytoplasmic mRNA degradation

Stabilisation

PAB1

Cap binding complex

PAB1

PAB1

m7GpppN

AAAAAAAAAAAA

From Tharun et al. 2001 & Thucker et al. 2002


Two hybrid links predicted a role for yel015w edc3 in mrna degradation l.jpg

Two-hybrid links predicted a role for YEL015W(EDC3) in mRNA degradation

EDC3

5’>3’

degradation

XRN1

Hsu and Stevens, MCB 1993

DCP2

Lsm1-7

mRNA

decapping

DCP1

PAT1

Beelman et al., Nature 1996

Dunckley et al., EMBO J 1999

mRNA decay

Tharun et al., Nature 2000; Bonnerot et al. MCB 2000

Fromont-Racine et al. 2000Yeast 17:95-110


Edc3 affects the levels of the rps28b mrna l.jpg

EDC3 affects the levels of the RPS28B mRNA...

∆edc3

RPS28B

WT


In both directions l.jpg

in both directions

EDC3 overexpressed

RPS28B

WT


Rps28b autoregulation l.jpg

RPS28B autoregulation

∆edc3

RPS28B mRNA/ACT1 normalized

∆edc3

wt

∆edc3

wt

wt

RPS28B

CEN

RPS28B

no plasmid


Rps28b mrna decay time course l.jpg

RPS28B mRNA decay time course

12.5

20

30

45

12.5

20

30

45

minutes

0

4

8

0

4

8

RPS28A

RPS28B

wild type

t1/2 = 11.5 minutes

∆edc3

t1/2 = 33 minutes


The 3 utr regulates in cis other transcripts l.jpg

The 3’ UTR regulates in cis other transcripts

∆edc3

∆edc3

wt

Beta galactosidase activity

wt

lacZ+3’UTR

lacZ


Slide62 l.jpg

The 3’UTR contains a conserved regionmultiple sequence alignment S cerevisiae, S kluyveri, Z rouxii, S bayanus, Sparodoxus(Souciet et al., 2000; Kellis et al., 2003)

204

548

600

850


Predicted hairpin structure in rps28b 3 utr l.jpg

Predicted hairpin structure in RPS28B 3’UTR

From 26 substitutions :

13 compensatory changes

4 conservative changes

7 mutations in loops

only one predicted pairing not conserved


The hairpin is the 3 utr regulatory element l.jpg

The hairpin is the 3’UTR regulatory element

∆edc3

∆edc3

RPS28B/RPS28A mRNA

wt

wt

-3’ UTR

+3’ hairpin

RPS28B


Rps28 physically interacts with the hairpin l.jpg

IRP or Rps28B

IRE or hairpin

Rps28 physically interacts with the hairpin

IRE-MS2

+AD-IRP

hairpin-MS2

+AD-IRP

IRE-MS2

+AD-RPS28B

hairpin-MS2

+AD-RPS28B

Beta galactosidase

assay


Edc3 is associated with the decapping complex l.jpg

Edc3 is associated with the decapping complex

EDC3

RPS28

XRN1

DCP2

Lsm1-7

DCP1

PAT1

Ito et al., 2001, PNAS

Gavin et al., 2001, Nature

Ho et al., 2001, Nature

Fromont-Racine et al. 2000Yeast 17:95-110


Rps28b mrna degradation a model l.jpg

Rps28

RPS28B mRNA degradation - a model

Edc3

Dcp2

Dcp1

m7Gppp

AAAAAAAAAAA

Decapping and 5’ to 3’ degradation

Badis et al., Mol. Cell, 2005


Slide68 l.jpg

Gwenaël

Badis-Bréard

Alain

Jacquier

Laurence

Decourty

The lab

Gwenaël

Badis-Bréard

Alice

Lebreton

Florence

Hantraye

Alain

Jacquier

Micheline

Fromont-Racine

Cosmin

Saveanu

Laurence

Decourty

Odile

Labouise

… and Claire Torchet


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