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

1. 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

2. 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

3. 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

4. Ski3 Ski8 Rrp41 Rrp4 Rrp45 Rrp41 Rrp4 Rrp45 Rrp47 Rrp6 Ski2 Mtr4 Exosome 3’->5’ exonucleases (complex) cytoplasm nucleus

5. 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

6. 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

7. Ski3 Ski8 Rrp41 Rrp4 Rrp45 Rrp41 Rrp4 Rrp45 Rrp47 Rrp6 Ski2 Mtr4 Other roles in a WT cell ? cytoplasm nucleus

8. ∆rrp6 snRNAs snoRNAs WT

9. snRNAs, rRNA and snoRNAs get poly(A) tails and become "visible" for the DNA chip WT TTTTTT rrp6∆ AAAAAAAAA van Hoof, 2000; Kuai, 2004

10. ∆rrp6 intergenic SAGE = short sequence known to be transcribed snRNAs snoRNAs SAGE Serial Analysis of Gene Expression WT

11. Intergenic regions are expressed in ∆rrp6 ∆rrp6 ∆rrp6 ∆rrp6 ∆rrp6 WT WT WT WT NEL025c NBL001c NGR060w NPL040w

12. Quantitative RT-PCR confirms transcription from intergenic regions Fold increase in ∆rrp6

13. Quantitative RT-PCR confirms transcription from intergenic regions Fold increase in ∆rrp6 1000 nt

14. 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

15. 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

16. 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

17. 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

18. 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?

19. 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

20. 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)

21. 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

22. Trf4, Air2 and Mtr4 form a complex ("TRAMP") Séraphin lab

23. 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 *

24. 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)+

25. ? exosome TRAMP complex (Trf4, Air2, Mtr4) Pap1 AAAAAAA AAAAAAA AAAAAAA AAAAAAA

26. ? exosome TRAMP complex Pap1 AAAAAAA How widespread are CUTs in yeast ? AAAAAAA AAAAAAA AAAAAAA

27. Non-polyadenylated CUTs accumulation in ∆rrp6 + ∆trf4 ∆rrp6 + ∆trf4 ∆rrp6 ∆trf4 wt total RNA NEL025C poly(A)+ total RNA RPS28A poly(A)+

29. ∆rrp6 intergenic SAGE = short sequence known to be transcribed snRNAs snoRNAs SAGE Serial Analysis of Gene Expression WT

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

31. 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

32. 3 to 200 fold induction for new CUTs in the double mutant: ∆rrp6 + ∆trf4

33. 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 ?

36. 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

37. new snoRNA

38. 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

39. - Almost no consensus sequences - guides consist of two short sequences - four specifically associated proteins

40. Il existe 44 sites  sur l’ARNr chez la levure 15 sites « orphelins » : sans snoARN guide associé

41. 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

42. 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

43. 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

44. 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

45. 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

46. 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

48. 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

49. 7 predictions verified (one was wrong) 15 entirely new guides identified Only one position (LSU 1051) with no guide identified

50. P. Schattner et al. (2004) Nucl. Acids Res. vol.32, 4281-4296 Use of a bioinformatic approach snR81 (new)