Aucun titre de diapositive

1
Caractérisation dun complexe de maturation des
ARNr chez les plantes

• Laboratoire Génome et Développement des Plantes
• UMR CNRS IRD 5096
• Université de Perpignan

2
Transcription and processing of rRNA genes
5.8S
18S
25S
IGS
IGS
rDNA
RNA
Pol I transcription
3'ETS
5'ETS
pre-
rRNA
P(A0)
ITS 1
ITS 2
Processing
mature
5.8
S
18
S
25
S
rRNA

100
ribosomal

5S rRNA
proteins
R
i
b
o
s
o
m
e
3
5 ETS processing complex forms on the nascent
rRNA
Chistmas tree-like structures with terminal balls
characteristically decorating the 5 ends of the
nascent rRNA branches (Miller and Beatty .,
1969).
4
Identification of two plants protein -rDNA
complexes
Electrophoretic Mobility Shift Assay (EMSA)
WCE
NF D
Protein- DNA Complex
NF B
NF B
Free- DNA
NF- B
NF
-D
rDNA
18
S
1
2
3
B
P(A0)
A
A
A
5
The A123BP cluster in crucifers
TIS
P
B
A1
A3
A2
180-183
Ra Bo Br Bj At
1275
1083 bp insertion
18S
Ra Bo Br Bj At
740 824 716 746 1836
6
Purification scheme of NF D
Cauliflower
Whole Cell Extract
DEAE
0.35 M KCl
0.2 M
Heparine
0.6 M KCl
0.175 M
Sephacryl S300
DNA Affinity
0.3 M KCl
0.1 M
NF D
7
NF D and NF B fractionation using S300
232
158
440
2000
669
Kda
fractions
36
40
38
44
42
46
48
50
52
54
56
58
60
62
64
66
NF D
NF B
Free- DNA
NF B
NF D
DNA affinity column
8
NF D purification using DNA affinity column
DNA affinity
S300
13
14
19
17
-
18
20
15
16
NF D
Free- DNA
9
PAGE and Western blot analysis of purified NF D
10
Fibrillarin and Nucleolin are two nucleolar
factors involved in pre-rRNA processing
Fibrillarin associates with snoRNA U3 to direct
specific pre -rRNA cleavages
Does NF D contains SnoRNA U3 ?
11
U3 snoRNA is detected in NF D
RNA
S300
--
NF D
RT/Taq - -
Taq - - - -
U3, 216 bp
300 200
U3
U6, 96 bp
U6
200 100
12

• We know
• NF D contains pre-rRNA processing factors
• Nucleolin
• Fibrillarin
• U3 snoRNA

• Question
• Is NF D a processing complex ?
• - Does NF D binds pre- rRNA ?
• - Is NF D able to cleavage pre-rRNA at P (A0)
  site ?

13
NF D binds A123BP-H rRNA
14
Does NF D cleave pre-rRNA in vitro ?
317
103
P (A0)
32P-CTP-RNA
NF D
132 b
82 b
Cleaved RNA
Denaturing PAGE
15
NF D cleaves pre- rRNA in vitro
rA123BP-H
A123BP
DNA Affinity
WCE
32P-CTP-RNA
13
14
19
17
18
20
15
16
214 b Precurseur
317
103
P (A0)
132 b
NF D fractions
132 b
82 b
82 b
Denaturing PAGE
16
NF D cleaves pre - rRNA at the P (A0) site as in
vivo
NF D
In vivo
R.s

-
-
G
A
T
C
317
103
P (A0)
Precursor
In vitro processing AND Primer extention
82 b
132 b
Processed
P32
17

• CONCLUSION
• NF D is an pre-rRNA processing complex
• Is around 600 kDa
• Contains rRNA processing factors
• Nucleolin
• Fibrillarin
• U3 snoRNA
• RNase (endonuclease) ?
• others
• Binds and cleaves pre-rRNA at the P(A0) site
• Binds specifically rDNA region encoding the
  primary rRNA processing site (A123BP) !!!

18
Model of Role of NF D in rRNA synthesis
18
S
1
2
3
A
A
A
B
P
19
Julio SAEZ -VASQUEZ David CAPARROS - RUIZ Fredy
BARNECHE Manuel ECHEVERRIA

• Laboratoire Génome et Développement des Plantes
• UMR CNRS IRD 5096
• Université de Perpignan