- Email: [email protected]
On a messenger ribonucleoprotein complex from rabbit reticulocytes
228
BIOCHIMICAET BIOPHYSICAACTA
PRELIMINARY NOTES BBA 91259 On a messenger ribonucleoprotein complex from rabbit reticulocytes We have previously reported the isolation from rabbit reticulocytes of an RNA fraction which nmst be a mixture of two messenger RNA's coding for the two chains of haemoglobin 1-4. This fraction was identified as the messenger RNA thread of the polyribosomes in view of its extreme susceptibility to ribonuclease (EC 2.7.7.16) z. Its sedimentation constant of about 9 S indicated a molecular weight in the expected range of x5o ooo (ref. 2). When rabbits were injected with radioactive phosphate I5 h before sacrifice, the 9-S RNA fraction was more highly labelled than rRNA 1. Furthermore, this fraction specifically associated with native 4o-S subparticles from reticulocytes (unpublished results); it had a poor secondary structure ~, as could be expected for a messenger. Recent work further supports the idea that the 9-S RNA fraction we obtained essentially consists of a mixture of the messenger RNA's of globin chains 5-v. On the other hand, the RNA isolated by another fractionation procedure and which directs haemoglobin synthesis in a bacterial cell-free system 8 is most probably identical with our 9-S fraction. It had already been shown in our laboratory that the same 9-S RNA can be released from polyribosomes by adding EDTA or by passing the polyribosomal suspension through a column of CM-cellulose 9. After such treatments, the 9-S RNA is never recovered as a naked fiber; it is always associated with protein materiaP °. Similar complexes between a rapidly labelled RNA and a protein have also been detected in other cells: L-cells n, liver TM, thyroid la and bacteria ~. It seems likely that when Mg2+ is removed, the polyribosonzes fall apart, the subparticles of the ribosomes unfold and the messenger remains associated with certain proteins. In most tissues, the messenger population is quite heterogeneous, making the purification of the ribonucleoprotein very difficult. In reticulocytes, on the contrary, there are only two messenger RNA's which are about the same size. The messenger ribonucleoprotein complexes of reticulocytes should therefore be more amenable to useful study. In the present note, we wish to report on the rapid isolation and the characterisation by CsC1 equilibrium centrifugation of the messenger ribonucleoprotein complex from reticulocytes. Rabbit reticulocyte polyribosomes were obtained as previously described 9. The stroma-free hemolysate was centrifuged at IOO ooo × g for 3 h through layers of 3o % sucrose. As a rule, anaemic rabbits received IO mC of !ia2Plphosphate 15 h before sacrifice. Their polyribosomes, suspended in o.o3 M EDTA, were layered on top of a I5-3o % sucrose gradient and spun for 4o h at 24 ooo rev./min in the SW-25.I rotor of a Spinco ultracentrifuge (see legend to Fig. I). Fig. I shows a typical sucrose gradient separation of the different particles obtained from EDTA-treated polyribosomes. The ribonucleoprotein containing the 9-S RNA sediments in the region of the gradient and is shown by the hatched area. Biochim. Biophys. dcta, i9o (I969) 228-23I
229
PRELIMINARY NOTES
i
i
I 2.0
"
f
.
,
~
12000
i ,
Q2 .
.
.
.
1
.
.
.....
r
L.
"
--
-
-"1000
p'
',
'
I
,
' I
.~
@ ~
ii
/I
•
c
: tRNA
't il
i
75o T
E
.
'
1.0
8
1000
2
i .
]
,i
!
:
b
!
'-
:
[
:
=E
I''
o.~
g o
250
5
3 5
Oo
? -
1 0
"
20 fractions
30
40
fractlons
4o
6_
%
Fig. I. 4 m g of p o l y r i b o s o m e s were s u s p e n d e d in 0.6 ml of IO mM s o d i u m p k o s p h a t e b u f f e r (pH 7.o), i o mM KC1. D i s s o c i a t i o n of t h e p a l y r i b o s o m e s w a s p e r f o r m e d b y a d d i t i o n of o. 3 ml of o . i M E D T A (pH 7.o). The s u s p e n s i o n w a s l a y e r e d on t o p of 15-3o % sucrose g r a d i e n t s ( i o mM s o d i u m p h o s p h a t e b u f f e r (pH 7), IO mM KC1) a n d c e n t r i f u g e d a t 4 ° in a S'W-25.I r o t o r (Spinco) a t 24 o ooo r e v . / m i n for 4 ° 11. 0 - - - O , 3~p r a d i o a t i v i t y . Fig. 2. S e d i m e n t a t i o n in sucrose g r a d i e n t of t h e m a t e r i a l c o r r e s p o n d i n g to t h e h a t c h e d zone in Fig. i. Tile s u s p e n s i o n w a s l a y e r e d on t o p of a 15-3o % sucrose g r a d i e n t ( i o mM s o d i u m phosp h a t e b u f f e r (pH 7.o), i o mM KC1) a n d c e n t r i f u g e d a t 4 ° in a S'W-25.I r o t o r (Spinco) a t 24 ooo r e v . / m i n for 4 ° h.
The fractions corresponding to this region were pooled and concentrated b y vacuum dialysis against IOO vol. of io mM triethanolamine buffer (pH 7.4), IO mM KCl. The suspension was then again centrifuged in a 15-3o% sucrose gradient at 24 ooo rev./min for 4 ° h (see Fig. 2). Most of the highly radioactive nucleic acid was found in the ribonucleoprotein complex; the slower moving peak which is much less radioactive was shown to be free 5-S RNA 9. By means of the method described above, it is thus easy to obtain a clean preparation of the messenger ribonucleoprotein complex. In order to further characterise this complex, we added 3o #g of unlabelled subribosomal particles as a marker to ioo fig of 9-S ribonucleoprotein. The mixture was dialysed and concentrated i n vacuo as described above, fixed with 6 % formaldehyde during 24 h at 4 ° and submitted to an equilibrium centrifugation in a preformed CsC1 gradient (p ~ 1.35-i.6; io mM triethanolamine (pH 7-4), IO mM KC1) as described under Fig. 3. The light and heavy ribosomal subparticles equilibrate at densities of 1.52 and 1.58, respectively. The ribonucleoprotein complex containing the 9-S RNA bands at a density of about 1.46. Such a buoyant density corresponds to a complex containing 45 % RNA and 55 % P roteinn. It is clear that the component which equilibrates at a density of 1.46 is not free 9-S RNA; indeed, if purified 32P-labelled 9-S RNA is Biochim. Biophys. Acta, 19o (1969) 228-231
230
PRELIMINARY NOTES
i
i I
i I
!
,
i
~2,50 1000
[
/
pi°,.4~
i
i
i
i
:
[
i 2.25.
'
F
8oo -700
0.1C
2,00"
I-7~o
! :
'
-600
!
[
', 6
I
1.75. " 5 0 0 ~
i
o
I
#= 1A6
0.5
i
['~ h/
d
E =
o
I .4oo
=1.5a c_
;~
0.0~
,.cc~
1.50-
"~-
-30O~ 8 •2 0 0 "~
!
i,,
D.5o
3
1.25"
._>
'~00 .~)_
O~-''~2"--
top
--
-'
O- 0
bottom
% top
! 10
20
fractions
30
a.
40 1 . 0 0 - 0
bottom
F i g . 3. CsC1 e q u i l i b r i u m s e d i m e n t a t i o n of t h e m e s s e n g e r r i b o n u c l e o p r o t e i n complex. R i b o s o m a l subparticles h a v e been a d d e d as i n t e r n a l markers. The c e n t r i f u g a t i o n w a s carried o u t at 4 ° for 18 h a t 36 o o o r e v . / m i n in a S W - 3 9 r o t o r (Spinco). The s e d i m e n t a t i o n p a t t e r n w a s a u t o m a t i c a l l y recorded at 260 n m , and f r a c t i o n s w e r e collected for r a d i o a c t i v i t y a n d refractive i n d e x m e a s u r e m e n t s , p is expressed in g / c m 3. F i g . 4. CsC1 e q u i l i b r i u m s e d i m e n t a t i o n of IOOffg of the m e s s e n g e r r i b o n u c l e o p r o t e i n complex. IO fig of free 3~P-labelled 9-S R N A h a v e been added to t h e fixed material. E q u i l i b r i u m centrifug a t i o n c o n d i t i o n s were t h e s a m e as for F i g . 3. P is expressed in g / c m 3.
added to the unlabelled fixed ribonucleoprotein and is submitted to a CsC1 centrifugation, all the radioactive material sediments at an apparent density of 1.66 (Fig. 4). The density of the messenger complex isolated from reticulocytes is very close to that found by other authors for the complexes from liver 1~ and from L-cellsn; the complex from thyroid was somewhat lighter (1.4o) 13. So far, the physiological significance of these ribonucleoprotein complexes remains uncertain. The purified messenger ribonucleoprotein complex from reticulocyte polyribosomes appears to be the best material available at present for further investigations in this field. We are grateful to Dr. R. P. Perry who communicated his results to us before publication and to Dr. P. Malpoix for her help in preparing the manuscript. G.H. and G.M. are fellows of the Fonds National de la Recherche Scientifique. This work was carried out under contract Euratom-U.L.B. oo7-61-1o BIAB.
Laboratory o/ Biological Chemistry, Faculty o/Sciences, University o/ Brussels, Brussels (Belgium)
A. BURNY G. HUEZ G. MARBAIX H. CHANTRENNE
I G. MARBAIX AND A. BURNY, Biochem. Biophys. Res. Commun., 16 (1964) 522. 2 A. BURNY AND G. MARBAIX, Biochim. Biophys. Acta, lO 3 (1965) 409 . 3 G. MARBAIX, A. BURNY, G. HUEZ .ANn H . CHANTRENN]~, Biochim. Biophys. Acta, 114 (1966) 404 • 4 H . CHANTRENNE,A. ]~URNY AND G. MARBAIX, Progr. Nucleic Acid Res. Mol. Biol., 7 (1967) 1735 F. LABRIE, Nature, 221 (1969) 1217.
Biochim. Biophys. Acla, 19o (1969) 228-231
PRELIMINARY NOTES
231
6 R. WILLIAMSON, G. LANYAN AND J. PAUL, Abstr. 6th Federation European Biochem. Soc. Meeting, Madrid, I969, p. 144. 7 M. J. EVANS AND J. B. LINGREL, Biochemistry, 8 (1969) 829. 8 D. G. LAYCOCK AND J. A. HUNT, Nature, 221 (1969) 1118. 9 G. HuEz, A. BURNY, G. MARBAIX AND a . LEBLEU, Biochim. Biophys. Acta, 145 (1967) 629. IO J. TEMMERMAN AND B. LEBLEU, Biochim. Biophys. Acta, 174 (1969) 544. i i R. P. PERRY AND n . E. KELLEY, J. Mol. Biol., 35 (1968) 37. 12 E. C. HENSHAW, J. Mol. Biol., 36 (1968) 4Ol. 13 G. CARTOUZOU, J.-C. ATTALI AND S. LlSSlTZK¥, European J. Bioehem., 4 (1968) 41. 14 R. P. PERRY AND D. E. KELLEY, Biophys. J . , 9 (1969) A-I32.
Received July 4th, 1969 Biochim. Biophys. Acta, 19o (1969) 228-231
BIOCHIMICAET BIOPHYSICAACTA
PRELIMINARY NOTES BBA 91259 On a messenger ribonucleoprotein complex from rabbit reticulocytes We have previously reported the isolation from rabbit reticulocytes of an RNA fraction which nmst be a mixture of two messenger RNA's coding for the two chains of haemoglobin 1-4. This fraction was identified as the messenger RNA thread of the polyribosomes in view of its extreme susceptibility to ribonuclease (EC 2.7.7.16) z. Its sedimentation constant of about 9 S indicated a molecular weight in the expected range of x5o ooo (ref. 2). When rabbits were injected with radioactive phosphate I5 h before sacrifice, the 9-S RNA fraction was more highly labelled than rRNA 1. Furthermore, this fraction specifically associated with native 4o-S subparticles from reticulocytes (unpublished results); it had a poor secondary structure ~, as could be expected for a messenger. Recent work further supports the idea that the 9-S RNA fraction we obtained essentially consists of a mixture of the messenger RNA's of globin chains 5-v. On the other hand, the RNA isolated by another fractionation procedure and which directs haemoglobin synthesis in a bacterial cell-free system 8 is most probably identical with our 9-S fraction. It had already been shown in our laboratory that the same 9-S RNA can be released from polyribosomes by adding EDTA or by passing the polyribosomal suspension through a column of CM-cellulose 9. After such treatments, the 9-S RNA is never recovered as a naked fiber; it is always associated with protein materiaP °. Similar complexes between a rapidly labelled RNA and a protein have also been detected in other cells: L-cells n, liver TM, thyroid la and bacteria ~. It seems likely that when Mg2+ is removed, the polyribosonzes fall apart, the subparticles of the ribosomes unfold and the messenger remains associated with certain proteins. In most tissues, the messenger population is quite heterogeneous, making the purification of the ribonucleoprotein very difficult. In reticulocytes, on the contrary, there are only two messenger RNA's which are about the same size. The messenger ribonucleoprotein complexes of reticulocytes should therefore be more amenable to useful study. In the present note, we wish to report on the rapid isolation and the characterisation by CsC1 equilibrium centrifugation of the messenger ribonucleoprotein complex from reticulocytes. Rabbit reticulocyte polyribosomes were obtained as previously described 9. The stroma-free hemolysate was centrifuged at IOO ooo × g for 3 h through layers of 3o % sucrose. As a rule, anaemic rabbits received IO mC of !ia2Plphosphate 15 h before sacrifice. Their polyribosomes, suspended in o.o3 M EDTA, were layered on top of a I5-3o % sucrose gradient and spun for 4o h at 24 ooo rev./min in the SW-25.I rotor of a Spinco ultracentrifuge (see legend to Fig. I). Fig. I shows a typical sucrose gradient separation of the different particles obtained from EDTA-treated polyribosomes. The ribonucleoprotein containing the 9-S RNA sediments in the region of the gradient and is shown by the hatched area. Biochim. Biophys. dcta, i9o (I969) 228-23I
229
PRELIMINARY NOTES
i
i
I 2.0
"
f
.
,
~
12000
i ,
Q2 .
.
.
.
1
.
.
.....
r
L.
"
--
-
-"1000
p'
',
'
I
,
' I
.~
@ ~
ii
/I
•
c
: tRNA
't il
i
75o T
E
.
'
1.0
8
1000
2
i .
]
,i
!
:
b
!
'-
:
[
:
=E
I''
o.~
g o
250
5
3 5
Oo
? -
1 0
"
20 fractions
30
40
fractlons
4o
6_
%
Fig. I. 4 m g of p o l y r i b o s o m e s were s u s p e n d e d in 0.6 ml of IO mM s o d i u m p k o s p h a t e b u f f e r (pH 7.o), i o mM KC1. D i s s o c i a t i o n of t h e p a l y r i b o s o m e s w a s p e r f o r m e d b y a d d i t i o n of o. 3 ml of o . i M E D T A (pH 7.o). The s u s p e n s i o n w a s l a y e r e d on t o p of 15-3o % sucrose g r a d i e n t s ( i o mM s o d i u m p h o s p h a t e b u f f e r (pH 7), IO mM KC1) a n d c e n t r i f u g e d a t 4 ° in a S'W-25.I r o t o r (Spinco) a t 24 o ooo r e v . / m i n for 4 ° 11. 0 - - - O , 3~p r a d i o a t i v i t y . Fig. 2. S e d i m e n t a t i o n in sucrose g r a d i e n t of t h e m a t e r i a l c o r r e s p o n d i n g to t h e h a t c h e d zone in Fig. i. Tile s u s p e n s i o n w a s l a y e r e d on t o p of a 15-3o % sucrose g r a d i e n t ( i o mM s o d i u m phosp h a t e b u f f e r (pH 7.o), i o mM KC1) a n d c e n t r i f u g e d a t 4 ° in a S'W-25.I r o t o r (Spinco) a t 24 ooo r e v . / m i n for 4 ° h.
The fractions corresponding to this region were pooled and concentrated b y vacuum dialysis against IOO vol. of io mM triethanolamine buffer (pH 7.4), IO mM KCl. The suspension was then again centrifuged in a 15-3o% sucrose gradient at 24 ooo rev./min for 4 ° h (see Fig. 2). Most of the highly radioactive nucleic acid was found in the ribonucleoprotein complex; the slower moving peak which is much less radioactive was shown to be free 5-S RNA 9. By means of the method described above, it is thus easy to obtain a clean preparation of the messenger ribonucleoprotein complex. In order to further characterise this complex, we added 3o #g of unlabelled subribosomal particles as a marker to ioo fig of 9-S ribonucleoprotein. The mixture was dialysed and concentrated i n vacuo as described above, fixed with 6 % formaldehyde during 24 h at 4 ° and submitted to an equilibrium centrifugation in a preformed CsC1 gradient (p ~ 1.35-i.6; io mM triethanolamine (pH 7-4), IO mM KC1) as described under Fig. 3. The light and heavy ribosomal subparticles equilibrate at densities of 1.52 and 1.58, respectively. The ribonucleoprotein complex containing the 9-S RNA bands at a density of about 1.46. Such a buoyant density corresponds to a complex containing 45 % RNA and 55 % P roteinn. It is clear that the component which equilibrates at a density of 1.46 is not free 9-S RNA; indeed, if purified 32P-labelled 9-S RNA is Biochim. Biophys. Acta, 19o (1969) 228-231
230
PRELIMINARY NOTES
i
i I
i I
!
,
i
~2,50 1000
[
/
pi°,.4~
i
i
i
i
:
[
i 2.25.
'
F
8oo -700
0.1C
2,00"
I-7~o
! :
'
-600
!
[
', 6
I
1.75. " 5 0 0 ~
i
o
I
#= 1A6
0.5
i
['~ h/
d
E =
o
I .4oo
=1.5a c_
;~
0.0~
,.cc~
1.50-
"~-
-30O~ 8 •2 0 0 "~
!
i,,
D.5o
3
1.25"
._>
'~00 .~)_
O~-''~2"--
top
--
-'
O- 0
bottom
% top
! 10
20
fractions
30
a.
40 1 . 0 0 - 0
bottom
F i g . 3. CsC1 e q u i l i b r i u m s e d i m e n t a t i o n of t h e m e s s e n g e r r i b o n u c l e o p r o t e i n complex. R i b o s o m a l subparticles h a v e been a d d e d as i n t e r n a l markers. The c e n t r i f u g a t i o n w a s carried o u t at 4 ° for 18 h a t 36 o o o r e v . / m i n in a S W - 3 9 r o t o r (Spinco). The s e d i m e n t a t i o n p a t t e r n w a s a u t o m a t i c a l l y recorded at 260 n m , and f r a c t i o n s w e r e collected for r a d i o a c t i v i t y a n d refractive i n d e x m e a s u r e m e n t s , p is expressed in g / c m 3. F i g . 4. CsC1 e q u i l i b r i u m s e d i m e n t a t i o n of IOOffg of the m e s s e n g e r r i b o n u c l e o p r o t e i n complex. IO fig of free 3~P-labelled 9-S R N A h a v e been added to t h e fixed material. E q u i l i b r i u m centrifug a t i o n c o n d i t i o n s were t h e s a m e as for F i g . 3. P is expressed in g / c m 3.
added to the unlabelled fixed ribonucleoprotein and is submitted to a CsC1 centrifugation, all the radioactive material sediments at an apparent density of 1.66 (Fig. 4). The density of the messenger complex isolated from reticulocytes is very close to that found by other authors for the complexes from liver 1~ and from L-cellsn; the complex from thyroid was somewhat lighter (1.4o) 13. So far, the physiological significance of these ribonucleoprotein complexes remains uncertain. The purified messenger ribonucleoprotein complex from reticulocyte polyribosomes appears to be the best material available at present for further investigations in this field. We are grateful to Dr. R. P. Perry who communicated his results to us before publication and to Dr. P. Malpoix for her help in preparing the manuscript. G.H. and G.M. are fellows of the Fonds National de la Recherche Scientifique. This work was carried out under contract Euratom-U.L.B. oo7-61-1o BIAB.
Laboratory o/ Biological Chemistry, Faculty o/Sciences, University o/ Brussels, Brussels (Belgium)
A. BURNY G. HUEZ G. MARBAIX H. CHANTRENNE
I G. MARBAIX AND A. BURNY, Biochem. Biophys. Res. Commun., 16 (1964) 522. 2 A. BURNY AND G. MARBAIX, Biochim. Biophys. Acta, lO 3 (1965) 409 . 3 G. MARBAIX, A. BURNY, G. HUEZ .ANn H . CHANTRENN]~, Biochim. Biophys. Acta, 114 (1966) 404 • 4 H . CHANTRENNE,A. ]~URNY AND G. MARBAIX, Progr. Nucleic Acid Res. Mol. Biol., 7 (1967) 1735 F. LABRIE, Nature, 221 (1969) 1217.
Biochim. Biophys. Acla, 19o (1969) 228-231
PRELIMINARY NOTES
231
6 R. WILLIAMSON, G. LANYAN AND J. PAUL, Abstr. 6th Federation European Biochem. Soc. Meeting, Madrid, I969, p. 144. 7 M. J. EVANS AND J. B. LINGREL, Biochemistry, 8 (1969) 829. 8 D. G. LAYCOCK AND J. A. HUNT, Nature, 221 (1969) 1118. 9 G. HuEz, A. BURNY, G. MARBAIX AND a . LEBLEU, Biochim. Biophys. Acta, 145 (1967) 629. IO J. TEMMERMAN AND B. LEBLEU, Biochim. Biophys. Acta, 174 (1969) 544. i i R. P. PERRY AND n . E. KELLEY, J. Mol. Biol., 35 (1968) 37. 12 E. C. HENSHAW, J. Mol. Biol., 36 (1968) 4Ol. 13 G. CARTOUZOU, J.-C. ATTALI AND S. LlSSlTZK¥, European J. Bioehem., 4 (1968) 41. 14 R. P. PERRY AND D. E. KELLEY, Biophys. J . , 9 (1969) A-I32.
Received July 4th, 1969 Biochim. Biophys. Acta, 19o (1969) 228-231