Volume 2 number 10 October 1975
Nucleic Acids Research
Isolation and in vitro translation of human placental lactogen messenger RNA from human term placenta
C.Hubert and L.Cedard
Laboratoire de Chimie Hormonale, Maternite 123, Bld de Port-Royal, Paris 75014, France
Received 1 September 1975 SUMMARY A messenger activity for HPL was identified in normal human term placentas. The mRNA was translated in rabbit reticulocyte cell-free system. The HPL synthesized was quantified by a specific immunoprecipitation and further identified by electrophoresis on sodium dodecyl sulfate polyacrylamide gel. The HPL synthesized in the reticulocyte lysate exhibited a molecular weight between 20, 000 and 22, 000 daltons similar to the active hormone. The messenger RNA activity for HPL corresponded to a sedimentation coefficient of 11-12 S. Furthermore the messenger activity for HPL was preferentially associated with membrane bound polyribosomes than with free polyribosomes.
INTRODUCTION Normal human pregnancy is characterized by multiple variations in the polypeptide hormone levels in the maternal blood I. Among these hormones, the Human Placental Lactogen (HPL) or Human Chorionic Somatomammotropin (HCS) proved to be important for a good outcome of the gestation 2 3. Though HPL secretion has been studied in vivo and in vitro 4 5, relatively little is known about the regulation of HPL synthesis and further studies are necessary to determine the factors involved in the control of HPL synthesis. In a previous paper, we pointed out that, in a cell-free system derived from human placenta, the "pH5 enzyme" fraction was responsible for the deficiency of amino-acid incorporation into proteins, whereas the polysomal preparations were quite active 6. These results led us to investigate further the messenger RNA activities in this human tissue. The isolation and the titration of HPL mRNA may be helpful to determine a possible regulation in the secretion level of this hormone during pregnancy. While this work was in progress BOIME et al 7 detected HPL synthesis in cell-free system from a total RNA preparation of human placenta. In this report, evidence is presented for the existence of HPL messenger RNA in polyribosomal preparation of normal term placentas, and the size of HPL mRNA is discussed. Furthermore the mRNA activities coding for HPL associated with free and membrane bound polyribosomes are compared.
1903
Nucleic Acids Research M&TERIALS AND METHODS Preparation of total, free, and membrane bound polyribosomes Fresh placentas (300-600 g) from full-term parturition delivered by vaginal or ceasarian outcome were processed within 30 minutes of delivery, as previously described 6, with a modification to inhibit RNase activities consisting in the addition of 1 mg/ml heparin to the homogenization buffer (50 mM Tris (pH 7.7) ; 50 mM KC1 ; 10 mM MgC12 ; 100 mM NH4Cl ; 0.5 mM EDTA). All chemicals used were reagent grade. Free and membrane bound polyribosomes were isolated on a discontinous sucrose gradient - 0.5 N - 1.35 M - 2.0 M sucrose - according to BLOBEL and POTTER 8. In some experiments, the free polyribosomes were first separated from most of the membrane bound polyribosomes : the microsomes were sedimented by centrifuging the post mitochondrial supernatant for 20 minutes at 40,000 g. The resulting supernatant was then layered over the 0.5 M-2M sucrose (Mann Research Laboratories) cushion. This method was expected to reduce a possible contamination of the free polyribosomes by the membrane bound polyribosomes.
Extraction of RNA RNA was obtained by extracting proteins with Tris sodium dodecyl sulfate (SDS) phenol solution (pH 9.0) as described by LEE et al 9. In the last extraction step, the mixture contained chloroform in addition to phenol. After the second precipitation by ethanol, the RNA pellet was washed with a solution of 2 M LiCI. 10 mM EDTA (pH 7.0) 10. Just before translation, the RNA pellet was washed several times with 80 Z ethanol and dissolved in autoclaved water.
Sucrose gradient analysis of RNA RNA were dissolved in HNES buffer (20 mM HEPES (pH 7.1), 150 mM NaCl, 0.1 m EDTA, 0.2 % SDS), then layered over 17 ml linear 15 to 35 x sucrose gradients in the same buffer. Centrifugation was performed for 17 hours at 22°C at 130,000 g in a SW 27 Beckman Rotor. The RNA fractions were pooled, adjusted to pH 5 and precipitated by ethanol at -20°C. For the localization of HPL mRNA, the gradients were centrifuged in the same condition but for 24 hours. Each RNA fraction (0.5 ml) after addition of E. Coli tRNA (50 rg) as carrier and adjustment to pH 5.0, was precipitated with 2 volumes of ethanol. Assay of mRNA activity
The HPL mRNA activity of RNA fractions was analyzed in a reticulo-
1904
Nucleic Acids Research cyte lysate. Preparation of the lysate was as described by LINGREL et al 11. The assay mixture contained in a total volume of 250 4l : 100 jl of lysate, 80 mM KC1, 2 mM (CH3 COO)2 Mg, 1 mM ATP, 0.2 mM GTP, 15 mM creatine phosphate, creatine phosphokinase (15 units per ml), a mixture of amino-acids 100 wM minus isoleucine, 40 lCi/mi 3H isoleucine (spec. act. 30 Ci/mM), (C.E.A. France),hemin 20-30 PM, 10-20 ig of mRNA containing fractions. Incubations were carried out for 90 minutes at 26°C, at the end of the incubation an aliquot was taken out to estimate the total radioactive proteins by precipitation with cold 10 % T.C.A.
Specific immunoprecipitation of the synthesized products After the translation, 200 4l of the reaction mixture were incubated at 37°C for 1 hour with 15 pg of HPL (Nutritional Biochemicals Corporation) in 10 mM phosphate buffer (pH 7.1), supplemented with 150 mM NaCl, 10 mM isoleucine and 0.5 % triton X 100, and addition of 100 p4 of specific anti HPL serum. The immunoprecipitation were performed according to RHOADS 12 by means of a centrifugation over a 0.6 M sucrose cushion.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis The immunoprecipitate was dissociated by SDS in presence of 0-mercapto-ethanol and submitted to electrophoresis on 12 % polyacrylamide gel according to WEBER and OSBORN 13 in phosphate-SDS buffer. After the run, gels were frozen, sliced and counted in a triton X 100 containing scintil-
lation fluid. RESULTS
Characterization and estimation of the HPL synthesized in the cellfree system The HPL synthesis directed by placental messenger RNA in the reticulocyte lysate was estimated quantitatively by immunoprecipitation. The specificity of rabbit anti-HPL serum was checked by the OUTCHERLONY immunodiffusion method. In this test no cross reaction with two possible contaminating proteins (human growth hormone or albumin) was detected. The non-specific immunoprecipitate was estimated with the complete translation system in the
absence of placental RNA resulting in a blank value which was subtracted from all samples counted. Synthesis of HPL was obtained for each RNA preparation (table I). 1905
Nucleic Acids Research cpm
3H
HPL /ig RNA
EXPERIMENT NUMBER TOTAL POLYRIBOSOMAL
RNIA
I II
III IV
V
300 160 455 170 230
5S - 18S FRACTION
695 270 1 280 260 550
HPL synthesis in rabbit reticulocyte cell-free system directed by various placental mRNA preparations from total and 5S-18S fraction polyribosomes. Incubations were performed as described in materials and methods during 90 mn at 260C. HPL synthesis was estimated by a specific immunoprecipitation and blank value has been subtracted. The observed variations can be attributed to the level of HPL synthesis by each placenta or to the variable delay between delivery and preparation of polyribosomes. In order to increase the messenger RNA activity, the 5S-18S RNA fraction was isolated in a linear sucrose gradient. This fraction was expected to contain the messenger RNA for a protein having a molecular weight of 22,000 daltons. Under these conditions the HPL synthesis was twice as high per pgRNA as with total polyribosome RNA (table I), and represented 3 % of total radioactive proteins synthesized in a reticulocyte system. The HPL synthesis directed by the 5S-18S RNA fraction was increased in a linear fashion up to 88 ig/ml RNA, over this concentration a plateau in HPL synthesis was observed. To investigate the nature of the radioactivity immunoprecipitable by the HPL anti-serum, the antigen antibody complex was dissociated an submitted to SDS acrylamide gel electrophoresis. Figure 1 indicates that the immuno-precipitated radioactivity migrated as one major peak concording with HPL marker stained with coomassie brilliant blue and corresponding to an apparent molecular weight of about 22,000 daltons by comparison with standard protein markers (ovalbumin, chymotrypsinogen A, HPL, myoglobin).
1906
Nucleic Acids Research HPL
a
a C
E
origin
tracking dye
0. 10
20
30
40
o0
60
70
fraction number
Fig. 1 - Distribution of radioactivity after electrophoresis in SDS 12 % polyacrylamide gel of immuno-precipitated proteins synthesized in vitro, in presence of placental polyribosomal mRNA. The direction of migration was from left to right. The position of HPL as marker is noted.
Distribution of mRNA activity for HPL in sucrose gradient In order to estimate the size of the mRNA coding for HPL, the distribution of HPL mRNA activity on sucrose gradient was examined. The gradients were run a longer time to improve the separation of the mRNA from 18S ribosomal RNA. RNA from each fraction (0.5 ml) was precipitated, redissolved in 100 p4 of autoclaved water. An aliquot of 40 p4 was tested for its HPL synthesis capacity. Figure 2 indicates that the messenger activity for HPL, giving rise to a single peak of HPL synthesis, had a sedimentation coefficient of 11-12S, determined by comparison with 5S and 18S RNA markers. The absence of a characteristic U.V. peak is not surprising, because of the small percentage of this hormone among placental proteins.
Synthesis of HPL by free and membrane bound polyribosomes The human placenta was demonstrated to contain free and membrane bound polyribosomes 14. The results of table II indicate that RNA isolated from membrane bound polyribosomes could direct the synthesis of HPL in a reticulocyte system, whereas those isolated from free polyribosomes exhibited only a poor activity. The translation of the 5S-18S RNA fraction isolated 1907
Nucleic Acids Research from both types of polyribosomes are even more discriminated.
I
E I1 o~~~~~~~~~~~~~
I o~~~~~~~~~
a
0i
CL
I
fractions
Fig.
2 -
SEDIMENTATION PROFILE OF HPL mRNA. Placental polyribosomal RNA in H.N.E.S. buffer was loaded on a linear 15-35 % sucrose gradient and centrifuged at 130,000 g at 22°C for 24 hours. Aliquots (40 pl) from each fraction were assayed for HPL mRNA activity.
RHA
MEMBRANE
FREE
BOUND
cp
TOTAL POLYRIBOSOKAL FRACTION
5S-18S FRACTION
3H
RATIO M.B. F
HPL /
Nucleic Acids Research
Isolation and in vitro translation of human placental lactogen messenger RNA from human term placenta
C.Hubert and L.Cedard
Laboratoire de Chimie Hormonale, Maternite 123, Bld de Port-Royal, Paris 75014, France
Received 1 September 1975 SUMMARY A messenger activity for HPL was identified in normal human term placentas. The mRNA was translated in rabbit reticulocyte cell-free system. The HPL synthesized was quantified by a specific immunoprecipitation and further identified by electrophoresis on sodium dodecyl sulfate polyacrylamide gel. The HPL synthesized in the reticulocyte lysate exhibited a molecular weight between 20, 000 and 22, 000 daltons similar to the active hormone. The messenger RNA activity for HPL corresponded to a sedimentation coefficient of 11-12 S. Furthermore the messenger activity for HPL was preferentially associated with membrane bound polyribosomes than with free polyribosomes.
INTRODUCTION Normal human pregnancy is characterized by multiple variations in the polypeptide hormone levels in the maternal blood I. Among these hormones, the Human Placental Lactogen (HPL) or Human Chorionic Somatomammotropin (HCS) proved to be important for a good outcome of the gestation 2 3. Though HPL secretion has been studied in vivo and in vitro 4 5, relatively little is known about the regulation of HPL synthesis and further studies are necessary to determine the factors involved in the control of HPL synthesis. In a previous paper, we pointed out that, in a cell-free system derived from human placenta, the "pH5 enzyme" fraction was responsible for the deficiency of amino-acid incorporation into proteins, whereas the polysomal preparations were quite active 6. These results led us to investigate further the messenger RNA activities in this human tissue. The isolation and the titration of HPL mRNA may be helpful to determine a possible regulation in the secretion level of this hormone during pregnancy. While this work was in progress BOIME et al 7 detected HPL synthesis in cell-free system from a total RNA preparation of human placenta. In this report, evidence is presented for the existence of HPL messenger RNA in polyribosomal preparation of normal term placentas, and the size of HPL mRNA is discussed. Furthermore the mRNA activities coding for HPL associated with free and membrane bound polyribosomes are compared.
1903
Nucleic Acids Research M&TERIALS AND METHODS Preparation of total, free, and membrane bound polyribosomes Fresh placentas (300-600 g) from full-term parturition delivered by vaginal or ceasarian outcome were processed within 30 minutes of delivery, as previously described 6, with a modification to inhibit RNase activities consisting in the addition of 1 mg/ml heparin to the homogenization buffer (50 mM Tris (pH 7.7) ; 50 mM KC1 ; 10 mM MgC12 ; 100 mM NH4Cl ; 0.5 mM EDTA). All chemicals used were reagent grade. Free and membrane bound polyribosomes were isolated on a discontinous sucrose gradient - 0.5 N - 1.35 M - 2.0 M sucrose - according to BLOBEL and POTTER 8. In some experiments, the free polyribosomes were first separated from most of the membrane bound polyribosomes : the microsomes were sedimented by centrifuging the post mitochondrial supernatant for 20 minutes at 40,000 g. The resulting supernatant was then layered over the 0.5 M-2M sucrose (Mann Research Laboratories) cushion. This method was expected to reduce a possible contamination of the free polyribosomes by the membrane bound polyribosomes.
Extraction of RNA RNA was obtained by extracting proteins with Tris sodium dodecyl sulfate (SDS) phenol solution (pH 9.0) as described by LEE et al 9. In the last extraction step, the mixture contained chloroform in addition to phenol. After the second precipitation by ethanol, the RNA pellet was washed with a solution of 2 M LiCI. 10 mM EDTA (pH 7.0) 10. Just before translation, the RNA pellet was washed several times with 80 Z ethanol and dissolved in autoclaved water.
Sucrose gradient analysis of RNA RNA were dissolved in HNES buffer (20 mM HEPES (pH 7.1), 150 mM NaCl, 0.1 m EDTA, 0.2 % SDS), then layered over 17 ml linear 15 to 35 x sucrose gradients in the same buffer. Centrifugation was performed for 17 hours at 22°C at 130,000 g in a SW 27 Beckman Rotor. The RNA fractions were pooled, adjusted to pH 5 and precipitated by ethanol at -20°C. For the localization of HPL mRNA, the gradients were centrifuged in the same condition but for 24 hours. Each RNA fraction (0.5 ml) after addition of E. Coli tRNA (50 rg) as carrier and adjustment to pH 5.0, was precipitated with 2 volumes of ethanol. Assay of mRNA activity
The HPL mRNA activity of RNA fractions was analyzed in a reticulo-
1904
Nucleic Acids Research cyte lysate. Preparation of the lysate was as described by LINGREL et al 11. The assay mixture contained in a total volume of 250 4l : 100 jl of lysate, 80 mM KC1, 2 mM (CH3 COO)2 Mg, 1 mM ATP, 0.2 mM GTP, 15 mM creatine phosphate, creatine phosphokinase (15 units per ml), a mixture of amino-acids 100 wM minus isoleucine, 40 lCi/mi 3H isoleucine (spec. act. 30 Ci/mM), (C.E.A. France),hemin 20-30 PM, 10-20 ig of mRNA containing fractions. Incubations were carried out for 90 minutes at 26°C, at the end of the incubation an aliquot was taken out to estimate the total radioactive proteins by precipitation with cold 10 % T.C.A.
Specific immunoprecipitation of the synthesized products After the translation, 200 4l of the reaction mixture were incubated at 37°C for 1 hour with 15 pg of HPL (Nutritional Biochemicals Corporation) in 10 mM phosphate buffer (pH 7.1), supplemented with 150 mM NaCl, 10 mM isoleucine and 0.5 % triton X 100, and addition of 100 p4 of specific anti HPL serum. The immunoprecipitation were performed according to RHOADS 12 by means of a centrifugation over a 0.6 M sucrose cushion.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis The immunoprecipitate was dissociated by SDS in presence of 0-mercapto-ethanol and submitted to electrophoresis on 12 % polyacrylamide gel according to WEBER and OSBORN 13 in phosphate-SDS buffer. After the run, gels were frozen, sliced and counted in a triton X 100 containing scintil-
lation fluid. RESULTS
Characterization and estimation of the HPL synthesized in the cellfree system The HPL synthesis directed by placental messenger RNA in the reticulocyte lysate was estimated quantitatively by immunoprecipitation. The specificity of rabbit anti-HPL serum was checked by the OUTCHERLONY immunodiffusion method. In this test no cross reaction with two possible contaminating proteins (human growth hormone or albumin) was detected. The non-specific immunoprecipitate was estimated with the complete translation system in the
absence of placental RNA resulting in a blank value which was subtracted from all samples counted. Synthesis of HPL was obtained for each RNA preparation (table I). 1905
Nucleic Acids Research cpm
3H
HPL /ig RNA
EXPERIMENT NUMBER TOTAL POLYRIBOSOMAL
RNIA
I II
III IV
V
300 160 455 170 230
5S - 18S FRACTION
695 270 1 280 260 550
HPL synthesis in rabbit reticulocyte cell-free system directed by various placental mRNA preparations from total and 5S-18S fraction polyribosomes. Incubations were performed as described in materials and methods during 90 mn at 260C. HPL synthesis was estimated by a specific immunoprecipitation and blank value has been subtracted. The observed variations can be attributed to the level of HPL synthesis by each placenta or to the variable delay between delivery and preparation of polyribosomes. In order to increase the messenger RNA activity, the 5S-18S RNA fraction was isolated in a linear sucrose gradient. This fraction was expected to contain the messenger RNA for a protein having a molecular weight of 22,000 daltons. Under these conditions the HPL synthesis was twice as high per pgRNA as with total polyribosome RNA (table I), and represented 3 % of total radioactive proteins synthesized in a reticulocyte system. The HPL synthesis directed by the 5S-18S RNA fraction was increased in a linear fashion up to 88 ig/ml RNA, over this concentration a plateau in HPL synthesis was observed. To investigate the nature of the radioactivity immunoprecipitable by the HPL anti-serum, the antigen antibody complex was dissociated an submitted to SDS acrylamide gel electrophoresis. Figure 1 indicates that the immuno-precipitated radioactivity migrated as one major peak concording with HPL marker stained with coomassie brilliant blue and corresponding to an apparent molecular weight of about 22,000 daltons by comparison with standard protein markers (ovalbumin, chymotrypsinogen A, HPL, myoglobin).
1906
Nucleic Acids Research HPL
a
a C
E
origin
tracking dye
0. 10
20
30
40
o0
60
70
fraction number
Fig. 1 - Distribution of radioactivity after electrophoresis in SDS 12 % polyacrylamide gel of immuno-precipitated proteins synthesized in vitro, in presence of placental polyribosomal mRNA. The direction of migration was from left to right. The position of HPL as marker is noted.
Distribution of mRNA activity for HPL in sucrose gradient In order to estimate the size of the mRNA coding for HPL, the distribution of HPL mRNA activity on sucrose gradient was examined. The gradients were run a longer time to improve the separation of the mRNA from 18S ribosomal RNA. RNA from each fraction (0.5 ml) was precipitated, redissolved in 100 p4 of autoclaved water. An aliquot of 40 p4 was tested for its HPL synthesis capacity. Figure 2 indicates that the messenger activity for HPL, giving rise to a single peak of HPL synthesis, had a sedimentation coefficient of 11-12S, determined by comparison with 5S and 18S RNA markers. The absence of a characteristic U.V. peak is not surprising, because of the small percentage of this hormone among placental proteins.
Synthesis of HPL by free and membrane bound polyribosomes The human placenta was demonstrated to contain free and membrane bound polyribosomes 14. The results of table II indicate that RNA isolated from membrane bound polyribosomes could direct the synthesis of HPL in a reticulocyte system, whereas those isolated from free polyribosomes exhibited only a poor activity. The translation of the 5S-18S RNA fraction isolated 1907
Nucleic Acids Research from both types of polyribosomes are even more discriminated.
I
E I1 o~~~~~~~~~~~~~
I o~~~~~~~~~
a
0i
CL
I
fractions
Fig.
2 -
SEDIMENTATION PROFILE OF HPL mRNA. Placental polyribosomal RNA in H.N.E.S. buffer was loaded on a linear 15-35 % sucrose gradient and centrifuged at 130,000 g at 22°C for 24 hours. Aliquots (40 pl) from each fraction were assayed for HPL mRNA activity.
RHA
MEMBRANE
FREE
BOUND
cp
TOTAL POLYRIBOSOKAL FRACTION
5S-18S FRACTION
3H
RATIO M.B. F
HPL /
