Molec. Biol. Rep. Vol. 5, 1-2: 59-64, 1979
FREE CYTOPLASMIC MESSENGER RIBONUCLEOPROTEIN COMPLEXES FROM RABBIT RETICULOCYTES
Hans M.G. PRINCEN, Chris A.G. van EEKELEN, Fred A.M. ASSELBERGS & Walther J. van VENROOLI*
Department of Biochemistry, University of Ni/megen, Geert Grooteplein Noord 21, Ni/megen, The Netherlands
Abstract
Free cytoplasmic globin mRNA containing mRNPparticles were isolated from rabbit reticulocytes by zonal sucrose gradient centrifugation and their properties were compared with mRNP particles isolated in the same way from EDTA-disso~iated reticulocyte polyribosomes. The average poly(A)length of 9S mRNA from free cytoplasmic mRNP was 17-20 nucleotides being about two times shorter than the average poly(A)-length of polysomal 9S mRNA. The protein composition of the free cytoplasmic mRNP particles disclosed the absence of the 76,000 dalton protein which is associated with the 3'poly(A)-segment of polysomal globin mRNA. It was concluded that free cytoplasmic mRNP-particles from rabbit reticulocytes can be classified as "old" mRNP in a post-translational phase. Free cytoplasmic mRNPs were translated in heterologous cell-free systems as well as in Xenopus laevis oocytes. Addition of hemin stimulated the synthesis of a-globin in all systems, while the presence of the cap analogue mTG(5')p inhibited translation of free cytoplasmic mRNA completely. The latter finding suggested that free cytoplasmic mRNA has a 5' terminal "cap". Shortening of the poly(A)-segment with concomitant loss of the 76,000 dalton protein may lead to less efficient translation of free cytoplasmic mRNP.
Introduction
Messenger RNA, released from polyribosomes by the action of EDTA or puromycin-high salt, is associated with a characteristic set of proteins. One of these proteins, with a molecular weight of about 76,000, has been found to be specifically associated with the 3'poly(A)-tail of mRNA in a wide variety of cells (1-3). Free cytoplasmic mRNA, not bound to ribosomes, has also been shown to be present in the form
of an mRNP** complex (4-11). However, the proteins bound specifically to the non-polysomal mRNA are not characterized very well mainly because purification of these particles is still hard to achieve (5). Furthermore, it is not generally realized that there are apparently several distinct functional forms of free mRNP particles which might well differ in the nature and function of thek proteins. For example, a class of mRNP particles "in transit", moving from the nucleus to the polyribosomes should be present, although their actual existence has not been proven yet. A second class might be the free mRNP particles which are present in the cytoplasm in an untranslatable, masked form. These "stored" mRNP particles have been shown to be present most abundantly in less active cells and in cells awaiting activation or fertilization (5-9). A third class of free mRNP particles has been shown to be present in a translatable, unmasked form. They are probably in equilibrium with the polysome bound mRNP (10, 11) and mostly are generated by less efficient initiation of the mRNA present in these particles. The impaired rate of initiation may be caused by an altered physiological condition of the cell (e.g. fasting) or by changes in the mRNA or mRNP complex itself. We have studied the free, globin mRNA containing mRNP particles in rabbit reticulocytes and compared their properties with the polysomal mRNP. Our results indicate that in the reticulocyte the mRNA of the free mRNP (predominantly a-globin mRNA (12-16)) contains a relatively short poly(A)-sequence and has a 5'terminal "cap". Furthermore, these mRNP lack the 76,000 dalton protein which binds to the poly(A) of polyribosomal mRNA.
*To whom correspondence should be sent. **Abbreviations: mRNP: Messenger r~onucleoprotein complex mTG(5')p: 7-methyl guanosine 5'monophosphate.
59
Materials and methods Anemia was induced in 2.5 kg rabbits by five daily injections of 1 ml 2.5% phenylhydrazine. Blood was collected at day 8 and polyribosomal and free cytoplasmic mRNP complexes were isolated and purified as described elsewhere (4). mRNA from polyriboseines or from free cytoplasmicc mRNP particles was isolated by thermal elution from poly(U)-Sepharose (12). The eluted mRNA was further purified by sucrose gradient centrifugation. The 9S globin mRNA was precipitated and stored at -20~ under ethanol. The length of the poly(A) segment of polysomal and free cytoplasmic globin mRNA was determined as follows: Known amounts of 9S polysomal and 9S free cytoplasmic .mRNA, purified by affinity chromatography and sucrose gradient centrifugation were diluted to 500 #1 giving a solution with the following final concentrations: 300 mM NaC1, 30 mM Na-citrate, 1 mM EDTA and 2 tzg x m1-1 polyvinyl sulphate. Then 1 /al of [aH]poly 0d) (0.1 /~g, 104 cpm) was added and hybrids v/ere allowed to form for 1 h at 41~ After cooling to 0~ unhybridized poly(U) was digested with ribonuclease A (25 #g x m1-1) for 20 min. Ribonuclease resistant ds-RNA was precipitated after addition of 12 /ag carrier yeast RNA with 7.5% TCA, collected on millipore filters and counted. A standard hybridization curve was prepared using poly(A)-solutions ranging from 0.001 ttg to 1/ag x m1-1 . Calculations were performed assuming that 1 mg POly(A) had an A26o of 30.1 (8) and RNA of 25, and that the length of globin mRNA without poly(A) corresponded to 589 nucleotides (I 7).
Results
a. Poly(A ) content of purified polysomal and free cytoplasmic mRNA Messenger RNA was purified from polysomal or free cytoplasmic mRNP preparations as described in the Methods section. Generally, between 12 and 20% of the total mRNA was found in the free mRNP fraction. The poly(U)-Sepharose elution profiles (not shown) revealed that 55% of the free cytoplasmic mRNA eluted at 25~ and the remaining 45% at 50~ In contrast only a small part of the polysomal globin mRNA (20%) eluted at 25~ while the remaining 80% eluted at the higher temperature. As judged by the sucrose gradient profiles, 60
neither polysomal nor free cytoplasmic globin mRNA could be detected in the run-through eluate of the column indicating that most if not all of the 9S mRNA had bound to the column. A third wash (at 55~ with 90% formamide) did not elute additional globin mRNA suggesting that all bound mRNA had been released after the 50~ wash. The thermal elution profiles from the poly(U)-Sepharose column were in good agreement with the results of Marbaix et al: (12) and suggested that the free cytoplasmic mRNA contained globin mRNA with relatively short poly(A)-segments as compared with the polyribosomal mRNA eluting at a higher temperature. This assumption was verified by direct measurement of the length of the poly(A)-segments. When the mRNA was isolated by poly(U)-Sepharose chromatography followed by sucrose gradient centrifugation, the free cytoplasmic 9S mRNA was found to contain poly(A)-tracts with an average length of 20-25 nucleotides. The average length of polysomal poly(A)-sequences was found to be 35-45 nucleotides. When 9S globin mRNA was isolated from free cytoplasmic mRNP by SDS treatment and repeated sucrose gradient centrifugation (thus omitting the poly(U)-Sepharose step) poly(A)-tracts with an average length of 17-20 nucleotides were found. All these experiments point to the conclusion that the free cytoplasmic globin mRNA has poly(A)tracts which, on the average, are considerably shorter than the poly(A)-tracts of globin mRNA present in polyribosomes.
b. Protein composition of free cytoplasmic mRNPcomplexes The protein composition of the 15-20S free mRNP (4) was examined by polyacrylamide gel electrophoresis in sodium dodecylsulphate (figure 1, slots 3-4) and compared with the protein composition of polyribosomal mRNP (figure 1, slot 2). As has been shown before by many investigators the polysomal globin mRNP contains a 76,000 dalton protein which is associated specifically with the 3'poly(A)sequence of the mRNA. In contrast, free mRNPparticles do not contain this protein (figure 1, slots 3-4) although, as we have shown above, a 3'poly(A)sequence is present in the globin mRNA of free mRNP. Another ol~vious feature of the protein composition of free mRNP are the proteins in the molecular weight range between 24,000 and 34,000 (slot 3). These proteins also appear to be present in the polysomal mRNP (compare slot 2). They must
4S
7S
11S
13S
18S
1
1
~
;
1
0.3.
? I -6O0 o'
E c
E
o 9
cO
40(3
.Q
O "O
FREE CYTOPLASMIC MESSENGER RIBONUCLEOPROTEIN COMPLEXES FROM RABBIT RETICULOCYTES
Hans M.G. PRINCEN, Chris A.G. van EEKELEN, Fred A.M. ASSELBERGS & Walther J. van VENROOLI*
Department of Biochemistry, University of Ni/megen, Geert Grooteplein Noord 21, Ni/megen, The Netherlands
Abstract
Free cytoplasmic globin mRNA containing mRNPparticles were isolated from rabbit reticulocytes by zonal sucrose gradient centrifugation and their properties were compared with mRNP particles isolated in the same way from EDTA-disso~iated reticulocyte polyribosomes. The average poly(A)length of 9S mRNA from free cytoplasmic mRNP was 17-20 nucleotides being about two times shorter than the average poly(A)-length of polysomal 9S mRNA. The protein composition of the free cytoplasmic mRNP particles disclosed the absence of the 76,000 dalton protein which is associated with the 3'poly(A)-segment of polysomal globin mRNA. It was concluded that free cytoplasmic mRNP-particles from rabbit reticulocytes can be classified as "old" mRNP in a post-translational phase. Free cytoplasmic mRNPs were translated in heterologous cell-free systems as well as in Xenopus laevis oocytes. Addition of hemin stimulated the synthesis of a-globin in all systems, while the presence of the cap analogue mTG(5')p inhibited translation of free cytoplasmic mRNA completely. The latter finding suggested that free cytoplasmic mRNA has a 5' terminal "cap". Shortening of the poly(A)-segment with concomitant loss of the 76,000 dalton protein may lead to less efficient translation of free cytoplasmic mRNP.
Introduction
Messenger RNA, released from polyribosomes by the action of EDTA or puromycin-high salt, is associated with a characteristic set of proteins. One of these proteins, with a molecular weight of about 76,000, has been found to be specifically associated with the 3'poly(A)-tail of mRNA in a wide variety of cells (1-3). Free cytoplasmic mRNA, not bound to ribosomes, has also been shown to be present in the form
of an mRNP** complex (4-11). However, the proteins bound specifically to the non-polysomal mRNA are not characterized very well mainly because purification of these particles is still hard to achieve (5). Furthermore, it is not generally realized that there are apparently several distinct functional forms of free mRNP particles which might well differ in the nature and function of thek proteins. For example, a class of mRNP particles "in transit", moving from the nucleus to the polyribosomes should be present, although their actual existence has not been proven yet. A second class might be the free mRNP particles which are present in the cytoplasm in an untranslatable, masked form. These "stored" mRNP particles have been shown to be present most abundantly in less active cells and in cells awaiting activation or fertilization (5-9). A third class of free mRNP particles has been shown to be present in a translatable, unmasked form. They are probably in equilibrium with the polysome bound mRNP (10, 11) and mostly are generated by less efficient initiation of the mRNA present in these particles. The impaired rate of initiation may be caused by an altered physiological condition of the cell (e.g. fasting) or by changes in the mRNA or mRNP complex itself. We have studied the free, globin mRNA containing mRNP particles in rabbit reticulocytes and compared their properties with the polysomal mRNP. Our results indicate that in the reticulocyte the mRNA of the free mRNP (predominantly a-globin mRNA (12-16)) contains a relatively short poly(A)-sequence and has a 5'terminal "cap". Furthermore, these mRNP lack the 76,000 dalton protein which binds to the poly(A) of polyribosomal mRNA.
*To whom correspondence should be sent. **Abbreviations: mRNP: Messenger r~onucleoprotein complex mTG(5')p: 7-methyl guanosine 5'monophosphate.
59
Materials and methods Anemia was induced in 2.5 kg rabbits by five daily injections of 1 ml 2.5% phenylhydrazine. Blood was collected at day 8 and polyribosomal and free cytoplasmic mRNP complexes were isolated and purified as described elsewhere (4). mRNA from polyriboseines or from free cytoplasmicc mRNP particles was isolated by thermal elution from poly(U)-Sepharose (12). The eluted mRNA was further purified by sucrose gradient centrifugation. The 9S globin mRNA was precipitated and stored at -20~ under ethanol. The length of the poly(A) segment of polysomal and free cytoplasmic globin mRNA was determined as follows: Known amounts of 9S polysomal and 9S free cytoplasmic .mRNA, purified by affinity chromatography and sucrose gradient centrifugation were diluted to 500 #1 giving a solution with the following final concentrations: 300 mM NaC1, 30 mM Na-citrate, 1 mM EDTA and 2 tzg x m1-1 polyvinyl sulphate. Then 1 /al of [aH]poly 0d) (0.1 /~g, 104 cpm) was added and hybrids v/ere allowed to form for 1 h at 41~ After cooling to 0~ unhybridized poly(U) was digested with ribonuclease A (25 #g x m1-1) for 20 min. Ribonuclease resistant ds-RNA was precipitated after addition of 12 /ag carrier yeast RNA with 7.5% TCA, collected on millipore filters and counted. A standard hybridization curve was prepared using poly(A)-solutions ranging from 0.001 ttg to 1/ag x m1-1 . Calculations were performed assuming that 1 mg POly(A) had an A26o of 30.1 (8) and RNA of 25, and that the length of globin mRNA without poly(A) corresponded to 589 nucleotides (I 7).
Results
a. Poly(A ) content of purified polysomal and free cytoplasmic mRNA Messenger RNA was purified from polysomal or free cytoplasmic mRNP preparations as described in the Methods section. Generally, between 12 and 20% of the total mRNA was found in the free mRNP fraction. The poly(U)-Sepharose elution profiles (not shown) revealed that 55% of the free cytoplasmic mRNA eluted at 25~ and the remaining 45% at 50~ In contrast only a small part of the polysomal globin mRNA (20%) eluted at 25~ while the remaining 80% eluted at the higher temperature. As judged by the sucrose gradient profiles, 60
neither polysomal nor free cytoplasmic globin mRNA could be detected in the run-through eluate of the column indicating that most if not all of the 9S mRNA had bound to the column. A third wash (at 55~ with 90% formamide) did not elute additional globin mRNA suggesting that all bound mRNA had been released after the 50~ wash. The thermal elution profiles from the poly(U)-Sepharose column were in good agreement with the results of Marbaix et al: (12) and suggested that the free cytoplasmic mRNA contained globin mRNA with relatively short poly(A)-segments as compared with the polyribosomal mRNA eluting at a higher temperature. This assumption was verified by direct measurement of the length of the poly(A)-segments. When the mRNA was isolated by poly(U)-Sepharose chromatography followed by sucrose gradient centrifugation, the free cytoplasmic 9S mRNA was found to contain poly(A)-tracts with an average length of 20-25 nucleotides. The average length of polysomal poly(A)-sequences was found to be 35-45 nucleotides. When 9S globin mRNA was isolated from free cytoplasmic mRNP by SDS treatment and repeated sucrose gradient centrifugation (thus omitting the poly(U)-Sepharose step) poly(A)-tracts with an average length of 17-20 nucleotides were found. All these experiments point to the conclusion that the free cytoplasmic globin mRNA has poly(A)tracts which, on the average, are considerably shorter than the poly(A)-tracts of globin mRNA present in polyribosomes.
b. Protein composition of free cytoplasmic mRNPcomplexes The protein composition of the 15-20S free mRNP (4) was examined by polyacrylamide gel electrophoresis in sodium dodecylsulphate (figure 1, slots 3-4) and compared with the protein composition of polyribosomal mRNP (figure 1, slot 2). As has been shown before by many investigators the polysomal globin mRNP contains a 76,000 dalton protein which is associated specifically with the 3'poly(A)sequence of the mRNA. In contrast, free mRNPparticles do not contain this protein (figure 1, slots 3-4) although, as we have shown above, a 3'poly(A)sequence is present in the globin mRNA of free mRNP. Another ol~vious feature of the protein composition of free mRNP are the proteins in the molecular weight range between 24,000 and 34,000 (slot 3). These proteins also appear to be present in the polysomal mRNP (compare slot 2). They must
4S
7S
11S
13S
18S
1
1
~
;
1
0.3.
? I -6O0 o'
E c
E
o 9
cO
40(3
.Q
O "O
