Molec. Biol. Rep. Vol. 5, 1-2: 71-78, 1979

REGULATION OF ALBUMIN SYNTHESIS IN RAT LIVER David A. SHAFRITZ, S.H. YAP & Roger K. STRAIR

The Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, U.S.A.

Abstract The present report reviews our findings on the subcellular distribution of albumin mRNA in rat fiver under normal and abnormal physiologic conditions, the identification of albumin mRNA in specific mRNP complexes in liver cytosol of starved rats, and evidence for albumin mRNA sequences in a higher molecular weight nuclear precursor to cytoplasmic albumin mRNA.

Introduction A number of years ago, various investigators reported. evidence in eukaryotic cells that secretory proteins are synthesized primarily on membrane-bound polysomes, whereas cytosol proteins are synthesized primarily on free polysomes (1-4). From these studies it was assumed that mRNAs for secretory versus non-secretory proteins are compartmentalized inside the cell, but whether this was indeed true and how such mRNA segregation might occur had not been established. We set about to examine these questions in rat liver, using albumin and ferritin as markers for secretory and cytosol proteins, respectively. Initially, we measured albumin and ferritin synthesis in cellfree systems, using isolated membrane-bound and free polysomes in comparison to their respective RNA extracts translated in a heterologous mRNAdependent system. With native liver polysomes, 5to 6- fold differences were found in the percentage synthesis of albumin and ferritin in these two polysomal subpopulations (5, 6). These results agreed with earlier findings (1-4) and were consistent with an interpretation that the mRNAs for these proteins are compartmentalized within the liver cell. However, when RNA extracts from the same preparations of membrane-bound and free liver polyribosomes were translated in a reticulocyte cell-free system,

little difference was found in the synthesis of either albumin or ferritin (6). These results were not consistent with mRNA segregation and suggested 1)that non-translated mRNAs for ferritin and albumin were present in membrane-bound and free liver polyribosomes, respectively, and 2) that the concentration of a given mRNA in a mixed mRNA preparation might not be the sole factor in governing translation of that specific mRNA. More recently, it has become clear that a variety of factors or conditions may influence in vitro translation of eukaryotic mRNAs (7), so that the precise level of a specific mRNA cannot be determined by cell-free translation. Therefore, we have developed techniques of molecular hybridization to determine albumin mRNA sequence content directly in liver RNA preparations without the need for measuring protein synthesis. Using albumin [3 HI eDNA prepared from purified albumin mRNA, we have reexamit~ed the distribution of albumin mRNA in membrane-bound and free liver polyribosomes (8, 9). The present report reviews our findings on the subcellular distribution of albumin mRNA in rat liver under normal and abnormal physiologic conditions, the identification of albumin mRNA in specific mRNP complexes in liver cytosol of starved rats, and evidence for albumin mRNA sequences in a higher molecular weight nuclear precursor to cytoplasmic albumin mRNA.

Development and utilization of an albumin eDNA probe

1) Purification of albumin mRNA and preparation of albumin [3H] -eDNA Procedures for purification of specific eukaryotic mRNAs have been rather limited. Success has been achieved generally in highly differentiated or induced 71

cells when the specific mRNA is present at very high levels (globin mRNA in reticulocytes, crystallin mRNA in lens fiber cells, immunoglobulin mRNA in myeloma cells, or ovalbumin mRNA in hormone stimulated oviduct) and[or when the mRNA has a unique property in terms of molecular weight (myosin mRNA or collagen mRNA) or base compesition (silk fibroin mRNA). For eukaryotic mRNAs without distinctive properties, immunological precipitation of polysomes bearing nascent chains for the specific protein has been employed. For complete purification and preparation of a specific cDNA probe, however, immunological precipitation alone may not be sufficient. To purify albumin mRNA our general strategy (Fig. 1) was to enrich for albumin mRNA sequences in total liver RNA by indirect' immunoprecipitation (lmmpt mRNA), and then to select for albumin mRNA in the enriched preparation by molecular hybridization to a Rot value in which only the most abundant mRNA species (hopefully albumin mRNA) and its DNA counterpart would form hybrids. In the procedure used (Fig. I), Immpt eDNA was prepared from the enriched DNA fraction

GENERAL PROCEDURE

APPLICATION FOR ALBUMIN mRNA. A Ib- mRNA . r ~ ' v ' , A ~ J v A A A

ccr .,;

on a solid matrix of oligo (dT)-cellulose primer. The properties of mRNA-cDNA hybridization on solid phase were then compared to hybridization in solution, an analytical hybridization was performed to determine the optimal RNA concentration and time to hybridize the most abundant component while excluding all other components, and a bulk reaction with Immpt cDNA cellulose was then performed. Specific albumin mRNA was eluted from the affinity column by denaturing hybrids with 85% formamide and was then used for characterization studies and for albumin [3 H]-cDNA synthesis. Fig. 2 shows the Rot curves for crude liver mRNA, Immpt mRNA and albumin mRNA. In each case the mRNA is hybridized to [aH]-cDNA prepared from itself. Crude mRNA shows the entire spectrum of abundancy classes, Immpt mRNA shows enrichment of the highly abundant component to 30-35%, and albumin mRNA shows a single complexity component with a Rot 1/2 of 9.8 x 10-4 mol-sec/liter. Compared to a,/~ globin mRNA standard, this material has a sequence complexity of 5.9 x lO s daltons, which correlates with the molecular mass of albumin mRNA on sucrose

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Figure 1. Schematic diagram for purification of albumin mRNA by Controlled Molecular Hybridization.

72

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Figure 2. Sequence complexity analysis of fiver mRNA fractions. Poly A+RNAs were prepared from total liver polysomes (etude mRNA), anti-albumin immunopreeipitated polysomes (Immpt mRNA) or mRNA eluted from an Immpt-cDNA cellulose affinity column (alb mRNA). [3 H[ -cDNAs were prepared to each of these mRNAs, using "reverse transetiptase", and in each case hybridization was performed using mRNA and ~ 500-600 clam of its [3H]-cDNA counterpart (8). A minimum of 10 fold excess of mRNA was used for each point. Table 1. Yield of Membrane-bound and Free Liver Polyribosomes from Fed Versus Fasted Rats Polyribosomal RNA Body weight

Fed Fasted % reduction

Liver weight

Total fiver RNA

g

g

mg/fiver

257.5 • 24.7 215 • 20.2 16.5

10.8 • 0.9 7.5 • 0.6 30.6

8.2 • 2.2 6.0 • 1.2 26.8

Free

Membrane-bound mg/g liver

1.1 • 0.09 0.85 • 0.05 22.7

4A8 • 1 A3 3.6 • 0.92 19.6

(Reproduced with permission of J. Biol. Chem.)

gradient centrifugation (17S or 6.0 x l0 s daltons). Within the limits o f this procedure, the shape o f the Rot curve for albumin m R N A suggests a single molecular species with a purification of > 95%. Upon cell-free translation of this mRNA, only albumin could be identified (8).

2)Cytoplasmic distribution of albumin mRNA sequences in rat liver and influence of fasting For all studies, Sprague Dawley rats were divided into two groups: fed and fasted, and experiments were performed simultaneously. As shown in Table I, a 24- to 30-h fast decre~ised body weight by 17%. The liver weight and total liver RNA were also reduced in fasting by approximately 25-30%. To determine quantitatively the amount of albumin mRNA in membrane-botmd and free polyribosomes and to study the influence o f the nutritional state on albumin m R N A content it was particularly important to utilize isolation procedures which provide

nigh yield of undegraded polyribosomes. Recently, amsey and Steele (10) have developed such methods ',~d we have been able to recover greater than 90% of total polysomal RNA by these procedures (9). As can be seen in Table I, there was a 20% decrease in total polysomal RNA/gm liver in fasted animals compared to fed controls. However, in fasted versus fed rats the proportion o f free and membrane-bound polyribosomes was not significantly different (membrane-bound polysomes in fed and fasted rats comprised 75 to 80% o f total hepatic polysomes). RNA extracts from membrane-bound and free polyribosomes were analyzed for albumin m R N A sequence content. In these experiments, varying amounts of RNA were titrated against a fixed amount of albumin [3I-I.]cDNA (saturation hybridization). The amount o f RNA required to protect a certain percentage of albumin [3Hj.cDNA measures the relative concentration o f albumin m R N A in each fraction. As can be seen in Fig. 3, the concentration o f albumin m R N A in liver membrane-bound polyribosomal RNA ( o - - e ) 73

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Figure 3. Hybridization of RNA extracted from membranebound and free liver polyribosomes from fed and fasted rats. RNA from liver membrane-bound polyribosomes of fed ( o - - o ) or fasted (A---A) rats and RNA from free polyribosomes of fed ( o - - o ) or fasted (z~--~) animals was hybridized to a given amount of albumin [3H]cDNA (~ 400 cpm;

specific activity, 7.5 x 106 cpm/ug). Reaction conditions were as described in ref. 11 (reproduced with permission of J. Biol. Chem.). is approximately 20 times greater than in liver-free polyribosomal RNA ( o - - o ) of fed animals. In fasted animals, the concentration of albumin mRNA in membrane-bound polyribosomal RNA (" A) is reduced 3.5 times, whereas free polyribosomes (A.._~) contain 67% more albumin mRNA as compared to free polyribosomes of fed rats. From these studies we calculate that the ratio of albumin mRNA concentration in membrane-bound versus free polyribosomal RNA in fasted rats is 3.5:1. We have also observed that very little albumin mRNA is present in the post-ribosomal fraction of fed rats, whereas the albumin mRNA content in the post-ribosomal fraction of fasted rat liver is increased dramatically (11). Table II shows the quantitative distribution of albumin mRNA sequences in the cytoplasm of fed and fasted rat liver based on the amount of RNA in each subcellular fraction, the

relative concentration of albumin mRNA sequences determiaed by molecular hybridization, and the finding that 1 p~, of purified albumin mRNA protects 5 cpm of albumin [a H] -cDNA (8). In fed rats, 97% of albumin mRNA sequences are present in membrane-bound polysomes, 2% are in free polysomes and 1% is in the poSt-ribosomal supernatant fraction. Upon fasting for 24-30 hrs (optimal during the Spring and Summer months), there is a shift of albumin mRNA sequences from membrane-bound to free polysomes and the bulk of albumin mRNA is found in the post-ribosomal supernatant (Table II). We have not determined what percentage of albumin mRNA in the free polysome fraction is actually present in polysomes, ollgosomes, monosomes or other rapidly sedimenting complexes. However, albumin mRNA in the post-ribosomal supernatant fraction of fasted ani~. . . . act 17S molecules (11). 3) Evidence for albumin mRNPs in liver cytosol of fasted rats and influence of a, ino acid refeeding To determine whether albumin mRNA in the cytosol of fasted rat liver was present in messenger ribonucleoprotein particles (mRNPs), sucrose gradient sedimentation and Cs2 SO4 equilibrium density centrifugation was performed, followed by hybridization of gradient fractions with albumin [3 H] -cDNA. Albumin mRNA sequences sedimented as a diffuse peak ranging from 30-60S [distinct from 40S ribosomal subunits (11)] and a portion of this material banded at a density in Cs2 SO4 of 1.33 gm/cc (Fig. 4), indicative of an RNP complex. Since glutaraldehyde fixation could not be performed prior to centrifugation (this_would render the albumin mRNA inactive in hybridization), we could not quantitative albumin mRNA in mRNPs. When fasted rats were refed a mixture of amino acids for one hr, albumin mRNA sequences were transferred from the mRNP fraction

Table II. Distribution of Albumin mRNA Sequences in Liver Subceilular Fractions from Fed Versus Fasted Rats Membranebound

Free

Postribosomal supernatant

fraction Albumin mRNA sequences (% total cytoplasmic albumin mRNA) Albumin mRNA content (ng/g liver) Albumin mRNA concentration (Pg/t~g RNA)

Fed Fasted Fed Fasted Fed Fasted

97 31 A 582 132 130 37

2 8.6 6.9 9.4 7 11

1 60 a 8.6 220 a 13 600 a

aThese values have been corrected for loss of albumin mRNA sequences during the isolation of the postribosomal supernatant fraction (reproduced with permission of the J. Biol. Chem.).

74

4)Evidence for a higher molecular weight nuclear precursor to albumin mRNA

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