Journal canadien

Pksb8islred by

Publie' gar

THE NialFEONAL RESEARCH COUNCIL OF CANADA

EE CONSEIL NATIONAL DE WECHEWGEES DKJ CANADA

Can. J. Biochem. Downloaded from www.nrcresearchpress.com by Santa Cruz (UCSC) on 11/13/14 For personal use only.

Canadian Journal of Biochemistry Volume 57

Number 8

August 1979

Volume 57

nurnkro 8

aoat 1 979

Isolation and characterization of the nonpolysomal cytoplasmic messenger ribonucleoproteincomplexes of rat liver Mentorial Univsrsify of Newfoursdland, Molecular Biology Laboratories, Faculty of Medicirte, Health Sciences Centre, St. John's, Aijct., Canada AIB 3V6 Received December 19, I978 Revised April 5, 1979 Bag, $. & Sells, B. H. (1979) Isolation and characterization sf the nonpolyssmal cytoplasmic messenger ribonucleoprotein complexes of rat liver. Can J. Biochem. 57, 1051-1057 Nompolysomal cytoplasmic mRNA protein complexes (free mRNP) from norn~aland regenerating rat liver were isolated by affinity chromatography on oligo (dT)-celiulosecolumn. The mRNP complexes bound to the oligoddT) cellulose were eluted in a three-step process by using low ionic strength buffer (25 mlWTris-HC1, pH 7.5) at (i) 2"C,(ii) 37'C, and (iii) containing 50% forrnamide. The protein content of these three mRNP fractions, calculated from their buoyant densities, was found to be 50, 63, and 7876, respectively. Sixteen polypeptides of 31 000 to 90 880 were present in these rnRNP fractions. These polypeptides were found to be of nonribosomal origin. The polypeptide complements of these mRNP fractions were qualitatively similar when both major and minor components were considered. Considerable diRerences in the major polypeptides of the diRerent mRNP fractions were observed. The major polypeptides of the mRNP eluted at 2°C (rnRNP-I) were of 1% equal to 51 000, 65 000, 74 000,78 000, 80 000, 84 000, 85 800, and 90 000. Five additional polypeptides of iaa, equal to 44 000, 46 000, 49 000. 60 000, and 64 000 were present as major components in mRNP eluted at 37°C (rnRNP-2). On the other hand, the relatively protein-rich mRNP fractiom eluted with 5OC,;:,formamide (mRNP-3) consists of only four major polypeptides of Mr equal to 49 080, 51 000, 57 000, and 66 008. The polypeptides present in mRNP-1 and mRNP-2 of both normal and regenerating rat liver were similar. However, the rnRNP-3 fraction of the regenerating rat liver contains an additional polypeptide s f M, equal t s 54 000. Bag, 5. & Sells, B. H. (1979) Isolation and characterization of the nonpolysomal cytophsmic messenger ribonucleoproteinzcomplexes of rat liver. Can. J. Biochern. 57, 1051-1057 Nous 8vons is016 les complexes mRNA-protCine (mRNP libre) cytoplasmiques nonpolysorniques du foie de rat normal et du. foie en rkgCnbation par chromatographie d'afinitC sur colonne d'oBigo (dT)-cellulose. Les complexes mRNP lies h l'oligo (dT)-cellulose ssnt Clu6 par un processus comportant trois Ctapes et utilisant un tampon de force ionique faible (TrisHCI 25 mM, pH 7.5) B (i) 2"C, (ii) 37°C et (iii) en prhence de 50% de formamide. Les teneurs protCiques de ces trois fractions mRNP, calculdes d9aprbs leur densit6 de flottaison, sont respectivement de 50, 63 et 78?;,. Seize polypeptides de poids msldculaires allant de 31 008 90 000 sont presents dam ces fractions mRNP. Ces polypeptides sont d90riginenoas ribosomique. Les complkments polypeptidiques de ces fractions mRNP soint qualitativemerat semblables quand nous considCrons Bes constitmnts majeurs et mineurs. Nous sbservons des ditT6remces consid6rables dans les polypeptides majeurs des diR6rerates fractions mRNP. Les poids mol&ulaires des polypeptides nnsajeurs du mRNP CluC ii 2°C QrnRNP-1) sont de 51 OQQ, 65 000,74 OOO,78 000,80 000,84 000,

ABBREVEATHQNS: cpm, counts per minute; mRNP, messenger ribonucleoprstein complex; Adr, relative mass; DTT, dithiothreitol. lAuthor to whom csrresponadence should be addressed. 0008-4018/79/08105 1-07$01.00/0 @ I979 National Research Council of Canada/Csnseil national de recherches du Canada

CAN. J. BIOCHEM. VOE. Sir, 1979

Can. J. Biochem. Downloaded from www.nrcresearchpress.com by Santa Cruz (UCSC) on 11/13/14 For personal use only.

85 080 et 90 000. Cinq polypeptides additionnels dont les poids molCsulaires sont de 44 000, 46 000,49 000,60 000 et 64 000 sont des constituants majeurs du mRNP CluC B 37'C (mKNP-2). D'autre part, la fraction mRNF, relativement rishe em grotCine, CluCe avec 50% de formarnide (mRNP-3) contient seulement qusstre polypeptides rnajeurs dont les poids mo8&ulaires sont de 49 000, 51 000, 57 000 et 66 000. Les polypeptides prCsentes daris mRNP-I et ma-RNP-2 du foie de rat normal et du foie en r6gCnCrsbtion ssnt semblabks. Cependant, la fraction mRNP-3 du foie de rat en rkg6nCsatIon contient urn polypeptide iadditionnel de 54 000. [Traduit par le journal]

Introduction

In the cytoplasm sf eukaryotic cells, rnRNAs are known to exist outside the polyssmes in the form s f mRNP (1, 2). The molecular weights of the different polypeptides present in these nonpolysomal cytoplasmic mRNA-protein complexes (free mRNP) have been determined for a number s f eukaryotic systems 63-10). In order to obtain further information about the nature of polypeptide complements of free mRNP of different eukaryotic cells, we have undertaken the investigation on the free mRNP of rat liver. Studies were also designed to determine whether the mRNP proteins were modified when the pattern s f liver proteins being synthesized was altered as is observed during liver regeneration ( 11-14). Bligo(dT) cellulose has been used to isolate the polysome-released mRNP complex of a large number sf eukaryotic cells ( 15-1 8 ) . These results suggested that the 3'-poly(A) region in the polysomal mRNP was not masked with proteins. Little is known concerning the use of similar methods for the isolation of relatively protein-rich mRNP complexes s f different eukaryotic systems. Recently, this method has been used for the isolation of free mRNP complex from embryonic chicken muscle (19) and Ehrlich ascites tumor cells ( 9 ) . Forrnamide was commonly used to elute the bound mRNP. This method has a disadvantage in that it renders the mRNA-associated proteins unsuitable for further study in a cell-free protein synthesizing system (Bag, J,, unpublished results). Employing a rnodzcation of the above method, we wish to report that free mRNP complexes of rat liver are capable of binding to affinity columns indicating that the 3'-poly(A) region of these complexes is free to interact with the oligo(dT) residues. Furthermore, our results also demonstrated that for the elution of the major portion of rat liver free mRNP from the affinity column, formamide can be substituted by a combination of low ionic strength buffer and elevation of temperature to 37°C. We also present the first report on the estimates of molecular weight of the polypeptide cornplements of normal and regenerating rat liver free rnRNP. MateaiJs and Methods Buflers

119 same as buffer E minus sucrose and containing 580 pg/ mL heparin; buffer 111, 25 mM Tris-HC1, 64.5 h4 KCl, 500 pg/mL heparin (pH 19.5); buffer PV, 25 rnM Tris-MCI, ( p H 4.5); buffer V, 25 m M Tris-HCl, 50% formamide (pH 7.5); buffer VH, 25 na~WTris-HC1, $ urea, 40 m.44 2-mercaptsethanol (pH 8.0) ; buffer VIH, 8.1 M Tris-acetate (pH 8.5), 1 % sodium dodecyl sulfate, 1% 2-mercaptoethansl; bufler VBII, 0.1 h4 Tris-acetate (pH 8.5), 0.1 % dodecyl sulfate; buffer IX, 20 mLVThiethanolamine-HCl9 25 m M MCl, 2 mM magnesium acetate, 0.1 mM EDTA, 0.5 mlW DTT (pH%7.0).

Subcellular FractionhlPion ofRat Liver

Female Sprague-Dawley rats, weighing 125-150 g (5-6 weeks old) were maintained on a diet of Pupina Rat Chow. Livers (50 g) from these rats were removed and rinsed with buffer I. Livers were then sliced into small portions and homogenized with two volumes sf the same buffer in a motor-driven, teflon homogenizer. The preparation of golysome-free supernatant is described in Fig. I . The [KC11 in the postpolysomal fraction was adjusted to 0.5 Ad. The mRNP and the native ribosomal subunits present in this fraction (5) were pelieted through a 5-mL cushion of 20% sucrose in buffer I1 by centrifugation at 59 000 rpm in a Spinco BBTi rotor. This postgolywmal pellet was used for the isolation of free rnRNP. isolation of mRNA-Proteiaa Complexes The mRNP complex from normal and regenerating rat liver was isolated by using oligo(dT) cellulose (type Tap Collaborative Research, Waltharn, Massachusetts). The oligss(dT)-celHulose chromatography was performed in a small column prepared in a Basteus pipette (4 x 0.5 cm) using 0.1 g dry powder. This column was equilibrated with buffer IIH. The high salt washed postpolysomal pellet was suspended in 58 mL of buffer III and this preparation was applied to the oligo(dT)-cellulose column. The flow rate was adjusted to 20 mL/h. The column was washed with 200 mL of buffer HI1 to bring the A,o of the effluent to zero. The material bound to the column was eluted by using buffer HV at 2 and 3'9°C followed by elution with buffer V as described in the legends of the figures, The absorbance of the eluted material was monitored at 260 md 280 nrn using a B e c h a n model 24 dual-beam spectrophotometer. Similar methods were used for the isolation of free mRNP from regenerating rat liver. Extraction of R N A from mRNP and Assay of mRNA Activity The mWNP fractions were adjusted to pH 5.6 using 100 mlW sodium acetate, 1064 m M NaC%,1 % sodium dodecyl sulfate, 580 pg/mL beparin (RNA extraction buRer). An equal volume of a mixture of phenol, chloroform, and isoarnyl alcohol (50:56: 1) was added (20, 21) and stirred for 15 min. The aqueous phase obtained by centrifugation

The following buffers were used for subcellular fractionation and isolation of mRNP complexes: buffer I, 25 mA.8 Tris-HC1, 10 mA4 KCB, 1.5 mM magnesium acetate, 2 mA4 Dm, 8.25 M sucrose, 1.5 mlW phenyImethyls~lfonylfBuoride, at 20 000 x g for 10 min was reextracted with the same 50 pg/m% cyclohexirnide, and 200 &miL heparin; buffer solvent until the interphase was clear. The RNA was pre-

BAG AND SELLS

Can. J. Biochem. Downloaded from www.nrcresearchpress.com by Santa Cruz (UCSC) on 11/13/14 For personal use only.

cipitated with 2.5 volumes sf absolute ethanol at -20°C for 16 h. The RNA was pelleted by centrifugation at 20 800 x g for 30min. After washing twice with 66% ethanol, the RNA was dissolved in buffer 111 and passed through an oligo(dT9-cellulose column. The bound RNA was eluted with water and precipitated with 2.5 volumes of ethanol after adjusting to 8.2 M potassium acetate (pH 5.0). The precipitated RNA was dissolved in small volume of Hz8 to give 100-200 pg RNA/mk. Micrococcal nuclease treated rabbit reticulocyte lysate cell free system (22) was used to assay mRNA activity. Incubation mixtures contained (in a total volume of 25 p L ) 18 pL rabbit reticulocyte lysate, 10 mM Tris-HC1 (pH 7.51, 2 mM magnesium acetate, 2 mlW BTT, BOO mM KC1, 1 rnM ATP, 8.2 mM GTP, 15 mM creatine phosphate, 50 pg/mk creatina kinase, 8 pM of each of 19 amino acid, and 10 pCi (1 Ci -- 37 GBq) of [=S]methionine (specific activity 600 Ci/mmol, New England Nuclear Corporation, (Canada)). Incubations were carried out at 37°C for 60 min. Twomicrolitre samples from the reaction mixture were processed to determine the CCLCBBK insoluble (95°C) radioactivity. Folyncrylamide Gel Electrcaghor.esL of snRNP Proteins The mRNP proteins were precipitated by 10% 6C1:ICQQH and the precipitate was washed with acetone, dried, and dissolved i n buffer VI. Iodoacetamide was used at a final concentration of 0.25 M to alkylate the proteins. After incubating for 2 h at 30°C, the solution was dialyzed against buffer VII. Electrophoresis of the alkylated proteins was performed in 7.5% polyacrylamide gels containing 0.2% N,N'-methylene-bis-acrylamide in the presence of 0.1 % sodium dodecyl sulfate (5). Separation was achieved after 3 h at 4 mA/gel using buffer VIII. The gels were stained for 12 h with coomassie brilliant blue using 0.1% dye in 10% acetic acid, 25% isopropanol, and destained by employing the same solvent in a diffusion destainer (Hoffer Scientific Instruments, San Francisco). Deteranination of Buoyant Density Buoyant densities of the mRNP complexes were determined by CsCl isopycnic gradient centrifugation (5). The fractions eluted from the sligo(dT)-cellulose column were dialyzed against buffer IX containing 4% formaldehyde. The sample was concentrated by vacuum drying to give approximately 5 Am unit/mka. Approximately 8.5 Aasounit of the fixed material in 100 pL was layered on the top of a preformed CsCl gradient of densities 1.2-1.7 g/mL prepared in buffer IX containing 2% formaldehyde. The preparation was then centrifuged at 35 000 rpm for 40 h in a Beckman SWS0.1 rotor at 20°C. The gradient was fractionated in an auto densi-flow apparatus (Buchkr Instruments) and the absorbance at 260 nm was recorded. Partial #egatecforny Two-thirds of the liver from female rats (158-160 g) was removed under ether anesthetic (23). Following partial hepatectomy, the rats were maintained on a diet of Purina Rat Chow and 5% glucose solution and fed ad libitum until sacrifice after 18 h.

Results Isolation ofmRNP

To avoid the presence sf adventitiously bound pro2Am~mo,nm unit, the quantity of material present in 1 rnL of a solution which has an absorbance of 1 at 260 or 280 nm when measured in a cell of 1-cm light path.

Ral Liver Mrnqlenate 13MOxp

20 min Post Mitochondrial Suparnatanl

I;:"'

Post hlysomal Supernatant KC1 l o 0. %\, Sucrose Cushion, ZMMOx~6h

m a I ~ R N P ,QS. 405. to5) Oflgo(dT)-cellulose Chromatography Bound Material

tnRNP-I

mide mRMB-3

mRNP-2

FIG.1. Scheme for the isolation of nonpolysornal cytoplasmic mRNA protein complexes from normal and regenerating rat liver. The details of the subcellular fractionation of rat liver homogenate and the subsequent oligo(dT)-cellulose chromatography of the postpolysomal pellet to fractionate m W P complexes have been described in Materials and Methods section. teins in the mRNP preparation, we have used 0.5 M KC1 treatment to reduce the nonspecific binding s f proteins to mRNA (Fig. 1) (5). After removal sf the polysomes, the mRNP and native ribosomal subunits ( 5 ) present in the high salt treated postpolysomal fraction were pelleted through a sucrose cushfon. T h e results of the oligo(dT) -cellulose chromatography of this fraction is shown in Fig. 2. The 268-nm absorbing material that remained bound to the column after extensive washing with a 0.5 M KC1 containing buifer (buifer III) was eluted in a three-step process by using: ( i ) buffer IV at 2°C; (ii) buffer PV at 37'C,

BUFFER V AT 20.C

1

5

10

15

FRACTION NLIMBEW

FIG. 2. Isolation and fractionation of poly (A) -containing free mRNP complexes by oligo(dT)-cellulose chromatography. Free mRNP complexes from 50 g of rat liver were isolated by affinity chromatography on an oligo(dT)cellulose column as described in Materials and Methods section. The mRNP complexes bound to the column were eluted in three steps and 2-mL fractions were collected. 0-0,absorbance at 260 nm; A - A, absorbance at 280 nm.

-

CAW. J. BIOCHEM. VOL. 57, 1949

TABLEI. Binding of RNA to oligo(dT)-cellulosecolumn after modification of interaction with excess poly(U). The suspension s f postpolysorna8 pellet was prepared from 10 g of rat liver in buffer IIH and mixed with 1 m g h k poly(U) for 1 h prior to chromatography on an oligo(d%)cellulose column. The bo~andRNA was eluted in one step with buffer %I

Can. J. Biochem. Downloaded from www.nrcresearchpress.com by Santa Cruz (UCSC) on 11/13/14 For personal use only.

Modification of interttction with oligo(dT)-cellulose None

Poly(U) saturated postpo8ysomal fraction and (iii) buffes containing 50% formamide (buffer V) . The elution profile (Fig. 2) shows that 50% of the total oligo(dT) cellulose bound material was eluted at 2°C (mRNP-1) while 30% was eluted when the temperature of the column and the elution buffer was raised to 37°C (mRNP-2). Finally, the remaining material was eluted using a 50% formamide-containing buffer (mRNP-3). The peak fractions of mRNP-1, rnRNP-2, and mRNP-3 exhibited A260:A280ratios of 1.75, 1.54, and I. I , respectively. These decreasing ratios indicate that the amount of proteins associated with these fractions are of the following order: rnRNP-3 > mWNP-2 > mRNP-1 ( 5 ) . Further studies on rebinding of the mRNP fractions have shown that 95% of the eluted rnRNP-1, rnRNP-2, and mRNP-3 can rebind to the affinity coluinn (results not shown). Control experiments were also performed to examine whether the binding of these fractions was mediated through the poly(A) of mRNAs. To examine this, we have modified the interactions between oligo(dT) cellulose and mRNP fractions by saturating the poly(A) of mRNP with 1 rng/mE of poly(U). The results described in Table 1 show that the amount of bound APoo absorbing material eluted with 50% formamide (all three mRNP fractions were eluted in one step) was appreciably reduced after this modification. These observations suggest, therefore, that the major contributing structure in the binding of mRNP fractions to the oligo(dT)-cellralose column was the poly(A) of mRNPs and the poly(A) regions of mRNP fractions were not completely masked with proteins. Furthermore, these results also show that the level of adventitious binding of nowpoly(A) -containing RNAs or proteins to the oligs(dT)-cellulose column was very low. Presence o f PolyqA) in mRNP-RNA and Translation in a Cell-free System In order to further examine whether binding of mRNP fractions was mediated by the poly(A) region of mRNAs, the binding of deproteinized mRNP-RNAs was studied. Eighty-five to ninety percent of these RNAs were able to bind to an oligo(dT)-cellulose column (results not shown). These observations show that tbe major portion of the isolated mRNP fractions contain poHy(A)+ mRNA. The poly(A)+ mRNA isolated from mRNP fractions were examined for their mRNA activity. The RNA isolated from all three fractions strongly stimulated [35S]methionine incorporation

Total eluted RNA ( A280 unit)

$%,cesnerol

control 3

into total proteins in the rnRNA-dependent rabbit reticulocyte lysate cell-free system (Table 2). The activity af these rnRNAs was comparable (albiet slightly lower) with that observed for rat liver polysomal poly(A)+ mRNAs anp to 20 pg/mL, indicating the absence of any significant amount of nontranslatable component. However, in contrast with golysomal mRNA, we have routinely observed an inhibitory effect of mWNP-mRNA on the translation when higher (40 pg/mL or above) concentrations were used (Table 2). These results suggest that probably an inhibitor of protein synthesis is copurified with these mRNAs. Buoyant Densities of mRNP Fractions The binding of 260-nm absorbing material to the oligo (dT)-cellulose column and the characteristic A 2 , 0 : A 2 , , ratios (5) have indicated that the different fractions eluted from the column contain both mRNA and proteins. In order to demonstrate the pesence of mRNP complexes in these fractions, the buoyant densities were determined by CsCl iospycnic gradient centrifugation. After fixing with formaldehyde and centrifugation ta equilibrium in preformed CsCl gradients, each fraction displayed a distinctive buoyant density. The major components present in rnRNP-1. rnRNP-2, and rnRNP-3 exhibited buoyant densities of 1.55, 1.47, and 1.38, respectively (Fig. 3 ) . These values correspond to a protein content sf 50, 63, and '7896, respectively (1 ). The buoyant densities of mRNP-2 and mRNP-3 were found to be lower than that observed for ribosomal subunits (5). In contrast, the buoyant density of mRNP-1 was very close to that of ribosomal subunit; however, analysis of the proteins has shown that (see Fig. 4) these fractions were free of ribosomal proteins. No detectable amount sf proteinfree poly(A)-containing mRNA was present in these fractions. Furthermore, the gradient profile shows that free proteins were also absent in these preparations. The Protein Moieties of Diflerent mRNB Fractions In order to characterize the nature of polypeptides associated with the different mRNP fractions, samples of alkylated proteins of rnRNP-1, mRNP-2, and mRNP-3 were separated by poIyacpgrlamide gel electrophoresis in the presence of sodium dodecyl sulfate. The electrophmetograms (Fig. 4) show that 16 polypeptides of M , 31 000 to 90 080 were associated with these mRNP complexes, Three diRerent mRNP fractions were qualitatively very similar when both major

1055

BAG AND SELLS

TABLE 2. Stimulation of [3SS)-methionineincorporation by free rnRNP-RNA and polysomal mRNA in an mRNA-dependent rabbit reticulocyte lysate eel%-free system. RNAs from mRNP1, rnRNP-2, and mRNP-3 were isolated by extraction with a mixture of phenol, chloroform, and isoamyl alcohol as described in Materials and Methods. RNAs were further purified by sligo(dT)-celBulose chromatography.The poly(A)-containing RNAs (907;) of input RNA) were

Can. J. Biochem. Downloaded from www.nrcresearchpress.com by Santa Cruz (UCSC) on 11/13/14 For personal use only.

tested in an rnRNA-dependent rabbit reticuloeyte Iysate dl-free system

Source of RNA Minus WNA Rat liver ribosomal RNA" Rat liver polysornal mRNAa

RNA used (micrograms)

-

CCl gCOC4H precipitable radioactivity (cpm x lo4)

Degree of stimulation control

1 0.5

B

-

-

- -

aRat liver R N A from total polysomes was extracted as previously described (20, 21) and poly(A)-containing WNAs were isolated by oligo(dT)-cellulose chromatography (209. The oligo(dT9-cellulose unbound WNA was used as ribosomal WNA.

and minor polypeptide components were considered. Considerable differences, however, were observed with respect to the relative proportion of the major polypeptide complements of these mRNP fractions. The major polypeptides of mRNP-l were of M , equal to 51 008,65 000,74 000,78 000,80 000, 84 000,85 000, and 90 000. Five additional polypeptides of 134, equal to 44 000, 46 000, 49 000, 60 000, and 64 000 were present as major eorngsnents in mRNP-2. In contrast, only four polypeptides of 134, equal to 49 800, 5 %000, 57 000, and 66 000 were the major components in mRNP-3. Further comparison of mRNP proteins with the proteins frsm the high salt washed ribosomal sub-

FIG.3. Buoyant densities of normal rat liver mRNP complexes. Formaldehyde-fixed mRNP csrnplexes were centrifuged in preformed CsCl gradient (for details see Materials and Methods section). -, mRNP-1; - -, mRNP-2; and - .-, mRNP-3.

units revealed that the characteristic ribosomal proteins of M , 15 000 to 30 000 were absent in these mRNP fractions, suggesting that these mRNP fractions were free from ribosomal subunits. The four polypeptides of A4, equal to 45 000 and 32 000, and 50 000 and 47 000 of the 40% and 405 ribosomal subunits, respectively, were very similar in moHecuIar weight to mRNP

FIG. 4. Sodium ddecyt sulfate - polyacrylarnide gel electrophoresis sf normal a d regenerating rat liver free mRNP csmplexes. Efectrsphoresis of alkylated mRNP proteins was carried out using 10 cm long 7.5% polyacrylamide gels (for details, see Materials and Methods section). a-e, normal rat liver mRNP complexes: a, mRNP-1 (6.4Anao unit); 6, mRNP-2 (8.2 unit); c, mRNP-3 (0.1 Aabo unit). d-f, regenerating rat liver mRNP complexes: 8, rnRNP-1 (0.15 Aloe unit); e, rnRNP-2 (8.15 Asa unit); f, mRNP-3 (0.1 Avro unit). g, rat liver high-salt washed 40s ribosomal subunit (0.6 L42,0unit).h, rat liver high-salt washed 60s ribosomal subunit (0.6 Azw unit).

CAN. 3. BIOCHEM. VOL. 57, 1979

Can. J. Biochem. Downloaded from www.nrcresearchpress.com by Santa Cruz (UCSC) on 11/13/14 For personal use only.

I056

proteins. It is not clear from these data whether these mRNP proteins represent stripped ribosomal proteins. Preliminary analysis, however, by two-dimensional gel electrophoresis has shown that these mRNP proteins are acidic proteins in contrast with those high molecular weight ribosomal proteins which migrated as basic proteins (results not shown). When mRNP complexes from regenerating rat liver were purified and fractionated on oligo(dT)-cellulose column, three different mRNP fractions were obtained (results not shown). The polypeptides associated with each of these t h e e fractions are shown in Fig. 4(d-f). The electrophoretograms show that the polypeptides associated with the regenerating rat liver mRNP fractions are similar to those observed in the mRNP fractions from normal rat liver. No new polypeptides were detected in regenerating rat liver mRNP-I and mRNP-2. Some differences, however, were observed in the polypeptides of mRNP-3 fraction between normal and regenerating rat liver. An additional polypeptide of M , equal to 54 000 was present as a major component in the mRNP-3 from regenerating rat liver. It should be noted that our method of analysis sf the mRNP proteins did not reveal whether modifications such as changes in the phosphorylation or acetylation of these proteins took place in regenerating rat liver mRNP. Further studies are necessary to examine these modifications.

Discussion The results described in this paper reveal that the poly(A) region of the nonpolysomal cytoplasmic mRNP complexes of rat liver is available for interaction with oligo(dT) cellulose and indicate that the mRNA-associated proteins do not entirely cover the 3'-poly(A) region of mRNAs. These studies also show that nonpolyssmal mRNP complexes can be fractionated by affinity chromatography on oligo(dT) cellulose. When protein-free poly (A) -containing mRNAs from either polysomes or postpolyssmal supernatant were bound to oligo(dT) cellulose, the bound mRNAs were eluted as a sharp peak by using buffer IV at 2'C (Bag, J. & Sells, B. H., unpublished observation). It has been noted, however, that a significant portion of the mRNP complexes could not be eluted under these conditions. Stronger denaturing conditions using buffer IV at 37°C or 50% formamide containing buffer (buffer V), were required to remove the remaining 50% of the mRNB complex. These findings, and the fact that mRNP-2 and mRNP-3 contain higher amounts of proteins than does mRNP-1, suggest that certain mRNP proteins fmction either to stabilize the hydrogen b ~ n d sbetween oligo(dT) and poly(A) or to interact with components of the oligo(dT) -cellulose column. Two possibilities exist to explain the nature of origin of these three different mRNP fractions: (a) these three mRNP fractions may have resulted from partial removal of proteins (24) from the mRNP complex during the affinity chromatogxaphy using high ionic

strength buffer; ( 6 ) alternatively, these may represent subpopulations of mRNAs having different afinity for the various mRNA binding proteins. The analysis of the length of the goly(A) chain, nature of cap structure, and the products of translation of the mRNA present in these mRNP fractions will be helpful to decide between these two possibilities. Since at present this information is not available, the heterogeneity of free mRNP complexes should be regarded as tentative. The result of translation of deproteinized RNAs from mRNP complexes showed that their RNAs are translatable. However, partial inhibition of protein synthesis was observed when higher concentrations of these RNAs were used. These observations suggest that an inhibitor of protein synthesis is copurified with mRNP-RNAs. Further studies are necessary to determine the nature of this inhibitor and its relationship to mRNP complexes. In this context, it is interesting to note that the presence of inhibitors has also been reported in free myosin heavy chain and globin mRNP complexes (25, 26). Fewer proteins were found associated with rat liver polysome released mRNP complexes (24) ; seven polypeptides of M , equal to 138 000, 109 000, 104 000, 73 000, 66 000, 62 000, and 52 000 were previously reported in the rat liver polysornal mRNP isolated by oligo (dT) -cellulose chromatography (24) . In contrast, our results show that the mRNB complexes of rat liver are protein-rich complexes and contain 16 polypeptides. It is quite likely, however, that these two kinds of cytoplasmic mRNP complexes share several common polypeptides. The polypeptides of M , equal to 74 000 and 51 000 present in free mRNP may be identical to M, equal to 73 000 and 52 000 polypeptides present in polysome released mRNP of rat liver and a variety of eukaryotic cells (2, 24). The M , 65 000 polypeptide of free mRNP may be similar to the M , 65 700 polypeptide of polysomal mRNP (24). Further work, however, is necessary to determine the relationship of these two groups of proteins present in polysomal and free mRNB complexes. The role of cytoplasmic mRNP proteins is still unknown. Recently, eukaryotic mRNAs have been found to form complexes with initiation factors,s eIF-2 (27, 28), eIF-3, eIF-5 (29), and eIF-4B (30) in an in vita0 reaction. However, the relationship between the initiation factor and mRNP proteins is still unknown. Further studies are now in progress in this and other laboratories to elucidate the role of mRNP proteins.

Acknowledgements This work was supported by a grant from the Medical Research Council of Canada, the March of Dimes (National Foundation), and the Muscular Dystrophy Association of Canada. It is our pleasure to acknowI-

-

T h e nomenclature adopted at the Fogarty Center, National Institutes of Health, United States Workshop held in Bethesda, Maryland, on October 18-20, 1976, has been used.

BAG AND SELLS

16. Kumar, A. d Pederson, T. (1975) J. Mol. Biol. 96, 353-365 17. Kish, V. M. & Pederson, T. (1975) J. Mol. Bisl. 95, 227-238 1. Spirin, A. S. (1972) The Mechartism o f Protein Synthesis arzd Its Regukation (Bosch, L., ed.), pp. 515- 18. Irwin, D., Kumar, A. & Malt, R. A. (1975) Cell 4, 157-165 537, Elsevier, Amsterdam 19. Bag, J., Jain, S. K., Coronado, R. & Sarkar, S. (1976) 2. Greenberg, J. R. (1975) B. Cell Bio!. 64, 269-288 Fed. Proc. Fed. Am. Soc. Exp. Biol. 35, 1440 (abstr.) 3. Gander, E. S., Stewart, A. G., Morel, C. M. & Scherrer, 20. Aviv, H. & Leder, P. (1972) Prsc. Natk. Acad. Sci. K. (1973) Eur. I . Biochern. 38,443-452 U.S.A. 69, 1408-1412 4. Barrieux, A., Ingraham, H. A., David, D. N. & Rosen21. Palmiter, R. D. (1974) Biochemistry 13, 3606-3615 feld, M. G. (1975) Biochemistry 14, 1815-1821 5. Bag, J. & Sarkar, S. (1975) Biochomistry 14, 3800- 22. Pelham, R. B. H. & Jackson, R. J. (1976) Elir. I . Bioehem. 67,247-256 3807 23. Higgins, G. M. & Anderson, R. M. (193 1) Arch. Pathol. 6. Bag, J. & Sarkar, S. (1976) 6. Biol. Chem. 251, 760812,186-202 7609 7. Liautard, J. P., Setyono, B., Spindler, E. & Kohler, K. 24. Cardelli, P. J. & Pitot, H. C. (1977) Biochemistry 16, 5127-5134 (1976) Biochim. Biophys. Asta 425, 373-383 8. Venrooij van, W. J., Eekelen van, C. A. G., Jansel, 25. Bester, A. J., Kennedy, D. S. & Heywood, S. M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1523-1527 R. T. P. & Princen, J. M. G. (1977) Nature (Londcan), 26. Civelli, Q., Vincent, A., Buri, J. F. & kherrer, K. (1976) 189-191 FEBS Lett. 72,71-76 9. Jeffery, W. R. (1977) 1. Biol. Chem. 3525-3532 16. Vincent, A., Civelli, O., Buri, J. F. & Scherrer, K. 27. Kaempfer, R., Hollendar, R., Abrams, W. R. & Israeli, R. (1978) Proc. Natl. Sci. U.S.A. 75, 209-21 3 (1977) FEBS Lett. 77,281-286 11. Buchner, N. L. & Malt, R. A. (1971) Regeneration o f 28. Kaempfer, R., Rosen, H., Israeli, R. ( 1978) Proc. Natl. Acad. Sci. U.S.A. 75,650-654 Liver and Kidney, Brown and Co., Boston 12. Schreiber, J., Urban, J., Zahringer, J., Reutter, W. & 29. Brown-Luedi, M. E., Benne, R., Yau, P. & Hershey, J. W. B. (1978) Fed. Proc. Fed. Anz. Soc. Exp. Biol. Fmsch, U. (1971) I . Biol. Chem. 246, 4531-4538 37, 1307 (abstr.) 13. Tominaga, T., Kitmura, Ma, Azurna, Y., Taguchi, T. 30. Padilla, M., Canaani, D., Groner, Y., Weinstein, J. A., & Takeda, Y. (1975) I . Biochem. 77, 1255-1259 Bar-Joseph, M., Merrick, W. & Shafritz, D. A. (1978) 14. Tsurugi, K. & Qgata, K. (1976) I . Biochem. 79, 883I . Biol. Chem. 253,5939-5945 893 15. Lindberg, U. & Sundquist, B. (1974) I . Mol. Biol. 86, 451-468

edge the skillful technical assistance of Mr. Philip Power.

Can. J. Biochem. Downloaded from www.nrcresearchpress.com by Santa Cruz (UCSC) on 11/13/14 For personal use only.

1057

Isolation and characterization of the nonpolysomal cytoplasmic messenger ribonucleoprotein complexes of rat liver.

Journal canadien Pksb8islred by Publie' gar THE NialFEONAL RESEARCH COUNCIL OF CANADA EE CONSEIL NATIONAL DE WECHEWGEES DKJ CANADA Can. J. Bioche...
751KB Sizes 0 Downloads 0 Views