Interaction of informational macromolecules with ribosomes: II. Binding of tissue-specific RNA's by ribosomes

196 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96470

INTERACTION OF INFORMATIONAL MACROMOLECULES W ITH RIBO-

SOMES

II. BINDING OF TISSUE-SPECIFIC RNA'S BY RIBOSOMES

H. N A O R A AND K. K O D A I R A

Research School o[ Biological Sciences, The Australian National University, Canberra, A .C.T. 26Ol (Australia)

(Received N o v e m b e r i 4 t h , 1969)

SUMMARY

I. The binding of rat liver, kidney and spleen nuclear RNA's by ribosomes prepared from these tissues has been studied.

2. Evidence suggesting the presence of a binding site, specific to tissue-specific RNA, on ribosomes or on a factor(s) associated with ribosomes, has been obtained.

3. The ability of ribosomes or factors to bind tissue-specific RNA is also species- specific.

INTRODUCTION

Experiments, reported previously, showed that ribosomes prepared from rat liver cells are capable of binding rat liver nuclear RNA (nRNA) preferentially in the presence of phage f2 RNA in excess 1,2. This observation may lead to speculation as to the role of ribosomes in cell differentiation. Indeed, qualitative ditferences in the RNA population of various tissues have been observed by the technique of DNA-RNA hybridization 3,4. There is a possibility that tissue-specific RNA molecules are preferentially bound by tissue-specific ribosome species, which are peculiar to the binding and translation of these genetic messages.

The present communication describes experiments involving the binding of nRNA's from rat liver, kidney and spleen cells by the crude preparation of ribosomes prepared from these tissues. The experiments suggest the presence of the binding site, specific to tissue-specific RNA, on the ribosomal particles or on a factor(s) associated with ribosomes.

MATERIALS AND METHODS

Materials

These were essentially the same as those used in the previous paper ~ except that [2-14C~uracil (61 mC/mmole) and E5-3Hlorotic acid (9.5 C/mmole) were pur- chased from New England Nuclear Corp, and Radioehemical Centre, respectively.

Biochim, Biophys. Acta, 209 (197 o) 196-2o6

RNA's AND RIBOSOMES 197

Preparation o/3zp_ or 3H-labeled and unlabeled nuclear R N A ]rom rat and mouse liver cells

The asP-labeled and unlabeled nRNA preparations were the same as those used in the experiments reported in the previous paper 2. For preparation oi all-labeled nRNA, 300/zC of ~5-aHlorotic acid was injected intravenously 60 rain prior to decapitation. Unlabeled nRNA was also prepared from C3H mouse liver cells by the same procedure as that used for rat liver cells.

Preparation o/ 32p-labeled and unlabeled nuclear R N A ]rom rat kidney, spleen and thymus cells

Nuclei were isolated from kidney cells either by the conventional method or by the modified method of CHAUVEAU et al. 5 and NAORA e/ al. 6.

The minced kidneys were suspended in a mixture of I vol. of 0. 5 M sucrose and 8 vol. of 0.25 M sucrose containing 3.3 mM MgC12 and 50 #g/ml polyvinyl sulfate. The suspension was homogenized in a Waring blendor at 7 ° V under conditions which gave 70-9 ° ~o cell breakage. The homogenate was centrifuged at IOOO ×g for 5 min. The cells that remained intact sedimented at the bot tom of the pellet. The upper half of the pellet was suspended in 0.25 M sucrose containing 3.3 mM MgC12 and 50/zg/ml polyvinyl sulfate and again homogenized in a glass homogenizer with lO-15 strokes of a tight-fitting teflon pestle at approx. IOOO rev./min. The preparation was re-centrifuged and re-homogenized 5-6 times until there was almost no contamination of the nuclei by cytoplasmic fragments. The final pellet was used as a nuclear fraction. In some cases, crude nuclear fraction was suspended in 2.3 M sucrose and centrifuged at 4 ° ooo ×g for 50 rain. The nuclear pellet thus obtained was suspended in 0.25 M sucrose containing IO/~g/ml polyvinyl sulfate and used for the preparation of nRNA.

The preparation of nuclei from rat spleen cells was carried out by the modified method of TsuzuKI AND NAORA 7.

The procedure used to prepare RNA from these isolated nuclei has already been described 2.

For the preparation of 32P-labeled nRNA, 5-12 mC of 3*Pt was injected intravenously 6o min prior to decapitation. Immediate ly after the animals were sacri- ficed, kidneys and spleens were removed and used for the isolation of nuclei. 32p_ Labeled and unlabeled RNA's prepared from kidney and spleen nuclei were then extracted 3 times with I M NaCI at 2 ° and the pellets were finally washed with 8o ~ ethanol. This fraction was used as I M NaCl-insoluble RNA. The I M NaCl-soluble fractions were precipitated with cold ethanol. The labeled nRNA's used in the present experiments were mainly i M NaCl-insoluble RNA fractions. These preparations had a specific activity of at least 13" lO 5 counts/min per mg.

Sucrose density centrifugation of 3*P-labeled kidney nRNA resulted in a poly- disperse distribution of radioactivity throughout the gradient with a prominent peak at approx. IO S and a small 28-S peak (Fig. I). A similar pat tern was also obtained with the preparation of 3*P-labeled spleen nRNA.

As in the case of 8*P-labeled liver nRNA, all the labeled materials used in the present experiments are ribonuclease-sensitive. After t rea tment of labeled nRNA with ribonucleases under the conditions described previously 2, no radioactivity remained after acid precipitation. Significantly, ribosomes did not bind label from

Biochim. Biophys. ~1eta, 2o9 (197 o) 196-2o6

198 H. NAORA, K. KODAIRA

1~ I 1.0 [ • i

0.5 i "~ .

~ ~ ' lop o

3

x

.c 2 E

o

or. ° 5

Bottom Fraction (ml)

Fig, I . Sedimenta t ion analysis of 8*P-labeled kidney nRNA. R N A was prepared from the isolated kidney nuclei of 82P-injected ra t s and t rea ted wi th i M NaC1 to remove low-molecular-weight RNA. - - . , absorbance a t 254 mp; O - - - 0 , radioactivi ty.

ribonuclease digest of labeled RNA's, indicating that the labeled materials which participate in binding are all RNA.

Unlabeled nRNA from rat thymus cells was the same as that used in the experiment reported in the previous paper 2.

Preparation o/14C-labeled and unlabeled phage/2 RNA Unlabeled phage f2 RNA was prepared by the method reported in the pre-

vious paper 2. 1*C-labeled phage f2 was prepared by exposing the cells of Escherichia coli (K38 strain), grown in synthetic medium, to E14C~uracil (20o #C/l).

Preparation o/ribosomes The preparation of ribosomes from rat livers was the same as tha t used in

the previous work 2. Rat liver ribosomes were also prepared by centrifugation through two layers

of sucrose (0. 5 and 1.8 M) and then further purified by sucrose density gradient (lO-3 ° %) centrifugation 2.

Ribosomes from kidney and spleen cells were prepared by the following method: minced kidneys and spleens were homogenized in a mixture of I vol. of 0.5 M sucrose and 3 vol. oi 0.25 M sucrose containing 20 mM Tris (pH 7-4), 3 mM MgC12 and 6 mM mercaptoethanol. The homogenate was centrifuged at I5 ooo ×g for 15 min to remove nuclei, cell debris and mitochondria. The supernatant was centrifuged at lO5 ooo ×g for 60 min. The microsomal pellet thus obtained was suspended in a suspension medium (I mM Tris (pH 7.4), 1.5 mM MgC12, 5 mM KC1 and 6 mM mercaptoethanol) and sodium deoxycholate was added to make a final concentration of I.O %. The deoxycholate-treated suspension was centrifuged for 9 ° min at lO 5 ooo ×g. The ribosomal pellet was gently resuspended in the suspension medium with a homogenizer and centrifuged at IOOO ×g for 5 min. The supernatant contaiving a homogeneous suspension of ribosomes was stored at --80 ° prior to use.

Biochim. Biophys. Acta, 209 (197 o) 196-2o6


Assay o/ the binding o/ labeled RNA to ribosomes Binding was studied in 0.2 ml of reaction mixtures under optimal conditions

and assayed by the Millipore filter method described previously 1,2. The blank, which was determined independently for each experiment under the same conditions but without ribosomes, was subtracted from the data. Sucrose sedimentation analysis showed that no apparent degradation of labeled RNA added was observed after incubation of labeled RNA with ribosomes under the assay condition.

In the experiments, in which the inhibitory effect of unlabeled RNA on the binding of labeled RNA by ribosomes was examined, the amount of labeled RNA bound by ribosomes in the presence of unlabeled RNA is expressed as per cent of that in the absence of unlabeled RNA.

RESULTS

RNA species bound by ribosomes In order to determine which species ot RNA are most readily bound by ribo-

somes the I M NaCl-soluble and I M NaCl-insoluble fractions of labeled kidney RNA's were incubated with kidney ribosomes.

Fig. 2 illustrates that ribosomes bind I M NaCl-insoluble RNA much more efficiently than I M NaCl-soluble RNA. It seems likely that the I M NaCl-soluble RNA bound by ribosomes is a degradation product of I M NaCl-insoluble RNA, although this has not been proved to be the case in these experiments. However, it can be concluded that the competent species of RNA is mainly I M NaCl-insoluble RNA.

25

~" o~-" ~o " 200 Ribosomes (IJg/tube)

9

u

.Q

< z ~: 0

j 0 50 150 250

Ribosomes (l~g/tube)

Fig. 2. The effect of f ract ionat ion on the binding of labeled kidney n R N A by ra t kidney ribosomes. The binding was assayed in o.2-ml react ion mix tures containing 35/zg of a2P-labeled i M NaCl-insoluble ( 0 - - - O ) or I IV[ NaCl-soluble ( × - - - × ) n R N A and varied a m o u n t s of kidney ribosomes. See MATERIALS AND METHODS for addit ional details.

Fig. 3- The effect of vary ing the a m o u n t of ra t kidney r ibosomes on the ex ten t of the binding of 32P-labeled kidney nRI~A. The binding was assayed in o.2-ml reaction mix tures containing 8. 5/*g of 3aP-labeled n R N A and varied a m o u n t s of ra t kidney ribosomes. See MATERIALS AND METHODS for addit ional details.

Binding profiles Figs. 3, 4 and 5 illustrate the binding profiles obtained when varied amounts

of rat kidney, spleen or liver ribosomes were added to 8. 5/zg of kidney nRNA, 6.5 #g

Biochim. Biophys. Acta, 2o9 (197 o) 196-2o6

200 t{. NAORA, K. KODAIRA

of spleen nRNA or lO.3 #g of phage f2 RNA, respectively, in o.2 ml of reaction mixtures. At low levels of ribosomes, i.e. up to 60 fig of kidney ribosomes, 7 ° fig of spleen ribosomes and 16o #g ot liver ribosomes, the amount of complex formed was essentially proportional to the concentration of ribosomes added. Continued addition of ribosomes resulted in the binding of more labeled RNA (Figs. 3, 4 and 5). However, the relationships were no longer linear.

9 ~3

c~

E

< z ~: 0

0 ' 160 ' 260 Ribosomes (Ng/tube)

9 i3

c~

c

m

8

< z

~8 16o ' Ribosomes (ug/tube)

2Jo

Fig. 4. The effect of vary ing the a m o u n t of ra t spleen r ibosomes on the ex ten t of the binding of asP-labeled spleen nRNA. The binding was assayed in o.2-ml reaction mixtures containing 6. 5 #g of a2P-labeled spleen n R N A and varied am oun t s of ra t spleen ribosomes. See MATERIALS AND METHODS for addit ional details.

Fig. 5. The effect of vary ing the a m o u n t of ra t liver r ibosomes on the extent of the binding of 14C-labeled phage f2 RNA. The binding was assayed in o.2-ml react ion mixtures containing lO.3 #g of t4C-labeled phage f2 RNA and varied am oun t s of ra t liver ribosomes. See MATERIALS AND METHODS for addit ional details.

At the fixed concentration of ribosomes, the amount of complex formed also increased with increasing concentrations of nRNA and finally reached a plateau. As in the case of the rat liver ribosome system, when spleen or kidney ribosomes were in excess the amount of complex formed was almost proportional to the concentration of nRNA added. Spleen ribosomes formed a complex with more than 4 ° °/o of the labeled spleen nRNA added when the mass ratio of RNA to ribosomes was I: > 3 ° in 0.2 ml of reaction mixture containing I9offg of spleen ribosomes. How- ever, the proportion of RNA bound by ribosomes decreased with increasing amount of RNA added (Fig. 6). An almost similar result was obtained with kidney nRNA and ribosomes. Therefore, the labeled nRNA, capable ot being bound by ribosomes, was not a small proportion of the labeled nRNA preparations used in this experiment. When 56 or 64 #g of spleen or kidney ribosomes were added to the reaction mixture, the concentrations of nRNA, which resulted in the complete saturation of ribosome binding sites were more than 25 ° fig per 0.2 ml (Fig. 6). However, these saturation curves varied slightly from preparation to preparation of ribosomes. Such a variation may be in part due to the availability of tree ribosome binding sites, since remov- al of endogenous mRNA from ribosomes resulted in an enhancement of the binding ~.



The profiles resulting from the binding of labeled liver nRNA and phage f2 RNA by rat liver ribosomes have been published previously 2. In this experiment, however, more than 400 #g of labeled phage f2 RNA was required for the complete saturation ot ribosome binding sites since 162 fig of liver ribosomes was used.

©

9 CL

~3 < o z 5 0 0

2 0

,oZ

~3 <

lOOO 32p- labeled RNA (Hg/ tube)

10(M

3oi

*E

0 I01

<

o Unlabeled RNA (#g / tube)

Fig. 6. The effect of vary ing the a m o u n t of 8*P-labeled n R N A added on the ex ten t of the binding by ra t spleen and kidney r ibosomes. The binding was assayed in o.2-ml reaction mixtures containing 56 or 64/zg of spleen (O) or kidney ( • ) r ibosomes and the varied amoun t s of a*P-labeled n R N A from spleen (O) or kidney ( • ) , respectively, s2P-Labeled n R N A was diluted wi th homologous unlabeled n R N A to the appropr ia te amounts . The ex ten t of the binding was expressed as bo th percent ( - - - ) and radioact iv i ty ( ) of RNA bound. The values of radioact ivi ty obtained wi th diluted labeled n R N A were corrected so as to make them comparable wi th those obtained wi th undi luted labeled nRNA. See MATERIALS AND METHODS for addit ional details.

Fig. 7. The effect of vary ing the a m o u n t of unlabeled n R N A on the binding of 3*P-labeled ra t liver n R N A by ra t liver ribosomes. The binding was assayed in o.2-ml reaction mixtures containing I 1.4/~g of 8~P-labeled ra t liver nRNA, 54/zg of ra t liver r ibosomes and varied amoun t s of unlabeled n R N A ' s prepared from ra t liver ( • - • ) , spleen ([~-[~), kidney ( 0 - 0 ) or t h y m u s cells ( x - 7, ), or from mouse liver cells ( A--/X ). The a m o u n t of labeled n R N A bound by r ibosomes in the presence of unlabeled nlRNA is expressed as a percent of t ha t in the absence of unlabeled nRNA. See MATERIALS AND METHODS f o r a d d i t i o n a l d e t a i l s .

Binding o/liver nuclear RNA by liver ribosomes In order to investigate the ability of ribosomes from one cell type to recognize

selectively genetic messages derived from the same cell type, the inhibition ot binding of labeled nRNA by unlabeled nRNA from various cell types was examined.

Fig. 7 shows the result obtained in an experiment in which the fraction of liver nRNA bound by liver ribosomes was examined as a function of the amount ot unlabeled rat liver, thymus, kidney or spleen nRNA or mouse liver nRNA added to the reaction mixture.

Some of these results have been reported in the previous paper 2. When a small amount of unlabeled rat kidney or thymus nRNA was added to

the reaction mixture, the binding of labeled liver nRNA by liver ribosomes was partially inhibited. However, addition of more than 600 fig of these unlabeled nRNA's did not result in further inhibition of binding. Nevertheless, unlabeled rat liver nRNA can efficiently inhibit the binding of labeled liver nRNA by liver ribosomes. A similar result was obtained with the liver ribosomes purified by sucrose density gradient centrifugation (Fig. 8). This inhibitory effect of unlabeled liver nRNA was not observed when RNA was treated with both 0.075 #g/ml pancreatic ribonuclease and 0.06 ffg/ml ribonuclease T 1 at 37 ° for 20 h before it was added to the reaction mixture.



These concentrations of ribonucleases did not result in digestion of labeled liver nRNA nor inhibition of binding under the assay conditions. This result clearly indicates the absence of non-specific inhibitors in the preparation of unlabeled liver nRNA added to the reaction mixture and supports the idea that ribosomes may be specialized such that they bind homologous RNA preferentially.

A more than 5o-fold excess of unlabeled nRNA prepared from mouse liver cells also failed to abolish the binding, suggesting that rat liver ribosomes can recognize the difference between rat and mouse liver nRNA.

Unlabeled nRNA from rat spleen cells inhibited the binding as efficiently as did homologous liver nRNA.

1°° I

o 1o

£

o

i

100

_~ 3c o

/3

C~

\

100 300 500 700 200 i 6~)0 ' ' ' 1000 Unlabeled RNA (#g/tube) Unlabeled RNA (pg/tube)

Fig. 8. The effect of v a r y i n g t he a m o u n t of un labe led n R N A on t h e b ind ing of 3H-labeled r a t l iver n R N A b y r a t l iver pur i f ied r ibosomes. The b ind ing was a s sayed in o .2-ml reac t ion m i x t u r e s con t a in ing 2.8 fig of 3H-labeled ra t l iver n R N A , 28/~g of r a t l iver r ibosomes purif ied b y sucrose d e n s i t y g r ad i en t cen t r i fuga t ion and va r ied a m o u n t s of un labe led n R N A ' s p repa red f rom ra t l iver ( O - O ) or t h y m u s cells ( × - × ) . See MATERIALS AND METHODS and Fig. 7 for add i t iona l detai ls .

Fig. 9. The effect of v a r y i n g t he a m o u n t of un labe led n R N A on t he b ind ing of 32P-labeled ra t k idney n R N A b y r a t k i dney r ibosomes . The b ind ing was a s sayed in o .2-ml reac t ion m i x t u r e s con t a in ing 8. 5 # g of 32P-labeled r a t k i dney n R N A , 64/~g of r a t k idney r ibosomes and var ied a m o u n t s of un labe led n R N A ' s p repa red f rom r a t k i dney ( O - O ) , t h y m u s ( × - x ) or l iver cells ( O - O ) . See MATERIALS AND METHODS and Fig. 7 for add i t iona l detai ls .

Bindin~ o/kidney nuclear RNA by kidney ribosomes To determine whether unlabeled homologous RNA is most effective as a com-

petitive inhibitor in the other systems, we carried out a series of reciprocal inhibition experiments between labeled and unlabeled nRNA's irom kidney and liver in the kidney ribosome system. If ribosomes can selectively recognize homologous RNA, unlabeled kidney nRNA, an inefficient inhibitor in the liver system, should be the most efficient inhibitor of the binding of labeled kidney nRNA by kidney ribosomes. The result presented in Fig. 9 shows that as predicted, unlabeled kidney nRNA was the most effective inhibitor in this system. It should be noted that the inhibition by unlabeled liver nRNA, an effective inhibitor in the liver system, was not as efficient as kidney nRNA in the kidney ribosome system. When more than 600 fig of unlabeled liver nRNA was added to the reaction mixture, no further significant inhibition of



the binding was observed. A similar result was obtained with thymus nRNA; addition of this RNA to the kidney system reaction mixture led only to a partial inhibition of binding.

These results clearly exclude the possibility that the efficient inhibition by unlabeled homologous RNA was caused by the presence of non-specific inhibitors in the preparations of unlabeled homologous RNA.

Binding o/spleen nuclear RNA by spleen ribosomes Fig. IO shows the result obtained in an experiment in which unlabeled spleen

or liver nRNA was added to a reaction mixture containing labeled spleen nRNA and spleen ribosomes. As can be seen in this figure, unlabeled spleen nRNA was a more efficient inhibitor than unlabeled liver nRNA. Again, this result clearly indicates that the homologous RNA was the most efficient inhibitor of binding and supports the hypothesis that ribosomes or factor(s) associated with ribosomes from one cell type preferentially bind to RNA derived from the same cell type.

1 3£

< z

100' 1

o

o o

13

2~0 ' 6~0 ' ~000' 2~0 ' 6 ; 0 ' ~ 0 Unlobeled RNA (~Jg/tube) Unlebeled RNA (IJg/tube)

Fig. IO. The effect of vary ing the a m o u n t of unlabeled n R N A on the binding of 82P-labeled ra t spleen n R N A by ra t spleen ribosomes. The binding was assayed in o.2-ml reaction mix tures containing 6. 5/zg of 8*P-labeled ra t spleen nRNA, 56/~g of ra t spleen r ibosomes and varied amoun t s of unlabeled n R N A ' s prepared f rom ra t spleen (V-I---[~) or liver cells ( 0 - 0 ) - See MATERIALS AND METHODS and Fig. 7 for addit ional details.

Fig. i i. The effect of vary ing the a m o u n t of unlabeled R N A on the binding of 14C-labeled phage f2 RNA by r a t liver r ibosomes. The binding was assayed in o.2-ml reaction mixtures containing IO. 3/zg of 14C-labeled phage f2 RNA, 162/zg of ra t liver r ibosomes and varied a moun t s of unlabeled RNA' s prepared f rom phage f2 (®-®) or isolated nuclei of ra t liver cells ( 0 - 0 ) . See MATERIALS AND METHODS and Fig. 7 for addit ional details.

Binding of phage/2 RNA by liver ribosomes To investigate the inhibitory effect of unlabeled RNA on binding in more de-

tail, an experiment in which unlabeled RNA was added to the reaction mixture containing rat liver ribosomes and labeled phage f2 RNA was carried out.

The coincidence of the inhibition curves resulting from the addition of preparations of unlabeled phage f2 and rat liver RNA's demonstrates that all the binding sites for phage f2 RNA can be occupied by rat liver nRNA (Fig. I I ) . Preliminary experiments have shown that other heterologous nRNA's, i.e. rat kidney, thymus and spleen nRNA, can inhibit the binding as efficiently as phage f2 RNA (H. NAORA AND K. KODAIRA, unpublished observations). Therefore, since phage f2 RNA failed



to abolish the binding of labeled rat liver nRNA by rat liver ribosomes 1,~, the liver- specific binding sites, if present, on the ribosomal particles or on a factor(s) associated with ribosomes cannot be occupied by non-liver nRNA's but occupied only by liver nRNA. The results obtained here also clearly indicate that the preparations of unlabeled RNA used in these experiments do not contain any non-specific inhibitors of the binding of labeled nRNA by ribosomes.

DISCUSSION

If competition experiments are carried out by incubating completely saturating amounts of labeled nRNA with ribosomes in the presence of increasing quantities of unlabeled nRNA from the same cell type as that of labeled nRNA, the radioac- t ivi ty of the complex formed should be reduced in a predictable manner. Indeed, when the amounts of unlabeled spleen or kidney nRNA added to the spleen or kidney ribosome system were more than 250/~g, the experimental values obtained in competition experiments with unlabeled nRNA homologous to labeled nRNA corre- sponded to a theoretical competition curve. However, such a correspondence was not observed at the low concentrations of unlabeled nRNA added. Since the addition of less than 250/~g of labeled spleen or kidney nRNA did not result in the complete saturation of binding sites, the deviation from the theoretical curve was interpreted as representing a partial saturation of binding sites at the low levels of RNA added. A similar interpretation could be made for the results obtained in competition experiments with liver nRNA and ribosomes, but less nRNA was required for the complete saturation of binding sites.

A method for assessing the extent of binding of labeled nRNA by ribosomes was reported in the previous papers J,~. This simple technique has been used to detect the existence of ribosomes which specifically bind certain classes of genetic messages. However, to arrive at this conclusion it has been necessary to exclude other possible explanations of the pat tern of inhibition of complex formation between ribosomes and labeled homologous RNA by unlabeled RNA.

I t is possible that non-specific inhibitory substances contaminated the unlabeled RNA preparations added to the reaction mixtures. However, the experiments reported here clearly reveal tha t this is not the case. I t is evident that unlabeled heterologous RNA's fail to abolish the binding of labeled nRNA by ribosomes in homologous systems. For example unlabeled rat liver nRNA is the most efficient inhibitor in the rat liver system but not in the other systems, and unlabeled kidney nRNA, an inefficient inhibitor in the rat liver system, is the most efficient inhibitor in the kidney system. The series of reciprocal inhibition experiments clearly indicates the absence of non-specific inhibitory substances in the preparations of the unlabeled RNA's. Furthermore, the finding that, following digestion with ribonucleases, unlabeled nRNA preparations no longer exhibited any inhibitory effect, on the binding of labeled nRNA by ribosomes, supports the above conclusion.

As discussed in the previous paper 2, it is certain that the partial inhibition of unlabeled heterologous RNA did not result from variation in the amount of mRNA present in the unlabeled RNA preparations or from alteration of the binding effi- ciency with the varied mass ratio of RNA to ribosomes. This was demonstrated in the series of reciprocal inhibition experiments with varied amounts of RNA added.


RNA's AND RIBOSOMES 2O5

There might be a possibility that the partial inhibition of unlabeled heterologous RNA is due to the alteration of the optimal binding condition, such as effective ionic concentrations, and of the enzymatic activity of ribonucleases contaminated ribosome preparations, resulting from the addition of the high concentration of unlabeled RNA to the reaction mixture. However, no apparent degradation of labeled RNA by endogenous ribonucleases was observed after incubation with ribosomes under the assay condition used, suggesting no selective degradation of heterologous or homologous RNA by ribonucleases during incubation. Furthermore the results observed in the series of reciprocal inhibition experiments and in the saturation experiments with increasing amounts of RNA suggest that this possibility is unlikely.

Another possibility that the labeled materials used were not RNA and hence not subject to competition by unlabeled RNA is excluded, since all the labeled materials were found to be Iibonuclease sensitive and the resulting digest was not bound by ribosomes.

The simplest explanation consistent with the results obtained here is that ribosomes or a factor(s) associated with ribosomes can selectively recognize homologous RNA. This hypothesis was propounded in the previous publications 1,2.

I t should be mentioned here that the ribosomes used in this experiment were not highly purified. Therefore, it is not clear at present whether ribosome particles themselves or factor(s) associated with the ribosomal particles are responsible for recognizing homologous RNA. If the latter is the case, there might be a possibility that a part of the factor(s) mentioned here is related, or similar to, the initiation and/or binding factors found on bacterial ribosomes. This possibility is now being studied. I t appears likely that labeled nRNA was bound by ribosomes or by factor(s) associated with ribosomes but not bound by similar or other materials, free of ribosomal particles. This was suggested in the previous experiments ~. The experiments with preincubated and washed ribosomes and with " m R N A " suggested that the binding indeed took place at the binding site for mRNA on the ribosomal particle or on a factor(s) associated with the particle 2. An interesting observation was that the ribosomes purified by sucrose density gradient centrifugation also exhibited the incomplete inhibition of binding by unlabeled heterologous RNA's.

The failure of heterologous RNA to fully inhibit the binding of labeled rat liver nRNA by rat liver ribosomes strongly suggests the presence, in the rat liver, of specific ribosomes or factor(s) which are incapable of binding to rat kidney or thymus nRNA but preferentially bind to rat liver nRNA. Similarly, the results indicate the presence of specific ribosomes or factor(s) in rat kidney and spleen cells. The inference that specialised ribosomes or factor(s) occur in different cell types may be pertinent to the possibility that ribosomes or factor(s) have an executive role in cell differentiation. Using a DNA-R NA hybridization technique, the specific RNA transcripts have been demonstrated in the cells of various tissues aA. Therefore, it seems likely that a fraction of the nRNA used in these experiments may specify par- ticular proteins which are unique to cells of a given tissue and hence may be only capable of being bound and translated by specific ribosomes or ribosomes containing a specific factor(s). Binding sites on ribosomes or factor(s) also appear to be species- specific, since rat liver ribosomes can distinguish the difference between DRNA's prepared from rat and mouse liver cells. However, the experiments do not provide information as to the number of ribosome or factor species present in a given type

Biochim. Biophys. Acta, 209 (x97 o) 196-2o6


of cell, nor the number of types of RNA binding site on each ribosomal particle or tactor.

It should be mentioned that although rat liver ribosomes or factor(s) cannot distinguish the difference between rat spleen and liver nRNA's rat spleen ribosomes or factor(s) are able to do so. This observation is not understood at present.

An attempt to demonstrate the ability of ribosomes to discriminate between genetic messages has failed 8-n. As has been mentioned in the previous paper 2, this iailure may be partially due to the use of a heterologous system. This contention is consistent with the obselvation that there are no quantitative diiferences in the inhibition, due to various unlabeled RNA's, of the binding of labeled phage f2 RNA by rat liver ribosomes. In fact, experiments with interferon a~-14 show that ribosomes do recognize specific RNA in homologous system and are, therefore, in good agree- ment with our observations. However, there is a need for further investigation in order to understand the nature of the differential affinities of ribosomes more clearly.

ACKNOWLEDGMENTS

The authors are indebted to Dr. D. E. A. Catcheside for help in the preparation of this manuscript. Part of this investigation was carried out at National Cancer Center Research Institute, Tokyo.

R E F E R E N C E S

I H. NAORA AND K. KODAIRA, Biochim. Bioflhys. Acta, 16I (1968) 276. 2 H. NAORA AND K. KODAIRA, Biochim. Biophys. Acta, 182 (1969) 469. 3 ]3. J. McCARTHY AND ]~. H. HOYER, Proc. Natl. Acad. Sci. U.S., 52 (1964) 915. 4 A. H. WHITELEY, ]3. J. MCCARTHY" AND H. R. WHITELEY, Proc. Natl. Acad. Sci. U.S., 55 (1966)

519. 5 J. CHAUVEAU, Y. MOULE AND C. ROUILLER, Exptl. Cell Res., I i (1956) 317 . 6 H. ~AORA, A. E. MIRSKY AND V. G. ALLFREY, J. Gen. Physiol., 44 (1961) 713 . 7 J. TsuzuKI AND H. NAORA, Biochim. Biophys. Acta, I69 (1968) 55 o. 8 J. E. DAHLBERG AND R. HASELKORN, Science, 149 (1965) 78. 9 P. ]3. MOORE, J. Mol. Biol., 18 (1966) 8.

IO J. E. DAHLBERG AND R. HASELKORN, J. Mol. Biol., 24 (1967) 83. I I M. ]~. WEKSLER AND H. V. GELBOIN, J. Biol. Chem., 242 (1967) 727 . 12 P. I. MARCUS AND J. M. SALB, Virology, 3 ° (1966) 5o2. 13 W. ~k. CARTER AND H. ]3. LEVY, Arch. Biochem. Biophys., 12o (1967) 562. 14 W. A. CARTER AND H. B. LEVY, Biochim. Biophys. Acta, 155 (1968) 437.

lBiochim. Biophys. Acta, 2o9 (197 o) 196-2o6

Documents

Interaction of informational macromolecules with ribosomes: II. Binding of tissue-specific RNA's by ribosomes