A microsomal endoribonuclease from rat liver

324

Biochimica et Biophysica Acta, 608 (1980) 324--331 © Elsevier/North-Holland Biomedical Press

BBA 99689

A MICROSOMAL E N D O R I B O N U C L E A S E FROM R A T L I V E R

HIROSHI KUMAGAI, KAZUEI IGARASHI, TAKEMI TAKAYAMA, KAORI WATANABE, KAZUE SUGIMOTO and SEIYU HIROSE

Faculty of Pharmaceutical Sciences, Chiba University, Yayoi-cho, Chiba 260 (Japan) (Received October 16th, 1979)

Key words: Endoribonuclease; RNAase; mRNA degradation; Spermine inhibition; (Rat liver microsome)

Summary An endoribonuclease has been purified a b o u t 320-fold from the microsomes of rat liver. The enzyme had an apparent molecular weight of 54 000--58 000 and produced oligonucleotides, each consisting of 3--7 nucleotides from poly(A) and poly(U). No mononucleotide was obtained by the enzymatic hydrolysis of poly(A) and poly(U) under standard conditions. The relative rates of breakdown of synthetic polynucleotides by the enzyme under standard conditions were in the order poly(U) - poly(A) > poly(C). Divalent cations (Mg 2+ or Mn 2÷) was required for the enzymatic activity, b u t monovalent cations (Na +, K ÷ or NH~) inhibited the enzyme. The breakdown of poly(C) and poly(U) by the enzyme was inhibited by spermine, b u t that of poly(A) was n o t influenced b y spermine. The enzyme was inhibited by p-chloromercuribenzoate and poly(G), b u t n o t by rat-liver ribonuclease-inhibitor and anti-RNAase A serum.

Introduction It is thought that m R N A of Escherichia coli is attacked first b y an endoribonuclease and then the degraded m R N A is hydrolyzed by a 3'-exonuclease (E. coli RNAase II) via a processive mechanism [1,2]. If m R N A of eukaryotic cells is degraded in a manner similar to m R N A degradation in E. coli, endoribonuclease and 3'-exoribonuclease are necessary for the breakdown of mRNA. Recently, Kwan [3] and our laboratory [4] have purified a 3'-exoribonuclease from cytoplasm of HeLa cells and microsomes of rat liver, respectively. In this communication, we have purified an endoribonuclease from the microsomes of rat liver and examined the properties of this enzyme. Abbreviation: 3'-exonuclease, an exonuclease which hydrolyzes nucleic acid in the 3' --* 5' direction.

325 Materials and Methods

Materials R a t liver microsomes were prepared as described previously [4]. RNAase inhibitor was purified a b o u t 1800-fold from the rat liver 100 000 X g supernatant fraction according to the m e t h o d of Gribnau et al. [5]. E. coli RNAase I was purified from the ribosomes by the m e t h o d of Sphar and Hollingworth [6]. Rabbit anti-RNAase A serum was prepared and purified as described previously [ 7]. Bovine pancreatic RNAase A (type II-A) and venom phosphodiesterase (type II) were purchased from Sigma Chemical Co. Poly(A) was obtained from Boehringer Mannheim GmbH. Poly(U) and poly(C) were purchased from Yamasa S h o y u Co. RNAase M from Aspergillus saitoi [8] was kindly gived by Dr. M. Irie of Hoshi College of Pharmacy.

Enzyme assay The reaction mixture (0.2 ml) for the assay of endoribonuclease activity contained 100 pg poly(A), 50 mM Tris-HC1 (pH 7.5), 1 mM magnesium acetate, 0.5 mM EDTA and enzyme. After incubation of the reaction mixture at 37°C for 30 rain, the reaction was terminated by the addition of 0.2 ml 5% HC104 containing 0.25% uranyl acetate. The mixture was centrifuged after being cooled in an ice bath for 30 rain. The resulting supernatant fraction was diluted with 4 vols. H20. The acid-soluble nucleotides were measured at 260 nm. One unit of RNAase activity was defined as the a m o u n t of enzyme which caused an increase in absorbance at 260 nm of 1.0. Percent degradation was calculated from the data of absorbance at 260 nm of acid-soluble nucleotides produced by enzymatic degradation and the absorbance at 260 nm of alkaline hydrolyzate o f the same substrate.

Identification of hydrolyzed products (a) Thin.layer chromatography of hydrolyzed products. This was carried o u t as described previously [4] using polyethyleneimine-cellulose thin-layer plates. (b) Sephadex G-I O0 chromatography of hydrolyzed products. After incubation, the reaction mixture (0.2 ml) was layered on a Sephadex G-100 column (1 X 33 cm) equilibrated with 0.1 M NH4HCO3. The column was eluted with the same solution at 4°C and 1.5-ml fractions were collected. Absorbance at 260 nm of each fraction was measured without dilution. (c) DEAE-Sephadex A-25 chromatography of hydrolyzedproducts. Size distribution of oligonucleotides after enzymatic hydrolysis was determined by the m e t h o d of Bishayee and Maitra [9] with a slight modification. After incubation, the reaction mixture (2.0 ml) was diluted with 8 ml water and solid urea was added to a final concentration of 7 M. The mixture was applied to a column of DEAE-Sephadex A-25 (1.2 X 30 cm) equilibrated with:20 mM TrisHC1, pH 8.0, containing 7 M urea. A f t e r the column was washed with 5 ml of the above column-equilibrating buffer, a linear gradient elution was made with 0--0.4 M NaC1 in a buffer containing 20 mM Tris-HC1, pH 8.0, and 7 M urea. A total volume of 400 ml of buffer was used for the gradient elution. The fractions were collected in 1.5-ml samples and absorbance at 260 nm of each fraction was measured w i t h o u t dilution. The elution positions of oligonucleotides

326

of varying chain length in this column was ascertained by determining the elution profile of products formed by exhaustive digestion (37°C, 20 h) of 5 mg of yeast RNA with 200 pg of pancreatic RNAase A in 10 mM Tris-HC1, pH 7.5, in a total volume of 1 ml. Estimation of molecular weight of the endoribonuclease. The molecular weight of the endoribonuclease was estimated by gel filtration according to the procedure of Andrews [10]. The endoribonuclease (1.3 mg) was applied to a Sephadex G-100 column (2 X 102 cm) equilibrated and eluted with a buffer containing 50 mM Tris-HC1, pH 7.5, and 100 mM KCI (3-ml fractions), and the endoribonuclease activity was measured. The markers used were cytochrome c (Ms 12400), ~-chymotrypsinogen (M~ 25 000), ovalbumin (Mr 45 000), and bovine serum albumin (M~ 67000}. All markers were purchased from Boehringer Mannheim GmbH.

Purification of rat liver microsomal endoribonuclease All purification steps were carried out at 4°C. Streptomycin treatment and (NH4)2SO4 fractionation were carried out as described previously [4]. DEAE-Sephadex A-25 column chromatography. The protein precipitated between 25 and 50% saturation of (NH4)2SO4 was dissolved in buffer B (10% glycerol, 10 mM Tris-HC1, pH 7.5, 5 mM magnesium acetate and 50 mM 2-mercaptoethanol), and was applied to a DEAE-Sephadex A-25 column (9 X 7 cm) equilibrated with buffer B. The active fractions eluted with buffer B were concentrated by ultrafiltration. Sephadex G-150 gel filtration. The enzyme solution was applied to a column of Sephadex G-150 (4.2 X 91 cm) equilibrated with buffer B/0.1 M KC1. The enzyme was eluted with the same buffer (8-ml fractions; flow rate 0.5 ml/min). The active fractions were concentrated by ultrafiltration and dialyzed against buffer B/30 mM KC1. CM-Sephadex C-25 column chromatography. The enzyme solution was applied to a column of CM-Sephadex C-25 (1.4 X 10 cm) equilibrated with buffer B/30 mM KC1. The column was washed with the same buffer and eluted with a linear gradient 0.03--0.25 M KC1 in buffer B (4-ml fractions). The active fractions were concentrated by ultrafiltration and dialyzed against buffer C (10% glycerol, 20 mM Tris-HC1 (pH 7.5) and 50 mM 2-mercaptoethanol). Results

A typical purification procedure of endoribonuclease is summarized in Table I. The enzyme had been purified about 320-fold at this stage.

Endonucleolytic cleavage The hydrolyzed products of poly(A) by the enzyme from the rat liver microsomes were identified by a polyethyleneimine-ceUulose thin-layer chromatography (Fig. 1). Neither 5'-AMP nor 3'-AMP was observed as the hydrolyzed product. The average size of the hydrolyzed products was then investigated by gel filtration on Sephadex G-100 during the course of poly(A) degradation by the enzyme (Fig. 2). In contrast to 3'-exoribonuclease [4], the enzymatic digests of poly(A) contained polynucleotides of intermediate length, as shown

327 TABLE I PURIFICATION

OF RAT LIVER MICROSOMAL ENDORIBONUCLEASE

Fraction

1. 2. 3. 4. 5.

Microsomes (NH4)2SO4 DEAE-Sephadex A-25 Sephadex G-150 CM-Sephadex C-25

Volume (ml)

Total protein (mg)

T o t a l activity

1250 670 276 17.5 1.44

50 271 19 028 1 899 177 3.34

23 17 4 1

(units) 530 688 554 480 512

Specific activity (units/rag) 0.47 0.93 2.40 12.6 153

by the significant absorbance in the fractions between the original substrate and AMP. Similar results were obtained using poly(U) as substrate. These results indicate that the enzyme is an endonuclease which cannot produce mononucleotide. The hydrolyzed products were also analyzed by DEAE-Sephadex A-25 chromatography in 7 M urea (Fig. 3). Under the conditions of the analysis, most of the untreated poly(U) remained absorbed. When the products after 100% hydrolysis of poly(U) with the enzyme were analyzed, there was a marked increase in the absorbance eluting from the column corresponding to tri-, tetra-, penta-, hexa- and heptanucleotide. When poly(U) was hydrolyzed 100% by pancreatic RNAase A, the main product was 3'-UMP. Therefore, it is clear that poly(U) was endonucleolytically cleaved by the enzyme from rat liver microsomes, and the main products of degradation of poly(U) were 3--7 nucleotides in length. The similar results were obtained with poly(A).

E

Poly(A)

3"~,~MP

o

5'-AMP

3'-AMP ,I,

G o

A

B

C

D

10 Fraction

20 Number

30

Fig. 1. I d e n t i f i c a t i o n o f h y d r o l y z e d products by polyethyleneimine-cellulose thin-layer chromatography. ( A ) 2 3 % d e g r a d a t i o n o f p o l y ( A ) w i t h v e n o m p h o s p h o d i e s t e r a s e 42 # g / 0 . 2 m l ) ; (B) 1 7 % d e g r a d a t i o n o f p o l y ( A ) w i t h m i c r o s o m a l e n d o r i b o n u c l e a s e 4 0 . 2 5 u n i t / 0 . 2 m l ) ; (C) 6 0 % d e g r a d a t i o n o f P o l y ( A ) with m i c r o s o m a l e n d o r i b o n u c l e a s e ( 0 . 9 u n i t / 0 . 2 m l ) ; (D) 2 3 % d e g r a d a t i o n o f p o l y ( A ) w i t h R N A a s e M 40.4

#g/0.2 ml). Fig. 2. S e p h a d e x G - 1 0 0 c h r o m a t o g r a p h y o f h y d r o l y z e d products o f p o l y ( A ) by microsomal endoribo-~, 8% d e g r a d a t i o n o f p o l y ( A ) ( 0 . 1 u n i t e n z y m e / 0 . 2 m l ) ; o o, 1 7 % d e g r a d a t i o n o f poly(A) (0.25 unit enzyme/0.2 ml); X X, 6 0 % d e g r a d a t i o n o f p o l y ( A ) 40.9 u n i t e n z y m e / 0 . 2 m l ) .

nuclease. •

328 r A 0.50}"

I

i

025

"~

(XP)2 (XP)4 (XP)6 [XP)8 (Xp)l I (Xp)3 t(Xp)51(xp)71

O2

O,~ . . . . . . . . .

0.50t

"

ill

I0

11111

oo. ,I

.Q

O0

- - 100 Fraction

- - "

'0

zo

6o

c

I

=o

.,_

t

;

E 2(}

.....

02

1.5

0

3

Endoribonuclease (~g)

Number

Fig. 3. Size distribution of hydrolyzed products of poly(U) (I m g / 2 ml) by micTosomal endozibonuclease. (A) 6 0 % degradation of poly(U) (7 units e n z y m e / 2 ml); ( B ) 1 0 0 % degradation of poly(U) (11 units e n z y m e / 2 ml). Fig. 4. A c t i v i t y o f m i c r o s o m a l e n d o r i b o n u c l e a s e a g a i n s t v a r i o u s s u b s t r a t e s . T h e assays w e r e c a r r i e d o u t u n d e r s t a n d a r d c o n d i t i o n s e x c e p t t h a t v a r i o u s s u b s t r a t e s w e r e u s e d as i n d i c a t e d . • ~, p o l y ( A ) (I00 #g/0.2 ml); o o, p o l y ( U ) ( I 0 0 # g / 0 . 2 m l ) ; X X, p o l y ( C ) ( I 0 0 ~ g / 0 . 2 m l ) ; • A yeast R N A (200/~g/0.2 ml).

Molecular weight The molecular weight of microsomal endoribonuclease was 54 000--58 000 as estimated by gel filtration. Substrate specificity and ion requirement Fig. 4 shows the relative rates of hydrolysis of different synthetic polynucleotides and yeast RNA by microsomal endoribonuclease under standard conditions. The relative rates of breakdown of substrates were in the order poly(U) - poly(A) > poly(C) > yeast RNA. Calf thymus DNA, heat-denatured

0.8 A

B

i°,'

o

,7 o.~ r-

. _

0.4

0.4

0.2

loo

Monovalent Cation(raM)

°0

2

4

Divalent Cation(raM)

Fig. 5. E f f e c t o f salts o n t h e a c t i v i t y o f m / c r o s o m a l e n d o r i b o n u e l e a s e . T h e a s s a y s w e r e c a r r i e d o u t u n d e l s t a n d a r d c o n d i t i o n s w i t h 0.6 u n i t e n z y m e e x c e p t t h a t v a r i o u s salts w e r e a d d e d as i n d i c a t e d . ( A ) • •, KCI; o o, N H 4 C I ; X X, NaCI. (B) • --,M g C l 2 ; o o, M n C I 2 ; X .... X, CaCl 2.

329 calf thymus DNA, double-stranded RNA (poly(A)+ poly(U)), and poly(G) were not degraded by the enzyme under our experimental conditions. As shown in Fig. 5A, the addition of NaC1, NH4C1 or KC1 inhibited the enzymatic activity in the order NaC1 > NH4C1 > KC1. When sulfates and phosphates were used instead of chlorides, similar results were obtained (data not shown). From these results it may be inferred that monovalent cations inhibited the enzymatic activity. Divalent cation (Mg~÷ or Mn 2÷) was essential for the activity, the optimal concentration being 1 mM (Fig. 5B). The further addition of Mg2÷ gradually inhibited the enzymatic activity, but that of Mn 2÷ drastically inhibited the activity. Ca 2÷ could not substitute for Mg2÷ or Mn 2÷. Recently, it has been reported that polyamines can stimulate the activity of RNAases and alter their base specificity [11--17]. Therefore, the effect of spermine on microsomal endoribonuclease activity was examined using various polynucleotides as substrates. As shown in Fig. 6, the breakdown of poly(C) and poly(U) was inhibited by spermine, and that of poly(A) and yeast RNA was not influenced significantly by spermine.

Other properties The enzymatic activity was inhibited by p-chloromercuribenzoate and the optimal pH was 7.5--8 (Fig. 7). Although poly(G) could inhibit the enzymatic activity, rat liver RNAase inhibitor and anti-RNAase A serum did not inhibit the enzyme (Fig. 8).

06

100

c~

c O.t,

¢n

.~

h. u

50

c

°o

o.'1 5permine

o:z (mM)

0.2

o's

6

7

8

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pH

Fig. 6. E f f e c t o f s p e r m i n e o n t h e a c t i v i t y o f m i c r o s o m a l e n d o r i b o n u c l e a s e o n varioUs s u b s t r a t e s . • "-, 1 0 0 ~tg p o l y ( A ) a n d 0.1 u n i t e n z y m e ; o o, 1 0 0 ~g p o l y ( U ) a n d 0.1 u n i t e n z y m e ; × - X, 1 0 0 ~tg p o l y ( C ) a n d 0 , 2 u n i t e n z y m e ; • • , 2 0 0 / ~ g y e a s t R N A a n d 1.5 u n i t e n z y m e . Fig. 7. E f f e c t o f p H a n d p - c h l o r o m e r c u r i b e n z o a t e o n t h e a c t i v i t y o f m i c r o s o m a l e n d o r i b o n u c l e a s e . The a s s a y s w e r e carried o u t u n d e r s t a n d a r d c o n d i t i o n s w i t h 0.6 u n i t e n z y m e e x c e p t t h a t v a r i o u s b u f f e r s w e r e u s e d in the a b s e n c e ( e =) a n d p r e s e n c e (o . . . . . . o) o f 0.1 m M p - c h l o r o m e r c u r i b e n z o a t e . T h e b u f f e r s u s e d w e r e 50 m M p o t a s s i u m a c e t a t e b u f f e r ( p H 5.0), 50 m M piperazine-N,N'-bis(2-ethanesulfonic a c i d ) ( P i p e s ) [ N a O H b u f f e r ( p H 6 . 0 a n d 7.0), 50 m M Tris-HCl b u f f e r ( p H 7.5, 8.0 a n d 9.0), a n d 50 m M g l y c i n e / N a O H buffer (pH 10.0).

330 .

~ _ _ ~ -

,L3 C

40 Poly(G)

80 (jug)

1.1 2.2 o 2 RNAase Anti-RNAase A Inhibitor(,ug) Serurn ()Jg)

4

Fig. 8. E f f e c t o f p o l y ( G ) , r a t liver R N A a s e i n h i b i t o r , a n d a n t i - R N A a s e A semam o n t h e activities o f m i c r o s o m a l e n d o r i b o n u c l e a s e a n d o t h e r R N A a s e s . T h e assays w e r e c a r r i e d o u t u n d e r s t a n d a r d c o n d i t i o n s w i t h m i c r o s o m a l e n d o r i b o n u c l e a s e (e e, 0.4 u n i t / 0 . 2 m l ) , p a n c r e a t i c R N A a s e A (o ©, 0.2 ~ g / 0 . 2 m l ) , o r E. coli R N A a s e I (X X, 0 . 2 5 u n i t [ 1 6 ] / 0 . 2 m l ) e x c e p t t h a t p o l y ( U ) ( 1 0 0 p g / 0 . 2 m l ) was u s e d as s u b s t r a t e a n d v a r i o u s e f f e c t o r s w e r e a d d e d as i n d i c a t e d .

Discussion It has been reported that globin m R N A was degraded into small molecules directly w i t h o u t any intermediates [18]. This suggests that eukaryotic m R N A is also degraded processively. If this is the case, there are t w o possible mechanisms. One is the same mechanism of E. coli m R N A degradation, that is, m R N A is attacked first by an endoribonuclease and then the degraded m R N A is hydrolyzed by a 3'-exonuclease (3' -* 5' direction). The other is that m R N A is degraded in the 5' -* 3' direction. Since we have purified 3'-exoribonuclease (3'-~ 5' direction) [4] and endoribonuclease from rat liver microsomes, we prefer the hypothesis of the degradation of m R N A in 3' -* 5' direction. Similar endoribonuclease which cannot produce mononucleotide has been found in the nuclei of mouse L cells and chick oviduct [19--21]. There is also a report that an endoribonuclease similar to our enzyme in terms of Mg 2÷ requirement and optimal pH is induced during the early larval development of Artemia salina [22]. In addition to the existence of 3'-exoribonuclease and endoribonuclease in rat liver microsomes, there is another ribonuclease which has smaller molecular weight and no ionic requirement in rat liver microsomes (unpublished data). These properties of this enzyme are similar to those of a ribonuclease which has been purified from reticulocyte membrane by Wreschner et al. [23]. The possible biological significance of the enzyme is n o w under investigation. Since our endoribonuclease was inhibited by monovalent cations and polyamines, the enzyme does n o t seem to work during protein synthesis. If the enzyme works as a nicking enzyme, it is of interest to k n o w when it works. We are n o w examining the other properties to elucidate the biological significance of the enzyme.

331 Acknowledgements The authors would like to express their thanks to Dr. M. Irie of Hoshi College of Pharmacy for his gift of RNAase M. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

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