Vol.

176,

May

15, 1991

No.

3, 1991

BIOCHEMICAL

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

Kxsayuki

Ryo Kominami,

Nashinnto*,

Department

of

Biochemistry,

+Departmentof

Received

AND

March

Biochemistry,

18,

Shinzo Nishi+

1163-1169

and Yukio Mishima

Niigata University Niigata, Japan Hokkaido University Sapporo, Japan

School

of

Medicine,

School

of Medicine,

1991

SLmARYt We have found a novel spennidine-dependent endoribonuclease activity in mouse F'M3A cell extracts. This endoribonuclease cleaves RNA substrates containing a sequence CCCCCGGWUGU in its middle. This activity is lost either by heat- or microccccal nuclease-pretreatment. When heat-pretreated extracts and micrococcal nuclease-pretreated ones are mixed, the activity is restored, suggesting that this activity requires both RNA and protein components. Testing the restoration of the lost endoribonuclease activity in micrococcal nuclease-pretreated extracts by addition of fractionated cellular RNAS, we identified an approximately 65 nucleotide RNA required for this 0 1991AcademicPress, 1°C. endoribonuclease activity.

RNA processing information. essential

A special RNAs for

RNase P(2)

is

subset

catalysis.

have catalytic

splicing(3),

histone

processing(5)

a crucial

of these

to

functions pre-mRNA

in

RNAs(1) vitro.

3'-end

For

RNA processing(6),

To further RNA substrates

*To whom correspondence Biochemistry, Hokkaido Sapporo 060, Japan. Abbreviations: phosphccellulose

nt,

genetic

requires

one or

and the

RNA moiety

events

formation(J), in vitro

the ribonucleolytic

cleavages

the sequence of CCNCCGGPyWGPy in the transcribed

short

cellular

more of

such

as pre-mRNA

first

pre-rRNA

catalysis

requires

components.

We have shown that

rRNA(7).

express

activities

Self-splicing

and mitochondrial

both RNA and protein

step

investigate containing

this

occur

spacer

preferentially

regions

sequence-specificity,

a sequence

should be addressed University School

nucleotide; PA fraction, column ChraMtcgraphy.

1163

of mouse prewe

synthesized

CCCCCGGUUUGU and assayed

at present address: of Medicine, Kita

Department 15, Nishi

M KC1 flow-through

0.1

All

Copyright 0 1991 rights of reproduction

in

fraction

the

of 7, in

0006-291X/91 $1.50 by Academic Press, Inc. in any form reserved.

Vol.

176,

cleavage happened

No.

BIOCHEMICAL

3, 1991

activity to

find

AND

in mouse FM3A cell that

BIOPHYSICAL

extracts.

a novel

activity

requires

In the course

endoribonucleolytic

CCCCCGGUUUGU in a spermidine-dependent endoribonuclease

RESEARCH

manner.

Here

a 65 nt RNA conponent

MATERIALS

AND

COMMUNICATIONS

of this

study,

cleavage

occurs

we report

that

as well

we in this

as protein.

METHODS

chlstnlctionof transcription templates: The following oligonucleotides were synthesized on an Applied Biosystems DNA Synthesizer:oligo C 5'M4 5'T-3',oligo C' 5'-CIAGAGGGSACAAA CCGGGGG-3',oligo 5'-CTAGAGGGGACAAACCGCG-3'. pSPMr TCGACGCTAGGTTTGTCCCCT-3',OliqO M4' containing a portion of mouse rDNA, SalI-EhmHI fragment of pMrSP/SE(18A),(8) between the SalI and BamHI sites in pSET18(Boehringer Mannheim) was used for transcription vector. Anealed oligo C and oligo C', which have the sticky ends of SalI and XbaI sites, were cloned into pSPMr digested by Sal1 and XbaI. The resultant plasmid designated as @PC contains synthesized oligo C preceded by SP6 promoter and 25 nt spacer in coding strand. Similarly, pSPCM4 was constructed using oligo M4 and oligo M4'. All restriction enzymes used here were obtained fram Takara Shuzo. Preparation of RNA substrates: RNA substrates were synthesized by transcription of templates digested with an appropriate restriction enzyme using SP6 RNA polymerase(Takara Shuzo). The transcription reaction was specified by the Mnufacturer using [d-j2P] performed under the condition UTP(Amersham Japan). FTeparation of dluhr extracts ard their fractionation: Mouse PM3A cells were cultured in ES medium(Nissui) containing 3% fetal calf serum(GIBCD) and were harvested at a density of 5~10~ cells/ml. Preparation of SIOO extracts of FM3A cells and their fractionation by phosphocellulose column chromatography were carried out as previously described(g). 0.1 M KC1 flow-through fraction of the extracts was designated PA fraction in this paper. The PA fraction of SlOO extracts was Micrococcal nuckase pretreatment: pretreated with 5 units/u1 microccocal nuclease at 3O'C for 30 min in the was then inactivated by adding 0.02 presence of 1 mM CaC12. The nuclease volume of 100 mM EGTA(pH8.0). EZ&&lxmuclease activity assay: The reaction mixture(25 ~1) contained 5 ul of the intact PA fraction or the pretreated one of SIOO extracts in a solution containing 10 mM Hepes(pH 7.9), 0.33 mM dithiothreitol, 3.2 mM spermidine, 0.002-0.005 pool RNA substrates. After an incubation at 37OC for the indicated times, RNA was extracted with phenol, precipitated with ethanol and analyzed on a 10% polyacrylamide-8 M urea gel. kactionation of cellular RNAs and their 3'-end labeling: RNAs in the PA fraction of SIOO extracts were extracted with phenol and then with chloroform, and precipitated with ethanol. These RNAs were separated by a 15 cm long 5% polyacrylamide-8M urea gel. The polyacrylamide gel between Bromophenol Blue and Xylene Cyan01 F'F dyes were sliced in approximately 2 rsn width vertically to the current direction. Eight fractions of RNAs(numbers 1 to 8; frcm large size RNA to small) were eluted frcrn each gel strip, extracted with phenol and then with chloroform, and precipitated with ethanol. 3'-end labeled RNAs were prepared by ligation of 32pCp to the fractionated RNAs using T4 RNA ligase(Takara Shuzo) (IO). RESULTS Amvelendcoriixmucleaseactivityinmxlse dependent:

50 nt RNA substrate

m43Acellextractsisspemidimcontaining 1164

the

12

nt wild

type

sequence

Vol.

176,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

C

B

A -

0

RESEARCH

606Omin

-

0

60

0

COMMUNICATIONS

0

60min

0

60

0

60min 0

6460-

so29- e

-

11-l

-

60

0

60 min

* -60

SZ

29-

w

*

4 ll-

z

-

12345

1234

am

1

1234

2

3

4

Fiq. 'l.~dine-&per&n t endrnibonuclease activity assay. (A) 50 nt RNA substrates containing the sequence -W were synthesized from XbaI-cut pSPC by SP6 RNA polymerase. These RNAs were incubated at 37'C with the PA fraction of SlOO extracts of mouse FM3A cells for the indicated times in the presence(lanes 2 and 4) and in the absence(lane 3) of spermidine. After incubation, RNAs were analyzed on a 10% polyacrylamide-8 M urea gel. 29 and 11 nt transcripts were generated by SP6 RNA polymerase fran SalI-cut and HindIIIcut pSP!t’l8, respectively and used as size markers(lane 1). Arrowheads indicate 35 and 15 nt products. (B) Spermidinedependent endoribonuclease activity was assayed by the incubation of 64 nt RNA substrates transcribed from PvuI-cut pSPC for the indicated times(lanes 4 and 5). Lanes 1, 2 and 3 are the same as the lanes 1, 2 and 4 in (A), respectively. 35, 29 and 15 nt products are indicated by arrowheads. (C),(D) 62 nt RNA without the sequence CCCCCGGUUUGU from EcoRI-cut pSFT18(lanes 3 and 4 in C) and mutant 50 nt RNA from XbaI-cut pSPCM4, containing the sequence CGCUAGGUUUGU (lanes 3 and 4 in D) were

incubated in the PA fraction with spennidine for the indicated times. Lanes 1

and 2 in (C) and (D) are the same as the lanes 2 and 4 in 35 and 15 nt products are indicated by arrowheads.

CCCCCGGUUUGU from nucleotide

site

pSPCby SP6 RNA polymerase(Fig. fraction

with or without

30 to

(A),

respectively.

was generated frcm XbaI-digested

41

2). The RNA substrate was incubated in the PA

spermidine.

Very efficient

cleavage occured to make

35 and 15 nt products in the presence of spermidine, while cleavage was not detected

in the absence of spermidine(Fig.

specificity

of the cleavage

synthesized

from PvuI-cut

RNA from PvuII-cut

activity,

three

pSPC, EcoRI-cut

pSPC, containing

as the 50 nt RNA from XbaI-cut

To characterize

1A).

other

during

incubation

three

pSPC, was cleaved

in the spermidine-containing

nucleotide

mutant

RNA

These

results

in the PA fraction

the

with

the

12

PA fraction(Fig.

nt sequence, 1C and 2).

pSPCM4contains the sequence EUJAGGUTJUGU which

substitutions

that

64 nt

IB and 2). No cleavage was

at

the

sites

2,

4 and

was not cleaved by the spennidine-dependent show

pSm4.

were

the same 12 nt sequence in the sameregion

from EcoRI-cut pSP!1?18 without

Mutant 50 nt RNAfrcun XbaI-cut has

RNA substrates

pSFTl8 and XbaI-cut

spermidine to generate 35 and 29 nt products(Fig. detected in 62 nt transcript

sequence

novel

cleaves RNA substrates in the middle

spermidine-dependent

of 1165

5(Fig.

2).

activity(Fig.

This ID).

endoribonuclease

the sequence CCECCGWUGU(Fig. 2).

Vol.

176,

No.

BIOCHEMICAL

3, 1991

AND

BIOPHYSICAL

RESEARCH

50 -

CCCCC

c! GUUUGU-

pSPC/PwJl

-

CCCCC

d

pSPT18lEcoRI

-

pSPC/Xbal

COMMUNICATIONS

nt

64

62 50 pSPCM4/Xbal

-

nt

GUUUGU nt

nt

C@Z~qGGUUUGU-

Fig. 2.RNA substrates ard their clea~ge sites. Four RNA substrates were svnthesized bv SP6 RNAwlvmerase. 50 nt and 64 nt RNAsubstrates from XbaIc& pSX and -&uI-cut pk, respectively, contain the wild type sequence 5'CJXCXUWGU-3'. 62 nt RNA without 5'-W-3' was fran FCoRI-cut 3' was fron pSpT18 and 50 nt RNAwith the mutant sequence 5'-WLWAGWUUGUXbaI-cut pSRX4. Vertical arrows indicate the cleavage site. Underlined bases in the mutant sequence are substituted ones compared with the wild type sequence.Ccmplete sequencesof 50 nt wild type, 64 nt wild type, 62 nt and 50 nt mutant RNASare as follows: 5'-GAAUAC!AAGUJUGC!AUGCCUGCAGGUGAAUACAAGaRTGCAUG (XXDXWWWmC aC?JAG-3', 5'GAAuAc?AAGcwGcAu~G AWXXKGAAUU-3', and -31, 5'-GAAUACAAGCUUGCAU~GGUGAAUACAAGCWGCAUGCCUGCAGGUCGACUCVAGAGG 5'-~~A~~~~AG-3', respectively.

Analysis

of two cleaved products of the 50 nt wild type RNA from X.&I-cut

on a 40 cm long 10%polyacrylamide-8 size markers proved that and

that

the

cleavage

ECOZXJWGJ(data %e

eribrmease

cellular

the exact

frcxn X.&I-cut micrococcal

both

requires

the PA fraction

activity

sequential

m

&

and

nt

15

guanosines

protein:

we pretreated

was pretreated

in

inactivation

was heat-pretreated

extracts(Fig. for

was canpletely

at 90°C, while the reduced activity

the PA fraction

EGTA, the

type RNA substrates

was not detectable 3,

10 min at

lane

3).

in the

Second, the

60°C or at

903C.

lost in the heat-pretreated

at 60nC(Fig. 3, lanes 4 and 6). When we assayed the activity

1166

extracts

and heat-pretreated

PA The

extracts

was maintained in the heat-pretreated

of micrococcal nuclease-pretreated

in

nuclease in

of the nuclease with

was assayed using 50 nt wild

nuclease-pretreated

To identify

with micrococcal

pSpC. The endoribonuclease activity

endoribonuclease activity

mixture

between

coanponentsof the endoribonuclease,

endorikonuclease

fraction

sizes of the products were 35

not shown).

the presence of CaCl2. After

fraction

M urea gel with an RNA sequence ladder as

occurred

activity

two ways. First,

pSPC

PA

in the ones,

Vol.

176,

No.

BIOCHEMICAL

3, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

B

A

5d

5:

3

2

2 5n

- MNase-treated PA-Fractionated RNA-

42-l

2345676

I

_ca

L

< +%tRNA-++

--66

50-

50-

50-

03

--MN-

4

0

4 Fig. 3.'Ihe exxkzribun~lease

1234567

4

12345 activity

inthepzWxeatedPAfractim.50ntRNAs containing the sequence CCCCCGGUUUGU,generated frcm XbaI-digested psPC,were used for substrates of the spermidine-dependent endoribonuclease activity assay. These RNAs were incubated in the presence of spermidine at 37% in the PA fraction for 0 min(lane 1) and for 60 min(lane 2), in the micrococcal nuclease-pretreated PA fraction(MN) for 60 min(lane 3), in the PA fraction heat-pretreated at 60OC(~60, lane 4) and at 90OC(H90, lane 6) for 60 min. was also assayed using the mixture(5 pl) of the micrococcal Cleavage activity nuclease pretreated PA fraction(2.5 ul) and the PA fraction(2.5 ul) heatpretreated at 60°C(lane 5) or at 90"C(lane 7). The RNAs were analyzed on a 10% denaturing polyacrylamide gel. Arrowheads indicate 35 and 15 nt products. Fiq. 4.A cellular RNA ampmentrequiredfor theembribnucleaseactivity. 32P-labeled 50 nt PNAs transcribed fran X&-cut p.SPC by sP6 RNA polymerase were used for substrates of the cleavage activity assay in the presence of spermidine. Two control assays(Input RNA and PA) were performed in the same conditions as in Fig. 3, lanes 1 and 2, respectively. Arrowheads indicate 35 and 15 nt products. (A) The RNA substrates were incubated at 37OC for 60 min in the micrococcal nuclease-pretreated PA fraction without(-) or with the additional RNA fractions of numbers 1 to 8(1 to 8). (B) Incubation of the RNA substrates at 37% for 60 min in the micrococcal nuclease-pretreated PA fraction without(lane 3) or with a 32pCp-labeled single 65 nt FtNA(lane 4) was performed. The 65 nt RNA was incubated at 37OC for 60 min in the micrococcal nuclease-pretreated PA fraction without the RNAsubstrates(lane 5).

however, it lost

was ccmpletely

by phenol-pretreatment,

shown). These results and protein

identify

3, lanes 5 and 7). The activity

but not lost

show that

by DBase I-pretreatment(data

the endoribonuclease activity

theendrnibornmcleaseisapZnoociroately65

the RNA ccmponent of the endoribonuclease,

RNA of the PA fraction Each fraction

polyacrylamide fractionated

was not

needs both RNA

ccxnponents.

IheI@@~ampmh?ntof

gel.

restored(Fig.

gel.

of

SIOO

containing

lysate

by a

multiple

15

ntlmg:

we fractionated

cm denaturing

FWAs and the micrococcal 1167

activity

the whole

polyacrylamide

RNA species was recovered

The endoribonuclease

To

from the

was assayed using these

nuclease-pretreated

PA fraction.

Vol.

176,

No.

Addition

BIOCHEMICAL

3, 1991

of

the

number

pretreated

PA fraction

substrates

from XbaI-cut

To further end of its polyacrylamide

gel.

this

A single

4B).

endoribonuclease

as the intact

RNA of

These

is the single

that

nuclease-

cleaves

PA fraction(Fig.

RNA fraction,

approximately

4A).

60

indicate

that

the

the 3'-

cm denaturing

65 nucleotides

restored

the RNA

we labeled

them on a

extracts

results

COMMUNICATIONS

micrococcal

the activity

number four

nuclease-pretreated

activity(Fiq.

the

and electrophoresed

32pcP

RESEARCH

to

restored

pSPC as well

fractionate

BIOPHYSICAL

RNA fraction

ccmpletely

RNAS with

micrococcal

four

AND

with

the

endoribonuclease

the RNA component

of

the

65 nt RNA. DISCUSSION

We have found a novel mouse FM3A cell middle

lysates.

of a sequence

nt RNA components. ribonuclease

we reported the

for

spennidine-independent cytidine-stretch

RNase 65 activity and its canponent

transcribed

The downstream

we conclude

of

that

in this

physiological

activity

F!NA substrates

requires

in

in the

both protein

spermidinedependent

the rikonucleolytic spacer

was spermidine-independent

shown).

points,

this

cleaves

activity

and 65

endoribonuclease

as

65).

not

upstream

endoribonuclease

endoribonuclease

We designate

CCNCCGGPyUUGPy in activity

This

CCCCCGGUUUGU. This

65(RNase

Previously

spermidine-dependent

cleavages

regions

of muse

and micrococcal

7 nucleotides

of the

activity(7),

sequence

of

pre-rRNA(7).

The

nuclease-insensitive(data 12 nt sequence

is

important

RNase 65 seems to recognize

the

in (XEZGUW~(Fig.

2). Taking

of these

two

previously

activity

fran

the

paper. role

are

while

in the

reported

A genuine not

cellular

known.

account is different

RNA substrates

Sequence

analysis

of RWase 65 of

the

RNA

RNase 65 is now in progress.

ACKN-: We thank Dr. Y. Takahashi work was supported in part by a Grant-in-Aid Science and Culture of Japan.

for his technical fran the Ministry

advice. This of Education,

1. &ch, T. R., and B. L. Bass. (1986) Annu. Rev. Biochem. %:599-629. 2. Guerrier-Takada, C., K. Gardiner, T. Marsh, N. Pace, and S. Albran. cell -35:849-857. 1168

(1983)

Vol.

176,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Steitz, J. A., D. L. Black, V. Gerke, K. A. Parker, A. Kramer, D. Frendewey, and W. Keller. (1988) In Structure and function of major and minor small nuclear ribonucleoprotein particles. pp. 115-154. SpringerVerlag, Berlin. 4. Bimstiel, M. L., and F. J. Schaufele. (1988) In Structure and function of major and minor small nuclear ribonucleoprotein particles. pp.155182. Springer-Verlag, Berlin. 5. Kass, S., K. Tyc., J. A. Steitz, and B. Sollner-Webb. (1990) Cell -69:897908. (1987) Science =:1178-1184. 6. Chang, D. D., and. D. A. Clayton. 7. Nashimoto, M., K. Ogata, and Y. Mishima. (1988) J. Biochem.(Tokyo) m:992997. 8. Mishima, Y., T. Mitsuma, and K. Cgata. (1985) m J. 4:3879-3886. R. Kcxninami, and M. Muramatsu. (1982) Nucleic 9. Mishima, Y., I. Financsek, Acids Res. 10:6659-6670. lO.Sngland, T.,- A. Bruce, and 0. Uhlenbeck. (1980) Methcds Rnzymol. -65:65-74. 3.

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