Vol. 107, No. 4, 1982

August 31, 1982

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1571-1576

THE NUCLEOTIDE SEQUENCE OF THE 5' REGION OF RAT 18S rDNA AND ADJOINING SPACER

Brandt G. Cassidy, Chirla S, Subrahmanyan and Lawrence I. Rothblum

Department of Pharmacology, Baylor College of Medicine Houston, Texas 77030

Received July 26, 1982

The first sixty-two nucleotides of rat 18S rDNA, as well as eighty-five nucleotides of the adjacent external transcribed spacer have been sequenced. The homologies between the rat se- quence and those of other species demonstrate the conservation of the 5' terminus of 18S rDNA and the divergence of the adja- cent external transcribed spacers. A secondary structure model depicting the possible interactions between the 5' and 3' termini of 18S rRNA and the adjacent transcribed spacers was constructed for rat and Xenopus preribosomal RNA. This model suggests a possible configuration for the processing of pre-18S rRNA which is apparently conserved despite the divergence of the sequences of the external and internal transcribed spacers.

INTRODUCTION

The temporal sequence of the cleavage reactions that produce

mature 18S and 28S rRNA has been found to vary when the pathways

were examined in different species (i) and within the same spe-

cies (2,3). Despite this variation, it has been suggested that

a primary cleavage site in 45S rRNA is at the 5' terminus of 18S

rRNA and that this step may be required for the subsequent pro-

cessing of pre-rRNA (4).

The nucleotide sequence of the 3' terminus of rat 18S rDNA,

the internal transcribed spacers I and II, 5.8S rRNA, and the 5'

terminus of 28S rRNA has previously been reported (5). In this

study, we have determined the sequence of the 5' terminus of 18S

rDNA and a portion of the adjacent external transcribed spacer.

From this new information, it was possible to construct a second-

ary structure model which suggests that the sequences that flank

18S rRNA are involved in the maturation process.

0006-291X/82/161571-06$01.00/0 Copyright © 1982 ~ Academw Press, ~c .

1571 AHrigh~ofreproduct~n m a ~ f o r m reserved.

Vol. 107, No. 4, 1982 BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

XChR-B4 XChR-B7E12

i t T

A

XChR-C4B9 ~,NR-42

n,TT i, / 18S ~\ 5.8S 28S I J

/ " ' ' . 2 Kb

ECO RI 1 8 S

! I Z SMA I B 100 bp ,& RSA

-80 -70 -60 -50 CCCCTACCCTCCCTCCCTCCCTCCTCTCGCTCTCTCTCTCT

-40 - 3 0 - 2 0 - 1 0 CTCTCTCCCGCCTCCCGCCGCGTCTCGGCTTCGCTCGCGCT

* I0 20 30 CCTTACCTGGTTGATCCTGCCGATAGCATATGCTTGTCTCA 40 50 60 AAGATTAAGCCATGCATGTCTAAG

Figure I. Restriction map of clon&d rat rDNA (see details re- ference 6). (A) The portion of the rDNA sequenced is shown in an expanded restriction map. (B) indicates the region sequenced and Figure IB shows the sequence of the noneoding strand of 85 nucleotides up-stream plus 62 nucleotides at 5' end of 18S (*) 5' nucleotide of rat 18S rRNA as determined by R, G. Williams (9).

MATERIALS AND METHODS

Rat ribosomal DNA cloned in Charon 4A (6) was subcloned into pBR-322, and plasmid DNA was isolated as described previously (6). The subclone used in this study was pB4-5.1, a 5.1 kilobase Barn HI fragment of ~ ChR-B4 (Figure i). DNA sequencing was performed as described by Maxam and Gilbert (7) and analyzed by electrophoresis on 8% or 20%, 7M urea, acrylamide gels. Restriction enzymes from New England Biolabs were used as suggested by the manufacturer. Bacterial alkaline phosphatase and polynucleotide kinase were from

35 Bethesda Research Labs and ~y P ] ATP was purchased from Amersham Corp. DNA sequences were analyzed using the computer program of Queen and Korn (8).

RESULTS AND DISCUSSION

Figure I depicts the region of the rat ribosomal gene se-

quenced in this study and the Sequence of that region. When this

region was compared for sequence homology with the analogous re-

gions of the Xenopus and yeast (I0,Ii), it was found that the

first sixty-two nucleotides of the small ribosomal RNAs were

highly conserved (>99%) (Fig. 2). In contrast, the nucleotide

sequence of the adjacent external transcribed spacers of the rat,

Xenopus, and yeast genes were not conserved. This finding is

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Vol. 107, No. 4, 1982 BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

XENOPUS

RAT

YEAST

-20 -I0 I I0 20 CGCGCCGGGCCCGGGAAAGGUGGCUACCUGGUUGAUCCUGCCAGU II I I I I II I I I I I I I I I I I I I I I I I I I I ~ I CGUCUCGGCUUCGCUCGCGCUCCUUACCUGGUUGAUCCUGCCGAU

11 I 11111 I I I I I I I I I I I I I I I I I I I I I I X I GUUGCUUCUUCUUUUAAGAUAGUUACCUGGUUGAUCCUGCCAGU

®

XENOPUS

RAT

YEAST

30 40 50 60 AG-CAUAUGCUUGUCUCAAAGAUUAAGCCAUGCAUGUGUAAG I I I I l l l l l ] l l l l l l l l l l l l l l ] l l l l t l ] l l l l j i l l AG-CAUAUGCUUGUCUCAAAGAUUAAGCCAUGCAUGUCUAAG ]l ] l l l l l l l l ] l l l l l l l l l l l l l l l l l l l l l l l l l l l l l AGUCAUAUGCUUGUCUCAAAGAUUAAGCCAUGCAUGUCUAAG

A RA~T

18S ETS CCCCTACCTCCCTCCCTCCCTCCTCTCGCTCTCTCTCTCTCTCTCTCCCGCCTCCCGCCGCGTCTCGGCTTCGCTCGCGCTCCTT~ACCT

28S ITS II CCCGTCGTTCTCGCTCTCGCTCTCCTCTCCTCTCCTCTCCTTCCGTCGCC~CGCGCGCGCC-~CACCTCTCCTCCTTCTCCTCCTCTGGA(~

B XENOPUS

18s ETS AGGGCG CCGACCC____G CCGCCCCCCCCCCCCGGCCGCCCCCGCGCCCGCCCGCCCGCGCCGGGAAAGGTGG(~-A-CCTGGT

28S ITS I I CCGCGGGCGGGAGCGGGCCCGGCCCCCCCCCCCGGGCCGCGGCCCCGCGCCCCCCCCCCCCCCCACGACTCAGCC~

® F i g u r e 2 . C o m p a r i s o n o f t h e n u c l e o t i d e s e q u e n c e s o f r a t , X e n o p u s , and y e a s t . C o m p a r i s o n o f t h e n u c l e o t i d e s e q u e n c e s o f t h e r i b o s o m a l RNA g e n e s o f r a t , X e n o p u s and y e a s t o f t h e a r e a s immediately surrounding the 5' terminus of 18S rRNA. The ver- tical lines designate homologies to the rat sequence.

Figure 3. Comparison of the external transcribed spacers adjacent to 18S rRNA (ETS) and the internal transcribed spacers (ITS II) adjacent to 28S rRNA. (A) Rat (B) Xenopus. Pyrimidine tracts of three or more are underlined.

consistent with previous reports that although the nucleotide

sequence of the 3' termini of the small RNA of yeast, Xenopus,

and rat are conserved, the internal transcribed spacer sequences

are not (5,12,13,14).

Although the nucleotide sequences are not identical, the 5'

flanking regions of rat 18S and 28S (5) are rich in pyrimidines,

consisting of alternating "C" and "T" residues (Fig. 3A). The

analogous regions in Xenopus are also very rich in pyrimidines

marked by the strings of "C" residues (Fig. 3B) (14). These poly

pyrimidine tracts may themselves be sufficient to direct the pro-

cessing of specific cleavage. This would suggest that the base

composition, rather than a more stringent, sequence homology, of

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Vol. 107, No. 4, 1982 BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

GGCGG C A ¢ G G G Gc_GG

C-G G-U G-U U-U G-C C-G C~G G-G U-A

A °-i. C-G C~G

G-U U~G

U-G G-U C-G G~G G-C U-A C-G

~,.~ ~.~. c:~ % o-u

g , gzg

C ~ ~c ~ o-c "C--G" C-G G A U-G C-G • " , l G-U U-G U-A C-G G-U G-C • C "A U-C G-C • "U-G" G-U A~U G-U C-G U-G C-G c.C'C- G G~C t~-U "U'G-U C~G • ~ (;-U G-C C~G # ~ A IG-C G-C U~A G-C U-G U-A / G-U G-C

-CAA U~G G-C G-C. A A GG" G A 'C G - C "G

P A 'G-U" C-G O u l u.C-. C-G C U G. c C ~" u.c. ~ .C C-G C" 'u C" "C u" u U "AUUAACGGAGA&G~ "CGC~GGAGGCGCGUC" "A' "C" "C" "U

A . UAGUUGGUCCU CCUCGCGCUCUUC GCG

, a , , , 7 . . . . . °% /% u.C°% o/° o:~.c / ~ "~ o" "c c "~ c" A-U "c" "u u ' "u u' u -A "C C" C-G G'A

~ ° - c " • p - A 2~

C "u U'A G -C AZA "U B AU_G-U

c:~" G:c G -C

00 ~ ~-~ ' AA C A A G U - A / %V-~,

CIA'G ,'1aS u G 3.Ac A A c A" ,~-" C AUCAUUA G G CC CC ,_, l ~ l l t l l l I IL ~I

AA "UAGUGGU C U GG GG A A G G - C C" " l ~ C U

A AU G -C A-U U-G'C C C-G. A U~A A -U U -A C-G U

Figure 4. One of the secondary structure models for the inter- action of the 5' and 3' termini of rat 18S rRNA and adjacent spacer regions. (A) The 5' and 3' termini of rat 18S rRNA are so labeled. (a) designates the stem formed by the free 5' and 3' ends of 18S rRNA (c) designates a highly conserved hairpin loop discussed in the text. (B) A similar secondary structure built from the corresponding Xenopus sequences to the boxed portion of "A".

the flanking regions might be an essential feature for processing

pre-ribosomal RNA.

An alternate model of the mechanism of processing would be

that the processing enzyme(s) recognizes the secondary structure

that results from the interaction of the sequences to be processed.

Figure 4A is one possible secondary structure that allows for the

interaction of the 5' and 3' termini of 18S rRNA as well as the

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Vo1.107, No. 4,1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

adjacent regions of the external and internal transcribed spacers.

This model is consistent with several features of the secondary

structure proposed for mature 18S rRNA (15): I) a hairpin loop

within the 18S rRNA, region C, which is conserved in yeast,

Xenopus, and rat; 2) the 5' and 3' termini of mature 18S rRNA

have not been found in hydrogen bonded structures and so are free

to interact; and 3) the stem region, a, is in a favored thermo-

dynamic structure in the precursor which becomes less likely after

a cleavage at the 5' end of 18S rRNA. If the enclosed regions of

this model are free to interact in pre-rRNA, the structure has a

Tenoco number (16) of -12.4 suggesting that it may be a stable

secondary structure. This secondary structure model may be con-

served as it can be constructed from the same regions of the pre-

ribosomal RNA of Xenopus laevis (Fig. 4B). When this is done,

this model does not disturb the secondary structure of the inter-

nal transcribed spacer proposed by Hall and Maden (4). These

models, although generally similar to that proposed by Veldman

et al (17) for the yeast 17S rRNA precursor are slightly differ-

ent, as the relative positions of the 5' and 3' termini have been

reversed. It should be pointed out that this model utilizes the

secondary structure model proposed for mature 18S rRNA (15). In

the nascent pre-rRNA, the external transcribed spacer sequences

could interact with 18S as it is being synthesized. Hence, the

secondary structure model proposed for mature 18S rRNA may not

exist during all the phases of the synthesis and processing of

pre-rRNA. The similarity of the secondary structure of this

model and that of Veldman et al (17) argues that the secondary

structure, rather than sequence, is important in the processing

of 18S rRNA.

ACKNOWLEDGEMENTS

These studies were supported by the Cancer Research Grant

CAI0893, P9, awarded by the National Cancer Institute, DHEW.

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Vol. 107, No. 4, 1 9 8 2 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES

1. 2.

3

4

5

6

7

8

9

i0

11

12.

13.

14.

15.

16.

17.

Perry, R.P. (1976) Ann. Rev. Biochem. 45, 605-629. Bowman, L.H., Robin, B., and Schlessinger, D. (1981) Nucleic Acid Research 9, 4951-4966. Busch, H., Reddy, R., Rothblum, L., and Choi, Y.C. (1982) Ann. Rev. Biochem. 51, 617-659. Hadjiolov, A., and Nikolaev, N. (1976) Prog. Biophys. Mol. Biol. 31, 95. Subrahmanyam, C.S., Cassidy, B., Busch, H,, and Rothblum, L.I. (1982) Nucleic Acid Research, I0, 3667-3680. Rothblum, L.I., Parker, D.L., and Cassidy, B. (1982) Gene 17, 75-77. Maxam, A.M., and Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA 74, 560-564. Queen, C., and Korn, L.J. (1979) Methods in Enzymology 65, 595-609. Eladari, M-E, and Galibert, F. (1975) Eur. J. Biochem. 55, 247-255. Salim, M., and Maden, B.E.H. (1980) Nucleic Acid Research 8, 2871-2884. Bayev, A.A., Drayev, A.S., Rubstov, P.M., Skryabin, K.G., and Zacharyev, V.M. (1979) Dokl. Acad. Nauk. USSR 247, 1275- 1277. Torezynski, R., Bollon, A., and Fuke, M. (1981) Mol. Gen. Genet. 184, 557-559. Gourse, R.L., and Gerbi, S.A. (1980) J. Mol. Biol. 140, 321-339. Hall, L., and Maden, B.E.H. (1980) Nucleic Acid Research 8, 5993-6005. Zweib, C., Glotz, C., and Brimacombe, R. (1981) Nucleic Acid Research 9, 3621-3639. Tenoco, I., Borer, P., Degler, B., Levine, M., Uhlenbeck, O., Crothers, D., and Gralla, J. (1973) New Biology 246, 40- 41. Veldman, G.M., Brand, R.C., Klootwijk, J., and Planta, R.J. (1980) Nucleic Acid Research 8, 2907-2920.

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