The Nucleotide Sequence of 8 S RNA Bound to Preribosomal RNA of ...

THEJOURNAI. OP BIOI.OGICAI.CHKYISTRY Vol. 2%. No. I . Issue of .January IO. pp. 5”.%9. 1983 Printed in 1J.S.A.

The Nucleotide Sequence of 8 S RNA Bound to Preribosomal RNA of Novikoff Hepatoma T H E 5’-END OF 8 S RNA IS 5.8 S RNA* (Received for publication, July 6, 1982)

Ram Reddy, Lawrence I. Rothblum, Chirala S. Subrahmanyam, Mei-Hua Liu, Dale Henning, Brandt Cassidy, and Harris Busch From the Baylor College of Medicine, Department of Pharmacology, Houston, Texas 77030

A

8 S RNA of Novikoff hepatoma was characterized by fingerprinting, sequencing gels, and by hybridization to rat ribosomal DNA clones. The data obtained show that 8 S RNA is 273 or 274 nucleotides long; ribosomal 5.8 S RNA is its 5”terminal 156 nucleotides. All the post-transcriptional modifications found in 5.8 S rRNA were also found in 8 S RNA; no other modifications were found. The 3”terminal 118 nucleotides were consistent with the adjoining internal transcribed spacer sequence in rDNA (Subrahmanyam, C. S., Cassidy, B., Busch, H., and Rothblum, L. (1982) Nucleic Acids Res. 10,3667-3680). Based on its nucleolar localization, the finding that all the 8 S RNA is hydrogen-bonded to preribosomal RNA and its consistency in sequence to the cloned rat ribosomal DNA sequence, it appears that 8 S RNA is a relatively stable intermediate in the formation of 5.8 S rRNA from 45 S pre-rRNA. This stable intermediate RNA may bea useful substrate for studies onrRNA processing andforstudies on eukaryotic rRNA-processing enzyme(s).

1

3

4

5

- ”1 - i

285

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1

0

-5

15

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rDNA 1

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B

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- -7 S

There are several small RNAs present in eukaryotic cells which are not directlyinvolved in protein synthesis(reviewed in Ref. 1).Many of the small RNAs appear tobe involved in important cellular functions. For example, U1 RNA, the most abundant of the cappedRNAs, is postulated tobe involved in transport (2) or splicing of pre-mRNAs (3, 4) and 7 S RNA (5), the most abundant of the noncapped RNAs, was shown to be part of a transport factor involved in the synthesis of secretory proteins (6). Previously, it was suggested that specific small RNAs are involved in processing preribosomal RNA inasmuch as prerRNA isolated a t 25 “C contained U 3 RNA, 7-1 RNA, and 8 S RNA(7, 8). Thisstudy wasdesigned toevaluatethe association between nucleolar preribosomal RNA and other small RNAs by hybridization of small RNAs to cloned ribosomal DNA fragments and by direct RNA sequencing. Previously, 5.8 S RNA, a product of 32 S RNA, was sequenced in this laboratory (9). The present study on the sequenceof 8 S RNA showed that it is an intermediate formed during the processing of 5.8 S rRNA from 45 S preribosomal RNA.

3

MATERIALSANDMETHODS

Novikoff hepatoma cells were labeled with [”Plphosphate as described before (10). Preparation of citric acid nuclei ( l l ) , isolation of

* This work was supported in part by Cancer Center Grant CA10893 awarded by theNationalCancerInstitute,Department of Health,Education,and Welfare. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

FIG. 1. Analysis of small RNAs hybridized to cloned rDNA fragments. Four cloned rDNA fragments corresponding to different portions of ribosomal gene were hybridizedto Novikoff nuclear 4-8 S RNA. Lanes 2 and 6 show the 4-8 S RNA used for hybridization. Lanes 1, 3, 4, and 5 show the analysis of hybridized RNAs to DNA Fragments 1,3,4, and 5, respectively. The hybridizations were carried out by the method of Kafatos et al. (18). and RNAs were analyzed on 10% polyacrylamide gels.

584

585

Nucleotide Sequenceof 8 S R N A RNA by the hot phenol method, and fractionation of 4-8 S RNA on 10%acrylamide gels were describedearlier (8).The 8 S RNA obtained wassufficientlyhomogeneous (8) to carry out sequencing studies. Fingerprinting of RNA and RNA fragments was carried out by the method of Brownlee et at. (12). RNA sequencing was carried out by the method of Peattie (13) and Donis-Keller et at. (14). The DNA sequencing wascarried outby the methodof Maxam and Gilbert (15). Plasmid subclones of cloned rat rDNA(16) were prepared as described previously (17). After linearization, the plasmids were denatured and bound to 13-mm diameter nitrocellulose and DNA-RNA hybridizations were carried out as described by Kafatos et at. (18). RESULTS

Fig. 1 shows the analysisof the small RNAswhich hybridized to several cloned ribosomal DNA fragments (16). Four different rDNA fragments were tested; they included most of the transcribed portion of the ribosomal gene (Fig. L4). Only one DNA fragment, which corresponded to the region containing the 3' terminus of 18 S rRNA, internal transcribed spacer I, 5.8 S rRNA, internal transcribed spacer 11, and the 5' terminus of 28 S rRNA (Fig. lB, Fragment 3), hybridized to 5.8 S RNA and 8 S RNA. Hybridization of 5.8 S RNA was expected since this region of DNA is known to contain the 5.8s RNA sequence. When the RNase A and TI RNase digests of 5.8 S RNA and 8 S RNA were analyzed, striking similarities were observed (Fig. 2). The fragments found in both 5.8 S and 8 S RNA were numbered 1-22 and oligonucleotides found only in 8 S RNA were numbered A-F. All the TI RNase oligonucleotides were analyzed by further digestion with U pRNase and RNase A the RNase A oligonucleotides were analyzed by digestion with TI RNase. Analysis of the results showed that all the oligonucleotides in 5.8 S RNA are also found in 8 S RNA. Analysis of 5'-End and 3'-Endsof 8 S RNA-The 5'-end of rat 5.8 S RNA had previously been shown to be heterogeneous, i.e.PC and pG were present in a ratio of 50:50 (9).The 5'-end of 8 S RNA was identical with that of 5.8 S RNA. For I

FIG.3. Analysis of 5'- and 3'-ends of 8 S RNA. A, oligonucleotide 21 from the RNase A map of 8 S RNA was digested with Tr RNase and analyzed on DEAE-paper a t pH 3.5. B , spots 20 and 21 from TI RNase fingerprint of 8 S RNA were digested with T? RNase and analyzed on DEAE-paper at pH 3.5. C, 3'-pCp-labeled 8 S RNA was digested with T1RNase and electrophoresed on 3MM paper at pH 3.5. TABLE I Analysis andquantitation of 5' and 3' termini and of modified nucleotides of 8 S RNA The values represent molarityof nucleotides obtained. 8 S RNA 5.8 S RNA RNA

5' Terminus 0.5 0.5 3' Terminus

4 a t 55 4 a t 69 0.25

FIG.2. Fingerprinting of 5.8 S and 8 S RNAs. A and B , T, RNasefingerprints of5.8 S and 8 S RNA, C and D, RNase A fingerprints of 5.8 S RNA and 8 S RNA. The fmt dimension was carried out on cellulose acetate a t pH 3.5, and the second dimension was carried out by homochromatography (12).

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PC pG COH AOH

0.5 0.5 0.7 0.3 1.o

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example, in the RNase A fingerprint of both 5.8 S and 8 S RNA, spot21 was pGACp. Analysis of RNase A spot 21 from 8 S RNA is shown in Fig. 3A. In addition, the5' terminus was in spots 20 and 21 in the TI RNase map of both 5.8 S and 8 S RNA. The analysis of TI RNase spots 20 and 21 from 8 S RNA is shown in Fig. 3B.

Nucleotide Sequenceof 8 S RNA

586

GACT

GACT

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GACT G ACT

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FIG. 4. Sequence of rDNA corresponding to the 3'-end of 8 S RNA.

B

Lanes A-D show the sequence of rDNA corresponding to the 3'-end of 8 S RNA from 143-274. The DNA sequencing was carried out by the methodof Maxam and Gilbert (15). The DNA used in lanes A. B , and C was the complementary strand.

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C

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FIG. 5. Nucleotide sequence of Novikoff hepatoma 8 S RNA. The J'-end portion of 8 S RNA from157-274 was derived from a combination of data derived from fingerprints, RNA-sequencing gels, and DNA sequencing.A-E fragments shown below the sequences are the TI RNase fragments found only in 8 S RNA (Fig. 2). and A-C fragments shown nhoce the sequence are the RNase A fragments found only in 8 S RNA (Fig. 2).

The 3'-ends of 8 S RNA and 5.8 S RNA differed. Fig. 3C shows the analysis of 3'-end-labeled 8 S RNA after it was labeled and digested with T2 RNase. Cp was found in 70% yield and Ap was found in 30% yield, indicating that the 3"

D

end, is heterogeneous. Digestion of 3"end-labeled 8 S RNA with RNase A, U r RNase, and TI RNase and analysis of the resulting labeled products showed the 3"end sequence of 8 S RNA to be -GUUC(A)OH. Modified Nucleotides in 8 S RNA-The RNase A and TI RNase oligonucleotides of 8 S RNA were analyzed for modified nucleotides (Table I). The base and sugar modifications were found in the sameoligonucleotides as in 5.8 S RNA and to the same extent as in 5.8 S RNA. These results showed that all the post-transcriptional modifications found in 5.8 S RNA are found in 8 S RNA. No additional modifications were detected. Nucleotide Sequence of 8 S RNA-Sequencing gels were obtained from 3'-end-labeled or 5"end-labeled partial TI RNase fragmentsof 8 S RNA. The G and A cleavages generated were consistent with those in the 5.8 S RNA sequence (9). The sequences from nucleotides 198-229 and 245-270 were identical with the DNA sequence (17). Fig. 4 shows the sequencing gels obtained from the rDNA corresponding to nucleotides 140-274of the 8 S RNA. The sequence of 8 S RNA in this region and the fingerprinting data obtainedfrom in vivo labeled 8 S RNA were consistentwith the DNA sequence. The TI RNase or RNase A fragments found in 8 S RNA and not in 5.8 S RNA were all in the 3'-end of 8 S RNA. The complete nucleotide sequence of 8 S RNA is shown in Fig. 5. The 5.8 S portion of 8 S RNA is shown in the box. The data show that the 5"terminal 156 nucleotides of 8 S RNA are identical with 5.8 S rRNA, and the remaining 118 nucleotides are the3'-adjoining sequence in pre-rRNA (17). Anomalous Behavior of 5.8 a n d 8 S RNAs on Acrylamide Gels-Initially the chain length of 8 S RNA was estimated at

Nucleotide Sequence of 8 S RNA

587

400 nucleotides since 8 S RNA migrated slower on the 10% polyacrylamide gels (Fig. 1) than 7 S RNA (301 nucleotides

U1 RNA (165 nucleotideslong) on 10% gel (Fig. l), but it migrates faster thanU1 RNA on 54 polyacrylamide gels (Fig. long) and 7-3 RNA (315 nucleotides long). When analyzed on 6). 5% polyacrylamide gels (Fig. 6), 8 S RNA migrated faster than Fig. 7 shows a possible secondary structure for 8 S RNA 7 S RNA, and the estimated size of 8 S RNA was consistent which is based on maximal base pairing and susceptibility of with the RNA length deduced from the sequence. Similarly, some single-strandedregions, marked by arrows, toTI RNase 5.8 S RNA which is 156 nucleotides long migrates slower than digestion. The 5'-terminal portion of the 8 S RNA structure is the 5.8 S rRNA structure determined previously in this laboratory by Nazar et al. (9). DISCUSSION

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FIG. 6. Mobility of 8 S RNA on 5% acrylamide gels. 8 S RNA isolated from 10% acrylamide gels (Fig. 1) was re-electrophoresed on 5% acrylamide-urea gels. Lanes A and C, 8 S RNA; lane E , 4-8 S

RNA.

Prestayko et al. (7) first observed that, in addition to 5.8 S RNA, U3 RNA and 8 S RNA were associated with nucleolar preribosomal RNA. Of these RNAs, only 5.8 S RNA is found as part of ribosomes in the cytoplasm. Later studies showed another RNA, designated 7-1, was also associated with nucleolar RNA (8). While only a fraction of the U3 RNA molecules were associated withpre-rRNA, all the 7-1 and 8 S RNA was associated with pre-rRNA. The present study indicates that 8 S RNA is a processing intermediate in the maturation of 5.8 S ribosomalRNA. This conclusion is based on the following observations. 1) The inferred sequence of 8 S RNA is identical with the DNA sequence derived from a cloned rat ribosomal genein the region corresponding to 5.8 S rDNA and its 3"adjoining spacer (17). 2) Sequence analysis showed the 5'-end of 8 S RNA was 5.8 S rRNA. 3) The modifications found in 8 S RNA are all found in the 5.8 S RNA portion and no additional modifications were found. 4) The 8 S RNA was found only inthe nucleolus and all of it was hydrogen-bonded to pre-rRNA. An RNA with similar characteristics was observed in yeast (19). The yeast RNA, referred to as 7 S RNA, is believed to be a processing intermediate (19). The present work establishes the likelihood of a similar processing step in the higher eukaryotes, suggesting that processing of ribosomal RNA may proceed by similar pathways inseveral species. Based on hybridization analysis of nuclear RNA, Bowman et al. (20) found that in mouse L-cells there is a 12 S RNA precursor to 5.8 S rRNA. This processing step may be rate-limiting in Novikoff hepatoma cells and may account for accumulation of the 8 S RNA intermediate (21). As there areapproximately 20,OOO-30,OOO copies of 8 S RNA/cell in Novikoff hepatoma, this RNAspecies may be useful for studies on rRNA processing and the enzyme(s)involved. Secondary Structure of 8 S RNA-The analysis of partial TI RNase products obtainedfrom 8 S RNA indicated that all the cleavage sites accessible in 5.8 S RNA are also available in 8 S RNA. This suggests that there may be two separate secondary structure domainsin the 8 S RNA, one containing the 5.8 S RNA portion and the second corresponding to the rest of the molecule (Fig. 7). The region around nucleotide 156 where processing occurs to generate mature 5.8 S RNA is highly sensitive to TI RNase digestion. These high yield cleavagesincludednucleotides 158 and 169 (Fig. 7). These data indicate that this putative processing site may be readily accessible to processing machinery. Sequence Conservation between Different Species-A 7 S

FIG.7. A possible model for the secondary structureof 8 S RNA.

Nucleotide Sequence

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FIG. 8. Comparison of RNA sequences beyond the 3'-end of 5.8 S RNA of rat (this report and Ref. 17) and Xenopus (22). The homologous nucleotides are indicated by asterisks, and the processing sites are indicated by arrows.

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FIG. 9. Possible secondary structure models for the RNA portions beyond the 3'-end of 5.8 S RNA of rat (17) and Xenopus (22). These were built to maximize hydrogen bonding and to accommodate T, RNase-sensitive sites. The stabiiity numbers are indicated next to each stem and were calculated according to Tinoco et al. (28).

G.c

c

c

u C C Novikoff hepatoma

E l

yeast

5'

C A A G U A C G G U C G U U U U A G

rat

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5'

C A A G G C C A G A C C C C C G G C

Xenopus

Xenopus

FIG. 10. Comparison of 3'-end sequences of yeast 7 S (19) and rat 8 S RNAs (this report) and the Xenopus 5.8 S precursor RNA (22). The processing sites are indicated by arrows, and the homologous sequences are shown in a box.

intermediate in yeast (19) contains 5.8 S RNA. Also, the DNA sequence beyond the 3'-end of 5.8 S RNA in Xenopus is known (22). Accordingly, comparisons were made to find homologies in primary sequence and secondary structures. The nucleotide sequence in yeast, beyond the 5.8 S RNA sequence, is A-Trich and, therefore, was not homologous to Xenopus and rat rDNA sequences. However, the Xenopus and rat RNA sequences in this region were G-C-rich and striking homologies were found in primary and in secondary structures (Fig. 9). Since the second internal transcribed spacer of Xenopus (22) is shorter (262 uersus 765 nucleotides) than in rat (17), the sequences were aligned to maximize homologies with deletions in Xenopus sequence; 78 out of 96 nucleotides (81%)were identical (Fig. 8). The secondary structure models built for the internal transcribed spacer region of 8 S RNA and the sequence beyond the 3'-end of 8 S RNA from both rat and Xenopus had striking similarities (Fig. 9). The stems had high stability numbers. From these secondary structures, it was possible to predict a processing site in Xenopus pre-rRNA (Figs. 8 and 9, arrows). Therefore, it is possible that these homologous regions in different species may contain similar primary and/or secondary structural features directing accurate processing of rRNA. The sequence around the site at which yeast 7 S RNA was processed was similar to the sequence in rat bordering the processing site (Fig. 10). Modified Nucleotides-The post-transcriptional modifications of rRNA may be an important step in processing (23, 24). The 5.8 S portion of 8 S RNA contained the corresponding

modified nucleotides reported for 5.8 S rRNA by Nazar et al. (25) and only the 5.8 S portion of the molecule contained modified nucleotides. This is consistent with previous studies on the maturation pathway of rRNA which showed that only the final products are modified and that the RNA moieties from the transcribed spacers are not modified (26, 27). Acknowledgments-We

wish to express our appreciation to Rose

K. Busch for the supply of tumor-bearing rats and to Dr. P. Epstein for useful discussions. REFERENCES 1. Busch, H., Reddy, R., Rothblum, L., and Choi, Y. C. (1982)Annu. Rev. Biochem. 51,617-654 2. Busch, H., Reddy, R., Henning, D., Spector, D., Epstein, P.,

3. 4. 5. 6. 7.

8. 9. 10.

11. 12. 13. 14.

Domae, N., Liu, M., Chirala, S., Schrier, W., and Rothblum, L. I. (1982) in Gene Regulation (O'Malley, B., and Fox, C. F., eds) Vol. XXVI, Academic Press, New York Lerner, M. R., Boyle, J. A., Mount, S. M., Wolin, S. L., and Steitz, J. A. (1980) Nature (Lond.) 283, 220-224 Rogers, J., and Wall, R. (1980) Proc. Natl. Acad.Sci. U. S. A . 77, 1877-1879 Li, W., Reddy, R., Henning, D., Epstein, P., and Busch, H. (1982) J. Biol. Chem. 257,5136-5142 Walters, P. (1982) Science (Wash. D. C.) 216, 1209 Prestayko, A. W., Tonato, M., and Busch, H. (1970) J.Mol. Biol. 47,505-515 Reddy, R., Li,W-Y., Henning, D., Choi, Y.C., Nohga, K., and Busch, H. (1981) J. Biol. Chem. 256,8452-8457 Nazar, R. N., Sitz, T. O., and Busch, H. (1975) J . Biol. Chem. 250,8591-8597 Mauritzen, C. M., Choi, Y. C., and Busch, H. (1970) Methods Cancer Res. 6,253-282 Higashi, K., Adams, H. R., and Busch, H. (1966) Cancer 26,21962201 Brownlee, G., Sanger, F., and Barrell, B. G. (1968) J.Mol. Biol. 34, 379-412 Peattie, D. A. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 17601764 Donis-Keller, H., Maxam, A. M., and Gilbert, W. (1977) Nucleic Acids Res. 4, 2527-2538

Nucleotide Sequence 15. Maxam, A. M., and Gilbert, W. (1977) Proc. Natl. Acad. Sei. U. S. A . 74,560-564 16. Rothblum, L. I., Parker, D. L., and Cassidy, B. (1982) Gene 17, 75-77 17. Subrahmanyam, C. S., Cassidy, B., Busch, H., and Rothblum, L. (1982) Nucleic Acids Res. (1982) 10,3667-3680 18. Kafatos, F. C., Jones, C. W., and Efstratiadis, A. (1979) Nucleic Acids Res. 7, 1541-1552 19. Veldman, G. M., Klootwijk, J., Heerikhuizen, H. V., and Planta, R. J. (1981) Nucleic Acids Res. 9,4847-4862 20. Bowman, L. H., Rabin, B., and Schlessinger, D. (1981) Nucleic Acids Res. 9,4951-4966 21. Perry, R. P. 1981) J . Cell Biol. 91, 28s-38s

of 8 S R N A

589

22. Hall, L. M.C., and Maden, B. E. (1980) Nucleic Acids Res. 8, 5993-6005 23. Vaughan, M. H., Soeiro, R., Warner, J. R., and Darnell, J. E. (1967) Proc. Natl. Acad.Sei. U. S. A . 58, 1527-1534 24. Perry, R. P. (1976) Annu. Rev. Biochem. 45, 605-629 25. Nazar, R. N.. Sitz, T. O., and Busch, H. (1975) FEBS Lett. 59, 83-87 26. Maden, B. E. H., and Salim, M. (1974) J . Mol. Biol. 88,133-164 27. Khan, M. S.N., and Maden, B. E. H. (1976) J . Mol. Biol. 101, 235-254 28. Tinoco, J. I., Borer, P. N., Dengler, B., Levine, M. D., Uhlenbeck, 0. C., Crothers, D. M., and Gralla, J. (1971) Nat. New B i d . 246, 40-41