Proc. Nadl. Acad. Sci. USA Vol. 82, pp. 5608-5611, September 1985 Biochemistry

Is the 5S RNA a primitive ribosomal RNA sequence? (ribosome/evolutlon// amplification) Ross N. NAZAR AND WILLIAM M. WONG Department of Molecular Biology and Genetics, University of Guelph, Guelph, Canada N1G 2W1 Communicated by J. Tuzo Wilson, April 22, 1985

ABSTRACT A tandemly arranged cluster of 5S RNA-like sequence. Further comparisons also suggest that similar sequences in the middle of ribosomal 26S to 28S rRNAs from sequences are found in all rRNAs, raising an intriguing divergent eukaryotic organisms raises the possibility that the question about the evolution of the larger ribosomal RNA larger ribosomal RNAs were built up, at least in part, by gene molecules. amplification events and suggests an intriguing evolutionary relationship between the 5S rRNA and the larger rRNA molecules. MATERIALS AND METHODS DNA Preparation and Cloning. Genomic DNA was extract- The advent of recombinant DNA technology and rapid gel ed and purified from Thermomyces lanuginosus (ATCC sequencing techniques has revolutionized our understanding 16455) as described (12); the DNA was isolated from mycelia of the eukaryotic . The organization and structure of essentially by using the method of Cryer et al. (13) and ribosomal has by no means been an exception; the purified on a cesium chloride/ethidium bromide gradient. genomic organization in many organisms has already been Saccharomyces cerevisiae (ATCC 26108) genomic DNA was determined (see ref. 1) and the primary nucleotide sequences also prepared by the method of Cryer et al. except that the of high molecular weight rRNA molecules (16S to 28S DNA was not repurified on a cesium chloride/ethidium rRNAs) appear almost monthly. More recently, comparative bromide gradient. A genomic library of EcoRI-digested T. studies have even given rise to working models for the lanuginosus DNA was prepared by using the X Charon 3A secondary structures ofthe larger rRNA molecules and have vector (12, 14). In addition, to isolate the intact repeating delineated both conserved and divergent regions or domains rDNA unit, a library in X Charon 4A was made by partially (e.g., see refs. 2-9). The results suggest a largely conserved digesting genomic DNA with EcoRI restriction endonuclease secondary structure core to which divergent domains have (digestion conditions, 0.5 unit of enzyme per ,g of genomic been added, especially in higher . DNA for 30 min at 37°C). Plaques were screened for frag- While this progress in our understanding of rRNA is very ments complementary to the cytoplasmic 5S rRNAs by impressive, little is yet known about the origins of the large plaque hybridization (15) and the complementary fragments RNA molecules. Indeed, the sequence comparisons that were subsequently subcloned into pBR322 (16). have been reported indicate relatively little sequence repe- DNA Sequence Analysis of Complementary Fragments. tition or duplication (10). Nevertheless, it is probably rea- Fragments that hybridized to the cytoplasmic SS rRNAs were sonable to speculate that the large RNAs must have gradually identified by electroblot hybridization techniques (17). built up in the course of evolution. Not surprisingly, se- from T. lanuginosus or S. cerevisiae were digested with quence comparisons between the smaller prokaryotic and EcoRI or HindIII restriction endonuclease, fractionated on larger eukaryotic species (e.g., see ref. 11) are consistent with 0.8% agarose gels (18), and electrophoretically transferred to this idea. nitrocellulose membranes or activated APT paper (15). The Recently, in attempting to isolate 5S rRNA genes from a 5S rRNA probe was prepared from whole cell RNA by eukaryotic thermophile (Thermomyces lanuginosus), we repeated purification using gel electrophoresis and end- found a tandemly arranged cluster of sequences that were labeled with [_y-32P]ATP by using polynucleotide kinase (19). '='50%o homologous in nucleotide sequence with the cytoplas- To prepare DNA for sequence analysis, hybrid plasmids mic 5S rRNA and selectable by hybridization techniques (12). containing complementary fragments were digested with Unlike typical pseudogenes, these sequences were not trun- EcoRI and the inserted DNA was purified on a 0.8% agarose cated but rather bore a limited sequence homology with the slab. The complementary fragments, identified by hybridiza- entire length of the 5S rRNA and were oriented end to end tion analysis, were further digested with restriction enzymes without significant intervening sequences. We suggested that (Taq I or Hinfl), 5' end-labeled with [y-32P]ATP and se- these were gene relic sequences that had been duplicated by quenced by the chemical degradation procedure of Maxam a rolling circle-like mechanism and had evolutionarily drifted and Gilbert (20). The fragments were ordered by using to their present state, perhaps playing the role of a DNA sequence overlaps in the two restriction enzyme digests and spacer. by comparisons with the published sequence for S. cerevisiae To more clearly define the role of these sequences, in the 26S rRNA (21). present study we determined the DNA sequence surrounding the cluster of 5S RNA-like sequences and examined their RESULTS AND DISCUSSION relationship to the other rRNAs. As might have been expect- ed, these 5S RNA-like sequences were present within the As shown in Fig. 1, hybridization experiments with T. highly repeated rDNA unit but, unexpectedly, they were not lanuginosus DNA indicate that an EcoRI digest of the situated in an intervening or spacer sequence region. Instead, genomic DNA contains three fragments, -6000, '-3000, and they have been localized in the middle of the 26S rRNA -900 base pairs, to which the cytoplasmic 5S rRNA readily hybridizes. These results are similar to those observed with as illustrated The publication costs of this article were defrayed in part by page charge DNA from other fungi except, with S. cerevi- payment. This article must therefore be hereby marked "advertisement" siae DNA (also shown in Fig. 1), hybridization to the in accordance with 18 U.S.C. §1734 solely to indicate this fact. 2.5-kilobase-pair (kbp) fragment predominates (23). In that 5608 Downloaded by guest on September 26, 2021 Biochemistry: Nazar and Wong Proc. Natl. Acad. Sci. USA 82 (1985) 5609 a b c d probe was twice repurified. Furthermore, sequence analyses of the more intense middle fragment with T. lanuginosus DNA did not reveal the 5S rRNA gene; instead, we found the unusual four-copy cluster of 5S RNA-like sequences (12). In our subsequent analyses, we have now shown that all three fragments are part of the rDNA in T. lanuginosus. As >20 indicated in Fig. 1, hybridization experiments have shown Wl f 6.0 that only one much larger (-20-kbp) fragment of a HindIII OM3.0 digest readily hybridizes with the cytoplasmic 5S rRNA, and ml 2.5 all three of the EcoRI-generated fragments could be isolated with the X Charon 4A vector from partially digested genomic DNA (X Ch 4A-TL rDNA). As indicated in Fig. 2, sequence analyses show that the two smaller fragments contained the 0.9 3' half of the 5.8S rRNA sequence and the entire 26S rRNA, while the largest fragment contained the 5' half of the 5.8S rRNA preceded by the entire 18S rRNA. More important, as indicated in Fig. 3, the cluster of 5S RNA-like sequences actually constitutes the middle portion of the 26S rRNA. Furthermore, the 26S rRNA sequence is a complementary copy to the mature 5S rRNA, the same orientation that is observed between the actual 5S rRNA gene and rDNA in yeast (24). In S. cerevisiae, these sequences are both present FIG. 1. Hybridization of cytoplasmic 5S rRNA to restriction in the same tandemly repeating unit. Unlike the yeast, a endonuclease EcoRI or HindIII digests of genomic or cloned DNAs from T. lanuginosus and S. cerevisiae. The DNA (0.2-2 ,ug) was strong signal for the actual 5S rRNA gene was not observed. digested with 1-5 units ofenzyme, fractionated on a 0.8% agarose gel Since the 5S rRNA genes of T. lanuginosus and some of the (18), and electrophoretically transferred to a nitrocellulose mem- other higher fungi (e.g., see refs. 25 and 26) have been shown brane or activated APT paper (15). The filters were hybridized (22) to be heterogeneous and dispersed, the actual copy number with 32P-labeled 5S rRNA at several different temperatures ofeach gene type is likely to be relatively low when compared (50°C-75°C) overnight in 50% formamide/0.75 M sodium to the four-copy cluster of 5S RNA-like sequences in the chloride/0.075 M sodium citrate, washed extensively with the same already highly repeated rDNA. Furthermore, the signals for solution and temperature, and washed twice with 0.30 M sodium 5S RNA-like sequences are particularly strong in T. chloride/0.030 M sodium citrate at room temperature prior to because of the G+C content in this autoradiography. Results for experiments at 50°C are shown. The lanuginosus higher molecular sizes (shown in kbp) of the fragments were determined thermophile. from marker fragments: lane a, EcoRI digest of yeast genomic DNA; Since the extended sequence homology and tandem ar- lane b, HindIlI digest of T. lanuginosus genomic DNA; lane c, EcoRI rangement make a coincidental relationship highly unlikely digest of T. lanuginosus genomic DNA; lane d, EcoRI digest of X Ch (12), we examined other known 23S to 28S rRNAs for similar 4A-TL rDNA. 5S RNA-like clusters (Fig. 3). Because the nucleotide se- quence of the rRNA from the thermophilic is highly case, however, Rutter and co-workers subsequently found homologous (-90%) to that ofyeast, the entire cluster offour that this 2.5-kbp fragment contained the 5S rRNA gene and 5S RNA-like sequences could be easily identified in the yeast suggested that the other less intense signals from other sequence. This readily explains the complementarity that genomic fragments were probably the result of slight con- was observed with the 2.7-kbp fragment from yeast (Fig. 1) tamination by the other rRNAs (22). In our case, contami- because this fragment is known to contain the equivalent 26S nation cannot explain the results. The hybridization ofthe 5S rRNA sequence (21). Similarly, the mammalian 28S rRNA is rRNA to the T. lanuginosus DNA remained unchanged even largely homologous throughout this region (e.g., see ref. 11) at substantially increased temperatures or when the 5S rRNA and the entire cluster, or much of it, could be identified. In

0 3 6 9 Kbp

Ch4A 18S 5.8S 26S Ch4A

A A A Eco Eco Eco Eco

Relic Relic Relic RellIc

A Hinf HA Hinf Hinf Hint FIG. 2. Structure of the cloned T. lanuginosus rDNA and an expanded map of the 3-kbp fragment containing a cluster of 5S RNA-like sequences. Genomic DNA was partially digested with restriction endonuclease EcoRI and cloned by using the X Charon 4A vector. A complete digest of this recombinant (X Ch 4A-TL rDNA) was subcloned into pBR322 and the three complementary clones were designated pTL560, pTL530, and pTL509 (12). Each clone was further digested with Hinfl and Taq I, the nucleotide sequences were determined by the chemical degradation techniques of Maxam and Gilbert (20), and the order ofthe fragments was determined from the published ribosomal RNA sequences of yeast (21). Downloaded by guest on September 26, 2021 5610 Biochemistry: Nazar and Wong Proc. Natl. Acad. Sci. USA 82 (1985)

2150 2200 *-&.. w~ Ft P..:. ,:F.:; -1".:.. Pa _-::1-1.:B.1 __F-_:.,,*_, ..xX'K * o Rat 28S rRNA GUAGCAAA uUqAAk Ak"-'UUUO"- Gi'$".GWAJL#AGAAGGGLW"' 40GAACf.1IG06-UUGA-A-tW';';"' ""C-AGWGdUCCbG AU...,6GG;.""'W"....C CO iN '.....UCG AAAGO.'.CU 16CA CCG$U4UUM-CAGIC(*-Ucuccc.*'.... CuA#C-I'low",CCC -..--C U AGCU toAGAVIUC GUAUAUgk0.....

1450 1500 1550 a """" ""'- a a a a.a. .. :QAi k;:*;aa-S*; 'H sA;i ------M .. W AM -. uUWkU CC Yeast 26S r,-RNA-.1- GUAGCAAAUAUUCAWGkACUUt'SAAGACU..:..... &A""'-UG'GWAAAGGUU CGVCAACAGNUU."'.'.'-ACON....40GW-".-...."'..-.CGAUCt-VA.W-- ...$ U;ftl'W"',. A.GG(P.-_ 10 1'.., 00'..' -,..'G0 ----- T. lan. 26S rRNA GUAGCAAAUACUCO-A".".."'..'...... G""' CUU..... NGACUI-l". AUG A""" GGUUWUGOGAACiG CACA UfC GGIG -- CGUNG---. ACW-CAACAGC"I.~~~~~~~~~~~~UUCID.CL .GU.XUCACCCACW-MACPACUAACM-.-..CCO CGL"U"MAM.-.'...ACGG-CftGPOIG-'AC"W;UqWCCACAqWW#-WAL"

1300 ::: 1350 E. col i 23S rRNA GUAACGAUA G U. 6G ""A * GUUA ..... b-"-AGkG"""""CC..... -GAAA AAUAUUC ACCAA. kA ...... ::-.- Io .. .'.' :.". '.-.GGUU..;: .11,11. ::." -.1 .. ---- ::::: ::: '.'. .1 , :, .:: -.1,'- ":: GUARCOUGG...',.' :.% ", ;.: '.,".GA..'GUU ... .-, X.: :::: ,. ::::. :::. "I"'.1", ::::: CA ,.,:C::::: C% u UU UC CC Co..iU;-- -GGq.."A4AC O- U o------wUI:,,:4.:,:L, CO U::;: P"" ...... AUGGOUCAG."..."G':' "CLACGOCCGCCAGGCA

Copy 2

Copy 3

Copy 4 1950 FIG. 3. A comparison of 5S RNA-like sequences in the 23S to 28S rRNA from diverse origins. The sequences for the 23S to 28S rRNAs from the thermophilic fungus, yeast, rat, and E. coli RNA are aligned overall for maximum sequence homology to each other as proposed by Hassouna et al. (11) and the residue number is indicated above; in each case, a complementary sequence to the 5S rRNA from that particular organism (rat, T. lanuginosus, and E. coli) is aligned below with the degree of sequence homology for the first gene copy being indicated by the shaded areas. A detailed analysis for the remaining regions in the T. lanuginosus DNA has been published (12) and is simply represented by the repeating boxes below. fact, as shown in Fig. 3, the first repeating unit was equally primitive nature of the 5S rRNA sequence and its role in the homologous with the rat 5S rRNA, although the homologous genesis of all ribosomal RNA. In time, further insights into residues were not necessarily equivalent in both organisms. the function of the 5S rRNA and the 5S RNA-like cluster as The comparison to prokaryotic organisms was more diffi- well as their interaction with ribosomal proteins may clarify cult but equally interesting. As recently noted by Hassouna this intriguing relationship. et al. (11), this homologous region of the 26S to 28S rRNA contains one of the largest variable domains when higher This research was supported by a grant from the Natural Sciences eukaryotic examples are compared to lower eukaryotes and and Engineering Research Council of Canada. prokaryotes. As indicated in Fig. 3, when the 23S rRNA of Escherichia coli is aligned for maximum sequence homology, 1. Busch, H. & Rothblum, L., eds. (1982) The Cell Nucleus (Academic, New York), Vol. 10, pp. 1-297. it contains most of the first 5S RNA-like sequence. The 2. Noller, H. F., Kop, J., Wheaton, V., Brosius, J., Gutell, nucleotides that follow, however, appear unrelated, much as R. R., Kopylov, A. M., Dohme, F., Herr, W., Stahl, D. A., the sequence comparison between the 23S and 26S rRNA. A Gupta, R. & Woese, C. R. (1981) Nucleic Acids Res. 9, sequence deletion or recombination seems to offer reason- 6167-6189. able explanation. 3. Carbon, P., Ebel, J. P. & Ehresmann, C. (1981) Nucleic Acids Because the large ribosomal RNAs are likely to have built Res. 9, 2325-2333. up from one or more much shorter sequences, it is tempting 4. Gupta, R., Lanter, J. M. & Woese, C. R. (1983) Science 221, to speculate that at least one of these shorter sequences may 656-659. have been 5S RNA-like and that some sort of duplication or 5. Atmadja, J., Brimacombe, R. & Maden, B. E. H. (1984) gene amplification event formed the central region of the 23S Nucleic Acids Res. 12, 2649-2667. to 28S rRNA and, perhaps, some of the other regions (e.g., 6. Michot, B., Hassouna, N. & Bachellerie, J. P. (1984) Nucleic the complementary region in the 6000 and 900 bp fragments). Acids Res. 12, 4259-4279. Subsequent base substitutions, insertions, and deletions 7. Spencer, D. F., Schmare, M. N. & Gray, M. W. (1984) Proc. would then result in the rRNA structures as we know them Natl. Acad. Sci. USA 81, 493-497. today. The amplification event would appear to have oc- 8. Brimacombe, R. (1984) Trends Biochem. Sci. 9, 1-5. curred at a very early stage, because at least a truncated 9. Veldman, G. M., Klootwijk, J., de Regt, V. C. H. F., Planta, structure even occurs in the 23S rRNA. R. J., Branlant, C., Krol, A. & Ebel, J. P. (1981) Nucleic Acids 18S rRNA Res. 9, 6935-6952. Although other regions within the (6-kbp frag- 10. Otsuka, T., Nomiyama, H., Yoshida, H., Kukita, T., Kuhara, ment) also readily hybridize with the 5S rRNA (e.g., see Fig. S. & Sakaki, Y. (1983) Proc. Natl. Acad. Sci. USA 80, 1), a careful examination of these regions after further 3163-3167. fragmentation with restriction enzymes did not reveal either 11. Hassouna, N., Michot, B. & Bachellerie, J. P. (1984) Nucleic clusters or complete 5S RNA-like sequences (27); at least one Acids Res. 12, 3563-3583. of these homologous regions has previously been suggested 12. Wong, W. M., Abrahamson, J. L. A. & Nazar, R. N. (1984) to play a role in the interaction of subunits or other as yet Proc. Natl. Acad. Sci. USA 81, 1768-1770. unrecognized function (28, 29). These additional regions of 13. Cryer, D. R., Eccleshall, R. & Marmur, J. (1975) Methods Cell sequence homology may be entirely coincidental or may Biol. 12, 39-44. represent other duplication events that have evolutionarily 14. Blattner, F. R., Williams, B. G., Blechl, A. E., Denniston- drifted gradually, becoming unrelated sequences. The cluster Thompson, K., Faber, H. E., Furlong, L.-A., Grunwald, within the 23S to 28S rRNA, however, is intriguing and more D. J., Kiefer, D. O., Moore, D. D., Schumm, J. W., Sheldon, forcefully raises a very interesting possibility about the E. L. & Smithies, O. (1977) Science 196, 161-169. Downloaded by guest on September 26, 2021 Biochemistry: Nazar and Wong Proc. Nati. Acad. Sci. USA 82 (1985) 5611

15. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular 22. Bell, G. I., DeGennaro, L. J., Gelfand, D. H., Bishop, R. J., Cloning: A Laboratory Manual (Cold Spring Harbor Labora- Valenzuela, P. & Rutter, W. J. (1977) J. Biol. Chem. 252, tory, Cold Spring Harbor, New York). 8118-8125. 16. Davis, R. W., Botstein, D. & Roth, J. R. (1980) Advanced 23. Nath, K. & Bollon, A. P. (1977) J. Biol. Chem. 252, 6562- Bacterial Genetics (Cold Spring Harbor Laboratory, Cold 6571. Spring Harbor, New York). 24. Maxam, A. M., Tizard, R., Skryabin, K. G. & Gilbert, W. 17. Bittner, M., Kupferer, P. & Morris, C. F. (1980) Anal. (1977) Nature (London) 267, 643-645. Biochem. 102, 459-471. 25. Selker, E. U. & Yanofsky, C. (1981) Cell 24, 819-828. 18: Studier, F. W. (19/3) J. Mol. Biol. 79, 237-248.: 26. Wildeman, A. G. & Nazar, R. N. (1982) J. Biol. Chem. 257, 19."Donis-Keller, H., Maxam, A. &XGilbert, W. (1977) Nucleic 11395-11404. Acids Res. 4, 2527-2538. 27. Abrahamson, J. L. A. (1984) Dissertation (University of 20. Maxam, A. M. & Gilbert, W. (1978) Proc. Natl. Acad. Sci. Guelph, Guelph, Ontario, Canada). USA 74, 560-564. 28. Azad, A. A. & Lane, B. G. (1973) Can. J. Biochem. 51, 21. Georgiev, 0. I., Nikolaev, N., Hadjiolov, A. A., Skryabin, 1669-1672. K. G., Zakharyev, V. M. & Bayev, A. A. (1981) Nucleic 29. Kennedy, T. D., Hanley-Bowdoin, L. K. & Lane, B. G. Acids Res. 9, 6953-6958. (1981) J. Biol. Chem. 256, 5802-5809. Downloaded by guest on September 26, 2021