Chapter 8 The Modified Nucleotides in Ribosomal RNA of Man and Other Eukaryotes

B265

CHAPTER 8 THE MODIFIED NUCLEOTIDES I N RIBOSOMAL RNA OF MAN AND OTHER EUKARYOTES B.E.H.

MADEN

Department o f Biochemistry. University o f Liverpool. Liverpool L69 3BX. United Kingdom

P . 0 . Box 147.

TABLE OF CONTENTS 8.1 I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . 8.2 E a r l y Analyses 8.2.1 2 -0-Methyl Groups . . . . . . . . . . . . . . C h a r a c t e r i z a t i o n o f A1 k a l i - S t a b l e Sequences 8.2.2 i n Wheat Germ r R N A . . . . . . . . . . . . . . 8.2.3 Pseudouridine 8.3 M o d i f i e d N u c l e o t i d e s and t h e rRNA M a t u r a t i o n Pathway 8.3.1 Use o f C u l t u r e d C e l l s 8.3.2 Methyl L a b e l l i n g o f r R N A and Ribosomal P r e c u r s o r RNA i n HeLa C e l l s 8.3.3 E s s e n t i a l Role o f M e t h y l a t i o n i n r R N A Maturation . . . . . . . . . . . . . . . . . . 8.3.4 Pseudouridine . . . . . . . . . . . . . . . . . 8.4 O l i g o n u c l e o t i d e Data 8.4.1 The M e t h y l a t e d N u c l e o t i d e Sequences i n HeLa C e l l r R N A and Ribosomal P r e c u r s o r RNA . . . . . 8.4.2 M e t h y l a t e d Sequences i n r R N A From O t h e r V e r t e b r a t e Sources . . . . . . . . . . . . . .

.

....................

B266 B267 B267 B268

. . . . . . . . . . . . . . . . . B269 . . B269 . . . . . . . . . . . . . B269 . . . . . . . . . . B270 .................

8.4.3

8.5

8.6

M e t h y l a t i o n i n Yeast r R N A and Ribosomal P r e c u r s o r RNA . . . . . . . . . . . . . . . . 8.4.4 I m p l i c a t i o n s o f t h e O l i g o n u c l e o t i d e Data Locating the Methylated Nucleotides 8.5.1 M o d i f i e d N u c l e o t i d e s i n 5.8s r R N A . . . . . . 8.5.2 M e t h y l a t i o n Map o f X e n o p u s L a e v i s r R N A 8.5.3 Exact L o c a t i o n s o f t h e Methyl Groups i n 18s r R N A o f X e n o p u s Laevis and Man . . . . . . . Problems and Prospects . . . . . . . . . . . . . . . 8.6.1 The Methyl Groups i n 28s r R N A . . . . . . . . 8.6.2 Pseudouridine and Other M o d i f i e d N u c l e o t i d e s .

B271 B272 B272 B272 B279

. B280 . . . 8280 . . . . . . . . . . B283 . B283 . . . . 8284 . B286 . B288 .

.

B288 B288

B266

8.6.3 Methylation S i t e s and Conformation: 18s rRNA 8.6.4 Closing Comments . . . . . . . . . . . . . . 8.7 Summary . . . . . . . . . . . . . . . . . . . . . . . . 8.8 Acknowledgements , .............. 8.9 References . . . . . . ..............

..

8.1

. 8293 . 8294 8295

. B296 . 8296

INTRODUCTION

I t i s a curious and poor y understood f a c t t h a t some types of RNA molecules contain r e l a t i v l y large numbers of modified nucleot i d e s whereas other types of RNA contain few or none. Analysis of rRNA from a variety of eukaryotes has revealed the presence o f numerous modified nucleotides. These can be c l a s s i f i e d i n t o three groups: 2'-O-methylated nucleotides, various base-modified nucleotides, and pseudouridine residues. Among the vertebrates, rRNA from human c e l l s and from the frog, X e n o p u s l a e v i s , have been studied i n d e t a i l . Among the lower eukaryotes, rRNA from the yeast, S a c c h a r o m y c e s c a r 7 s b e r g e n s i s has been studied in detail and some data are available from other sources including plants. O f particular significance has been the recent determination of the exact locations of the 40 or so methyl groups i n 18s rRNA of X . 7 a e v i s and man. The picture t h a t i s beginning t o emerge from these studies i s t h a t the modified nucleotides f i t into the functional architecture of rRNA i n a precise and i n t r i c a t e manner. Moreover, most of the nucleotide modifications are made while the ribosomal sequences are within ribosomal precursor RNA in the nucleolus. Therefore, a fundamental aim of work in t h i s area of research i s t o unravel the detailed relationship between the modified nucleotides, rRNA maturation and the overall structure of rRNA. Current knowledge of the modified nucleotides in eukaryotic rRNA has come from the application of successive new techniques, several of which have been basic t o the development of broad areas i n molecular and c e l l biology. To i l l u s t r a t e the relationship between advances in general methods and in s p e c i f i c knowledge of the modified nucleotides, I have adopted a broadly historical approach, in which key references t o the major developments are cited and the main findings and conclusions are summarized. I have emphasized work on modified nucleotides in rRNA from ver-

B267

tebrates, but have also outlined data from other eukaryotes where they have contributed importantly to the overall picture. 8.2 EARLY ANALYSES 8.2.1 2I-O-Methvl Grouos 2'-O-methylated nucleotides in RNA were first identified by Smith and Dunn (ref. 1 ) . The discovery came from finding a small proportion of a1 kali-stable dinucleotides in a1 kal ine hydrolysates of RNA from a number of sources, including wheat embryo, rat liver microsomes and rat liver "soluble" RNA. Modern methods for fractionating cell ul ar RNA species were not then avai 1 able, but it is clear in retrospect that the "rnicrosomal fraction contained mainly rRNA whereas the "soluble" RNA fraction consisted largely of tRNA. From the chemical standpoint the analysis by Smith and Dunn was fundamental. The a1 kal i-stable dinucleotides were resolved into groups by a two-stage procedure o f paper chromatography fol 1 owed by el ectrophoresis. The recovered di nucl eoti des were subjected to degradation by phosphomonoesterase, phosphodiesterase, and the Whitfield procedure (ref. 2). The products were characterized by further paper chromatography and electrophoresis. The results established the general structure NxpNp for the alkali-stable dinucleotides, where Nx is a nucleoside with a blocked 2'-OH group, linked via its 3'-OH group through a standard phosphodiester bond to the adjacent nucleoside. Evidence on the existence and nature of the blocked 2'-OH group came from a number of observations: (i) The initial fact of alkali-stability implied the absence of a normal 2'-OH group, since this group participates in the mechanism of hydrolysis by alkali. (ii) Upon electrophoresis in borate buffer (at pH 9.2) the Nx nucleosides failed to form complexes with borate, whereas ordinary ribosides form complexes due to the presence of two c i s OH groups. (iii) The RF yalues of Nx on paper chromatography (in acid and alkaline solvents) were greater than for the standard ribosides or deoxyri bosides, implying the presence of a hydrophobic group. The absorption spectra of the Nx nucleosides were as for the corresponding standard nucleosides, indicating that the bases were probably unmodified. All these observations led to the tentative identification o f the Nx compounds as 2'-0-methyl "

B268

nucleosides. Smith and Dunn also noted that a1 kal i-stable dinucleotides appeared to be absent from alkaline hydrolysates of RNA from Aerobacter aerogenes and turnip-yellow mosaic virus. Hall (ref. 3) provided further evidence that the modified sugar in the Nx nucleosides is 2'-0-methyl ribose. Each of the four presumed 2'-0-methyl nucleosides was isolated from a large preparation of "soluble yeast RNA" following enzymic digestion. The compounds were characterized by methods which were fairly similar to those used by Smith and Dunn. In addition, the nucleosides were hydrolyzed by HC1, and in each case the recovered sugar was shown to co-migrate chromatographically with chemically synthesized 2'-0-methyl ribose. 8.2.2 Characterization of Alkali-Stable Seauences i n Wheat Germ

m.

Lane and coworkers (refs. 4-6) then made a detailed study of alkali-stable sequences i n wheat germ rRNA. This study was important both from the standpoint of methodology and from the results obtained. A1 kal ine hydrolysates of RNA were separated into mononucleotides, a1 kali-stable dinucleotides and small amounts of a1 kal i-stable trinucleotides by chromatography on DEAE cellulose (refs. 4-6). The separation method was suitable for large quantities of starting material ( > lg) and hence yielded several mg of a1 kal i -stab1 e materi a1 for analysis. The materi a1 i n the a1 kal i -stab1 e di nucl eoti de peak was dephosphoryl ated with a1 kal ine phosphatase and was fractionated into individual components or isomeric pairs of compounds by paper chromatography. The a1 kal i-stable trinucleotide peak was simi larly dephosphorylated and resolved into several distinct compounds. These various compounds were then digested with snake venom phosphodiesterase to yield products as indicated:- ------- > Nm + pN di nucl eoti des: NmpN --------> Nm + pNm + pN trinuc1eotides:Nmp Nmp N These products were in turn separated by paper chromatography using a borate-saturated developing system (system 3 of ref. 4 ) , in which the 2'-0-methylated compounds were clearly distinguished from their non-methyl ated counterparts due to complexi ng o f the latter with borate. This degradation procedure, which was a

B269

r e f i n e m e n t o f t h a t o r i g i n a l l y used by Smith and Dunn, enabled t h e s t r u c t u r e s o f a l l t h e a l k a l i - s t a b l e compounds t o be determined. A p p l i c a t i o n o f t h e procedure t o t h e a1 k a l i - s t a b l e components i n wheat germ rRNA l e d t o t h e f o l l o w i n g main c o n c l u s i o n s ( r e f s 5 (i)A l l 16 p o s s i b l e a l k a l i - s t a b l e d i n u c l e o t i d e s were and 6): p r e s e n t i n wheat germ rRNA, amounting t o g e t h e r t o 3% o f t h e t o t a l (ii) The r e l a t i v e amounts o f t h e f o u r 2'-O-methyl nucleotides. r i b o s i d e s d i f f e r e d d i s t i n c t l y from t h e r e l a t i v e amounts o f t h e four normal r i b o s i d e s , i m p l y i n g t h a t a non-random s e t o f nucleo(iii)The s e v e r a l a l k a l i - s t a b l e t r i n u c l e o t i d e s i s methylated. t i d e s were a l s o c h a r a c t e r i z e d ( r e f . 6); a l l o f t h e s e were found subsequently t o be i n 28s rRNA ( r e f . 7). ( i v ) Pseudouridine was a l s o p r e s e n t , i n a q u a n t i t y which was r o u g h l y equal t o t h e t o t a l number o f 2 ' -0-methyl groups. One o f t h e a1 k a l i-stab1 e tri n u c l eoA c o r r e l a t i o n was t i d e s c o n t a i n e d pseudouridine:- UmpGmp$p. p o i n t e d o u t ( r e f . 6) between t h e occurrence o f Z'-O-methyl r i b o s i d e s and p s e u d o u r i d i n e i n RNA species f o r which a n a l y t i c a l d a t a were then a v a i l a b l e .

8.2.3 Pseudouridine Pseudouridine was f i r s t c l e a r l y d e f i n e d as an e x t r a , unident i f i e d component i n h y d r o l y s a t e s o f RNA by Davis and A l l e n ( r e f . 8) and then by Smith and Dunn ( r e f . l), a l t h o u g h t h e f i r s t s i g h t i n g had been made e a r l i e r (peak l a b e l l e d ? i n r e f . 9 and quoted i n Cohn d e f i n i t i v e l y c h a r a c t e r i z e d t h e unknown r e f s . 10 and 11). compound as 5 - r i b o s y l u r a c i l ( r e f s . 10 and 11). I t s main chemical f e a t u r e s d e r i v e from t h e carbon-carbon g l y c o s i d i c bond 1 i n k i n g C-5 o f u r a c i l w i t h C-1' o f r i b o s e , and t h e consequent a l t e r e d o r i e n t a As mentioned above ( r e f . 6) and t i o n o f the pyrimidine ring. f u r t h e r d e s c r i bed be1 ow, pseudouri d i ne i s r e 1 a t i v e l y abundant i n e u k a r y o t i c rRNA.

8.3 MODIFIED NUCLEOTIDES AND THE rRNA MATURATION PATHWAY 8.3.1 Use o f C u l t u r e d C e l l s The s t u d i e s o u t l i n e d i n t h e p r e v i o u s s e c t i o n were c a r r i e d o u t w i t h l a r g e amounts o f m a t e r i a l , and t h e p r o d u c t s a r i s i n g from t h e v a r i o u s d e g r a d a t i o n procedures were recovered i n s u f f i c i e n t q u a n t i t i e s f o r d e t e c t i o n and c h a r a c t e r i z a t i o n by u l t r a v i o l e t a b s o r p t i o n o r comparable methods. These e a r l y s t u d i e s were, o f

B270

n e c e s s i t y , p r i m a r i l y chemical i n n a t u r e . The next advances were made by studying RNA metabolism i n c u l t u r e d c e l l s . Cultured c e l l s a f f o r d a number o f advantages f o r metabolic s t u d i e s . F i r s t , they comprise a homogeneous population of growing c e l l s . Second, r a d i o a c t i v e l a b e l l i n g o f n u c l e i c a c i d s i s r e a d i l y performed. T h i r d , rRNA and i t s n u c l e o l a r p r e c u r s o r molecules can be i s o l a t e d and f r a c t i o n a t e d r e l a t i v e l y e a s i l y . The major steps i n the maturation pathway f o r rRNA i n the n u c l e o l i of animal c e l l s were worked o u t l a r g e l y by t h e use of cultured c e l l s . 45s RNA i s the primary t r a n s c r i p t and common p r e c u r s o r t o 18s and 28s rRNA i n mammal i an cel Is, and 32s RNA i s an i n t e r m e d i a t e i n the maturation of 28s rRNA. Refs. 12 and 13 a r e e a r l y reviews. Nucleotide m o d i f i c a t i o n s f e a t u r e prominently i n the rRNA maturation pathway, and a l s o f e a t u r e d i n i t s e l u c i d a t i o n , a s summari zed i n t h e f o l 1 owing paragraphs. Methyl Labellina of rRNA and Ribosomal P r e c u r s o r RNA in HeLa C e l l s Brown and A t t a r d i ( r e f . 14) were among the f i r s t t o apply methyl l a b e l l i n g t o the study of n u c l e i c a c i d s i n animal c e l l s . They grew HeLa c e l l s (a l i n e of c u l t u r e d human c e l l s ) f o r two g e n e r a t i o n s i n E a g l e ’ s medium which had been prepared f r e e of methionine and supplemented with l%-methyl methionine. The RNA was then i s o l a t e d and 28s and 18s rRNA were s e p a r a t e d by s u c r o s e gradient centrifugation. The r e s u l t s showed i n c o r p o r a t i o n of methyl l a b e l i n t o both 28s and 18s r R N A , the 18s rRNA being l a b e l l e d t o a somewhat higher s p e c i f i c a c t i v i t y than 28s rRNA. P a r t i a l a n a l y s i s of the methylated components suggested t h a t most of the r a d i o a c t i v i t y was i n 2’-O-methyl r i b o s e with some l a b e l l i n g a1 so of speci f i c bases. Greenberg and Penman ( r e f . 15) then made the important discovery t h a t methylation t a k e s p l a c e a t t h e l e v e l of 45s r i b o somal p r e c u r s o r RNA i n the nucleolus. By using s h o r t pulse l a b e l l i n g with 1%-methyl methionine, followed by c e l l f r a c t i o n a t i o n and s u c r o s e g r a d i e n t c e n t r i f u g a t i o n of RNA, t h e y obtained evidence t h a t methylation t a k e s p l a c e on nascent c h a i n s of 45s RNA. During longer l a b e l l i n g p e r i o d s , o r following a chase with 8.3.2

B271

excess unl abel 1 ed methi oni ne, 1abel passes i n t o nucl eol a r 32s RNA and cytoplasmic 18s and 28s rRNA. S i m i l a r general conclusions were reached by Zimmerman and H o l l e r ( r e f . 1 6 ) , using s l i g h t l y d i f f e r e n t methods. When HeLa c e l l s were l a b e l l e d f o r 30 min. with 14C-methyl methionine, and actinomycin was added t o block f u r t h e r RNA s y n t h e s i s , the methyl l a b e l was chased i n t o 32s and 18s RNA. When actinomycin was added 5 minutes p r i o r t o 14C-methyl methionine no l a b e l was taken up i n t o 45s RNA, implying t h a t methylation o f 45s RNA i s c l o s e l y coupled t o s y n t h e s i s . However, methyl l a b e l was taken u p i n t o dimethyladenosine i n 18s rRNA under these c o n d i t i o n s ( r e f . 1 7 ) . This was t h e f i r s t evidence f o r a small group of l a t e m e t h y l a t i o n s i n rRNA m a t u r a t i o n , f u r t h e r discussed below ( s e c t i o n 1 7 . 4 . 1 ) . Wagner e t . a l . ( r e f . 18) then c a r r i e d o u t a chemical a n a l y s i s of the a1 kal i - s t a b l e sequences i n HeLa c e l l rRNA and i t s nucl eol a r precursors. The methyl -1 abel 1 i ng approach was appl i e d , i n cont r a s t t o the e a r l i e r s t u d i e s ( r e f s . 4-6) on l a r g e - s c a l e preparat i o n s of u n l a b e l l e d rRNA. A f t e r s e p a r a t i o n of the RNA s p e c i e s by s u c r o s e g r a d i e n t c e n t r i f u g a t i o n , a1 kal i n e h y d r o l y s i s was c a r r i e d o u t , followed by dephosphorylation and s e p a r a t i o n of the r e s u l t i n g compounds by chromatography on DEAE Sephadex. The recovered compounds were i d e n t i f i e d by t h e i r e l e c t r o p h o r e t i c m o b i l i t i e s a t pH3 and by cleavage with venom phosphodiesterase t o y i e l d the methyl-label l e d n u c l e o s i d e s . The important conclusions from t h i s work were: ( i ) t h e methylation p a t t e r n s of HeLa c e l l 18s and 28s rRNA d i f f e r e d from each o t h e r , ( i i ) t h e 2'-0-methylation p a t t e r n of 45s RNA resembled t h a t of an equimolar mixture of 18s and 28s rRNA and ( i i i ) t h e 32s p a t t e r n resembled t h a t of 28s rRNA. T h u s , t h e s e d a t a provided chemical confirmation of t h e evidence from RNA l a b e l l i n g k i n e t i c s and i n h i b i t o r s t u d i e s t h a t most of the methyl groups o f rRNA a r e added t o ribosomal p r e c u r s o r RNA i n the nucleolus. (Subsequent work has shown t h a t the e s t i m a t e s o f the abs o l u t e numbers of methyl groups i n this s t u d y were n o t q u i t e a c c u r a t e , b u t t h e general conclusions ( i ) t o ( i i i ) remain v a l i d , a s f u r t h e r d e t a i l e d i n s e c t i o n 8 . 4 . 1 , below). 8.3.3

E s s e n t i a l Role of Methvlation i n rRNA Maturation A key q u e s t i o n which a r o s e from t h e s e s t u d i e s was whether the methylation of ribosomal p r e c u r s o r RNA plays an e s s e n t i a l r o l e i n

ribosome m a t u r a t i o n . T h i s q u e s t i o n was addressed i n a s e r i e s o f HeLa c e l l s were susexperiments by Vaughan e t . a l . ( r e f . 19). pended i n m e t h i o n i n e - f r e e medium f o r s e v e r a l hours and were then Analysis o f l a b e l l e d w i t h 14C u r i d i n e o r adenosine f o r 2.5 h r . RNA from t h e v a r i o u s c e l l f r a c t i o n s on sucrose g r a d i e n t s showed t h a t 45s RNA c o n t i n u e d t o be s y n t h e s i z e d and processed i n t o 32s RNA i n t h e nucleolus, b u t no newly formed rRNA appeared i n t h e cytoplasm. I n a p a r a l l e l c u l t u r e d e p r i v e d o f v a l i n e , an e s s e n t i a l amino a c i d which has no s p e c i a l r o l e i n rRNA m e t h y l a t i o n , r R N A c o n t i n u e d t o appear i n t h e cytoplasm a t a reduced r a t e (see a l s o r e f s . 20 and 21). The 45s and 32s RNA f r o m m e t h i o n i n e - d e p r i v e d c e l l s were s u b j e c t e d t o a1 k a l i n e h y d r o l y s i s f o l l o w e d by chromatography on DEAE c e l l u l o s e and were found t o be s e v e r e l y d e f i c i e n t i n 2'-0-methyl groups: 45s RNA possessed o n l y 20% o f t h e 2'-0m e t h y l a t i o n found i n c o n t r o l c e l l s , and 32s RNA possessed about 30-40% o f t h e normal l e v e l o f 2 ' - 0 - m e t h y l a t i o n . These r e s u l t s s t r o n g l y i m p l i e d t h a t m e t h y l a t i o n o f ribosomal p r e c u r s o r RNA was t h e f a c t o r l i m i t i n g ribosome m a t u r a t i o n d u r i n g m e t h i o n i n e d e p r i vation. Moreover, t h e continued p r o d u c t i o n o f 45s and 32s RNA t o g e t h e r w i t h t h e 1ack o f appearance o f c y t o p l a s m i c rRNA i n d i c a t e d t h a t t h e m e t h y l - d e f i c i e n t RNA was b e i n g degraded a t a r e l a t i v e l y I t was shown i n an acl a t e stage i n t h e m a t u r a t i o n pathway. t i n o m y c i n chase experiment t h a t a t l e a s t some o f t h e m e t h y l d e f i c i e n t RNA c o u l d be rescued by a d d i t i o n o f m e t h i o n i n e . 8.3.4

Pseudouridi ne The presence o f pseudouridine i n HeLa c e l l rRNA and ribosomal p r e c u r s o r RNA was r e p o r t e d by A t t a r d i and co-workers ( r e f s . 22 and 23). Thus, t h i s t y p e o f m o d i f i c a t i o n a l s o t a k e s p l a c e a t t h e l e v e l o f 45s RNA. The amounts o f pseudouridine i n h y d r o l y s a t e s o f t h e v a r i o u s RNA species were o f t h e o r d e r o f 1-2% o f t h e t o t a l n u c l e o t i d e s , t h e h i g h e s t r e l a t i v e c o n t e n t b e i n g i n 18s r R N A . R e f i n e d e s t i m a t e s o f t h e pseudouridine c o n t e n t o f r R N A a r e g i v e n l a t e r , below.

8.4 OLIGONUCLEOTIDE DATA 8.4.1 The M e t h v l a t e d N u c l e o t i d e Seouences i n HeLa C e l l r R N A and R i bosomal Precursor RNA The n e x t major advances came from a n a l y s i s o f t h e m e t h y l a t e d

B273

oligonucleotides released by enzymic digestion of rRNA and ribosomal precursor RNA. The methods which had been developed by Sanger and Brownlee for electrophoretic separation and sequence analysis of radioactive oligonucleotides (refs. 24, 25) were first applied to the modified nucleotides in rRNA by Fellner (ref. 26), who analyzed the methylated sequences in E . c o 7 f rRNA. E . c o 7 i rRNA contains a relatively small number of methyl groups, most of which occur on bases. (See also the chapter by Ebel in this treatise.) By contrast, the methylation patterns of HeLa cell rRNA and its nucleolar precursors are quite complex when analyzed at the oligonucleotide level. Successive stages i n the description and analysis of the methylated oligonucleotides in rRNA and its precursors from HeLa cells were reported in refs. 27-32, and some additional findings were reported later. The following account summarizes the main features and findings of the analysis. (i) Methods. Ref. 32 contains a full account of the methods that were employed for preparing rRNA and nucleolar RNA after i n v i v o labelling of HeLa cells with 14C-methyl methionine or 32P04, and a1 so the procedures for enzymic hydrolysis of RNA, separation of the ol igonucl eotides by two-dimensional electrophoresis ('fingerprinting') and characterization of the oligonucleotides. Two methodological points may be mentioned here. First, to obtain RNA that is radioactively labelled exclusively in its methyl groups it is necessary to carry out labelling in the presence of adenosine, guanosine (2 x 10-5M each) and sodium formate (10-2M). In the absence of these compounds, small amounts of carbon label enter the "one-carbon pool" and purine biosynthetic pathway. Cells labelled in the presence of purine nucleosides and formate yielded RNA with no trace of purine ring labelling, as shown by the complete absence of label in nonmethylated but abundant T, ribonuclease products such as Gp and APGP. Secondly, the procedure for purifying nucleol i and obtaining nucleolar RNA was developed for HeLa cells (ref. 33) and has worked well for these cells, but may not necessarily work well for other cell types. Moreover, the nucleolar processing pathway is slower in HeLa cells than in many other cell types; hence the steady-state levels of 45s and 32s RNA are higher and good yields of these molecules can be obtained. These considerations underlay

B274

,

the choice of HeLa c e l l s f o r the work in r e f . 32 as well as f o r many e a r l i e r studies, including those outlined i n section 8.3, above. ( i i ) Numbers of methyl aroutx in rRNA. When T, ribonuclease fingerprints of methyl labelled rRNA were f i r s t obtained ( r e f s . 2 7 , 28) i t was apparent t h a t many methylated oligonucleotides were approximately equally labelled. The simplest i n t e r p r e t a t i o n was t h a t each of these equally labelled products was present once per molecule of rRNA. However, Fellner ( r e f . 26) had o r i g i n a l l y inferred t h a t most of the methylated oligonucleotides i n f . c o 7 i rRNA occurred twice per molecule. (The frequencies were corrected l a t e r t o once per molecule: r e f . 34). Conversely, the numbers of methyl groups per HeLa rRNA molecule, assuming unimol a r frequenc i e s , were i n e x c e s s of the numbers previously estimated by Wagner e t . a 1 . ( r e f . 18). I t was c l e a r l y necessary t o resolve the question of the numbers of methyl groups. The following approach made t h i s possible ( r e f s . 29, 30). When 32P labelled rRNA was digested w i t h combined T, plus pancreatic ri bonucleases, not o n l y were the expected s t a n d a r d products obtained (Up, Gp, Cp and t r a c t s of A residues terminated by Up, Gp, or Cp) b u t also an array of e x t r a products. Nearly a l l of the extra products possess Z'-O-rnethylated nucleotides which confer resistance t o cleavage by the respective enzymes. Several of these extra products were completely separated from non-methylated products in fingerprints ( r e f . 29). The s t r u c t u r e s of these products were analyzed, and t h e i r mol ar y i e l d s were determi ned with reference t o the t o t a l 32P label in the f i n g e r p r i n t and the known s i z e s of the rRNA molecules. Several of the T, p l u s panc r e a t i c RNase products were indeed present once per RNA molecule. 14C methyl fingerprints were then prepared, and the molar yields of a l l the labelled products were determined with reference t o the products which had a1 ready been characterized in the P fingerprints. This gave an estimate of the t o t a l numbers of methyl groups of T, pl us pancreatic r i bonucl ease f i n g e r p r i n t s of rRNA ( r e f . 30). I t was then possible t o identify the T, plus panc r e a t i c RNase products as "derivatives" of the T, products ( r e f s . 32, 35). The r e s u l t s of t h i s correlation c l e a r l y indicate t h a t most of the T, products occur once per molecule of rRNA, with a

small number of s i t e s which a r e incompletely methylated. The numbers of methyl groups c a l c u l a t e d i n t h i s way a r e : 18s rRNA: approximately 47 28s rRNA: approximately 70 5.8s rRNA: 1.2 (One s i t e i n 5 . 8 s rRNA i s f r a c t i o n a l l y methylated.) ( i i i ) Comoarison of rRNA with ribosomal p r e c u r s o r RNA. Comparison of the methyl f i n g e r p r i n t s of rRNA with t h o s e of ribosomal p r e c u r s o r RNA ( r e f s . 28, 32) immediately confirmed t h e r e l a t i o n s h i p s between the r e s p e c t i v e molecules, which had been i n f e r r e d e a r l i e r from k i n e t i c l a b e l l i n g experiments and a n a l y s i s of a1 kal i - s t a b l e products ( r e f . 18). The f i n g e r p r i n t o f 45s RNA i s very s i m i l a r t o t h a t of 28s p l u s 18s rRNA; t h a t of pure 32s RNA i s the same a s t h a t of 28s p l u s 5 . 8 s rRNA ( s e e Fig. 8.1). Two a s p e c t s of t h i s r e s u l t r e q u i r e f u r t h e r comment. F i r s t , ribosomal p r e c u r s o r RNA c o n t a i n s e x t e n s i v e t r a n s c r i b e d s p a c e r s which a r e e l i m i n a t e d d u r i n g ribosome m a t u r a t i o n . Electron microscopy revealed t h a t t h e t r a n s c r i b e d s p a c e r s amount t o some 40% of the o v e r a l l l e n g t h o f HeLa c e l l 45s RNA ( r e f . 3 6 ) . Howe v e r , t h e methyl f i n g e r p r i n t s o f ribosomal p r e c u r s o r RNA do not c o n t a i n any e x t r a , methylated ol i g o n u c l e o t i d e s which cannot be accounted f o r i n t h e rRNA f i n g e r p r i n t s . T h u s , although t h e g r e a t m a j o r i t y of m e t h y l a t i o n s occur r a p i d l y on ribosomal p r e c u r s o r R N A , the methyl groups a r e a l l l o c a t e d w i t h i n the rRNA sequences i n t h e p r e c u r s o r molecules: t h e t r a n s c r i b e d s p a c e r s a r e unmethyl a t e d . This important conclusion w i l l be d i s c u s s e d l a t e r , below. Second, following the a n a l y s i s of Zimmerman ( r e f . 17, above), experiments were c a r r i e d o u t t o d i s t i n g u i s h between e a r l y and l a t e methylations ( r e f s . 31, 32). I t was found t h a t t h r e e charact e r i s t i c 18s methylated o l i g o n u c l e o t i d e s a r e a b s e n t from t h e 45s f i n g e r p r i n t , and a r e s e l e c t i v e l y l a b e l l e d i n 18s rRNA a f t e r RNA s y n t h e s i s has been blocked by actinomycin ( F i g u r e 8 . 2 ) . These l a t e methylations e v i d e n t l y comprise a small c l a s s of events which a r e d i s t i n c t from the many e a r l y m e t h y l a t i o n s , and probably occur a t about the time when t h e nascent small ribosomal s u b u n i t emerges from t h e nucleolus i n t o the cytoplasm. ( i v ) Nature of the methvlated seauences. Analysis of t h e methylated ol i g o n u c l e o t i d e s showed t h a t t h e g r e a t m a j o r i t y a r e 2 ' O-methylated and a few c o n t a i n methylated b a s e s . The f i r s t major

B276

data compilation was given in r e f . 32. Some additional data were obtained and a few corrections made in a f.urther compilation ( r e f . 37), which also contains data f o r several other vertebrate species (see below). Table 8.1 summarizes d a t a on the modified components i n HeLa c e l l rRNA, together w i t h d a t a from X e n o p u s and yeast (see also below). A l l of the 2'-O-methylations occur rapidly on 45s rRNA, with one exception i n the 28s sequence. There i s great d i v e r s i t y among the methylated sequences. A l l of the 16 possible 2'-O-methylated dinucleotides are present i n HeLa c e l l rRNA, most of them occurri n g in both 18s and 28s rRNA, i n d i f f e r e n t frequencies (Table 8 . 1 ) . A few oligonucleotides contain more t h a n one methyl group, e i t h e r on adjacent nucleotides (see note g t o Table 8.1) or separated by one or more unmethylated nucleotides (see r e f . 37 f o r detai 1 s ) . The l a t e methylations i n 18s rRNA a r e a l l base methylations. There i s a l s o a hypermodified nucleoside i n 18s rRNA, which was f i r s t characterized by Saponara and Enger ( r e f . 38) and designated 3- (3-ami no-3-carboxypropyl ) -1-methyl pseudouri d i ne (m1cap3$, sometimes a l s o abbreviated am$). This nucleoside receives both i t s methyl group and the 3-amino-3-carboxypropyl group from methionine (via S-adenosyl methionine) i n separate reactions, and therefore has the unusual property t h a t i t can be labelled w i t h l - 1 4 C or 214C labelled methionine as well as with methyl labelled methionine. These c h a r a c t e r i s t i c s enabled the t i m i n g of the various steps i n the modification of this nucleoside t o be deduced w i t h respect t o the overall rRNA maturation pathway ( r e f . 39). The methyl group i s added in the nucleolus whereas the bulkier a l i phatic group appears t o be added a f t e r 18s rRNA has l e f t the nucleolus, probably a t about the same time as the other l a t e base methyl ations. There a r e also 5 base methylations in 28s rRNA. These 28s base methyl groups a r e already present i n 4 5 s rRNA and t h e i r timing i s not e a s i l y distinguishable from the majority of 2'-0methylations, although there may be very s l i g h t delays (M.S.N. Khan and B . E . H . Maden, unpublished d a t a ) . However, one "semi1 a t e " methyl a t i on was detected: the sequence UmpGmp$p, indicated in note g of Table 8.1, i s incompletely methylated a t the level of

B277

RNA. This multiply modified component occurs in a large T, similarity between the methylation patterns of rRNA from these

45s

Figure 8.1. T, ribonuclease fingerprints of HeLa cell methyl labelled rRNA and nucleolar 45s and 32s RNA, showin the relationships between the methylation patterns of rRNA an] the nucleolar precursors. Spots above and to the left of the diagonal line in the key are better resolved in fingerprints obtained by the Such combined T, plus phosphatase procedure (ref. 32). fin er rints also show correspondence between the ribosomal and nuc!eo!?ar methylation patterns. Spots 29a and 36a are not seen in fingerprints of mature rRNA. Spot 36a is an incompletely modified form of the hypermodified mlcap311, spot see the text and Table 8.1). Spot 29a may be an incompletely mo ified form of the UmGm11, sequence. Reproduced from ref. 32.

d

B278

Fi ure 8.2. Fingerprints showing the late methylation sites in l8! rRNA, within T, products 30, 34, and 49. These oligonucleotides contain, respectively, the following methylated bases: m$A (two residues), m6A, m7G. The spots are absent from methyl fingerprints o f nucleol ar 18S-20s RNA (the immedi ate recursor to cytoplasmic RNA). They are selectively labelled in 1 S rRNA when methyl labelled methionine is added to cells 5 minutes after RNA

l

B279

Figure 8.2 (cant i n u e d ) synthesis has been blocked by actinomycin. There i s v i r t u a l l y no labelling of 28s rRNA under these condit i o n s , except for a very f a i n t , submolar component designated 2a. Reproduced from r e f . 32. several vertebrate sources ( r e f . 37). Various f u r t h e r ri bonuclease ol igonucleotide whose sequence was determined Eladari e t . a 7 . ( r e f . 40).

by

Methvlated Sequences in r R N A from Other Vertebrate Sources I t was of i n t e r e s t t o extend the analysis of methylated ol igonucleotides t o rRNA from other vertebrate sources. Accordi ngly, methyl -1 abel 1 ed rRNA was prepared from the fol1 owing sources: mouse L c e l l s , hamster C13 c e l l s , freshly cultured chick embryo fibroblasts and a l i n e of X . l a e v i s c e l l s . T 1 ribonuclease fingerprints were prepared, and revealed a very high level of similarity between the methylation patterns o f rRNA from these several vertebrate sources ( r e f . 37). Various f u r t h e r analytical procedures were carried o u t using 14C-methyl-1 abell ed or 32P-labe11ed material, and the data on the oligonucleotides were tabulated i n r e f . 37. (T, plus pancreatic RNase fingerprints also yielded useful data f o r X e n o p u s and chick; r e f . 41). The r e s u l t s of t h i s comparative analysis may be summarized as follows. 14C-methyl fingerprints of 18s rRNA from the three mammalian sources were indistinguishable. The chick and X e n o p u s 18s fingerprints resembled the mammalian fingerprints closely b u t differed in a few respects. (See Table 8.1 f o r a summary of Xenopus d a t a a t the mono- and dinucleotide l e v e l , and r e f . 37 f o r the ol igonucleotide d a t a . ) The various 28s methyl fingerprints a l s o differed only s l i g h t l y between species. Some of the d i f ferences between fingerprints appeared t o be due t o single base substitutions; others appeared t o be due t o the presence or absence of a methyl g r o u p a t a particular point in the sequence. (Both of these inferences were confirmed i n subsequent work: see below.) All of the differences were between 2'-O-methylated sequences. The base methyl ated sequences were identical between species.

8.4.2

B280

At about this time Choi and Busch (ref. 42) published a catalogue of T, 01 i gonucl eoti des from rat hepatoma (asci tes cell ) 18s rRNA. Their partial sequence data and quantification of methylated 01 igonucleotides were in practically complete agreement with our data for mammalian 18s rRNA (ref. 37), with the same numbers of the 16 different 2'-O-methylated dinucleotides as summarized for HeLa cell 18s rRNA in Tab1 e 8.1, 8.4.3 Methvlation in Yeast rRNA and Ribosomal Precursor RNA While the above work on vertebrate rRNA was in progress, Klootwijk and Planta carried out a similar analysis on the methylated ol igonucleotides in rRNA of the yeast, S a c c h a r o m y c e s c a r l s b e r g e n s i s (refs. 39, 43, 44, and 45). The general conclusions were very similar to those obtained from vertebrate rRNA, although there were many differences in detail. As in vertebrates, the majority o f methylations are 2'-O-substituents (ref. 44). These are added rapidly to ribosomal precursor RNA, with the exception of semi-late methylation of UmpGmp$p in the 28s sequence (ref. 45). There are, however, considerably fewer 2'-O-methyl groups in yeast rRNA than in vertebrate rRNA (Table 8.1). The base methylations are also similar, but not quite identical, to those in vertebrate rRNA. The timing of the base methylations is also similar with respect to the rRNA maturation pathway, the 18s base methylations occurring late in the pathway (ref. 45). 8.4.4 ImPlications of the Oliaonucleotide Data It was evident from the methyl fingerprints that rRNA methylation is a highly specific process. The great majority o f methylations, including all the 2'-O-methylations, occur rapidly upon ribosomal precursor RNA, and all of the methylation sites are within the ribosomal sequences of the precursor molecule. The oligonucleotide data imply that most of the methylation sites are highly conserved in rRNA from different vertebrates, but the numbers of 2'-O-methyl groups differ considerably between vertebrates and yeast (Table 8.1). Despite the highly specific patterns of the methyl fingerprints, sequence analysis of the methylated oligonucleotides revealed no common motif in the methylation sites at the level of primary structure; indeed, there is great diversity at this level among the methylated sequences.

B281

TABLE 8 . l a Modified Nucleotides i n 18s rRNA

The HeLa and X e n o p u s d a t a on methyl groups a r e summarized from t h e o l i g o n u c l e o t i d e d a t a i n r e f . 37 a n d the sequence d a t a i n r e f s . 61 and 62. The y e a s t ( S a c c h a r o m y c e s c a r l s b e r g e n s i s ) d a t a on methyl groups a r e summarized from the o l i g o n u c l e o t i d e d a t a i n ref. 44. HeLa

X. l a e v i s

S.carlsbergensis

Notes

A1 kal i -stab1 e d i n ucl eo t i des : UmpU UmpG UmpA UmpC Total Um

2 4 2 3 11

2 3 1 3

0

GmpU GmpG GmpA GmpC Total Gm

1 6 1 1 9

1 5 0 1 7

2 2 1 0

AmpU AmpG AmpA AmpC Total Am

2 4 2 13

5

4 2 4 2 12

2 1 4 1

CmpU CmpG CmpA CmpC

Total Cm Total 2'-O-methyl

9

1 1

a

0 2

a a

5

a

0

3 7

0 2 0 3 5

40

33

ia

1 1 1 2 7

1 1 1 2 7

1 1 0 2 6

b

37

46

14

d

1 2 1

1

0 2 3

a

Methylated bases: rn1cap3fl

m7G

m6A

m$A Total base methyl Pseudouridi ne (approx. 1 a. b.

c.

C

In HeLa c e l l 18s rRNA t h e r e a r e f o u r p a r t i a l 1 meth l a t e d UmpG, &npG, &PA, a n d s i t e s , one f o r each of t h e CmpC (see r e f . 62 f o r Thi s a b b r e v i a t i o n denotes -1methyl pseudouri d i n e ; see the t e x t . Each m$A c a r r i e s 2 methyl groups.

B282

TABLE 8 . l b M o d i f i e d N u c l e o t i d e s i n 28s rRNA HeLa

X . laevis

S.carlsbergensis

Notes

A1 k a l i-stab1 e d i nucleotides: UmpU UmpG UmpA UmpC T o t a l Um GmpU GmpG GmpA GmpC T o t a l Gm AmpU AmpG AmpA AmpC T o t a l Am

0 3

1

3 7 6

10

3 0

19

5 7 2 4

18

0 3 1 3 7

2 3 1 1 7

e

f f

5 9 3 1 18 5 7 2 4 18

f

14

5 2 3 3 13

A1 k a l i-stab1 e 01 igonucl e o t i des (2 ' -0-methyl groups)

7

7

8

9

T o t a l 2'0-methyl

65

63

37

f

1 2 2 5

1 2 2 5

2 2 2 6

h h

56

62

32

d

CmpU CmpG CmpA CmpC T o t a l Cm

4

3

3 4

Methyl a t e d bases:

m3 U mA mC T o t a l base methyl Pseudouri d i ne (approx. 1

B283

TABLE 8 . l c

Modified Nucleotides i n 5.8s rRNA GmpC Um G

Otler methyl Pseudouri di ne

1 0.2 0 2

1 0.4 0 2

0 0 0 1

i

Notes t o T a b l e 8.1 ( c o n ' t )

d.

e. f.

g.

h.

i.

The v a l u e s f o r pseudouridine a r e from r e f s . 61 and 65 (HeLa and X e n o p u s ) and 43 ( S a c c h a r o m y c e s ) , and a r e probably a c c u r a t e t o w i t h i n + 10% of the s t a t e d v a l u e s . I n c l u d e s . oiie dmpG. The p r e c i s e numbers o f the i n d i c a t e d d i n u c l e o t i d e s i n HeLa and Xenopus a r e s l i g h t 1 u n c e r t a i n from o l i g o n u c l e o t i d e d a t a , b u t the u n c e r t a i n t i e s a t f e c t t h e t o t a l numbers of methyl groups by o n l y about + 2. The a l k a l i - s t a b l e o l i g o n u c l e o t i d e s i n HeLa and X e n o p u s a r e : UmpGmpU, UmpGmp , and AmpGmpCmpA; i n S a c c h a r o y m c e s t h e y a r e : UmpGmpd, AmpGmp8 AmpCmpG, and AmpAmpU. The s i t e s of s u b i t i t u t i o n i n mA and mC i n HeLa and X e n o p u s 28s rRNA have not a l l been determined. Yeast 5 . 8 s d a t a a r e from S . c e r e v i s i a e , r e f . 46.

To account f o r t h e s e v a r i o u s f i n d i n g s i t was proposed ( r e f . 32) t h a t the s p e c i f i c i t y f o r methylation i s determined by conformation w i t h i n t h e rRNA sequences: i n p a r t i c u l a r , t h a t the many 2'-O-methylation s i t e s p r e s e n t some common conformational f e a t u r e t o a n u c l e o l a r enzyme o r a small number of enzymes which c a r r y o u t t h i s t y p e of methylation on ribosomal p r e c u r s o r R N A . I t was now becoming apparent t h a t f u r t h e r p r o g r e s s would r e q u i r e knowledge of the l o c a t i o n s of t h e methyl groups i n t h e complete primary s t r u c t u r e of rRNA. 8.5 LOCATING THE METHYLATED NUCLEOTIDES 8 . 5 . 1 Modified Nucleotides i n 5.8s rRNA A s t a r t t o l o c a t i n g t h e modified n u c l e o t i d e s i n rRNA came from the sequence a n a l y s i s of 5.8s rRNA. This small (160 nucleot i d e ) molecule i s non-covalently a t t a c h e d t o 28s rRNA and i s t r a n s c r i b e d a s p a r t of 45s ribosomal p r e c u r s o r R N A . I t becomes a s e p a r a t e e n t i t y a s a r e s u l t of e x c i s i o n o f the second i n t e r n a l t r a n s c r i b e d s p a c e r region (ITS 2) during the f i n a l m a t u r a t i o n of 32s t o 28s rRNA. When 28s rRNA i s i s o l a t e d by non-denaturing

B284

procedures, 5.8s r R N A can be r e l e a s e d subsequently by h e a t denat u r a t i o n o r comparable methods and recovered i n p u r e form. Because o f i t s r e l a t i v e l y small s i z e i t was t h e f i r s t f u n c t i o n a l r e g i o n o f t h e e u k a r y o t i c ribosomal t r a n s c r i p t i o n u n i t t o be s u b j e c t e d t o complete sequence a n a l y s i s . 5.8s sequences were r e p o r t e d f o r y e a s t ( S a c c h a r o m y c e s c e r e v i s i a e , r e f . 46), r a t ( r e f . 47), o t h e r v e r t e b r a t e sources ( r e f s . 48, 49) and o t h e r non-vert e b r a t e s . Ref. 50 g i v e s a summary o f c u r r e n t l y known 5.8s sequences. ( T h i s c o m p i l a t i o n a l s o i n c o r p o r a t e s c o r r e c t i o n s t o some small e r r o r s which appeared i n some o f t h e o r i g i n a l r e p o r t s . ) Each of t h e 5.8s sequences c o n t a i n s one o r more m o d i f i e d nucleotides. The v e r t e b r a t e 5.8s sequences c o n t a i n two 2 ' - 0 m e t h y l a t i o n s i t e s , one o f which i s f r a c t i o n a l l y m e t h y l a t e d , and two pseudouridines. Thus 5.8s rRNA, which i s f u n c t i o n a l l y p a r t o f 28s rRNA b u t i s separated from t h e r e s t o f t h e 28s sequence by ITS 2 i n ribosomal p r e c u r s o r RNA, c o n t a i n s s p e c i f i c r e c o g n i t i o n s i t e s f o r 2 ' - 0 - m e t h y l a t i o n and p s e u d o u r i d i n e f o r m a t i o n . Although t h e 5.8s and 28s sequences a r e separated from each o t h e r i n t h e p r i m a r y s t r u c t u r e o f ribosomal p r e c u r s o r RNA i t seems probable t h a t t h e y e s t a b l i s h t h e i r mutual i n t e r a c t i o n w h i l e t h e y a r e w i t h i n t h e p r e c u r s o r molecule. O l i g o n u c l e o t i d e d a t a show t h a t t h e p r i n c i p a l 2 ' - 0 - m e t h y l a t i o n s i t e i n HeLa c e l l 5.8s rRNA i s a l r e a d y m o d i f i e d w i t h i n ribosomal p r e c u r s o r RNA ( r e f s . 32, 51). I t i s an i n t e r e s t i n g and c u r r e n t l y unanswered q u e s t i o n whether m e t h y l a t i o n o f t h e 5.8s sequence occurs b e f o r e o r a f t e r i t s i n t e r a c t i o n w i t h t h e 28s sequence. T h i s p r i n c i p a l 5.8s m e t h y l a t i o n s i t e occurs i n a 10 nucleot i d e h a i r p i n l o o p near t h e m i d d l e o f t h e sequence, and i s conserved i n v e r t e b r a t e 5.8s rRNA ( r e f s . 47, 48). The c o r r e s p o n d i n g l o o p i n S a c c h a r o m y c e s 5.8s rRNA i s s m a l l e r and unmethylated. Comparative d a t a o f t h i s k i n d may p r o v i d e a s t a r t i n g p o i n t from which t o i d e n t i f y t h e s t r u c t u r a l determinants o f m e t h y l a t i o n sites. 8.5.2

M e t h v l a t i o n Map o f X e n o u u s 7 a e v i s rRNA By t h e l a t e 1970s i t had become c l e a r t o many workers t h a t sequenci ng 1arge RNA mol ecul es by t h e c l a s s i c a l procedures which were then a v a i l a b l e ( r e f s . 25, 34, 46) was l i k e l y t o be e x t r e m e l y

B285

l a b o r i o u s and a l s o e r r o r - p r o n e . Thus an impasse appeared t o have been reached i n t h e a n a l y s i s o f e u k a r y o t i c r R N A and i t s m o d i f i e d nucleotides. T h i s impasse was surmounted w i t h t h e a i d o f recomb i n a n t DNA. The DNA encoding t h e m a j o r r R N A species (rDNA) f r o m Xenopus l a e v i s was t h e f i r s t e u k a r y o t i c DNA t o be c l o n e d ( r e f s . 52, 53). The a v a i l a b i l i t y of c l o n e d X e n o p u s rDNA enabled a s e r i e s o f experiments t o be c a r r i e d o u t i n which t h e d i s t r i b u t i o n o f m e t h y l a t i o n s i t e s w i t h i n X e n o p u s 18s and 28s rRNA was e x p l o r e d . The e x p l o r a t i o n was c a r r i e d o u t by repeated a p p l i c a t i o n o f t h e f o l l o w i n g experimental p r o t o c o l ( r e f . 54). Methyl -1 abel l e d o r 32P-1abelled 18s o r 28s r R N A was h y b r i d i z e d t o c l o n e d fragments o f rDNA, o r t o s m a l l e r r e s t r i c t i o n fragments which had been p u r i f i e d from t h e clones. I n each h y b r i d i z a t i o n , o n l y p a r t o f t h e r R N A was represented by t h e rDNA c l o n e o r fragment, and was t h e r e f o r e bound i n t h e RNA:DNA h y b r i d . The n o n - h y b r i d i z e d r e g i o n o f RNA was removed by t r i m m i n g w i t h T, RNase. The h y b r i d i z e d r e g i o n was t h e n recovered by h e a t i n g t h e h y b r i d , and was analyzed by f i n g e r p r i n t i n g f o r i t s content o f methylated oligonucleotides (using t h e T, o r t h e combined T, p l u s p a n c r e a t i c RNase f i n g e r p r i n t i n g procedure). I n t h i s way each o f t h e m e t h y l a t e d o l i g o n u c l e o t i d e s i n Xenopus 18s r R N A was l o c a l i z e d t o one o f e i g h t r e g i o n s d e f i n e d by S i m i l a r l y , each o f t h e m e t h y l a t e d r e s t r i c t i o n s i t e s i n rDNA. o l i g o n u c l e o t i d e s i n 28s rRNA was l o c a l i z e d t o one o f eleven r e g i o n s d e f i n e d by r e s t r i c t i o n s i t e s i n rDNA. The r e s u l t s y i e l d e d a “ m e t h y l a t i o n map” o f X e n o p u s r R N A ( r e f . 54). The map y i e l d e d a u s e f u l overview o f t h e d i s t r i b u t i o n o f m e t h y l a t e d sequences a l o n g 18s and 28s rRNA. The d i s t r i b u t i o n i s non-uniform. I n 18s rRNA, 18 o f t h e 33 2’-O-methyl groups (54%) were found i n t h e 5 ’ 40% o f t h e molecule. The base m e t h y l a t i o n s i t e s were i n t h e 3 ’ h a l f o f t h e molecule. I n 28s r R N A t h e r e was a much lower m e t h y l a t i o n d e n s i t y i n t h e 5 ‘ r e g i o n , and 40 o u t o f t h e 68 o r so methyl groups (60%) were found i n t h e 3 ’ 30% o f t h e mol ecul e. Meanwhile Gerbi and co-workers had s t a r t e d t o e x p l o r e t h e d i s t r i b u t i o n of p h y l o g e n e t i c a l l y conserved and v a r i a b l e r e g i o n s along t h e rRNA sequences ( r e f . 55) and had a l s o c a r r i e d o u t some h y b r i d i z a t i o n experiments w i t h m e t h y l - l a b e l l e d r R N A ( a l t h o u g h n o t

B286

a t t h e l e v e l o f f i n g e r p r i n t i n g a n a l y s i s , r e f . 56). The r e s u l t s suggested t h a t m e t h y l a t i o n i s concentrated i n t o rRNA r e g i o n s which show h i g h p h y l o g e n e t i c sequence c o n s e r v a t i o n . To e x p l o r e f u r t h e r t h i s s u g g e s t i v e r e l a t i o n s h i p i t was n e c e s s a r y t o o b t a i n f u l l sequence d a t a w i t h t h e e x a c t l o c a t i o n s o f t h e rRNA m e t h y l g r o u p s . 8.5.3

E x a c t L o c a t i o n s o f t h e M e t h y l Grouos i n 18s rRNA o f X e n o p o s l a e v i s and Man The n u c l e o t i d e sequence o f a c o m p l e t e X . l a e v i s gene e n c o d i n g 18s r R N A was d e t e r m i n e d b y S a l i m and Maden ( r e f . 5 7 ) . The m e t h y l groups were l o c a t e d i n t h e i n f e r r e d r R N A sequence, i n m o s t i n s t a n ces e x a c t l y , i n t h e r e m a i n i n g few i n s t a n c e s t o w i t h i n a few n u c l e o t i d e s ( t h e u n c e r t a i n t i e s b e i n g r e s o l v e d l a t e r , see b e l o w ) . The 18s gene sequence o f S a c c h a r o m y c e s c e r e v i s i a e had been d e t e r mined a s h o r t t i m e p r e v i o u s l y ( r e f . 58), a l t h o u g h t h e RNA m e t h y l Comparison o f t h e t w o g r o u p s i n S a c c h a r o m y c e s were n o t l o c a t e d . sequences r e v e a l e d e x t e n s i v e t r a c t s o f h i g h homology i n t e r s p e r s e d w i t h t r a c t s h a v i n g l i t t l e o r no homology. Most o f t h e X e n o p u s r R N A m e t h y l groups were f o u n d t o be l o c a t e d i n t h e h i g h l y cons e r v e d t r a c t s , as d e p i c t e d s c h e m a t i c a l l y i n F i g . 8.3, w h i c h i s f r o m r e f . 57. R e s u l t s f r o m f u r t h e r s e q u e n c i n g o f c l o n e d and u n c l o n e d x . l a e v i s rDNA i n d i c a t e d t h a t t h e X . l a e v i s 18s gene p o o l i s homogeneous, and hence t h a t 18s r R N A a l s o c o m p r i s e s a homogeneous A single, small c o r r e c t i o n t o p o p u l a t i o n o f m o l e c u l e s ( r e f . 59). t h e o r i g i n a l sequence was made ( r e f . 60) and a s e c o n d a r y s t r u c t u r e model was proposed ( r e f . 60, see b e l o w ) . The human 18s rDNA sequence was d e t e r m i n e d and t h e m e t h y l g r o u p s were l o c a t e d i n t h e i n f e r r e d 18s rRNA sequence ( r e f . 61). D e t a i l e d e v i d e n c e on t h e l o c a t i o n s o f t h e i n d i v i d u a l m e t h y l g r o u p s i n X . l a e v i s and human 18s r R N A was p u b l i s h e d i n r e f . 62. I n most i n s t a n c e s t h e l o c a t i o n s were e s t a b l i s h e d u n a m b i g u o u s l y by c o r r e l a t i o n o f d a t a from t h r e e l i n e s o f evidence: o l i g o n u c l e o t i d e d a t a ( r e f . 37 and f u r t h e r d a t a i n r e f . 62), t h e X e n o p o s m e t h y l a Some a d d i t i o n a l t i o n map ( r e f . 54) and t h e rDNA sequence d a t a . i n f o m a t i o n came f r o m t h e c a t a l o g u e o f m e t h y l a t e d 01 i g o n u c l e o t i d e s f r o m r a t 18s rRNA ( r e f . 42), w h i c h complemented o u r own d a t a . F i n a l y , Connaughton e t . a l . ( r e f . 63) c a r r i e d o u t a d i r e c t

X

x

5'

xx

X

Y

Y

x xxxx

x

200

A

400

x

x

X

x x x x x

I

boo

a

X

X

800

1

1

1000

1

x

1200

x

X

0 0 x

x

x

1400

--

B

x

C

@

X

x

X

1600

0 m

1800

3'

D

Figure 8.3. Summary o f m e t h y l a t i o n s i t e s i n X . 7 a e v i s 18s rRNA and p a t t e r n o f homology with y e a s t (5. c e r e v i s i a e ) 18s rRNA. Up e r s e c t i o n : p l a i n a s t e r i s k s d e n o t e p o s i t i o n s o f 2'-0methyl roups a l o n g the X . 7 a e v i s 1 l S rRNA sequence- c i r c l e d a s t e r i s k s d e n o t e base methyl groups ?see a l s o F i g . 8 . 4 ) . Lower s e c t i o n : the h i 'h blocks i n d i c a t e r e g i o n s o f the x . l a e v i s sequence showing 85-100% homology w i t h y e a s t ; ow blocks i n d i c a t e r e g i o n s showing 7085% homology. A , B , C , D d e n o t e the r e g i o n s o f g r e a t e s t v a r i a b i l i t y between the two 18s sequences. Reproduced by permission from Nature, Vol. 291, No. 5812, p p . 205-208. Copyr i g h t ( c ) 1981, Macmillian J o u r n a l s Limited.

B

B288

sequence a n a l y s i s o f 18s r R N A from r a b b i t r e t i c u l o c y t e s u s i n g endl a b e l l i n g and p a r t i a l d e g r a d a t i o n methods, w i t h s e p a r a t i o n o f t h e p r o d u c t s on sequencing g e l s . Z'-O-methyl groups gave r i s e t o gaps a t s p e c i f i c p o i n t s i n the gels. By c o r r e l a t i n g t h e l o c a t i o n s o f these gaps w i t h known o l i g o n u c l e o t i d e d a t a ( r e f . 37) and sequence d a t a (e.g. r e f . 57) these authors i n f e r r e d t h e l o c a t i o n s o f 2'-0methyl groups i n 18s rRNA from r a b b i t r e t i c u l o c y t e s . The r e s u l t s a f f o r d e d a u s e f u l cross-check on t h e d a t a c o m p i l a t i o n i n r e f . 62, and enabled t h e l a s t few u n c e r t a i n t i e s i n t h e X e n o p u s and human d a t a t o be resolved, as d e t a i l e d i n r e f . 62. The Xenopus 18s sequence w i t h t h e l o c a t i o n s o f a l l t h e methyl groups as f i n a l l y i n f e r r e d ( r e f . 62) i s shown i n F i g . 8.4. In F i g . 8.5 t h e human sequence i s shown a l o n g w i t h s i t e s o r r e g i o n s where X e n o p u s d i f f e r s from t h e human sequence. Human 18s rRNA i s m e t h y l a t e d a t a l l t h e same l o c a t i o n s as i n X e n o p u s , and a l s o a t a few a d d i t i o n a l l o c a t i o n s , m a i n l y i n t h e 5 ' r e g i o n o f t h e sequence.

8.6 PROBLEMS AND PROSPECTS 8.6.1 The Methyl Groups i n 28s rRNA The t a s k o f l o c a t i n g a l l o f t h e m e t h y l a t i o n s i t e s i n 28s r R N A a t t h e n u c l e o t i d e sequence l e v e l has n o t y e t been completed. However, about 50 o f t h e 70 methyl groups have been l o c a t e d i n t h e Xenopus and human 28s sequences on t h e b a s i s o f v a r i o u s l i n e s o f a v a i l a b l e evidence (B.E.H.M., manuscript i n preparation). T h i r t y o f t h e 43 methyl groups i n y e a s t 28s rRNA were l o c a t e d i n t h e A c o n s i d e r a b l e number o f t h e s e occur a t sequence ( r e f . 64). homologous l o c a t i o n s i n t h e y e a s t and v e r t e b r a t e sequences, as w i l l be d e s c r i b e d elsewhere i n d e t a i l (B.E.H. Maden, i n preparat i o n ) . Meanwhile, t h e X e n o p u s 28s " m e t h y l a t i o n map" which was o b t a i n e d by h y b r i d i z a t i o n experiments ( r e f . 54) i s shown here ( F i g . 8.6) t o d e p i c t t h e heavy c l u s t e r i n g o f methyl groups i n t h e 3 ' r e g i o n o f t h e molecule. 8.6.2 Pseudouridine and Other M o d i f i e d N u c l e o t i d e s The approximate numbers o f p s e u d o u r i d i ne r e s i d u e s i n human, X e n o p u s and S a c c h a r o m y c e s r R N A a r e summarized on t h e bottom l i n e s The values i n r e f s . 6 1 and 65 were from base o f Table 8.1. composition a n a l y s i s f o l l o w e d by chromatographic s e p a r a t i o n o f

B289 m

UACCUCCUUC AUCCUCCCAC UACCAUAUCC UUCUCUCAAA GAUUAACCCA UCCACCUCUA

m

60

ACUACCCACC CCCCCUACAC UCAAACUCCG AAUCCCUCAU UAAAUCAGUU AUCCUUCCUU

120

UCAUCCCUCC AUCUCUUACU UCCAUAACUC U C C U A A U U C U C C U A A U A

CAUCCCCACC

180

ACCCCUCACC CCCAGGCAUC CGUCCAUUUA

UCACACCAAA ACCAAUCCCC CCCCCCCCCC

240

CCCCCCCCCC UUUCCUGAC-UAACC

UCGCCCCCAU C C C A C G U C C C CWGACCCCC

300

XbaI

XbaI

ACCAUACAUU CCCAUGUCUC CCCUAUCAAC UUUCGAUCCU ACUUUCUGCC CCUACCAUCC

360

m m UCACCACGCC UAACCCCGAA UCACCCUUCC AUUCCCCACA GCCACCCUCA CAAACGGCUA

420

r n m AUUACCCACU CCCGACCCCC CCACCUACUC

480

n m 111 CCACAUCCAA CCAACCCACC ACCCGCCCAA

..

.

..

. .

m ACCAAAAAUA A C A A U A C A C C U U U C C A CCCCCUCUAA UUCCAAUGAC UACACUUUAA HlCIt-1 .m nl .m. ..m AUCCUUUAAC CACCAUCUAU UGCACGCCAA CucuCCuCcc A C C A ~ C C C C C

.

-..

.

540

CUAAUUCCAC

600

m m U U A A A A A C C U CCUACUUGCA

UCUUCCCAUC

660

CACCUCCCCC UCCCCCGCCA CGCCACCUAC CCCCUCUCCC ACCCCCUGCC

UCUCGCCCCC

720

UCCCCCAUCC UCUUGACUCA GUCUCCCGCG CCCCCCAACC CUUUACUUUC A A A A A A U U A C

780

. . PStI

m . CUCCAAUAGC GUAUAUUAAA CUUC-

m

m

?,ma1 -

AGUCUUCCAA G C A C C C C C C C

uccccuccnu

ACUUCACCUA CCAAUAAUCC AAUACCACUC

840

CCGUUCUAUU UUCUUCCUUU UCCCAACUGC CCCCAUCAUU AAGACCCACC CCCCGCCCCA

900

UUCCUAUUCU CCCCCUACAC GUCAAAUUCU UCCACCGCCC CAACACCAAC C A A A C C C A A A

960

C C A U U U C C C A A C A A U G U U U U C A U U A A U C A A G A A C C A A A C U CCGACCUUCC AACACCAUCA

1020

GAUACCCUCG UACUUCCCAC C A U A A A C C A U CCCCACUACC CAUCCGCCCG CGUUAUUCCC

1080

AUGACCCCCC CACCAGCUUC CCCCAAACCA AAGUCUUUCC GUUCCCCGGC CACUAUCCUU

1140

CCAAACCUCA AACUUAAAGC AAUUGACCCA ACCGCACCAC CACCACUGCA CCCUGCGCCU

1200

M

m UAAUUUCACU CAACACCCCA AACCUCACCC CCCCCGGACA C C G A A A C C A U UCACACAUUC m m AUAGCUCUUU CUCCAUUCUC UCCCUCCUGC UCCAUCCCCC UUCUUACUUC GUCGACCCAU -1 m UUCUCUGGUU AAUUCCCAUA A C C A A C C A ~ C U C C A U C CUAACUAGUU ACCCCACCCC

m

Hint-1

1260 1320 1380

CCCCGGUCCC CCUCCAACUU CUUACACCCA CAACUCCCGU UCACCCACAC CACAUCGACC

1440

m AAUAACACCU CUCUGAUCCC CUUACAUCUC CCCCCCUCCA CCCGCGCUAC ACUGAACGCA

1500

UCACCCUCUC UCUACCCUCC CCCCACACCU CCCCGUAACC CCCUCAACCC CCUUCGUGAU

1560

M ACCGAUCGCC CAUUCCAAUU AUUUCCCAUG AACCAC-CCACUAAG

UGCGCGUCAU

1620

m m AAGCUCCCCU UCAUUAACUC CCUCCCCUUU CUACACACCC CCCCUCCCUA CUACCGAUUC

1680

CAUCCUUUAC UCACCUCCUC GCAUCCCCCC CCCCCCCCUC CCCCACCCCC CUCCCCCAGC

1740

m M X C G A G A A C A CGAUCAAACU UCACUAUCUA GACCAACUAA AAClJCCUAAC AAGCUUUCCC

1800

MM UACCUCAACC UGCCCAACCA UCAUUA

1826

&RI

Figure 8.4. Nucleotide sequences of X . l a e v i s 18s rRNA, w i t h t h e l o c a t i o n s of the 2I-O-rneth 1 groups (rn) and base methyl g r o u p s (M). (Legend continued folfowing).

B290 m UACCUGCUUC AUCCUCCCAC UACCAUAUCC UUCUCUCAAA CAUUAACCCA UCCAUCUCUA C C

60 60

ACUACCCACG CCCCCUACAC UCAAACUCCC AAUCCCUCAU UAAAUCACUU AUCCUUCCUU

120 120

..

m

"

rn

UGCUCCCUCC CUCCUCUCCC ACUUCGAUAA CUCUCCUAAU UCUACACCUA A A CUU

- --

____

ACGGCCCCUC ACCCCCUUCC CCGGCCCGAU GCGUCCAUUU AUCACAUCAA A ____A C

"

mnl AUACAUCCCC

240 229

CUCAGCCCCU CUCCCCCCCC CCCCCCCGCC CGGCCCCCCC CCCCUUUCGU CACUCUACAU

300 266

AACCUCGCCC CCAUCCCACC CCCCCCCUCG CCCCCACCAC CCAUUCCAAC CUCUGCCCUA U A U A C U

360 325

UCAACUUUCC AUCCUACUCC CCCUGCCUAC CAUCCUCACC ACCCCUCACC CCCAAUCACC cull u c A

420 385

m m m CUUCCAUUCC GGACAGCCAC CCUCACAAAC GCCUACCACA UCCAACCAAC CCAGCACGCC

480

c

--c

----- --- - ----_- ------

m

m

u

c

m

m

540 505

UUCGACGCCC UCUAAUUCCA

AUCACUCCAC UUUAAAUCCU UUAACCACGA UCCAUUGCAG A U

600 565

CCCAAGUCUC CUGCCAGCAC CCCCGCUAAU UCCACCUCCA AUAGCGUAUA UUAAAGUUCC

660 625

m

"

m

X.D.

UCCACUUAAA AAGCUCCUAC UUCGAUCUUC CCACCCCCCC GCCCCUCCGC CCCGACGCCA U A U c A CCCACCCCCC CUCCCCCCCC CUUGCCUCUC CCCGCCCCCU CGAUCCUCUU ACCUCAGUCU U U A u c GA

rnm

-

720 685 780 744

CCCCCCCCCC CCCAACCGUU UACUUUGAAA AAAUUACACU CUUCAAACCA CGCCCCACCC c u C

840 802

CCCUGCAUAC CGCAGCUACG AAUAAUCCAA UACCACCCCC CUUCUAUUUU CUUCGUUUUC

900 862

CCAACUCACC CCAUCAUUAA CACCCACCGC CCGGCCCAUU CGUAUUGCCC CCCUACACGU G U

960 922

CAAAUUCUUC CACCGCCCCA ACACCGACCA GACCCAAACC AUUUCCCAAC AAUGUUUUCA A A

1020 982

UUAAUCAACA ACCAAACUCC GAGCUUCGAA GACCAUCACA UACCCUCGUA GUUCCCACCA

1080 1042

UAAACGAUCC CCACCCCCCA UOCCCCCCCG UUAUUCCCAU CACCCCCCCG CCACCUUCCC UA C A

1140 1102

uc

uu

CCAAACCAAA GUCUUJCCCU UCCGGCGCCA CUAUCCUUCC AAACCUCAAA

CUUAAACCAA

1200 1162

UUCACCCAAC CCCACCACCA CCACUCCACC CUCCCCCUUA AUUUCACUCA ACACCCCAAA

1260 1222

CCUCACCCCC CCCCCACACC CACACCAUUC ACACAUUCAU ACCUCUUUCU CCAUUCCCUC A U

1320 1282

CCUCCUCCUC CAUCCCCCUU CUUACUUCCU CCACCCAUUU CUCUCCUUAA UUCCGAUAAC

1380 1342

ti

m m

m

,

m

-__

CAACCACACU CUCCCAUCCU AACUACUUAC CCCACCCCCC ACCCGUCCCC CUCCCCCAAC

m

m

cuc

m

1440 1398

UUCUUACAGC CACAAGUCCC CUUCACCCAC CCCAGAUUCA GCAAUAACAC CUCUCUCAUC A C

1500 1458

CCCUUACAUC UCCCCCCCUC CACCCCCCCU ACACUCACUC CCUCAGCCUG UCCCUACCCU AC A U

1560 1518

ACGCCCCCAC CCGCCCCUAA CCCGUUGAAC CCCAUUCCUC AUCGCCAUCC CCCAUUGCAA C A U C c H A * UUAUUCCCCA UCAACGACGA AUUCCCACUA ACUCCCCCUC AUAACCUUCC GUUCAUUAAC U C

1620 1578 1680 1638

"

Y.1. X.b.

445

CCCAAAUUAC CCACUCCCCA CCCCCCCACG UACUCACGAA AAAUAACAAU ACACCACUCU G

m

x.1.

180 177

AACCAACCCC

UCCCUGCCCU UUCUACACAC CCCCCCUCCC UACUACCGAU UCCAUCCUUU ACUCACGCCC U

1740 1698

UCCCAUCCCC CCCGCCCGCC UCCCCCCACC GCCCUCCCGG ACCCCUGACA ACACCCUCCA CC A A A

1800 1157

ACUUGACUAU CUACACGAAG UAAAAGUCCU AACAACCUUU CCCUACCUCA ACCUCCCGAA

1860 1817

GGAUCAUUA

1869 1826

M

M M

F i g u r e 8.5. N u c l e o t i d e sequence o f human 18s r R N A w i t h l o c a t i o n s o f methyl groups and comparison w i t h X e n o p u s . The f i r s t s u b s c r i p t l i n e shows t h e p o s i t i o n s a t which t h e X . 7 a e v i s se uen.ce d i f f e r s from t h a t of human ( o r from x). (legend c o n t i n u e d f o l y o w i n g ) .

B291

F i g u r e 8.4 l e g e n d c o n t i n u e d . R e s t r i c t i o n s i t e s i n rDNA which were used f o r ma p i n g t h e methyl groups t o w i t h i n s p e c i f i c regions of

\

rRNA r e f . !4) a r e shown as s u b s c r i p t s . S u p e r s c r i p t d o t s repreRNase cleava e s i t e s i n one such rRNA region, between sent nucleo2ides 500 and 6j5. The base modified n u c l e o t i d e s a r e : t h e hypermodified m'ca 3$ ( p o s i t i o n 1210), m7G (1597), m6A (1789) and two mqA r e s i d u e s (f.807, 1808). Reproduced from r e f . 62.

b o r e a l i s , i n d i c a t e d in the second sugscript line). Where e x t r a n u c l e o t i d e s occur i n the human sequence dashes a r e shown i n t h e aligned X . l a e v i s sequence. The l o c a t i o n s of Z'-O-methyl groups (m) and base methyl qroups ( M a r e i n d i c a t e d . A s t e r i s k s denote n u c l e o t i d e s which a r e 2 -0-methy a t e d i n human 18s rRNA b u t unmethylated i n X e n o p u s . The same base methyl groups occur a t homologous l o c a t i o n s i n the X e n o p u s and human 18s sequences, and a r e defined i n the l e end t o Figure 18.4. One o l i g o n u c l e o t i d e containing CmpC i n human 18s rRNA i s u n laced Reprinted by permission from Biochem. J . , Vol. 232, pp. 755-733: Copyright ( c ) 1985. F i u r e 8.5 l e g e n d c o n t i n u e d .

1

pseudouridine from u r i d i n e . The human 18s value obtained i n this way was i n very good agreement w i t h r a t 18s d a t a from oligonucleot i d e a n a l y s i s (38 pseudouridines, r e f . 42) , suggesting t h a t most of the values i n r e f . 65 a r e f a i r l y a c c u r a t e . The numbers of pseudouridines a r e f a i r l y s i m i l a r t o the numbers of 2'-O-methyl groups i n the r e s p e c t i v e rRNA s p e c i e s . There a r e about 95 pseudouri d i nes per human ribosome. Few of t h e s e pseudouridines have yet been l o c a t e d i n p u b l i s h ed primary s t r u c t u r e s of 18s o r 28s rRNA. This i s the g r e a t e s t unsolved problem a t the level of primary s t r u c t u r e determi,nation of rRNA from eukaryotes. Locating the pseudouridines w i l l provide i n s i g h t i n t o t h e r o l e of t h i s i n t r i g u i n g c l a s s of modified nucleotides i n the maturation and function of rRNA. Very r e c e n t l y we have succeeded i n c o r r e l a t i n g most of the pseudouridine-containing o l i g o n u c l e o t i d e s i n r e f . 42 w i t h s p e c i f i c l o c a t i o n s i n the mammal ian 18s sequence (Maden and Wakeman, manuscript i n preparat i o n ) . Further experiments d i r e c t e d toward l o c a t i n g the pseudouridines a r e i n progress i n our l a b o r a t o r y . 18s rRNA analyzed from a v a r i e t y of e u k a r y o t i c s p e c i e s c o n t a i n s N4-acetylcytidine i n an apparent y i e l d of 1.4 mol per mol of rRNA ( r e f . 66). A t t h e time of w r i t i n g this compound has not been l o c a t e d i n the 18s sequence.

,

X

x

9

x ;}x

x

x

x 9 x i l x

xxx ; z x

0.5kb

x x x x x x x x @X X X @ X X @ X X x x x x x x x x x x x x x x x } x @ x x x l x x l

x

Figure 8.6. General distribution of methyl groups in X . l a e v i s 28s rRNA. Methyl groups were identified as oligonucleotides within rRNA regions that hybridize between the indicated restriction sites in rDNA (ref. 54 . The analysis shows a heavy cluster of methyl groups in the 3 ’ region of 28s rRNA. T e precise locations o f individual methyl grou s within the indicated regions are currently under analysis (unpublished data of B.E.H.M.! Reprinted by permission from Nature, Vol. 288, No. 5788, pp. 293-296. Copyright (c) 1980; Macmi 1 1 i an Journals Limited

b

B293

8.6.3

Methvlation S i t e s and Conformation: 18s rRNA How do the modified nucleotides f i t i n t o the conformation of rRNA? This question has been addressed i n two ways f o r the methylated nucleotides in 18s rRNA. The f i r s t approach r e l a t e s t o secondary structure modelling. A number of attempts have been made t o construct secondary s t r u c t u r e models f o r eukaryotic 18s rRNA t o which the various published primary structures can be accommodated (e.g. r e f s . 60, 67-69). The various models agree i n many of the proposed interactions although some differences i n detail remain t o be resolved. In most cases the locations of the However, the X e n o p u s model methyl groups were n o t considered. ( r e f . 60) includes the methyl groups. The 5 ' one third of the molecule i s believed t o form a functional domain, i n which various segments from nucleotides 2253 i n t e r a c t w i t h t r a c t s between nucleotides 440 and 617 ( i n the X e n o p u s numbering system), the whole region forming an array of local hairpin arms and 1 onger range interactions. The model brings together many of the 2'-O-methylation s i t e s i n the f i r s t 620 nucleotides i n t o a rather complex conformational core. (Revisions are currently being made t o d e t a i l s o f the model i n ref. 60 t o allow a b e t t e r f i t w i t h the human 18s sequence: B . E . H . Maden, i n preparation.) The second approach involves d i r e c t p r o b i n g of the accessib i l i t y of specific features of rRNA t o enzymic or chemical probes, particularly w i t h methyl-labelled rRNA as substrate. Simple conformational hypotheses f o r rRNA methylation m i g h t envisage t h a t the methylation s i t e s are i n exposed regions of the molecule. Khan and Maden explored t h i s p o s s i b i l i t y some years ago, before the primary structure of 18s rRNA had been determined, by p r o b i n g the a c c e s s i b i l i t y of methylated sequences i n HeLa c e l l rRNA t o m i l d digestion by the single strand s p e c i f i c nuclease S,. An i n i t i a l s t u d y on 5.8s rRNA ( r e f . 70) showed t h a t the loop containi n g the principle methylation s i t e (see above) i s highly suscept i b l e t o S, nuclease digestion. The r e s u l t s from 18s rRNA were more complicated. After m i l d predigestion w i t h S, nuclease some o f the methylated o l igonucleot i d e s were almost eliminated from subsequent fingerprints whereas others were recovered i n good yields and had evidently n o t been

B294

a c c e s s i b l e t o S, nuclease ( r e f . 7 1 ) . In p a r t i c u l a r , the base methylation s i t e s were highly s u s c e p t i b l e t o S, nuclease, i n accord w i t h the f a c t t h a t t h e s e methylations occur l a t e d u r i n g ribosome maturation; hence t h e s e s i t e s would be expected t o be i n exposed l o c a t i o n s i n rRNA (and i n nascent ribosomes). By contrast, only some of t h e 2'-O-methylation s i t e s were s u s c e p t i b l e t o S, nucl e a s e whereas o t h e r s were r e s i s t a n t and were presumably "buried" i n the conformation. Re-examination of the S, d a t a i n the l i g h t of knowledge of t h e l o c a t i o n s of the methyl groups i n the primary s t r u c t u r e gives an i n d i c a t i o n a s t o which methylated regions a r e i n a c c e s s i b l e t o S, nuclease. The i n a c c e s s i b l e methylation s i t e s i n c l u d e several i n the 5 ' domain. For example the ol i gonucl e o t i des encompassing t h e methyl groups a t p o s i t i o n s 428, 462 and 468 (Fig. 8.5) in t h e human sequence (corresponding t o p o s i t i o n s 393, 427 and 433 i n X e n o p u s , F i g . 8.4) a r e i n a c c e s s i b l e t o S, nuclease, whereas those encompassing p o s i t i o n s 484, 512 and 517 i n the human sequence ( p o s i t i o n s 449, 477 and 482 i n X e n o p u s ) a r e in exposed regions. T h i s d i f f e r e n t i a l e f f e c t could not have been p r e d i c t e d from the secondary s t r u c t u r e model ( r e f . 60), which is e s s e n t i a l l y twodimensional. The d a t a suggest t h a t t h e r e a r e t e r t i a r y intera c t i o n s i n t h i s region whereby t h e methyl groups a t p o s i t i o n s 428, 462 and 468 a r e buried i n mature rRNA. A f u l l e r account of t h e s e c o r r e l a t i o n s i s i n preparation (B.E.H.M., unpublished d a t a ) . Further e x p l o r a t i o n s with conformational probes should a f f o r d unique o p p o r t u n i t i e s f o r continuing t o unravel the s t r u c t u r a l organization of rRNA, w i t h special r e f e r e n c e t o the p o s i t i o n s and c o n t r i b u t i o n s of the modified nucleotides i n the rRNA a r c h i t e c t u r e and the r i bosome assembly process. 8.6.4

Closinq Comments The thrust of t h i s account has been t o o u t l i n e how f i n d i n g s from successive experimental approaches have c o n t r i b u t e d t o the unfolding of knowledge of the modified n u c l e o t i d e s i n rRNA from man and o t h e r eukaryotes. The a n a l y s i s i s unfinished, b u t the avai 1 a b l e d a t a can be summari zed and provisional general i z a t i o n s can be drawn.

B295

Among t h e many thousands of n u c l e o t i d e s i n 45s rRNA, about 200 ( i n v e r t e b r a t e s ) a r e recognized and modified, probably by a l i m i t e d number of s p e c i f i c enzymes. All of the methylations and probably a l l of the o t h e r modifications occur i n t h e ribosomal sequences of the precursor molecule, and most of them occur i n regions where primary s t r u c t u r e i s highly (but not a b s o l u t e l y ) conserved among eukaryotes. The d i v e r s i t y of sequences which a r e 2'-O-methylated suggests t h a t t h e s p e c i f i c i t y f o r t h i s ty p e of methylation i s determined by conformation ( r e f . 32). A t least some of the methylations seem t o be i n regions where conformation may be more complex than i s evident from standard secondary s t r u c t u r e model s. Perhaps small i n t e r s p e c i e s v a r i a t i o n s between primary and hence t e r t i a r y s t r u c t u r e may determine the presence o r absence of p a r t i c u l a r methyl a t i on s i t e s . Given t h a t 2'-O-methylation i s i n i t i a t e d upon nascent 45s RNA ( r e f . 15), i t w i l l be of i n t e r e s t t o discover how l a r g e a region of t h e RNA i s required t o generate the conformation t h a t i s recognized by t h e methylase. Although h i t h e r t o i t has only been p o s s i b l e t o c o n j e c t u r e upon the r e l a t i o n s h i p between rRNA methylat i o n , f o l d i n g , and o t h e r i n t e r a c t i o n s d u r i n g ribosome maturation, i t may soon become p o s s i b l e t o approach these q u e s t i o n s d i r e c t l y by u s i n g recombinant DNA t o produce i n i t i a1 l y unmodi f i ed rRNA t r a n s c r i p t s ( r e f . 72), and then u s i n g t h e t r a n s c r i p t s i n assays f o r secondary modification and f u r t h e r s t e p s i n ribosome product i o n . T h i s approach i s c u r r e n t l y being explored i n our laboratory. 8.7

SUMMARY

Ribosomes of man and o t h e r v e r t e b r a t e s contain more than 200 modified n u c l e o t i d e s . In p a r t i c u l a r , ribosomes from human c e l l s contain about 110 2'-0-methylated n u c l e o t i d e s , a small number of base-modified nucleotides and about 95 pseudouridine r e s i d u e s . Approximately 80 of t h e modified n u c l e o t i d e s occur i n 18s rRNA i n the small ribosomal subunit; f o u r occur i n 5.8s rRNA and t h e rest (about 130) a r e i n 28s r R N A i n t h e l a r g e ribosomal s u b u n i t . All of t h e 2'-0-methyl groups a r e added t o ribosomal precursor RNA i n the nucleolus soon a f t e r t r a n s c r i p t i o n . Available d a t a suggest t h a t many o r a l l of the pseudouridine modifications a l s o t a k e

B296

p l a c e on r i b o s o m a l p r e c u r s o r RNA. Some o f t h e base m o d i f i c a t i o n s o c c u r l a t e d u r i n g r i b o s o m e m a t u r a t i o n . The e x a c t l o c a t i o n s o f a l l t h e m e t h y l groups i n t h e p r i m a r y s t r u c t u r e o f 18s r R N A f r o m X e n o p u s 7 a e v i s and man have been d e t e r m i n e d . The m e t h y l groups are widely b u t non-uniformly d i s t r i b u t e d , w i t h a major c l u s t e r o f 2'-O-methyl groups i n a s e c t i o n o f t h e 5 r e g i o n o f t h e m o l e c u l e w h i c h shows h i g h p h y l o g e n e t i c sequence c o n s e r v a t i o n . Some o f t h e s e m e t h y l groups a r e i n sequence t r a c t s w h i c h a r e r e l a t i v e l y i n a c c e s s i b l e t o S, n u c l e a s e , s u g g e s t i n g t h a t t h e y a r e b u r i e d w i t h i n complex t e r t i a r y s t r u c t u r e . P a r t i a l d a t a on t h e l o c a t i o n s o f t h e m e t h y l g r o u p s i n 28s rRNA a l s o i n d i c a t e c l u s t e r i n g i n p h y l o g e n e t i c a l l y c o n s e r v e d sequences, w i t h a m a j o r c l u s t e r i n t h e 3 ' r e g i o n o f t h e m o l e c u l e . O n l y a few p s e u d o u r i d i n e s i n rRNA have so f a r been l o c a t e d . C o m p l e t i o n o f t h e mapping o f t h e m o d i f i e d n u c l e o t i d e s i s t h e o u t s t a n d i n g problem i n t h e t o t a l d e t e r m i n a t i o n o f t h e p r i m a r y s t r u c t u r e o f r R N A f r o m human and o t h e r e u k a r y o t i c sources. A t t a i n m e n t o f t h i s o b j e c t i v e w i l l be a m a j o r s t e p towards understanding t h e h i g h l y s p e c i f i c m o l e c u l a r r e c o g n i t i o n p r o c e s s e s i n v o l v e d i n t h e m o d i f i c a t i o n s , and t h e i r b i o l o g i c a l r o l e i n r i b o s o m e b i o s y n t h e s i s and f u n c t i o n .

8.8

ACKNOWLEDGEMENTS I t h a n k a l l my f o r m e r c o l l e a g u e s who have c o n t r i b u t e d t o i m p o r t a n t p a r t s o f t h e work d e s c r i b e d i n t h i s c h a p t e r : i n parKhan and F.S. McCallum. The a u t h o r ' s t i c u l a r M. S a l i m , M.S.N. work was s u p p o r t e d b y g r a n t s f r o m t h e M e d i c a l Research C o u n c i l . 8.9 1.

2. 3. 4.

REFERENCES

J . D. S m i t h and D. B. Dunn, An a d d i t i o n a l s u g a r

component o f r i b o n u c l e i c acids, Biochim. Biophys. Acta, 3 1 (1959) 573-575. P. R. W h i t f i e l d , A method f o r t h e d e t e r m i n a t i o n o f n u c l e o t i d e sequence i n p o l y r i b o n u c l e o t i d e s , Biochem. J . , 5 8 (1954) 390-396. R. H. H a l l , Method f o r i s o l a t i o n o f 2 ' - O - m e t h y l r i b o n u c l e o s i d e s and Nl-methyladenosine f r o m r i b o n u c l e i c a c i d , B i o c h i m . B i o p h y s . Acta, 6 8 1963) 278-283. H. S i n g h and B. Lane The s e p a r a t i o n , e s t i m a t i o n , and c h a r a c t e r i z a t i o n o f a1 k a l i - s t a b l e o l i g o n u c l e o t i d e s d e r i v e d r ib o n u c l e a t e p r e p a r a t i o n s , Canadian J . f r o m commerci a1 Biochem., 42 (1964) 87-93.

di .

B297

5.

H. Singh and B. G. Lane The a l k a l i - s t a b l e d i n u c l e o t i d e sequences i n 18s and 28s r i b o n u c l e a t e s from wheat germ, Canadian J. B i ochem. 42 (1964) 1011-1021. 6. B. G. Lane, The a l k a l i - s t a b l e t r i n u c l e o t i d e sequences and t h e c h a i n t e r m i n i i n 18s and 28s r i b o n u c l e a t e s from wheat germ, Biochemistry, 4 (1965) 212-221. 7. R. Y . Lau, T . D. Kennedy and B. G. Lane, Wheat-embryo r i bonucleates. 111. M o d i f i e d n u c l e o t i d e c o n s t i t u e n t s in each o f t h e 5.8S, 18s and 28s r i b o n u c l e a t e s , Canadian J. Biochem., 52 (1974 1110-1123. 8. F. F. D a v i s - a n d W. A l l e n , R i b o n u c l e i c a c i d s from y e a s t which c o n t a i n a f i ? t h n u c l e o t i d e , J. B i o l . Chem., 227 (1957)

1

907-915. 9. W. E. Cohn and E. 10. 11.

Vol k i n , Nucleoside-5'-phosphates from r i b o n u c l e i c a c i d , Nature, 167 (1951) 483-484. W. E . Cohn, 5-Ribos 1 u r a c i l , a carbon-carbon r i b o f u r a n o s 1 n u c l e o s i d e i n r i b o n u c e i c acids, Biochim. Biophys. Acta., $2

(1959) 569-571.

r

W. E. Cohn, Pseudouridine, a carbon-carbon l i n k e d r i b o n u c l e o s i d e i n r i b o n u c l e i c a c i d s : i s o l a t i o n , s t r u c t u r e , and chemical c h a r a c t e r i s t i c s , J. B i o l . Chem., 235 (1960) 1488-

1498. 12. J. E. D a r n e l l R i b o n u c l e i c a c i d s from animal c e l l s , Bact. Rev., 32 (19681 262-290. 13. B. E. H. Maden, The s t r u c t u r e and f o r m a t i o n of ribosomes i n 22 (1971) animal c e l l s , Prog. Biophys. Molec. B i o l . , 127-177. 14. G. M. Brown and G. A t t a r d i , M e t h y l a t i o n o f n u c l e i c a c i d s i n HeLa c e l l s , Biochem. Biophys. Res. Comm., 20 (1965) 298-302. 15. H. Greenberg and S. Penman, M e t h y l a t i o n and p r o c e s s i n o f ribosomal RNA i n HeLa c e l l s , J. Mol. B i o l . , 21 (1966) !27535. 16. E. F. Zimmerman and B.H. H o l l e r , Meth l a t i o n of 45s ribosomal RNA p r e c u r s o r i n HeLa c e l l s , J . Mor. B i o l . , 23 (1967) 149161. 17. E. F. Zimmerman, Secondary meth l a t i o n o f ribosomal r i b o n u c l e i c a c i d i n HeLa c e l l s , B i o c e m i s t r y , 7 (1968) 31563164. 18. E. K. Wagner, S. Penman and V.M. Ingram, M e t h y l a t i o n p a t t e r n s

E

19.

o f HeLa c e l l ribosomal RNA and i t s n u c l e o l a r p r e c u r s o r s , J. Mol. B i o l . , 29 (1967) 371-387. M. H. Vau han, R. Soeiro, J. R. Warner and J . E. D a r n e l l , The m e t h i o n i n e d e p r i v a t i o n on ribosome s y n t h e s i s i n effects HeLa c e l l s , Proc. Nat. Acad. S c i . U.S.A., 58 (1967) 1527-

07

1534. 20. B. E. H. 21. 22.

Maden, M. H. Vaughan, .J.R. Warner and J.E. D a r n e l l , E f f e c t s o f v a l i n e d e p r i v a t i o n on ribosome f o r m a t i o n i n HeLa c e l l s , J. Mol. B i o l . , 45 (1969) 265-275. B . E. H. Maden, E f f e c t s o f amino a c i d s t a r v a t i o n on ribosome f o r m a t i o n i n HeLa c e l l s : ribosomal l a b e l l i n g p a t t e r n s i n c e l l s d e p r i v e d o f d i f f e r e n t i n d i v i d u a l amino a c i d s , Biochim. Biophys. Acta, 281 (1972) 396-398. F. Amaldi and G. A t t a r d i P a r t i a l sequence a n a l y s i s o f ribosomal RNA from HeLa ce! 1s . I. 01 igonucl e o t i de p a t t e r n o f 28s and 18s RNA a f t e r a n c r e a t i c r i b o n u c l e a s e d i g e s t i o n , J. Mol B i o l , 33 (1968) 735-755.

.

.

B298

23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.

P h . J e a n t e u r , F. Amaldi and G . A t t a r d i , P a r t i a l sequence a n a l y s i s of ribosomal RNA from HeLa c e l l s . 11. Evidence f o r sequences of non-ribosomal t pe i n 45s and 32s ribosomal RNA p r e c u r s o r s , J . Mol Biol . , 33 (19681 757-775. F. Sanger, G . G . Brownlee and B . G . Barre 1 , A two-dimensional f r a c t i o n a t i o n rocedure f o r r a d i o a c t i v e n u c l e o t i d e s , J . Mol. Biol , 13 (196g) 373-398 G. G. Brownlee, Determination of sequences i n RNA, NorthHol l a n d / E l s e v i e r , Amsterdam, 1972. P . F e l l n e r , Nucleotide sequences from s e c i f i c a r e a s of t h e 16s and 23s ribosomal RNAs of E . c o l i , fur. J . Biochem., 11 (1969) 12-27, M. Salim, R. Williamson and B. E. H . Maden, Methylated o l i onucleotides from HeLa c e l l ribosomal and n u c l e o l a r RNA, FEB! L e t t e r s , 12 (1970) 109-113 B . E . H . Maden, M. Salim and 0. i.Summers Maturation pathway f o r ribosomal RNA in the HeLa c e l l n u c j e o l u s , Nature New Biolog 237 (1972) 5-9. B. E. Maden and J Forbes, Standard and non standard products i n combined f , pl us p a n c r e a t i c RNAase f i ngerpri nts of HeLa c e l l rRNA and i t s p r e c u r s o r s , FEBS Letters, 28 (1972) 289-292. B. E. H . Maden, C. D. Lees and M. Salim, Some methylated se uences and the numbers of methyl groups i n HeLa c e l l rRiA FEBS L e t t e r s , 28 (1972) 293-296 M . Salim and B. E . H . Maden, Early a n d l a t e meth l a t i o n s i n HeLa c e l l ribosome maturation, Nature, 244 (1973) 334-336 B. E. H . Maden and M . Salim, The methylated n u c l e o t i d e sequences i n HeLa c e l l ribosomal RNA and i t s p r e c u r s o r s , J . Mol. B i o l . , 88 (1974) 133-164. S. Petpan, Preparation of u r i f i e d nuclei and n u c l e o l i from mammalian c e l l s , i n K . Rabel and N . P. Salzman (Eds;, Fundamental techniques i n Virology, Academic Press, New Yor , 35-48, b96g~e~!,er, S t r u c t u r e of the 16s and 23s ribosomal RNAs, i n Ribosomes, in M . Nomura, A . T i s s i e r e s and P. Lengyel (Eds), Cold S r i n g Harbor Laboratory, 1974, 169-191. B. E . Maden and M . S . N. Khan, ethylated nucleotide sequences i n HeLa c e l l r i bosomal ri bonucl e i c . aci d : c o r r e l at i on between the r e s u l t s of f i n g e r p r i n t i n g hydro1 s a t e s obtained by d i g e s t i o n with T, ribonuclease and with plus a n c r e a t i c ribonuclease, Biochem. J . 167 (1977) 211-221. K . Wellauer and I . B. Dawid, Secondary s t r u c t u r e maps of RNA: processin of HeLa ribosomal RNA, Proc. Nat. Acad. S c i . U.S.A., 70 1973) 2827-2831. M.S.N. Khan, M. alim and B . E . H . Maden, Extensive.homologies between t h e methyl a t e d nucl e o t i de sequences 1 n s e v e r a l v e r t e b r a t e r i bosomal r i bonucl e i c a c i d s , Biochem. J . , 169 1978) 531-542. . G . Saponara and M.D. Enger, The i s o l a t i o n from r i b o n u c l e i c a c i d of s u b s t i t u t e d u r i d i n e s containing a-aminobutyrate m o e i t i e s derived from methionine, Biochim. Biophys. Acta, 349 (1974) 61-77. R. C. Brand, J . Klootwijk, R. J . P l a n t a and.B. E. H . Maden, Biosynthesis of a hypermodified n u c l e o t i d e i n S a c c h a r o m y c e s c a r l s b e r g e n s i s 17s and HeLa c e l l 18s ribosomal r i b o n u c l e i c a c i d , Biochem. J . , 169 (1978) 71-77.

.

.

fi:

pi.

R.

$:

36. 37. 38. 39.

P

d

B299

40.

E. E l a d a r i , A. Hampe and F. G a l i b e r t , N u c l e o t i d e sequence n e i g h b o u r i n g a l a t e m o d i f i e d g u a n y l i c r e s i d u e w i t h i n t h e 28s ribosomal RNA o f s e v e r a l e u k a r y o t i c c e l l s , Nucl. Acids Res.,

41.

M. S . N. Khan and B. E. H. Maden, N u c l e o t i d e sequences w i t h i n t h e ribosomal r b o n u c l e i c a c i d s o f HeLa c e l l s , X e n o p u s j a e v i s and c h i c k embryo f i b r o b l a s t s , J . Mol. B i o l . , 101

42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57.

4 (1977) 1759-1767. 1976) 235-254.

. C. Choi and H. Busch, M o d i f i e d n u c l e o t i d e s i n T RNase o l i g o n u c l e o t i d e s o f 18s ribosomal RNA o f t h e Nhikoff Hepatoma, Biochem s t r y , 17 (1978) 2551-2560. J . K l o o t w i 'k and R. J . Planta, M o d i f i e d sequences i n y e a s t ribosomal ANA, Mo B i o l . Reports, 1 (1973) 187-191. J . K l o o t w i j k and R. J . P l a n t a , A n a l y s i s . o f t h e m e t h y l a t i o n 39 (1973) s i t e s i n y e a s t ribosomal RNA, Eur. J . Biochem., 325-333.

R. C. Brand, J . K l o o t w i j k , T. J . M. Van Steenbergen, A. J. De Kok and R. J. P l a n t a , Secondary meth l a t i o n o f y e a s t ribosomal r e c u r s o r RNA, Eur. J . Biochem., $5 (1977) 311-318 G. M . RuEin The n u c l e o t i d e sequence o f S a c c h a r o m y c e ; c e r e v i s i a e 5:8S ribosomal r i b o n u c l e i c a c i d , J. B i o l . Chem.,

248 (1973) 3860-3875.

R. N. Nazar, T. 0. S i t z and H. Busch, S t r u c t u r a l a n a l y s i s o f mammal ian ribosomal r i bonucl e i c a c i d and i t s p r e c u r s o r s . N u c l e o t i d e sequence o f ribosomal 5 . 8 s r i b o n u c l e i c a c i d , J . B i o l Chem., 250 (1975 8591-8597. M. S . N. Khan and B.E. !I Maden, N u c l e o t i d e sequence r e l a t i o n s h i p s between v e r t e b r a t e 5 . 8 s ribosomal RNAs, Nucl. Acids Res., 4 (1977) 2495-2505. P. J . Ford and T. Mathieson, The n u c l e o t i d e sequences o f 5 . 8 s ribosomal RNA from X e n o p u s l a e v i s and X e n o p u s b o r e a l i s , Eur. J . Biochem., 87 (1978) 199-214. V . A. Erdmann and J . Wolters, C o l l e c t i o n of p u b l i s h e d 5S, 5 . 8 s and 4 . 5 s ribosomal RNA sequences, Nucl Acids Res., 14, Su p l ement (1986) r l - r 5 9 . B. E. Maden and J. S . Robertson, Demonstration o f t h e 5 . 8 s r i bosomal sequence i n HeLa c e l l ribosomal p r e c u r s o r RNA, J . Mol B i o l , 87 (1974 227-235. J . F. Morrow, S . N. ohen, A. C. Y. Chang, H. W . Boyer, H. M. Goodman and R. B. H e l l i n g , R e p l i c a t i o n and t r a n s c r i p t i o n o f eukar o t i c DNA i n E s c h e r i c h i a c o l i , Proc. Nat. Acad. S c i . U.S.A.. $1 (1974) 1743-1747. P.K. Wellauer, 1:B. Dawid, D. D. Brown and R: H. Reeder The m o l e c u l a r b a s i s f o r l e n t h h e t e r o e n e i t i n ribosomaf DNA from X e n o u s l a e v i s , J . Mo?. B i o l . , 185 (19$6) 461-486 B. E. H. kaden Meth l a t i o n map o f X e n o p u s l a e v i s ribosomal RNA, Nature, 2b8 (1986 293-296 R.L. Gourse and S . A. k e r b i FiAe s t r u c t u r e o f ribosomal RNA. 111. L o c a t i o n s o f e v o l u t i o n a r i l y conserved r e g i o n s w i t h i n ribosomal DNA, J . Mol B i o l , 140 (1980) 321-339. R. C. Brand and S . A. Gerbi, F i n e s t r u c t u r e o f ribosomal RNA. 11. D i s t r i b u t i o n o f m e t h y l a t e d sequences w i t h i n X e n o p u s l a e v i s rRNA, Nucl. Acids Res., 7 (1979) 1497-1511. M. S a l i m and B. E. H. Maden, N u c l e o t i d e sequence o f X e n o p u s gene sequence, l a e v i s 18s ribosomal RNA i n f e r r e d from Nature, 291 (1981) 205-208.

.

.

.

1.

.

.

i

.

.

B300

58. P.M. Rubstov, M.M. Musakhanov, V. M. Zakharyev, A. S. Krayev,

K. G. Skr a b i n and A. A. Ba ev, The s t r u c t u r e o f t h e y e a s t I. T i e complete n u c l e o t i d e sequence ribosomal 8 N A genes o f t h e 18s ribosomai RNA ene from S a c c h a r o m y c e s c e r e v i s i a e , Nucl Acids Res., 8 19803 5779-5794 59. B. E. H. Maden, J. borbes .M: A. S t e w a r t and R . Eason, 18s coding sequences i n amp! if i ed ribosomal DNA from X e n o p u s l a e v i s oocytes a r e h i g h l y homogeneous, unmethylated and l a c k m a j o r open r e a d i n g frames, The EMBO J o u r n a l , 1 (1982) 597-

.

601. 60. J. Atmadja, R. Brimacombe and B. E. H. Maden, X e n o p u s l a e v i s 18s ribosomal RNA: experimental d e t e r m i n a t i o n o f secondary s t r u c t u r e elements, and l o c a t i o n s o f secondary s t r u c t u r e model, Nucl Acids

.

2667. 61. F. S. McCallum and B. E. H. Maden,

Human 18s ribosomal RNA sequence i n f e r r e d from DNA se uence. V a r i a t i o n s i n 18s sequences and secondary modi c a t i o n atterns between v e r t e b r a t e s , B i ochem. J. , 232 (1985 725-73g B. E. H. Maden, I d e n t i f i c a t i o n o f t e l o c a t i o n s o f t h e methyl groups i n 18s ribosomal RNA from X e n o p u s 7 a e v i s and man, J. Mol. B i o l ., 189 (1986) 681-699. J. F. Connaughton, A . R a i r k e r , R. E. Lockard and A. Kumar, Primary s t r u c t u r e o f r a b b i t 18s ribosomal RNA b d i r e c t se uence a n a l y s i s , Nucl Acids Res. , 12 1984) 4731-4f45 Veldman, J. K l o o t w i j k , V.C.H.F. de e g t and R.J. P l a n t a r The p r i m a r y and secondary s t r u c t u r e o f y e a s t 26s rRNA, Nucl. Acids Res., 9 1981 6935-6952. D.G. Hughes an6 B. H. Maden, The p s e u d o u r i d i n e c o n t e n t s o f t h e ribosomal r i b o n u c l e i c a c i d s o f t h r e e v e r t e b r a t e species: numerical correspondence between p s e u d o u r i d i n e r e s i d u e s and Z’-O-methyl roups i s n o t always conserved, Biochem. J., 171

Ti

62. 63. 64. 65.

66. 67. 68.

methyl groups i n t h e Res. , 12 (1984) 2649-

b

.

G.1.

6

k.

781-7!6 h.v 1978) Thomas,.J. Gordon and.H. Rogg, N4-Acet l c y t i d i n e , a p r e i o u s l y u n i d e n t i f i e d l a b i l e component o f x e s m a l l s u b u n i t o f eukar o t i c ribosomes, J. B i o l . Chem., 253 1978) 1101-1105. G.J. g l s e n , R. McCarroll and L. So i n , I econdary s t r u c t u r e o f the smal ! s u b u n i t ribosomal RNA, Nucl . Acids Res., 11 (1983 8037-8049. L. N e l l e s , B-L. Fang, 6 . V o l c k a e r t , A. Vandenberghe and R. De Wachter, N u c l e o t i d e sequence of a crustacean 18s t

Dictyostelium

M.

discoideum

ribosomal RNA and secondar structure s u b u n i t ribosomal RNAs, f i u c l . Acids

8768. 69. Y . L. Chan,

o f e u k a r y o t i c small Res., 12 (1984) 8749-

R. G u t e l l , H. F. N o l l e r and I.G. Wool, n u c l e o t i d e sequence o f a r a t 18s r i bosomal r i bonucl e i c gene and a roposa! f o r t h e secondar structure o f “-In ribosomal r i l o n u c l e i c a c i d , J. B i o l . Cxem., 259 (1984)

L5V.

The acid

18s 224-

Khan and B.E.H. Maden, Conformation o f mammalian 5.8s ribosomal RNA: S, nuclease as a probe, FEBS L e t t e r s , 72

70. M.S.N.

(1976) 105-110. Khan and B. E. H. 71. M.S.N. sequences i n HeLa c e l l probe, Eur. J. Biochem.,

Maden,

Conformation of m e t h y l a t e d

18s ribosomal RNA: nuclease S , as a 84 (1978) 241-250.

B301

72.

Y . A k h t a r and B. E. H. Maden, A n a l y s i s o f X e n o p o s ribosomal RNA s y n t h e s i z e d by t r a n s c r i p t i o n i n v i t r o , Biochem. SOC. Trans. , 14 (1986) 269-270.

Note added i n press. Since t h i s c h a p t e r was s u b m i t t e d t h e work on t h e l o c a t i o n s o f t h e 28s methyl g r o u s and on t h e p s e u d o u r i d i n e s .in 18s. rRNA, mentioned i n S e c t i o n s 8.g.l and 8.6.2, has been p u b l i s h e d i n t h e f o l l o w i n g references:

73. 74.

B. E. H . Maden,

L o c a t i o n s o f methyl groups i n 28s r R N A of clusterin i n t h e conserved c o r e of t h e molecule, J . Mol B i o l , 2 0 1 (!!988) 289-314 B.E.H. Maden and J . A. Wakeman, Pseudouridine d j s t r i b u t i o n i n mammalian 18s ribosomal RNA- a m a j o r c l u s t e r i n t h e c e n t r a l r e g i o n o f t h e molecule, Biochem. J . , 249 (1988) 459-464. X e n o p u s l a e v i s and man:

.

.