J. Mol. Biol. (1992) 223, 899-910

Functional Analysis of Internal Transcribed Spacer 2 of Saccharomyces cerevisiae Ribosomal DNA Carine A. F. M. van der Sande, Marcel Kwa, Rob W. van Nues Harm van Heerikhuizen, Hendrik A. Rau& and Rude J. Plantat Department of Biochemistry and Molecular Biology Vrije Universiteit, De Boelelaan 1083 1081 HV Amsterdam, The Netherlands (Received 4 September 1991; accepted 18 November 1991) Using the previously described “tagged ribosome” (pORCS) system for in viva mutational analysis of yeast rDNA, we show that small deletions in the 5’-terminal portion of ITS2 completely block maturation of 26 S rRNA at the level of the 29 SB precursor (58 S rRNAITS2-26 S rRNA). Various deletions in the 3’-terminal part, although severely reducing the efficiency of processing, still allow some mature 26 S rRNA to be formed. On the other hand. none of the ITS2 deletions affect the production of mature 17 S rRNA. Since all of the deletions severely disturb the recently proposed secondary structure of ITS2, these findings suggest an important role for higher order structure of ITS2 in processing. Analysis of the effect of complete or partial replacement of S. cerevisiae ITS2 with its counterpart sequences from Saccharomyces rosei or Hansen&a wingei, points to helix V of the secondary structure model as an important element for correct and efficient processing. Direct mutational analysis shows that disruption of base-pairing in the middle of helix 1 does not detectably affect 26 S rRNA formation. In contrast, introduction of clustered point mutations at the apical end of helix V that both disrupt base-pairing and change the sequence of the loop, severely reduces processing. Since a mutant containing only point mutations in the sequence of the loop produces normal amounts of mature 26 S rRNA, we conclude that the precise (secondary and/or primary) struture at the lower end of helix V. but excluding the loop, is of crucial importance for efficient removal of ITS2. Keywords:

ribosome;

ribosomal

RNA;

RNA

1. Introduction

t Author to whom all correspondence should be addressed. $ Abbreviations used: ETS, external transcribed spacer; ITS, internal transcribed spacer; kb, kilobase; M-MLV, mouse Moloney leukemic virus; nt, nucleotide; pre-rRNA, precursor ribosomal RNA; rDNA, ribosomal DNA; rRNA. ribosomal RNA: PCR, polymerase chain reaction. $03.00/O

transcribed

spacer; yeast

processing, ribosomal proteins associate with the pre-rRNA, finally resulting in the 40 S and 60 S ribosomal subunits. Virtually all of eukaryotic ribosome biogenesis occurs in the nucleolus (Jordan, 1987; Reeder, 1990; Sollner-Webb & Mougey, 1991), though the finishing touches are applied in the cytoplasm (Klootwijk & Planta, 1989; Rau& & Planta, 1991). In contrast to the rapidly expanding knowledge concerning the mechanism and regulation of pre-rRNA transcription (Sollner-Webb & Tower, 1986; Reeder, 1990; Raue BE Planta, 1991), information on the subsequent steps leading to mature ribosomal subunits is still limited mainly to the pathway of rRNA processing (Klootwijk & Planta, 1989; Raue & Planta, 1991). Only recently has progress been made in identifying components of the machinery involved in this processing (Tollervey, 1987; Savino & Gerbi, 1990; Li et al., 1990; Tollervey & Hurt, 1990; Fabian et aZ., 1990; Ghisolfi et al., 1990; Jarmolowski et al., 1990; Kass &

In all eukaryotes the two high-molecular-mass cytoplasmic rRNA species, as well as the 5.8 S are transcribed as a single precursor rRNA, molecule by RNA polymerase I, which is subsequently converted into the mature molecules by an ordered series of specific endonucleolytic cleavages that remove the external (ETS$) and internal (ITS) transcribed spacer regions (see Fig. 1; for reviews, see Hadjiolov, 1985; Sollner-Webb & Mougey, 1991; Ra& & Planta, 1991). Simultaneously with this

0022-2836/92/040899-12

processing;

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0 1992 Academic Press Limited

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Sollner-Webb, 1990; Kass et al., 1990). One important question concerns the efficiency and specificity of the endonucleolytic cleavages occurring during rRNA maturation. Obviously both these aspects must, at least in part, be determined by elements of primary and/or secondary structure within the various pre-rRNA molecules acting as substrates for the processing endonucleases. So far, however, only one of these &-acting elements, namely that determining the site of the primary processing event occurring in the 5’-ETS of mouse pre-rRNA has been identified by means of mutational analysis in an in vitro cell extract (Craig et al., 1991). We have recently developed a system in the yeast Saccharomyces cerevisiae, which allows us to identify &s-acting elements involved in processing, assembly and function of rRNA by in vivo mutational analysis (Musters et al.? 1989). This “tagged ribosome” system (also called pORCS system for Oligonucleotide-tagged Ribosomal Centromeric rDNA unit of Saccharomyces cerevisiae) is based upon the use of plasmid-encoded yeast rDNA units, the transcripts of which can be distinguished from the chromosomally derived rRNAs due to the presence of a small oligonucleotide insertion (“tag”) in the 17 S and/or 26 S rRNA gene. In a first series of experiments we showed that deletion of various parts of the 5’-ETS or part of the ITS1 (which separates the 17 S and 5.8 S rRNA sequences; Fig. 1) prevents the formation of tagged 17 S rRNA from such a mutant rDNA unit, whereas production of tagged 26 S rRNA is unaffected (Musters et aZ., 1990a). Deletion of large parts of ITS2 (which separates the 5.8 S and 26 S rRNA sequences; Fig. 1) was found to block 26 S rRNA maturation completely or almost completely at the stage immediately preceding removal of the (shortened) spacer (Musters et al., 1990b). Even precise deletion of the complete ITS2 region, resulting in a fusion of the 58 S and 26 S rRNA sequences, a situation similar to that found in prokaryotes (Ram? et al., 1988), abolished the assembly of the fusion rRNA into mature 60 S ribosomal subunits (Musters et al., 1990b). These results unequivocally demonstrate that each of the three transcribed spacer regions of yeast pre-rRNA contains elements that are essential for their correct removal from this transcript. and efficient Moreover, at least ITS2 also contains elements that participate directly in some other step of the maturation process apart, from its own removal. In order to further delineate the cis-acting elements present in S. cerevisiae ITS2, we have embarked upon a more detailed functional analysis of this region, involving replacement by ITS2 more or less regions from other, evolutionarily closely related, yeast species as well as the introduction of small deletions and clustered point mutations at various positions. The interpretation of the results has profited significantly from the fact that during the course of this work a secondary structure model for S. cerevisiae TTSS, based on chemical and enzymatic probing, became available (Yeh & Lee, 1990).

2. Materials and Methods (a) Enzymes, strains and transformation DNA procedures

and recombinant

Restriction enzymes were purchased from Bethesda Research Laboratories (Rockville. MD. c:.S.A.) and Boehringer (Mannheim, Germany). Polynucleotide kinase. Escherichia coli DNA polymerase I (Klenow fragment). phage T4 DNA ligase, T4 DNA polymerase and M-MLV reverse transcriptase were obtained from Bethesda Research Laboratories. Zymolyase-1OOT was from Seikagaku Kogyo Co. (Tokyo, Japan). Helicase was from Industrie Biologique Francaise (Clichy, France). E. coli DHl (F-: recA1, endAl, gyrA96, t&l, hsdR17, supE44. &Al, A-) and JMlOl (A(lac-proAB), supE, thi[F’ : traD36 proAB Zac14 ZAM15]) were used for construction and propagation of plasmid and Ml3 phage DNAs. respectively. S. cerevisiac! MG34 (leu2, trpl, rad2. cir+) was used for transformation of pORCS derivatives. Transformation of yeast cells, isolation of DNA and RNA from yeast cells, recombinant DNA procedures. Northern hybridization and primer extension analysis were performed essentially as described (Musters et al.. 1989, 1990a).

(b) Construction of ITS2 deletion mutants The various plasmids used in this study and t,heir construction is schematically depicted in Fig. I. Plasmids pTZ-ITS and pORCS(26 S*), which carries a tagged 26 S rRNA gene, have been described (Musters et al.. 1989, 1990a) as has the construct,ion of pORCS( 17 S*+26 S*) carrying a tag in both the 17 S and 26 S rRNA gene (Musters et al., 1990a). In order to be able to construct small ITS2 deletions, we first cloned the @4 kb SphI-BamHI fragment from pTZ-ITS, encompassing the entire ITS2 into pTZ19U (USB, Cleveland, U.S.A.), resulting in plasmid pTZl9-ITS2. Using this subclone, 2 additional restriction sites were created by replacing the original ITS2 sequence between the HpaI and CZaI sites with a double-stranded oligonucleotide containing a point mutation at both position +88 and + 156 (relative to the 3’-end of the 5.8 S rRNA gene). The resulting pTZ19-ITS2AP plasmid contains a unique AsuII and PstI site within the ITS2 (Fig. I). Partial ITS2 deletions were constructed by digestion of pTZ19-ITS2AP with various combinations of HpaI, AsuII. PstI and ClaI followed by religation. If necessary, sticky ends were made blunt pTZ19-ITS2-A5’H was using T4 DNA polymerase. constructed by replacing the SphI-HpaI fragment from with an oligonucleotide that exactly pTZ19-ITS2AP restores the 3’-terminal sequence of the 5.8 S rRNA gene. A precise deletion of ITS2 was created by replacing the SphI-A$11 fragment of pTZ-ITS with a synthetic oligonucleotide that restores the 3’-terminal region of the 5.8 S and the 5’-terminal region of the 26 S rRNA gene (Musters et at.. 199Ob). The various mutant ITS2 regions were transferred to pORCS(26 S*) or pORCS( 17 S* + 26 S*) by first replacing the @4 kb SphI-XhoI wild-type fragment of pTZ-ITS with the corresponding fragment from the appropriate pTZ19-ITS2 mutant. The 1.4 kb SacI&XhoI fragments of the resulting pTZ18-ITS2 mutants were then used to fragment of wild-type SacI-XhoI replace the pORCS(26 S*) or pORCS(17 S*+26 S*).

Mutational

Andy&

qf yeast ITS2

KS2 deletion mutants

901

ITS2 replacement mutants pTZ1 E-ITS2-Sr

pTZl8-ITS2-Hw pTi’19-ITS245’H

m pTZl8-ITS27Sr3

pTZ19-ITS2-HP pTi’lE-ITS2-Hw5’

pTZ19-ITS2-PC pTZ19-IT.%?-AP

plZl8-ITS2-Hw3

pTi!lE-ITS24lll

poRCS(26S’) pORCS(17S1+26S*) -

37s pre-rRNA SETS

ITS1 ITS2

3’ ETS

Figure 1. Plasmids used in this study. Parent constructs pORCS(26 S*) and pORCS(17 S*+26 S*) have been described (Musters et al., 1989, 199Ou). Filled bars represent mature. open bars non-transcribed spacer and other bars transcribed spacer sequences (stippled: S. cerevisiae; hatched: S. rosei or H. wingei). The position of the oligonucleotide tags in 17 8 and 26 S rRNA is indicated by n and 0, respectively. The primary precursor rRNA transcript is shown at the bottom together with the sequences present in the 29 SB precursor molecule. The 2 subclones used for construction of the various ITS2 mutations are pTZ-ITS (Musters et al., 199Ou) and pTZ19-ITSB. pTZ19-ITS2AP containing an extra ilusI and PstT site was obtained by site-directed mutagenesis of pTZl9-ITSZ. The sequences into which the mutations were introduced are shown with the pertinent mutations shown in bold faced type (see also Fig. 4). The structures of the ITS2 deletion mutants are depicted at the left of this Figure. Numbers indicate the end points of the deletions, the 5’-end of ITS:! being position + 1. Complete and partial (chimeric) replacement ITS2 mutants are depicted at’ the right. Hatched bars represent ITS2 sequences of other yeast strains (Sr, S. rosei; Hw, H. wingei). Asterisks mark the positions of clustered point mutations present in pTZ18ITS2-1 and -11 (see Fig. 7). The broken lines indicate the segments exchanged between the pORCS plasmid and the pTZ18 or pTZ19 series. Abbreviations used for restriction enzyme sites are as follows: Af. ;?flII: As. ~4.~11; Bl, BaZI; Cl. ClaI; Hi, HindIII; Hp. HpaI; Ps, P&I; SC. SacI; Sp, SphI; Xh, XhoI.

(c) Cloning and sequencing of ITS2 regions from S. rosei and Hansenula wingei

In order to sequence the ITS2 region of S. rosei and wingei. we subcloned the 3.5 kb XbuI-XbaI fragment of each of the 2 strains, spanning ITSl, the 58 S rRNA gene and ITS2. into pUC19, yielding pUC19Sr and pUC19-Hw, respectively. Sequence analysis was carried out on double-stranded DNA using a primer complementary to a universally conserved part of 26 S rRNA (positions + 52 to + 67, relative to the 5’-end of the 26 S rRNA) by means of a Sequenase@ 2.0 kit from USB (Cleveland, Ohio. U.S.A.). Reactions were performed according to the supplier’s instructions. Hansen&a

(d) Construction

of mutants carrying chimeric ITS2 regions

heterologous

or

In order to replace the ITS2 of S. cerevisiae present in pORCS(26 S*) with its counterpart from S. rosei we substituted the 93 kb SphI-A$11 fragment from pUC19Sr for the SphI-A$11 fragment of pTZ-ITS (Fig. 1). The resulting plasmid was named pTZ18ITS2-Sr. For a similar replacement, with the ITS2 from H. wingei we first

carried out a triple ligation of the 0.2 kb HindIII-A$11 fragment from pUC19-Hw, which contains most of ITS2 of H. wingei, the 2.9 kb AJIII-SphI fragment from pTZl9-ITS2 and an oligonucleotide that rest,ores the missing 3’-terminal part of the 5.8 S rRNA gene as well as the 5’-terminal sequence of the H. wingei ITS2. The 94 kb SphI-A$11 fragment of the resulting plasmid was then used to replace its counterpart in pTZ-ITS, yielding pTZ18-ITS2-Hw. The heterologous ITS2 regions were finally transferred to pORCS(26 S*) by substitution of its SacI-XhoI fragment with the corresponding fragment from the appropriate pTZ18-ITS2 construct (Fig. 1). To construct a chimeric ITS2 composed of the 5’-terminal half of the S. cerevisiue ITS2 and the 3’-terminal half of its S. rosei counterpart, we first. created a PstI site in the S. rosei sequence at a position identical to that in pTZ19-ITS2AP by performing a polymerase chain reaction using the 93 kb HpaI-SmuI (the latter site is part of the polylinker) fragment from pTZ18-ITS2Sr as the template. The same 26 S rRNA primer as employed in ITS2 sequencing (section (c), above) and an oligonucleotide spanning positions + 138 to + 168 of the S. rosei ITS2 were used as primers for the reaction, which was carried out with the aid of 95 unit of Ampli Taq polymerase (Perkin Elmer, The Netherlands). The sequence of

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the latter primer was chosen in such a way that point mutations were introduced at positions + 151 (U-G) and + 153 (G=>A) to create the desired PatI site (see Figs 1 and 4). The PCR product was cut with P&I and A$11 and the resulting oligonucleotide was substituted for the Ps&A$II fragment of pTZ18-ITS2AP, yielding pTZl8-ITS2-Sr3’. Chimeras between S. cerevisiae and H. wingei ITS2 were constructed either by ligation of the @3 kb AsuII-XhoI fragment from pTZ18-ITS2AP with the 3.9 kb XhoILBaZI fragment from pTZ18-ITS2-Hw, yielding pTZl%ITS2-Hw5’ or by ligation of the @3 kb BaZI-XhoI fragment from pTZ18-ITS2-Hw with the 3.9 kb XhoILAsuII fragment from PTZ~~-ITS~*~, resulting in pTZ18-ITS2-Hw3’. Prior to both ligations the AsuII site was made blunt with the aid of T4 DNA polymerase. Finally, the 1.4 kb SacI-XhoI fragment from each of the pTZ18-ITS2 constructs described in this section was substituted for the corresponding fragment of pORCS(26 S*). (e) Construction

of

ITS2 mutants point mutations

carrying

clustered

pTZ18-ITS2 mutants carrying clustered point mutations were constructed by replacing the AsuII-PstI fragment of pTZ 18-ITS2AP with 1 of 2 double-stranded oligonucleotides containing 10 consecutive point mutations. In pTZ18-ITS2-I.1 the sequence from position + 117 to + 126 (relative to the 3’-end of the 58 S rRNA gene) and in pTZ18-ITS2-II.1 that from position + 138 to + 147 was mutated by changing every A to a U and every G to a C or vice-versa. pTZ18-ITS2-II.2 was obtained with the aid of the pSELECT system (Promega) for in vitro mutagenesis using an oligonucleotide spanning positions + 134 to + 158, which introduced U*C and A*G eransitions at position + 145 and + 147, respectively. The corresponding ~0:~: derivatives were obtained by substituting 1.4 kb SacI-XhoI fragment of pORCS(26*) with the same fragment from the pTZ18ITS2 constructs.

3. Results (a) ITS2

does not play a role in formation mature 17 S rRNA

of

Previous analysis of the effect of partial or complete deletion of ITS2 was carried out with a version of the pORCS system in which only the 26 S rRNA gene carried a “tag” inserted into variable region V2 of domain I. Consequently we were unable to ascertain whether these deletions had any effect on the formation of 17 S rRNA (Musters et al., a deletion 199Ob). Therefore, we introduced ITS2 into comprising the entire pORCS (17 S* + 26 S*), which contains a yeast rDNA unit carrying a tag in both the 17 S and 26 S rRNA gene, the former having been inserted into variable region V8 of domain III (Musters et al., 1990a). Figure 2 depicts the results of a Northern hybridization experiment in which RNA isolated from cells transformed with the resulting pORCS(17 S* +26 S*)AITS2 plasmid was probed with the two oligonucleotides complementary to the 26 S (a) and 17 S (b) tag, respectively. As a control we analyzed

Figure 2. Effect of deletion of ITS2 on production of 17 S and 26 S rRNA. Total RNA isolated from cells with transformed pORCS(17 S*+26 S*) (lane 1). untransformed host cells (lane 2) or pORCS(17 S* +26 S*)AITS2 (lane 3) was analyzed by Northern blotting with an oligonucleotide complementary to either the 26 S rRNA (a) or the 17 S rRNA (b) tag.

RNA isolated from cells carrying the same number of copies of a wild-type, doubly tagged, rDNA unit. Consistent with our earlier findings (Musters et al., 1990a), the accumulation of tagged 26 S rRNA is severely reduced by the deletion. In fact no 26 S rRNA having the correct 5’-end could be detected even by reverse transcription analysis (data not shown). The small amount of material detected by Northern hybridization with the probe complementary to the 26 S rRNA tag most likely represents the 5.8 S-26 S fusion transcript. This transcript, corresponding to the 29 &-type precursor that accumulates in all ITS2 deletion mutants analyzed so far (Musters et al., 19906; see also Fig. 3(c)), would only be about 150 nt longer than the mature 26 S rRNA and, thus, would migrate at practically the same position. In contrast to the severe reduction in tagged 26 S rRNA levels, the amount of tagged 17 S RNA produced from pORCS(17 S*+26 S*)AITS2 is virtually the same as that present in the pORCS( 17 S* + 26 S*) control transformant (Fig. 2(b)). From these data it is clear that the involvement of ITS2 in yeast rRNA processing is limited to the formation of 5.8 S/26 S rRNA and does not extend to the production of 17 S rRNA or its assembly into 40 S subunits.

Mutational

Analysis

qf yeast

903

ITS2

1 AAP

WT

AP

A5'H

AHA

AAP

APC

G ATC AP A5’H AHA DAP OPC

- 29SB

- 26s

(b)

(cl

Figure 3. Effect of small deletions within ITS2 on production of 26 S rRNA. (a) Schematic representation of the various lTS2 deletions superimposed upon the secondary structure model for S. cerevisiae ITS2 proposed by Yeh & Lee (1999) depicted schematically at the left. The restriction sites used in the introduction of the various deletions are indicated, together with the code used for naming the deletion mutants. Mature 58 S and 26 S rRNA sequences are represented by the thick bars. (b) Northern analysis, using the oligonucleotide complementary to the 26 S rRNA tag as probe, of total RNA isolated from cells transformed with pORCS(26 S*) (lane WT); pORCS(26 S*)ITS2AP (lane Al?) and pORCS(26 S*) derivatives carrying the various ITS2 deletions (a). In all cases transformants carrying the same number of copies of the plasmid were used. (c) Primer extension analysis of the same RNA samples analyzed in (b) using the same oligonucleotide as the primer (right-hand lanes). In each of the primer extension experiments in vitro synthesized RNA from the corresponding pTZ18-ITS2 construct (Fig. 2) was analyzed as a control (left-hand lanes). The dideoxy sequencing shown at the left was carried out on total RNA from pORCS(26 S*) transformants using the oligonucleotide complementary to the 26 S rRNA tag as the primer. The sequence of the non-transcribed strand spanning the $-end of mature 26 S rRNA is indicated. The extra bands visible in t,he primer extension analysis of the APC mutant have been observed previously (Musters et aZ., 1990b). They probably arise by degradation or, possibly by alternative processing of a minor portion of the pre-tRNA molecules.

(b) Deletion

analysis

of ITS2

In order to gain further insight into the distribution of elements within ITS2 essential for proper rRNA processing, we constructed a set of mutants

carrying small adjacent deletions. These deletions were obtained by first creating additional unique restriction sites for the enzymes AsuII and PstI, respectively, via the introduction of two point mutations within the ITS2 that is part of

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pORCS(26 S*) (Fig. 1). Northern blot analysis of total RNA isolated from transformants that contain the resulting pORCS-ITS2AP (Fig. 3(b)) clearly demonstrates that the amount of tagged 26 S rRNA accumulating in these cells is indistinguishable from that produced in cells carrying the same number of copies of the control pORCS(26 S*) plasmid. Reverse transcription analysis showed the tagged 26 S rRNA to have the correct 5’-end (Fig. 3(c)). Thus, the presence of the two point mutations in ITS2 does not interfere with the production of 26 S rRNA from the mutant rDNA unit. Csing the newly introduced AsuII and PstI sites in combination with sites already present in the wild-type ITS2 sequence (HpaI and CZaI), we removed various parts of the ITS2 region and introduced the mutant spacers into the pORCS(26 S*) plasmid for testing in viva. Figure 3(a) depicts the various deletions superimposed upon the secondary structure of ITS2 as recently proposed by Yeh & Lee (1990). As shown in Figure 3(b), we were unable to detect tagged 26 S rRNA by Northern analysis in any of the deletion mutants. Since each of the deletions would severely affect the (proposed) secondary structure of ITS2 (Fig. 3(a)) these results suggest that correct folding of (parts of) the spacer is crucial for the efficient production of 26 S rRNA and its assembly into 60 S subunits. In order to ascertain at what stage formation of 26 S rRNA is blocked by the structural alterations in ITSB, we also studied the RNA transcribed from the ITS2 deletion mutants by reverse transcription analysis using the oligonucleotide complementary to the tag in the 26 S rRNA gene as the primer. In vitro synthesized RNA was used as a control to distinguish between artificial stops and bona-$dr 5’-ends of processing intermediates. The results (Fig. 3(c)) confirm the absence of mature 26 S rRNA molecules in all of the deletion mutants except pORCS-ITSB-APC, which lacks a portion of the 3’-terminal region of the spacer (Fig. 3(a)). This mutant is seen to be still capable of producing a small amount of tagged 26 S rRNA that possesses the correct 5’-end. The cellular amount of this mature, tagged 26 S rRNA apparently is below the detection level of the Northern hybridization (Fig. 3(a)) but can be detected by the more sensitive primer extension analysis. The production of a small amount of correctly processed 26 S rRNA by the pORCS-ITS2-APC mutant is in agreement with earlier studies from our laboratory, which showed that’ a mutant having a deletion of the 3’-terminal 52 nucleotides of ITS2 also still generated a small amount of correctly processed tagged 26 S rRNA (Musters et al., 19906). Thus, sequences in the 3’-terminal part of ITS2 (positions 154 to 233) appear not to be essential for correct 5’-end formation of 26 S rRNA, although they exert a strong influence on the efficiency of processing. Sequences in the 5’-terminal part’ (positions 1 to 153), on the other hand, are indispensable for formation of mature 26 S rRNA. The primer extension analysis reveals the pre-

sence of increased amounts of the 29 SB-type precursor (5.8 S rRNA-(A)ITS2-26 S rRNA; see Fig. 1) in all ITS2 deletion mutants studied except A5’H, where the 5’-end of this precursor is at the limit of detection (Fig. 3(c)). This confirms our earlier conclusions (Musters et aZ., 1990b) that removal of part or all of the ITS2 blocks. or at least severely retards, maturation of the large subunit rRNA at the stage immediately preceding the severance of the covalent link between the 5.8 S and 26 8 rRNA sequences. (c) Mutants

carrying

heterologous

ITS2

regions

Phylogenetic comparison of equivalent RNA molecules has proved to be a powerful tool for highlighting possible functional elements, in particular when information on the secondary structure of the RNA is available (Gutell & Woese, 1990). In order to apply this tool to the analysis of the yeast ITS2 we determined the nucleotide sequence of the corresponding spacer of two additional members of the subfamily Saccharomycetoideae namely Sacehuromyces rosei, which is fairly closely related to S. cerevisiae, and Hansen&a wingei, a more distant relative (Kreger-Van Rij, 1973). Comparison of the resulting sequences to that of S. cerevisiae ITS2 revealed a relatively low homology in ITS2 primary structure between the three strains, consistent wit,h the observation that eukaryotic transcribed spacer regions are subject to only limited evolutionary constraints (Gerbi, 1985). In agreement with the previously established evolutionary relationships, the S. cerevisiae ITS2 shows a larger homology t,o its counterpart from S. rosei than to that from H. wingei (Fig. 4). However, when we modeled t,he 8. rosei and H. wingei ITS2 sequences according to the recently published secondary structure model for S. cerevisiae ITS2 (Yeh & Lee, 1990) we observed a more extensive overall resemblance (Fig. 4). The major differences between 8. cerevisiae and S. rosei are found in helices IV and VI, the latter being considerably larger in S. rosei. The structure of the H. wingei ITSS, while still conforming to the model, diverges to a larger extent. Helices IV, V and VI are all significantly reduced in size, while helix III shows more irregularities than its counterpart from either of the other two strains. Nevertheless, the ability of the S. rosei and H. wingei ITS2 sequences to fold into secondary structures similar to that proposed for S. cerevisiae ITS2 constitutes additional support for this secondary structure model, originally derived from chemical and enzymatic probing data in combination with computer folding (Yeh & Lee, 1990). In order to analyze the functional equivalence of constructed ITS2 regions we the three pORCS(26 S*) derivatives in which the TTSZ of S. cerevisiae had been replaced with its counterpart from either S. rosei or H. wingei and determined the production of tagged 26 S rRNA in cells transformed wit’h the resulting pORCSITS2Sr and pORCS-ITS2-Hw plasmids. The Northern blot

H. wingei

Figure 4. Secondary structure models for the ITS2 regions of 8. rosei and H. ukagei. The sequences of the ITS2 regions of the 2 yeast strains were determined and modeled according to the secondary structure model for the S. cerevisiae ITS2 proposed by Yeh $ Lee (1990) shown at the left. Sequence conservation between each of the 2 ITS2 regions and its 8. cereviviae counterpart is indicated in reversed contrast. The cleavage site for processing of the A. c~reviniae 29 SR precursor to mature 5.8 8 and 26 S rRNA is shown, as are the positions of the restriction sites (natural as well as newly created) used in constructing the ITS2 deletion mutants and chimeric ITS2 regions. Broken lines indicate a possible tertiary interaction between the loops of helices IV and VT discussed in the text. Note that in the case of S. cerevisiaf: additional tertiary base-pairs can be formed between the 2 loops.

S. rosei

C. A. F. M. van der Sande et al. WT

Sr

WT Sr

Hw

Hw

b’

Figure 5. Complete replacement of S. cerevisiae ITS2 by its counterpart from 8. rosei (Sr) or H. wingei (Hw). Total RNA from 8. cerevisiue cells transformed with pORCS(26 S*) (lane WT), pORCS(26 S*)-ITS2-Sr or pORCS(26 S*)-ITS2-Hw (see Fig. 2) was analyzed by Northern hybridization (a) and reverse transcription (b) using the oligonucleotide complementary to the 26 S rRNA tag as the probe. In each of the primer extension from the experiments in vitro synehesized RNA (Fig. 2) was corresponding pTZ18-ITS2 construct analyzed as a control (left-hand lanes). In all cases transformants carrying the same number of copies of the plasmid were used. The bands corresponding to mature 26 S rRNA and 29 SB-type precursor rRNA are indicated.

analysis clearly shows that in the first case formation of tagged 26 S rRNA is not affected, whereas in the second type of transformant no tagged 26 S rRNA is detectable (Fig. 5(a)). However, a small amount of correctly processed tagged 26 S rRNA transcripts can be observed in cells transformed with pORCS-ITS2-Hw by primer extension analysis (Fig. 5(b)). Consequently, we conclude that the S. rosei ITS2 is functionally fully equivalent with its A“. cerevisiae counterpart, containing all elements required for correct and efficient rRNA maturation and assembly by the S. cerevisiae processing machinery. The H. wingei ITS2, on the other hand, must lack one or more elements essential for efficient processing in the heterologous environment, though the small amount of 26 S rRNA that is allowed to form has the correct 5’ terminus. (d) Mutants

carrying

chimeric

ITS2

regions

We next extended the type of analysis described in the previous section by constructing mutants that carried chimeric ITS2 regions consisting of (1) the 5’-terminal part of the S. cerevisiae ITS2 (nt 1 to 158) linked to the 3’-terminal part (nt 156 to 258) of its S. rosei counterpart (pORCS-ITS2-Sr3), (2) nt 85 to 234 of S. cerevisiae fused to nt 1 to 84 of H. wingei (pORCS-ITS2-Hw~‘), and (3) nt 1 to 88 of

S. cerevisiae fused to nt 85 to 179 of H. wingei (pORCS-ITS2-Hw3’). Clearly none of these three chimeric ITS2 regions, the proposed structure of which is shown in Figure 6(a), allows efficient production of tagged 26 S rRNA, as indicated by the results of the Northern hybridization experiments carried out on total RNA from the respective transformants (Fig. 6(b)). Primer extension analysis (Fig. 6(c)) demonstrates that the pORCS-ITS2-Sr3’ and pORCS-ITS2-Hw5’ chimeras support production of only a barely detectable amount of correctly processed 26 S rRNA. Cells transformed with the pORCS-ITS2-Hw3’ chimera, on the other hand, do contain a significant, though still considerably reduced, amount of tagged 26 S rRNA that has the correct 5’ terminus. Again the amount of this mature 26 S rRNA is too low to be clearly detectable by the less sensitive Northern analysis, although a faint signal was visible on the original autoradiogram, which is lost in reproduction. The reverse transcription data again show the presence of the 29 SB-type precursor in all cases. The amount of this precursor is likely to be underestimated in this experiment because its 5’-end is at the limit of detection using the primer complementary to the 26 S rRNA tag. The results obtained in the analysis of the two S. cerevisiae/H. wingei chimeras are in agreement with those presented above for the ITS2 deletion mutants since they once more indicate a greater importance of the B’-proximal region of the S. cerevisiae ITS2, compared to the 3’-proximal region, for processing. The inability of the S. cerevisiaels. rosei chimera to support significant ITS2 processing is somewhat surprising in view of the full functional equivalence of the two spacers demonstrated in the previous section. This result indicates that some form of interaction between the two “halves” of ITS2 is crucial for processing and that this interaction must involve nucleotides that have not been conserved between the two yeast strains. A close inspection of the structure of the chimera (Fig. 6(a)) does not reveal any gross differences of individual structural elements with their counterparts in one or another of its two parents. The most tangible structural variations are present in helix V and concern the distribution of irregularities (bulged nucleotides and internal loops) in particular in the upper part of this helix. A second significant difference between the chimera and its parents is the disruption of a possible tertiary interaction between the loops of helices IV and VI which in both S. cerevisiae and S. rosei show complementarity (Fig. 4). (e) Mutants carrying clustered point mutations of helix V

In order to examine the importance features

of helix

V further,

we first

of structural replaced

two

ten-nucleotide-long regions within this helix by linkers of the same length but having a totally unrelated sequence (Fig. 7(a)). In pORCS-ITSZ-I

Mutational

WT

Sr3’

907

Analysis of yeast ITS2

Hw5’

Hw3’

WT

Sr3’

Hw5’ Hw3’

26s

(b) Figure 6. Partial replacement of S. cerevisiae ITS2 with the corresponding region of its counterparts from 8. rosei (Sr) or H. tingei (Hw). (a) Schematic representation of the proposed secondary structure of the various chimeric ITS2 regions tested, according to the secondary structure model proposed by Yeh 6 Lee (1990). Mature 58 S and 26 S rRNA sequences are indicated by the thick bars. The “foreign” part of the ITS sequence is represented by the closed connected circles, the S. cerevisiue part by the open circles. (b) Northern analysis of total RNA isolated from cells transformed with pORCS(26 S*) (lane WT) and pORCS(26 S*) derivatives carrying the chimeric ITS2 regions (indicated as in (a)). In all cases transformants carrying the same number of copies of the plasmid were used. (c) Primer extension analysis of the same RNA samples analyzed in (b) using the same oligonucleotide as the primer (right-hand lanes). In each of the primer extension experiments in vitro synthesized RNA from the corresponding pTZ18-ITS2 construct (Fig. 2) was analyzed as a control (left-hand lanes). The bands corresponding to mature 26 S rRNA and 29 SB-type precursor rRNA are indicated. this replacement completely abolishes the predicted base-pairing in the middle of helix V. In pORCSITSS-11.1, on the other hand, introduction of the linker both disrupts the double-stranded region at the apical end of helix V and alters the nucleotide sequence of the loop. The results of Northern blot

hybridization (Fig. 7(b)) and reverse transcription analysis (data not shown) demonstrate that the mutations present in pORCS-ITSS-I have no detectable effect. Normal amounts of correctly processed tagged 26 S rRNA are present in pORCS-ITSB-I transformants. The clustered point mutations intro-

C. A. F. M. van der Sande et al.

908

(a)

xl.2

gr:

g-z

A-U U A

KUA

i

ir

A-U

t

i

A,

uG U- AC

i k-l!i AUC A U-G’ C-G G-U

Au A

CG-:

8:: G-u u u ‘AU A-U C- GA C-G A U UUU

AP

ITS-I

ITS-II.1

‘A U-GC C-G

Et :

U A- U A- UA C- G C-G BU “W

ITS-II.2

Figure 7. Effect of clustered point mutations in ITS2 on the production of 26 S rRNA. (a) Structure of the mutant, ITS2 regions. To the left a schematic representation of the S. cerevisiae ITS2 is given. The mature 5.8 S and 26 S rRNA sequences are indicated by the stippled bars. The regions of helix V that are changed by the mutations are boxed. The structure of the pertinent region of helix V in the various mutants is depicted in detail. Changed nucleotides are shown in reversed contrast. (b) Northern analysis of total RNA isolated from cells transformed with pORCS(26 S*)-ITS2AP (lane AP) and the derivatives ITS2-I and ITS2-II.1 using the oligonucleotide complementary to the 26 S rRNA tag as probe. (c) Northern analysis of total RNA isolated from cells transformed with pORCS(26 S*) (lane WT) and its derivative ITS2-11.2. Transformants containing the same number of copies of the plasmid were used in each of the 2 experiments.

duced into the bottom part of domain V (pORCSITSB-II.l), on the other hand, strongly interfere with the efliciency of processing reaction (Fig. 7(b)), although some tagged 26 S rRNA having the correct 5’-end could still be observed in this mutant (data not shown). Thus, the secondary and/or primary structure at the apical end of helix V is important for efficient ITS2 processing. Because the sequence of the loop of helix V is absolutely conserved between S. cerevisiae and S. rosei but differs at two positions in the H. wingei ITS2 we introduced the latter two changes into S. cerevisiae ITS2 (Fig. 7(a)). As judged from the Northern analysis (Fig. 7(c)) the resulting pORCS-ITS2-II.2 construct produces normal amounts of 26 S rRNA, which possesses the correct $-end (data not shown). This result demonstrates that at least a certain amount of flexibility in the nucleotide sequence of the loop of helix V is tolerated by the processing machinery.

4. Discussion The yeast “tagged ribosome” system developed in our laboratory (Musters et al., 1989, 1990a) consti-

tutes an excellent tool for characterizing primary and/or secondary structural elements of (pre-)rRNA involved in rRNA processing, assembly and ribosomal function. Since the presence of the tags does not noticeably interfere with ribosomal biogenesis or function it allows in vivo mutational analysis of the pre-rRNA which, so far, is not possible by other means. Applying this system to a functional study of the transcribed spacers of pre-rRNA, we have recently demonstrated each of these spacers (5’-ETS, ITS1 and ITS2) to be indispensable for prerRNA processing (Musters et aZ., 1990a,b). The sphere of influence of each of the spacers is circumscribed, however. Deletions of (part of) the 5’-ETS and ITS1 abolish the production of mature 17 S, but not 26 S rRNA (Musters et al., 1990a). Similarly, as shown in this paper, complete removal of ITS2 blocks the maturation of 26 S rRNA but does not affect the formation of mature 17 S rRNA. Thus, despite the fact that during the first stages in yeast ribosome biogenesis the 17 S, 5.8 S and 26 S rRNA sequences are still part of a single molecule (Klootwijk & Planta, 1989), maturation and assembly of the rRNA molecules that end up in

Mutational

Analysis

different subunits appears to proceed more or less independently. Independent assembly of the two ribosomal subunits is also apparent from the fact that (partial) inactivation of a yeast ribosomal protein gene affects the formation of only the subunit of which the protein in question forms part and does not interfere with biogenesis of the other subunit (Himmelfarb et al., 1984; Rotenberg et al., 1988; Finley et al., 1989). The main portion of the studies presented in this paper concerns a more detailed identification of the &s-acting elements with ITS2 required for processing. Analysis of the effect of small adjacent deletions indicates that the information for efficient removal of ITS2 from its precursor is dispersed throughout the entire ITS2 region, since all of the deletion mutants show severely reduced levels of tagged 26 S rRNA (Fig. 3(b)). Localization of the 5’-ends of processing intermediates by primer extension analysis demonstrates that in these mutants processing is blocked at the level of the 29 Sn-type having the structure 5.8 S rRNAprecursor (mutant)ITS2-26 S rRNA (see Fig. 1). In all but one case this block is complete. No band corresponding to the mature 5’-end of 26 S rRNA is observed by reverse transcription using a primer that detects only transcripts from the tagged rDNA unit (Fig. 3(c)). One of the mutants, however, carrying a deletion of part of the 3’-terminal portion of the spacer (Fig. 3(a)) still allows some correctly processed 26 S rRNA to be produced. This result, together with a similar observation on an ITS2 mutant containing an overlapping deletion within the 3’-terminal region (Musters et al., 1990b), shows that correct 5’-end formation of 26 S rRNA does not depend upon the presence of sequences between positions 153 and 234 of the ITSB. Nevertheless, these sequences are required for optimal efficiency of ITS2 processing. The region between positions 1 and 153, on the other hand, is indispensable for correct and efficient, processing as shown by the complete absence of any mature tagged 26 S rRNA in mutants carrying deletions covering various parts of this region. The most plausible explanation for the drastic reduction caused by the various ITS2 deletions in the efficiency of processing seems to be disruption of secondary structure elements since each of the deletions tested would severely affect the folding of the spacer according to the recently proposed model of Yeh & Lee (1990) (Fig. 3(a)). Secondary structure appears to play little or no role in directing the processing nucleases to their correct sites, at least in the case of the enzyme producing the 5’ terminus of 26 S rRNA, since the deletions in the 3’-terminal region also destroy most of the (proposed) base-pairing within ITS2 but still allow formation of some correctly processed 26 S rRNA (Fig. 3(a)). The secondary structure model for S. cerevisiae ITS2 developed by Yeh and Lee on the basis of chemical and enzymatic probing of in vitro synthesized 37 S pre-rRNA (Yeh & Lee, 1990), gains further support from the fact that both the S. rosei and II. wingei ITS2 regions can be folded into

of yeast ITS2

909

similar structures (Fig. 4). A number of compensating base-pair mutations are present in helices III to VI, although the detailed structure of each of these helices varies from one species to another. The largest deviations are present in helices TV and VI, which differ in both length and sequence. Roth the primary and secondary structure of helix III is very well conserved in S. rosei, while in H. wingei this helix contains more extensive irregularities. Helices IV, V and VI have all been shortened considerably in H. wingei. All in all, the extent of structural conservation is in agreement with the evolutionary distance between S. cerevisiae on the one hand and S. rosei and H. wingei on the other (Kreger-Van Rij, 1973). Taking into account the structural similarities and differences between the three ITS2 regions, the ability of the S. rosei ITS2 to support efficient rRNA processing in S. cerevisiae cells while the H. wingei ITS2 lacks this ability (Fig. 4), points to helices II, TII and V as the most important elements required. Close inspection of the structure of the S. cerevisiaelh’. rosei chimera (Fig. 6(a)), which almost completely disallows processing (Fig. 6(b) and (c)), narrows this selection down to helix V, although a possible tertiary interaction between the helices IV and VI, which would be disrupted in the chimera, cannot be excluded. The results depicted in Figure 7 demonstrate that not all of the structure of helix V is crucial for efficient processing. In fact, considerable disruption of base-pairing is allowed in the middle of the stem of this helix without any detectable deleterious effect (pORCS-ITSB-1.1). Surprisingly. local disruption of base-pairing at the cleavage site used to produce the 7 S precursor of 5.8 S rRNA also has no effect on maturation of 26 S rRNA, as demonstrated by the normal amounts of tagged 26 S rRNA transcripts produced by the pORCS-ITS2AP mutant (Fig. 3), which contains an A. C mismatch in the immediate neighborhood of the internal cleavage site (Fig. 4). It remains to be seen whether 5.8 S rRNA maturation is affected in this mutant. In contrast to the two mutations just described, changing positions 138 to 147 at the apical end of helix V drastically reduces formation of mature 26 S rRNA, though it does not completely block processing (pORCS-ITSB-11.1; Fig. 7). The production of normal amounts of tagged mature 26 S rRNA by pORCS-ITS2-II.2 containing two point mutations in the loop closing helix V (Fig. 7) makes it likely that the disruption of base-pairing at the lower end of helix V is the primary cause of the failure of mutant pORCS-ITS2-II.1 to support efficient processing. A specific sequence requirement in this region, however, cannot be excluded. In this respect it should be noted that both the primary and secondary structure of this part of helix V has been strongly conserved in all three yeast species (Fig. 4). Experiments are in progress to test the importance of both primary and secondary structure in this region. Recently evidence has been obtained for an important role of various nucleolus-associated small

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RNA molecules in yeast pre-rRNA processing, notably snRl0 (Tollervey & Guthrie, 1985; Tollervey, 1987), snR17 (U3; Hughes et al., 1987) and snR128 (U14; Zagorski et al., 1988; Li et al., 1990). These snRNAs have been suggested to exert their function in rRNA processing through specific base-pairing interactions with pre-rRNA. The tagged ribosome system should allow us to trace these interactions by mutating &-acting elements in the pre-rRNA and then searching for compensating mutations in one of the snRNAs. We gratefully acknowledge the assistance of Dr Alex Yorkin during part of these experiments. This work was supported in part by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for Scientific Research (N.W.O.).

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Sollner-Webb, B. & Tower, J. (1986). Transcription of cloned eukaryotic ribosomal RNA genes. Anna. Rev. Biochem. 55, 801-830. Tollervey, D. (1987). A yeast small nuclear RNA is required for normal processing of pre-ribosomal RNA. EMBO J. 6, 41694175. Tollervey, D. 6 Guthrie, C. (1985). Deletion of a yeast small nuclear RNA gene impairs growth. EMBO J. 4, 3873-3878. Tollervey, D. & Hurt, E. C. (1990). The role of small nucleolar ribonucleoproteins in ribosome synthesis. Mol. Biol. Reports, 14, 103-106. Yeh, L.-C. C. BELee, J. C. (1990). Structural analysis of the Internal Transcribed Spacer 2 of the precursor RNA from Sacchawnnyces cerevisiae. ribosomal J. Mol. Biol. 211, 699712. Zagorski, J., Tollervey, D. 6 Fournier, M. J. (1988). Characterization of an SNR locus in Saccharomyces cerevisiae that specifies both dispensable and essential small nuclear RNAs. Mol. Cell. Biol. 8, 3282-3290. by S. Reed