Different Mechanisms for Pseudouridine Formation in Yeast 5S and 5.8S rRNAs

The selection of sites for pseudouridylation in eukaryotic cytoplasmicrRNA occurs by the base pairing of the rRNA with specific guidesequences within the RNA components of box H/ACA small nucleolarribonucleoproteins (snoRNPs). Forty-four of the 46 pseudouridines( ${Psi}$ s) in the cytoplasmic rRNA of Saccharomyces cerevisiae havebeen assigned to guide snoRNAs. Here, we examine the mechanismof ${Psi}$ formation in 5S and 5.8S rRNA in which the unassigned ${Psi}$ soccur. We show that while the formation of the ${Psi}$ in 5.8S rRNAis associated with snoRNP activity, the pseudouridylation of5S rRNA is not. The position of the ${Psi}$ in 5.8S rRNA is guidedby snoRNA snR43 by using conserved sequence elements that alsofunction to guide pseudouridylation elsewhere in the large-subunitrRNA; an internal stem-loop that is not part of typical yeastsnoRNAs also is conserved in snR43. The multisubstrate synthasePus7 catalyzes the formation of the ${Psi}$ in 5S rRNA at a site thatconforms to the 7-nucleotide consensus sequence present in othersubstrates of Pus7. The different mechanisms involved in 5Sand 5.8S rRNA pseudouridylation, as well as the multiple specificitiesof the individual trans factors concerned, suggest possibleroles in linking ribosome production to other processes, suchas splicing and tRNA synthesis.

INTRODUCTION

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INTRODUCTION

RNA harbors a large array of structurally diverse, modifiednucleosides (33, 93). Pseudouridine (5-ribosyluracil; ${Psi}$ ) wasthe first discovered and is the most abundant (for a review,see reference 16). The isomerization of U to ${Psi}$ involves the recognitionof the specific U followed by the cleavage of the N-glycosidicbond, the rotation of the base, and the formation of a C-glycosidicbond (82). At the level of local RNA structure, ${Psi}$ contributesto greater stability relative to that of U by providing an additionalhydrogen-bonding capability that allows a water-mediated bridgeto form between the base and the sugar-phosphate backbone aswell as improved base stacking (for a review, see reference16).

In eukaryotes, ${Psi}$ is found at specific sites in the rRNAs, tRNAs,and snRNAs. The cytoplasmic rRNA precursor (pre-rRNA) of eukaryotesis abundantly modified. As a result, in addition to about 54ribose-methylated and 10 base-methylated nucleosides (61, 89,108), 46 ${Psi}$ s are reported for the mature cytoplasmic rRNA of Saccharomycescerevisiae (6, 7, 65, 83, 85). The pseudouridylation sites ineukaryotic, cytoplasmic rRNA are selected by the base pairingof the cytoplasmic rRNA with specific guide sequences presentin the box H/ACA family of small nucleolar RNAs (snoRNAs) (forreviews, see references 10, 21, 54, and 114). These guide RNAsare associated with protein and function as ribonucleoprotein(RNP) particles, termed small nucleolar RNPs (snoRNPs).

Each box H/ACA snoRNP contains four core proteins in additionto the guide RNA (71). One of the core proteins, Cbf5 in yeast(dyskerin in humans), has significant similarity to TruB, aknown Escherichia coli tRNA ${Psi}$ synthase (40, 55), and experimentaland structural evidence supports the inference that the proteinis indeed the catalytic component of box H/ACA snoRNPs (17,60, 88, 115). Defective ribosome activity due to mutations indyskerin has been implicated in the human disease dyskeratosiscongenita (97, 112). The three other core box H/ACA snoRNP proteinsalso are essential for pseudouridylation, as demonstrated withyeast (12, 37), contributing to a stable, efficient complexcapable of productive association with the substrate in a mannerthat establishes the characteristic spacing (14 to 16 nucleotides[nt]) between the active site and box H or ACA sequence element(5, 36, 38, 51, 60, 66, 81, 88, 90).

The goal of several initiatives is to identify and assign functionsto the entire catalog of snoRNAs from various model organisms(14, 54, 59, 99, 100, 102). Presently, the population of snoRNAsin S. cerevisiae is the best characterized. Seventy-six snoRNAshave been identified in yeast (86). Remarkably, not only havethe guide modification site pairings for most of the 2'-O-methylated(box C/D snoRNA targets) and ${Psi}$ sites in yeast been worked out,the guide assignments also have been experimentally verifiedby showing the specific loss of a particular modification(s)following the deletion of each snoRNA gene. Specifically, inthe case of the box H/ACA type, 28 snoRNAs have been assignedto 44 of the 46 known ${Psi}$ s in yeast rRNA (4, 102, 107).

The formation of the numerous ${Psi}$ s in the spliceosomal snRNAs ofvertebrates is guided by a class of small RNAs that reside inthe nucleoplasmic Cajal bodies and are closely related to thebox H/ACA snoRNAs (44, 114). The box H/ACA small-Cajal-bodyRNAs also function as dyskerin-containing RNPs and contain thesame small sequence motifs as the typical box H/ACA snoRNAs,as well as an additional sequence element implicated in thecharacteristic localization (92). Three of the six ${Psi}$ s in andnear the branch point recognition region of vertebrate U2 snRNAhave counterparts in yeast U2 snRNA (63). Whereas the ${Psi}$ s of humanU2 snRNA are assigned box H/ACA small-Cajal-body RNA guides(59), initial work had shown that yeast U2 snRNA modificationdoes not depend on the Cbf5 guide RNA system (63, 68). Instead,the classic protein-only enzymes Pus1 and Pus7 were shown toeach catalyze the formation of individual ${Psi}$ s in yeast U2 snRNA(63, 68). These proteins also pseudouridylate tRNAs in yeast(9, 68) and are homologs of eubacterial tRNA modification enzymes(49, 55, 76). However, recently a snoRNA was shown to guidethe formation of the last of the three ${Psi}$ s in yeast U2 snRNA (62).Since U2 snRNA is transcribed by RNA polymerase II (RNAP II),the finding revealed that box H/ACA snoRNP-mediated modificationin yeast is not limited to RNAP I transcripts (18S, 5.8S, and25S rRNAs).

In this work, we investigate the factors involved in the pseudouridylationof the two small rRNAs that are found in the large subunit ofthe S. cerevisiae cytoplasmic ribosome. To address the mechanismsinvolved in the pseudouridylation of 5S and 5.8S rRNAs, we examinedthe formation of ${Psi}$ s in a strain impaired for Cbf5 activity. Weshow that 5.8S rRNA pseudouridylation requires functional Cbf5and is guided by the snoRNA snR43. We demonstrate that the formationof ${Psi}$ in 5S rRNA is not dependent on a Cbf5-containing RNP andinstead is catalyzed by Pus7, which also forms ${Psi}$ in U2 snRNAand tRNAs. Thus, the pseudouridylation of 5S and 5.8S rRNA iscarried out by different mechanisms, with each of the transfactors involved displaying intriguing multispecificity.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Yeast strains. The deletion of the snR43 gene was performed in the haploidYS602 (MAT ${alpha}$ ade2-101 his3-11,15 trp ${Delta}$ 901 ura3-52 leu2-3,112) S.cerevisiae strain by the replacement of the entire coding regionwith the TRP1 auxotrophic marker from Kluyveromyces lactis thatwas amplified from plasmid pBS1479 (87) with the primers WD440(5'-CACGCGTGTTTTGTGCAGTGTATTACCAACTTGCGCATGCAAGGATATCATGATATCGAATTCCTGC-3')and WD441 (5'-AGTATAAGAAAAATAAGCAAAATAAGTATACATAAAAATAAAT ACGAATAGTACGACTCACTATAGGG-3'),which add homologous sequences flanking snR43 to the ends ofthe marker gene. Following transformation with the PCR product(31), positive transformants (snR43 ${Delta}$ ::TRP1) were selected onyeast nitrogen base plates lacking tryptophan, and the integrationof the TRP1 marker at the correct location was verified in individualisolates by the isolation of genomic DNA followed by PCR withthe primers WD328 (5'-CTCTAGAACTAGTGGATCC-3') and WD442 (5'-GTCTAATGTGCTTGTTTGTGC-3').

The haploid MAT ${alpha}$ pus ${Delta}$ S. cerevisiae strains were purchased fromOpen Biosystems, Inc. (Huntsville, AL). The cbf5-D95A S. cerevisiaestrain was described previously (115).

Complementation of the synthase gene deletion. To generate an S. cerevisiae plasmid expressing the proteinencoded by the PUS7 gene in a functional form, a region containingthe entire PUS7 gene, as well as 261 bp upstream and 134 bpdownstream, was amplified from S. cerevisiae genomic DNA usingthe primers WD446 (5'-ATTTCCGAGCTCGGTAGCACAGGAAGCGTCTAAAG-3')and WD447 (5'-GGCTTTCTCGAGGTGTGCGATGCGCAGATACATATTTAC-3') andPlatinum Taq high-fidelity polymerase (Invitrogen, Carlsbad,CA). The PCR product was cloned into pCR2.1-TOPO (Invitrogen)and propagated in E. coli TOP10 (Invitrogen). The resultingplasmid was digested with XhoI and SacI restriction enzymes(New England Biolabs, Ipswich, MA), the sites for which wereadded in the initial PCR as part of the primers, and the PUS7gene region was cloned into the polylinker of XhoI/SacI-cutpRS415 (103) to generate pPUS7(RS415). The plasmid was isolatedfrom E. coli TOP10 and transformed into the haploid yeast pus7 ${Delta}$ strain.

Reverse transcription-based RNA sequencing and detection of ${Psi}$ in RNA. Primer labeling and RNA sequencing were done as described previously(61, 70). The rRNA ${Psi}$ sites were detected by primer extensionafter the treatment of the RNA to create carbodiimide adductsat the sites of ${Psi}$ as described previously (102). The primersto sequence rRNA and detect the various ${Psi}$ s are as follows: WD387(102) for 25S rRNA, WD443 (5'-GCGTTCAAAGATTCGATGATTC-3'; purifiedby polyacrylamide gel electrophoresis) for 5.8S rRNA, and WD444(5'-CCACTACACTACTCGGTCAGGC-3') for 5S rRNA.

Sequence analysis. The snoGPS Web server was described previously (101); the querysequence used in the search for guides to the 5S and 5.8S rRNAtargets consisted of the known H/ACA snoRNA collection (4, 86,99, 102, 107) in FASTA format. snR43 homologs were identifiedusing the fungal BLAST search provided by the SaccharomycesGenome Database (43); the alignment of snR43 homologs was doneusing the EMMA multiple-sequence alignment program that is partof EMBOSS (91) with initial formatting applied by PrettyPlotMulSeqs, provided via http://biotools.umassmed.edu/. Mfold (116)was used to consider local folding.

RESULTS

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RESULTS

Different types of molecular machinery pseudouridylate 5S and 5.8S rRNAs. A single ${Psi}$ is known to occur in each of the two small rRNAs (5Sand 5.8S) that are components of the large subunit of S. cerevisiaecytoplasmic ribosomes (Fig. 1) (75, 85, 94). To determine thefactors involved in the pseudouridylation of these rRNAs, wefirst examined whether the snoRNP-associated ${Psi}$ synthase Cbf5catalyzes the formation of either of these ${Psi}$ s. We did this byusing a mutant yeast strain (cbf5-D95A) that only expressesa form of Cbf5 that harbors a point mutation (D95A) in the identifiedcatalytic domain. This mutant strain lacks the detectable pseudouridylationof rRNA in vivo and exhibits severely impaired cell growth (115).Total RNA was isolated from the mutant strain and a controlstrain with a wild-type CBF5 gene, and the ${Psi}$ sites in 5S and5.8S rRNA were examined to see if there was a loss of pseudouridylation.RNA was treated with the chemical N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimidemethyl-p-toluenesulfonate (CMC) followed by mild alkali treatment,and ${Psi}$ s were detected as strong, CMC-dependent primer extensionstop bands at positions one nucleotide before the ${Psi}$ sites. Notethat in our experiments there also were template-specific backgroundladders that served as internal controls to show that sufficienttemplate RNA was present in each reaction. As illustrated inFig. 2, the formation of ${Psi}$ 73 of 5.8S rRNA is abrogated in thecbf5-D95A strain but is present in the wild-type control (lanes5 to 8), while the ${Psi}$ is observed at position 50 of 5S rRNA inboth the cbf5-D95A mutant strain and a control strain (lanes1 to 4). This result suggests that ${Psi}$ formation in 5.8S rRNA isdependent on the catalytic activity of the ${Psi}$ synthase Cbf5 ina box H/ACA snoRNP, whereas surprisingly ${Psi}$ formation in 5S rRNAis dependent on another ${Psi}$ synthase, presumably a protein enzyme.

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FIG. 1. Pseudouridylated nucleotides of the small rRNAs of the yeast cytoplasmic large ribosomal subunit. (A) S. cerevisiae 5.8S rRNA has a ${Psi}$ at position 73. The secondary structure includes portions of 25S rRNA (light gray) that interact with 5.8S rRNA (15). (B) Alignment of the yeast 5.8S rRNA sequence with those of human (Homo sapiens) (50, 78) and wheat (Triticum aestivum) (64) shows that the ${Psi}$ in yeast occurs in a commonly modified stretch of 5.8S rRNA (59, 72, 83). The number of nucleotides upstream of each 5.8S portion is shown in parentheses; the numbering for yeast is for the short (major) version (28, 39). S. cer., S. cerevisiae; T. aes., T. aestivum; H. sap., H. sapiens. (C) The S. cerevisiae cytoplasmic 5S rRNA secondary structure (15, 53) showing the position of the ${Psi}$ .

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FIG. 2. Formation of the ${Psi}$ in 5.8S rRNA, but not the ${Psi}$ in 5S RNA, is lost in the strain severely compromised for the activity of the snoRNP-associated ${Psi}$ synthase Cbf5. Primer extension reactions were performed using appropriate primers (complementary to 5S rRNA in lanes 1 to 4 and to 5.8S rRNA in lanes 5 to 8) with CMC-treated (+) and mock-treated RNA (–) from the haploid control laboratory strain YS602 (WT) and a strain in which the Cbf5 protein harbors a point mutation in the active site (cbf5-D95A). The site of the CMC-dependent pause in the ladder of extension products is indicated for the respective ${Psi}$ s.

snR43 guides 5.8S rRNA pseudouridylation. Given that ${Psi}$ formation in 5.8S rRNA is dependent on Cbf5 functioningin a putative box H/ACA snoRNP, we sought to identify the particularguide RNA in the RNP particle. Recent experimental and computationalapproaches have most likely revealed the complete collectionof S. cerevisiae snoRNAs (4, 20, 25, 61, 69, 102, 107). Particularlyunderscoring the comprehensive nature of the current collection,a recent experimental approach to purify and identify the fullarray of S. cerevisiae box H/ACA snoRNAs based on associationswith H/ACA snoRNPs discovered only one novel snoRNA, yet theset lacked only a single box H/ACA snoRNA that was previouslyrecognized (107). Therefore, we began our search by examiningthe pseudouridylation pockets of the known H/ACA snoRNAs forcomplementarity to the sequences immediately flanking the 5.8S ${Psi}$ site. As shown in Fig. 3A, snR43 displays a significant potentialfor base pairing with the region modified in 5.8S rRNA, positioningU73 at the pocket for isomerization. snR43 previously had beenpredicted, and subsequently shown, to guide ${Psi}$ 966 of yeast 25SrRNA (84, 107); in fact, the same guide elements are involved(Fig. 3B).

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FIG. 3. snR43 is responsible for the formation of both the ${Psi}$ in 5.8S rRNA and ${Psi}$ 966 in 25S rRNA. (A) The predicted secondary structure of snR43 is shown paired with a segment of pre-rRNA corresponding to 5.8S rRNA to guide the appropriate U into the pseudouridylation pocket. (B) The same domain functions in the pseudouridylation of 25S rRNA. The putative base pairing between the 5' domain of snR43 and the 966 region of 25S rRNA is illustrated in a condensed manner. Primer extension reactions detecting ${Psi}$ s in 5.8S rRNA (C) and the 960 to 990 region of 25S rRNA (D) show that the formation of ${Psi}$ in 5.8S rRNA and ${Psi}$ 966 in 25S rRNA is abolished in an snR43 deletion strain (snr43 ${Delta}$ ). (D) Primer extension sequencing ladder reactions (lanes 1 to 4) were performed with the same primer as that for the adjacent lanes using total RNA. Each panel is labeled in the same manner as that described in the legend to Fig. 2, with the RNA in this case being isolated from the haploid control strain (WT) and the snR43 deletion strain; the numbering scheme for rRNA positions matches that for the yeast genome (43, 46).

As an independent technique to identify putative guides for5.8S rRNA that is not limited to considering the previouslyidentified pseudouridylation pockets of the box H/ACA snoRNAs,we turned to the computational method described by Schattnerand colleagues (101, 102). This approach, on the snoGPS server,identified only snR43 as a candidate and suggested the sameguide RNA-target pairing revealed by a manual inspection ofthe known pockets.

With two analytical approaches indicating snR43 as the guide,we investigated whether snR43 is involved in forming ${Psi}$ in 5.8SrRNA in vivo. To do this, we generated a strain in which thegene for snR43 is deleted by gene replacement with a selectablemarker. RNA from the deletion strain and the control parentalstrain in which snR43 is intact was prepared and used for CMCtreatment, and this was followed by primer extension to examineseveral ${Psi}$ sites in rRNA to see if pseudouridylation is altered.In the parent strains, the ${Psi}$ is seen in 5.8S rRNA (Fig. 3C, lanes1 and 2). The formation of ${Psi}$ 73 in 5.8S rRNA is indeed abolishedin the snR43 deletion strain (Fig. 3C, lanes 3 and 4), demonstratingthat snR43 is involved in the formation of the ${Psi}$ of 5.8S rRNA.Primer extension was performed with the same RNA samples usingprimers specific for a region in 25S rRNA, and, consistent withthe reported role for snR43 as the guide for 25S- ${Psi}$ 966 (107),the formation of ${Psi}$ 966 is abrogated in the snR43 deletion strain(Fig. 3D, lanes 7 and 8). Importantly, CMC-dependent signalscorresponding to several nearby ${Psi}$ s persist, showing that isomerizationto ${Psi}$ still occurs in the snR43 deletion strain for surroundingsites in the region of 25S rRNA examined in Fig. 3D (lanes 6and 8). Taken together, the results show that snR43 serves asa guide for specific ${Psi}$ sites in both 5.8S and 25S rRNA usingthe same guide elements.

snR43 targets 5.8S and 25S rRNA sites in several hemiascomycetes. The secondary structure of a typical eukaryotic box H/ACA snoRNAconsists of two primary extended stem-loops, each separatedby a single-stranded hinge region containing the box H motif,all followed by the single-stranded ACA motif three nucleotidesfrom the 3' end of the RNA (10, 21, 54, 114). If an extended5' or 3' stem-loop functions as a guide domain, then an internalbulged region is present forming the pseudouridylation pocket.snR43 contains an additional 53-nt stem-loop after the box Hsequence that we refer to here as the insert stem-loop (Fig.4). While not unique, such an arrangement is not observed inmost eukaryotic snoRNAs. Other S. cerevisiae snoRNAs (snR42,snR46, and snR191) contain a similarly positioned, additionalstem-loop, yet in two of the three cases the insert stem-loopis much smaller, i.e., 36 nt for snR42 and 28 nt for snR46.With regard to the extended nature of the insert stem-loop,snR43 resembles the S. cerevisiae snR191 and Euglena gracilissnoRNA h1, both of which have significant stem-loops (98 and48 nt, respectively) between flanking box H and ACA domains(4, 98).

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FIG.4. snR43 is predicted to maintain both guide functions in the hemiascomycetes. (A) The DNA encoding the S. cerevisiae snR43 RNA is shown aligned with the sequences encoding putative snR43 in other hemiascomycetes. Conserved nucleotides are emphasized in boldface; asterisks along the bottom indicate positions identical in all sequences. D. hansenii, Debaryomyces hansenii. In the bottom right, S. cerevisiae snR43 is shown with the secondary structure labeled according to the annotation along the top of the alignment; the guides are illustrated paired with the 25S rRNA target. (B) Potential pairings between the putative guides of yeast snR43 orthologs and the respective rRNA regions are shown, with differences from the nucleotides present in S. cerevisiae highlighted (shaded boxes). As drawn, the last base pair in the 5.8S diagrams would compete with pairing in the P1:P1' helix; several other yeast snoRNAs (e.g., snR5, snR9, snR35, snR36, and snR46) display this feature.

Recently, based on comparisons of sequenced yeast genomes, ithas been possible to ascertain and add support for the existenceof small structured RNAs (67, 69, 102). In an effort to providesupport for the predicted secondary structure and to gain furtherinsight into the function of snR43 in related yeasts, we searchedfor snR43 homologs from the available genomes of several hemiascomycetes(30). These putative snR43 homologs were manually inspectedfor the potential to form box H/ACA snoRNA-like secondary structures.Our alignment, based upon primary sequences and secondary structures,is presented in Fig. 4A. The 5' portion of the snR43 species,specifically the guide elements and portions of the insert stem-loop,is more conserved than the 3' end. The high conservation atthe sequence level of a substantial portion of the insert stem-loop,in particular the 3' portion of the loop, is unexpected; however,the Yarrowia lipolytica sequence shows considerable variationin this region. Sequences of the hemiascomycete snR43 homologsalso can be readily modeled using Mfold (116) to form secondarystructures reminiscent of the entire S. cerevisiae snR43, withthe H and ACA boxes similarly configured relative to the threeprimary stem-loops (data not shown).

Given the high conservation of the guide elements and overallstructural similarity to the functional S. cerevisiae snoRNA,the aligned sequences likely correspond to orthologs of snR43in the other yeasts analyzed. In contrast to the variation seenin the orthologs of snR43, the rRNA is highly conserved (30).The region of 25S rRNA proposed to interact with snR43 is 100%conserved among these hemiascomycetes, while the correspondingregions in the 5.8S rRNAs display only a few differences. Thereis some variation in the snoRNA 3' guide element distal to thesite of pseudouridylation and outside of the region proposedto interact with S. cerevisiae 25S rRNA; these sequence changesare not always accompanied by compensating changes in rRNA sequenceand, thus, tend to weaken the potential interaction with 5.8SrRNA and, in some cases, strengthen the proposed interactionwith 25S rRNA (Fig. 4B). Overall, the high degree of conservationin the rRNA and the base-pairing potential displayed by thehomologs to the known sites of snR43 action suggest that thesesnoRNAs also function as guides for the sites correspondingto 5.8S- ${Psi}$ 73 and 25S- ${Psi}$ 966 in these other organisms. Thus, thisdual action of snR43 does not appears to be unique to S. cerevisiaeor even simply to the members of the closely related Saccharomycessensu stricto group.

Screening for the 5S rRNA ${Psi}$ synthase. As shown in Fig. 2 (lanes 1 to 4), ${Psi}$ formation in 5S rRNA dependson a ${Psi}$ synthase other than Cbf5, presumably a protein enzymeacting without a guide RNA. Unlike the case for the 5.8S rRNA ${Psi}$ , using the known collection of yeast H/ACA snoRNAs, no well-ranking,convincing candidate is proposed by the snoGPS server for the5S rRNA ${Psi}$ . Nine ${Psi}$ synthases, including Cbf5, were identified inthe yeast genome based on sequence and motif analyses (2, 3,19, 55); later, a synthase with a highly divergent sequencewas added to bring the total to 10 (63). In order to identifythe synthase catalyzing 5S rRNA pseudouridylation, we beganinvestigating the status of 5S rRNA pseudouridylation in strainsin which the other nonessential ${Psi}$ synthase genes are deleted.The disruption of the gene corresponding to the 5S rRNA ${Psi}$ synthasewould result in the loss of the 5S rRNA ${Psi}$ , presuming that theactivity is nonredundant. As illustrated in Fig. 5A, primerextension analysis following CMC treatment shows the loss ofthe CMC-dependent stop in the pus7 ${Delta}$ strain (lanes 9 and 10) andnot in the pus1 ${Delta}$ and pus4 ${Delta}$ strains (lanes 5 to 8), suggestingthat Pus7 catalyzes the formation of ${Psi}$ 50 in 5S rRNA of S. cerevisiae.

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FIG. 5. Formation of the 5S rRNA ${Psi}$ is dependent on Pus7. (A) RNA from several strains was screened for the presence of the 5S rRNA ${Psi}$ as described in the legend to Fig. 2 and with the panel labeled in a similar manner. The primer extension sequencing ladder reactions (lanes 1 to 4) were performed with the same primer as that used for the adjacent lanes using total RNA. The following deletion strains were examined: pus1 ${Delta}$ (Open Biosystems yeast clone no. 11080), pus4 ${Delta}$ (clone no. 11152), and pus7 ${Delta}$ (clone no. 12499) strains. (B) Restoring the expression of Pus7 to the deletion strain rescues the formation of the 5S rRNA ${Psi}$ . Primer extension reactions to detect the 5S rRNA ${Psi}$ were performed with RNA from a pus7 ${Delta}$ strain harboring either an empty yeast shuttle plasmid pRS415 (+ empty vector) or the plasmid bearing the PUS7 gene [+ pPUS7(RS415)]. Two independent isolates are shown for each set.

Pus7 pseudouridylates 5S rRNA. To verify that the absence of Pus7 is responsible for the lossof the formation of the 5S rRNA ${Psi}$ in the deletion strain, werestored Pus7 expression to the pus7 ${Delta}$ strain. The PUS7 gene,along with 261 bp upstream and 134 bp downstream, was amplifiedfrom the yeast genome and cloned into a LEU2/CEN-based, low-copy-numberyeast shuttle plasmid, which subsequently was transformed intothe pus7 ${Delta}$ strain; the transformation of an empty plasmid servedas a control. RNA from both strains was analyzed by CMC treatmentfollowed by primer extension to determine the pseudouridylationstate of position 50 in the 5S rRNA. Whereas the pus7 ${Delta}$ strainharboring the empty vector fails to generate the 5S rRNA ${Psi}$ (Fig.5B, lanes 5 to 8), the addition of the PUS7 gene to the plasmidrescues the formation of the 5S rRNA ${Psi}$ (Fig. 5B, lanes 9 to 12).These results confirm that PUS7 gene deficiency is responsiblefor the loss of the 5S rRNA ${Psi}$ in the deletion strain.

Pus7 is a multisite, multisubstrate ${Psi}$ synthase in vivo, and theactivity of recombinant Pus7 in the absence of other factorshas been demonstrated with U2 snRNA and tRNA substrates in vitro(9, 63). In those substrate RNAs, the seven nucleotides encompassingthe modification site conform to a consensus sequence (9). Presumably,these sequences form, at least in part, the synthase recognitionsite (9); however, for U2 snRNA it was shown that activity alsodepends on an additional element located downstream of the 7-ntconsensus sequence critical for recognition and/or modificationby Pus7 (63). Strikingly, the alignment of the seven nucleotidessurrounding the sites of S. cerevisiae Pus7 activity (9, 104)demonstrates that the modification site in 5S rRNA matches theconsensus sequence seen in other Pus7 substrates (Fig. 6). Hence,the observed Pus7 dependency of the formation of the 5S rRNA ${Psi}$ is reasonable in light of the previously characterized targetsof Pus7 activity. Taken together, the results concerning 5SrRNA and the prior characterization of Pus7 indicate that Pus7directly catalyzes the pseudouridylation of 5S rRNA, and catalysismost likely is not dependent on a guide RNA or additional proteinfactors.

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FIG. 6. Alignment of the seven nucleotides encompassing the sites of characterized S. cerevisiae Pus7 activity (9, 104). The consensus was established previously (9). R, purine; S, G/C.

DISCUSSION

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DISCUSSION

By using a strain in which Cbf5, the ${Psi}$ synthase enzyme associatedwith the box H/ACA snoRNP complexes, is compromised for activity,we have shown that while the formation of the ${Psi}$ in S. cerevisiae5.8S rRNA is associated with snoRNP activity, the formationof the ${Psi}$ in S. cerevisiae 5S rRNA is independent of Cbf5. Ouranalysis suggests that 5.8S rRNA modification at this positionis not unique to S. cerevisiae and most likely occurs in a widerange of hemiascomycetes, a group that covers an evolutionarybreadth comparable to that of the entire chordate phylum (24).Likewise, the formation of the ${Psi}$ in 5S rRNA has been reportedfor other hemiascomycetes (75, 105).

The synthesis of 5.8S rRNA in yeast is complex (27-29), andwe have now shown that an snoRNA is involved; snR43 uses thesame guide sequences that function elsewhere (position 966 in25S rRNA) to direct the pseudouridylation of 5.8S rRNA. snR43adds to the list of yeast snoRNAs that guide pseudouridylationat more than one site in rRNA, bringing the total to 13, nearlyhalf of the 28 guide H/ACA snoRNAs. snR43 and snR49 are theonly S. cerevisiae snoRNAs known to use the same guide domainto target the pseudouridylation of two different species ofmature rRNA.

The nucleotide equivalent to yeast 25S- ${Psi}$ 966 in human 28S rRNAalso is a ${Psi}$ (83), and the site is one of two assigned to the133-nt snoRNA ACA9 (54, 59). Only the guide elements in ACA9show any resemblance to snR43, constrained by the need to basepair with a homologous site in 28S rRNA; ACA9 does not targethuman 5.8S rRNA. A similar phenomenon has been noted for theyeast and human snoRNAs guiding the highly conserved ${Psi}$ s in helix69 of large-subunit rRNA (4). However, in that case the overallsecondary structure and length both are similar, unlike thecase of yeast snR43 and its human counterpart, for which eventhose characteristics differ markedly due to the insert stem-loopand the greater than 70-nt difference in size. Human and plant5.8S rRNAs do not contain a ${Psi}$ at a position equivalent to thatof yeast 5.8S- ${Psi}$ 73, yet neighboring nucleotides are modified (Fig.1B) and assigned to particular snoRNAs (14, 59).

Given that guide H/ACA snoRNAs are involved in the formationof ${Psi}$ s in transcripts produced by polymerases other than RNAPI, the fact that no snoRNA is involved in the modification of5S rRNA, a product of RNAP III, was not obvious a priori. Bytesting known RNA guide-independent ${Psi}$ synthases of yeast, wehave identified Pus7 as the synthase catalyzing the formationof the ${Psi}$ in 5S rRNA. This is the first report of a ${Psi}$ in cytoplasmiceukaryotic rRNA that is not snoRNA guided. Importantly, nowall 46 U's known to be converted to ${Psi}$ s in S. cerevisiae rRNAare associated with a modifying trans-acting factor.

On the basis of transcriptional coregulation, Pus7 was includedin a set of genes that function in ribosome and rRNA biosynthesis(109). Here, we have directly linked Pus7 activity to the modificationof 5S rRNA. The targets of Pus7 all share a 7-nt consensus atthe site of modification (9). How Pus7 recognizes its naturaltargets still is not known. Presumably, there is more determininga Pus7 substrate than the short 7-nt consensus encompassingthe modification site, because matches are moderately frequentin RNA (data not shown). Moreover, even within a recognizedsubstrate, only particular sites are modified, while other matchesare not. Particularly illuminating is the fact that roughly30 nt downstream of the site pseudouridylated by Pus7 in S.cerevisiae 5S rRNA (nt 79 to 85) is a perfect match to the particularversion of the 7-nt sequence found at the site of Pus7 actionin U2 snRNA, yet position 50 is the only ${Psi}$ in S. cerevisiae 5SrRNA (75, 85). Similarly, U2 snRNA has a match downstream (nt52 to 58) as well, within a region previously shown to be importantfor modification by Pus7, yet this site is not modified either(62, 63). The inability of Pus7 to modify substrate RNAs atadditional sites that match the consensus may be due to themasking of these sites by a protein(s). Alternatively, onlycertain portions of substrates may be suitable for modificationby Pus7, and perhaps only at certain stages of RNA folding,as seen with other ${Psi}$ synthases and enzymes modifying uridine(41). Consistent with this possibility, only an altered conformationof tRNA, called the lambda form (42) and featuring an unfoldedD-stem/loop, could be docked computationally into the presumedactive site of E. coli TruD (26). Until now, natural substratesfor studying Pus7 have been in short supply; our discovery thatthe very abundant 5S rRNA is a Pus7 substrate means that thepus7 ${Delta}$ strain should provide large amounts of 5S rRNA/RNP substrate,thereby leading to a better understanding of recognition andcatalysis by Pus7.

Until recently, the prevailing view was that the pseudouridylationand 2'-O methylation of eukaryotic cytoplasmic rRNA were guidedby snoRNAs in snoRNP complexes. Now, for each of the two classesof nucleotide modification that are introduced primarily bysnoRNPs, an exception has been identified in which rRNA modificationdepends on classic protein-only enzymes. Our elucidation ofPus7 as the synthase responsible for the pseudouridylation of5S rRNA, combined with the revelation that the protein Spb1acts in the 2'-O methylation of a site in yeast 25S rRNA (58),means that snoRNP-independent mechanisms need to be consideredfor both types of rRNA modification. The reason for the differentmechanisms and particular targets is not obvious. Possible reasonsfor different mechanisms are that (i) separate machinery enablescoupling to separate regulatory networks, (ii) they assure thatcertain modifications are formed irrespective of the bulk ofrRNA modifications, and (iii) the bacterium-like site-specificprotein mechanism may have been retained from a common ancestor(Spb1 is related to RrmJ, the enzyme that modifies the neighboringsite in bacteria) (58). While there presently is a paucity ofknowledge on their exact role, no single snoRNP-catalyzed modificationhas been identified as being overly critical in yeast (8, 52).Perhaps the rRNA modifications introduced by guide RNA-independentenzymes are more important and necessitate separate machinery.Whereas the Spb1-guided modification has been shown to be essentialfor normal ribosome production and translation (58), the significanceof the Pus7-catalyzed ${Psi}$ in 5S rRNA is not obvious from its position,and a critical role is not supported by phylogenetic conservationoutside of ascomycetes.

The pseudouridylations of the RNAP II-transcribed U2 snRNA andthe RNAP I-transcribed pre-rRNA are known to be linked by ansnoRNP-dependent pathway; snR81 uses different guide domainsto target a ${Psi}$ site in rRNA and one in U2 snRNA (62, 102). Wehave now demonstrated a link between the pseudouridylation ofU2 snRNA and the RNAP III-transcribed 5S rRNA by the snoRNA-independentsynthase Pus7, which also participates in the modification oftRNAs (RNAP III transcripts). The 5S rRNA genes are interspersedbetween the rRNA repeats in S. cerevisiae and are, by definition,nucleolar. The observed spatial juxtaposition of pre-tRNAs andthe nucleolus indicates that a major part of tRNA biogenesisalso is compartmentalized in the nucleolus with rRNA synthesisand ribosome assembly (11). In fact, additional experimentshave shown that tRNA genes are proximal to the nucleolus inyeast (34, 35, 106, 110). Finally, snRNAs have been detectedin the Xenopus laevis nucleolus, in which some of the modificationsoccur (57, 113). These observed associations suggest the possibilitythat Pus7 activity is connected to the nucleolus and, more importantly,point to the possibility that the modification status providesa signal allowing the coregulation of the production and turnoverof rRNAs, spliceosomes, and tRNAs. These observations also suggesta link to the production of ribosomal proteins, since the Pus7-catalyzed ${Psi}$ 35 in U2 snRNA is important for the high-efficiency splicingof S. cerevisiae introns (79, 80, 111), which are preferentiallylocated in ribosomal protein genes (32). Indeed, the interdependenciesare consistent with the concept that the production of RNAPII and RNAP III transcripts is regulated in response to pre-rRNAsynthesis via an extensive network of interactions (18, 56,74, 95, 96).

Recent work has shown that the modification of tRNA is importantfor its stability and that specific degradation pathways aretriggered by undermodification (1, 45, 47); thus, the hypomodification-triggereddegradation of RNA could provide a means of coupling modificationactivity to a regulatory network. Interestingly, Pus7 is oneof several factors directly linked to a rapid tRNA degradationpathway that acts on specific hypomodified tRNAs (1). AnotherRNA degradation pathway triggered by tRNA hypomodification alsoshows links to 5S rRNA synthesis in yeast (48). Furthermore,the interaction of 5S rRNA and ribosomal protein L5 is criticalfor 5S rRNA stability and incorporation into the pre-60S ribosomalparticle (22, 23, 73, 77), and it has been proposed that theunstable nature of unbound 5S rRNA probably is connected toa lack of modification and that this is part of a network keepingpre-rRNA processing linked to RNA polymerase III activity (13).Interestingly, altered 5S rRNA stability also is observed whenRNAP I activity is uncoupled genetically from attenuating itsactivity in response to stress (18, 56). Our elucidation ofPus7 as the factor involved in the pseudouridylation of yeast5S rRNA will facilitate the further examination of the possiblelink between 5S rRNA stability and modification.

The specificity for multiple sites demonstrated by the factorsstudied here has important implications for assigning functionsto enzymes or guide RNAs in other organisms. Pus7 shows multisite,multisubstrate specificity that is unparalleled. Furthermore,snR43 shows dual specificity; the same guide elements of snR43are used to modify pre-rRNA at different sites that correspondto mature 5.8S and 25S rRNA sequences. The exact base-pairingpotential seems to differ slightly, with the pairings for the5.8S rRNA target being lengthier (Fig. 3A and B). The high degreeof flexibility in the guide pairings (Fig. 4B) makes the assigningof function difficult for organisms in which the exact modificationshave not been mapped or in which the stringent verificationof the guide RNA by effecting the disruption of its activityis not convenient. Likewise, the increasing number of targetsfor bacterium-like ${Psi}$ synthases makes assigning the exact extentof their functions more tentative. Moreover, for enzymes orguide RNAs for which the disruption of activities causes observedphenotypes, one has to be cautious in assigning roles to particularmodifications when unidentified ones may yet exist.

ACKNOWLEDGMENTS

We thank Maurille J. Fournier and Michael W. Gray, in whoselaboratories this study was completed. We thank Jeremy Ho forassistance in the preparation of the snR43-deletion strain.

The manuscript is dedicated to the memory of James Ofengand,a pioneer in the field of ${Psi}$ and the synthases.

This work was supported by U.S. Public Health Service grantnumber GM19351 to Maurille J. Fournier.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, 903 Lederle Graduate Research Tower, University of Massachusetts, Amherst, MA 01003. Phone: (413) 545-0566. Fax: (413) 545-3291. E-mail: wdecatur{at}biochem.umass.edu

Published ahead of print on 10 March 2008.

REFERENCES

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