Genome-Wide Prediction and Analysis of Yeast RNase III-Dependent snoRNA Processing Signals

In Saccharomyces cerevisiae, the maturation of both pre-rRNAand pre-small nucleolar RNAs (pre-snoRNAs) involves common factors,thereby providing a potential mechanism for the coregulationof snoRNA and rRNA synthesis. In this study, we examined theglobal impact of the double-stranded-RNA-specific RNase Rnt1p,which is required for pre-rRNA processing, on the maturationof all known snoRNAs. In silico searches for Rnt1p cleavagesignals, and genome-wide analysis of the Rnt1p-dependent expressionprofile, identified seven new Rnt1p substrates. Interestingly,two of the newly identified Rnt1p-dependent snoRNAs, snR39 andsnR59, are located in the introns of the ribosomal protein genesRPL7A and RPL7B. In vitro and in vivo experiments indicatedthat snR39 is normally processed from the lariat of RPL7A, suggestingthat the expressions of RPL7A and snR39 are linked. In contrast,snR59 is produced by a direct cleavage of the RPL7B pre-mRNA,indicating that a single pre-mRNA transcript cannot be splicedto produce a mature RPL7B mRNA and processed by Rnt1p to producea mature snR59 simultaneously. The results presented here reveala new role of yeast RNase III in the processing of intron-encodedsnoRNAs that permits independent regulation of the host mRNAand its associated snoRNA.

INTRODUCTION

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Introduction

Bacterial pre-rRNA processing is carried out by a defined setof nucleases (3-5, 43, 52). Key among this set is RNase III,initially isolated by its ability to bind and cleave duplexRNA (47, 48). RNase III generates the immediate precursors tothe mature 16S and 23S rRNAs from the primary transcripts bycleaving within two extended RNA duplexes formed by long-rangeinteractions that pair the termini of each rRNA (7, 63). Theselong-range interactions provide a simple method of coordinatingthe processing events at both ends of the transcript. In eukaryotes,pre-rRNA processing is more complex and requires many more smallnucleolar RNAs (snoRNAs) and protein components with overlappingfunctions (13, 15, 16, 41, 46). For example, the removal ofthe 5' external-transcribed spacer requires 4 snoRNAs (U3, snR30,U14, and snR10) and about 64 snoRNAs are required for rRNA modifications(24, 57). snoRNAs are divided into two major subclasses: thefirst includes box C/D snoRNAs that function mostly as a guidefor the methylation of rRNA (6, 20, 21, 55), while the secondincludes H/ACA snoRNAs that guide RNA pseudouridine formation(25, 39, 59). Most mammalian snoRNAs are encoded within intronsequences and are processed from either unspliced precursorsor lariat species (18, 19, 64). In Saccharomyces cerevisiae,most snoRNAs are transcribed either as independent units oras a part of polycistronic transcript, while only 7 of the 66known snoRNAs are located in the introns of mRNAs (14, 44, 53).Several polycistronic snoRNAs, and a few monocistronic ones,are processed by Rnt1p, the orthologue of the bacterial RNaseIII (30), which is also required for the processing of the pre-rRNA's3' end (2, 10, 11, 23, 33). Following processing by Rnt1p, theRNAs are trimmed by exonucleases producing the mature ends (22,56).

Unlike other RNase IIIs, Rnt1p recognizes substrates with conservedstem-loop structures and has a low affinity for generic RNAduplexes (27). Most Rnt1p substrates exhibit a conserved AGNNtetraloop structure (9, 28, 32, 61). Rnt1p cleaves at a fixeddistance from the conserved loop, generating a product withstaggered ends (28). Mutations (28), chemical protection assays(27), chemical interference (9), and nuclear magnetic resonanceanalysis (28) indicate that Rnt1p binding and cleavage are regulatedby reactivity epitopes grouped into three boxes (see Fig. 1A).These are the initial binding and positioning box (IBPB), locatedat the tetraloop; the binding stability box (BSB), located adjacentto the tetraloop; and the cleavage efficiency box (CEB), locatednear the cleavage site. Alteration of the sequences of boththe IBPB and the BSB inhibits cleavage and reduces binding,while alteration of the CEB sequence inhibits cleavage withoutaffecting the binding efficiency. Thus, despite the lack ofuniversally conserved residues, the nucleotide composition ofthe reactivity epitopes contributes to substrate selectivity.The second nucleotide of the IBPB is believed to be universallyconserved, and changing it to any nucleotide other than G reducesbinding to known substrates and blocks cleavage (9, 27, 28,38). The solution structure of the Rnt1p/substrate complex indicatesthat the enzyme interacts with the minor groove adjacent tothe 3' end of the tetraloop, suggesting that substrate recognitiondepends on the shape of the groove (60). However, accurate identificationof the universal features of Rnt1p substrates requires the identificationof a large set of substrates that allows statistical analysisof the cleavage signals.

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FIG. 1. A combined in silico and in vitro approach identifies Rnt1p cleavage signals near known snoRNAs. (A) Schematic representation of a model RNA substrate illustrating the features used for the selection of new Rnt1p substrates. Arrows indicate the sites of cleavage. N represents any nucleotide, and N' is its counterpart. W-C indicates a position where base pairing is predominant and is required for optimal activity (28). The IBPB indicates nucleotides that position the enzyme for cleavage. The BSB indicates nucleotides that stabilize the binding of Rnt1p and enhance cleavage. The CEB indicates nucleotides that directly contribute to the Rnt1p cleavage efficiency without affecting substrate binding. (B) Methods used for the selection of Rnt1p substrates in silico. The illustrated procedure takes as input two independent compilations: one contains snoRNAsequences, and the other contains known Rnt1p substrates. P1 indicates the search performed using RNAMotif (34). P2 and P3 indicate RNA folding obtained with the Vienna RNA package (51) and the minimum free-energy folding algorithm (65) and using the dynamic programming algorithm (36). P4 and P5 indicate comparisons performed using an algorithm developed during this study. P6 and P7 indicate an evaluation performed with the Vienna RNA package (51). P8 indicates the evaluation performed using an algorithm developed in the course of this study. S1 indicates a score for which the highest value 1 is for structures with a long and stable stem having few bulges and an internal loop. The lowest value of S1 is given to a sequence that, when unconstrained, does not fold into an AGNN tetraloop and has a high stability difference compared to the constrained structure. S2 indicates a score for which the highest value of 1 is given to primary sequences that shares the characteristics common to all known substrates and is highly similar to at least one of the known substrates. The lowest value of S2 (0) is given to a sequence that does not resemble any of the known substrates. (C) Summary of the data obtained from the prediction algorithm, the microarray data, and both in vitro and in vivo validation. Details of the experimental data and the references of previously published results are indicated in Table S1 in the supplemental material. A score was given to each of the potential Rnt1p cleavage sites within 1 kb of all known snoRNAs. The score was assigned as described for panel B. In vitro cleavage was tested by the incubation of total RNA with purified Rnt1p as described in the Material and Methods section. Expression level detected upon the inactivation of the Rnt1p temperature-sensitive mutant (TS Expression) was recorded after a 4-h shift to the nonpermissive temperature. In vivo processing was assessed by Northern blot analysis of RNA extracted from ${Delta}$ rnt1 cells, and a defect in processing was scored by the accumulation of a snoRNA precursor in the absence of Rnt1p. Information about the snoRNA families and gene organization was obtained from the snoRNA database (49). The snoRNAs were organized either according to the prediction score (left) or according to the snoRNA gene family (right). Notice that most C/D-box snoRNAs are processed by Rnt1p, while only a few H/ACA-box snoRNAs reside near Rnt1p cleavage signals.

In this study, we searched for new Rnt1p substrates by examiningthe expression profiles of all known snoRNAs in the presenceand absence of Rnt1p. In parallel, we developed a program thatidentifies potential Rnt1p cleavage signals near known snoRNAsequences. All newly identified substrates were tested for cleavagein vitro, and their contributions to snoRNA processing wereverified in vivo. Our combined in silico and in vitro approachidentified all known substrates of Rnt1p and revealed sevennew snoRNA substrates. In general, monitoring the expressionof snoRNAs was most effective when Rnt1p cleavage was not redundantwith other processing events that could lead to the maturationof the snoRNA in question. In contrast, the in silico screenwas most effective in identifying snoRNAs that harbored conservedprocessing signals, regardless of their processing pathway invivo.

MATERIALS AND METHODS

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Materials and Methods

Strains and plasmids. Yeast cells were grown and manipulated using standard procedures(1, 31). The effect of Rnt1p depletion was studied using thestrains W303-1A and ${Delta}$ rnt1 (12). The ${Delta}$ rnt1 ${Delta}$ dbr1 strain (40) wasconstructed by crossing ${Delta}$ rnt1 cells with ${Delta}$ dbr1 cells kindly providedby J. D. Boeke, Johns Hopkins University. The temperature-sensitivestrain was a recreation of the rnt1-ts strains described previously(38). The temperature-sensitive strain prp2ts was a kind giftfrom Ren-Jang Lin, City of Hope.

Microarray-based analysis of snoRNA expression. The microarray experiment was conducted and analyzed at theGenome Quebec Innovation Center (Montreal, Quebec, Canada).The RNA was extracted from W303, ${Delta}$ rnt1, and RNT1-TS cells (31,38) grown at either 26 or 37°C in either yeast complete(YC) medium or YC medium without leucine (54).

In silico screening of Rnt1p substrates. The sequence homology score was calculated using two methods.One looked for homology to the sequences conserved in all knownsubstrates, and the other used an algorithm that searched forthe best sequence homology to any single known substrate. Forboth methods, a nucleotide probability matrix was generatedfrom the alignment of known substrates by using their tetraloopsas anchor points. In the second method, a score was given toeach substrate based on its sequence homology to all known substratesand the sum of the probability of its nucleotides in relationto the distance to the tetraloop. Higher significance was givento the nucleotides near the tetraloop. In order to identifythe best sequence homology to a known substrate, an intermediatescore was weighted for each known substrate. Only the highestintermediate score was kept. The intermediate scores were calculatedby comparing the nucleotides of potential substrates to thoseof known substrates. For each nucleotide comparison, when the2 nucleotides (nt) were identical, the intermediate score wasraised by a distance-weighted factor. When the 2 nt were notidentical, the intermediate score was raised only accordingto the probability matrix multiplied by the weight factor. Inaddition to thermostability, an evaluation of the secondarystructure of the potential substrates was based on the qualityof its stem. In this study, this was calculated by giving apositive score to nucleotides downstream of the tetraloop thatwere paired to upstream nucleotides and vice versa. This scorewas weighted according to the distance from the nucleotide tothe tetraloop, with higher significance being given to the nucleotidesclose to the tetraloop. For any potential substrate, the sumof its nucleotide scores represented the quality of its stem.

In vitro RNA cleavage. Cleavage reactions were performed essentially as described previously(28) using either 0.2 pmol of purified Rnt1p (26) or total cellextracts (29). For the in vitro cleavage assay, 3 fmol of internallylabeled RNA was incubated in the presence of 0.2 pmol of Rnt1pfor 20 min at 30°C in 20 µl of reaction buffer (30mM Tris [pH 7.5], 5 mM spermidine, 10 mM MgCl₂, 0.1 mM dithiothreitol,0.1 mM EDTA [pH 7.5]). Yeast extracts were prepared using 3liters of yeast culture (W303 or ${Delta}$ RNT1 strain) grown to an opticaldensity at 600 nm of 0.8 at 26°C in yeast extract-peptone-dextrose.Cells were harvested by centrifugation, washed, and resuspendedin 0.4 times the cell pellet's volumes of AGK buffer (10 mMHEPES [pH 7.9], 1.5 mM MgCl₂, 200 mM KCl, 10% glycerol, 1 mMphenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 µgof leupeptin/ml, 1 µg of aprotinin/ml, 1 µg of pepstatinA/ml, 1 µg of antipain/ml). Following cell lysis in liquidnitrogen, the frozen powder was transferred to a centrifugetube and centrifuged at 18,900 x . for 30 min. The supernatantwas then centrifuged at 94,000 x . for 30 min and dialyzed for3 h against 2 liters of dialysis buffer (20 mM HEPES [pH 7.0],0.2 mM EDTA, 0.5 mM dithiothreitol, 50 mM KCl, 20% glycerol).Finally, the extract was centrifuged at 18,900 x . for 20 minand the supernatant was stored at –80°C. The modelsnR55 substrate was generated by T7 RNA polymerase (1). 5'-End-labeledRNA was produced as described previously (27). The snR55, snR56,and snR48 templates were prepared by in vitro transcriptionusing PCR products as templates. Each PCR product was obtainedfrom genomic DNA by using a forward primer carrying a T7 promoterlocated 200 to 300 nt upstream of the mature 5' end of snoRNAand a reverse primer located 200 to 300 nt downstream of themature 3' end of the snoRNA. The oligonucleotides are listedin Oligonucleotide List S1 in the supplemental material. Cleavageof total RNA extracted from both wild-type and ${Delta}$ rnt1 cells wasconducted as described previously (8) using total RNA (50 µg)incubated with purified Rnt1p (10 pmol) in the reaction bufferdescribed above.

Northern blot analyses. Northern blot analyses were performed with total RNA (10 to15 µg) run on 4 to 8% denaturing polyacrylamide gels asdescribed previously (1). The RNA was visualized using eitherrandomly labeled probes or 5'-end-labeled oligonucleotides.The oligonucleotides used are listed in Oligonucleotide ListS2 in the supplemental material.

Primer extension. Primer extension reactions were performed as described previously(2). The oligonucleotides used for the primer extensions ofsnR50, snR52, snR54, snR56, snR57, snR58, snR59 snR60, snR62,snR64, snR67, snR68, snR69, and snR71 are listed in OligonucleotideList S2 in the supplementary material. Oligonucleotides specificto snR67, snR55, and snR39 are listed in Oligonucleotide ListS3 in the supplemental material.

RESULTS

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Results

In silico and in vivo search for Rnt1p-dependent snoRNA. In order to identify snoRNAs that require Rnt1p cleavage fortheir maturation, we screened the sequences within 1 kb upstreamand downstream of all known yeast snoRNAs for Rnt1p cleavagesignals (Fig. 1A). As indicated in Fig. 1B, the sequences werescanned for the presence of an AGNN tetraloop followed by threeWatson and Crick base pairs. Scores were given for each substratebased on sequence homology, secondary structure stability, andsecondary structure similarity to known substrates. Based onthe scores of known substrates, we set the score cutoff to 0.8.Above this cutoff, the identified RNA structures were expectedto be cleaved by Rnt1p. A total of 64 snoRNAs with potentialstem-loops were identified, but only 26 obtained a score above0.8 and 2 did not associate with any predicted structure (Fig.1C). The other 38 snoRNAs were found near structures with ascore lower than 0.8. As expected, all tested structures withscores higher than 0.8 were cleaved by Rnt1p in vitro, validatingthe efficiency of the selection scheme (see Table S1 in thesupplemental material). Interestingly, most of Rnt1p substrateswith scores higher than 0.8 were found near C/D-box snoRNAsand only three were found near H/ACA-box snoRNAs (Fig. 1C; seealso Table S1 in the supplemental material). The 38 snoRNA-associatedstructures with scores lower than 0.8 include two cleaved byRnt1p in a loop-independent but Nop1-dependent manner and fiveassociated with snoRNAs expressed as part of polycistronic unitsand processed using a stem associated with an adjacent snoRNAwithin the polycistronic unit. On the other hand, 22 other snoRNAsassociated with structures that were not cleaved by Rnt1p andtheir processing were not affected by RNT1 deletion in vivo(see Table S1 in the supplemental material and references therein).The snoRNA snR17b (U3) carried a cleavage signal identical tothat of snR17a and thus was presumed to be processed by Rnt1p(22) but was not directly tested. Finally, four snoRNAs wereprocessed by Rnt1p by using structures that differ from thecanonical AGNN tetraloop motif that was used in the search forRnt1p cleavage sites (Fig. 1). Only four AGNN stem-loop structureswith scores between 0.675 and 0.8 were cleaved by Rnt1p in vitro.Therefore, by using a score cutoff of 0.8, we missed four substrateswith the canonical AGNN tetraloop, resulting in a false-negativerate of 11%. In silico search using NGNN as the starting motifdid not identify new snoRNA-associated motifs that are affectedby the deletion of RNT1 in vivo (data not shown).

In order to assess the efficiency of the AGNN tetraloop as indicatorfor Rnt1p-dependent snoRNAs, we examined the expression profileof snoRNAs in the presence and absence of Rnt1p. The microarray-basedexpression profile of wild-type cells was compared to that ofcells carrying a complete deletion of Rnt1p. In parallel, thesnoRNA expression profile of cells carrying a temperature-sensitiveallele of Rnt1p grown at the permissive temperature was comparedto that of cells grown at the restrictive temperature. It isimportant to note that the microarray analysis will not differentiatebetween precursor and mature snoRNA, and therefore, the increasein the expression level of any snoRNA could reflect the accumulationof a pre-snoRNA, a mature snoRNA, or both. The expression levelof most snoRNAs located near stem-loops that were cleaved byRnt1p in vitro was increased more than 1.5-fold upon deletionof Rnt1p (Fig. 1C). The expression level of only five snoRNAsnear the cleavable Rnt1p processing signal did not increasein the absence of Rnt1p, probably due to rapid degradation ofthe unprocessed RNA transcript. All but nine of the snoRNAsthat were cleaved in vitro were affected 4 h after a shift tothe restrictive temperature. Most of the in vitro substratesthat were not overexpressed in ${Delta}$ rnt1 cells were associated withmonocistronic or intron-encoded snoRNAs. The most sensitivesubstrates to Rnt1p deletion or inactivation were those expressedas polycistronic units. All independently transcribed snoRNAsand intron-encoded snoRNAs except snR42 with scores inferiorto 0.8 did not exhibit Rnt1p-dependent expression. The resultof the in silico and in vivo screens identified 7 new substratesand indicated that all but 22 snoRNAs, mostly H/ACA snoRNAs,are processed by Rnt1p.

Identification of Rnt1p substrates that form through long-range base pairing. The ideal Rnt1p substrate (Fig. 1A) is a perfect uninterruptedA-U-rich stem capped with an AGNN tetraloop, a feature whichis very easy to identify by searching for a stable structuralmotif. However, most Rnt1p substrates are interrupted stemsthat in many cases are not structurally stable when taken outof their native RNA context. Furthermore, it has been previouslyshown that some Rnt1p substrates can form through long-rangeinteractions that are very difficult to identify using conventionalmotif-based searches or folding programs like mfold (65-67).In contrast, the in silico screen we have developed is capableof identifying AGNN tetraloops with 3-bp stems regardless ofeither the context or the global folding of the targeted RNA.Consequently, we were able to identify a hidden 3-bp stem cappedwith an AGGA tetraloop that could not form a stable local stemwithin the polycistronic unit of snR67/snR53 (Fig. 2A). Northernblot analysis of RNA extracted from either wild-type or ${Delta}$ rnt1cells hybridized to a probe corresponding to the mature sequenceof snR67 revealed an accumulation of a large RNA precursor in ${Delta}$ rnt1 cells and a decrease in the level of the mature snR67 (Fig.2B). Hybridization to a probe corresponding to snR53 also showedan accumulation of a precursor corresponding to the size ofthe unprocessed polycistronic transcript. However, the levelof the mature snR53 was less affected than that of snR67, suggestingthat the localization of snR53 near the 3' end of the primarytranscript makes it less sensitive to Rnt1p deletion. Extensionof a primer hybridized to the mature snR67 sequence confirmedthe accumulation of an RNA species that extends to the predicted5' end of the polycistronic subunit in the absence of Rnt1p(Fig. 2C). The capacity of Rnt1p to directly cleave the primarytranscript of snR67/snR53 was tested in vitro using purifiedRnt1p and total RNA extracted from ${Delta}$ rnt1 cells. Northern blotanalysis of total RNA cleaved by Rnt1p in vitro confirmed thatthe primary transcript of snR67/snR53 is a direct substrateof Rnt1p (data not shown). Primer extension of total RNA incubatedwith purified Rnt1p revealed three cleavages, two near the 5'end of snR67 and one between snR53 and snR67 (Fig. 2D and E).This indicates the presence of two redundant cleavages at the5' end of snR67. We concluded that the snR67/snR53 polycistronictranscript is a direct substrate of Rnt1p in vitro and thatRnt1p is required for the efficient processing of both snR53and snR67 in vivo.

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FIG. 2. Rnt1p is required for the maturation of the polycistronic snR67/snR53 unit. (A) Schematic representation of the predicted stem-loop structure that forms through long-range interactions between the sequences surrounding snR67 and snR53. The arrowheads indicate the positions of the cleavage sites identified in vitro. (B) Northern blot analysis of the snR67 and snR53 expression patterns both in the presence and in the absence of Rnt1p. RNA was extracted from either wild-type or ${Delta}$ rnt1 cells, separated on a 6% acrylamide gel, and hybridized to radioactive probes corresponding to the mature sequence of either snR67 or snR53. The positions of the mature snoRNAs (M), the processing intermediates (I1 and I2), and the primary transcripts (PT) are indicated on the right. (C) Primer extension mapping of the mature and extended termini of snR67. RNA extracted from both wild-type and ${Delta}$ rnt1 cells was subjected to primer extension using primer A, which was complementary to the coding sequence of snR67. Positions of the mature RNA (snR67) and the extended forms detected in the absence of Rnt1p (5' end) are indicated on the right. (D) Mapping Rnt1p cleavages upstream of snR67. RNA extracted from either wild-type or ${Delta}$ rnt1 cells was incubated with purified Rnt1p enzyme. The cleaved RNA was subjected to primer extension using primer B that hybridized to the sequence upstream of snR67. The positions of the cleavage sites (C2 and C3) and of the 5' ends are indicated on the right. (E) Mapping of the Rnt1p cleavage sites downstream of snR67. RNA was treated as described for panel D, but the primer extension was performed using a primer (primer C) that hybridized downstream of snR67.

A single stem-loop structure directs cleavage in two independent stems. The in silico prediction assay identified a short stem cappedwith an AGUU tetraloop located in the middle of the snR57/snR55/snR61polycistronic unit. This single tetraloop was predicted to directcleavages in two independent stems; one released snR61, andthe other released both snR55 and snR57. An additional canonicalstem was also identified at the 5' end of snR57 (Fig. 3A). Verylittle signal of the mature snR57, snR61, and snR55 was detectedupon deletion of RNT1 (Fig. 3B and C and data not shown). Incontrast, an RNA species corresponding to the size of the unprocessedprecursor was detected, indicating that Rnt1p is required forthe processing of this transcript (Fig. 3B). Extension of aprimer corresponding to the sequence at the 5' end of snR57(primer A) confirmed that in the absence of Rnt1p, the processingof the stem at the 5' end does not occur (data not shown). Incubationof purified Rnt1p with a T7-transcribed model substrate correspondingto the predicted stem-loop structure upstream of snR57 resultedin a specific cleavage at the predicted distance from the AGGAloop (Fig. 3D). This indicated that the predicted stem is adirect substrate of Rnt1p. In order to test whether or not thepredicted structure located between snR55 and snR57/snR61 iscleaved by Rnt1p, and to map the cleavage site, we produceda T7 transcript that corresponded to the entire region locatedbetween snR57 and snR61 and incubated it with purified Rnt1p.Incubation of the T7 transcript with Rnt1p revealed six differentcleavage products consistent with four cleavage events in twoindependent stems at the right distance from the tetraloop (Fig.3E). Two cleavages released snR55, one released the 3' end ofsnR57, and one liberated the 5' end of snR61 (Fig. 3E and datanot shown). Mutation of the AGUU tetraloop to GAAA blocked allcleavages in both stems (data not shown). These data clearlydemonstrate that the predicted AGUU tetraloop is required forcleavage events that occur in the sequence that separate snR57/snR55from snR61.

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FIG. 3. Rnt1p uses a single tetraloop to release three different snoRNAs. (A) Illustration of the predicted cleavage sites associated with the snR57/snR55/snR61 cluster is given. Arrowheads indicate the cleavage sites identified in vitro. The positions of the primers used are indicated by letters. The positions of the mutations used in G are indicated in black boxes. (B) Northern blot analysis of snR57 is shown. Northern analysis was performed as described in the legend to Fig. 2B. (C) Northern blot analysis of snR61 processing intermediates is shown. The hybridization was carried out using probe C, which corresponds to the mature sequence of snR61 (indicated in panel A). The asterisk indicates truncated RNA that accumulated in the absence of Rnt1p. (D) Cleavage of a model substrate representing the stem-loop structure found upstream of snR57 is shown. The T7-transcribed RNA representing the 45-nt-long stem-loop structure near the 5' end of snR57 was 5' end labeled and incubated with purifiedRnt1p. The cleavage product corresponding to a cleavage in position C1 is indicated on the left. (E) An internally labeled T7-transcribed substrate corresponding to the entire snR57/snR55/snR61 cluster was incubated in the presence of Rnt1p. The cleaved RNA was separated using a 4% polyacrylamide electrophoresis gel and directly visualized by autoradiography. The bands corresponding to the different cleavage sites are indicated on the right. (F) Primer extension mapping of total RNA cleaved by Rnt1p in vitro is shown. The RNA was cleaved to completion and then incubated with primer B that corresponded to the sequence near the 3' end of snR55 (indicated in panel A). Unrelated DNA sequence was used as a marker. (G) In vitro cleavage of an artificial substrate confirmed Rnt1p's capacity to direct four cleavage events by using a single binding site. A T7 transcript of an RNA harboring the AGUU stem-loop structure found in the snR57/snR55/snR61 cluster was tested for cleavage in vitro. The snR55 sequence of this RNA was replaced by a UAUU tetraloop, and the snR57 and snR61 sequences were replaced by terminating the RNA with a G-C base pair. The RNA was labeled either at the 3' or the 5' end and was incubated in the presence of either purified Rnt1p, wild-type cell extracts, or ${Delta}$ rnt1 cell extracts. The cleavage products are indicated on the right. Exo indicates products produced by exonucleases found in cell extracts. The asterisks indicate unspecific cleavages that occurred under low-salt conditions.

The predicted cleavage sites at positions C4 and C6 were confirmedby reverse transcription using a primer corresponding to themature sequence of snR55 (data not shown). The cleavage sitespredicted 5' to snR55 were determined by extending a primercorresponding to the sequence at the 3' end of snR55 (primerB) after incubation of total RNA with purified Rnt1p (Fig. 3F).In the context of the native RNA subunit that accumulates inthe absence of Rnt1p (Fig. 3B and C), we detected in vitro cleavageat the 5' end of snR55 (C5) and at the 3' end of snR57 (C2).These same cleavage sites were also detected using a primercorresponding to the mature sequence of snR61 (data not shown).Failure to detect cleavage at C3 by using a primer at the 3'end of C5 (Fig. 3A and F) indicated that under this cleavagecondition, all substrates that were cleaved at C3 were alsocleaved at C5. In contrast, cleavage at C3 was detected usinginternally labeled and 5'-end-labeled RNA transcripts (Fig.3E and G and data not shown). Together, these data suggest thata single tetraloop may direct Rnt1p cleavages within two separatestems. In order to directly examine this possibility, we produceda short T7 transcript representing a model substrate that maintainsthe two stems linked to the AGUU tetraloop binding site. Thefirst stem ends with the 2 nt located below the C4 cleavagesite, and the other terminates with a UAUU tetraloop that replacesthe naturally occurring snR55 sequence. The UAUU tetraloop cannotdirect cleavage by itself, and no tetraloop other than the AGUUwas found within this RNA transcript. Thus, any cleavage detectedin the adjacent stem would be directed by the AGUU tetraloop.The model T7 substrate was labeled at either the 3' or the 5'ends and incubated with purified Rnt1p either at a low, monovalentsalt concentration or at physiological salt concentration. Asshown in Fig. 3G, all four cleavages were detectable at bothsalt concentrations. The cleavage sites were mapped based onthe sizes of the released fragments (Fig. 3G). The simultaneousdetection of cleavages at C3/C5 and C4/C6 within a single-end-labeledRNA species suggested that the four cleavages are not producedfrom a single binding event. Incubation of the model substratein either a wild-type or a ${Delta}$ rnt1 cell extract confirmed thatthe native Rnt1p could also cleave substrates with bifurcatedstems. Consequently, kinetic analysis using similar bifurcatedRNA substrate showed that each stem is cleaved by a distinctbinding event (B. Lamontagne and S. Abou Elela, unpublishedresults).

The AGNN tetraloop is not conserved near all Rnt1p-dependent snoRNAs. Analysis of the expression profiles of snoRNAs suggested thatthe monocistronic snR56 and snR48 snoRNAs are Rnt1p dependent.However, we failed to identify AGNN tetraloops near the terminiof these snoRNAs. Northern blot analysis confirmed that thedeletion of Rnt1p impairs the processing of these two snoRNAs(Fig. 4B and 5B). In vitro cleavage of a T7-transcribed RNAcorresponding to the 5' end of snR56 indicated that Rnt1p coulddirectly cleave the RNA near snR56 despite the absence of anAGNN tetraloop (Fig. 4C). Extension of a primer correspondingto a portion of the snR56 mature sequence confirmed cleavageat a stem capped with UGGU that occurs at the predicted distancefrom the loop (Fig. 4D). While this study was in progress, itwas also reported that Rnt1p could cleave a stem in the intronof RPL18A that is capped with UGGU (12). Thus, Rnt1p does notrequire A in the first position of the tetraloop for cleavage.Similarly, in vitro cleavage of a model substrate near the 5'end of snR48 confirmed that Rnt1p could cleave this substrate.However, no canonical stem capped with an NGNN tetraloop wasfound in the vicinity of the cleavage. Primer extension analysisindicated cleavage near an AAGU-terminal tetraloop (Fig. 5D).Since all known substrates to date contain a G in the secondposition of the tetraloop and it has previously been shown thatchanging this G to any other nucleotide blocks cleavage in vitro(9), we presume that a special feature of either this RNA stemor a combination of the loop and stem sequence permits Rnt1pcleavage in this case. We concluded that the AGNN tetraloopis not essential for the Rnt1p-dependent maturation of snoRNA.

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FIG. 4. Rnt1p does not require the presence of an AGNN tetraloop for the maturation of snR56. (A) A schematic representation of the predicted cleavage sites associated with snR56. The cleavage sites identified in vitro are indicated by arrowheads. (B) Northern blot analysis of snR56. RNA extraction and Northern analysis were performed as described in the legend to Fig. 2B. (C) In vitro cleavage of a model substrate representing the cleavage signals found near snR56. Internally labeled T7-transcribed RNA possessing the stem-loop structure indicated for panel A was incubated with purified Rnt1p. The cleavage products were separated by polyacrylamide gel electrophoresis and visualized by autoradiography. (D) Mapping of the Rnt1p cleavage site by using primer extension. The primer extension was performed as described in the legend to Fig. 2C. The mature RNA, extended ends, and cleavage site are indicated on the right.

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FIG. 5. Rnt1p is required for the processing of snR48 in the absence of any detectable NGNN tetraloop. (A) A schematic representation of the predicted stem near the 5' end of snR48. The cleavage sites identified in vitro are indicated by arrowheads. (B) Northern blot analysis of snR48. RNA extraction and Northern analysis were performed as described in the legend to Fig. 2B. (C) In vitro cleavage of a model substrate representing the cleavage signals found near snR48. Internally labeled T7-transcribed RNA exhibiting the stem-loop structure indicated for panel A was incubated with purified Rnt1p, and the cleavage products were separated by polyacrylamide gel electrophoresis and visualized by autoradiography. (D) Mapping of the Rnt1p cleavage site by using primer extension. The primer extension was conducted as described in the legend to Fig. 2C. The mature RNA, extended ends, and cleavage site are indicated on the right.

Direct processing of intron-embedded snoRNAs by Rnt1p. Two of the predicted Rnt1p cleavage signals were located inthe introns of the pre-mRNA of the ribosomal protein (r-protein)RPL7A and that of its nearly identical isoform RPL7B near snR39and snR59, respectively. Consistently, the microarray expressionprofile indicated that the expression of both snoRNAs was inducedupon the deletion of RNT1 (see Table S1 in the supplementalmaterial). In order to examine the impact of Rnt1p on the processingof snR39 and snR59, we monitored the RNA profiles of these twoRNAs in both the absence and the presence of Rnt1p. As shownin Fig. 6B, the deletion of Rnt1p did not inhibit the accumulationof mature snR39 or of the mature RPL7A mRNA (Fig. 6C) but causedmild accumulation of the unspliced pre-mRNA precursor. The Northernblot analysis suggests that the increase in the expression ofsnR39 observed by microarray analysis (see Table S1 in the supplementalmaterial) is due to either an accumulation of the unsplicedmRNA or a partially degraded snoRNA and not to the accumulationof snR39 or its immediate precursor. In contrast, deletion ofRnt1p causes the accumulation of a precursor of snR59, a significantaccumulation of the pre-mRNA, and a reduction in the level ofmature snR59 (Fig. 7B). The accumulation of RPL7B pre-mRNA didnot result in a decrease in the level of the mature mRNA, suggestingthat splicing is not impaired, as is observed upon inactivationof the essential splicing factor Prp2p (Fig. 7B).

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FIG. 6. Rnt1p assists in releasing the intron-encoded snR39 from the lariat of RPL7A pre-mRNA. (A) Schematic representation of the structure found near snR39 within the intron of RPL7A. The cleavage sites identified in vitro are indicated by arrowheads. (B) Northern blot analysis of snR39 and the associated mRNA sequence. RNA extraction and Northern analysis were performed as described in the legend to Fig. 2B. Probes corresponding to the snR39 mature sequence (intron 2), intron 1, or exon 3 were used. A probe corresponding to snR10 that was notaffected by Rnt1p deletion is shown as a control for both loading and RNA quality. NT indicates the nascent transcript. wt indicates the wild type. (C) Northern blot analysis using a probe specific to exon 3 of RPL7A. The RNA used was extracted and manipulated as described for panel B except that it was fractionated on 1% agarose gel. (D) In vitro cleavage of total RNA. RNA extracted from either wild-type cells or ${Delta}$ rnt1 cells was incubated with purified Rnt1p. Northern blot analysis using probes specific to the sequence near either the 3' end of snR39 or the mature sequence was used to display the cleavage products. The positions of the different cleavages are indicated on the right. L designates the position of the lariat sequence. (E) Primer extension mapping of Rnt1p cleavage in vitro. The experiment was conducted as described in the legend to Fig. 2C.

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FIG. 7. Rnt1p is required for the processing of the intron-encoded snR59 from the pre-mRNA of RPL7B. (A) Schematic representation of the structure found near snR59 within the intron of RPL7B. The cleavage sites identified in vitro are indicated by arrowheads. (B) Northern blot analysis of snR59 and the associated mRNA sequence. Probes corresponding to either intron 1 or exon 3 were used. A probe corresponding to snR10 that was not affected by Rnt1p deletion is shown as a control for both loading and RNA quality. E1 and E2 indicate exons 1 and 2, respectively, I1 indicates intron 1, and I2 indicates intron 2. L indicates the splice lariat. NT indicates the nascent transcript. C indicates the site of cleavage. TI2 indicates truncated fragments of intron 2. (C) In vitro cleavage of total RNA. RNA was extracted from either wild-type or ${Delta}$ rnt1 cells incubated with purified Rnt1p. Northern blot analysis using probes specific to the sequences near either the 3' end of snR59 or the mature sequence were used to display the cleavage products. The positions of the different cleavages are indicated on the right. L designates the position of the lariat sequence. (D) Primer extension mapping of Rnt1p cleavage in vitro. The experiment was conducted as described in the legend to Fig. 2C.

In order to examine the possibility of a lariat-dependent processingpathway of snR39 and snR59, we studied the accumulation of thesetwo snoRNAs in cells lacking the debranching enzyme Dbr1p andin cells containing deletions in the DBR1 and RNT1 genes. Inthe case of snR39, the deletion of DBR1 resulted in the accumulationof a lariat containing snR39 and in a severe reduction in themature form of snR39. Deletion of both DBR1 and RNT1 completelyabolished the production of mature snR39 (Fig. 6B). These dataindicate that Rnt1p plays a minor role in releasing snR39 fromthe lariat, while the major processing pathway of snR39 is throughthe trimming of the debranched lariat. In vitro cleavage assaysand primer extension mapping of the cleavage site revealed thatRnt1p could cleave the lariat containing the snR39 but not theprimary mRNA transcript. Eight different cleavage sites weremapped near both the 5' and 3' ends of snR39 (Fig. 6D and E).Two of these were located at the predicted distance from theidentified AGUU tetraloop, while the six others were not nearany recognizable tetraloop motif. It is possible that an alternativefold brings these cleavage sites close to the identified terminaltetraloop. In contrast, in vitro cleavage indicated that Rnt1ptargets RPL7B pre-mRNA and not the produced lariat for cleavage(Fig. 7C and D). The cleavage of Rnt1p occurred at the predicteddistance from the AGUU tetraloop (Fig. 7A and D). These dataindicate that Rnt1p is required for the maturation of snR59.We concluded that, while the processing of snR39 is dependenton the splicing of RPL7A pre-mRNA, the splicing of RPL7B pre-mRNAis inhibited when Rnt1p-dependent snR59 RNA processing occurs.

DISCUSSION

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Discussion

In this study, we presented a combined in silico, in vitro,and in vivo approach for the detection of Rnt1p substrates anddemonstrated its utility for the identification of key processingevents. Most Rnt1p processing signals were found near box C/DsnoRNAs, while only three were found near box H/ACA snoRNAs,indicating either a distinct evolutionary origin or a distinctregulatory pathway for each class of snoRNA. The location andorganization of Rnt1p cleavage signals were found to vary fromone snoRNA transcript to another. Rnt1p-cleaved substrates formedthrough base pairing between distantly located RNA sequences,thereby ensuring the maturation of both ends of the targetedsnoRNA (Fig. 2 and 3), as previously suggested for the bacterialRNase III (63). In other cases, a single NGNN tetraloop directedcleavages at two distinct cleavage sites, thereby relating theprocessing of two adjacent snoRNAs (Fig. 3). The cleavage signalsof all monocistronic snoRNAs that are processed by Rnt1p werefound near the 5' end of the precursor except the two isoformsof U3 snoRNA that contain introns, which were instead maturedthrough splicing (22). Finally, depending on the nature andcontext of the Rnt1p cleavage signal, the processing of intron-encodedsnoRNAs could be either linked to or separated from the splicingof the host pre-mRNA, allowing a flexible control of the snoRNA-associatedredundant r-protein isoforms (Fig. 6 and 7). The data presentedhere indicate the capacity of Rnt1p processing signals to providea flexible tool to relate rRNA, snoRNA, and r-protein production.

In yeast, most intron-containing pre-mRNAs encode r-proteinsand are redundant (45). It has been suggested that this organizationis important for fine-tuning the expression of r-proteins andlinking it to rRNA production (58). Indeed, in several cases,the expression of one r-protein isoform regulates the splicingor the mRNA transport of another. However, it is not clear howthe production of two r-protein isoforms might be regulatedif they harbor a functionally distinct snoRNA, as is the casefor RPL7A and RPL7B. The two proteins are nearly identical andhave similarity to the Escherichia coli L30 and rat L7 r-proteins(35, 62). Deletion of RPL7A that harbors snR39 within its intronsmoderately impairs growth and affects budding (37); however,deletion of RPL7B that harbors snR59 in its intron has no effecton growth (37). Deletion of both genes is lethal, reflectingtheir housekeeping function as part of the ribosome. Like otherr-proteins, the expression of these two isoforms needs to beregulated in order to achieve an equimolar production of boththe protein and the rRNA it binds. For example, the expressionof these two proteins is shut down along with that of all otherr-proteins when no rRNA is produced (58). However, controllingthe transcriptional level will also affect the snoRNAs encodedwithin the introns of these proteins, which are required forthe production of normal, mature rRNA. Although the snoRNAsembedded in the introns of both RPL7 isoforms are not essentiallike most snoRNAs, they are conserved among fungi (49, 50) andtheir expression is expected to be controlled like most componentsof the ribosome biogenesis machinery (42). In this study, weshow that in the case of RPL7B, cleavage by Rnt1p could releasesnR59 while preventing the production of RPL7B. In contrast,snR39 production appears to be linked to RPL7A since Rnt1p couldcleave only the splicing by-product of RPL7A and not the matureRNA. Therefore, the cells could control the overall level byslowing the splicing of RPL7B, which would lead to an increasein cleavage by Rnt1p, producing snR59 and reducing the amountof RPL7B. This is consistent with the fact that the deletionof RPL7A has a greater effect on growth than the deletion ofRPL7B. Introns of r-protein mRNA were previously searched forRnt1p cleavage sites, and no cleavage was detected other thanthat identified within the introns of RPS22B and RPL18 pre-mRNAs(12). We have searched all other mRNAs containing introns inyeast (17) for potential Rnt1p cleavage sites, and we did notfind mRNAs with intronic stem-loops above 0.8 that are significantlyoverexpressed upon Rnt1p deletion other than those previouslyidentified (J. Gagnon and S. Abou Elela, unpublished results).This indicates that Rnt1p cleavage within pre-mRNA introns mightbe restricted to r-proteins, possibly due to the specific needto regulate ribosome biogenesis. However, we cannot excludea more general but redundant role of Rnt1p in the regulationof intron-containing mRNAs that cannot be easily detected bythe deletion of RNT1.

ACKNOWLEDGMENTS

We thank J. D. Boeke for the ${Delta}$ dbr1 strain and Ren-Jang Lin forthe prp2 temperature-sensitive strain. We also thank StéphanieLarose for preparing the yeast cell extract used in this study.We are indebted to all members of the Abou Elela lab for stimulatingdiscussions and critical reading of the manuscript.

This work was supported by a grant from the Natural Sciencesand Engineering Research Council of Canada. S.A. is a Chercheur-BoursierJunior II of the Fonds de la Recherche en Santé du Québec.

FOOTNOTES

* Corresponding author. Mailing address: Université de Sherbrooke, Département de Microbiologie et d'Infectiologie, 3001 12e Ave nord, Sherbrooke, Québec J1H 5N4, Canada. Phone: (819) 564-5275. Fax: (819) 564-5392. E-mail: Sherif.Abou.Elela{at}Usherbrooke.ca.

Supplemental material for this article may be found at http://mcb.asm.org/.

REFERENCES

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