Rrp6p, the Yeast Homologue of the Human PM-Scl 100-kDa Autoantigen, Is Essential for Efficient 5.8 S rRNA 3' End Formation*

Michael W. Briggs, Karina T. D. Burkard, and J. Scott ButlerDagger

From the Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14618

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The eukaryotic 25 S, 18 S, and 5.8 S rRNAs are synthesized as a single transcript with two internal transcribed spacers (ITS1 and ITS2), which are removed by endo- and exoribonucleolytic steps to produce mature rRNA. Genetic selection for suppressors of a polyadenylation defect yielded two cold-sensitive alleles of a gene that we named RRP6 (ribosomal RNA processing). Molecular cloning of RRP6 revealed its homology to a 100-kDa human, nucleolar PM-Scl autoantigen and to Escherichia coli RNase D, a 3'-5' exoribonuclease. Recessive mutations in rrp6 result in the accumulation of a novel 5.8 S rRNA processing intermediate, called 5.8 S*, which has normal 5' ends, but retains ~30 nucleotides of ITS2. Pulse-chase analysis of 5.8 S rRNA processing in an rrp6- strain revealed a precursor-product relationship between 5.8 S* and 5.8 S rRNAs, suggesting that Rrp6p plays a role in the removal of the last 30 nucleotides of ITS2 from 5.8 S precursors. A portion of 5.8 S* rRNA assembles into 60 S ribosomes which form polyribosomes, suggesting that they function in protein synthesis. These findings indicate that Rrp6p plays a role in 5.8 S rRNA 3' end formation, and they identify a functional intermediate in the rRNA processing pathway.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The maturation of the three major classes of eukaryotic RNA (mRNA, rRNA, and tRNA) requires extensive processing events, including endonucleolytic and exonucleolytic cleavages, intron removal, and exon splicing, as well as nucleotide additions and removals exemplified by polyadenylation and editing. Three of the four eukaryotic rRNAs are synthesized as a single precursor RNA and must undergo a complex series of processing events to produce mature 18 S, 5.8 S, and 25 S rRNAs. Central to this process is the removal of the two internal transcribed spacers (ITS)1 that separate 18 S from 5.8 S (ITS1) and 5.8 S from 25 S (ITS2) (Fig. 1). In the yeast Saccharomyces cerevisiae, the removal of ITS1, which is coupled to the removal of the 5' external transcribed spacer, features a series of cleavage events requiring proteins, small nucleolar RNAs and the ribonucleoprotein RNase MRP (reviewed in Refs. 1 and 2). Cleavage within ITS1 separates 20 S RNA from 27 S RNA. 20 S RNA is processed in the cytoplasm to form mature 18 S rRNA, while 27 S RNA is processed by two alternative pathways. Some 15% of 27 S undergoes cleavage at a site that appears to correspond to the 5' end of the long form of 5.8 S rRNA, while RNase MRP, in conjunction with Rrp5p cleaves the remaining 27 S molecules, which then undergo 5'-3' exonucleolytic processing to generate the 5' end of the short form of 5.8 S (3-6).


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Fig. 1.   Diagram of the major processing events involved in removal of ITS1 and ITS2 from pre-rRNA.

The events required for processing of ITS2 remain less well characterized than those for ITS1. After processing by alternative pathways during the latter stages of ITS1 removal, both types of 27 S rRNAs undergo cleavages within ITS2 that separate 25 S molecules, with either mature or extended 5' ends, from 5.8 S precursors (7 SL and 7 SS) with 3' extensions. These 3' extensions must be removed to produce mature 5.8 SL and 5.8 SS rRNAs. Recessive mutations in the S. cerevisiae RRP4 gene result in the accumulation of 3' extended forms of 5.8 S rRNA suggesting a role for Rrp4p in 5.8 S rRNA 3' end processing. Indeed, immunoprecipitates of Rrp4p demonstrated 3'-5' riboexonuclease activity in vitro (7). More extensive analysis of Rrp4p immunoprecipitates revealed the existence of Rrp4p in a multisubunit complex, called the exosome (8). In addition to Rrp4p, the exosome contains three 3'-5' riboexonucleases required for efficient maturation of 5.8 S rRNA 3' ends. Each of these enzymes show strong homology to 3'-5' riboexonucleases found in Escherichia coli.

In this report, we present the identification and characterization of RRP6, the product of which plays a role in the proper 3' end processing of 5.8 S rRNA. Mutations in RRP6 result in the accumulation of 3' extended 5.8 S rRNAs. RRP6 encodes a protein highly homologous to the human PM-Scl 100-kDa autoantigen, as well as proteins of unknown function from Caenorhabditis elegans and Schizosaccharomyces pombe. Each of these eukaryotic proteins share homology in their central domains with the E. coli 3'-5' exoribonuclease RNase D, suggesting the conservation of some RNase D function from bacteria to humans.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Strains, Media, and Genetic Techniques-- The experiments reported here were performed using the yeast strains described in Table I. The suppressor isolation has been described by Briggs and Butler (9).

                              
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Table I
Yeast strains used in this study

Yeast strains were grown in either YEPD or synthetic complete medium (10) lacking uracil (to maintain plasmids). Genetic techniques were performed as described (10, 11). Transformation of yeast was performed as described by (12). E. coli strain DH5alpha was used for all recombinant DNA manipulations.

Plasmids and Oligonucleotides-- The plasmids and deoxyoligonucleotides utilized in this study are described in Table II. Restriction enzymes were purchased from Life Technologies, Inc., Promega, or New England Biolabs, and digestions were performed as per manufacturers' instructions. Double-stranded DNA probe templates were prepared by diethylaminoethyl paper purification from 1% agarose gels and radiolabeled by random hexamer priming with 5'-[alpha -32P]deoxyCTP (NEN Life Science Products, 3000 Ci/mmol) and the Klenow fragment of DNA polymerase (Boehringer Mannheim), according to the manufacturer's instructions. Deoxyoligonucleotide probes (25 pmol; Oligos Etc., Inc.) were radiolabeled by incubation for 60 min at 37 °C in 15-µl reactions containing 50 mM Tris-Cl, pH 7.5, 10 mM MgCl2, 5 mM dithiothreitol with 1 unit of T4 polynucleotide kinase (Life Technologies, Inc.) and 50 µCi of 5'-[gamma -32P]ATP (NEN Life Science Products, 6000 Ci/mmol). Unincorporated nucleotides were removed from probes by chromatography on Sephadex G-25.

                              
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Table II
Plasmids and oligonucleotides

Molecular Cloning of RRP6-- Twenty µg of library DNA (9) were transformed into strain 1BC1 (Table I), and transformants were selected on URA- plates at 25 °C for 36 h, after which the plates were incubated at 14 °C until putative RRP6-containing complementing plasmid-bearing strains grew above background levels. One colony out of approximately 100,000 transformants displayed reproducible complementation of the cold-sensitive phenotype after rescue of the plasmid from yeast cells and retransformation into 1BC1. The apparent low number of RRP6 transformants is likely due to its tight centromere linkage, since E. coli colony blot hybridization experiments indicate that its representation in the library is similar to other genes (9). A 3.5-kilobase RRP6-specific XbaI/EcoRI fragment was cloned into the same sites in YIplac211 (13) to produce pUNC9. CEN15 was removed from pUNC9 by digestion with XbaI and BglII, filling in the 5' overhangs with the Klenow fragment DNA polymerase and ligation of the plasmid with T4 DNA ligase to produce p10dXB. Analysis of the linkage between rrp6-1 and UNC733 was carried out by transformation of ABC1-2D with p10dXB linearized with BamHI.

RNA Analyses-- Total RNA was prepared and Northern analysis carried out as described previously (14). Levels of specific RNAs were quantitated by storage PhosphorImager analysis (Molecular Dynamics) and normalized to 5 S rRNA levels, which were quantitated by FluorImager analysis (Molecular Dynamics) of ethidium bromide-stained gels or by storage PhosphorImager analysis after hybridization of 5'-32P-labeled deoxyoligonucleotide probes to 5.8 S rRNA or to the RNA polymerase III-transcribed ScR1 (Table II).

For analyses of small rRNA transcripts, 3 µg of total RNA were separated on a 12% polyacrylamide, 8 M urea gel, and RNAs were transferred to GeneScreen membranes (DuPont) by electroblotting at 10 V for 16 h at 4 °C. Hybridizations were performed at 42 °C as described previously (14) except that formamide was omitted when oligonucleotide probes were used. Length determinations of 5.8 S* rRNA were made by linear regression from a semilog plot of the mobilities of 7 S, 5.8 S, and 5 S rRNA as a function of their known lengths.

Primer extension analysis of 5.8 S rRNA was performed as described (15). Polyribosomes were prepared and analyzed as described (16).

DNA Sequence Analyses-- The accession numbers of the nucleotide and protein sequences analyzed in this study are as follows: Rrp6p, Z74909; Homo sapiens, Q01780; S. pombe, Q10146; C. elegans, P34607; E. coli, P09155; Haemophilus influenza, P44442. Sequence data derived from complementing plasmid pC114 were compared with the yeast genome sequence data base using the BLAST program at the National Center for Biotechnology Information2 and the Stanford Genome Resource Data Base.3 Pairwise comparisons of Rrp6p and proteins from other species were made using BESTFIT, and the protein sequences were aligned using Pileup and MSAShade. Putative nuclear localization signals were determined using the Psort program.

Construction and Analysis of a rrp6 Null Allele-- The URA3 portion of pUNC9 was deleted by digestion with AatII and NarI, followed by filling the overhanging ends with the Klenow fragment of DNA polymerase and subsequent ligation to produce pUN9d4. URA3 was removed from pJJ244 (17) with BamHI and PvuII and inserted into BamHI/EcoRV-digested pBR322 to produce pBRU2. The internal BglII fragment of RRP6 in pUN9d4 was replaced with the BamHI fragment containing URA3 from pBRU2 to produce pdRRP6F. Disruption of chromosomal RRP6 was carried out by digestion of pdRRP6F with BamHI and PvuII to liberate rrp6::URA3 followed by transformation of the digestion mixture into a diploid made by crossing T481 and T581 (Table I). Disruptants were selected on synthetic complete URA- plates at 30 °C. Disruption of RRP6 was verified by PCR using primers flanking the RRP6 BglII sites (Table II, Fig. 6).

Pulse-Chase Analysis of 5.8 S rRNA Processing-- Cultures of BPO2 and BPO2-12F (Table I) were grown at 30 °C in synthetic complete glucose medium supplemented with uracil (20 mg/ml) to an A600 of 1.7. Cells were harvested by centrifugation, resuspended in 5 ml of fresh medium lacking uracil and labeled for 3 min by the addition of 50 µCi of [5,6-3H] uracil/ml (NEN Life Science Products, 39 Ci/mmol). Following the initial pulse labeling, the radioactive precursor was chased for up to 1 h by the addition of unlabeled uracil at a final concentration of 300 µg/ml. At various times, samples were taken, the cells collected by centrifugation and frozen in dry ice. Total RNA was isolated from yeast cells as described (14). Small RNAs were separated by electrophoresis on 6% polyacrylamide gels containing 7.5 M urea in 50 mM Tris borate (pH 8.3). Gels were fixed in 30% methanol, 10% acetic acid for 1 h, incubated in EN3HANCE (NEN Life Science Products) for 1 h and washed with water for 20 min. The gels were then dried and subjected to autoradiography. Quantitation of the amount of [5,6-3H]uracil incorporated into specific RNAs was determined by cutting the appropriate bands from the dried gels and determining the amount of radioactivity present by scintillation counting.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Isolation of rrp6 Mutations as Suppressors of a Polyadenylation Defect-- Pseudoreversion analysis of the pap1-1 temperature sensitive mutation in S. cerevisiae was carried out to identify gene products that play roles in the mechanism or function of polyadenylation (9). We isolated five independent suppressors that exhibited a cold-sensitive growth phenotype at 14 °C from an initial population of about 5,000 spontaneously arising pseudorevertants that allowed growth of a pap1-1 strain at the restrictive temperature of 30 °C (Table III). Two of the five cold-sensitive suppressor strains isolated represented alleles of the RRP6 complementation group (previously named PDS1) (9). Tetrad analysis after sporulation of a diploid homozygous for pap1-1 and rrp6-1 demonstrated independent, 2:2 segregation of both the cold-sensitive and suppressor phenotypes indicating that the rrp6-1 mutation occurred in a single-copy nuclear gene, extragenic to pap1-1. Normal growth at low temperature of a diploid heterozygous for rrp6-1 or rrp6-2 indicated that these mutations are recessive, suggesting loss of function mutations. Delineation of the mechanism of suppression of pap1-1 by alleles of rrp6 will be presented elsewhere.

                              
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Table III
Growth phenotypes of isogenic sets of RRP6 and rrp6 strains
Growth phenotypes were determined by incubation on YEPD or synthetic complete, URA- plates at the indicated temperatures.

Molecular Cloning and Sequence Analysis of RRP6-- The recessivity of the rrp6-1 cold-sensitive phenotype allowed us to clone the wild-type gene by complementation. We transformed an rrp6-1 strain with a yeast centromere-based genomic library (9), and isolated one plasmid that conferred wild-type growth at 14 °C (pC114, Fig. 2). Plasmid rescue and retransformation confirmed plasmid linkage to complementation. Sequence determination of the ends of the pC114 insert and comparison with the European Molecular Biology Laboratory (EMBL) data base mapped the insert to chromosome XV. We performed a number of deletions to isolate the complementing open reading frame and found that complementation required the presence of a previously uncharacterized open reading frame, UNC733 (SCYOR001W, GenBankTM accession no. Z74909). For linkage analysis of rrp6-1 and UNC733, we targeted integration of an UNC733,URA3 plasmid (p10dXB; Fig. 2) into the chromosomal UNC733 locus in an rrp6-1,ura3-52 strain. This strain was crossed to an RRP6,ura3-52 strain, the resultant diploid was sporulated, and the progeny were analyzed. All cold-sensitive progeny were URA+ (36/36) and all URA- spores were cold-resistant (25/25), thus demonstrating tight linkage of rrp6-1 to the UNC733,URA3 plasmid. Furthermore, we constructed a strain with YIpRRP6 integrated at RRP6, which lost the integrated plasmid spontaneously in the absence of selection, probably due to the presence of CEN15 on the plasmid. Half of the resultant URA- isolates demonstrated normal growth at 14 °C, suggesting that homologous recombination between the chromosomal rrp6-1 and the plasmid-borne UNC733 had removed the chromosomal lesion. Taken together, these data indicate that the previously cloned, but hitherto uncharacterized gene UNC733 is allelic to rrp6-1 and will hereafter be referred to as RRP6.


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Fig. 2.   Complementation analysis of subclones of the RRP6 region. A, schematic representation of the RRP6 locus on chromosome XV (Bg, BglII; X, XbaI; K, KpnI; E, EcoRI). Plasmids containing the indicated genomic fragments were transformed into an rrp6-1 strain, and complementation was determined by the ability of the transformants to grow on synthetic complete dextrose, URA- plates at 14 °C and 25 °C.

The RRP6 open reading frame encodes a polypeptide with a predicted mass of 84,038 Da and an isoelectric point of 7.14. The sequence is relatively rich in leucine (11%), serine (9%), and lysine (8%), but a search for patterns found in the PROSITE Dictionary of Protein Sites and Patterns revealed no motifs in common with other characterized proteins. Comparison of the sequence with the Swissprot data base revealed significant homology to the human nucleolar PM-Scl 100-kDa autoantigen (18, 19), E. coli and H. influenza RNases D (20, 21), as well as to predicted proteins from S. pombe and C. elegans (22). Figure 3 illustrates the alignment of the Rrp6p sequence with these sequences and shows a consensus sequence which serves to illustrate the regions of greatest homology among these polypeptides. BESTFIT analysis using a gap weight of 3.0 and a length weight of 0.1 revealed Rrp6p to be most closely related to the proteins from S. pombe (32% identity, 57% similarity), H. sapiens (32% identity, 52% similarity), and C. elegans (19% identity, 42% similarity). Rrp6p is also homologous, over a smaller region, to RNase D from E. coli (24% identity, 45% similarity) and H. influenza (22% identity, 48% similarity). The greatest region of homology among these proteins lies between amino acids 280 and 640 of Rrp6p within which lie six stretches of amino acids conserved in both sequence and relative position among all these species. These primary sequence homologies suggest the possibility that this core region represents the conservation of protein function among these organisms. Indeed, Hidden Markov computer modeling of E. coli endo- and exoribonucleases recently suggested that RRP6 and PM-Scl 100-kDa are members of a family of structurally related proteins including RNase D and the 3'-5' deoxyriboexonuclease domains of DNA polymerases I (23). Domains I, III, and V indicated in Fig. 3 contain, respectively, motifs homologous to the ExoI, ExoII, and ExoIII domains shown to be essential for the two metal ligand mechanism of catalysis required for the 3'-5' deoxyriboexonuclease activity of DNA polymerase I (24). Putative nuclear localization signals are found in the C-terminal portions of the eukaryotic proteins (Fig. 3). Immunofluorescence experiments have shown that human PM-Scl 100 kDa exists in the nucleus and the nucleolus (25), but no characterization of the function of the protein has been reported.


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Fig. 3.   Amino acid sequence of Rrp6p and comparison with the amino acid sequences of homologues from other organisms. The sequences were aligned using the Pileup program. The consensus sequence was defined as positions where all (uppercase) or 3/4 (lowercase) sequences have identical amino acids. The boxed areas labeled I-VI indicate regions of greatest homology shared among all of the sequences. The boxes on individual sequences indicate the positions of putative nuclear localization signals. The GenBank accession numbers are as follows: Rrp6, Z74909; H. sapiens, Q01780 (18, 19); S. pombe, Q10146; C. elegans, P34607 (22); E. coli, P09155 (20); H. influenza, P44442 (21).

The rrp6-1 Mutation Causes the Accumulation of a 3' Extended 5.8 S rRNA Processing Intermediate-- In light of the striking homology between Rrp6p and a known nucleolar protein, we reasoned that rrp6-1 might cause a defect in some aspect of rRNA processing or ribosome biogenesis. Northern analysis of total cellular RNA from normal, rrp6-1, and rrp6-1 cells harboring the complementing plasmid YCpRRP6 revealed no obvious defects in the processing of the large rRNA precursors (data not shown). However, Northern blot analysis of total RNA separated on a polyacrylamide gel, using an oligonucleotide probe complementary to an internal portion of 5.8 S rRNA (o5.8S in Table II), revealed the accumulation of a novel 5.8 S rRNA intermediate in rrp6-1 strains (Fig. 4A). This intermediate (labeled 5.8S* in Fig. 4) is a doublet ~30 nucleotides longer than mature 5.8 S rRNA, and accounts for half of the 5.8 S rRNA in rrp6-1 strains. As expected, the presence of the complementing plasmid, YCpRRP6, reduces the accumulation of 5.8 S* by 95%, suggesting semidominance of rrp6-1 over RRP6. To determine if 5.8 S* represents a 5' or 3' extension of mature 5.8 S rRNA, we probed the same Northern blot with random-primed probes complementary to portions of ITS1 or ITS2 (Fig. 4, B and C, respectively). The ITS2 probe, but not the ITS1 probe, hybridized to 5.8 S*, suggesting that this molecule carries an extension at its 3' end.


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Fig. 4.   Analysis of 5.8 S rRNA lengths in RRP6 (A364A) and rrp6-1 (ABC1-2D) strains. Total RNA from indicated strains was separated on a 12% denaturing gel and analyzed by Northern blotting with probes specific for regions including or surrounding 5.8 S rRNA (bottom). A, o5.8S is complementary to positions +48 through +24 of 5.8 S rRNA, where +1 represents the mature 5' end of the "5.8 S S" form. The positions of precursor rRNA intermediates are indicated at the left. B, the ITS1 probe is complementary to the last 8 nucleotides of 18 S rRNA, the entire ITS1 region, and the first 19 nucleotides of 5.8 S rRNA. C, the ITS2 probe is complementary to the last 23 nucleotides of 5.8 S rRNA, all of ITS2, and 194 nucleotides of 25 S rRNA.

If 5.8 S* rRNA accumulates solely due to a defect in 3' end processing, then analysis of steady-state 5.8 S rRNA 5' ends should reveal no differences between RRP6 and rrp6-1 strains. Indeed, primer extension analysis of the 5' ends of 5.8 S rRNAs from the normal and rrp6-1 strains revealed no significant differences in the pattern or amounts of the short (5.8S S) and long (5.8S L) forms of 5.8 S rRNAs found in normal cells (Fig. 5; Refs. 26 and 27). The detection of primer extension products ending at the A2 cleavage site in ITS1 (corresponding to the 27 S A2 precursor; see schematic in Fig. 1) with RNAs from normal and rrp6-1 strains indicates that reverse transcriptase was able to transcribe past the mature 5' ends of 5.8 S rRNA to the 5' end found in pre-5.8 S rRNAs. Since we detected no rrp6-1-specific 5'-extended intermediates, we conclude that the rrp6-1 mutation causes a defect in 5.8 S rRNA 3' end processing.


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Fig. 5.   Primer extension analysis of the 5' end points of mature 5.8 S rRNA in RRP6 (A364A) and rrp6-1 (ABC1-2D) strains. RNA samples used in the experiments shown in Fig. 4 were hybridized to 5'-32P-labeled o5.8S and analyzed by primer extension with M-MLV reverse transcriptase as described under "Experimental Procedures." Indicated to the right are the positions of the input oligonucleotide (o5.8S), the reverse transcription product ending at the two normal 5' ends of mature 5.8 S rRNA (5.8S L and S) and at the 5' end of the 27 S A2 pre-rRNA (27S). The positions of pBR322-MspI-digested molecular length markers is shown at the left of the figure.

We assessed the impact of a chromosomal deletion of RRP6 by replacing a 1.2-kilobase BglII fragment of RRP6 with URA3 on a plasmid, followed by allelic exchange of chromosomal RRP6 with rrp6::URA3 in a diploid homozygous for ura3-52. PCR analysis of DNA from this diploid using primers adjacent to the insertion junctions of RRP6 and URA3 verified disruption of one copy of RRP6 (Fig. 6, A and B). Tetrad analysis after sporulation of this diploid revealed that disruption of RRP6 leads to a slow growth at 30 °C and an inability to grow at 37 °C, indicating that the RRP6 plays an important role in cell viability. Since the disruption did not lead to inviability we verified that the URA+ progeny indeed carried the disrupted rrp6::URA3 allele (Fig. 6, A and B). Analysis of 5.8 S rRNA from the disruptants showed a similar ratio of 5.8 S to 5.8 S* seen in a rrp6-1 strain, suggesting that rrp6-1 confers a null phenotype with respect to 5.8 S rRNA processing (Fig. 6C).


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Fig. 6.   Characterization of rrp6::URA3 disruptants. A, diagram of the RRP6 locus before and after disruption with URA3 (Bg, BglII). oRRP6-2 and oRRP6-3 indicate approximate hybridization positions and orientations of the two oligonucleotide primers used to verify disruption of RRP6 by PCR. B, PCR products generated from genomic DNA isolated from strains with the indicated genotypes as well as from progeny from an ascospore derived from sporulation of TT45dR5. Values to the left of the figure indicate the lengths of lambda  HindIII markers. C, Northern blot detection of 5.8 S rRNAs in normal and rrp6-1 strains compared with those from the ascospore progeny analyzed in B.

Rrp6p Defects Block the Major Pathway of 5.8 S rRNA Processing-- The results presented above cannot distinguish between a role for Rrp6p in normal 5.8 S 3' end formation, or a role in destroying 5.8 S rRNA molecules with improperly processed 3' ends. The former model predicts that 5.8 S* rRNA should appear before 5.8 S rRNA, thereby slowing its rate of formation compared with that in normal cells. To test this, we analyzed 5.8 S rRNA processing by pulse-chase labeling with [3H]uracil in normal and rrp6::URA3 cells. The results, shown in Fig. 7, illustrate two interesting aspects of 5.8 S rRNA processing in mutant cells. First, the rate and extent of formation of total 5.8 S (5.8 S + 5.8 S*) is similar in normal and rrp6::URA3 cells. Second, the rates of formation of 5.8 S and 5.8 S* are vastly different in the two strains. In rrp6::URA3 cells, 5.8 S rRNA appears at one-twentieth the rate in normal cells. Importantly, 5.8 S* formation precedes that of 5.8 S rRNA in rrp6::URA3 cells and 5.8 S* levels fall as 5.8 S levels begin to rise. These findings suggest that 5.8 S* rRNA is an intermediate in the major pathway leading to the production of mature 5.8 S rRNA.


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Fig. 7.   Pulse-chase analysis of small RNA synthesis in RRP6 and rrp6::URA3 cells. A, autoradiograph of polyacrylamide gel electrophoretic analysis of small RNAs pulse labeled for 3 min with [5,6-3H]uracil and chased for the indicated lengths of time with an excess of unlabeled uracil. The labels to the right of the autoradiograph indicate the identity of each of the major small RNAs labeled. B, graphic representation of the rates of production and loss of the 5.8 S rRNA species monitored in A. The data were collected by cutting out the appropriate bands from the gel displayed in A and counting the radioactivity in a scintillation counter.

Assembly of 5.8 S* rRNA into Active 60 S Ribosomes-- The viability and incomplete loss of 5.8 S rRNA 3' end formation displayed by the rrp6::URA3 disruptant could result from partial Rrp6p function or from activity produced from a duplicate copy of Rrp6p. However, a search of the open reading frames in the S. cerevisiae genome data base revealed no significant matches to the Rrp6p amino acid sequence other than Rrp6p itself. Partial Rrp6p activity from the rrp6::URA3 disruptant seems unlikely since it would result from a polypeptide with only 39 amino acids of Rrp6p. Finally, rrp6::URA3 cells grow surprisingly well considering that they produce mature 5.8 S rRNA 20-fold more slowly than normal cells. This led us to consider whether 5.8 S* rRNA may function in ribosomes, thereby accounting for the viability of rrp6::URA3 cells. Accordingly, we assessed the ability of 5.8 S* to assemble into 60 S ribosomal subunits and polyribosomes. Total RNA samples prepared from fractions of rrp6-1 and rrp6-1,YCpRRP6 polyribosomal gradients were analyzed by Northern blotting. Hybridization of o5.8S to samples derived from rrp6-1 cells revealed a distribution of 5.8 S* rRNA across the polyribosomal gradient identical to that for normal 5.8 S rRNA (Fig. 8). On the other hand, 7 S pre-5.8 S rRNA assembles into 60 S ribosomes, but does not form polyribosomes, consistent with previous findings (28). These results suggest that some portion of 5.8 S* rRNA assembles into active 60 S subunits and may account in part for the ability of rrp6- strains to survive despite slow production of mature 5.8 S rRNA.


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Fig. 8.   Polyribosome analysis of extracts from rrp6-1 strains carrying the complementing plasmid YCpRRP6 or the control plasmid YCplac33. A, Northern blot analysis of the distribution of 5.8 S rRNA forms on polyribosomal gradients. RNAs were prepared from the rrp6-1 polyribosomal fractionations depicted in B, separated by denaturing polyacrylamide gel electrophoresis, and transferred to a membrane. The position and amount of 5.8 S rRNA were determined by Northern blotting with radiolabeled o5.8S and subsequent storage PhosphorImager analysis. B, polyribosome gradient tracings from rrp6-1 strains. Polyribosomal extracts were prepared from the indicated strains and fractionated over 15-50% sucrose density gradients for UV analysis (the orientation of the respective profiles is from right to left). The positions of the 80 S monosomes, 60 S and 40 S ribosomal subunits, and halfmer polyribosomes are indicated.

The fact that rrp6 cells produce 5.8 S* 20-fold faster than 5.8 S rRNA, yet accumulate similar amounts of the two molecules at steady state, indicates the loss of a significant fraction the 5.8 S* rRNA synthesized. The appearance of equal amounts of 5.8 S* and 5.8 S rRNA in all polyribosome and ribosomal subunit fractions suggests that 5.8 S* rRNA may be lost prior to, or during, ribosomal subunit assembly. Indeed, absorbance profiles from these gradients reveal a decrease in the amount of 80 S and free 60 S ribosomal subunits, consistent with a defect in 60 S assembly (Fig. 8B).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The experiments described here provide evidence that RRP6 encodes a protein essential for efficient 3' end formation of 5.8 S rRNA. Recessive mutations in this gene cause a block in 5.8 S rRNA processing, resulting in the accumulation of 5.8 S molecules with normal 5' ends but with ~30-nucleotide extensions at their 3' ends. The homologies among Rrp6p, the human nucleolar PM-Scl 100 kDa autoantigen, and the E. coli 3'-5' exoribonuclease RNase D suggest that RRP6 may encode a nuclear and/or nucleolar exoribonuclease that plays a direct role in 5.8 S 3' end formation. Moreover, RNase D shows greater similarity to Rrp6p, PM-Scl 100 kDa, and predicted proteins from C. elegans and S. pombe than it does to any of the other four E. coli exoribonucleases, suggesting conservation of a specific RNase D function from bacteria to humans.

RNase D exhibits a non-processive 3'-5' exoribonuclease activity in vitro and may play a role in 3' end processing of tRNAs in E. coli (29, 30). Considering the similarity between Rrp6p and RNase D, we looked in rrp6 mutants for evidence of defects in tRNA processing, but observed no changes in the pattern of tRNATyr nor tRNALeu3 precursors (data not shown). These results indicate that rrp6 defects differ from those caused by a mutation in the RNA component of RNase P, which results in the accumulation of a similarly 3' extended form of 5.8 S rRNA, but which also causes defects in tRNA processing (31).

Loss of Rrp6p activity causes a 20-fold decrease in the rate of production of mature 5.8 S rRNA (Fig. 7B), suggesting that it plays an important role in the major pathway of 5.8 S rRNA 3' end processing. Recently, Mitchell et al. (8) identified and characterized a complex named the exosome, containing three or possibly four 3'-5' riboexonucleases required for efficient 5.8 S 3' end processing activity (8). Rrp6p is distinct from the proteins found in the exosome, yet our findings suggest that full 5.8 S rRNA processing activity by the exosome may require Rrp6p activity. Whether Rrp6p exists in a complex distinct from the exosome, or is a weakly associated subunit remains to be determined. Each of the exosomal riboexonucleases are required for cell growth, while Rrp6p is only required for growth at elevated temperature. The viability of rrp6 strains may result from the accumulation of 5.8 S* and its apparent ability to assemble into functional 60 S subunits.

Rrp6p shows strong similarity to PM-Scl 100 kDa, a protein to which autoantibodies are found in patients suffering from polymyositis, scleroderma, and an overlap of these two diseases (18, 32, 33). Immunofluorescence studies using autoantibodies to PM-Scl 100 kDa indicated that the human protein resides mostly in the nucleolus, with the remainder spread throughout the rest of the nucleus (34, 35). Immunoprecipitation experiments indicated that PM-Scl 100 kDa exists in large complexes containing 11-16 other proteins, but no small RNAs (35, 36). The cold-sensitive phenotype of rrp6-1 cells, as well as its semidominance over RRP6 is consistent with function of Rrp6p in a multisubunit complex. Whether Rrp6p acts alone, in concert with the exosome, or as part of a separate complex is currently under investigation.

We embarked on these studies in an effort to shed light on poly(A) function in yeast. The observation that mutations producing specialized ribosomes can suppress a polyadenylation defect is reminiscent of the effects of ski and mak mutations on the stability and translation of poly(A)- killer RNAs, as well as the ability of dcp1, mrt, xrn1 and spb mutations to suppress poly(A)-binding protein defects (37-42). However, we have found that loss of function mutations in XRN1, as well as in genes such as SPB2(RPL46) and RPL16B that increase the ratio of 40 S to 60 S ribosomes, do not suppress pap1-1 (43).4 Instead, our unpublished results suggest that RRP6 may act at an early step in mRNA biogenesis by limiting the levels of slowly, or improperly processed precursor mRNAs.

    ACKNOWLEDGEMENTS

We thank Lasse Lindahl for providing plasmids and for helpful discussions, and Terry Platt and the members of our laboratory for comments on the manuscript.

    FOOTNOTES

* This work was supported by United States Public Health Service Predoctoral Training Grant 5-T32-AI070362 (to M. W. B.) and National Science Foundation Grants MCB-931664 and MCB-9603893 (to J. S. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Z74909.

Dagger To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Box 672, Rochester, NY 14618. Tel.: 716-275-7921; Fax: 716-473-9573; E-mail: btlr{at}uhura.cc.rochester.edu.

1 The abbreviations used are: ITS, internal transcribed spacer; PCR, polymerase chain reaction.

2 The BLAST program is available via the World Wide Web (http://www.ncbi.nlm.nih.gov/Recipon/bs_seq.html).

3 The Stanford Genome Resource Data Base is available via the World Wide Web (http://genome-www.stanford.edu).

4 M. W. Briggs, K. T. D. Burkard, and J. S. Butler, unpublished results.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
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