The 5′ end of yeast 5.8S rRNA is generated by exonucleases from an upstream cleavage site.

Abstract

We have developed techniques for the detailed analysis of cis-acting sequences in the pre-rRNA of Saccharomyces cerevisiae and used these to study the processing of internal transcribed spacer 1 (ITS1) leading to the synthesis of 5.8S rRNA. As is the case for many eukaryotes, the 5' end of yeast 5.8S rRNA is heterogeneous; we designate the major, short form 5.8S(S), and the minor form (which is seven or eight nucleotides longer) 5.8S(L). These RNAs do not have a precursor/product relationship, but result from the use of alternative processing pathways. In the major pathway, a previously unidentified processing site in ITS1, designated A3, is cleaved. A 10 nucleotide deletion at site A3 strongly inhibits processing of A3 and the synthesis of 5.8S(S); processing is predominantly transferred to the alternative 5.8S(L) pathway. Site A3 lies 76 nucleotides 5' to the end of 5.8S(S), and acts as an entry site for 5'-3' exonuclease digestion which generates the 5' end of 5.8S(S). This pathway is inhibited in strains mutant for XRNlp and RATlp. Both of these proteins have been reported to have 5'-3' exonuclease activity in vitro. Formation of 5.8S(L) is increased by mutations at A3 in cis or in RATlp and XRNlp in trans, and is kinetically faster than 5.8S(S) synthesis.

Two mutant forms of the S1/TPR-containing protein Rrp5p affect the 18S rRNA synthesis in Saccharomyces cerevisiae

“…Effect of the bipartite allele on pre-rRNA processing+ Strains BMA41 (WT [lanes 1 to 3]) and YCJL7 (RRP5-N2 ϩ RRP5-C1 [lanes 4 to 6]) were grown in glucose-rich medium at 30 8C (lanes 1 and 4) or shifted at 37 8C during 8 h or 16 h (lanes 2 and 5 or 3 and 6, respectively)+ Total RNA was extracted and subjected to Northern analyses+ About 10 mg of total RNAs were loaded in each lane+ The panels represent consecutive hybridizations of the same membrane with the different probes drawn in Figure 1A+ Fig+ 4B,C, lanes 4 to 6) although 32S accumulation appears nearly equivalent at both temperatures (Fig+ 4B-F, lanes 4 to 6)+ This could either suggest that the direct cleavage of the 32S pre-rRNA at site A 3 might be a temperature-sensitive event or that the 21S species is unstable at 37 8C+ We favor the latter hypothesis since the direct cleavage of 32S species at site A 3 also produces the 27SA 3 species (Fig+ 1C) whose specific derivative product 5+8S S rRNA accumulates normally (Fig+ 5A; see below)+ The 21S pre-rRNA was first reported as accumulating in a yeast strain deleted for the snoRNA snR10 (Tollervey, 1987;see Fig+ 1B) and was also detected in three rrp5 mutant strains, but not in the Rrp5p-depleted strain (Venema & Tollervey, 1996)+ The fact that the RRP5 bipartite-allele-expressing strain does not contain detectable 20S precursor but does contain mature 18S rRNA strongly suggests that the aberrant 21S species can be used as a precursor for 18S synthesis and that this 21S pre-rRNA can be cleaved at site D (Fig+ 1C)+ There is a modest accumulation of the 35S rRNA in the mutant YCJL7 strain (Fig+ 4B-G) indicating that the early cleavages at sites A 0 and A 1 are weakly affected+ Nevertheless, the excised A 0 A 1 fragment accumulates at a wild-type level as shown in Figure 5B, thus indicating that the cleavages occur in the YCJL7 strain+ Furthermore, hybridization with probe in 59 ETS failed to reveal any accumulation of 33S pre-rRNA species (Fig+ 4G)+ While the 27SB pre-rRNAs were found to be strongly reduced in a Rrp5p-depleted strain (Venema & Tollervey, 1996), these species accumulate near a wild-type level in the bipartite-allele-containing strain (Fig+ 4E,F)+ Consistently, the mature 25S and 5+8S derivative products were normally detected in that strain (Fig+ 4A and Fig+ 5A)+ The apparent lower level of 27SB and 25S rRNAs detected at 37 8C (Fig+ 4A and 4E-F, lanes 5 and 6) is rather because of a lower loading of the corresponding lanes than to a real accumulation defect+ Moreover, the two forms of 5+8S rRNA, the major short form (5+8S S ) and the minor long form (5+8S L ; see Henry et al+, 1994), were both detected at normal levels in YCJL7 strain (Fig+ 5A) and wild-type reported ratios ranging from 1:7 to 1:10 (5+8S L :5+8S S ) were determined by densitometry analyses (Eichler & Craig, 1994;Allmang et al+, 1996) + Venema & Tollervey (1996) have shown that the Rrp5p-depleted strain strongly underaccumulated the major short form 5+8S S rRNA due to an inhibition of the A 3 cleavage in that strain+ No such defect was observed in the YCJL7 strain which is consistent with an efficient cleavage event occurring at site A 3 + We conclude that despite its unusual genetic organization the so-call...…”

“…The early pre-rRNA cleavages at sites A 0 , A 1 , and A 2 are known to be required for the synthesis of 18S rRNA (Venema & Tollervey, 1995) whereas cleavage at site A 3 is required for the normal formation of 5+8S rRNA (Henry et al+, 1994) + Venema & Tollervey (1996) have recently demonstrated that Rrp5p is the first rRNA-processing component required for both snoRNP-dependent cleavages A 0 /A 1 /A 2 and RNase MRP cleavage A 3 (see Fig+ 1B)+ This was shown by genetic depletion of Rrp5p, which results in a synthesis defect of both 18S and 5+8S rRNAs+ We show here that two conditional alleles of RRP5 cause a defect only in 18S rRNA formation without altering the 5+8S rRNA synthesis+ These results indicate that the Rrp5p functions in 18S and 5+8S rRNA-processing can be separated+ It also suggest that our modified forms of Rrp5p could be affected for relationship with the snoRNP complex while putative functions within the RNase MRP complex could take place normally+…”

Functional equivalence of hairpins in the RNA subunits of RNase MRP and RNase P in Saccharomyces cerevisiae

“…RNase MRP (mitochondrial RNA processing) was first identified as a ribonucleoprotein enzyme capable of in vitro formation of primers for mitochondrial DNA replication (Chang & Clayton, 1987;Schmitt & Clayton, 1992;)+ However, most of the enzyme is localized to the nucleolus (Reddy et al+, 1981;Reimer et al+, 1988;Kiss & Filipowicz, 1992), with only minute amounts found in the mitochondria (Topper et al+, 1992;Li et al+, 1994)+ Its cellular location suggested that the primary function of RNase MRP is in ribosome biogenesis+ Indeed, recent genetic evidence has established that RNase MRP is required for normal processing of 5+8S rRNA in Saccharomyces cerevisiae (Schmitt & Clayton, 1993b;Chu et al+, 1994;Lygerou et al+, 1994Lygerou et al+, , 1996+ The 5+8S rRNA of S. cerevisiae exists in short (5+8S S ) and long (5+8S L ) versions, differing by 6-7 nt at their 59 ends (Rubin, 1974;Lindahl et al+, 1992)+ The ratio of 5+8S S :5+8S L in wild-type cells is 5-10:1+ However, a single base change in the RNA subunit of RNase MRP results in a substantially decreased ratio of 5+8S S to 5+8S L , as well as accumulation of a very long version of 5+8S rRNA with 149 bases of the ITS1 sequence attached at the 59 end (Shuai & Warner, 1991;Lindahl et al+, 1992;Chu et al+, 1994)+ These observations together with other experiments (Henry et al+, 1994) have led to the conclusion that RNase MRP is responsible for initiating the processing of the short form of 5+8S rRNA by cleaving the pre-rRNA in the transcribed spacer between the 18S and 5+8S moieties+ RNase MRP is structurally related to RNase P, the endonucleolytic enzyme that creates the mature 59 end of tRNAs+ Both enzymes have one RNA subunit (here called MRP RNA and P RNA) that share secondarystructure features (Forster & Altman, 1990;Schmitt & Clayton, 1993a)+ Furthermore, RNases MRP and P of S. cerevisiae contain eight common protein subunits (Lygerou et al+, 1994;Chu et al+, 1997;Dichtl & Tollervey, 1997;Stolc & Altman, 1997;Chamberlain et al+, 1998;Stolc et al+, 1998); only one protein specific to each of the two nucleases has been identified (Schmitt & Clayton, 1994;…”

The structure of the ITS2-proximal stem is required for pre-rRNA processing in yeast

“…Secondary structure of a portion of the 59 half of the large subunit rRNA+ Shown is the entire sequence of 5+8S rRNA in bold type, with 59 and 39 ends indicated+ The ITS2 proximal stem is indicated and is formed by the base pairing of sequences near the 39 end of 5+8S with those near the 59 end of 25S+ The ITS2 sequence, represented here by dashed lines, is 234 nt in yeast and joins the 39 end of 5+8S and the 59 end of 25S in pre-rRNA+ For a schematic of the predicted ITS2 structure, see Figure 6B+ The sequence of the unique tag (shown in italics; after Musters et al+, 1989) and the structural alterations induced in that stem by the presence of the tag inserted into the KpnI site is shown below+ Structure is modeled after that proposed by Gutell et al+ (1993)+ tional for RNA polymerase I activity+ Transcription of the plasmid-borne rDNA gene is driven by Pol II and is repressed by growth in glucose and induced in response to galactose+ The Pol II transcripts from this construct are processed accurately and efficiently and incorporated into ribosomes which are fully functional (Nogi et al+, 1991a,b;Henry et al+, 1994)+ The yeast strain (NOY504) used in these experiments carries a disruption in the rrn4 (rpa12) gene encoding a Pol I subunit (A12+2) (Nogi et al+, 1993), which renders this strain temperature sensitive for Pol I transcription and cell growth+ Use of this strain has two advantages+ First, processing of plasmid-encoded rRNA transcripts can be studied under conditions of reduced competition with endogenous, chromosomal rDNA transcripts+ Second, the ability of mutated rDNA genes to support processing, ribosome assembly, and translational function can be readily tested by their ability to confer galactose-dependent growth at a restrictive temperature (37 8C) (Nogi et al+, 1991a,b;Henry et al+, 1994)+ A third component of the system used here was a unique; 18-base-tag sequence inserted within the mature 25S coding sequence (Musters et al+, 1989;Beltrame & Tollervey, 1992) for use in Northern analyses and primer extensions+ The sequence of the tag is indicated in Figure 2 and the insertion site is depicted schematically in Figure 3A+ Although it had been shown previously that the tag inserted at this site had no effect on processing or translational function (Musters et al+, 1989), we have analyzed tagged and untagged versions of each of the mutations described below+ The results were identical for the two versions and only the results for tagged constructs are presented below+ This system was used to directly test the proposed requirement for the 5+8S:25S interaction in the ITS2-proximal stem+ This was carried out by constructing single and compensatory double-clustered mutations in the plasmid-encoded yeast rDNA template (pNOY102) as outlined in Figure 3A+ Sequences for each of the mu-FIGURE 3. Mutagenesis of yeast rDNA template+ A: Schematic showing the mutagenesis strategy+ Using PCR directed mutagenesis (as described in Materials and Methods) with yeast rDNA plasmid pNOY102 as the template, four constructs wer...…”

“…Pre-rRNA processing scheme in yeast+ Schematic of the genomic organization of rDNA and processing pathways of the pre-rRNA transcript+ The full-length precursor in yeast, 35S, is processed by alternative coexisting pathways, utilizing a variety of processing sites (denoted by gray letters and thin arrowheads on the precursor, 35S)+ Some of the first cleavages separate 18S rRNA from the 5+8S/25S+ Subsequent cleavages within ITS2 yield mature 5+8S and 25S and are shown by the heavy lines with arrows+ The sequence and structural requirements for these ITS2 processing events are focused upon in this work+ ITS: Internal Transcribed Spacer+ ITS2-proximal stem, a near perfect 15-base-pair helix in Xenopus, is the longest of the 3 base paired interactions between 5+8S and 28S+ This model for U8 snoRNA function and the role of the ITS2-proximal stem is based on four lines of evidence+ First, extensive mutagenesis studies and phylogenetic conservation of regions within the U8 sequence implicate the 59 end of U8 in critical sequence-dependent interactions (Peculis & Steitz, 1994;Peculis, 1997b)+ Second, 29-O-methyl oligoribonucleotides complementary to the 59 end of U8 RNA inhibit processing of the large subunit rRNAs in Xenopus oocytes (Peculis, 1997b)+ This inhibition is sequence specific, does not result in degradation of U8 RNP and can be rescued by subsequent injection of U8 snoRNA+ Thus, inhibition in this oligonucleotide competition experiment is presumably because of interference with base pairing of the 59 end of U8 RNA to its target+ Third, complementarity between the 59 end of U8 RNA and the 59 end of 28S rRNA is phylogenetically conserved, implicating this segment of 28S rRNA as a potential U8 target site (Peculis, 1997b)+ Fourth, 29-O-methyl oligoribonucleotides complementary to the 5+8S or 28S rRNA sequences in the ITS2-proximal stem inhibit processing of the large subunit rRNAs (Peculis, 1997b)+ While this fourth result is consistent with a requirement for the ITS2-proximal stem in processing, a direct test of this key feature of the model was lacking+ To provide such a test, we have taken advantage of the observation that this stable, base paired structure involving 28S and 5+8S rRNA is conserved in mature ribosomes across evolution (see Figs+ 2 and 6A)+ Thus, this interaction also occurs in yeast pre-rRNA, for which a genetic system to analyze mutations affecting rRNA processing is readily available (Nogi et al+, 1991a,b;Henry et al+, 1994)+ The results of our analyses in yeast demonstrate that the structure of the ITS2-proximal stem is essential for processing, whereas the sequence of the stem affects efficiency+…”