Evolutionarily Conserved Structural Elements are Critical for Processing of Internal Transcribed Spacer 2 fromSaccharomyces cerevisiaePrecursor Ribosomal RNA

Identification of cis-acting elements involved in 3′-end formation of Saccharomyces cerevisiae 18S rRNA

“…In a first series of experiments, we introduced three different sets of clustered point mutations, T, M1, and M2 (Fig+ 2), which alter the upstream and downstream flanking sequences of site D, into the GAL-driven rDNA unit of plasmid pJV12, which carries a neutral tag in the 18S as well as the 25S rRNA sequence (Musters et al+, 1989;Beltrame et al+, 1994)+ We then performed an initial characterization of these mutations by analyzing the growth of YJV100 cells transformed with the mutant rDNA units on glucose-based medium, which allows them to express only the mutant rDNA (Venema et al+, 1995a)+ The results showed that the M1 mutation reduced the growth rate about threefold compared to control cells transformed with wild-type rDNA units, whereas the M2 mutation was lethal (data not shown)+ The T mutation, however, did not have any detectable effect on growth+ This indicates that this mutation, even though it destroys the same set of base pairs as the M2 mutation (Fig+ 2B), is neutral with respect to ribosome biogenesis and can be used as a tag to mark the ITS1 of the mutant rDNA unit+ This conclusion was confirmed by northern analysis of total RNA isolated from the YJV100 transformants 16 h after the shift to glucosebased medium+ Using probe F complementary to the tag in 18S rRNA and probe E complementary to a wild-type sequence in the 59 portion of ITS1 (Fig+ 1B,C) we detected normal levels of both mature 18S rRNA and its 20S precursor in glucose-grown T mutant cells ( Fig+ 3A,B, cf+ lane 3 to lane 2)+ The effect of the various mutations on processing at site D was therefore analyzed using rDNA units carrying the additional tag in the ITS1 sequence+ As shown in Figure 3, the M1 mutation, which alters nt Ϫ2 through Ϫ5 of the mature 18S rRNA sequence relative to site D, has a negative effect on the efficiency of processing: after being shifted to glucose-based medium, TM1 transformants contain a significantly reduced level of mature 18S rRNA, whereas the level of the 20S precursor is clearly increased (lane 4)+ The processing phenotype and growth rate of these transformants is identical to that of cells expressing M1 mutant rDNA units that lack the ITS1 tag (data not shown)+ The M2 mutation, which changes nt Ϫ4 through Ϫ8 of 18S rRNA, and thus overlaps mutation M1 by 2 nt (Fig+ 2B), causes a completely different and rather peculiar phenotype+ TM2 transformants grown on glucose lack detectable amounts of both 18S rRNA and the 20S precursor species (Fig+ 3A,B, lane 5)+ Again, YJV100 cells expressing M2 mutant rDNA units that lack the ITS1 tag show an identical phenotype (data not shown)+ It should be noted that the T and M2 mutations were chosen in such a way that complementarity between the two sequences is maintained (Fig+ 2B)+ Restoration of the base-pairing potential in the upper portion of the proposed hairpin containing site D, therefore, does not reverse the effect of the M2 mutation+ The neutral character of the T mutation, on the other hand, shows that abolishing base pairing, in itself, does not affect production of mature 18S rRNA+ Therefore, we conclude that secondary structure in the upper region of the helix containing site D is not important for fully efficient processing at this site+ Neither is it required for production of functional 40S ribosomal subunits+ Next we analyzed two additional clustered point mutations+ Mutation M3 spans site D, altering nt Ϫ2 through ϩ2, whereas mutation M4 changes the 4 nt of the loop of the hairpin containing site D (Fig+ 2B)+ The first of these two mutations severely inhibits processing+ After having been shifted to glucose, YJV100 cells transformed with the TM3 mutant rDNA units contain a very low...…”

“…In eukaryotes, three of the four rRNAs (18S, 5+8S, and 25/28S rRNA) are encoded by a large number of tandemly repeated rDNA units (in yeast, 150-200 copies)+ These units are transcribed by RNA polymerase I into a single, large precursor RNA that contains external transcribed spacers, the 59-ETS and the 39-ETS, at either end as well as two internal transcribed spacers, ITS1 and ITS2, that separate the mature rRNA sequences (Fig+ 1)+ The transcribed spacers are removed by a series of endonucleolytic cleavages and exonucleolytic degradation steps (Eichler & Craig, 1994;Venema & Tollervey, 1995Raué & Planta, 1995) to produce the mature rRNA species+ So far, the pre-rRNA processing pathway has been studied most extensively in the yeast Saccharomyces cerevisiae (for reviews see Venema & Tollervey, 1995Raué & Planta, 1995)+ In this organism, the first detectable pre-rRNA species containing the 18S, 5+8S, and 25S sequences is the 35S precursor, which has already lost most of the 39-ETS+ Processing of 35S pre-rRNA starts with endonucleolytic cleavages at sites A 0 (within the 59-ETS), A 1 (the 59 end of 18S rRNA), and A 2 (within ITS1) to yield the small subunit 20S pre-rRNA and the large subunit 27SA 2 pre-rRNA (cf+ Fig+ 1B)+ Whereas further processing of the latter into 5+8S and 25S rRNA occurs in the nucleolus, the 20S pre-rRNA is exported as part of a 43S preribosomal subunit to the cytoplasm, where cleavage at site D removes the remaining portion of ITS1 to produce mature 18S rRNA (Udem & Warner, 1973;Trapman & Planta, 1976;Stevens et al+, 1991;Moy & Silver, 1999)+ Accurate and efficient processing of yeast pre-rRNA involves a large number of trans-acting factors as well as specific primary and secondary structural features (cis-acting elements) within the pre-rRNA (reviewed in Van Nues et al+, 1995a;Kressler et al+, 1999;Venema & Tollervey, 1999)+ In vivo mutational analysis has shown that removal of most of the 39-ETS by Rnt1p, the yeast homolog of RNase III, requires a hairpin located within this spacer (Kufel et al+, 1999)+ Production of the 20S and 27SA 2 species by cleavage of the 35S pre-rRNA at sites A 0 , A 1 , and A 2 depends upon a single-stranded region of 10 nt, located in the 59-ETS (Beltrame & Tollervey, 1992Beltrame et al+, 1994), as well as sequences at the 59 end of 18S rRNA (Hughes, 1996;Sharma & Tollervey, 1999), both of which are recognized by the U3 snoRNA+ Cleavage at A 1 is guided by two additional cis-acting signals, a conserved sequence upstream from A 1 in the 59-ETS and the 59-terminal stem-loop/pseudoknot structure in 18S rRNA )+ The 59 region of ITS1 contains several further cis-acting elements involved in cleavage at site A 2 (Lindahl et al+, 1994;…”

A new putative Zygosaccharomyces yeast species isolated from traditional balsamic vinegar

“…Van der Sande et al (1992) and Musters et al (1990) have demonstrated that both ITS regions play a primary role in rRNA processing. Deletions or mutations within ITS2, complete omission of ITS2 or replacement of ITS2 in Saccharomyces cerevisiae and in other yeasts result in failure to produce mature 5.8S and 26S rRNAs (Musters et al,1990;van der Sande et al, 1992;van Nues et al, 1995;Cote and Peculis, 2001). The occurrence of mutations impairing the functional role of some ITS regions could act as a source of pseudogenes in addition to functional rDNA copies.…”

Genetic diversity analysis in South African taro (Colocasia esculenta) accessions using molecular tools

“…This motif conserved in all the accessions is probably due to a functional significance of the region. Van Nues et al (1995) suggested that the nature of the conserved motifs may probably have a function in the regulation of the transcription of active ribosomal subunits as this provides structural elements necessary for the correct pre-rRNA processing. Besides the common motif found in all accessions at positions (48-73), other motifs were identified in some accessions (Figure 3).…”