The primary transcript of the ribosomal repeating unit in yeast

Klootwijk, J.; jonge, Petrus de; Planta, Rudi J.

doi:10.1093/nar/6.1.27

Cited by 49 publications

(28 citation statements)

References 29 publications

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“…These observations suggest that the mechanism of spacer-sequencedependent stimulation of 35S rRNA synthesis in yeast cells is likely to be similar to the readthrough enhancement mechanism proposed for mice (7,10) and Xenopus species (3,4,24,25). It is unlikely that sequences within the EcoRI-HindIII spacer rDNA fragment represent the gene promoter, since the 35S rRNA precursor appears to possess a 5' triphosphate indicative of a transcriptional initiation event (18,26) and since 35S fusion transcript was synthesized from pribl derivatives that lack the EcoRI-HindIII fragment. A more attractive hypothesis is that sequences within the EcoRI-HindIII fragment mediate termination and reinitiation of readthrough RNA polymerase I and perhaps de novo initiation of transcription by RNA polymeraseI.…”

Section: Constructionmentioning

confidence: 53%

“…The 5' terminus of 35S rRNA has been mapped in both S. cerevisiae (1, 17) and Saccharomyces carlsbengensis (19). RNA sequencing analysis showed that 35S rRNA molecules contain a 5' triphosphate, suggesting that transcription is initiated with the 5'-terminal sequences of 35S rRNA (18,26). Analysis of transcripts synthesized in isolated yeast nuclei in the presence of -y-sulfhydryl nucleoside triphosphates supports this conclusion (19,23).…”

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confidence: 94%

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Sequences within the spacer region of yeast rRNA cistrons that stimulate 35S rRNA synthesis in vivo mediate RNA polymerase I-dependent promoter and terminator activities.

Mestel¹,

Yip²,

Holland³

et al. 1989

Mol. Cell. Biol.

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Sequences within the spacer region of yeast rRNA cistrons stimulate synthesis of the major 35S rRNA precursor in vivo 10-to 30-fold (E. A. Elion and J. R. Warner, Cell 39:663-673, 1984). Spacer sequences that mediate this stimulatory activity are located approximately 2.2 kilobases upstream from sequences that encode the 5' terminus of the 35S rRNA precursor. By utilizing a centromere-containing plasmid carrying a 35S rRNA minigene, a 160-base-pair region of spacer rDNA was identified by deletion mapping that is required for efficient stimulation of 35S rRNA synthesis in vivo. A 22-base-pair sequence, previously shown to support RNA polymerase I-dependent selective initiation of transcription in vitro, was located 15 base pairs upstream from the 3' boundary of the stimulatory region. A 77-base pair region of spacer DNA that mediates transcriptional terminator activity in vivo was identified immediately downstream from the 5' boundary of the stimulatory region. Deletion mutations extending downstream from the 5' boundary of the 160-base-pair stimulatory region simultaneously interfere with terminator activity and stimulation of 35S rRNA synthesis from the minigene. The terminator region supported termination of transcripts initiated by RNA polymerase I in vivo. The organization of sequences that support terminator and promoter activities within the 160-base-pair stimulatory region is similar to the organization of rDNA gene promoters in higher organisms. Possible mechanisms for spacer-sequence-dependent stimulation of yeast 35S rRNA synthesis in vivo are discussed.rRNA genes in the yeast Saccharomyces cerevisiae are tandemly repeated approximately 120 times in one cluster located on chromosome 12 (27). Each cistron contains the information for all of the mature rRNAs. RNA polymerase I is responsible for the synthesis of a 35S rRNA precursor that is processed to form the 18S, 5.8S, and 25S rRNAs. The 5S rRNA gene is located in the spacer region between 35S rRNA transcription units and is transcribed by RNA polymerase III. Synthesis of 35S rRNA has been studied in several laboratories using both in vitro (9,11,19,23,32,33,35, 36) and in vivo (1,5,15,17,19,28) approaches. The 5' terminus of 35S rRNA has been mapped in both S. cerevisiae (1, 17) and Saccharomyces carlsbengensis (19). RNA sequencing analysis showed that 35S rRNA molecules contain a 5' triphosphate, suggesting that transcription is initiated with the 5'-terminal sequences of 35S rRNA (18,26). Analysis of transcripts synthesized in isolated yeast nuclei in the presence of -y-sulfhydryl nucleoside triphosphates supports this conclusion (19,23).We showed previously that cloned yeast rDNA templates support RNA polymerase I-dependent selective initiation of transcription with a whole-cell extract (32). Transcription of this in vitro reaction initiates from a site 2.2 kilobases (kb) upstream from sequences that encode the 5' terminus of 35S rRNA. Deletion mapping studies defined a 22-base-pair rDNA sequence as being both necessary and sufficient for selecti...

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Section: Constructionmentioning

confidence: 53%

mentioning

confidence: 94%

Sequences within the spacer region of yeast rRNA cistrons that stimulate 35S rRNA synthesis in vivo mediate RNA polymerase I-dependent promoter and terminator activities.

Mestel¹,

Yip²,

Holland³

et al. 1989

Mol. Cell. Biol.

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“…When M1 was supported by the L-A cDNA clone, even though the copy number of To test whether this result could be an effect of the SKI system on the processing or secretion of the K1 killer toxin, we tested the effect of the sAi2::HIS3 mutation on toxin production from either of two cDNA clones of M1: pP-T316, in which the preprotoxin gene is controlled by the PGKI promoter (74), or pVT100-U/KT, in which its transcription is driven by the ADHI promoter (68). In each case, there was no detectable effect of the sh2 mutation (Table 4) (38,50) from an rDNA promoter was affected by a shi2::HIS3 mutation (Table 5). We examined six independent shi2::HIS3 disruptants, testing four transformants of each with the rDNA-lacZ plasmid.…”

Section: Downloaded Frommentioning

confidence: 99%

Evidence that the SKI antiviral system of Saccharomyces cerevisiae acts by blocking expression of viral mRNA.

1993

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The SKI2 gene is part of a host system that represses the copy number of the L-A double-stranded RNA (dsRNA) virus and its satellites M and X A system of six yeast chromosomal genes (SK12, -3, -4, -6, -7, and -8) lowers the copy numbers of the L-A, M, X, and L-BC dsRNA viruses and of the ssRNA replicon 20S RNA (3,17,44,54,65 (70), and the ski mutations also suppress mkt mutations (54).The SK13 and SK18 genes have been cloned and characterized. The SK13 product is a 163-kDa nuclear protein (52) with several copies of an amino acid repeat pattern, the TPR repeat, of unknown function (62). The SK18 protein has two copies of a different repeat amino acid sequence pattern first identified in ,B-transducin (45). Deletion mutations of either SK13 or SK18 had no effect on cell growth unless an M replicon was present. In that case, the cells were cold sensitive for growth as discussed above. For this reason, we view the SKI system as a dedicated antiviral system. However, the mechanism by which the SKI proteins interfere with the propagation of the RNA replicons is completely unknown.We report here that Ski2p resembles helicases and nucleolar proteins and present evidence that Ski2p acts by blocking the translation of viral mRNA. MATERIALS AND METHODSStrains and media. The yeast strains used in this study are shown in Table 1. Escherichia coli DHSa was used to propagate plasmid DNA, strain MV1190 and M13 helper phage K07 were used for the isolation of single-stranded plasmid DNA for DNA sequencing, and strain CJ236 was used for the production of uracil-containing single-stranded plasmid DNA for site-directed mutagenesis (40). Yeast strains were grown on YPAD, SD, H-His, H-Trp, H-Ura, or 4.7MB medium (70). E. coli strains were grown on LB, TB, or M9 medium (43).The strains in which M or X is supported by an L-A cDNA clone were constructed as follows: a strain derived from strain 3221 harboring L-A and either M1 or X (produced by cytoduction into strain 3221) was transformed with the L-A 4331

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“…In the yeast Saccharomyces cerevisiae, the largest detectable transcript from the rDNA genes is a 35S pre-rRNA (17), which is rapidly processed into three molecules: the 18S rRNA assembled into 40S ribosomal subunits and the 5.8S and 25S rRNAs found in 60S subunits (Fig. 1).…”

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confidence: 99%

NSR1 is required for pre-rRNA processing and for the proper maintenance of steady-state levels of ribosomal subunits.

Lee¹,

Zabetakis²,

Melese³

1992

Mol. Cell. Biol.

118

104

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NSR1 is a yeast nuclear localization sequence-binding protein showing striking similarity in its domain structure to nucleolin. Cells lacking NSR1 are viable but have a severe growth defect. We show here that NSR1, like nucleolin, is involved in ribosome biogenesis. The nsrl mutant is deficient in pre-rRNA processing such that the initial 35S pre-rRNA processing is blocked and 20S pre-rRNA is nearly absent. The reduced amount of 20S pre-rRNA leads to a shortage of 18S rRNA and is reflected in a change in the distribution of 60S and 40S ribosomal subunits; there is no free pool of 40S subunits, and the free pool of 60S subunits is greatly increased in size. The lack of free 40S subunits or the improper assembly of these subunits causes the nsrl mutant to show sensitivity to the antibiotic paromomycin, which affects protein translation, at concentrations that do not affect the growth of the wild-type strain. Our data support the idea that NSR1 is involved in the proper assembly of pre-rRNA particles, possibly by bringing rRNA and ribosomal proteins together by virtue of its nuclear localization sequence-binding domain and multiple RNA recognition motifs. Alternatively, NSR1 may also act to regulate the nuclear entry of ribosomal proteins required for proper assembly of pre-rRNA particles.In eukaryotic cells, the nucleolus is a specialized subcompartment in which ribosome biogenesis occurs. Pre-rRNA first is transcribed from rDNA genes located in the nucleolus and then undergoes a series of modifications. Ribosomal proteins are imported from the cytoplasm and packed onto the RNA molecule. At the same time, the primary transcript goes through sequential cleavages to generate mature forms of rRNA. Finally, the newly formed ribosomal particles are exported to the cytoplasm.In the yeast Saccharomyces cerevisiae, the largest detectable transcript from the rDNA genes is a 35S pre-rRNA (17), which is rapidly processed into three molecules: the 18S rRNA assembled into 40S ribosomal subunits and the 5.8S and 25S rRNAs found in 60S subunits (Fig. 1). During or immediately after transcription of the rDNA genes, the pre-rRNA is modified, mainly by methylation (32, 34). Meanwhile, a large number of ribosomal proteins and nonribosomal components (including proteins and RNA) associate with the pre-rRNA to form a 90S preribosomal particle. This particle is then split into 66S and 43S preribosomal particles, which are the precursors of the 60S and 40S subunits, respectively (31). While the 66S particles mature completely within the nucleus, the final maturation steps of the 43S particles, including the processing of 20S to 18S rRNA, are completed in the cytoplasm (31,33).Only a few mutations that block specific steps in the pre-rRNA processing pathway have been found. One example is a temperature-sensitive mutation in a gene designated RRPJ that was shown to prevent the 27S-to-25S rRNA cleavage step specifically (1). In another mutant, CLP-8, the efficiency of the processing of 20S to 18S rRNA is greatly reduced, apparently because...

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The primary transcript of the ribosomal repeating unit in yeast

Cited by 49 publications

References 29 publications

Sequences within the spacer region of yeast rRNA cistrons that stimulate 35S rRNA synthesis in vivo mediate RNA polymerase I-dependent promoter and terminator activities.

Sequences within the spacer region of yeast rRNA cistrons that stimulate 35S rRNA synthesis in vivo mediate RNA polymerase I-dependent promoter and terminator activities.

Evidence that the SKI antiviral system of Saccharomyces cerevisiae acts by blocking expression of viral mRNA.

NSR1 is required for pre-rRNA processing and for the proper maintenance of steady-state levels of ribosomal subunits.

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