A Polymerase Switch in the Synthesis of rRNA in <i>Saccharomyces cerevisiae</i>

Conrad-Webb, Heather; Butow, Ronald A.

doi:10.1128/mcb.15.5.2420

Cited by 78 publications

(49 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…There is a significant reduction of rhis4s mRNA detected in the rpa135⌬ strain (lanes 3 and 4), suggesting that at least the majority of rhis4s transcription is promoted by pol I. The remaining rhis4s transcript is most likely a result of transcription from an overlapping pol II promoter in the rDNA pol I promoter region as previously reported (9). The rate of rhis4 transcription is higher than that of HIS4.…”

Section: Vol 18 1998 Functions Of the 5ј Cap In S Cerevisiaementioning

confidence: 57%

RNA Polymerase I-Promoted HIS4 Expression Yields Uncapped, Polyadenylated mRNA That Is Unstable and Inefficiently Translated in Saccharomyces cerevisiae

Huang

Donahue

1998

Molecular and Cellular Biology

View full text Add to dashboard Cite

The HIS4 gene in Saccharomyces cerevisiae was put under the transcriptional control of RNA polymerase I to determine the in vivo consequences on mRNA processing and gene expression. This gene, referred to as rhis4, was substituted for the normal HIS4 gene on chromosome III. The rhis4 gene transcribes two mRNAs, of which each initiates at the polymerase (pol) I transcription initiation site. One transcript, rhis4s, is similar in size to the wild-type HIS4 mRNA. Its 3 end maps to the HIS4 3 noncoding region, and it is polyadenylated. The second transcript, rhis4l, is bicistronic. It encodes the HIS4 coding region and a second open reading frame, YCL184, that is located downstream of the HIS4 gene and is predicted to be transcribed in the same direction as HIS4 on chromosome III. The 3 end of rhis4l maps to the predicted 3 end of the YCL184 gene and is also polyadenylated. Based on in vivo labeling experiments, the rhis4 gene appears to be more actively transcribed than the wild-type HIS4 gene despite the near equivalence of the steady-state levels of mRNAs produced from each gene. This finding indicated that rhis4 mRNAs are rapidly degraded, presumably due to the lack of a cap structure at the 5 end of the mRNA. Consistent with this interpretation, a mutant form of XRN1, which encodes a 5-3 exonuclease, was identified as an extragenic suppressor that increases the half-life of rhis4 mRNA, leading to a 10-fold increase in steady-state mRNA levels compared to the wild-type HIS4 mRNA level. This increase is dependent on pol I transcription. Immunoprecipitation by anticap antiserum suggests that the majority of rhis4 mRNA produced is capless. In addition, we quantitated the level of His4 protein in a rhis4 xrn1⌬ genetic background. This analysis indicates that capless mRNA is translated at less than 10% of the level of translation of capped HIS4 mRNA. Our data indicate that polyadenylation of mRNA in yeast occurs despite HIS4 being transcribed by RNA polymerase I, and the 5 cap confers stability to mRNA and affords the ability of mRNA to be translated efficiently in vivo.RNA transcribed by RNA polymerase (pol) II undergoes a number of covalent modifications before being exported to the cytoplasm as mature mRNA and subsequently translated. Two such modifications, capping at the 5Ј end and polyadenylation at the 3Ј end of mRNA, are believed to be limited to the RNA pol II transcriptional machinery. The addition of the unique cap structure to the 5Ј end of all mature eukaryotic mRNAs is tightly coupled to RNA pol II transcription as the cap can be detected when the 5Ј end of mRNA emerges from the pol II transcriptional machinery (22,32,49). It has been shown that the cap structure is important for RNA transport, pre-RNA splicing, and mRNA stability (16,23,30,40).One of the best-understood functions of the cap structure is its role in translation initiation. According to the ribosomal scanning model (34), the eukaryotic initiation factor eIF-4F complex is required for the binding of the ribosomal preinitiation complex to ...

show abstract

Section: Vol 18 1998 Functions Of the 5ј Cap In S Cerevisiaementioning

confidence: 57%

RNA Polymerase I-Promoted HIS4 Expression Yields Uncapped, Polyadenylated mRNA That Is Unstable and Inefficiently Translated in Saccharomyces cerevisiae

Huang

Donahue

1998

Molecular and Cellular Biology

View full text Add to dashboard Cite

show abstract

“…Although its expression was shown to be responsive to Sir2, we did not observe high levels of K4-trimethylated H3 over the TAR1 ORF. Previous studies have characterized mutants that lack subunits of Pol I and survive by transcribing the 35S rRNA genes with Pol II (Conrad-Webb and Butow, 1995;Vu et al, 1999). However, in RNA from sir2⌬ cells, Pol II-derived 35S rRNA transcripts have not been detected (Oakes et al, 1999(Oakes et al, , 2006.…”

Section: Other Pol II Transcription Units In the Rdnamentioning

confidence: 87%

Sir2 Represses Endogenous Polymerase II Transcription Units in the Ribosomal DNA Nontranscribed Spacer

Mueller

Bryk

2006

MBoC

101

View full text Add to dashboard Cite

Silencing at the rDNA, HM loci, and telomeres in Saccharomyces cerevisiae requires histone-modifying enzymes to create chromatin domains that are refractory to recombination and RNA polymerase II transcription machineries. To explore how the silencing factor Sir2 regulates the composition and function of chromatin at the rDNA, the association of histones and RNA polymerase II with the rDNA was measured by chromatin immunoprecipitation. We found that Sir2 regulates not only the levels of K4-methylated histone H3 at the rDNA but also the levels of total histone H3 and RNA polymerase II. Furthermore, our results demonstrate that the ability of Sir2 to limit methylated histones at the rDNA requires its deacetylase activity. In sir2⌬ cells, high levels of K4-trimethylated H3 at the rDNA nontranscribed spacer are associated with the expression of transcription units in the nontranscribed spacer by RNA polymerase II and with previously undetected alterations in chromatin structure. Together, these data suggest a model where the deacetylase activity of Sir2 prevents euchromatinization of the rDNA and silences naturally occurring intergenic transcription units whose expression has been associated with disruption of cohesion complexes and repeat amplification at the rDNA. INTRODUCTIONModified histones at silent genomic domains contribute to a chromatin environment that is refractory to gene expression and genetic recombination (reviewed in Strahl and Allis, 2000;Turner, 2000;Jenuwein and Allis, 2001). In Saccharomyces cerevisiae, chromatin at the homothallic mating-type loci HML and HMR, telomeres, and the ribosomal DNA locus (rDNA) silences genetic recombination and expression of native and ectopic genes transcribed by RNA polymerase (Pol) II. Silencing at the HM loci and telomeres has been studied extensively, whereas the mechanisms of Pol II silencing at the rDNA are not well characterized (reviewed in Moazed, 2001;Rusche et al., 2003). Increasing our understanding of the factors and mechanisms that regulate silencing at the rDNA will provide insight into the pathways that regulate gene expression and genome stability.In S. cerevisiae, the rDNA contains ϳ150 -200 tandem copies of a 9.1-kilobase (kb) repeat, with each repeat containing a Pol I-transcribed 35S rRNA gene and a nontranscribed spacer (NTS) that is subdivided into NTS1 and NTS2 by the Pol III-transcribed 5S rRNA gene (reviewed in Warner, 1999; Figure 1). Despite high levels of transcription by Pol I and Pol III in the rDNA locus, Pol II-transcribed genes integrated into the rDNA are silenced (referred to as rDNA silencing) (Bryk et al., 1997;Fritze et al., 1997;Smith and Boeke, 1997). Additionally, silent chromatin at the rDNA is essential for repression of genetic recombination (Gottlieb and Esposito, 1989;Davis et al., 2000;Kobayashi et al., 2004) and extension of replicative life span (reviewed in Guarente, 2000).Chromatin-associated proteins and modified histones regulate silencing of Pol II transcription at the rDNA (Bryk et al., 1997;Fritze et al., 199...

show abstract

“…This finding, together with the fact that transcription is initiated in the DNA fragment which contains the RNA polymerase I core promoter, suggests that the ribosomal transgene ends up in a nucleoluslike environment. However, in the simple eukaryote, S. cerevisiae, extrachromosomal copies of rDNA, or rRNA gene copies equiped with an RNA polymerase II promoter, do not form a proper nucleolus (Conrad-Webb and Butow, 1995;Oakes et aL, 1993). It was therefore of interest to determine whether transferred DNA can be located within the nucleus of a transformed A. thaliana cell.…”

Section: Nucleolar Environment Of Rdna Transgenesmentioning

confidence: 99%

Ribosomal transcription units integrated via T‐DNA transformation associate with the nucleolus and do not require upstream repeat sequences for activity in Arabidopsis thaliana

et al. 1997

View full text Add to dashboard Cite

SummaryRibosomal repeat units of Arabidopsis thaliana were introduced into the A. thaliana genome via Agrobacteriurnmediated transformation. Ribosomal transgenes integrated into chromosomal regions outside the nucleolus organizers. Cytological data suggest that the transgenes associate with a nucleolus. To allow detection of transgenic rRNA, a short extension was inserted into the V1 variable region of the 25S ribosomal gene. The RNA transcript from the transgene undergoes a series of maturation steps, including correct processing of the 5' end of 25S rRNA. Using primer extension analysis, expression of a complete rDNA repeat unit was compared with the activity of a repeat unit lacking a sequence called "upstream Sal repeats'. No qualitative or quantitative differences were detected, suggesting that upstream repeat sequences of the rDNA intergenic region do not act as transcriptional enhancers for RNA polymerase I in A. thaliana.

show abstract

A Polymerase Switch in the Synthesis of rRNA in Saccharomyces cerevisiae

Cited by 78 publications

References 56 publications

RNA Polymerase I-Promoted HIS4 Expression Yields Uncapped, Polyadenylated mRNA That Is Unstable and Inefficiently Translated in Saccharomyces cerevisiae

RNA Polymerase I-Promoted HIS4 Expression Yields Uncapped, Polyadenylated mRNA That Is Unstable and Inefficiently Translated in Saccharomyces cerevisiae

Sir2 Represses Endogenous Polymerase II Transcription Units in the Ribosomal DNA Nontranscribed Spacer

Ribosomal transcription units integrated via T‐DNA transformation associate with the nucleolus and do not require upstream repeat sequences for activity in Arabidopsis thaliana

Contact Info

Product

Resources

About