[29] Measurement of mRNA decay rates in Saccharomyces cerevisiae

Parker, Roy; Herrick, David; Peltz, Stuart W.; Jacobson, Allan

doi:10.1016/0076-6879(91)94032-8

Cited by 112 publications

(97 citation statements)

References 6 publications

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“…Routinely, 9 cells were frozen within 15 sec of removal of the culture aliquot. Total yeast RNA was isolated as described previously Parker et al 1991). Equal amounts (usually 10-20 ~g) of total RNA from each time point of an experiment were analyzed by Northern blotting (Thomas 1980).…”

Section: Methodsmentioning

confidence: 99%

“…The numbers at left correspond to the various PGK1 alleles represented in B: (1) the wild-type PGK1 gene; (2) the PCK1 gene with a nonsense mutation at 92.6% of the coding region (inserted at the BgIII site; 385 PGK1 codons translated); {3) the PGKI gene with a nonsense mutation at 76.2% of the coding region (inserted at the XbaI site; 317 PGK1 codons translated); (4) the PGK1 gene with a nonsense mutation at 67.7% of the coding region [inserted at the H2(1) site; 282 PGK1 codons translated[; [5) the PGK1 gene with a nonsense mutation at 55% of the coding region [inserted at the H2{2) site; 229 PGK1 codons translated]; (6) the PGK1 gene with a nonsense mutation at 39% of the coding region (inserted at the Asp718 site; 164 PGK1 codons translated); (7) the PGK1 gene with a nonsense mutation at 5.6% of the coding region [inserted at the H2{3) site; 23 PGK1 codons translated]. The decay assay, RNA blotting, hybridization, and quantitation of the blots were performed as described previously Parker et al 1991). (B) mRNA abundance and decay rates for the wild-type and mutant PGK1 alleles in SWP154( + ) (wild-type for the UPF1 gene) or SWP154( -) (UPFI(-) (a deletion of the UPF1 gene), mRNA abundance was measured by RNA blot analysis of equal amounts of RNA from the time zero points from each of the mRNA decay measurements, normalizing to the abundance of the CYH2 transcript.…”

Section: Destabilization Of the Pgk1 Transcript Is Dependent On The Pmentioning

confidence: 99%

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mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one trans-acting factor.

Peltz¹,

Brown²,

Jacobson³

1993

Genes Dev.

283

349

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Nonsense mutations in a gene can accelerate the decay rate of the mRNA transcribed from that gene, a phenomenon we describe as nonsense-mediated mRNA decay. Using amber (UAG) mutants of the yeast PGK1 gene as a model system, we find that nonsense-mediated mRNA decay is position dependent, that is, nonsense mutations within the initial two-thirds of the PGKl-coding region accelerate the decay rate of the PGK1 transcript ~<12-fold, whereas nonsense mutations within the carboxy-terminal third of the coding region have no effect on mRNA decay. Moreover, we find that this position effect reflects (1) a requirement for sequences 3' to the nonsense mutation that may be necessary for translational reinitiation or pausing, and (2) the presence of an additional sequence that, when translated, inactivates the nonsense-mediated mRNA decay pathway. This stabilizing element is positioned within the coding region such that it constitutes the boundary between nonsense mutations that do or do not affect mRNA decay. Rapid decay of PGK1 nonsense-containing transcripts is also dependent on the status of the UPF1 gene. Regardless of the position of an amber codon in the PGK1 gene, deletion of the UPF1 gene restores wild-type decay rates to nonsense-containing PGK1 transcripts.

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Section: Methodsmentioning

confidence: 99%

Section: Destabilization Of the Pgk1 Transcript Is Dependent On The Pmentioning

confidence: 99%

mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one trans-acting factor.

Peltz¹,

Brown²,

Jacobson³

1993

Genes Dev.

283

349

View full text Add to dashboard Cite

show abstract

“…The strains to be tested were grown overnight at 23°C to log phase. Cell cultures were concentrated fivefold and then shifted to 37°C (43). At the indicated time points 2-ml aliquots of the cultures were quickly collected and frozen in dry ice-ethanol.…”

Section: Strainsmentioning

confidence: 99%

Transcriptional Elements Involved in the Repression of Ribosomal Protein Synthesis

Nierras

Warner

1999

Molecular and Cellular Biology

103

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The ribosomal proteins (RPs) of Saccharomyces cerevisiae are encoded by 137 genes that are among the most transcriptionally active in the genome. These genes are coordinately regulated: a shift up in temperature leads to a rapid, but temporary, decline in RP mRNA levels. A defect in any part of the secretory pathway leads to greatly reduced ribosome synthesis, including the rapid loss of RP mRNA. Here we demonstrate that the loss of RP mRNA is due to the rapid transcriptional silencing of the RP genes, coupled to the naturally short lifetime of their transcripts. The data suggest further that a global inhibition of polymerase II transcription leads to overestimates of the stability of individual mRNAs. The transcription of most RP genes is activated by two Rap1p binding sites, 250 to 400 bp upstream from the initiation of transcription. Rap1p is both an activator and a silencer of transcription. The swapping of promoters between RPL30 and ACT1 or GAL1 demonstrated that the Rap1p binding sites of RPL30 are sufficient to silence the transcription of ACT1 in response to a defect in the secretory pathway. Sir3p and Sir4p, implicated in the Rap1p-mediated repression of silent mating type genes and of telomere-proximal genes, do not influence such silencing of RP genes. Sir2p, implicated in the silencing both of the silent mating type genes and of genes within the ribosomal DNA locus, does not influence the repression of either RP or rRNA genes. Surprisingly, the 180-bp sequence of RPL30 that lies between the Rap1p sites and the transcription initiation site is also sufficient to silence the Gal4p-driven transcription in response to a defect in the secretory pathway, by a mechanism that requires the silencing region of Rap1p. We conclude that for Rap1p to activate the transcription of an RP gene it must bind to upstream sequences; yet for Rap1p to repress the transcription of an RP gene it need not bind to the gene directly. Thus, the cell has evolved a two-pronged approach to effect the rapid extinction of RP synthesis in response to the stress imposed by a heat shock or by a failure of the secretory pathway. Calculations based on recent transcriptome data and on the half-life of the RP mRNAs suggest that in a rapidly growing cell the transcription of RP mRNAs accounts for nearly 50% of the total transcriptional events initiated by RNA polymerase II. Thus, the sudden silencing of the RP genes must have a dramatic effect on the overall transcriptional economy of the cell.The Saccharomyces cerevisiae cell invests enormous resources in the synthesis of ribosomes. In a rapidly growing cell, the 100 or more rRNA genes are responsible for at least 60% of total transcription (59). The 137 ribosomal protein (RP) genes are among the most active in the genome (57). As a result, the cell has evolved mechanisms that tightly control the synthesis of the components of the ribosome. The 78 RPs are synthesized in nearly equimolar amounts that are matched to the synthesis of rRNA. Furthermore, the synthesis of these components is coo...

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“…To determine whether the differences we observed are due to a difference in the splicing rate, we measured the half-lives of the spliced intron RNA and each pre-rRNA after inhibition of GAL transcription by glucose at 30°C (Parker et al 1991). Addition of glucose inhibits the expression of GAL genes within 1-2 min (Ronen and Botstein 2006).…”

Section: Rrna Improves Fidelity Of Foldingmentioning

confidence: 99%

“…The cells were harvested and resuspended in 15 mL of minimal medium with 2% glucose at 30°C to prevent further transcription from the GAL promoter (Parker et al 1991). Aliquots (1 mL) were harvested at various times by spinning in a microcentrifuge for 10 sec at 14,000g.…”

Section: Rna Decay Ratesmentioning

confidence: 99%

Self-splicing of a group I intron reveals partitioning of native and misfolded RNA populations in yeast

Jackson¹,

Koduvayur²,

Woodson³

2006

RNA

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Stable RNAs must form specific three-dimensional structures, yet many RNAs become kinetically trapped in misfolded conformations. To understand the factors that control the accuracy of RNA folding in the cell, the self-splicing activity of the Tetrahymena group I intron was compared in different genetic contexts in budding yeast. The extent of splicing was 98% when the intron was placed in its natural rDNA context, but only 3% when the intron was expressed in an exogenous pre-mRNA. Further experiments showed that the probability of forming the active intron structure depends on local sequence context and transcription by Pol I. Pre-rRNAs decayed at similar rates, whether the intron was wild type or inactivated by an internal deletion, suggesting that most of the unreacted pre-rRNA is incompetent to splice. Northern blots and complementation assays showed that mutations that destabilize the intron tertiary structure inhibited self-splicing and processing of internal transcribed spacer 2. The data are consistent with partitioning of pre-rRNAs into active and inactive populations. The misfolded RNAs are sequestered and degraded without refolding to a significant extent. Thus, the initial fidelity of folding can dictate the intracellular fate of transcripts containing this group I intron.

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[29] Measurement of mRNA decay rates in Saccharomyces cerevisiae

Cited by 112 publications

References 6 publications

mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one trans-acting factor.

mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one trans-acting factor.

Transcriptional Elements Involved in the Repression of Ribosomal Protein Synthesis

Self-splicing of a group I intron reveals partitioning of native and misfolded RNA populations in yeast

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