Determination of mRNA half-lives inCandida albicansusing thiolutin as a transcription inhibitor

Genome ◽  
2006 ◽  
Vol 49 (8) ◽  
pp. 894-899 ◽  
Author(s):  
Bessie W Kebaara ◽  
Lindsey E Nielsen ◽  
Kenneth W Nickerson ◽  
Audrey L Atkin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Mrna Stability ◽  
18S Rrna ◽  
Mrna Decay ◽  
Defined Medium ◽  
Northern Blotting ◽  
Decay Rates ◽  
Transcription Inhibitor

A method for determining mRNA half-lives in the polymorphic fungus Candida albicans is described. It employs growth in a defined medium, the inhibition of transcription with thiolutin (10–20 µg/mL), and quantitative Northern blotting. The method is effective for the A72, SC5314, and CAI-4 strains of C. albicans, and for mRNAs that have a wide variety of decay rates and steady-state abundances. The range of half-lives detected (from 4–168 min) shows that this method is effective for mRNAs with widely varying half-lives. The mRNA decay rates obtained are compared with those for orthologous mRNAs from Saccharomyces cerevisiae. This procedure should work for a broad range of C. albicans strains and can be adapted to other fungal species.Key words: comparative mRNA stability, ACT1, ADH1, EFG1, PGK1, 18S rRNA, mRNA decay.

scholarly journals Identification and comparison of stable and unstable mRNAs in Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (5) ◽  
pp. 2269-2284 ◽  
Author(s):  
D Herrick ◽  
R Parker ◽  
A Jacobson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Mrna Decay ◽  
Thermal Inactivation ◽  
Decay Rates ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mrna Decay Rate ◽  
Approach To Steady State

We developed a procedure to measure mRNA decay rates in the yeast Saccharomyces cerevisiae and applied it to the determination of half-lives for 20 mRNAs encoded by well-characterized genes. The procedure utilizes Northern (RNA) or dot blotting to quantitate the levels of individual mRNAs after thermal inactivation of RNA polymerase II in an rpb1-1 temperature-sensitive mutant. We compared the results of this procedure with results obtained by two other procedures (approach to steady-state labeling and inhibition of transcription with Thiolutin) and also evaluated whether heat shock alter mRNA decay rates. We found that there are no significant differences in the mRNA decay rates measured in heat-shocked and non-heat-shocked cells and that, for most mRNAs, different procedures yield comparable relative decay rates. Of the 20 mRNAs studied, 11, including those encoded by HIS3, STE2, STE3, and MAT alpha 1, were unstable (t1/2 less than 7 min) and 4, including those encoded by ACT1 and PGK1, were stable (t1/2 greater than 25 min). We have begun to assess the basis and significance of such differences in the decay rates of these two classes of mRNA. Our results indicate that (i) stable and unstable mRNAs do not differ significantly in their poly(A) metabolism; (ii) deadenylation does not destabilize stable mRNAs; (iii) there is no correlation between mRNA decay rate and mRNA size; (iv) the degradation of both stable and unstable mRNAs depends on concomitant translational elongation; and (v) the percentage of rare codons present in most unstable mRNAs is significantly higher than in stable mRNAs.

scholarly journals Identification and comparison of stable and unstable mRNAs in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (5) ◽  
pp. 2269-2284 ◽  
Author(s):  
D Herrick ◽  
R Parker ◽  
A Jacobson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Mrna Decay ◽  
Thermal Inactivation ◽  
Decay Rates ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mrna Decay Rate ◽  
Approach To Steady State

We developed a procedure to measure mRNA decay rates in the yeast Saccharomyces cerevisiae and applied it to the determination of half-lives for 20 mRNAs encoded by well-characterized genes. The procedure utilizes Northern (RNA) or dot blotting to quantitate the levels of individual mRNAs after thermal inactivation of RNA polymerase II in an rpb1-1 temperature-sensitive mutant. We compared the results of this procedure with results obtained by two other procedures (approach to steady-state labeling and inhibition of transcription with Thiolutin) and also evaluated whether heat shock alter mRNA decay rates. We found that there are no significant differences in the mRNA decay rates measured in heat-shocked and non-heat-shocked cells and that, for most mRNAs, different procedures yield comparable relative decay rates. Of the 20 mRNAs studied, 11, including those encoded by HIS3, STE2, STE3, and MAT alpha 1, were unstable (t1/2 less than 7 min) and 4, including those encoded by ACT1 and PGK1, were stable (t1/2 greater than 25 min). We have begun to assess the basis and significance of such differences in the decay rates of these two classes of mRNA. Our results indicate that (i) stable and unstable mRNAs do not differ significantly in their poly(A) metabolism; (ii) deadenylation does not destabilize stable mRNAs; (iii) there is no correlation between mRNA decay rate and mRNA size; (iv) the degradation of both stable and unstable mRNAs depends on concomitant translational elongation; and (v) the percentage of rare codons present in most unstable mRNAs is significantly higher than in stable mRNAs.

scholarly journals Rapid Hypothesis Testing with Candida albicans through Gene Disruption with Short Homology Regions

1999 ◽  
Vol 181 (6) ◽  
pp. 1868-1874 ◽  
Author(s):  
R. Bryce Wilson ◽  
Dana Davis ◽  
Aaron P. Mitchell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Gene Function ◽  
Gene Disruption ◽  
Genomic Sequence ◽  
Selectable Marker ◽  
Genome Project ◽  
Gene Products ◽  
Pcr Products

ABSTRACT Disruption of newly identified genes in the pathogen Candida albicans is a vital step in determination of gene function. Several gene disruption methods described previously employ long regions of homology flanking a selectable marker. Here, we describe disruption of C. albicans genes with PCR products that have 50 to 60 bp of homology to a genomic sequence on each end of a selectable marker. We used the method to disrupt two known genes,ARG5 and ADE2, and two sequences newly identified through the Candida genome project,HRM101 and ENX3. HRM101 and ENX3are homologous to genes in the conserved RIM101 (previously called RIM1) and PacC pathways ofSaccharomyces cerevisiae and Aspergillus nidulans. We show that three independenthrm101/hrm101 mutants and two independentenx3/enx3 mutants are defective in filamentation on Spider medium. These observations argue that HRM101 andENX3 sequences are indeed portions of genes and that the respective gene products have related functions.

scholarly journals A small segment of the MAT alpha 1 transcript promotes mRNA decay in Saccharomyces cerevisiae: a stimulatory role for rare codons.

1993 ◽  
Vol 13 (9) ◽  
pp. 5141-5148 ◽  
Author(s):  
G Caponigro ◽  
D Muhlrad ◽  
R Parker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mrna Decay ◽  
Decay Rates ◽  
Coding Region ◽  
Rapid Decay ◽  
Small Segment ◽  
Rare Codons ◽  
Discrete Sequence ◽  
Chimeric Transcripts ◽  
Sequence Elements

Differences in decay rates of eukaryotic transcripts can be determined by discrete sequence elements within mRNAs. Through the analysis of chimeric transcripts and internal deletions, we have identified a 65-nucleotide segment of the MAT alpha 1 mRNA coding region, termed the MAT alpha 1 instability element, that is sufficient to confer instability to a stable PGK1 reporter transcript and that accelerates turnover of the unstable MAT alpha 1 mRNA. This 65-nucleotide element is composed of two parts, one located within the 5' 33 nucleotides and the second located in the 3' 32 nucleotides. The first part, which can be functionally replaced by sequences containing rare codons, is unable to promote rapid decay by itself but can enhance the action of the 3' 32 nucleotides (positions 234 to 266 in the MAT alpha 1 mRNA) in accelerating turnover. A second portion of the MAT alpha 1 mRNA (nucleotides 265 to 290) is also sufficient to destabilize the PGK1 reporter transcript when positioned 3' of rare codons, suggesting that the 3' half of the MAT alpha 1 instability element is functionally reiterated within the MAT alpha 1 mRNA. The observation that rare codons are part of the 65-nucleotide MAT alpha 1 instability element suggests possible mechanisms through which translation and mRNA decay may be linked.

A small segment of the MAT alpha 1 transcript promotes mRNA decay in Saccharomyces cerevisiae: a stimulatory role for rare codons

1993 ◽  
Vol 13 (9) ◽  
pp. 5141-5148
Author(s):  
G Caponigro ◽  
D Muhlrad ◽  
R Parker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mrna Decay ◽  
Decay Rates ◽  
Coding Region ◽  
Rapid Decay ◽  
Small Segment ◽  
Rare Codons ◽  
Discrete Sequence ◽  
Chimeric Transcripts ◽  
Sequence Elements

Differences in decay rates of eukaryotic transcripts can be determined by discrete sequence elements within mRNAs. Through the analysis of chimeric transcripts and internal deletions, we have identified a 65-nucleotide segment of the MAT alpha 1 mRNA coding region, termed the MAT alpha 1 instability element, that is sufficient to confer instability to a stable PGK1 reporter transcript and that accelerates turnover of the unstable MAT alpha 1 mRNA. This 65-nucleotide element is composed of two parts, one located within the 5' 33 nucleotides and the second located in the 3' 32 nucleotides. The first part, which can be functionally replaced by sequences containing rare codons, is unable to promote rapid decay by itself but can enhance the action of the 3' 32 nucleotides (positions 234 to 266 in the MAT alpha 1 mRNA) in accelerating turnover. A second portion of the MAT alpha 1 mRNA (nucleotides 265 to 290) is also sufficient to destabilize the PGK1 reporter transcript when positioned 3' of rare codons, suggesting that the 3' half of the MAT alpha 1 instability element is functionally reiterated within the MAT alpha 1 mRNA. The observation that rare codons are part of the 65-nucleotide MAT alpha 1 instability element suggests possible mechanisms through which translation and mRNA decay may be linked.

scholarly journals Non-invasive measurement of mRNA decay reveals translation initiation as the major determinant of mRNA stability

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Leon Y Chan ◽  
Christopher F Mugler ◽  
Stephanie Heinrich ◽  
Pascal Vallotton ◽  
Karsten Weis
Keyword(s):  
Translation Initiation ◽  
Mrna Stability ◽  
Mrna Decay ◽  
Decay Rates ◽  
Major Determinant ◽  
Processing Bodies ◽  
Non Invasive ◽  
Tightly Coupled ◽  
Mrna Half Life ◽  
Dose Dependent

The cytoplasmic abundance of mRNAs is strictly controlled through a balance of production and degradation. Whereas the control of mRNA synthesis through transcription has been well characterized, less is known about the regulation of mRNA turnover, and a consensus model explaining the wide variations in mRNA decay rates remains elusive. Here, we combine non-invasive transcriptome-wide mRNA production and stability measurements with selective and acute perturbations to demonstrate that mRNA degradation is tightly coupled to the regulation of translation, and that a competition between translation initiation and mRNA decay -but not codon optimality or elongation- is the major determinant of mRNA stability in yeast. Our refined measurements also reveal a remarkably dynamic transcriptome with an average mRNA half-life of only 4.8 min - much shorter than previously thought. Furthermore, global mRNA destabilization by inhibition of translation initiation induces a dose-dependent formation of processing bodies in which mRNAs can decay over time.

scholarly journals Scavenger Decapping Activity Facilitates 5′ to 3′ mRNA Decay

2005 ◽  
Vol 25 (22) ◽  
pp. 9764-9772 ◽  
Author(s):  
Hudan Liu ◽  
Megerditch Kiledjian
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mrna Stability ◽  
Mrna Decay ◽  
Hydrolytic Activity ◽  
Mrna Degradation ◽  
Mrna Turnover ◽  
Threefold Increase ◽  
Mechanistic Analysis ◽  
Decapping Enzyme ◽  
Mrna Half Life

ABSTRACT mRNA degradation occurs through distinct pathways, one primarily from the 5′ end of the mRNA and the second from the 3′ end. Decay from the 3′ end generates the m7GpppN cap dinucleotide, which is subsequently hydrolyzed to m7Gp and ppN in Saccharomyces cerevisiae by a scavenger decapping activity termed Dcs1p. Although Dcs1p functions in the last step of mRNA turnover, we demonstrate that its activity modulates earlier steps of mRNA decay. Disruption of the DCS1 gene manifests a threefold increase of the TIF51A mRNA half-life. Interestingly, the hydrolytic activity of Dcs1p was essential for the altered mRNA turnover, as Dcs1p, but not a catalytically inactive Dcs1p mutant, complemented the increased mRNA stability. Mechanistic analysis revealed that 5′ to 3′ exoribonucleolytic activity was impeded in the dcs1Δ strain, resulting in the accumulation of uncapped mRNA. These data define a new role for the Dcs1p scavenger decapping enzyme and demonstrate a novel mechanism whereby the final step in the 3′ mRNA decay pathway can influence 5′ to 3′ exoribonucleolytic activity.

scholarly journals Non-invasive measurement of mRNA decay reveals translation initiation as the major determinant of mRNA stability

2017 ◽  
Author(s):  
Leon Y Chan ◽  
Christopher F Mugler ◽  
Stephanie Heinrich ◽  
Pascal Vallotton ◽  
Karsten Weis
Keyword(s):  
Translation Initiation ◽  
Mrna Stability ◽  
Mrna Decay ◽  
Decay Rates ◽  
Major Determinant ◽  
Processing Bodies ◽  
Non Invasive ◽  
Tightly Coupled ◽  
Mrna Half Life ◽  
Dose Dependent

AbstractThe cytoplasmic abundance of mRNAs is strictly controlled through a balance of production and degradation. Whereas the control of mRNA synthesis through transcription has been well characterized, less is known about the regulation of mRNA turnover, and a consensus model explaining the wide variations in mRNA decay rates remains elusive. Here, we combine non-invasive transcriptome-wide mRNA production and stability measurements with selective and acute perturbations to demonstrate that mRNA degradation is tightly coupled to the regulation of translation, and that a competition between translation initiation and mRNA decay -but not codon optimality or elongation- is the major determinant of mRNA stability in yeast. Our refined measurements also reveal a remarkably dynamic transcriptome with an average mRNA half-life of only 4.8 minutes - much shorter than previously thought. Furthermore, global mRNA destabilization by inhibition of translation initiation induces a dose-dependent formation of processing bodies in which mRNAs can decay over time.

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