scholarly journals Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

10.3791/52240 ◽  
2014 ◽  
Author(s):  
Megan Peccarelli ◽  
Bessie W. Kebaara
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mrna Decay ◽  
Decay Rates

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.

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 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 Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates.

1988 ◽  
Vol 8 (5) ◽  
pp. 1957-1969 ◽  
Author(s):  
R A Shapiro ◽  
D Herrick ◽  
R E Manrow ◽  
D Blinder ◽  
A Jacobson
Keyword(s):  
Steady State ◽  
Dictyostelium Discoideum ◽  
Mrna Decay ◽  
Decay Rates ◽  
Time Dependent ◽  
Translational Efficiency ◽  
Cdna Clones ◽  
Dependent Manner ◽  
Significant Difference ◽  
Kinetics Of

As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32PO4 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.

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