The relationship between mRNA stability and length in<i>Saccharomyces cenvisiae</i>

Santiago, T.C.; Purvis, Ian J.; Bettany, A. J. E.; Brown, Alistair J. P.

doi:10.1093/nar/14.21.8347

Cited by 109 publications

(77 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, studies of mRNAs with coding region deletions (20,22) and the data of Table 1. individual mRNA and its stability. Our observations are not consistent with the results of similar experiments in Saccharomyces cerevisiae (72), but this discrepancy may be attributable to differences in the respective methods used to measure mRNA half-lives. Santiago et al (72) inhibited transcription with high concentrations of 1,10-phenanthroline, a chelating agent which affects a number of cellular processes (15,32,37) The possibility that poly(A) tail lengths directly determine mRNA stabilities has been suggested by microinjection experiments with adenylated and deadenylated mRNAs (17,29,46,56) and by experiments with cordycepin-treated cells (90).…”

Section: Discussioncontrasting

confidence: 99%

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.

Shapiro¹,

Herrick²,

Manrow³

et al. 1988

Mol. Cell. Biol.

View full text Add to dashboard Cite

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 32P04 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.We have previously demonstrated that the mRNA population in vegetatively growing amoebae of the cellular slime mold Dictyostelium discoideum decays with complex kinetics, consisting of at least two major components: a rapidly decaying component with a half-life of approximately 50 min, and a long-lived component with a half-life of approximately 10 h (14, 58). Similar complex kinetics have been observed for the decay of poly(A)+ RNA in a variety of other eucaryotic cells (5,25,39,57,77,78,81). It is likely that these complex decay kinetics reflect the sum of the decay rates of individual mRNAs (e.g., in D. discoideum, the most stable mRNAs have half-lives of approximately 10 h and the least stable mRNAs have half-lives which are approximately 10-fold shorter). It has been s...

show abstract

Section: Discussioncontrasting

confidence: 99%

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.

Shapiro¹,

Herrick²,

Manrow³

et al. 1988

Mol. Cell. Biol.

View full text Add to dashboard Cite

show abstract

“…The experiments were done in duplicate and the error bars represent the standard deviation between replicates. All samples were normalized using ethidium bromide staining of the 25S rRNA of thiolutin generates estimations of mRNA halflives that are in agreement with those previously known (31-77 min for ACT1 and 11-24 min for RPL24 ; Santiago et al, 1986;Herrick et al, 1990;Wang et al, 2002). However, the addition of higher concentrations of thiolutin leads to the estimation of apparently longer mRNA half-lives in a concentration-dependent manner.…”

Section: Apparent Mrna Decay Rates Depend On the Thiolutin Concentratsupporting

confidence: 75%

The transcriptional inhibitor thiolutin blocks mRNA degradation in yeast

Pelechano

Pérez-Ortín

2007

Yeast

View full text Add to dashboard Cite

Thiolutin is commonly used as a general inhibitor of transcription in yeast. It has been used to calculate mRNA decay rates by stopping the transcription and then determining the relative abundance of individual mRNAs at different times after inhibition. We report here that thiolutin is also an inhibitor of mRNA degradation, and thus its use can lead to miscalculations of mRNA half-lives. The inhibition of mRNA decay seems to affect the mRNA degradation pathway without impeding poly(A) shortening, given that the decay rate of total poly(A) amount is not reduced by thiolutin. Moreover, the thiolutin-dependent inhibition of mRNA degradation has variable effects on different functional groups of genes, suggesting that they use various degradation pathways for their mRNAs.

show abstract

“…However once partial degradation of RNA occurs, it affects distinct genes differentially with 18S being least affected thus confirming previous reports. 22,23 Consequently, genes that exhibit marked resistance to degradation compared to most other genes may not serve as a good endogenous control.…”

Section: Discussionmentioning

confidence: 99%

Optimization of quantitative real-time RT-PCR parameters for the study of lymphoid malignancies

Lossos

Czerwinski

Wechser³

et al. 2003

Leukemia

135

114

View full text Add to dashboard Cite

Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) is a powerful method for measurement of gene expression for diagnostic and prognostic studies of nonHodgkin's lymphomas (NHL). In order for this technique to gain wide applicability, it is critically important to establish a uniform method for normalization of RNA input. In this study, we have determined the best method to quantify the RNA/cDNA input per reaction and searched for the most useful endogenous control genes for normalization of the measurements, based on their abundance and lowest variability between different types of lymphoid cells. To accomplish these aims, we have analyzed the RNA expression of 11 potential endogenous control genes (glyceraldehyde-3-phosphate dehydrogenase, b-actin, peptidylprolyl isomerase A, b2 microglobulin, protein kinase cGMPdependent, type I, hypoxanthine phosphoribosyltransferase 1, TATA box binding protein, transferrin receptor, large ribosomal protein, b-glucoronidase and 18S ribosomal RNA). In all, 12 different B-and T-cell lymphoma/leukemia cell lines, 80 B-and T-cell NHL specimens, and resting and activated normal B and T lymphocytes were screened. Normalization of the nucleic acid input by spectrophotometric OD 260 measurement of RNA proved more reliable than spectrophotometric or fluorometric measurements of cDNA or than electrophoretic estimation of the ribosomal and mRNA fractions. The protein kinase cGMP-dependent, type I (PRKG1) and the TBP genes were expressed at common abundance and exhibited the lowest variability among the cell specimens. We suggest that for further lymphoma studies based on the real-time RT-PCR quantification of gene expression, that RNA input in each reaction be equalized between the specimens by spectrophotometric OD 260 measurements. The expression of the gene of interest in different samples should be normalized by concomitant measurement of the PRKG1 and/or the TBP gene products.

show abstract

The relationship between mRNA stability and length inSaccharomyces cenvisiae

Cited by 109 publications

References 55 publications

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.

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.

The transcriptional inhibitor thiolutin blocks mRNA degradation in yeast

Optimization of quantitative real-time RT-PCR parameters for the study of lymphoid malignancies

Contact Info

Product

Resources

About