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 rpbl-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 alters 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 MATal, were unstable (t112 < 7 min) and 4, including those encoded by ACT) and PGKI, were stable (t112 > 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.Differences in the decay rates of individual mRNAs can have profound effects on the overall levels of expression of specific genes (80,93). Although the potential importance of mRNA stability as a mechanism for regulating gene expression has been recognized (7, 86), the structures and mechanisms involved in the determination of individual mRNA decay rates have yet to be elucidated. As an approach to understanding the determinants of mRNA stability, we have begun to compare the properties of mRNAs in Dictyostelium discoideum which differ significantly in their respective decay rates (94). In this report, we describe our initial efforts to perform a similar analysis of mRNAs in the yeast Saccharomyces cerevisiae. Our objective was the identification of both stable and unstable yeast mRNAs that were encoded by genes which had already been well characterized. Success in such an endeavor would make it possible to explore the structural determinants of mRNA stability, for example, by analyzing the decay rates of mRNAs transcribed from chimeric genes (25,42,43,81,96,97).Decay rates for both the poly(A)+ RNA population and for individual yeast mRNAs have been measured previously by several different functional or chemical assays. Half-lives ranging from 16 to 23 min have been measured for the average turnover rate of the poly(A)+ RNA population, whereas half-lives of individual mRNAs span a broader range from 1 to over 100 min (3,18,19,35,36,46,47,50,52,54,...
Polysome-associated c-tnyc mRNA is degraded relatively rapidly in cells and in an in vitro mRNA decay system containing extracts from cultured mammalian cells. Using this system, a competition/screening assay was devised to search for factors that bind to specific regions of polysome-associated c-myc mRNA and thereby alter its half-life. mRNA stability was first assayed in reactions containing exogenous competitor RNAs corresponding to portions of c-myc mRNA itself. The addition of a 182-nucleotide sense strand fragment from the carboxy-terminal portion of the c-myc-coding region destabilized c-myc mRNA by at least eightfold. This RNA fragment had no effect on the stability of other mRNAs tested. Moreover, c-myc mRNA was not destabilized in reactions containing unrelated competitor RNAs or sense strand RNA from the c-myc 5' region. Polysome-associated globin mRNA containing the c-myc-coding region segment in-frame was also destabilized in vitro by the 182-nucleotide RNA. As determined by UV-cross-linking experiments, the 182-nucleotide RNA fragment was recognized by and bound to an ~75-kD polysome-associated protein. On the basis of these data plus Northern blotting analyses of c-myc mRNA decay products, we suggest that the ~75-kD protein is normally bound to a c-myc-coding region determinant and protects that region of the mRNA from endonuclease attack. Possible links between the protective protein, translation, ribosome pausing, and c-myc mRNA turnover are discussed.
IMP-3, a member of the insulin-like growth factor-II (IGF-II
To elucidate the role of specific proinflammatory cytokines in regulating airway responsiveness, we examined the effects and mechanisms of action of IL-1  , TNF-␣ , and IL-2 on the  -adrenoceptor-and postreceptor-coupled transmembrane signaling mechanisms regulating relaxation in isolated rabbit tracheal smooth muscle (TSM) segments. During half-maximal isometric contraction of the tissues with acetylcholine, relaxation responses to isoproterenol, PGE 2 , and forskolin were separately compared in control (untreated) TSM and tissues incubated for 18 h with IL-1  (10 ng/ml), TNF-␣ (100 ng/ml), or IL-2 (200 ng/ml). Relative to controls, IL-1  -and TNF-␣ -treated TSM, but not IL-2-treated tissues, depicted significant attenuation of their maximal relaxation and sensitivity (i.e., Ϫ log dose producing 50% maximal relaxation) to isoproterenol ( P Ͻ 0.001) and PGE 2 ( P Ͻ 0.05); whereas the relaxation responses to direct stimulation of adenylate cyclase with forskolin were similar in the control and cytokine-treated tissues. Further, the attenuated relaxation to isoproterenol and PGE 2 was ablated in the IL-1  -treated TSM that were pretreated with either the muscarinic M 2 -receptor antagonist, methoctramine (10
c-myc mRNA contains at least two discrete sequence elements that account for its short half-life, one in the 3' untranslated region and the other in the carboxy-terminal coding region (coding-region determinant). To investigate the function of each determinant, one or both were fused in frame to portions of a gene encoding long-lived I8-globin mRNA. Each chimeric gene was stably transfected into HeLa and NIH 3T3 cells and was transcribed from a constitutive cytomegalovirus promoter or from a serum-regulated c-fos promoter, respectively. The steady-state levels of the chimeric mRNAs in exponentially growing HeLa cells were compared, and their half-lives were measured by two independent methods: (i) in actinomycin D-treated HeLa cells and (ii) after serum addition to starved 3T3 cells. By each method, mRNAs containing either instability determinant were less stable than P-globin mRNA. mRNA containing only the c-myc 3' untranslated region was not significantly more stable than mRNA with both determinants. In a cell-free mRNA decay system containing polysomes from transfected HeLa cells, mRNA containing the coding-region determinant was destabilized by addition of a specific RNA competitor, whereas mRNA containing only the 3' untranslated region was unaffected. When a stop codon was inserted upstream of the coding-region determinant, the chimeric mRNA was stabilized approximately twofold. These and other data suggest that degradation involving the codingregion determinant occurs most efficiently when ribosomes are translating the determinant.The c-myc proto-oncogene encodes phosphoproteins that influence cell proliferation, differentiation, and neoplastic transformation (reviewed in references 9, 10, 27, and 34). During cell growth and differentiation, c-myc gene expression is regulated both transcriptionally and posttranscriptionally. Depending on cell growth conditions, the mRNA half-life can vary from approximately 30 to 120 min. To map c-myc mRNA instability determinants, investigators have expressed modified c-myc genes in transfected cells and measured the half-lives of the modified mRNAs by various techniques. In some cases, mRNA lacking a particular region was analyzed; in other cases, a chimeric mRNA, in which a segment from c-myc mRNA was inserted within a stable mRNA, was expressed. Both approaches revealed at least two instability determinants, one in the 3' untranslated region (3'-UTR) and the other in the carboxy-terminal portion of the coding region (the codingregion determinant [CRD]). Deleting either segment prolonged the mRNA half-life but only by a factor of 5 or less. The CRD was first detected indirectly through the analysis of 3'-UTR deletions. c-myc mRNA lacking most of its 3'-UTR was more stable than wild-type c-myc mRNA but was still significantly less stable than most other mRNAs. Therefore, the 3'-truncated mRNA retained an instability determinant (1, 4, 17, 22, 24, 27a, 39
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.