GSBS Dissertations and Theses

Approval Date

January 1989

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences

Subjects

Microbiology; RNA, Messenger; Saccharomyces cerevisiae; Academic Dissertations; Dissertations, UMMS

Abstract

Large differences exist in the decay rates of individual mRNAs yet the molecular basis for such differences is substantially unknown. We have developed a procedure for the measurement of individual mRNAs in the yeast Saccharomyces cerevisiae which utilizes northern or dot blotting to quantitate the levels of individual mRNAs after thermal inactivation of RNA polymerase II in an rpb1-1 temperature-sensitive mutant strain (RY260). To assess the reliability of half-life measurements obtained in this manner, we have compared the results of this procedure to results obtained by three other procedures (pulse-chase analysis, approach to steady-state labeling, and inhibition of transcription with thiolutin) and also evaluated whether heat-shock alters mRNA decay rates. We find that: i) for most mRNAs, all four procedures yield comparable relative decay rates and ii) there are no significant differences in the mRNA decay rates measured in heat-shocked or non-heat-shocked cells. Of the 20 mRNAs studied, 11, including those encoded by HIS3, STE2, STE3, and MATα1, were unstable (t1/2<7min) and 4, including those encoded by ACT1 and PGK1, were stable (t1/2> 25 min). We have begun to assess the basis and significance of such differences in the decay rates of these two classes of mRNA. The following parameters have been analyzed to determine their role in mRNA decay: i) mRNA size; ii) poly(A) tail metabolism; iii) translational status; iv) relative content of rare codons; and v) structures and sequences within the 3'-untranslated region (UTR).

To identify the structural determinants responsible for the rapid decay of the unstable HIS3 and STE2 mRNAs, recombinants of their respective genes were constructed and transformed into strain RY260 on centromere-containing vectors, and the half-lives of the resulting chimeric mRNAs were measured in vivo. Chimeric genes were constructed in which the 3'-UTR of ACT1 was replaced with the corresponding region of the unstable HIS3 or STE2 mRNAs. The decay rate of the ACT1-5'-HIS3-3' mRNA was very similiar to that of the stable endogenous ACT1 mRNA, implying that the 3'-end of HIS3 is not sufficient to transfer the instability phenotype of the HIS3 mRNA. The HIS3-5'-ACT1-3' mRNA from the reciprocal construct was unstable, suggesting that HIS3 instability determinants are located within its 5'-UTR or coding sequence. A 411 nucleotide (nt) deletion within the HIS3 coding region (with either the HIS3 or ACT1 3'-UTR) was stabilized 3-fold suggesting this region is necessary for the rapid decay of HIS3 mRNA. Insertion of these 411 nts in-frame into the entire ACT1 gene had no significant effect on the stability of the hybrid mRNA implying that these HIS3 sequences are not sufficient to function on their own and that they may have to interact with HIS3 5'- sequences. The ACT1-5' -STE2-3' hybrid mRNA decayed with an intermediate half-life of 12 min. Furthermore, an 82% deletion of the STE2 coding region increased the half-life by nearly 2-fold. Both results suggest that instability determinants of STE2 mRNA are not restricted to the 3'-UTR. Our overall conclusion is that mRNA stability is not dictated by simple, transferable elements (sequences or structures), but may involve interactions between multiple determinants in the mRNA.

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