Abstract
Translation and mRNA decay constitute key players in the post-transcriptional control of gene expression. We examine the mechanisms by which the 5'-untranslated region (UTR) of nonaberrant mRNAs acts to modulate both these processes in Saccharomyces cerevisiae. Two classes of functional relationship between ribosome-5'-UTR interactions and mRNA decay are identifiable. In the first of these, elements in the main open reading frame (ORF) dictate how the decay process reacts to inhibitory structures in the 5'-UTR. The same types of stability modulation can be elicited by trans-regulation of translation via inducible binding of the iron-regulatory protein to an iron-responsive element located 9 nucleotides from the 5' cap. A eukaryotic translational repressor can therefore modulate mRNA decay via the 5'-UTR. In contrast, translational regulation mediated via changes in the activity of the cap-binding eukaryotic translation initiation factor eIF-4E bypasses translation-dependent pathways of mRNA degradation. Thus modulation of mRNA stability via the 5'-UTR depends on disruption of the scanning process, rather than changes in translational initiation efficiency per se. In the second class of pathway, an upstream ORF (uORF) functions as a powerful destabilizing element, inducing termination-dependent degradation that is apparently independent of any main ORF determinants but influenced by the efficiencies of ribosomal recognition of the uORF start and stop codons. This latter mechanism provides a regulatable means to modulate the stability of nonaberrant mRNAs via a UPF-dependent pathway.
Highlights
The steady-state abundance of mRNA in the eukaryotic cell is determined by the relative rates of its transcription and degradation. mRNA decay rates are not uniform, but rather vary over at least a 100-fold range [1,2,3,4,5,6], influencing significantly the rates of expression of individual genes
An upstream open reading frame1 in the 5Ј-untranslated region (UTR) of a bacterial cat gene expressed in yeast destabilizes the whole mRNA [25]
In the present paper we focus on the 5Ј-UTR as a mediator of posttranscriptional control for diverse nonaberrant mRNAs in the eukaryotic cell
Summary
Strains and Media—We used the following yeast strains: YPM156B (a ade leu leu112 trp ura rpb1–1), a segregation product from a cross involving RY262 [38]; SWP154 (a trp1-⌬1 upf1::URA3 leu his – ura rpb1–1; 4); SWP154(ϩ) (a trp1-⌬1 upf1::URA3 leu his – ura rpb ϽUPF1 TRP1 CENϾ; 19); JMC 1/2 (a leu ura cdc33::LEU2 trp 1–1 Ͻcdc33⌬196 URA3 CENϾ; 39). Each of these leader sequences was synthesized in the form of an oligodeoxyribonucleotide pair using an Applied Biosystems DNA synthesizer. Half-lives were determined using the strain YPM156B, which has a temperature-sensitive polymerase II. Culture samples were taken at various times subsequent to the temperature shift and used for the preparation of RNA. Glyoxylation of RNA samples, electrophoresis through agarose gels, Northern blotting, membrane hybridization, and autoradiography were performed according to standard procedures [42]. Polysomal Gradient Analysis—Polysomal gradients and subsequent analysis of the fractions were performed as described previously [25]
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