Efficient translation of poly(A)-deficient mRNAs in Saccharomyces cerevisiae.

Abstract

The polyadenylate tail of eukaryotic mRNAs is thought to influence various metabolic phenomena including mRNA stability^ translation initiation, and nucleo-cytoplasmic transport. We have analyzed the fate of mRNAs following inactivation of poly(A) polymerase in Saccharomyces cerevisiae containing a temperature-sensitive, lethal mutation {papl-1) in the gene for poly (

Replacement of the yeast TRP4 3??? untranslated region by a hammerhead ribozyme results in a stable and efficiently exported mRNA that lacks a poly(A) tail

“…Comparisons of mRNA levels, enzyme assays, and analyses of mRNA association with polyribosomes demonstrated that translation of the TRP4-ribozyme mRNA was reduced approximately 50-75%+ Impaired utilization of this mRNA may well reflect the important role of the poly(A) tail in translation initiation (Munroe & Jacobson, 1990;Tarun & Sachs, 1995;)+ Under most circumstances, it would be expected that a poly(A)-deficient mRNA would initiate poorly in yeast (Gallie, 1991;Proweller & Butler, 1994), an event consistent with the shift of approximately half of the ribozymecontaining mRNA to the nonpolysomal fractions of the cytoplasm (Fig+ 3)+ It is likely that the TRP4-ribozyme construct used here will prove valuable in further analyses of the mechanism of translation of poly(A) Ϫ mRNAs in yeast+ An additional translational regulatory phenomenon uncovered by these studies is suggested by the specific translational activity of the TRP4 ⌬39 UTR mRNA (Fig+ 2)+ Whereas the level of this mRNA is reduced more than threefold, relative to wild-type, PR-transferase activity is reduced less than half+ These results imply that the deletion has removed a TRP4 39 UTR sequence element normally capable of repressing translation of this mRNA+ The existence of such an element is not without precedent (Gray & Wickens, 1998)+ As summarized above, cells expressing the TRP4-ribozyme construct have decreased levels of Trp4p and are starved for tryptophan+ In light of the ability of yeast cells to alter their patterns of gene expression to ensure survival in different environments, it was not surprising that the TRP4-ribozyme construct elicited the activation of the general control system of amino acid biosynthesis+ The partial activation observed undoubtedly reflects the ability of cells harboring these constructs to synthesize modest amounts of tryptophan sufficient to support growth of trp4 cells+…”

Ribosome Concentration Contributes to Discrimination against Poly(A)− mRNA during Translation Initiation in Saccharomyces cerevisiae

“…41). We showed previously that, at 35°C in a pap1-1 background, PAB1 mRNA exists without detectable poly(A) tails, while PGK1 mRNA exists as a mixture of mostly poly(A)-deficient and poly(A) ϩ species as determined by oligo(dT) cellulose selection (31) . Fig.…”

“…Our laboratory recently showed that yeast cells with a conditional mutation (pap1-1) in the poly(A) polymerase gene accumulated poly(A)-deficient and poly(A) Ϫ mRNA following inactivation of the enzyme by cell growth at a nonpermissive temperature (35°C) (31). A 1-h incubation at 35°C reduced the total amount of mRNA approximately 2-fold, consistent with an important role for poly(A) tails in mRNA stability.…”

“…Following 4°C centrifugation at 40,000 rpm (Beckman SW40Ti rotor) for 2.5 h, gradient fractions (20 ϫ 0.6 ml) were collected while recording an A 254 polyribosome profile by continuous flow measurement using an ISCO model UA-5 absorbance/fluorescence monitor. Isolation of total RNA from polyribosome gradient fractions and subsequent ribonuclease protection and Northern analyses were carried out as described previously (31). Note that for each gradient RNA fractions 18, 19, and 20 were pooled and are proportionally represented in fraction 18 in the experiments illustrated here.…”

Initiation of Protein Synthesis in Eukaryotic Cells

“…The effects are often small in in vitro translation systems, but more pronounced differences have been seen in experiments where mRNAs were microinjected into Xenopus oocytes [343] or electroporated into cells 13441 (see, however, [345] which describes rather different behaviour in yeast). In the reticulocyte lysate, a rather small increment in efficiency of recruitment of poly(A)-bearing mRNA was localized to the joining of the 60s ribosomal subunit to the 43s .…”