Messenger RNA degradation in Saccharomyces cerevisiae

The analysis of 17 functional mRNAs and two recombinant mRNAs in the yeast Saccharomyces cerevisiae suggests that the length of an mRNA influences its half-life in this organism. The mRNAs are clearly divisible into two populations when their lengths and half-lives are compared. Differences in ribosome loading amongst the mRNAs cannot account for this division into relatively stable and unstable populations. Also, specific mRNAs seem to be destabilized to differing extents when their translation is disrupted by N-terminus-proximal stop codons. The analysis of a mutant mRNA, generated by the fusion of the yeast PYK1 and URA3 genes, suggests that a destabilizing element exists within the URA3 sequence. The presence of such elements within relatively unstable mRNAs might account for the division between the yeast mRNA populations. On the basis of these, and other previously published observations, a model is proposed for a general pathway of mRNA degradation in yeast. This model may be relevant to other eukaryotic systems. Also, only a minor extension to the model is required to explain how the stability of some eukaryotic mRNAs might be regulated.     

Messenger RNA guanylyltransferase from Saccharomyces cerevisiae. I. Purification and subunit structure

GTP:mRNA guanylyltransferase, an enzyme that catalyzes the transfer of the GMP moiety from GTP to the 5' end of the RNA to form a cap structure (G(5')pppN-), has been purified to an apparent homogeneity from Saccharomyces cerevisiae. The mRNA 5'-triphosphatase activity hydrolyzing the gamma-phosphoryl group from pppN-RNA was co-purified with mRNA guanylyltransferase activity through column chromatographies on CM-Sephadex and poly(U)-Sepharose, and centrifugation through glycerol gradients, suggesting that these two activities are physically associated. An 820,w value of 7.3, and Mr = 140,000 were estimated from the sedimentation behavior in glycerol gradients. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two major polypeptides, Mr = 45,000 (alpha) and 39,000 (beta), were detected with the purified enzyme preparation. Their molar ratios were close to unity when estimated by the relative density of silver staining. These results suggest that the yeast mRNA-capping enzyme is an oligomeric protein which may consist of two alpha and two beta chains (alpha 2 beta 2).     

Role of poly(A) tail length in Alu retrotransposition

Alu are mobile noncoding Short INterspersed Elements (SINEs) present at a million copies in the human genome. Using marked Alu sequences in an ex vivo assay, we previously showed that they are mobilized through diversion of the LINE (Long INterspersed Elements) retrotransposition machinery, with the poly(A) tail of the Alu being required for their mobility. Here we show that other homopolymeric tracts cannot functionally replace the Alu poly(A) tail, and that the Alu transposition rate varies over a two-log range depending on the poly(A) tail length. Variation is according to a sigmoid-shaped curve with a lag observed for tails shorter than 15 nt and a plateau reached for tails longer than 50 nt, consistent with the binding of a limited number of a protein component requiring multiple contacts for a productive interaction with the poly(A) stretch. This analysis indicates that most of the naturally occurring genomic Alu, owing to their pA tail length, should be poor substrates for the LINE machinery, a feature possibly "selected" for the host sake.     

Factor-free translation of poly(dT) by 80S ribosomes from Saccharomyces cerevisiae

The conditions for preparation of 80S ribosomes from S. cerevisiae are suggested. The ribosomes can bind Phe-tRNAPhe in poly(U)-or poly(dT)-directed manner and are shown to be able to translate poly(dT) in the absence of elongation factor and GTP. Effects of different antibiotics on the factor-free translation have been studied.     

Size of poly (A) +-mRNA in Saccharomyces cerevisiae polysomes.

The size of poly (A) +-mRNA in different classes of yeast polysomes is estimated. The average molecular weight of long-term labelled polysomal poly (A) +-mRNA is about 0,65 x 10(6) daltons. Approximately 60% of the poly (A) +-mRNA polynucleotide chains located at the 5' end, are unprotected by ribosomes and degraded by nucleases upon incubation of cell lysates, to yield a population of poly (A) +-mRNA with an average molecular weight of 0,25 x 10(6) daltons.     

Purification and characterization of poly(A) polymerase from Saccharomyces cerevisiae

Poly(A) polymerase was purified 22,000-fold to homogeneity from a whole cell extract of Saccharomyces cerevisiae with a yield of 22%. The enzyme is a monomeric polypeptide with a denatured molecular weight of 63,000. Incorporation of labeled ATP into acid-precipitable material by the purified enzyme proceeds faster with manganese than with magnesium ions. Various RNA homopolymers as well as Escherichia coli tRNA or rRNA can serve as primers. An RNA that terminates at the natural poly(A) site of the CYC1 gene is not more efficiently elongated than several nonspecific substrates, indicating the requirement for additional factors to provide specificity. Elongation of the primer is distributive. Covering of a poly(A) primer with poly(A)-binding protein reduces the enzyme's activity more than 10-fold.     

Molecular dissection of mRNA poly(A) tail length control in yeast

In eukaryotic cells, newly synthesized mRNAs acquire a poly(A) tail that plays several fundamental roles in export, translation and mRNA decay. In mammals, PABPN1 controls the processivity of polyadenylation and the length of poly(A) tails during de novo synthesis. This regulation is less well-detailed in yeast. We have recently demonstrated that Nab2p is necessary and sufficient for the regulation of polyadenylation and that the Pab1p/PAN complex may act at a later stage in mRNA metabolism. Here, we show that the presence of both Pab1p and Nab2p in reconstituted pre-mRNA 3′-end processing reactions has no stimulating nor inhibitory effect on poly(A) tail regulation. Importantly, the poly(A)-binding proteins are essential to protect the mature mRNA from being subjected to a second round of processing. We have determined which domains of Nab2p are important to control polyadenylation and found that the RGG-box work in conjunction with the two last essential CCCH-type zinc finger domains. Finally, we have tried to delineate the mechanism by which Nab2p performs its regulation function during polyadenylation: it likely forms a complex with poly(A) tails different from a simple linear deposit of proteins as it has been observed with Pab1p.     

Immobilization of bacteria and Saccharomyces cerevisiae in poly(tetrafluoroethylene) membranes.

A novel method for immobilization of bacteria and Saccharomyces cerevisiae cells is described. Microorganisms may be entrapped in a matrix of poly(tetrafluoroethylene) (PTFE) fibrils. Cells are blended with an aqueous emulsion of PTFE stabilized with Triton X-100 surfactant to form a thick paste. The paste is calendered biaxially in a standard rubber mill. This process causes fibrillation of the PTFE and formation of the fibril matrix, which serves only to impart physical integrity to the composite microporous membrane. The cells trapped in the membrane were shown to be viable by incubation of the membrane on solid media and in broth culture. This bioactive membrane represents a new means of immobilization of cells for bioprocessing.