We report here the characterization of a bypass suppressor of pab1⌬ which leads to a fourfold stabilization of the unstable MFA2 mRNA. Cloning of the wild-type gene for that suppressor reveals that it is identical to PAT1 (YCR077c), a gene whose product was reported to interact with Top2p. PAT1 is not an essential gene, but its deletion leads to a thermosensitive phenotype. Further analysis has shown that PAT1 is allelic with mrt1-3, a mutation previously reported to affect decapping and to bypass suppress pab1⌬, as is also the case for dcp1, spb8, and mrt3. Coimmunoprecipitation experiments show that Pat1p is associated with Spb8p. On sucrose gradients, the two proteins cosediment with fractions containing the polysomes. In the absence of Pat1p, however, Spb8p no longer cofractionates with the polysomes, while the removal of Spb8p leads to a sharp decrease in the level of Pat1p. Our results suggest that some of the factors involved in mRNA degradation could be associated with the mRNA that is still being translated, awaiting a specific signal to commit the mRNA to the degradation pathway.A general pathway of mRNA degradation in the yeast Saccharomyces cerevisiae has been described in which mRNAs are deadenylated prior to decapping and then degraded in the 5Ј-to-3Ј direction by the exonuclease Xrn1p (4, 12). The decapping enzyme has been identified in yeast and named Dcp1p (5). In most yeast strains, depletion of Dcp1p leads to a slowgrowth phenotype and to stabilization of mRNAs that are accumulated in a capped and oligoadenylated form (5). However, to become functional, Dcp1p requires the activity of Dcp2p, a putative pyrophosphatase (14). Recently, a polypeptide (M r 70,000) copurifying with Dcp1p has been identified as Ssa1p or Ssa2p, Hsp70 family members (50). Interestingly, two mutations that inhibit decapping, vps16 and mrt1, enhance the interaction of Ssa1p or Ssa2p with Dcp1p. This observation suggests that Ssa1p or Ssa2p could be involved in regulating the activity of Dcp1p.In addition to Dcp1p and Dcp2p, other factors might regulate the decapping activity, since several mutations have been isolated that lead to stabilization of capped, oligoadenylated mRNAs. The best characterized of these factors is Spb8p (6), a protein containing an Sm-like domain that has also been referred to as Lsm1p (40). The Sm motif is found in a set of proteins that interact with the small nuclear RNAs involved in mRNA splicing. However, Spb8p is distinguished from other Sm or Lsm proteins by not being associated with any known small nuclear RNA (29, 38). Mutations within three other loci named mrt1, mrt3, and vps16 lead to an accumulation of capped, oligoadenylated mRNAs (21, 50). While VPS16 has been characterized (22, 50), the genes for mrt1 and mrt3 have not yet been cloned.Several lines of evidence indicate the existence of a link between mRNA translation and degradation (23). It is now well established that the two structures present on the ends of an mRNA, the cap at the 5Ј end and the poly(A) tail at the 3Ј end, act s...

The Escherichia coil lacZ gene has been used as an indicator gene for the study of cell lineage in vivo. To adapt this marker for gene expression studies, a sequence encoding a modified 13-galactosidase and including the simian virus 40 large tumor nuclear location signal (nls-(3-Gal) has been introduced into vectors. In differentiated cells, multipotential cells, and embryos, the constructs led to the expression of an enzymatically active protein. Its location was examined by its P-galactosidase activity or by using antibodies and electron microscopy. The results show that the nls-i8-Gal protein remains mainly located at the nuclear periphery (probably at the nuclear pores) but does not reach the nucleoplasm. It suggests that an interaction with the nuclear membrane is necessary but not sufficient for protein uptake into the nucleus. In multipotential cells, the expression of nuclear location signal LacZ (nls-LacZ) interferes neither with cell growth nor with differentiation. Using various 1acZ constructs, the transcriptional activity of embryos was studied. At the two-cell stage, the promoters of the Rous sarcoma virus, simian virus 40, and the fi-actin gene are functional but the Moloney murine leukemia virus long terminal repeat is not. Thus, transcriptional specificity must already be present at the stage of activation of the embryonic genome.

Mitochondria of the yeast Saccharomyces cerevisiae assemble their ribosomes from ribosomal proteins, encoded by the nuclear genome (with one exception), and rRNAs of 15S and 21S, encoded by the mitochondrial genome. Unlike cytoplasmic rRNA, which is highly modi®ed, mitochondrial rRNA contains only three modi®ed nucleotides: a pseudouridine (Y 2918 ) and two 2¢-O-methylated riboses (Gm 2270 and Um 2791 ) located at the peptidyl transferase centre of 21S rRNA. We demonstrate here that the yeast nuclear genome encodes a mitochondrial protein, named Mrm2, which is required for methylating U 2791 of 21S rRNA, both in vivo and in vitro. Deletion of the MRM2 gene causes thermosensitive respiration and leads to rapid loss of mitochondrial DNA. We propose that Mrm2p belongs to a new class of three eukaryotic RNA-modifying enzymes and is the orthologue of FtsJ/RrmJ, which methylates a nucleotide of the peptidyl transferase centre of Escherichia coli 23S rRNA that is homologous to U 2791 of 21S rRNA. Our data suggest that this universally conserved modi®ed nucleotide plays an important function in vivo, possibly by inducing conformational rearrangement of the peptidyl transferase centre.