Claire Bonnerot

The Two Proteins Pat1p (Mrt1p) and Spb8p Interact In Vivo, Are Required for mRNA Decay, and Are Functionally Linked to Pab1p

ABSTRACT We report here the characterization of a bypass suppressor ofpab1Δ 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 thatPAT1 is allelic with mrt1-3, a mutation previously reported to affect decapping and to bypass suppresspab1Δ, 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.

Trm7p catalyses the formation of two 2′‐O‐methylriboses in yeast tRNA anticodon loop

The genome of Saccharomyces cerevisiae encodes three close homologues of the Escherichia coli 2′‐O‐rRNA methyltransferase FtsJ/RrmJ, designated Trm7p, Spb1p and Mrm2p. We present evidence that Trm7p methylates the 2′‐O‐ribose of nucleotides at positions 32 and 34 of the tRNA anticodon loop, both in vivo and in vitro. In a trm7Δ strain, which is viable but grows slowly, translation is impaired, thus indicating that these tRNA modifications could be important for translation efficiency. We discuss the emergence of a family of three 2′‐O‐RNA methyltransferases in Eukaryota and one in Prokaryota from a common ancestor. We propose that each eukaryotic enzyme is located in a different cell compartment, in which it would methylate a different RNA that can adopt a very similar secondary structure.

MRM2 encodes a novel yeast mitochondrial 21S rRNA methyltransferase

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 modified, mitochondrial rRNA contains only three modified nucleotides: a pseudouridine (Ψ2918) and two 2′‐O‐methylated riboses (Gm2270 and Um2791) 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 U2791 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 U2791 of 21S rRNA. Our data suggest that this universally conserved modified nucleotide plays an important function in vivo, possibly by inducing conformational rearrangement of the peptidyl transferase centre.

Changes in permissiveness for the expression of microinjected DNA during the first cleavages of mouse embryos

LacZ DNA and LacZ RNA were microinjected during the first cleavages of embryos. LacZ DNA was not expressed before 18-19 h post insemination (hpi) but LacZ RNA was translated. Before 22 hpi LacZ DNA was expressed in the pronuclei of the one-cell embryos and the polypeptides of the minor, but not the major activation period of the genome were synthesized. This suggests a negative control of transcription before 18-19 hpi and demonstrates that its resumption is independent of the first cleavage and of the major activation of the genome. At the time of the minor activation the eggs contain the trans-acting elements to express a variety of genes that they do not express. It may indicate that, the minor and the major activation of the genome are differently controlled.

Functional Redundancy of Spb1p and a snR52-Dependent Mechanism for the 2′-O-Ribose Methylation of a Conserved rRNA Position in Yeast

In yeast, guide snoRNAs have been assigned to 51 of the 55 rRNA ribose methylation sites. LSU-Um2918 is one of the four remaining positions. This residue is highly conserved and located in the peptidyl transferase center of the ribosome. The equivalent position on the E. coli 23S rRNA is methylated by FtsJ/RrmJ which has three yeast homologs: Spb1, involved in biogenesis of LSU; Trm7, a tRNA methyltransferase; and Mrm2, a mitochondrial 21S rRNA methyltransferase. We demonstrate that a point mutation in the Ado-Met binding site of Spb1p affects cell growth but does not abolish methylation of U2918. When this mutation is combined with disruption of snR52 (a snoRNA C/D), cell growth is severely impaired and U2918 is no longer methylated. In vitro, Spb1p is able to methylate U2918 on 60S subunits. Our results reveal the importance of this methylation for which two mechanisms coexist: a site-specific methyltransferase (Spb1p) and a snoRNA-dependent mechanism.

Purification of a NADH-ferricyanide reductase from plant microsomal membranes with a zwitterionic detergent

A microsomal NADH-ferricyanide reductase was purified to homogeneity from potato tubers. A zwitterionic detergent (CHAPS) was used for the extraction of this reductase which is the first to be purified from plant microsomal membranes. The successive steps of purification included an anion exchange column (DEAE-cellulose or DEAE-Trisacryl), a blue-Ultrogel affinity column and a gel filtration on Sephadex G75. The purification factor was 280 and the yield was 1.6%. The protein has an apparent molecular weight of 44,000 +/- 1,000 as estimated from SDS-PAGE. This successful purification opens new perspectives in the study of oleate desaturase of higher plants which is assumed to contain NADH-ferricyanide reductase as an essential component.

Expression of microinjected DNA and RNA in early rabbit embryos: Changes in permissiveness for expression and transcriptional selectivity

Gene expression in rabbit early development was investigated by microinjecting LacZ DNA and LacZ RNA in 1-cell and 2-cell embryos. Expression of LacZ DNA could not be obtained before 30-36 hpf, although synthetic LacZ RNA was translated from 12 hpf at the least. The onset of expression of microinjected DNA correlated with the 8- to 16-cell stage. This suggests that before this stage, there is a general negative control of gene expression. The arrest of in vitro development at the 2- to 8-cell stages did not inhibit LacZ expression, which still occurred at 33 hpf. In addition the inhibition of the first cleavage by nocodazole resulted in LacZ expression in 1-cell embryos. Expression of microinjected DNA thus occurs at a fixed time after fertilization and is independent of cleavages and of the second and subsequent DNA replications. Therefore, the changes in permissiveness for the expression of microinjected DNA in rabbit embryos are reminiscent of those in mouse embryos. Transcriptional selectivity in rabbit embryos was compared to that in early mouse embryos. In both species, Sp1-sensitive promoters were active and the promoter of simian virus 40 did not require far upstream enhancers before late cleavage stages; genes driven by the -447, +563 region of murine leukemia virus were repressed. In rabbit, however, the H-2Kb promoter active in mouse was silent. Altogether, the results illustrate a remarkable conservation of the characteristics of the transcription in early rabbit and mouse embryos and the independence of its resumption from the pattern of cleavage.

Xenotropic and amphotropic pseudotyped recombinant retrovirus to transfer genes into cells from various species

The ability to transfer genes into cells from different species with murine recombinant retroviruses was evaluated with the SVnls LacZ reporter gene. Mouse and cat packaging cell lines can be used to transfer amphotropic pseudotype, in human, mouse, cat, rabbit, sheep, horse and beef cells and with a very low efficiency in pig and avian cells. Xenotropic pseudotype recombinant retroviruses, produced in cat and rabbit packaging cell lines, transferred genes with the same efficiency as amphotropic retroviruses in human, cat, rabbit and sheep cells. In contrast to amphotropic retroviruses, xenotropic retroviruses infect beef, pig and horse cells with a high efficiency. These results emphasize the need to determine carefully the producer cell line (the type of helper virus and the species origin of the cell) for efficient transfer of genes in cells and embryos.