Chris L. Greer

PRE-TRNA SPLICING : VARIATION ON A THEME OR EXCEPTION TO THE RULE ?

There has been a growing recognition that there are many conserved features among apparently diverse RNA splicing systems, suggesting that they may have a common origin. However, pre-tRNA splicing is an apparent exception in nearly all respects. Features of this unique class should be considered in any comprehensive discussion of the origin(s) of splicing and its implications for the evolution of gene structure.

Location of N2, N2-dimethylguanosine-specific tRNA methyltransferase

Most steps in the maturation of nuclear coded tRNAs occur in the nucleus in eukaryotic cells, but little is known as to the intranuclear location of this RNA maturation pathway. Indirect immunofluorescence experiments using antibody to N2,N2 dimethylguanosine-specific tRNA methyltransferase, a tRNA processing enzyme, and to Nup1p, a nuclear pore protein, show that both locate to the nuclear periphery in wild type cells. Staining of the nuclear membrane is more uniform with anti-Trm1p than the punctate staining observed with antibodies recognizing Nup1p. Biochemical fractionation experiments comparing fractionation of Trm1p with Nup1p, tRNA splicing ligase, and tRNA splicing endonuclease show that Trm1p behaves more like the known peripheral nuclear membrane proteins, Nup1p and tRNA splicing ligase, than like the integral membrane protein, tRNA splicing endonuclease. Cells overproducing Trm1p also concentrate it to the nuclear periphery. Thus, the site(s) of interaction of Trm1p are not easily saturable and are likely to be in excess to Trm1p. Trm1p is shared by mitochondria and the nucleus. Cells transformed with a gene coding Trm1p with a mutant nuclear targeting signal display cytoplasmic staining and an enzyme with increased solubility when compared to the solubility of wild type enzyme. Thus, mutations that prevent the enzyme from entering the nucleus result in an increase in its cytosolic but not mitochondrial concentration suggesting that the mitochondrial/nuclear distribution of Trm1p is not due solely to competition of mitochondrial and nuclear targeting information.

Copy number and stability of yeast 2μ-based plasmids carrying a transcription-conditional centromere

For certain yeast plasmids, the presence of a centromere segment (CEN) enhances mitotic stability and results in low copy number. Transcription from an inducible promoter adjacent to a CEN segment has been shown to alter centromere function. The rate of loss of a conditional CEN-ARS plasmid was examined and the results suggest that segregation control was immediately and effectively inactivated upon shift to inducing conditions. The effect of a conditional centromere on stability and copy number of hybrid CEN3-2 mu plasmids was also examined. When transcription was repressed, copy number was low. Mitotic stability varied and was correlated with the presence of an intact 2 mu recombination system. When transcription was induced, plasmid copy number increased. However, plasmids became highly unstable. These results indicate that while centromere function is affected by transcription from an adjacent promoter the centromere remains incompatible with the 2 mu maintenance system and may retain partial function.

The functional analysis of nonsense suppressors derived from in vitro engineered Saccharomyces cerevisiae tRNA Trp genes

Nonsense suppressors derived from Saccharomyces cerevisiae tRNA(Trp) genes have not been identified by classical genetic screens, although one can construct efficient amber (am) suppressors from them by making the appropriate anticodon mutation in vitro. Herein, a series of in vitro constructed putative suppressor genes was produced to test if pre-tRNA(Trp) processing difficulties could help to explain the lack of classical tRNA(Trp)-based suppressors. It is clear that inefficient processing of introns from precursor tRNA(Trp), or inaccurate overall processing, may explain why some of these constructs fail to promote nonsense suppression in vivo. However, deficient processing must be only one of the reasons why classical tRNA(Trp)-based suppressors have not been characterized, as suppression may still be extremely weak or absent in instances where the in vitro construct can lead to an accumulation of mature tRNA(Trp). Furthermore, suppression is also very weak in strains transformed with an intronless derivative of a putative tRNA(Trp) ochre (oc) suppressor gene, wherein intron removal cannot pose a problem.

Purification of yeast transfer RNA ligase.

The splicing of yeast tRNA precursors requires the action of at least two proteins, an endonuclease which cleaves the two splice junctions to release the linear intron and a ligase which joins the resulting tRNA half molecules together. Each of the activities of tRNA ligase can be assayed independent of the overall reaction. Researchers have routinely assayed the enzyme activity during purification by the ligation of tRNA half-molecules produced by the action of tRNA endonuclease. The pre-tRNA phe substrate can now conveniently be synthesized in vitro by T7 RNA polymerase transcription of a synthetic gene. One microliter of each reaction mixture is applied to a cellulose plate which is developed in solvent containing saturated ammonium sulfate, 1 M sodium acetate, 2-propanol (40:9: 1) and then analyzed by autoradiography. The amounts of cyclic GMP and 2′-GMP are quantitated by cutting the spots from the thin-layer plate and counting them in a scintillation counter.