Isolation of a yeast gene involved in species-specific pre-tRNA processing.

To identify genes involved in pre-tRNA processing, we searched for yeast DNA sequences that specifically enhanced the expression of the SUP4(G37) gene. The SUP4(G37) gene possesses a point mutation at position 37 of suppressor tRNA(Tyr). This lesion results in a reduced rate of pre-tRNA splicing and a decreased level of nonsense suppression. A SUP4(G37) strain was transformed with a yeast genomic library, and the transformants were screened for increased suppressor activity. One transformant contained a plasmid that encoded an unessential gene, STP1, that in multiple copies enhanced the suppression of SUP4(G37) and caused increased production of mature SUP4(G37) product. Disruption of the genomic copy of STP1 resulted in a reduced efficiency of SUP4-mediated suppression and the accumulation of pre-tRNAs. Not all intron-containing pre-tRNAs were affected by the stp1-disruption. At least five of the nine families of pre-tRNAs were affected. Two other species, pre-tRNA(Ile) and pre-tRNA(3Leu), were not. We propose that STP1 encodes a tRNA species-specific product that functions as a helper for pre-tRNA splicing. The STP1 product may interact with pre-tRNAs to generate a structure that is efficiently recognized by splicing machinery.     

PTA1, an essential gene of Saccharomyces cerevisiae affecting pre-tRNA processing.

We have identified an essential Saccharomyces cerevisiae gene, PTA1, that affects pre-tRNA processing. PTA1 was initially defined by a UV-induced mutation, pta1-1, that causes the accumulation of all 10 end-trimmed, intron-containing pre-tRNAs and temperature-sensitive but osmotic-remedial growth. pta1-1 does not appear to be an allele of any other known gene affecting pre-tRNA processing. Extracts prepared from pta1-1 strains had normal pre-tRNA splicing endonuclease activity. pta1-1 was suppressed by the ochre suppressor tRNA gene SUP11, indicating that the pta1-1 mutation creates a termination codon within a protein reading frame. The PTA1 gene was isolated from a genomic library by complementation of the pta1-1 growth defect. Episome-borne PTA1 directs recombination to the pta1-1 locus. PTA1 has been mapped to the left arm of chromosome I near CDC24; the gene was sequenced and could encode a protein of 785 amino acids with a molecular weight of 88,417. No other protein sequences similar to that of the predicted PTA1 gene product have been identified within the EMBL or GenBank data base. Disruption of PTA1 near the carboxy terminus of the putative open reading frame was lethal. Possible functions of the PTA1 gene product are discussed.     

A steroid/thyroid hormone receptor superfamily member in Drosophila melanogaster that shares extensive sequence similarity with a mammalian homologue

A gene in Drosophila melanogaster that maps cytologically to 2C1-3 on the distal portion of the X-chromosome encodes a member of the steroid/thyroid hormone receptor superfamily. The gene was isolated from an embryonic cDNA library using an oligonucleotide probe that specifies the consensus amino acid sequence in the DNA-binding domain of several human receptors. The conceptual amino acid sequence of 2C reveals at least four regions of homology that are shared with all identified vertebrate receptors. Region I includes the two cysteine-cysteine zinc fingers that comprise a DNA-binding domain which typifies all members of the superfamily. In addition, three regions (Regions II-IV) in the carboxy-terminal portion of the protein that encode the putative hormone-binding domain of the 2C gene product resemble similar sequences in vertebrate steroid/thyroid hormone receptors. The similarity suggests that this Drosophila receptor possesses many of the regulatory functions attributed to these regions in vertebrate counterparts. A portion of Region II also resembles part of the human c-jun oncoprotein's leucine zipper, which in turn, has been demonstrated to be the heterodimerization site between the jun and fos oncoproteins. The 2C receptor-like protein most resembles the mouse H2RII binding protein, a member of the superfamily which has been implicated in the regulation of major histocompatibility complex (MHC) class I gene expression. These two gene products are 83% identical in the DNA-binding domain and 50% identical in the putative hormone-binding domain, although no ligand has been identified for either protein. The high degree of similarity in the hormone-binding domain between the 2C protein and the H2RII binding protein outside regions II-IV suggests specific functional roles which are not shared by other members of the superfamily.     

A potential membrane protein involved in pre-tRNA splicing of Schizosaccharomyces pombe

We had previously isolated six pre-tRNA splicing mutants of Schizosaccharomyces pombe named ptp1 to ptp6. To investigate the molecular mechanism of tRNA splicing, we cloned the ptp4(+) gene by complementation of the temperature-sensitive growth defect. The ptp4(+) gene consists of three exons and encodes a putative protein of 218 amino acids with a molecular mass of 24.4 kDa. Analysis of the amino acid sequence reveals that the protein is a potential membrane protein with four membrane-spanning regions. The ptp4(+) shows significant similarity to the Saccharomyces cerevisiae putative protein YOR311C. Expression of the ptp4(+) gene in the ptp4(-) mutant restores the ability to splice tRNA. Northern blot analysis showed that the ptp4(+) gene is expressed in both mating-type cells of S. pombe. These results suggest that the Ptp4(+) could be a component involved in tRNA splicing.     

The second cholera toxin, Zot, and its plasmid-encoded and phage-encoded homologues constitute a group of putative ATPases with an altered purine NTP-binding motif.

It is shown that the second cholera toxin, Zot, ORF3 product of Pseudomonas plasmid pKB740, and ORF424 product of bacteriophage Pf1 are a group of closely related proteins containing a modified version of the purine NTP-binding motif, with a drastic substitution of tyrosine for a conserved glycine. They are distantly but reliably related to the product of gene I of filamentous bacteriophages which is a putative ATPase containing the classical NTP-binding motif and is involved in bacteriophage assembly and exit from the bacterial cell. Hydropathy analysis suggests that the Zot and gene I product may have a similar transmembrane topology. It is hypothesized that Zot may possess ATPase activity and modify the membrane structure of its target cells in an ATP-dependent fashion. Genes for Zot and the related protein of pKB740 are likely to have evolved from gene I of a Pf1-like bacteriophage.     

A yeast nuclear gene, MRS1, involved in mitochondrial RNA splicing: nucleotide sequence and mutational analysis of two overlapping open reading frames on opposite strands.

We have cloned a 1.6-kb fragment of yeast nuclear DNA, which complements pet- mutant MK3 (mrs1). This mutant was shown to be defective in mitochondrial RNA splicing: the excision of intron 3 from the mitochondrial COB pre-RNA is blocked. The DNA sequence of the nuclear DNA fragment revealed two open reading frames (ORF1 with 1092 bp; ORF2 with 735 bp) on opposite strands, which overlap by 656 bp. As shown by in vitro mutagenesis, ORF1, but not ORF2, is responsible for complementation of the splice defect. Hence, ORF1 represents the nuclear MRS1 gene. Disruption of the gene (both ORFs) in the chromosomal DNA of the respiratory competent yeast strain DBY747 (long form COB gene) leads to a stable pet- phenotype and to the accumulation of the same mitochondrial RNA precursors as in strain MK3. The amino acid sequence of the putative ORF1 product does not exhibit any homology with other known proteins, except for a small region of homology with the gene product of another nuclear yeast gene involved in mitochondrial RNA splicing, CBP2. The function of the MRS1 (ORF1) gene in mitochondrial RNA splicing and the significance of the overlapping ORFs in this gene are discussed.     

A novel mitochondrial DEAD box protein (Mrh4) required for maintenance of mtDNA in Saccharomyces cerevisiae

In a screen of nuclear genes that assist splicing of mitochondrial localized group II introns in yeast we isolated low-copy number suppressors of splicing and respiratory-deficient point mutants of intron aI5gamma, the last intron of the gene encoding cytochrome c oxidase subunit I. One of the genes found contains the open reading frame (ORF) YGL064c that has previously been proposed to encode a putative RNA helicase of the DEAD box family. Deletion of the ORF gives rise to 100% cytoplasmic petites, indicating that the protein plays an essential role in the mitochondrial RNA metabolism. Overexpression of YGL064c-GFP fusions clearly revealed a mitochondrial localization of the protein. The gene encodes the fourth putative RNA helicase of Saccharomyces cerevisiae implicated in a mitochondrial function and was therefore termed MRH4 (for mitochondrial RNA helicase).     

A cell-free plant extract for accurate pre-tRNA processing, splicing and modification.

An intron-containing tobacco tRNA(Tyr) precursor synthesized in a HeLa cell nuclear extract has been used to develop a cell-free processing and splicing system from wheat germ. Removal of 5' and 3' flanking sequences, accurate excision of the intervening sequence, ligation of the resulting tRNA halves, addition of the 3'-terminal CCA sequence and modification of seven nucleosides were achieved in appropriate wheat germ S23 and S100 extracts. The maturation of pre-tRNA(Tyr) in these extracts resembles the pathway observed in vivo for tRNA biosynthesis in Xenopus oocytes and yeast in that processing of the flanks precedes intron excision. Most of the modified nucleosides (m2(2) G, psi 35, psi 55, m7G and m1A) are introduced into the intron-containing pre-tRNA with mature ends, whereas two others (m1G and psi 39) are only found in the mature tRNA(Tyr). Processing and splicing proceed very efficiently in the wheat germ extracts, leading to complete maturation of 5' and 3' ends followed by about 65% conversion to mature tRNA(Tyr) under our standard conditions. The activity of the wheat germ endonuclease is stimulated 3-fold by the non-ionic detergent Triton X-100. All previous attempts to demonstrate the presence of a splicing endonuclease in wheat germ had failed (Gegenheimer et al., 1983). Hence, this is the first cell-free plant extract which supports pre-tRNA processing and splicing in vitro.     

Structure and functional expression of a cloned Xenopus thyroid hormone receptor.

A clone corresponding to a thyroid hormone receptor was isolated from a Xenopus laevis cDNA library prepared from folliculated oocytes. The cDNA encodes a protein of 418 amino acid residues with a domain structure, including a putative DNA binding region with two zinc fingers, similar to other members of the v-erbA-related superfamily of receptors. The encoded protein resembles the TR alpha 1-type receptor of the rat. When expressed in COS cells the protein product binds triiodothyronine with a Kd of 0.12 nM. The receptor mediates thyroid-hormone-inducible expression of a reporter gene which includes a thyroid hormone response element in its upstream region.     

A cold-regulated nucleic acid-binding protein of winter wheat shares a domain with bacterial cold shock proteins

The molecular mechanisms of cold acclimation are still largely unknown; however, it has been established that overwintering plants such as winter wheat increases freeze tolerance during cold treatments. In prokaryotes, cold shock proteins are induced by temperature downshifts and have been proposed to function as RNA chaperones. A wheat cDNA encoding a putative nucleic acid-binding protein, WCSP1, was isolated and found to be homologous to the predominant CspA of Escherichia coli. The putative WCSP1 protein contains a three-domain structure consisting of an N-terminal cold shock domain with two internal conserved consensus RNA binding domains and an internal glycine-rich region, which is interspersed with three C-terminal CX(2)CX(4)HX(4)C (CCHC) zinc fingers. Each domain has been described independently within several nucleotide-binding proteins. Northern and Western blot analyses showed that WCSP1 mRNA and protein levels steadily increased during cold acclimation, respectively. WCSP1 induction was cold-specific because neither abscisic acid treatment, drought, salinity, nor heat stress induced WCSP1 expression. Nucleotide binding assays determined that WCSP1 binds ssDNA, dsDNA, and RNA homopolymers. The capacity to bind dsDNA was nearly eliminated in a mutant protein lacking C-terminal zinc fingers. Structural and expression similarities to E. coli CspA suggest that WCSP1 may be involved in gene regulation during cold acclimation.     

Splicing of a yeast proline tRNA containing a novel suppressor mutation in the anticodon stem

The intron-containing proline tRNAUGG genes in Saccharomyces cerevisiae can mutate to suppress +1 frameshift mutations in proline codons via a G to U base substitution mutation at position 39. The mutation alters the 3' splice junction and disrupts the bottom base-pair of the anticodon stem which presumably allows the tRNA to read a four-base codon. In order to understand the mechanism of suppression and to study the splicing of suppressor pre-tRNA, we determined the sequences of the mature wild-type and mutant suppressor gene products in vivo and analyzed splicing of the corresponding pre-tRNAs in vitro. We show that a novel tRNA isolated from suppressor strains is the product of frameshift suppressor genes. Sequence analysis indicated that suppressor pre-tRNA is spliced at the same sites as wild-type pre-tRNA. The tRNA therefore contains a four-base anticodon stem and nine-base anticodon loop. Analysis of suppressor pre-tRNA in vitro revealed that endonuclease cleavage at the 3' splice junction occurred with reduced efficiency compared to wild-type. In addition, reduced accumulation of mature suppressor tRNA was observed in a combined cleavage and ligation reaction. These results suggest that cleavage at the 3' splice junction is inefficient but not abolished. The novel tRNA from suppressor strains was shown to be the functional agent of suppression by deleting the intron from a suppressor gene. The tRNA produced in vivo from this gene is identical to that of the product of an intron+ gene, indicating that the intron is not required for proper base modification. The product of the intron- gene is a more efficient suppressor than the product of an intron+ gene. One interpretation of this result is that inefficient splicing in vivo may be limiting the steady-state level of mature suppressor tRNA.     

Zinc finger protein gene complexes on mouse chromosomes 8 and 11

Two murine homologs of the Drosophila Krüppel gene, a member of the gap class of developmental control genes that encode a protein with zinc fingers, were mapped to mouse chromosomes 8 and 11 by using somatic cell hybrids and an interspecific backcross. Surprisingly, both genes were closely linked to two previously mapped, Krüppel-related zinc finger protein genes, suggesting that they are part of gene complexes.