Box C/D small nucleolar RNA trafficking involves small nucleolar RNP proteins, nucleolar factors and a novel nuclear domain.

Nucleolar localization of box C/D small nucleolar (sno) RNAs requires the box C/D motif and, in vertebrates, involves transit through Cajal bodies (CB). We report that in yeast, overexpression of a box C/D reporter leads to a block in the localization pathway with snoRNA accumulation in a specific sub-nucleolar structure, the nucleolar body (NB). The human survival of motor neuron protein (SMN), a marker of gems/CB, specifically localizes to the NB when expressed in yeast, supporting similarities between these structures. Box C/D snoRNA accumulation in the NB was decreased by mutation of Srp40 and increased by mutation of Nsr1p, two related nucleolar proteins that are homologous to human Nopp140 and nucleolin, respectively. Box C/D snoRNAs also failed to accumulate in the NB, and became delocalized to the nucleoplasm, upon depletion of any of the core snoRNP proteins, Nop1p/fibrillarin, Snu13p, Nop56p and Nop5p/Nop58p. We conclude that snoRNP assembly occurs either in the nucleoplasm, or during transit of snoRNAs through the NB, followed by routing of the complete snoRNP to functional sites of ribosome synthesis.

Functional characterization of nuclear localization signals in yeast Sm proteins.

In mammals, nuclear localization of U-snRNP particles requires the snRNA hypermethylated cap structure and the Sm core complex. The nature of the signal located within the Sm core proteins is still unknown, both in humans and yeast. Close examination of the sequences of the yeast SmB, SmD1, and SmD3 carboxyl-terminal domains reveals the presence of basic regions that are reminiscent of nuclear localization signals (NLSs). Fluorescence microscopy studies using green fluorescent protein (GFP)-fusion proteins indicate that both yeast SmB and SmD1 basic amino acid stretches exhibit nuclear localization properties. Accordingly, deletions or mutations in the NLS-like motifs of SmB and SmD1 dramatically reduce nuclear fluorescence of the GFP-Sm mutant fusion alleles. Phenotypic analyses indicate that the NLS-like motifs of SmB and SmD1 are functionally redundant: each NLS-like motif can be deleted without affecting yeast viability whereas a simultaneous deletion of both NLS-like motifs is lethal. Taken together, these findings suggest that, in the doughnut-like structure formed by the Sm core complex, the carboxyl-terminal extensions of Sm proteins may form an evolutionarily conserved basic amino acid-rich protuberance that functions as a nuclear localization determinant.

Interactions within the yeast Sm core complex: from proteins to amino acids.

Sm core proteins play an essential role in the formation of small nuclear ribonucleoprotein particles (snRNPs) by binding to small nuclear RNAs and participating in a network of protein interactions. The two-hybrid system was used to identify SmE interacting proteins and to test for interactions between all pairwise combinations of yeast Sm proteins. We observed interactions between SmB and SmD3, SmE and SmF, and SmE and SmG. For these interactions, a direct biochemical assay confirmed the validity of the results obtained in vivo. To map the protein-protein interaction surface of Sm proteins, we generated a library of SmE mutants and investigated their ability to interact with SmF and/or SmG proteins in the two-hybrid system. Several classes of mutants were observed: some mutants are unable to interact with either SmF or SmG proteins, some interact with SmG but not with SmF, while others interact moderately with SmF but not with SmG. Our mutational analysis of yeast SmE protein shows that conserved hydrophobic residues are essential for interactions with SmF and SmG as well as for viability. Surprisingly, we observed that other evolutionarily conserved positions are tolerant to mutations, with substitutions affecting binding to SmF and SmG only mildly and conferring a wild-type growth phenotype.

The nucleotide sequence of Saccharomyces cerevisiae chromosome XV.

Chromosome XV was one of the last two chromosomes of Saccharomyces cerevisiae to be discovered. It is the third-largest yeast chromosome after chromosomes XII and IV, and is very similar in size to chromosome VII. It alone represents 9% of the yeast genome (8% if ribosomal DNA is included). When systematic sequencing of chromosome XV was started, 93 genes or markers were identified, and most of them were mapped. However, very little else was known about chromosome XV which, in contrast to shorter chromosomes, had not been the object of comprehensive genetic or molecular analysis. It was therefore decided to start sequencing chromosome XV only in the third phase of the European Yeast Genome Sequencing Programme, after experience was gained on chromosomes III, XI and II. The sequence of chromosome XV has been determined from a set of partly overlapping cosmid clones derived from a unique yeast strain, and physically mapped at 3.3-kilobase resolution before sequencing. As well as numerous new open reading frames (ORFs) and genes encoding tRNA or small RNA molecules, the sequence of 1,091,283 base pairs confirms the high proportion of orphan genes and reveals a number of ancestral and successive duplications with other yeast chromosomes.

Analysis of a 35.6 kb region on the right arm of Saccharomyces cerevisiae chromosome XV.

We report the sequence of a 35,600 bp fragment covering the PET123 region on the right arm of chromosome XV from Saccharomyces cerevisiae. This region contains 19 possible open reading frames (ORFs) of which 16 are non-overlapping ORFs. Eight ORFs correspond to the SPP2, SMP3, PDR5, NFI1, PUP1, PET123 and MTR10 loci, described previously. Two ORFs correspond to yeast homologues of genes from other organisms: O3530 is a member of the large ribosomal subunit protein L13 family and O3560 (SME1 gene) is a 94-codon ORF and is a homologue of the mammalian SmE spliceosomal core protein. Three ORFs (O3513, O3521, O3548) present significant similarities to proteins of unknown function and three ORFs (O3510, O3536, O3545) lack homology to sequences within the databases screened.

The yeast SME1 gene encodes the homologue of the human E core protein.

Removal of introns from pre-messenger RNA (pre-mRNA) requires small nuclear RNAs (snRNAs) packaged into stable small ribonucleoprotein particles (snRNP). These snRNPs contain specific and common proteins also called Sm proteins. Correct assembly of the snRNAs with the common proteins is an essential step for the biogenesis of snRNP particles. We have identified a new Saccharomyces serevisiae gene, SME1 whose product shows 45% identity with the E core protein of human snRNP. The Sme1p contains the evolutionary conserved residues found in all Sm proteins. Combining genetic and biochemical experiments, we show that SME1 is an essential gene required for pre-mRNA splicing, cap modification and U1, U2, U4 and U5 snRNA stability. We show also that the human E core protein complements a yeast SME1 disruption demonstrating the functional equivalence of Sme1p and the human E core protein.

RPK1, an essential yeast protein kinase involved in the regulation of the onset of mitosis, shows homology to mammalian dual-specificity kinases.

We report here the sequence of RPK1 (for Regulatory cell Proliferation Kinase), a new Saccharomyces cerevisiae gene coding for a protein with sequence similarities to serine/threonine protein kinases. The protein sequence of 764 amino acids includes an amino-terminal domain (residues 1-410), which may be involved in regulation of the kinase domain (residues 411-764). The catalytic domain of Rpk1 is not closely related to other known yeast protein kinases but exhibits strong homology to a newly discovered group of mammalian kinases (PYT, TTK, esk) with serine/threonine/tyrosine kinase activity. Null alleles of RPK1 are lethal and thus this gene belongs to the small group of yeast protein kinase genes that are essential for cell growth. In addition, eliminating the expression of RPK1 gives rise to the accumulation of non-viable cells with less than a 1 N DNA content suggesting that cells proceed into mitosis without completion of DNA synthesis. Therefore, the Rpk1 kinase may function in a checkpoint control which couples DNA replication to mitosis. The level of the RPK1 transcript is extremely low and constant throughout the mitotic cycle. However it is regulated during cellular differentiation, being decreased in alpha-factor-treated a cells and increased late in meiosis in a/alpha diploids. Taken together, our results suggest that Rpk1 is involved in a pathway that coordinates cell proliferation and differentiation.

Human and human-yeast chimeric U6 snRNA genes identify structural elements required for expression in yeast.

U6 is the most highly conserved spliceosomal snRNA. Previous mutational studies have shown that the majority of essential residues in U6 are located in a region of 35 nucleotides encompassing a conserved hexanucleotide and stem I and stem II of the U4-interaction domain. Although the yeast and human U6 RNAs are 80% identical in this region, the human U6 gene cannot functionally replace the yeast gene in vivo. The human gene is not transcribed when placed in the context of yeast flanking sequences. Transcription of the human gene, but not its function, can be stimulated by the introduction of an A block promoter element in the U6 coding region. Using a set of human-yeast chimeras, we show that the 5' domain and the 3' terminal region of the human U6 gene can each functionally replace the corresponding yeast domains. However, a combination of both domains in a single molecule is lethal. The basis of the inability of the human U6 snRNA to function in yeast cells is discussed.

Multiple roles for U6 snRNA in the splicing pathway.

U6 is the most highly conserved of the five spliceosomal RNAs. It is associated with U4 by an extensive base-pairing interaction, which is disrupted immediately prior to the first nucleolytic step of splicing. It has been proposed that this event activates catalysis by unmasking U6. Using a combination of doped synthesis and site-directed mutagenesis to generate point mutations in U6, we have now identified 12 positions, in three domains, at which single nucleotide substitutions or deletions result in lethal or temperature-sensitive phenotypes. Biochemical analysis demonstrates that most of these mutants retain the ability to assemble into U4/U6 and U4/U5/U6 snRNPs. Notably, although mutations at three positions in U6 that base-pair with U4 are lethal, mutations in the complementary residues in U4 are fully viable. Furthermore, compensatory mutations in U4 that restore base-pairing fail to suppress the phenotypes of the U6 mutations. This demonstrates a function for U6 independent of its role in base-pairing. Remarkably, two of the three essential regions in U6 identified genetically correspond to intron insertion points in two yeast species. A temperature-sensitive mutation at one of these sites is defective in the second step of splicing in vitro.

Domains of yeast U4 spliceosomal RNA required for PRP4 protein binding, snRNP-snRNP interactions, and pre-mRNA splicing in vivo.

U4 small nuclear RNA (snRNA) contains two intramolecular stem-loop structures, located near each end of the molecule. The 5' stem-loop is highly conserved in structure and separates two regions of U4 snRNA that base-pair with U6 snRNA in the U4/U6 small nuclear ribonucleoprotein particle (snRNP). The 3' stem-loop is highly divergent in structure among species and lies immediately upstream of the binding site for Sm proteins. To investigate the function of these two domains, mutants were constructed that delete the yeast U4 snRNA 5' stem-loop and that replace the yeast 3' stem-loop with that from trypanosome U4 snRNA. Both mutants fail to complement a null allele of the yeast U4 gene. The defects of the mutants have been examined in heterozygous strains by native gel electrophoresis, glycerol gradient centrifugation, and immunoprecipitation. The chimeric yeast-trypanosome RNA does not associate efficiently with U6 snRNA, suggesting that the 3' stem-loop of yeast U4 snRNA might be a binding site for a putative protein that facilitates assembly of the U4/U6 complex. In contrast, the 5' hairpin deletion mutant associates efficiently with U6 snRNA. However, it does not bind the U4/U6-specific protein PRP4 and does not assemble into a U4/U5/U6 snRNA. Thus, we propose that the role of the PRP4 protein is to promote interactions between the U4/U6 snRNP and the U5 snRNP.

Expression of the oxi1 and maturase-related RF1 genes in yeast mitochondria.

Transcription of the yeast mitochondrial oxi1 gene (cytochrome oxidase subunit 2) is initiated at a variant non anucleotide sequence, TTAAAAGTA, located 54 bp upstream from the protein-coding gene. Transcriptional initiation at this site gives rise to a 2,500 nucleotide primary transcript containing both the oxi1 gene and the downstream maturase-related reading frame, RF1. Precise transcript mapping has revealed that the 3'-end of the mature oxi1 mRNA is generated by an endonucleolytic cleavage which takes place after the conserved dodecamer sequence, AAUAAUAUUCUU (End-of-Messenger signal), 75 nucleotides downstream from the oxi1 stop codon. Since the RF1 5'-terminal coding region overlaps the oxi1 3'-terminal coding sequence, cleavage at this motif truncates the RF1 message suggesting that the expression of the putative RF1 protein is controlled at the level of dodecamer processing.

Transcription initiation and RNA processing of a yeast mitochondrial tRNA gene cluster.

Expression of 5 yeast mitochondrial tRNA genes (Ala, Ile, Tyr, Asn and Metm), localized upstream from the oxil gene has been analyzed by in vitro capping using guanylyltransferase, northern hybridization and S1 nuclease mapping in the wild type and a rho-strain. The 5 tRNA sequences belong to the same transcriptional unit which is initiated 133 bp upstream from the tRNA(Ala) gene at a promoter sequence TTATAAGTA. Furthermore, a truncated tRNA(Tyr) transcript, 2 nucleotides shorter than mature tRNA(Tyr) has been found, only in the rho-strain. This minor transcript may result from secondary transcription initiation at a variant nonanucleotide sequence, ATATAAGGA, which overlaps the tRNA(Tyr) coding sequence by 3 nucleotides. The polycistronic precursor has proven to be useful in investigation of the mechanisms of tRNA processing. Maturation of this primary transcript proceeds exclusively by precise endonucleolytic cleavages at the 5' and 3'-ends of tRNA sequences.

A single base change in the extra-arm of yeast mitochondrial tyrosine tRNA affects its conformational stability and impairs aminoacylation.

The mitochondrial temperature-sensitive mutation tsm-8 maps on a 1.8 kb HpaII fragment of mitochondrial DNA (mt DNA) which contains genes for tRNA(Ala), tRNA(Ile) and tRNA(Tyr). The phenotype of this mutation is, among multiple pleiotropic defects, a temperature-induced reduction of mitochondrial translation. DNA sequencing of the HpaII fragment from the wild type and mutant tsm-8 revealed a single transversion from T to A in position 56 of the mutant tRNA(Tyr) gene. This nucleotide change disrupts a base pairing in the long extra arm of the tRNA cloverleaf. Revertants of the tsm-8 mutant restore correct base pairing in the extra arm by a second-site mutation in the tRNA(Tyr) gene. Analysis of the tRNA(Tyr) transcripts revealed that neither transcription nor processing of the tRNA is affected in the mutant. However, the base alteration destabilizes the conformation of the tRNA and affects its charging parameters. At the non-permissive temperature, the Michaelis-Menten constant of the mitochondrial tyrosyl-tRNA synthetase for the mutant tRNA is increased over 20-fold when compared to the wild-type tRNA. As a consequence, mitochondrial protein synthesis is drastically reduced at the restrictive temperature. Moreover, synthesis of apocytochrome b and of cytochrome oxidase subunit 3 is decreased relative to the other mitochondrially synthesized polypeptides.

[Yeast mitochondrial transfer RNA. Structure, coding properties and genome organization]. / Transportnye RNK mitokhondrii drozhzhei. Struktura, kodovye svoistva i organizatsiia genoma.

The up-to-date data on mitochondrial tRNAs of yeast, their structures and peculiarities of these structures, anomalies of the mitochondrial genetic code and anticodons of tRNAs, the structure and number of tRNA genes are reviewed in the present paper. New information concerning 17 types of yeast mitochondrial tRNAs, deciphered by the authors of the paper are given; among them 8 types are first published. The likeness and differences of yeast mitochondrial tRNAs from their cytoplasmic counterparts are discussed by comparison with other organisms.

[Primary structure of yeast mitochondrial tryptophan-tRNA capable of translating the termination U-G-A codon]. / Structure primaire d'un tryptophane-tRNA de mitochondrie de levure capable de traduire le codon de terminaison U-G-A.

The primary structure of Yeast Saccharomyces cerevisiae mitochondrial tryptophane-tRNA, isolated by reversed phase chromatography and two-dimensional gel electrophoresis has been determined. It is as follows: pA-A-G-G-A-U-A-U-A-G-U-U-U-A-A-D-G-G-D-A-A-A-A-C-A-G-U-U-G-A-psi-U-U-C-A-i6 A (ms2i6 A)-A-psi-C-A-A-U-C-A-U-U-A-G-G-A-G-T-psi-C-G-A-A-U-C-U-C-U-U-U-A-U-C-C-U-U-G-C-C-A. Owing to its anticodon U-C-A-, where U represents a modified uridine, this tRNA can translate the opal codon U-G-A, which is usually a protein synthesis termination codon in cytoplasmic systems.