Evidence for Related Functions of the RNA Genes of Saccharomyces cerevisiae

The yeast genes RNA2-RNA11 are necessary for splicing of nuclear intron-containing pre-mRNAs. We investigated the relationships among these genes by asking whether increased expression of one RNA gene leads to suppression of the temperature-sensitive lethality of a mutation in any other RNA gene. The presence of extra plasmid-borne copies of the RNA3 gene relieves the lethality of temperature-sensitive rna4 mutations. A region of the yeast genome (SRN2) is described that suppresses temperature-sensitive rna2 mutations when it is present on either medium or high-copy number plasmids. Neither suppression occurs via a bypass of RNA gene function since null alleles of rna2 and rna4 are not suppressed by elevated dosage of SRN2 and RNA3, respectively. These results suggest that the SRN2 and RNA2 gene products have related functions, as do the RNA3 and RNA4 gene products.     

The yeast RNA gene products are essential for mRNA splicing in vitro

The yeast rna mutations (rna2-rna11) are a set of temperature-sensitive mutations that result in the accumulation of intron-containing mRNA precursors at the restrictive temperature. We have used the yeast in vitro splicing system to investigate the role of products of the RNA genes in mRNA splicing. We have tested the heat lability of the in vitro mRNA splicing reaction in extracts isolated from mutant and wild-type cells. Extracts isolated from seven of the nine rna mutants demonstrated heat lability in this assay, while most wild-type extracts were stable under the conditions utilized. We have also demonstrated that heat inactivation usually results in the specific loss of an exchangeable component by showing that most combinations of heat-inactivated extracts from different mutants complement one another. In three cases (rna2, rna5, and rna11), the linkage of the in vitro defect to the rna mutations was ascertained by a combination of reversion, tetrad, and in vitro complementation analyses. Furthermore, each heat-inactivated extract was capable of complementation by at least one fraction of the wild-type splicing system. Thus many of the RNA genes are likely to code for products directly involved in and essential for mRNA splicing.     

Identification and nuclear localization of yeast pre-messenger RNA processing components: RNA2 and RNA3 proteins

Temperature-sensitive mutations in the RNA2 through RNA11 genes of yeast prevent the processing of nuclear pre-mRNAs. We have raised antisera that detect the RNA2 and RNA3 proteins in immunoblots of extracts of yeast containing high copy number RNA2 and RNA3 plasmids. Subcellular fractionation of yeast cells that overproduce the RNA2 and RNA3 proteins has revealed that these proteins are enriched in nuclear fractions. Indirect immunofluorescence results have indicated that these proteins are localized in yeast nuclei. These localization results are consistent with the fact that these genes have a role in processing yeast pre-mRNA.     

RNA11 protein is associated with the yeast spliceosome and is localized in the periphery of the cell nucleus.

The yeast rna mutations (rna2 through rna10/11) are a set of temperature-sensitive mutations that result in the accumulation of pre-mRNAs at the nonpermissive temperature. Most of the yeast RNA gene products are involved in and essential for mRNA splicing in vitro, suggesting that they code for components of the splicing machinery. We tested this proposal by using an in vitro-synthesized RNA11 protein to complement the temperature-sensitive defect of the rna11 extract. During the in vitro complementation, the input RNA11 protein was associated with the 40S spliceosome and a 30S complex, suggesting that the RNA11 protein is indeed a component of the spliceosome. The formation of the RNA11-associated 30S complex did not require any exogenous RNA substrate, suggesting that this 30S particle is likely to be a preassembled complex involved in splicing. The RNA11-specific antibody inhibited the mRNA splicing in vitro, confirming the essential role of the RNA11 protein in mRNA splicing. Finally, using the anti-RNA11 antibody, we localized the RNA11 protein to the periphery of the yeast nucleus.     

Deletion mapping of mitochondrial transfer RNA genes in Saccharomyces cerevisiae by means of cytoplasmic petite mutants

Summary Mitochondrial transfer RNA genes have been ordered relative to the position of five mitochondrial drug resistance markers, namely, chloramphenicol (C), erythromycin (E), oligomycin I and II (O_I, O_II), and paromomycin (P). Forty-six petite yeast clones that were genetically characterized with respect to these markers were used for a study of these relationships. Different regions of the mitochondrial genome are deleted in these individual mutants, resulting in variable loss of genetic markers. Mitochondrial DNA was isolated from each mutant strain and hybridized with eleven individual mitochondrial transfer RNAs. The following results were obtained: i) Of the seven petite clones that retained C, E, and P resistance markers (but not O_I or O_II), four carried all eleven transfer RNA genes examined; the other three clones lost several transfer RNA genes, probably by secondary internal deletion; ii) Prolyl and valyl transfer RNA genes were located close to the P marker, whereas the histidyl transfer RNA gene was close to the C marker; iii) Except for a glutamyl transfer RNA gene that was loosely associated with the O_I region, no other transfer RNA genes were found in petite clones retaining only the O_I and/or the O_II markers; and iv) Two distinct mitochondrial genes were found for glutamyl transfer RNA, they were not homologous in DNA sequence and were located at two separate loci.The data indicate that the petite mitochondrial genome is the result of a primary deletion followed by successive additional deletions. Thus an unequivocal gene arrangement cannot be readily established by deletion mapping with petite mutants alone. Nevertheless, we have derived a tentative circular map of the yeast mitochondrial genome from the data; the map indicates that all but one of the transfer RNA genes are found between the C and P markers without forming a tight cluster. The following arrangement is suggested:-P-pro-val-ile-(phe, ala, tyr, asp)-glu₂-(lys-leu)-his-C-E-O_I-glu₁-O_II-P-.Supported in part by Cancer Center CCRC 111B-3. Present address: Laboratoire de Biologie Generale, Universite Paris-Sud Orsay, 91405, FranceThe Franklin McLean Memorial Research Institute is operated by the University of Chicago for the U.S. Energy Research and Development Administration under Contract E(11-1)69     

Yeast H3 and H4 histone messenger RNAs are transcribed from two non-allelic gene sets

The genes coding for the H3 and H4 histones of Saccharomyces cerevisiae have been isolated by recombinant DNA cloning. The genes were detected in a bacteriophage lambda library of the yeast genome by hybridization with plasmids containing the cloned Psammechinus miliaris sea urchin histone genes (pCH7) and the cloned Drosophila histone genes (cDM500). Two non-allelic sets of the H3 and H4 genes have been isolated. Each set consists of one H3 gene and one H4 gene arranged as a divergently transcribed pair separated by an intergene spacer DNA. The histone genes were located on the cloned yeast fragments by S1 nuclease mapping, as was a gene (SMT1) of unknown function that does not code for a histone but is closely linked to one of the histone sets. Sequence homology between the two non-allelic sets is confined to the coding regions of the respective genes while the flanking DNA and intergene spacer DNA are extensively divergent. Cellular RNA homologous to the histone genes, including transcribed non-coding sequences unique to each of the four genes, was detected by S1 mapping, thus demonstrating that all four genes are transcribed in vegetative cells.     

Characterization of the intron-encoded U19 RNA, a new mammalian small nucleolar RNA that is not associated with fibrillarin.

We have characterized a new member (U19) of a group of mammalian small nuclear RNAs that are not precipitable with antibodies against fibrillarin, a conserved nucleolar protein associated with most of the small nucleolar RNAs characterized to date. Human U19 RNA is 200 nucleotides long and possesses 5'-monophosphate and 3'-hydroxyl termini. It lacks functional boxes C and D, sequence motifs required for fibrillarin binding in many other snoRNAs. Human and mouse RNA are 86% homologous and can be folded into similar secondary structures, a finding supported by the results of nuclease probing of the RNA. In the human genome, U19 RNA is encoded in the intron of an as yet not fully characterized gene and could be faithfully processed from a longer precursor RNA in HeLa cell extracts. During fractionation of HeLa cell nucleolar extracts on glycerol gradients, U19 RNA was associated with higher-order structures of approximately 65S, cosedimenting with complexes containing 7-2/MRP RNA, a conserved nucleolar RNA shown to be involved in 5.8S rRNA processing in yeast cells.     

Molecular cloning and analysis of the CRY1 gene: a yeast ribosomal protein gene.

Using cloned DNA from the vicinity of the yeast mating type locus (MAT) as a probe, the wild type allele of the cryptopleurine resistance gene CRY1 has been isolated by the technique of chromosome walking and has been shown to be identical to the gene for ribosomal protein 59. A recessive cryR1 allele has also been cloned, using the integration excision method. The genetic distance from MAT to CRY1 is 2.2 cM, while the physical distance is 21 kb, giving a ratio of about 10 kb/cM for this interval. The phenotypic expression of both plasmid borne alleles of the gene can be detected in vivo. The use of this gene as a hybridization probe to examine RNA processing defects in the rna 2, rna 3, rna 4, rna 8, and rna 11 mutants is also discussed.     

A panoramic view of yeast noncoding RNA processing

Predictive analysis using publicly available yeast functional genomics and proteomics data suggests that many more proteins may be involved in biogenesis of ribonucleoproteins than are currently known. Using a microarray that monitors abundance and processing of noncoding RNAs, we analyzed 468 yeast strains carrying mutations in protein-coding genes, most of which have not previously been associated with RNA or RNP synthesis. Many strains mutated in uncharacterized genes displayed aberrant noncoding RNA profiles. Ten factors involved in noncoding RNA biogenesis were verified by further experimentation, including a protein required for 20S pre-rRNA processing (Tsr2p), a protein associated with the nuclear exosome (Lrp1p), and a factor required for box C/D snoRNA accumulation (Bcd1p). These data present a global view of yeast noncoding RNA processing and confirm that many currently uncharacterized yeast proteins are involved in biogenesis of noncoding RNA.     

Determination of the sequence homology between the four RNA species of cucumber mosaic virus by hybridization analysis with complementary DNA

The method of Taylor etal., (11) has been used to transcribe complementary DNA probes from the four major RNA species of cucumber mosaic virus (RNAs 1 - 4 in order of decreasing molecular weight). Analysis of the kinetics of hybridization of these probes in homologous and heterologous complementary DNA-RNA hybridization reactions has shown that the sequence of the smallest RNA (RNA 4), which contains the coat protein gene, is present within RNA 3. RNAs 1 and 2 are unique RNA molecules while each has a region of approximately 300 nucleotides in common with RNA 4.     

Transcription and processing of intervening sequences in yeast tRNA genes.

Genes for yeast tRNATyr and tRNAPhe have been sequenced (Goodman, Olson and Hall, 1977; Valenzuela et al., 1978) which contain additional nucleotides (intervening sequences) within the middle of the gene that are not present in the mature tRNA. We have isolated precursors to rRNATyr and tRNAPhe from a yeast temperature-sensitive mutant (at the rna1 locus) which accumulates only certain precursor tRNAs at the nonpermissive temperature. The tRNATyr and tRNAPhe precursors were analyzed by oligonucleotide mapping; they each contain the intervening sequence and fully matured 5' and 3' termini. Furthermore, these precursors were used as substrates to search for an enzymatic activity which can remove the intervening sequences and religate the ends. We have shown that wild-type yeast contains such an activity, and that this activity specifically removes the intervening sequences to produce mature-sized RNAs.