Characterization of an SNR gene locus in Saccharomyces cerevisiae that specifies both dispensible and essential small nuclear RNAs.

A genetic locus is described that specifies two Saccharomyces cerevisiae small nuclear RNAs (snRNAs). The genes specifying the two snRNAs are separated by only 67 base pairs and are transcribed in the same direction. The product RNAs contain 128 and 190 nucleotides and are designated snR128 and snR190, respectively. These RNAs resemble snRNAs of other eucaryotes in nuclear localization and possession of a 5' trimethylguanosine cap. Neither snRNA is related in sequence to previously described vertebrate or yeast snRNAs. Both RNAs exhibit properties consistent with nucleolar organization and hydrogen bonding to pre-rRNA species, suggesting possible roles in ribosome biogenesis. The snR128 species cosediments with deproteinized 27S pre-rRNA, whereas snR190 is associated with a 20S intermediate. Gene disruption in vitro followed by replacement of the chromosomal alleles reveals that SNR128 is essential, whereas SNR190 is not.     

An essential yeast snRNA with a U5-like domain is required for splicing in vivo

Yeast contains at least 24 snRNAs, many of which are dispensable for viability. We recently demonstrated that a small subset of these RNAs has a functional binding site for the Sm antigen, a hallmark of metazoan snRNAs involved in mRNA processing. Here we show that one of these snRNAs, snR7, is required for growth. To determine the biochemical basis of lethality in cells lacking snR7, we engineered the conditional synthesis of snR7 by fusing the snRNA coding sequences to the yeast GAL1 control region. Cells depleted for the SNR7 gene product by growth on glucose for five generations show marked accumulation of unspliced mRNA precursors from the four intron-containing genes tested. In some cases, intron-exon 2 lariats also accumulate. We have identified a 70 nucleotide domain within snR7 with limited sequence-specific but striking structural homology to the mammalian snRNA U5. We conclude that mRNA splicing in yeast requires the function of a U5-like snRNA.     

RNA B is the major nucleolar trimethylguanosine-capped small nuclear RNA associated with fibrillarin and pre-rRNAs in Trypanosoma brucei.

RNA B is one of three abundant trimethylguanosine-capped U small nuclear RNAs (snRNAs) of Trypanosoma brucei which is not strongly identified with other U snRNAs by sequence homology. We show here that RNA B is a highly diverged U3 snRNA homolog likely involved in pre-rRNA processing. Sequence identity between RNA B and U3 snRNAs is limited; only two of four boxes of homology conserved between U3 snRNAs are obvious in RNA B. These are the box A homology, specific for U3 snRNAs, and the box C homology, common to nucleolar snRNAs and required for association with the nucleolar protein, fibrillarin. A 35-kDa T. brucei fibrillarin homolog was identified by using an anti-Physarum fibrillarin monoclonal antibody. RNA B and fibrillarin were localized in nucleolar fractions of the nucleus which contained pre-rRNAs and did not contain nucleoplasmic snRNAs. Fibrillarin and RNA B were precipitated by scleroderma patient serum S4, which reacts with fibrillarins from diverse organisms; RNA B was the only trimethylguanosine-capped RNA precipitated. Furthermore, RNA B sedimented with pre-rRNAs in nondenaturing sucrose gradients, similarly to U3 and other nucleolar snRNAs, suggesting that RNA B is hydrogen bonded to rRNA intermediates and might be involved in their processing.     

Deletion of a yeast small nuclear RNA gene impairs growth.

We have cloned and sequenced the single copy gene SNR10 which encodes the yeast small nuclear RNA, snR10. This species does not show obvious primary sequence homology to any previously identified small nuclear RNA. As an inital step towards determining the function of snR10, we have introduced insertions and deletions into the chromosomal copy of the gene. Strains lacking an intact copy of SNR10 are viable but considerably imparied in growth, particularly at elevated osmotic strengths or low temperatures; at 25 degrees C the doubling time of snr10- strains is 47% greater than that of otherwise isogenic SNR10 strains. As judged by the incorporation of radioactive precursors, snr10- strains are impaired in net RNA synthesis at low temperatures. The identification of a leaky, conditional phenotype associated with the deletion of this small nuclear RNA gene was entirely unexpected since the defect in snR10 synthesis is complete and non-conditional.     

An essential snRNA from S. cerevisiae has properties predicted for U4, including interaction with a U6-like snRNA

Three yeast snRNAs (snR20, snR7, and snR14) have been implicated in pre-mRNA splicing. snR20 and snR7 contain domains of homology to U2 and U5, respectively, and each is required for viability. These RNAs are found associated with the spliceosome, as is snR14. We show here that snR14 is also an essential gene product. Sequence analysis reveals that, like snR7 and snR20, snR14 contains a consensus binding site for the Sm antigen, a feature common to all mammalian snRNAs involved in splicing. Moreover, snR14 exhibits several blocks of sequence and structural homology to U4, which in metazoans is found in association with U6. Native gel electrophoresis demonstrates that snR14 is in fact base-paired with another yeast snRNA, designated snR6, which has primary sequence homology to U6. We conclude that snR14 is the yeast analog of U4.     

Quantitative analysis of snoRNA association with pre-ribosomes and release of snR30 by Rok1 helicase

In yeast, three small nucleolar RNAs (snoRNAs) are essential for the processing of pre-ribosomal RNA—U3, U14 and snR30—whereas 72 non-essential snoRNAs direct site-specific modification of pre-rRNA. We applied a quantitative screen for alterations in the pre-ribosome association to all 75 yeast snoRNAs in strains depleted of eight putative helicases implicated in 40S subunit synthesis. For the modification-guide snoRNAs, we found no clear evidence for the involvement of these helicases in the association or dissociation of pre-ribosomes. However, the DEAD box helicase Rok1 was required specifically for the release of snR30. Point mutations in motif I, but not in motif III, of the helicase domain of Rok1 impaired the release of snR30, but this was less marked than in strains depleted of Rok1, and resulted in a dominant-negative growth phenotype. Dissociation of U3 and U14 from pre-ribosomes is also dependent on helicases, suggesting that release of the essential snoRNAs might differ mechanistically from release of the modification-guide snoRNAs.     

18S rRNA processing requires base pairings of snR30 H/ACA snoRNA to eukaryote‐specific 18S sequences

The H/ACA RNAs represent an abundant, evolutionarily conserved and functionally diverse class of non‐coding RNAs. Many H/ACA RNAs direct pseudouridylation of rRNAs and snRNAs, while members of the rapidly growing group of ‘orphan’ H/ACA RNAs participate in pre‐rRNA processing, telomere synthesis and probably, in other nuclear processes. The yeast snR30 ‘orphan’ H/ACA snoRNA has long been known to function in the nucleolytic processing of 18S rRNA, but its molecular role remained unknown. Here, we provide biochemical and genetic evidence demonstrating that during pre‐rRNA processing, two evolutionarily conserved sequence elements in the 3′‐hairpin of snR30 base‐pair with short pre‐rRNA sequences located in the eukaryote‐specific internal region of 18S rRNA. The newly discovered snR30‐18S base‐pairing interactions are essential for 18S rRNA production and they constitute a complex snoRNA target RNA transient structure that is novel to H/ACA RNAs. We also demonstrate that besides the 18S recognition motifs, the distal part of the 3′‐hairpin of snR30 contains an additional snoRNA element that is essential for 18S rRNA processing and that functions most likely as a snoRNP protein‐binding site.     

S. cerevisiae U1 RNA is large and has limited primary sequence homology to metazoan U1 snRNA

We have cloned and sequenced the yeast SNR19 gene and show here that snR19 is the yeast homolog of metazoan U1 snRNA. sn R19 is 569 nucleotides long, strikingly larger than its metazoan counterpart. The two molecules resemble each other closely in the predicted secondary structure of their first 50 nucleotides. Primary sequence homology is restricted to some of their single-stranded regions, including 11 consecutive nucleotides at the 5' end of the two molecules, the region that interacts with pre-mRNA 5' splice junctions. snR19 is spliceosome-associated and required for in vitro pre-mRNA splicing. We also note that 8 sequences in snR19 have extensive complementarity to snR20, the large yeast U2 RNA, suggesting that yeast U1 may interact with yeast U2 by base-pairing.     

Cloning of Schizosaccharomyces pombe genes encoding the U1, U2, U3 and U4 snRNAs

Schizosaccharomyces pombe contains a group of five relatively abundant small nuclear RNAs (snRNAs) which are immunoprecipitated by human autoimmune antibodies of Sm serotype. The S. pombe RNAs hybridise to probes specific for human U1, U2, U4, U5 and U6 and in each case are similar in size to the human species. A further group of snRNAs from S. pombe are precipitated by antibodies against U3 containing ribonucleoprotein; the most abundant of these species hybridises to a probe specific for human U3. We have cloned the genes encoding U1, U2, U3 and U4 from S. pombe, together with that encoding another abundant snRNA, previously designated SPU43. U2 and U4 are encoded by single-copy genes, while two genes encode U3. The latter are not clustered, since a chromosomal Southern transfer shows them to lie on different chromosomes.     

Characterization of three new snRNAs from Saccharomyces cerevisiae: snR34, snR35 and snR36.

Genes for three novel snRNAs of Saccharomyces cerevisiae have been isolated, sequenced and tested for essentiality. The RNAs encoded by these genes are designated snR34, snR35 and snR36 respectively and contain 203, 204 and 182 nucleotides. Each RNA is derived from a single copy gene and all three RNAs are believed to be nucleolar, i.e. snoRNAs, based on extraction properties and association with fibrillarin. SnR34 and snR35 contain a trimethylguanosine cap, but this feature is absent from snR36. The novel RNAs lack elements conserved among several other snoRNAs, including box C, box D and long sequence complementarities with rRNA. Genetic disruption analyses showed each of the RNAs to be dispensable and a haploid strain lacking all three RNAs and a previously characterized fourth snoRNA (snR33) is also viable. No differences in the levels of precursors or mature rRNAs were apparent in the four gene knock-out strain. Possible roles for the new RNAs in ribosome biogenesis are discussed.     

Isolation and sequence of four small nuclear U RNA genes of Trypanosoma brucei subsp. brucei: identification of the U2, U4, and U6 RNA analogs.

Trypanosomes use trans splicing to place a common 39-nucleotide spliced-leader sequence on the 5' ends of all of their mRNAs. To identify likely participants in this reaction, we used antiserum directed against the characteristic U RNA 2,2,7-trimethylguanosine (TMG) cap to immunoprecipitate six candidate U RNAs from total trypanosome RNA. Genomic Southern analysis using oligonucleotide probes constructed from partial RNA sequence indicated that the four largest RNAs (A through D) are encoded by single-copy genes that are not closely linked to one another. We have cloned and sequenced these genes, mapped the 5' ends of the encoded RNAs, and identified three of the RNAs as the trypanosome U2, U4, and U6 analogs by virtue of their sequences and structural homologies with the corresponding metazoan U RNAs. The fourth RNA, RNA B (144 nucleotides), was not sufficiently similar to known U RNAs to allow us to propose an identify. Surprisingly, none of these U RNAs contained the consensus Sm antigen-binding site, a feature totally conserved among several classes of U RNAs, including U2 and U4. Similarly, the sequence of the U2 RNA region shown to be involved in pre-mRNA branchpoint recognition in yeast, and exactly conserved in metazoan U2 RNAs, was totally divergent in trypanosomes. Like all other U6 RNAs, trypanosome U6 did not contain a TMG cap and was immunoprecipitated from deproteinized RNA by anti-TMG antibody because of its association with the TMG-capped U4 RNA. These two RNAs contained extensive regions of sequence complementarity which phylogenetically support the secondary-structure model proposed by D. A. Brow and C. Guthrie (Nature [London] 334:213-218, 1988) for the organization of the analogous yeast U4-U6 complex.     

U17/snR30 is a ubiquitous snoRNA with two conserved sequence motifs essential for 18S rRNA production

Saccharomyces cerevisiae snR30 is an essential box H/ACA small nucleolar RNA (snoRNA) required for the processing of 18S rRNA. Here, we show that the previously characterized human, reptilian, amphibian, and fish U17 snoRNAs represent the vertebrate homologues of yeast snR30. We also demonstrate that U17/snR30 is present in the fission yeast Schizosaccharomyces pombe and the unicellular ciliated protozoan Tetrahymena thermophila. Evolutionary comparison revealed that the 3'-terminal hairpins of U17/snR30 snoRNAs contain two highly conserved sequence motifs, the m1 (AUAUUCCUA) and m2 (AAACCAU) elements. Mutation analysis of yeast snR30 demonstrated that the m1 and m2 elements are essential for early cleavages of the 35S pre-rRNA and, consequently, for the production of mature 18S rRNA. The m1 and m2 motifs occupy the opposite strands of an internal loop structure, and they are located invariantly 7 nucleotides upstream from the ACA box of U17/snR30 snoRNAs. U17/snR30 is the first identified box H/ACA snoRNA that possesses an evolutionarily conserved role in the nucleolytic processing of eukaryotic pre-rRNA.     

SnR31, snR32, and snR33: three novel, non-essential snRNAs from Saccharomyces cerevisiae.

Genes for three novel yeast snRNAs have been identified and tested for essentiality. Partial sequence information was developed for RNA extracted from isolated nuclei and the respective gene sequences were discovered by screening a DNA sequence database. The three RNAs contain 222, 188 and 183 nucleotides and are designated snR31, snR32 and snR33, respectively. Each RNA is derived from a single copy gene. The SNR31 gene is adjacent to a gene for an unnamed protein associated with the cap-binding protein eIF-4E. The SNR32 gene is next to a gene for ribosomal protein L41 and the gene for SNR33 is on chromosome III, between two open reading frames with no known function. Genetic disruption analyses showed that none of the three snRNAs is required for growth. The new RNAs bring the number of non-spliceosomal snRNAs characterized thus far in S. cerevisiae to 14, of which only three are essential.