Saccharomyces Cerevisiae RNA Research Articles

Investigating the Role of Metal Ions in the Catalytic Mechanism of the Yeast RNA Triphosphatase

The Saccharomyces cerevisiae RNA triphosphatase (Cet1) requires the presence of metal ion cofactors to catalyze its phosphohydrolase activity, the first step in the formation of the 5'-terminal cap structure of mRNAs. We have used endogenous tryptophan fluorescence studies to elucidate both the nature and the role(s) of the metal ions in the Cet1-mediated phosphohydrolase reaction. The association of Mg2+, Mn2+, and Co2+ ions with the enzyme resulted in a decrease in the intensity of the tryptophan emission spectrum. This decrease was then used to determine the apparent dissociation constants for these ions. Subsequent dual ligand titration experiments demonstrated that the metal ions bind to a common site, for which they compete. The kinetics of real-time metal ion binding to the Cet1 protein were also investigated, and the effects on RNA and nucleotide binding were evaluated. To provide additional insight into the relationship between Cet1 structure and metal ion binding, we correlated the effect of ion binding on protein structure using both circular dichroism and guanidium hydrochloride-induced denaturation as structural indicators. Our data indicate that binding of RNA, nucleotides, and metal ion cofactors does not lead to significant structural modifications of the Cet1 architecture. This suggests a model in which Cet1 possesses a preformed active site, and where major domain rearrangements are not required to form an active catalytic site. Finally, denaturation studies demonstrate that the metal ion cofactors can act by stabilizing the ground state binding of the phosphohydrolase substrate.

Subunit Communications Crucial for the Functional Integrity of the Yeast RNA Polymerase II Elongator (γ-Toxin Target (TOT)) Complex

In response to the Kluyveromyces lactis zymocin, the gamma-toxin target (TOT) function of the Saccharomyces cerevisiae RNA polymerase II (pol II) Elongator complex prevents sensitive strains from cell cycle progression. Studying Elongator subunit communications, Tot1p (Elp1p), the yeast homologue of human IKK-associated protein, was found to be essentially involved in maintaining the structural integrity of Elongator. Thus, the ability of Tot2p (Elp2p) to interact with the HAT subunit Tot3p (Elp3p) of Elongator and with subunit Tot5p (Elp5p) is dependent on Tot1p (Elp1p). Also, the association of core-Elongator (Tot1-3p/Elp1-3p) with HAP (Elp4-6p/Tot5-7p), the second three-subunit subcomplex of Elongator, was found to be sensitive to loss of TOT1 (ELP1) gene function. Structural integrity of the HAP complex itself requires the ELP4/TOT7, ELP5/TOT5, and ELP6/TOT6 genes, and elp6Delta/tot6Delta as well as elp4Delta/tot7Delta cells can no longer promote interaction between Tot5p (Elp5p) and Tot2p (Elp2p). The association between Elongator and Tot4p (Kti12p), a factor that may modulate the TOT activity of Elongator, requires Tot1-3p (Elp1-3p) and Tot5p (Elp5p), indicating that this contact requires a preassembled holo-Elongator complex. Tot4p also binds pol II hyperphosphorylated at its C-terminal domain Ser(5) raising the possibility that Tot4p bridges the contact between Elongator and pol II.

Characterization of human RNA polymerase III identifies orthologues for Saccharomyces cerevisiae RNA polymerase III subunits.

Unlike Saccharomyces cerevisiae RNA polymerase III, human RNA polymerase III has not been entirely characterized. Orthologues of the yeast RNA polymerase III subunits C128 and C37 remain unidentified, and for many of the other subunits, the available information is limited to database sequences with various degrees of similarity to the yeast subunits. We have purified an RNA polymerase III complex and identified its components. We found that two RNA polymerase III subunits, referred to as RPC8 and RPC9, displayed sequence similarity to the RNA polymerase II RPB7 and RPB4 subunits, respectively. RPC8 and RPC9 associated with each other, paralleling the association of the RNA polymerase II subunits, and were thus paralogues of RPB7 and RPB4. Furthermore, the complex contained a prominent 80-kDa polypeptide, which we called RPC5 and which corresponded to the human orthologue of the yeast C37 subunit despite limited sequence similarity. RPC5 associated with RPC53, the human orthologue of S. cerevisiae C53, paralleling the association of the S. cerevisiae C37 and C53 subunits, and was required for transcription from the type 2 VAI and type 3 human U6 promoters. Our results provide a characterization of human RNA polymerase III and show that the RPC5 subunit is essential for transcription.

Deletion of the RNA Polymerase Subunit RPB4 Acts as a Global, Not Stress-specific, Shut-off Switch for RNA Polymerase II Transcription at High Temperatures

We used whole genome expression analysis to investigate the changes in the mRNA profile in cells lacking the Saccharomyces cerevisiae RNA polymerase II subunit RPB4 (Delta RPB4). Our results indicated that an essentially complete shutdown of transcription occurs upon temperature shift of this conditionally lethal mutant; 98% of mRNA transcript levels decrease at least 2-fold, 96% at least 4-fold. This data was supported by in vivo experiments that revealed a rapid and greater than 5-fold decline in steady state poly(A) RNA levels after the temperature shift. Expression of several individual genes, measured by Northern analysis, was also consistent with the whole genome expression profile. Finally we demonstrated that the loss of RNA polymerase II activity causes secondary effects on RNA polymerase I, but not RNA polymerase III, transcription. The transcription phenotype of the Delta RPB4 mutant closely mirrors that of the temperature-sensitive rpb1-1 mutant frequently implemented as a tool to inactivate the RNA polymerase II in vivo. Therefore, the Delta RPB4 mutant can be used to easily design strains that enable the study of distinct post-transcriptional cellular processes in the absence of RNA polymerase II transcription.

An Essential Function of Saccharomyces cerevisiae RNA Triphosphatase Cet1 Is to Stabilize RNA Guanylyltransferase Ceg1 against Thermal Inactivation

Saccharomyces cerevisiae RNA triphosphatase (Cet1) and RNA guanylyltransferase (Ceg1) interact in vivo and in vitro to form a bifunctional mRNA capping enzyme complex. Here we show that the guanylyltransferase activity of Ceg1 is highly thermolabile in vitro (98% loss of activity after treatment for 10 min at 35 degrees C) and that binding to recombinant Cet1 protein, or a synthetic peptide Cet1(232-265), protects Ceg1 from heat inactivation at physiological temperatures. Candida albicans guanylyltransferase Cgt1 is also thermolabile and is stabilized by binding to Cet1(232-265). In contrast, Schizosaccharomyces pombe and mammalian guanylyltransferases are intrinsically thermostable in vitro and they are unaffected by Cet1(232-265). We show that the requirement for the Ceg1-binding domain of Cet1 for yeast cell growth can be circumvented by overexpression in high gene dosage of a catalytically active mutant lacking the Ceg1-binding site (Cet1(269-549)) provided that Ceg1 is also overexpressed. However, such cells are unable to grow at 37 degrees C. In contrast, cells overexpressing Cet1(269-549) in single copy grow at all temperatures if they express either the S. pombe or mammalian guanylyltransferase in lieu of Ceg1. Thus, the cell growth phenotype correlates with the inherent thermal stability of the guanylyltransferase. We propose that an essential function of the Cet1-Ceg1 interaction is to stabilize Ceg1 guanylyltransferase activity rather than to allosterically regulate its activity. We used protein-affinity chromatography to identify the COOH-terminal segment of Ceg1 (from amino acids 245-459) as an autonomous Cet1-binding domain. Genetic experiments implicate two peptide segments, (287)KPVSLYVW(295) and (337)WQNLKNLEQPLN(348), as likely constituents of the Cet1-binding site on Ceg1.

Importance of Homodimerization for the in Vivo Function of Yeast RNA Triphosphatase

Saccharomyces cerevisiae RNA triphosphatase Cet1 is an essential component of the yeast mRNA capping apparatus. The active site of Cet1 resides within a topologically closed hydrophilic beta-barrel (the triphosphate tunnel) that is supported by a globular hydrophobic core. The homodimeric quaternary structure of Cet1 is formed by a network of contacts between the partner protomers. By studying the effects of alanine-cluster mutations, we highlight the contributions of two separate facets of the crystallographic dimer interface to Cet1 function in vivo. One essential facet of the interface entails hydrophobic cross-dimer interactions of Cys(330) and Val(331) and a cross-dimer hydrogen bond of Asp(280) with the backbone amide of Gln(329). The second functionally relevant dimer interface involves hydrophobic side-chain interactions of Phe(272) and Leu(273). Ala-cluster mutations involving these residues elicited lethal or severe temperature-sensitive phenotypes that were suppressed completely by fusion of the mutated triphosphatases to the guanylyltransferase domain of mammalian capping enzyme. The recombinant D279A-D280A and F272A-L273A proteins retained phosphohydrolase activity but sedimented as monomers. These results indicate that a disruption of the dimer interface is uniquely deleterious when the yeast RNA triphosphatase must function in concert with the endogenous yeast guanylyltransferase. We also identify key residue pairs in the hydrophobic core of the Cet1 protomer that support the active site tunnel and stabilize the triphosphatase in vivo.

Topology of yeast RNA polymerase II subunits in transcription elongation complexes studied by photoaffinity cross-linking.

The subunits of Saccharomyces cerevisiae RNA polymerase II (RNAP II) in proximity to the DNA during transcription elongation have been identified by photoaffinity cross-linking. In the absence of transcription factors, RNAP II will transcribe a double-stranded DNA fragment containing a 3'-extension of deoxycytidines, a "tailed template". We designed a DNA template allowing the RNAP to transcribe 76 bases before it was stalled by omission of CTP in the transcription reaction. This stall site oriented the RNAP on the DNA template and allowed us to map the RNAP subunits along the DNA. The DNA analogue 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUTP (N(3)RdUTP) [Bartholomew, B., Kassavetis, G. A., Braun, B. R., and Geiduschek, E. P. (1990) EMBO J. 9, 2197-205] was synthesized and enzymatically incorporated into the DNA at specified positions upstream or downstream of the stall site, in either the template or nontemplate strand of the DNA. Radioactive nucleotides were positioned beside the photoactivatable nucleotides, and cross-linking by brief ultraviolet irradiation transferred the radioactive tag from the DNA onto the RNAP subunits. In addition to N(3)RdUTP, which has a photoreactive azido group 9 A from the uridine base, we used the photoaffinity cross-linker 5N(3)dUTP with an azido group directly on the uridine ring to identify the RNAP II subunits closest to the DNA at positions where multiple subunits cross-linked. In cross-linking reactions dependent on transcription, RPB1, RPB2, and RPB5 were cross-linked with N(3)RdUTP. With 5N(3)dUTP, only RPB1 and RPB2 were cross-linked. Under certain circumstances, RPB3, RPB4, and RPB7 were cross-linked. From the information obtained in this topological study, we developed a model of yeast RNAP II in a transcription elongation complex.

Identification of the Saccharomyces cerevisiae RNA:pseudouridine synthase responsible for formation of psi(2819) in 21S mitochondrial ribosomal RNA.

So far, four RNA:pseudouridine (Psi)-synthases have been identified in yeast Saccharomyces cerevisiae. Together, they act on cytoplasmic and mitochondrial tRNAs, U2 snRNA and rRNAs from cytoplasmic ribosomes. However, RNA:Psi-synthases responsible for several U-->Psi conversions in tRNAs and UsnRNAs remained to be identified. Based on conserved amino-acid motifs in already characterised RNA:Psi-synthases, four additional open reading frames (ORFs) encoding putative RNA:Psi-synthases were identified in S.cerevisiae. Upon disruption of one of them, the YLR165c ORF, we found that the unique Psi residue normally present in the fully matured mitochondrial rRNAs (Psi(2819)in 21S rRNA) was missing, while Psi residues at all the tested pseudo-uridylation sites in cytoplasmic and mitochondrial tRNAs and in nuclear UsnRNAs were retained. The selective U-->Psi conversion at position 2819 in mitochondrial 21S rRNA was restored when the deleted yeast strain was transformed by a plasmid expressing the wild-type YLR165c ORF. Complementation was lost after point mutation (D71-->A) in the postulated active site of the YLR165c-encoded protein, indicating the direct role of the YLR165c protein in Psi(2819)synthesis in mitochondrial 21S rRNA. Hence, for nomenclature homogeneity the YLR165c ORF was renamed PUS5 and the corresponding RNA:Psi-synthase Pus5p. As already noticed for other mitochondrial RNA modification enzymes, no canonical mitochondrial targeting signal was identified in Pus5p. Our results also show that Psi(2819)in mitochondrial 21S rRNA is not essential for cell viability.

Identification of a approximately 30S size non-ribosomal Saccharomyces cerevisiae RNA that is rapidly labeled on its 3' end by ATP or UTP.

Cell-free extracts prepared from S. cerevisiae cells were incubated in the presence of [alpha-32P]-labeled ATP, CTP, GTP or UTP. An RNA larger than ribosomal 25S RNA with an apparent size of approximately 30S was prominently labeled on its 3' end in the presence of ATP or UTP but not with CTP or GTP. This labeled RNA was not hybrid-selected by cloned yeast ribosomal DNA; in addition, this approximately 30S RNA was not cleaved by RNase H in the presence of complementary deoxyribooligonucleotides to rRNA. These two lines of evidence show that this approximately 30S RNA is not structurally related to ribosomal RNA gene repeat. The cell-free extracts prepared from yeast cells containing temperature-sensitive poly(A) polymerase adenylated this novel yeast RNA at restrictive temperature with efficiency similar to extracts prepared from wild-type yeast cells. These data show that the enzyme responsible for adenylation of this approximately 30S RNA is distinct from mRNA poly(A) polymerase. While the human SRP RNA 3' adenylating enzyme in the HeLa cell extract adenylated human SRP or Alu RNAs, the yeast adenylating enzyme did not adenylate the human SRP or Alu RNAs in vitro; these data indicate species specificity for this adenylating enzyme.

A Saccharomyces cerevisiae RNA 5'-triphosphatase related to mRNA capping enzyme.

The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: the RNA 5'-triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1). Using computer homology searching, a S. cerevisiae gene was identified that encodes a protein resembling the C-terminal region of Cet1. Accordingly, we designated this gene CTL1 (capping enzyme RNAtriphosphatase-like 1). CTL1 is not essential for cell viability and no genetic or physical interactions with the capping enzyme genes were observed. The protein is found in both the nucleus and cytoplasm. Recombinant Ctl1 protein releases gamma-phosphate from the 5'-end of RNA to produce a diphosphate terminus. The enzyme is specific for polynucleotide RNA in the presence of magnesium, but becomes specific for nucleotide triphosphates in the presence of manganese. Ctl1 is the second member of the yeast RNA triphosphatase family, but is probably involved in an RNA processing event other than mRNA capping.

Structure-function analysis of the triphosphatase component of vaccinia virus mRNA capping enzyme.

The N-terminal 60 kDa (amino acids 1 to 545) of the D1 subunit of vaccinia virus mRNA capping enzyme is an autonomous bifunctional domain with triphosphatase and guanylyltransferase activities. We previously described two alanine cluster mutations, R77 to A (R77A)-K79A and E192A-E194A, which selectively inactivated the triphosphatase component. Here, we characterize the activities of 11 single alanine mutants-E37A, E39A, Q60A, E61A, T67A, T69A, K75A, R77A, K79A, E192A, and E194A-and a quadruple mutant in which four residues (R77, K79, E192, and E194) were replaced by alanine. We report that Glu-37, Glu-39, Arg-77, Glu-192, and Glu-194 are essential for gamma-phosphate cleavage. The five essential residues are conserved in the capping enzymes of Shope fibroma virus, molluscum contagiosum virus, and African swine fever virus. Probing the structure of D1(1-545) by limited V8 proteolysis suggested a bipartite subdomain structure. The essential residue Glu-192 is the principal site of V8 cleavage. Secondary cleavage by V8 occurs at the essential residue Glu-39. The triphosphatase-defective quadruple mutant transferred GMP to the triphosphate end of poly(A) to form a tetraphosphate cap structure, GppppA. We report that GppppA-capped RNA is a poor substrate for cap methylation by the vaccinia virus and Saccharomyces cerevisiae RNA (guanine-7) methyltransferases. The transcription termination factor activity of the D1-D12 capping enzyme heterodimer was not affected by mutations that abrogated ATPase activity. Thus, the capping enzyme is not responsible for the requirement for ATP hydrolysis during transcription termination.

Gene organization and protein sequence of the small subunits of Schizosaccharomyces pombe RNA polymerase II

RNA polymerase II purified from the fission yeast Schizosaccharomyces pombe contains 10 different species of polypeptides. Previously, we cloned and sequenced both cDNA and the genes encoding the four large subunits, Rpb1, Rpb2, Rpb3 and Rpb5. Later, other groups isolated the genes for Rpb6 and Rpb12 and cDNA for Rpb10. Here, we cloned both cDNA and the genes encoding four small subunits, Rpb7, Rpb8, Rpb10 and Rpb11. These genes were found to encode Rpb7, Rpb8, Rpb10 and Rpb11 consisting of 172 (19 103 Da), 125 (14 300 Da), 71 (8276 Da) and 123 (14 127 Da) amino acid residues, respectively. All these four subunits are homologous to the corresponding subunits of Saccharomyces cerevisiae RNA polymerase II. The rpb7 gene contains one intron, whereas the rpb8, rpb10 and rpb11 genes contain two introns. Taken altogether, the gene organization and the predicted protein sequence have been determined for all 10 subunits of the S. pombe RNA polymerase II.

Mapping the Contacts of Yeast TFIIIB and RNA Polymerase III at Various Distances from the Major Groove of DNA by DNA Photoaffinity Labeling

The structure of the Saccharomyces cerevisiae RNA polymerase III transcription complex on the SUP4 tRNATyr gene was probed at distances of approximately 10 to approximately 23 A from the C-5 methyl of thymidine in the major groove of DNA using photoreactive aryl azides attached to deoxyuridine by variable chain lengths. The nucleotide analogs contained an azidobenzoyl group attached with chain lengths that were incrementally increased by approximately 4. 3 A by inserting 1-3 glycine residues into the chain. Another photoreactive deoxyuridine analog was made that contained a butyl chain (ABU-dUMP) to assess the effect of the chain's hydrophobicity on its ability to photoaffinity label the transcription complex. These nucleotide analogs were incorporated at base pairs (bp) -26/-21, -17, or -3/-2 on the nontranscribed strand of the SUP4 tRNATyr gene along with an [alpha-32P]dNMP by primer extension using an immobilized single-stranded DNA template annealed to specific oligonucleotides. The 27-kDa subunit of TFIIIB or the TATA box binding protein was photoaffinity labeled at bp -26/-21 with nucleotide analogs containing a approximately 19- or approximately 23-A chain and not with shorter chains of approximately 10 to approximately 15 A in length. The B" subunit of TFIIIB (Mr = 90 kDa) was photoaffinity labeled at bps -26/-21 with DNA containing a approximately 14-A chain and not with shorter or longer chains. Cross-linking of the B" subunit was inhibited by binding of RNA polymerase III (Pol III) to the TFIIIB-DNA complex and suggested that Pol III binding causes a conformational change in the TFIIIB-DNA complex resulting in the displacement of the 90-kDa subunit at bps -26/-21. Next, the chain length dependence of photoaffinity labeling the 34-kDa subunit of Pol III at bps -17 and -3/-2 indicated that the 34-kDa subunit of Pol III is slightly removed from the major groove at bp -17 in the initiation complex and makes closer contact at bps -3/-2 in a stalled elongation complex.

Cloning, expression, and function of TFC5, the gene encoding the B" component of the Saccharomyces cerevisiae RNA polymerase III transcription factor TFIIIB.

TFC5, the unique and essential gene encoding the B" component of the Saccharomyces cerevisiae RNA polymerase III transcription factor (TF)IIIB has been cloned. It encodes a 594-amino acid protein (67,688 Da). Escherichia coli-produced B" has been used to reconstitute entirely recombinant TFIIIB that is fully functional for TFIIIC-directed, as well as TATA box-dependent, DNA binding and transcription. The DNase I footprints of entirely recombinant TFIIIB, composed of B", the 67-kDa Brf, and TATA box-binding protein, and TFIIIB reconstituted with natural B" are indistinguishable. A truncated form of B" lacking 39 N-terminal and 107 C-terminal amino acids is also functional for transcription.

Characterization of the Components of Reconstituted Saccharomyces cerevisiae RNA Polymerase I Transcription Complexes

We have reconstituted specific RNA polymerase I transcription from three partially purified chromatographic fractions (termed A, B, and C). Here, we present the chromatographic scheme and the initial biochemical characterization of these fractions. The A fraction contained the RNA polymerase I transcription factor(s), which was necessary and sufficient to form stable preinitiation complexes at the promoter. Of the three fractions, only fraction A contained a significant amount of the TATA binding factor. The B fraction contributed RNA polymerase I, and it contained an essential RNA polymerase I transcription factor that was specifically inactivated in response to a significant decrease in growth rate. The function of the C fraction remains unclear. This reconstituted transcription system provides a starting point for the biochemical dissection of the yeast RNA polymerase I transcription complex, thus allowing in vitro experiments designed to elucidate the molecular mechanisms controlling rRNA synthesis.

RNA polymerase II subunit RPB9 is required for accurate start site selection.

The diverse functions of Saccharomyces cerevisiae RNA polymerase II are partitioned among its 12 subunits, designated RPB1-RPB12. Although multiple functions have been assigned to the three largest subunits, RPB1, RPB2, and RPB3, the functions of the remaining smaller subunits are unknown. We have determined the function of one of the smaller subunits, RPB9, by demonstrating that it is necessary for accurate start site selection. Transcription in the absence of RPB9 initiates farther upstream at new and previously minor start sites both at the CYC1 promoter in vitro and at the CYC1, ADH1, HIS4, H2B-1, and RPB6 promoters in vivo. Immunoprecipitation of RNA polymerase II from cells lacking the RPB9 gene revealed that all of the remaining 11 subunits are assembled into the enzyme, suggesting that the start site defect is attributable solely to the absence of RPB9. In support of this hypothesis, we have shown that addition of wild-type recombinant RPB9 completely corrects for the start site defect seen in vitro. A mutated recombinant RPB9 protein, with an alteration in a metal-binding domain required for high temperature growth and accurate start site selection in vivo, was at least 10-fold less effective at correcting the start site defect in vitro. RPB9 appears to play a unique role in transcription initiation, as the defects revealed in its absence are distinct from those seen with mutants in RNA polymerase subunit RPB1 and factor e (TFIIB), two other yeast proteins also involved in start site selection.

Covalent catalysis in nucleotidyl transfer reactions: essential motifs in Saccharomyces cerevisiae RNA capping enzyme are conserved in Schizosaccharomyces pombe and viral capping enzymes and among polynucleotide ligases.

Formation of the 5' cap structure of eukaryotic mRNAs occurs via transfer of GMP from GTP to the 5' terminus of the primary transcript. RNA guanylyltransferase, the enzyme that catalyzes this reaction, has been isolated from many viral and cellular sources. Though differing in molecular weight and subunit structure, the various guanylyltransferases employ a common catalytic mechanism involving a covalent enzyme-(Lys-GMP) intermediate. Saccharomyces cerevisiae CEG1 is the sole example of a cellular capping enzyme gene. In this report, we describe the identification and characterization of the PCE1 gene encoding the capping enzyme from Schizosaccharomyces pombe. PCE1 was isolated from a cDNA library by functional complementation in Sa. cerevisiae. Induced expression of PCE1 in bacteria and in yeast confirmed that the 47-kDa Sc. pombe protein was enzymatically active. The amino acid sequence of PCE1 is 38% identical (152 of 402 residues) to the 52-kDa capping enzyme from Sa. cerevisiae. Comparison of the two cellular capping enzymes with guanylyltransferases encoded by DNA viruses revealed local sequence similarity at the enzyme's active site and at four additional collinear motifs. Mutational analysis of yeast CEG1 demonstrated that four of the five conserved motifs are essential for capping enzyme function in vivo. Remarkably, the same motifs are conserved in the polynucleotide ligase family of enzymes that employ an enzyme-(Lys-AMP) intermediate. These findings illuminate a shared structural basis for covalent catalysis in nucleotidyl transfer and suggest a common evolutionary origin for capping enzymes and ligases.

Replacement of the Saccharomyces cerevisiae RPR1 gene with heterologous RNase P RNA genes.

Phylogenetic studies of yeast nuclear RNase P RNA genes have shown a striking conservation of secondary structure for the Saccharomyces and Schizosaccharomyces RNase P RNAs, yet much of the primary sequence and many substructures vary among the RNAs examined. To investigate which sequences and structural features can be varied and still allow function in a heterologous organism, RNase P genes from several yeast species were tested for the ability to substitute for the Saccharomyces cerevisiae RNA. The RNase P genes from Saccharomyces carlsbergensis and Saccharomyces kluyveri could act as the sole source of RNase P RNA within S. cerevisiae cells, whereas the genes from Saccharomyces globosus and Schizosaccharomyces pombe could not. Although heterologous RNase P RNAs were synthesized by the cells in all cases, the RNAs that complemented tended to be processed from longer precursor transcripts into mature-sized RNase P RNA, while the RNAs that did not complement tended to accumulate as the longer precursor form. The results identified sequences and structures in the RNA that are not essential for interaction with species-specific proteins, processing or localization, and suggested other positions that may be candidates for such processes.

Yeast snR30 is a small nucleolar RNA required for 18S rRNA synthesis.

Subnuclear fractionation and coprecipitation by antibodies against the nucleolar protein NOP1 demonstrate that the essential Saccharomyces cerevisiae RNA snR30 is localized to the nucleolus. By using aminomethyl trimethyl-psoralen, snR30 can be cross-linked in vivo to 35S pre-rRNA. To determine whether snR30 has a role in rRNA processing, a conditional allele was constructed by replacing the authentic SNR30 promoter with the GAL10 promoter. Repression of snR30 synthesis results in a rapid depletion of snR30 and a progressive increase in cell doubling time. rRNA processing is disrupted during the depletion of snR30; mature 18S rRNA and its 20S precursor underaccumulate, and an aberrant 23S pre-rRNA intermediate can be detected. Initial results indicate that this 23S pre-rRNA is the same as the species detected on depletion of the small nucleolar RNA-associated proteins NOP1 and GAR1 and in an snr10 mutant strain. It was found that the 3' end of 23S pre-rRNA is located in the 3' region of ITS1 between cleavage sites A2 and B1 and not, as previously suggested, at the B1 site, snR30 is the fourth small nucleolar RNA shown to play a role in rRNA processing.

Yeast snR30 is a small nucleolar RNA required for 18S rRNA synthesis.

Subnuclear fractionation and coprecipitation by antibodies against the nucleolar protein NOP1 demonstrate that the essential Saccharomyces cerevisiae RNA snR30 is localized to the nucleolus. By using aminomethyl trimethyl-psoralen, snR30 can be cross-linked in vivo to 35S pre-rRNA. To determine whether snR30 has a role in rRNA processing, a conditional allele was constructed by replacing the authentic SNR30 promoter with the GAL10 promoter. Repression of snR30 synthesis results in a rapid depletion of snR30 and a progressive increase in cell doubling time. rRNA processing is disrupted during the depletion of snR30; mature 18S rRNA and its 20S precursor underaccumulate, and an aberrant 23S pre-rRNA intermediate can be detected. Initial results indicate that this 23S pre-rRNA is the same as the species detected on depletion of the small nucleolar RNA-associated proteins NOP1 and GAR1 and in an snr10 mutant strain. It was found that the 3' end of 23S pre-rRNA is located in the 3' region of ITS1 between cleavage sites A2 and B1 and not, as previously suggested, at the B1 site, snR30 is the fourth small nucleolar RNA shown to play a role in rRNA processing.