Differential sensitivity of 16S rRNA targeted oligonucleotide probes used for fluorescence in situ hybridization is a result of ribosomal higher order structure

The use of 16S rRNA targeted gene probes for the direct analysis of microbial communities has revolutionized the field of microbial ecology, yet a comprehensive approach for the design of such probes does not exist. The development of 16S rRNA targeted oligonucleotide probes for use with fluorescence in situ hybridization (FISH) procedures has been especially difficult as a result of the complex nature of the rRNA target molecule. In this study a systematic comparison of 16S rRNA targeted oligonucleotide gene probes was conducted to determine if target location influences the hybridization efficiency of oligonucleotide probes when used with in situ hybridization protocols for the detection of whole microbial cells. Five unique universal 12-mer oligonucleotide sequences, located at different regions of the 16S rRNA molecule, were identified by a computer-aided sequence analysis of over 1000 partial and complete 16S rRNA sequences. The complements of these oligomeric sequences were chemically synthesized for use as probes and end labeled with either [gamma-32P]ATP or the fluorescent molecule tetramethylrhodamine-5/-6. Hybridization sensitivity for each of the probes was determined by hybridization to heat-denatured RNA immobilized on blots or to formaldehyde fixed whole cells. All of the probes hybridized with equal efficiency to denatured RNA. However, the probes exhibited a wide range of sensitivity (from none to very strong) when hybridized with whole cells using a previously developed FISH procedure. Differential hybridization efficiencies against whole cells could not be attributed to cell wall type, since the relative probe efficiency was preserved when either Gram-negative or -positive cells were used. These studies represent one of the first attempts to systematically define criteria for 16S rRNA targeted probe design for use against whole cells and establish target site location as a critical parameter in probe design.     

Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis

The Saccharomyces cerevisiae temperature-sensitive (ts) allele nip7-1 exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes. The cloned NIP7+ gene complemented the nip7-1 ts growth defect, the paromomycin hypersensitivity, and the halfmer defect. NIP7 encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Arabidopsis thaliana, indicating that Nip7p function is evolutionarily conserved. Gene disruption analysis demonstrated that NIP7 is essential for growth. A fraction of Nip7p cosedimented through sucrose gradients with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Nip7p was found evenly distributed throughout the cytoplasm and nucleus by indirect immunofluorescence; however, in vivo localization of a Nip7p-green fluorescent protein fusion protein revealed that a significant amount of Nip7p is present inside the nucleus, most probably in the nucleolus. Depletion of Nip7-1p resulted in a decrease in protein synthesis rates, accumulation of halfmers, reduced levels of 60S subunits, and, ultimately, cessation of growth. Nip7-1p-depleted cells showed defective pre-rRNA processing, including accumulation of the 35S rRNA precursor, presence of a 23S aberrant precursor, decreased 20S pre-rRNA levels, and accumulation of 27S pre-rRNA. Delayed processing of 27S pre-rRNA appeared to be the cause of reduced synthesis of 25S rRNA relative to 18S rRNA, which may be responsible for the deficit of 60S subunits in these cells.     

Functional analysis of Rrp7p, an essential yeast protein involved in pre-rRNA processing and ribosome assembly

During the functional analysis of open reading frames (ORFs) identified during the sequencing of chromosome III of Saccharomyces cerevisiae, the previously uncharacterized ORF YCL031C (now designated RRP7) was deleted. RRP7 is essential for cell viability, and a conditional null allele was therefore constructed, by placing its expression under the control of a regulated GAL promoter. Genetic depletion of Rrp7p inhibited the pre-rRNA processing steps that lead to the production of the 20S pre-rRNA, resulting in reduced synthesis of the 18S rRNA and a reduced ratio of 40S to 60S ribosomal subunits. A screen for multicopy suppressors of the lethality of the GAL::rrp7 allele isolated the two genes encoding a previously unidentified ribosomal protein (r-protein) that is highly homologous to the rat r-protein S27. When present in multiple copies, either gene can suppress the lethality of an RRP7 deletion mutation and can partially restore the ribosomal subunit ratio in Rrp7p-depleted cells. Deletion of both r-protein genes is lethal; deletion of either single gene has an effect on pre-rRNA processing similar to that of Rrp7p depletion. We believe that Rrp7p is required for correct assembly of rpS27 into the preribosomal particle, with the inhibition of pre-rRNA processing appearing as a consequence of this defect.     

The exosome subunit Rrp43p is required for the efficient maturation of 5.8S, 18S and 25S rRNA

The Saccharomyces cerevisiae protein Rrp43p co-purifies with four other 3'-->5' exoribonucleases in a complex that has been termed the exosome. Rrp43p itself is similar to prokaryotic RNase PH. Individual exosome subunits have been implicated in the 3' maturation of the 5.8S rRNA found in 60S ribosomes and the 3' degradation of mRNAs. However, instead of being deficient in 60S ribosomes, Rrp43p-depleted cells were deficient in 40S ribosomes. Pulse-chase and steady-state northern analyses of pre-RNA and rRNA levels revealed a significant delay in the synthesis of both 25S and 18S rRNAs, accompanied by the stable accumulation of 35S and 27S pre-rRNAs and the under-accumulation of 20S pre-rRNA. In addition, Rrp43p-depleted cells accumulated a 23S aberrant pre-rRNA and a fragment excised from the 5' ETS. Therefore, in addition to the maturation of 5.8S rRNA, Rrp43p is required for the maturation 18S and 25S rRNA.     

Optimization of excimer-forming two-probe nucleic acid hybridization method with pyrene as a fluorophore

A previously presented homogeneous assay method, named the excimer-forming two-probe nucleic acid hybridization (ETPH) method, is based on specific excimer formation between two pyrenes attached at the neighboring terminals of two sequential probe oligonucleotides complementary to a single target. In this study, we investigated assay conditions and optimal molecular design of probes for intense excimer emission using a pyrenemethyliodoacetamide-introduced 16mer probe, a pyrene butanoic acid-introduced 16merprobe and a target 32mer. The length of the linker between the pyrene residue and the terminal sugar moiety remarkably influenced the quantum efficiency of excimer emission; the pair of linker arms of these two probes was optimal. The quantum efficiency was also dependent upon the concentrations of dimethylformamide and NaCl added to the assay solution. Spectroscopic measurements and T m analysis showed that an optimal configuration of the two pyrene residues for intense excimer emission might be affected by pyrene-pyrene interaction, pyrene-duplex interaction (intercalation/stacking) and solvent conditions as a whole. We then demonstrated the practicality of the ETPH method with the optimal hybridization conditions thus attained by determining that the concentration of 16S rRNA in extracts from Vibrio mimicus ATCC 33655 cells in exponential growth phase is 18 500 16S rRNA molecules/cell on average.     

Variable regions V13 and V3 of Saccharomyces cerevisiae contain structural features essential for normal biogenesis and stability of 5.8S and 25S rRNA

The homologous ribosomal RNA species of all organisms can be folded into a common "core" secondary structure. In addition, eukaryotic rRNAs contain a large number of segments, located at fixed positions, that are highly variable in size and sequence from one organism to another. We have investigated the role of the two largest of these variable regions in Saccharomyces cerevisiae 25S rRNA, V13, and V3, by mutational analysis in a yeast strain that can be rendered completely dependent on the synthesis of mutant (pre-)rRNA. We found that approximately half of variable region V13 can be deleted without any phenotypic effect. The remaining portion, however, contains multiple structural features whose disturbance causes serious growth defects or lethality. Accumulation of 25S rRNA is strongly reduced by these mutations, at least in part because they inhibit processing of ITS2. Removal of even a relatively small portion of V3 also strongly reduces the cellular growth rate and larger deletions are lethal. Interestingly, some of the deletions in V3 cause accumulation of 27S(A) pre-rRNA and, moreover, appear to interfere with the close coupling between the processing cleavages at sites A3 and B1(S). These results demonstrate that both variable regions play an important role in 60S subunit formation.     

The structure of the ITS2-proximal stem is required for pre-rRNA processing in yeast

Accurate and efficient processing of pre-rRNA is critical to the accumulation of mature functional ribosomal subunits for maintenance of cell growth. Processing requires numerous factors which act in trans as well as RNA sequence/ structural elements which function in cis. To examine the latter, we have used directed mutagenesis and expression of mutated pre-rRNAs in yeast. Specifically, we tested requirements for formation of an ITS2-proximal stem on processing, a structure formed by an interaction between sequences corresponding to the 3' end of 5.8S rRNA and the 5' end of 25S. Pre-rRNA processing is inhibited in templates encoding mutations that prevent the formation of the ITS2-proximal stem. Compensatory, double mutations, which alter the sequence of this region but restore the structure of the stem, also restore processing, although at lower efficiency. This reduction in efficiency is reflected in decreased levels of mature 5.8S and 25S rRNA and increased levels of 35S pre-rRNA and certain processing intermediates. This phenotype is reminiscent of the biochemical depletion of U8 snoRNA in vertebrates for which the ITS2-proximal stem has been proposed as a potential site for interaction with U8 RNP. Thus, formation of the ITS2-proximal stem may be a requirement common to yeast and vertebrate pre-rRNA processing.     

Topography of the E site on the Escherichia coli ribosome

Three photoreactive tRNA probes have been utilized in order to identify ribosomal components that are in contact with the aminoacyl acceptor end and the anticodon loop of tRNA bound to the E site of Escherichia coli ribosomes. Two of the probes were derivatives of E. coli tRNA(Phe) in which adenosines at positions 73 and 76 were replaced by 2-azidoadenosine. The third probe was derived from yeast tRNA(Phe) by substituting wyosine at position 37 with 2-azidoadenosine. Despite the modifications, all of the photoreactive tRNA species were able to bind to the E site of E. coli ribosomes programmed with poly(A) and, upon irradiation, formed covalent adducts with the ribosomal subunits. The tRNA(Phe) probes modified at or near the 3' terminus exclusively labeled protein L33 in the 50S subunit. The tRNA(Phe) derivative containing 2-azidoadenosine within the anticodon loop became cross-linked to protein S11 as well as to a segment of the 16S rRNA encompassing the 3'-terminal 30 nucleotides. We have located the two extremities of the E site-bound tRNA on the ribosomal subunits according to the positions of L33, S11 and the 3' end of 16S rRNA defined by immune electron microscopy. Our results demonstrate conclusively that the E site is topographically distinct from either the P site or the A site, and that it is located alongside the P site as expected for the tRNA exit site.     

Nop5p is a small nucleolar ribonucleoprotein component required for pre-18 S rRNA processing in yeast

We have identified a novel nucleolar protein, Nop5p, that is essential for growth in Saccharomyces cerevisiae. Monoclonal antibodies B47 and 37C12 recognize Nop5p, which has a predicted size of 57 kDa and possesses a KKX repeat motif at its carboxyl terminus. Truncations that removed the KKX motif were functional and localized to the nucleolus, but conferred slow growth at 37 degreesC. Nop5p shows significant sequence homology with yeast Sik1p/Nop56p, and putative homologues in archaebacteria, plants, and human. Depletion of Nop5p in a GAL-NOP5 strain lengthened the doubling time about 5-fold, and selectively reduced steady-state levels of 40 S ribosomal subunits and 18 S rRNA relative to levels of free 60 S subunits and 25 S rRNA. Northern blotting and primer extension analyses showed that Nop5p depletion impairs processing of 35 S pre-rRNA at the A0 and A2 cleavage sites. Nop5p is associated with the small nucleolar RNAs U3, snR13, U14, and U18. Depletion of Nop5p caused the nucleolar protein Nop1p (yeast fibrillarin) to be localized to the nucleus and cytosol. Also, 37C12 co-immunoprecipitated Nop1p. These results suggest that Nop5p functions with Nop1p in the execution of early pre-rRNA processing steps that lead to formation of 18 S rRNA.     

Regulation of ribosomal RNA synthesis and processing during inhibition of protein synthesis by 1,3-bis(2-chloroethyl)-1-nitrosourea

The antineoplastic agent BCNU (1,3-bis(2-chloroethyl)-1nitrosourea) at a concentration of 25 mug/ml inhibits initiation of protein synthesis in HeLa cells. At this low concentration of the drug, the rate of synthesis of 45S ribosomal precursor RNA (pre-rRNA) is selectively inhibited without a marked inhibition of nucleoplasmic RNA. The inhibitory effects of the drug are readily reversible upon removal of BCNU from the growth medium. Pulse-chase analysis of the labeled nucleolar RNA in sucrose-gradients and acrylamide gels indicated that the 45S pre-rRNA synthesized before the addition of BCNU matures normally in the presence of the inhibitor. However, the processing of precursor RNA molecules synthesized following the addition of the drug is inhibited when incubation is continued on in the presence of 25 mug/ml BCNU. Since the formation of mature ribosomes is blocked by BCNU, the data would suggest that the effectiveness of the drug as a potent cell growth inhibitor may result from its inhibition of ribosome formation induced by inhibition of protein synthesis.     

Spb4p, an essential putative RNA helicase, is required for a late step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae

Spb4p is a putative ATP-dependent RNA helicase that is required for synthesis of 60S ribosomal subunits. Polysome analyses of strains genetically depleted of Spb4p or carrying the cold-sensitive spb4-1 mutation revealed an underaccumulation of 60S ribosomal subunits. Analysis of pre-rRNA processing by pulse-chase labeling, northern hybridization, and primer extension indicated that these strains exhibited a reduced synthesis of the 25S/5.8S rRNAs, due to inhibition of processing of the 27SB pre-rRNAs. At later times of depletion of Spb4p or following transfer of the spb4-1 strain to more restrictive temperatures, the early pre-rRNA processing steps at sites A0, Al, and A2 were also inhibited. Sucrose gradient fractionation showed that the accumulated 27SB pre-rRNAs are associated with a high-molecular-weight complex, most likely the 66S pre-ribosomal particle. An HA epitope-tagged Spb4p is localized to the nucleolus and the adjacent nucleoplasmic area. On sucrose gradients, HA-Spb4p was found almost exclusively in rapidly sedimenting complexes and showed a peak in the fractions containing the 66S pre-ribosomes. We propose that Spb4p is involved directly in a late and essential step during assembly of 60S ribosomal subunits, presumably by acting as an rRNA helicase.     

Dbp6p is an essential putative ATP-dependent RNA helicase required for 60S-ribosomal-subunit assembly in Saccharomyces cerevisiae

A previously uncharacterized Saccharomyces cerevisiae open reading frame, YNR038W, was analyzed in the context of the European Functional Analysis Network. YNR038W encodes a putative ATP-dependent RNA helicase of the DEAD-box protein family and was therefore named DBP6 (DEAD-box protein 6). Dbp6p is essential for cell viability. In vivo depletion of Dbp6p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase labeling of pre-rRNA and steady-state analysis of pre-rRNA and mature rRNA by Northern hybridization and primer extension show that Dbp6p depletion leads to decreased production of the 27S and 7S precursors, resulting in a depletion of the mature 25S and 5.8S rRNAs. Furthermore, hemagglutinin epitope-tagged Dbp6p is detected exclusively within the nucleolus. We propose that Dbp6p is required for the proper assembly of preribosomal particles during the biogenesis of 60S ribosomal subunits, probably by acting as an rRNA helicase.     

Variable region V1 of Saccharomyces cerevisiae 18S rRNA participates in biogenesis and function of the small ribosomal subunit

The role of helix 6, which forms the major portion of the most 5'-located expansion segment of Saccharomyces cerevisiae 18S rRNA, was studied by in vivo mutational analysis. Mutations that increased the size of the helical part and/or the loop, even to a relatively small extent, abolished 18S rRNA formation almost completely. Concomitantly, 35S pre-rRNA and an abnormal 23S precursor species accumulated. rDNA units containing these mutations did not support cell growth. A deletion removing helix 6 almost completely, on the other hand, had a much less severe effect on the formation of 18S rRNA, and cells expressing only the mutant rRNA remained able to grow, albeit at a much reduced rate. Disruption of the apical A.U base pair by a single point mutation did not cause a noticeable reduction in the level of 18S rRNA but did result in a twofold lower growth rate of the cells. This effect could not be reversed by introduction of a second point mutation that restores base pairing. We conclude that both the primary and the secondary structure of helix 6 play an important role in the formation and the biological function of the 40S subunit.     

Synthetic lethal interactions with conditional poly(A) polymerase alleles identify LCP5, a gene involved in 18S rRNA maturation

To identify new genes involved in 3'-end formation of mRNAs in Saccharomyces cerevisiae, we carried out a screen for synthetic lethal mutants with the conditional poly(A) polymerase allele, pap1-7. Five independent temperature-sensitive mutations called Icp1 to Icp5 (for lethal with conditional pap1 allele) were isolated. Here, we describe the characterization of the essential gene LCP5 which codes for a protein with a calculated molecular mass of 40.8 kD. Unexpectedly, we found that mutations in LCP5 caused defects in pre-ribosomal RNA (pre-rRNA) processing, whereas mRNA 3'-end formation in vitro was comparable to wild-type. Early cleavage steps (denoted A0 to A2) that lead to the production of mature 18S rRNA were impaired. In vivo depletion of Lcp5p also inhibited pre-rRNA processing. As a consequence, mutant and depleted cells showed decreased levels of polysomes compared to wild-type cells. Indirect immunofluorescence indicated a predominant localization of Lcp5p in the nucleolus. In addition, antibodies directed against Lcp5p specifically immunoprecipitated the yeast U3 snoRNA snR17, suggesting that the protein is directly involved in pre-rRNA processing.     

Nip7p interacts with Nop8p, an essential nucleolar protein required for 60S ribosome biogenesis, and the exosome subunit Rrp43p

NIP7 encodes a conserved Saccharomyces cerevisiae nucleolar protein that is required for 60S subunit biogenesis (N. I. T. Zanchin, P. Roberts, A. DeSilva, F. Sherman, and D. S. Goldfarb, Mol. Cell. Biol. 17:5001-5015, 1997). Rrp43p and a second essential protein, Nop8p, were identified in a two-hybrid screen as Nip7p-interacting proteins. Biochemical evidence for an interaction was provided by the copurification on immunoglobulin G-Sepharose of Nip7p with protein A-tagged Rrp43p and Nop8p. Cells depleted of Nop8p contained reduced levels of free 60S ribosomes and polysomes and accumulated half-mer polysomes. Nop8p-depleted cells also accumulated 35S pre-rRNA and an aberrant 23S pre-rRNA. Nop8p-depleted cells failed to accumulate either 25S or 27S rRNA, although they did synthesize significant levels of 18S rRNA. These results indicate that 27S or 25S rRNA is degraded in Nop8p-depleted cells after the section containing 18S rRNA is removed. Nip7p-depleted cells exhibited the same defects as Nop8p-depleted cells, except that they accumulated 27S precursors. Rrp43p is a component of the exosome, a complex of 3'-to-5' exonucleases whose subunits have been implicated in 5.8S rRNA processing and mRNA turnover. Whereas both green fluorescent protein (GFP)-Nop8p and GFP-Nip7p localized to nucleoli, GFP-Rrp43p localized throughout the nucleus and to a lesser extent in the cytoplasm. Distinct pools of Rrp43p may interact both with the exosome and with Nip7p, possibly both in the nucleus and in the cytoplasm, to catalyze analogous reactions in the multistep process of 60S ribosome biogenesis and mRNA turnover.