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R N Nazar

Publications and source records attributed to R N Nazar.

At least 19 recordsLinked to original sources
PubMed2001

A chaperone for ribosome maturation.

The nascent pre-rRNA of eukaryotic ribosomes is fully transcribed and assembled into an 80-90 S nucleolar particle before being cleaved into mature ribosomal RNA. The interdependence of steps in the processing of this precursor RNA indicates that RNA processing, at least in part, acts as a quality control mechanism that helps ensure that only functional RNA is incorporated into mature ribosomes. In search of structural components that underlie this interdependence using the Schizosaccharomyces pombe internal transcribed spacer 1 (ITS) as a ligand for affinity chromatography of ITS1-specific proteins, we have isolated a large spliceosome-like protein complex, a ribosome assembly chaperone (RAC) of 20 or more polypeptides (Lalev, A. I., Abeyrathne, P. D., and Nazar, R. N. (2000) J. Mol. Biol. 302, 65-77). When the ITS2 spacer was used in the present study to isolate ITS2-specific proteins, the same proteins were identified consistent with a complex containing multiple specific binding sites. Subsequent competition binding studies indicated that the protein complex actually contains independent binding sites for all four of the transcribed spacers in the pre-rRNA. Because disruption of protein-binding sites in these spacer RNAs is known to severely affect rRNA processing, taken together these results suggest that the RAC complex is a chaperone for ribosome maturation acting as a "rack" on which critical structure is organized.

Binding Sites↗
PubMed2000

Ribosomal RNA maturation in Schizosaccharomyces pombe is dependent on a large ribonucleoprotein complex of the internal transcribed spacer 1.

The interdependency of steps in the processing of pre-rRNA in Schizosaccharomyces pombe suggests that RNA processing, at least in part, acts as a quality control mechanism which helps assure that only functional RNA is incorporated into mature ribosomes. To determine further the role of the transcribed spacer regions in rRNA processing and to detect interactions which underlie the interdependencies, the ITS1 sequence was examined for its ability to form ribonucleoprotein complexes with cellular proteins. When incubated with protein extract, the spacer formed a specific large RNP. This complex was stable to fractionation by agarose or polyacrylamide gel electrophoresis. Modification exclusion analyses indicated that the proteins interact with a helical domain which is conserved in the internal transcribed spacers. Mutagenic analyses confirmed an interaction with this sequence and indicated that this domain is critical to the efficient maturation of the precursor RNA. The protein constituents, purified by affinity chromatography using the ITS1 sequence, retained an ability to form stable RNP. Protein analyses of gel purified complex, prepared with affinity-purified proteins, indicated at least 20 protein components ranging in size from 20-200 kDa. Peptide mapping by Maldi-Toff mass spectroscopy identified eight hypothetical RNA binding proteins which included four different RNA-binding motifs. Another protein was putatively identified as a pseudouridylate synthase. Additional RNA constituents were not detected. The significance of this complex with respect to rRNA maturation and interdependence in rRNA processing is discussed.

Base Sequence↗
PubMed2000

Cotranscription of 5S rRNA-tRNA(Arg)(ACG) from Brassica napus chloroplasts and processing of their intergenic spacer.

S1 mapping showed that at least a significant portion of the 5S rRNA and tRNA(Arg)(ACG) is co-transcribed in canola chloroplast, making trnR the last gene transcribed in an operon of which the final sequence is 5'-16S-tRNA(Ile)-tRNA(Ala)-23S-4.5S-5S-tRNA(Arg)-3'. Various RNA termini representing RNA processing sites at several parts of the 5S rRNA-tRNA(Arg) area were detected. This gene spacer is substantially conserved among various species compared here, and a secondary structure model for this chloroplast region in canola applies to other plant sequences. The conservation of this intergenic sequence suggests a functional role, possibly by providing recognition structures for endogenous RNases involved in its maturing process.

Base Sequence↗
PubMed1999

Structural equivalence in the transcribed spacers of pre-rRNA transcripts in Schizosaccharomyces pombe.

The structure of the internal transcribed spacer 2 (ITS2) in Schizosaccharomyces pombe was re-evaluated with respect to phylogenetically conserved features in yeasts, features in other transcribed spacer regions as well as the binding of transacting factors which potentially play a role in ribosomal maturation. Computer analyses and probes for nuclease protection indicate a very simple core structure consisting of a single extended hairpin which includes the interacting termini of the mature 5.8S and 25S rRNAs. Comparisons with ITS2 sequences in greatly diverging organisms indicate that the same feature also can be recognized. This is especially clear in organisms that contain very short sequences in which the putative structures are much less ambiguous. Diversity between organisms is the result of changes in hairpin length as well as the addition of branched helices. Protein binding and gel retardation studies with the S.pombe ITS2 further indicate that, as observed in the 3" external transcribed spacer (ETS) and ITS1 regions, the extended hairpin is not only the site of intermediate RNA cleavage during rRNA processing but also a site for specific interactions with one or more soluble factors. Taken together with other analyses on transcribed spacer regions, the present data suggest that the spacer regions all may act in a similar fashion, not only to organize the maturing terminal sequences, but also serve to organize specific soluble factors possibly acting with snoRNAs or in a manner which is analogous with that of the free snoRNPs.

Base Pairing↗
PubMed1999

Terminator element mutations affect both the efficiency and position of RNA polymerase I termination in Schizosaccharomyces pombe.

RNA polymerase I transcripts, purified from Schizosaccharomyces pombe cells, terminate at three sites that precede 'Sal box'-like termination element (TE) sequences. Essential features in these elements were investigated by the in vivo expression of targeted mutations. RNA analyses confirmed a functional significance for two of the elements (Boxes 1 and 3), but indicated that the third, less related, sequence (Box 2) does not function as a termination signal. The results further indicated that the most conserved residues in the two active TEs, as well as adjacent regions, are also most critical to function. Furthermore, some mutations in these elements or in immediately flanking sequences affect not only the efficiency of termination, but also alter the position of termination by as much as 35 nt. Since the element is able to influence the site of termination over a surprisingly long stretch of DNA sequence, these observations suggest that the TE does not act simply as a pause element by fixing the termination factor.

Base Sequence↗
PubMed1999

Essential structural features in the Schizosaccharomyces pombe pre-rRNA 5' external transcribed spacer.

The proximal region in the 5' external transcribed spacer (5'ETS) of the genes encoding ribosomal RNAs in Schizosaccharomyces pombe was examined with respect to structural features which underlie rRNA maturation. Computer analyses and partial digestion with nuclease probes indicate a crucifix-like structure composed primarily of three extended hairpins which are more highly ordered than previously proposed in Saccharomyces cerevisiae. A re-evaluation of the same region in S. cerevisiae indicates a conserved core structure, including the U3 snoRNA binding site within this higher-order structure. The sequences encoding the individual hairpins were deleted by PCR-mediated mutagenesis and the mutant rDNAs were expressed in vivo to determine the effect of these features on rRNA maturation. Quantitative hybridization analyses indicate that the first hairpin only has modest effects on 18 S rRNA maturation, but the other two regions are critical and no mature 18 S rRNA was observed. When smaller changes were systematically introduced into the critical regions, strong correlations were observed with known or putative events in rRNA maturation. Changes associated with an intermediate cleavage site in helix II and with the putative U3 snoRNA binding site were again critical to 18 S rRNA production. In each case, the effects were sequence dependent and not simply the result of disrupted structure. Further analyses of the 5.8 S rRNA indicate that the large ribosomal subunit RNA can be properly processed in each case but the efficiency is reduced by as much as 60 %, an observation which provides new evidence of interdependency in the maturation process. The results illustrate that rRNA processing is more critically dependent on the 5'ETS than previously believed.

Base Sequence↗
PubMed1998

Conserved core structure in the internal transcribed spacer 1 of the Schizosaccharomyces pombe precursor ribosomal RNA.

The structure of the internal transcribed spacer 1 (ITS1) in Schizosaccharomyces pombe was examined with respect to phylogenetically conserved features in yeasts as well as the binding of transacting factors that potentially play a role in ribosomal maturation. Computer analyses and probes for nuclease protection indicate a compact, more highly organized structure than previously proposed in Saccharomyces cerevisiae, with distinct structural features which can be recognized in S. cerevisiae. These include a central extended hairpin structure as well as smaller hairpins immediately adjacent to the maturing termini. Comparisons with ITS sequences in more diverse organisms indicate that the same features also can be recognized. This is especially clear in organisms which contain very short sequences in which the putative structures are much less ambiguous. Again nuclease protection analyses in one of these, Verticillium albo-atrum, confirm a central hairpin with additional hairpins linked to the maturing termini. Protein binding and gel retardation studies with the S. pombe ITS1 further indicate that, as observed in the 3' external transcripted spacer (ETS) region, the extended hairpin is not only the site of intermediate RNA cleavage during rRNA processing, but also a site for specific interactions with one or more soluble factors. Taken together with other analyses on transcribed spacer regions, the present data provide evidence that the spacer regions act not only to organize the maturing terminal sequences but also may serve to organize specific soluble factors, possibly acting in a manner which is analogous with that of the free small nucleolar ribonucleo protein particles (snoRNPs).

Animals↗
PubMed1997

In vivo analyses of RNA polymerase I termination in Schizosaccharomyces pombe.

Recent studies on the termination of rDNA transcription by RNA polymerase I in Saccharomyces cerevisiae and Schizosaccharomyces pombe have suggested a more complex mechanism then previously described in higher eukaryotes. Termination appears to occur when a DNA-bound Reb1 protein molecule induces polymerase to pause in the context of a release element [see Reeder,R.H. and Lang,W. (1994) Mol. Microbiol ., 12, 11-15]. Because these conclusions in yeast were based entirely on in vitro analyses, we have examined the same termination process in S.pombe by expressing targeted mutations in vivo . S1nuclease protection studies indicate three tandemly arranged termination sites with most transcripts very efficiently terminated at the first site, 267 nt after the 3' end of the mature 25S rRNA sequence. Termination at each site is mediated by conserved terminator elements which bear limited sequence homology with that of mouse and also can be identified in S.cerevisiae . Removal of the first terminator element transfers dominance to the second site and construction of a new single terminator element at +150 still results in efficient termination and rRNA processing without a need for an additional upstream element. Genomic 'footprint' analyses and gel retardation assays confirm a process mediated by a strongly interacting protein factor but implicate an alternate binding site. Removal of the 5' flanking sequence or structure also had no effect on the site or efficiency of termination. Taken together the results in vivo suggest that the termination process in this fission yeast more strongly resembles the single element-mediated mechanism initially reported in mouse and is not dependent on additional upstream sequence as first reported in S.cerevisiae and postulated to function in general.

Base Sequence↗
PubMed1997

Structural features in the 3' external transcribed spacer affecting intragenic processing of yeast rRNA.

A highly conserved extended hairpin structure in the 3' external transcribed spacer (3' ETS) region of nascent eukaryotic rRNA transcripts is essential for the maturation of the large ribosomal subunit RNAs (5.8 S and 25 to 28 S rRNAs). Systematic changes were introduced into this structure by PCR-mediated mutagenesis and the mutant rDNAs were expressed in vivo to determine the structural features that are essential for rRNA maturation. Changes in the lower half of the stem or the large loop at the end had little or no effect on the maturation of either the 5.8 S or 25 S rRNA, but changes that disrupted secondary structure in the upper half of this stem had equal and dramatic effects on both RNAs. When the RNA stem was incubated with a cellular protein extract, gel retardation studies indicated that the stem forms a ribonucleoprotein complex, and a comparison with mutant RNA indicated that protein binding could be compromised by changes that were critical for rRNA maturation. Sequence comparisons with other spacer regions as well as snRNAs reveal some structural analogy, which, when taken together with the mutational studies, raise the possibility that this hairpin functions during RNA processing in a manner that may be analogous with that of free snRNPs.

Base Sequence↗
PubMed1997

Interdependence in the processing of ribosomal RNAs in Schizosaccharomyces pombe.

Eukaryotic rRNAs are produced by cleavage of a large 35 to 45 S pre-rRNA transcript which initially must be fully transcribed and assembled into an 80 to 90 S nucleolar ribonucleoprotein particle. Despite this need for a completed transcript, several investigations have reported a split processing scheme for independent maturation of the large and small subunit rRNAs. Here, an efficiently expressed rDNA plasmid was used to quantitatively analyze the effects of mutations in the internal transcribed spacer (ITS) region in the yeast, Schizosaccharomyces pombe. The results show that substitution of ITS regions inhibits the processing of distant external transcribed spacers (ETS) and that deletion of the ITS2 spacer not only prevents the maturation of the large subunit, but severely affects maturation of the small subunit rRNA. This indicates that the processing mechanisms are not fully split and, when taken together with other evidence of interdependences in rRNA maturation, the results suggest that the interdependences act as a quality control mechanism to help ensure that only functional rRNA is incorporated into ribosomes.

Nucleic Acid Conformation↗
PubMed1997

The ribosomal 5.8S RNA as a target site for p53 protein in cell differentiation and oncogenesis.

Previous studies have shown that the ribosomal 5.8S rRNA of human cells is partially methylated in a tissue-specific fashion, a modification which occurs largely or entirely in the cytoplasm. More recent studies have shown that the 5.8S rRNA forms a covalent linkage with tumor suppressor p53 protein and have suggested that this RNA plays a functional role in protein elongation. We now show that the expression of p53 protein in Schizosaccharomyces pombe results in cells which: morphologically resemble transformed cells expressing mutant 5.8S rRNA; are equally compromised in their ability to sustain protein synthesis, in vitro; and contain polyribosome profiles which strongly resemble the elevated profiles which also are observed with mutated 5.8S rRNA. Taken together, these results provide new physiological evidence of the possibility that the 5.8S rRNA is an important target in the control of ribosome function during cell differentiation and oncogenesis.

Cell Differentiation↗
PubMed1997

The ribosomal-RNA-processing pathway in Schizosaccharomyces pombe.

In all cells, a long precursor RNA is processed into mature rRNAs for ribosome biogenesis. In eukaryotes, the complexity and speed of the overall process often has made it difficult to establish finer details of the maturation pathway. Since phylogenetic comparisons can provide evidence for critical events, the major rRNA processing pathway for the yeast Schizosaccharomyces pombe was determined using primer extension, nuclease protection and Northern-hybridisational analyses. Transcript mapping of the 5' external transcribed spacer revealed six cleavage sites which occur upstream of the mature 18S termini. Two of these sites as well as a site adjacent to the 18S termini are complementary to conserved Box sequences in the S. pombe U3 small nucleolar RNA. Transcript mapping of the internal transcribed spacers (1 and 2) suggest similar maturation schemes for the two spacers, in which an initial endonuclease cleavage is followed by processing to the mature termini. The mature 5' termini of 25S rRNA appear to be heterogeneous in S. pombe, as has been demonstrated for 5.8S rRNA, suggesting an essential limiting structure in the ribosome-integrated mature RNA. Together with our previous analysis of the 3' external spacer region, the results reveal the major processing pathway for S. pombe and further support a maturation process which acts as a quality assurance mechanism.

Base Sequence↗
PubMed1997

In vivo analyses of upstream promoter sequence elements in the 5 S rRNA gene from Saccharomyces cerevisiae.

Upstream promoter elements of the Saccharomyces cerevisiae 5 S rRNA gene have been characterized by genomic DNase I "footprinting" and by in vivo mutational analyses using base substitutions and deletions. A high copy shuttle-vector was used to efficiently express the mutant 5 S rRNA genes in vivo and a structural mutation in the 5 S rRNA, which was previously shown to be functionally neutral but easily detected by gel electrophoresis, allowed for an accurate measure of gene expression. The results provide direct evidence for upstream regulatory elements which confirms a start site element (sse) from -1 to -8 and identifies a new independent upstream promoter element (upe) centered from about -17 to -20. In contrast to previous reports with reconstituted systems, both elements dramatically affect the efficiency of gene expression and suggest that the saturated conditions which are used in reconstituted studies mask sequence dependence; a dependency that could be physiologically significant and play a role in the regulation of 5 S rRNA expression. The footprint analyses support an extended region of protein interaction as recently observed in reconstituted systems but again provide evidence of significant structural rearrangements when the upstream sequence is changed.

Base Sequence↗
PubMed1997

Ribosomal 5 S rRNA maturation in Saccharomyces cerevisiae.

The maturation of the ribosomal 5 S RNA in Saccharomyces cerevisiae is examined based on the expression of mutant 5 S rRNA genes, in vivo, and a parallel analysis of RNA processing, in vitro. Both types of analysis indicate that 5 S rRNA processing is not dependent on the nucleotide sequence of either the external transcribed spacer or the mature 5 S rRNA. The results further indicate the RNA is processed by an exonuclease activity which is limited primarily or entirely by helix I, the secondary structure formed between the mature and interacting termini. The 5 S RNA binding protein (YL3) also appears not to influence directly the maturation process, but rather to play a role in protecting the rRNA from further degradation by "housekeeping" nucleases. Taken together, the results continue to support a "quality control" function which helps to ensure that during maturation only normal precursors are processed and assembled into active ribosomes.

Nucleic Acid Conformation↗
PubMed1997

Role of the 5.8S rRNA in ribosome translocation.

Studies on the inhibition of protein synthesis by specific anti 5.8S rRNA oligonucleotides have suggested that this RNA plays an important role in eukaryotic ribosome function. Mutations in the 5. 8S rRNA can inhibit cell growth and compromise protein synthesis in vitro . Polyribosomes from cells expressing these mutant 5.8S rRNAs are elevated in size and ribosome-associated tRNA. Cell free extracts from these cells also are more sensitive to antibiotics which act on the 60S ribosomal subunit by inhibiting elongation. The extracts are especially sensitive to cycloheximide and diphtheria toxin which act specifically to inhibit translocation. Studies of ribosomal proteins show no reproducible changes in the core proteins, but reveal reduced levels of elongation factors 1 and 2 only in ribosomes which contain large amounts of mutant 5.8S rRNA. Polyribosomes from cells which are severely inhibited, but contain little mutant 5.8S rRNA, do not show the same reductions in the elongation factors, an observation which underlines the specific nature of the change. Taken together the results demonstrate a defined and critical function for the 5.8S rRNA, suggesting that this RNA plays a role in ribosome translocation.

Biological Transport↗