Ribosomal Precursor Research Articles

Stereochemistry of RNA Cleavage by the Tetrahymena Ribozyme and Evidence that the Chemical Step Is Not Rate-Limiting

The intervening sequence of the ribosomal RNA precursor of Tetrahymena is a catalytic RNA molecule, or ribozyme. Acting as a sequence-specific endoribonuclease, it cleaves single-stranded RNA substrates with concomitant addition of guanosine. The chemistry of the reaction has now been studied by introduction of a single phosphorothioate in the substrate RNA at the cleavage site. Kinetic studies show no significant effect of this substitution on kcat (rate constant) or Km (Michaelis constant), providing evidence that some step other than the chemical step is rate-limiting. Product analysis reveals that the reaction proceeds with inversion of configuration at phosphorus, consistent with an in-line, SN2 (P) mechanism. Thus, the ribozyme reaction is in the same mechanistic category as the individual displacement reactions catalyzed by protein nucleotidyltransferases, phosphotransferases, and nucleases.

9] Isolation and characterization of yeast ribosomal RNA precursors and preribosomes

This chapter describes the identification and characterization of the precursor RNAs and ribonucleoproteins (RNPs) from the 35 S pre-rRNA operon. In addition to 35 S prerRNA, 20 and 27 S pre-rRNAs are the predominant intermediates. The 32 S pre-rRNA is found in variable yields depending on the genetic background of the particular strain and the conditions of growth and harvesting. The major part of the ribosome biogenesis pathway is located in the nucleolus. Shortly after, or possibly already during transcription, 35 S pre-rRNA is subjected to a large number of modifications, including methylation and pseudouridylation. High-molecular-weight pre-rRNAs (20-35 S) are fractionated on agarose gels after glyoxylation or on standard formamide–formaldehyde gels and then are blotted onto a solid support. Filters are hybridized with probes containing ETS, ITS1, or ITS2 sequences, but lacking sequences corresponding to the mature and S rRNAs. Spheroplasts have to be poured into a 10-fold excess of ice-cold 1 M sorbitol containing unlabeled uracil or methionine.

A model for regulation of mammalian ribosomal DNA transcription. Co-ordination of initiation and termination.

Based on recent experimental data about transcription initiation and termination, a model for regulation of mammalian ribosomal DNA transcription is developed using a simple kinetic scheme. In this model, the existence of the transition pathway from the terminator to the promoter increases the rate of ribosomal RNA precursor synthesis. In addition to this 'non-transcribed spacer' traverse of RNA polymerase I, the co-ordination of initiation and termination allows a rapid on/off switch transition from the minimum to the maximum rate of ribosomal RNA precursor synthesis. Furthermore, taking account of the participation of two factors in the termination event, we propose a plausible molecular mechanism for the co-ordination of initiation and termination. This co-ordination is emphasized by repetition of the terminator unit.

Molecular genetics of group I introns: RNA structures and protein factors required for splicing — a review

In vivo and in vitro genetic techniques have been widely used to investigate the structure-function relationships and requirements for splicing of group-I introns. Analyses of group-I introns from extremely diverse genetic systems, including fungal mitochondria, protozoan nuclei, and bacteriophages, have yielded results which are complementary and highly consistent. In vivo genetic studies of fungal mitochondrial systems have served to identify cis-acting sequences within mitochondrial introns, and trans-acting protein products of mitochondrial and nuclear genes which are important for splicing, and to show that some mitochondrial introns are mobile genetic elements. In vitro genetic studies of the self-splicing intron within the Tetrahymena thermophila nuclear large ribosomal RNA precursor ( Tetrahymena LSU intron) have been used to examine essential and nonessential RNA sequences and structures in RNA-catalyzed splicing. In vivo and in vitro genetic analysis of the intron within the bacteriophage T4 td gene has permitted the detailed examination of mutant phenotypes

A Specific Amino Acid Binding Site Composed of RNA

A specific, reversible binding site for a free amino acid is detectable on the intron of the Tetrahymena self-splicing ribosomal precursor RNA. The site selects arginine among the natural amino acids, and prefers the L- to the D-amino acid. The dissociation constant is in the millimolar range, and amino acid binding is at or in the catalytic rG splicing substrate site. Occupation of the G site by L-arginine therefore inhibits splicing by inhibiting the binding of rG, without inhibition of later reactions in the splicing reaction sequence. Arginine binding specificity seems to be directed at the side chain and the guanidino radical, and the alpha-amino and carboxyl groups are dispensable for binding. The arginine site can be placed within the G site by structural homology, with consequent implications for RNA-amino acid interaction, for the origin of the genetic code, for control of RNA activities, and for further catalytic capabilities for RNA.

Reversion from Erythromycin Dependence in Escherichia coli: Strains Altered in Ribosomal Sub-unit Association and Ribosome Assembly

A mutant of Escherichia coli dependent on erythromycin for growth spontaneously gives erythromycin-independent strains with altered or missing ribosomal proteins. strains with defects in ribosome assembly were sought and obtained from among these revertants. Two organisms in which ribosomal protein L19 is altered and absent respectively have 70S ribosomes whose dissociation into sub-units is particularly sensitive to pressures generated during centrifuging. The mutant that lacks protein L19 also accumulates ribosome precursor particles during exponential growth as do others including mutants that lack proteins S20 or L1. These strains also show unbalanced synthesis of RNA and so will be useful in investigating both the pathways and the regulation of ribosome assembly.

The synergistic cytocidal effect produced by immune interferon and tumor necrosis factor in HT-29 cells is associated with inhibition of rRNA processing and (2′,5′)oligo(A) activation of RNase L

The cytocidal activity of human immune interferon (IFN-tau) in combination with recombinant tumor necrosis factor-alpha (TNF) was assessed in human colon carcinoma cell line HT-29. IFN-tau or TNF alone reduced cell viability by 15-20%, but in combination produced a synergistic cytotoxic effect which inhibited colony formation by 55-85%. TNF impaired the processing of ribosomal precursor 45S RNA to 32S RNA which resulted in the selective inhibition of 28S rRNA. Both TNF and IFN-tau induced the activity of (2',5') oligo (A) synthetase and produced selective degradation of 28S rRNA, effects possibly related to the (2',5') oligo (A)-dependent activation of RNase L. The combination of IFN-tau and TNF resulted in a marked reduction in the labeling of ribosomal precursor RNAs as well as mature rRNA. These results demonstrate that the posttranscriptional and processing effects of IFN-tau and TNF are related to the enhanced cytotoxicity by this combination of cytokines.

Differential effects of axotomy on immature and mature hamster facial neurons: a tritiated-uridine autoradiographic study.

In this study, tritiated-uridine incorporation was autoradiographically examined following axotomy of hamster facial motor neurons (HFMN) at the critical development age of 15 days postnatal and in the adult. The postoperative times selected were 0.5, 1, 2, and 4 days. In the 15-day operative series, no changes in incorporation were observed at any of the postoperative times, except at 4 days postoperative, when there was a decrease in tritiated-uridine incorporation in the axotomized neurons relative to the controls. In the adult operative series there were no changes in incorporation at 0.5 or 1 day postoperative, relative to the controls. At 2 days postoperative in the adult, there was a transient increase in tritiated-uridine incorporation that returned to control levels by 4 days postoperative. When axotomized and control cytoplasmic/nuclear grain densities were compared, no changes were found in either operative series. These results of the time course of axotomy-induced changes in RNA synthesis in HFMN corroborate our previous findings of an age-dependent reactive sequence in HFMN and lend support to the hypothesis that the young neurons are synthesizing at peak capacity related to final growth and cannot be stimulated further by axotomy. As discussed, the transient increase in RNA levels in the adult, the lack of any changes in the rate of transfer of RNA from the nucleus to the cytoplasm, and the decrease in RNA levels in the 15-day neurons may be related to the presence of an unusual intranucleolar body within the nucleolus of HFMN that contains ribosomal precursors.

ChemInform Abstract: The Chemistry of Self‐Splicing RNA and RNA Enzymes

Proteins are not the only catalysts of cellular reactions; there is a growing list of RNA molecules that catalyze RNA cleavage and joining reactions. The chemical mechanisms of RNA-catalyzed reactions are discussed with emphasis on the self-splicing ribosomal RNA precursor of Tetrahymena and the enzymatic activities of its intervening sequence RNA. Wherever appropriate, catalysis by RNA is compared to catalysis by protein enzymes.

Self-splicing RNA and an RNA enzyme in Tetrahymena.

The RNA molecules transcribed from many eukaryotic genes are interrupted by intervening sequences, which are removed by a process called RNA splicing. One structurally related group of intervening sequences, the group I intervening sequences, are found in a variety of microorganisms. Some of these, including the group I intervening sequence from the ribosomal RNA precursor of Tetrahymena thermophila, have been shown to mediate their own splicing in an RNA-catalyzed reaction. Following its excision from the ribosomal RNA precursor, the Tetrahymena intervening sequence acts as an enzyme, cutting and rejoining RNA substrates.

Activation of double-stranded RNA dependent protein kinase by ribosomal RNA precursors

Two major changes in RNA metabolism occured in macrophages expressing tumoricidal activity: a down regulation of total RNA synthesis and an imbalanced accumulation of ribosomal RNA precursors with double stranded secondary structure. The aim of this study was to investigate a possible role of endogenous ds rRNA precursors in the activation of the dsRNA dependent protein kinase. Using a cell free transcription system purified rRNA precursors were obtained from the murine rRNA gene. The results indicate that rRNA precursors are potent activators of the dsPK. The effects are dose dependent and are not affected by treatment of rRNA precursors with proteinase K or RNase A. The treatment with RNase V(1), specific for dsRNA, completely abrogates the activity of transcripts. The results suggest that endogenous RNA, namely rRNA, could control dsPK activation and it can be speculated that dsPK activation may be involved in the control of tumoricidal activity by macrophages.

Detection of sugar-binding sites in the fibrillar and the granular components of the nucleolus: An experimental study in cultured mammalian cells

The intranucleolar distribution of sugar-binding sites (i.e., lectin-like molecules) was analyzed in segregated nucleoli of actinomycin D-treated HeLa cells. The detection of sugar-binding sites was performed by incubation either of permeabilized nuclei in the presence of fluorescein-labeled neoglycoproteins or of ultrathin sections cut through in situ-fixed nuclei in the presence of gold-labeled neoglycoproteins. In the former case, the fluorescent nucleolar components were identified by comparison with the nucleolar components of similarly treated cells observed in electron microscopy. For the first time, this study reveals the presence of sugar-binding sites in both the fibrillar and the granular components of the nucleolus. In view of the data already reported on the biochemical composition of the nucleolus, some of our results led us to conclude that the nucleolar sugar-binding sites are lectin-like proteins. These proteins could be associated with preribosomes since the nucleolus is the site of both synthesis and stockage of ribosomal precursors. Some results from this study, however, show that the possibility of a relationship between some lectins and a structural component cannot be excluded.

Regulation of ribosome synthesis during compensatory renal hypertrophy in mice.

Ribosomal synthesis was studied at the transcriptional and translational levels to investigate the mechanisms of ribosome accretion during compensatory renal hypertrophy. As measured by in vitro transcriptional runoff comparisons 6-48 h after surgery, nuclei from the kidney remaining after contralateral nephrectomy show an increase of up to 150% in the rate of synthesis of ribosomal precursor RNA. The rate of rDNA transcription is 40-50% greater than control values as early as 6 h after nephrectomy; by 48 h, the rate returns to normal. In contrast to the stimulated transcription of rDNA and accretion of rRNA, the steady-state levels and the cytoplasmic distribution of ribosomal protein mRNAs S16 and L10 remain unchanged during induced renal growth. Thus coordinate production of adequate protein for increased assembly of ribosomes during induced renal growth appears to be accomplished by increasingly efficient translation of existing ribosomal protein mRNAs or by post-translational stabilization of ribosomal proteins. The rate of rDNA transcription may be regulated by accelerating the transcription of already functioning genes or, more likely, by recruiting transcription units that are transcriptionally inactive in the normal kidney.

5′ exon requirement for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA and identification of a cryptic 5′ splice site in the 3′ exon

The intervening sequence (IVS) of the Tetrahymena thermophila ribosomal RNA precursor undergoes accurate self-splicing in vitro. The work presented here examines the requirement for Tetrahymena rRNA sequences in the 5′ exon for the accuracy and efficiency of splicing. Three plasmids were constructed with nine, four and two nucleotides of the natural 5′ exon sequence, followed by the IVS and 26 nucleotides of the Tetrahymena 3′ exon. RNA was transcribed from these plasmids in vitro and tested for self-splicing activity. The efficiency of splicing, as measured by the production of ligated exons, is reduced as the natural 5′ exon sequence is replaced with plasmid sequences. Accurate splicing persists even when only four nucleotides of the natural 5′ exon sequence remain. When only two nucleotides of the natural exon remain, no ligated exons are observed. As the efficiency of the normal reaction diminishes, novel RNA species are produced in increasing amounts. The novel RNA species were examined and found to be products of aberrant reactions of the precursor RNA. Two of these aberrant reactions involve auto-addition of GTP to sites six nucleotides and 52 nucleotides downstream from the 3′ splice site. The former site occurs just after the sequence GGU, and may indicate the existence of a GGU-binding site within the IVS RNA. The latter site follows the sequence CUCU, which is identical with the four nucleotides preceding the 5′ splice site. This observation led to a model where the CUCU sequence in the 3′ exon acts as a cryptic 5′ splice site. The model predicted the existence of a circular RNA containing the first 52 nucleotides of the 3′ exon. A small circular RNA was isolated and partially sequenced and found to support the model. So, a cryptic 5′ splice site can function even if it is located downstream from the 3′ splice site. Precursor RNA labeled at its 5′ end, presumably by a GTP exchange reaction mediated by the IVS, is also described.

Ribosomal precursor 3′ end formation requires a conserved element upstream of the promoter

In Xenopus laevis the 3′ end of the longest intact ribosomal RNA precursor is formed by a processing event at site T2, which is located 7860 bp downstream of the site of transcription initiation. Processing at T2 is eliminated by mutations within the T2 box, a 7 nucleotide conserved element, GACTTGC, located 15 bp downstream of the 3′ ends. The same conserved box is also present at T3, a site 60 bp upstream of the gene promoter and that is part of a termination site. Surprisingly, mutations within the T3 box also eliminate processing at T2. To obtain proper T2 function, T3 can be at any distance but must be in the correct orientation upstream of a ribosomal gene promoter.

The Chemistry of Self-Splicing RNA and RNA Enzymes

Proteins are not the only catalysts of cellular reactions; there is a growing list of RNA molecules that catalyze RNA cleavage and joining reactions. The chemical mechanisms of RNA-catalyzed reactions are discussed with emphasis on the self-splicing ribosomal RNA precursor of Tetrahymena and the enzymatic activities of its intervening sequence RNA. Wherever appropriate, catalysis by RNA is compared to catalysis by protein enzymes.

The ribosomal DNA transcription unit of the house cricket, Acheta domesticus.

A composite map representing a single ribosomal DNA repeat unit of the house cricket, Acheta domesticus, was constructed from overlapping cloned fragments. Sites in the repeat unit for nine restriction enzymes were mapped. R-loop mapping of sequences coding for 18 S and 28 S RNA demonstrates that the 58-kb ribosomal DNA repeat unit contains a novel-sized internal transcribed spacer of 8.4 kb. The existence of this large spacer was confirmed in genomic DNA, most if not all of the genomic repeat units containing such a spacer. A 15- to 17-kb ribosomal RNA precursor transcript is synthesized as predicted on the basis of the size of the internal transcribed spacer. The 5.8 S RNA gene is localized to a 1-kb sequence immediately 5' to the 28 S gene. The coding regions examined contain no intervening sequences analogous to those described within ribosomal DNA of other eukaryotes. Only 11% of the repeat unit codes for mature ribosomal RNA, while the remainder is nontranscribed (71%) and transcribed (18%) spacer DNA.

Recombination-stimulating sequences in yeast ribosomal DNA correspond to sequences regulating transcription by RNA polymerase I

A DNA sequence ( HOT1) from the repeated ribosomal RNA gene cluster of Saccharomyces cerevisiae can stimulate genetic exchange when inserted at novel locations in the yeast genome. Localization of the sequences required for HOT1 activity demonstrates that two noncontiguous fragments of DNA are required for the stimulation of recombination. One of these fragments contains the transcription initiation site for the major 35S ribosomal RNA precursor. The other contains an enhancer of RNA polymerase I transcription. We suggest that transcription by RNA polymerase I initiating in the inserted rDNA and proceeding through the adjacent sequences is responsible for the stimulation of exchange. Consistent with this interpretation, insertion of the putative termination site for RNA polymerase I transcription between HOT1 and the adjacent recombining DNA abolishes the recombination stimulation. Transcription through both copies of the homologous recombining sequences appears to be necessary for enhanced exchange.

Transcription termination and RNA processin in the 3‘-end spacer of mouse ribosomalo RNA genes

The 3' termini of ribosomal RNA precursors from mouse FM3A cultured cells are mapped to eight sites within 625 bp downstream from the 3' terminus of 28 S rRNA. Three additional sites are mapped in liver RNA from C3H/He strain mice. Two of them, the sites at 570 bp and 625 bp are assumed to be termination sites in vivo, because they correspond to in vitro termination sites of RNA polymerase I, and 45 S RNAs having these 3' termini decay with kinetics distinct from others. The amount of 45 S RNA having the 3' terminus at other sites is variable among several mouse strains, despite their having the same DNA sequence in these regions. The ability to produce 3' termini in these sites seems to follow Mendel's law of inheritance. Therefore, we postulate that these nine sites are RNA processing sites which are controlled genetically.

Heat shock stabilizes highly unstable transcripts of the Xenopus ribosomal gene spacer.

We have shown recently that, in Xenopus laevis oocytes, the 3' end of the longest detectable ribosomal precursor RNA is not formed by transcription termination but by RNA processing and that RNA polymerase I continues to transcribe through the intergenic spacer region. In oocytes, these spacer transcripts are turned over rapidly, and the only apparent transcription termination site is located 215 base pairs upstream of the 5' end of the next transcription unit. In this paper we show that, at heat shock temperature (34 degrees C), processing at the 3' end of the precursor, rapid turnover of spacer transcripts, and termination are all severely impaired. In contrast, transcription initiation and chain elongation are not significantly affected by heat shock. This results in the appearance of large RNA in the range of 10-20 kilobases and longer.