Ribosomal Precursor Research Articles

Characterization of an authentic intermediate in the self-splicing process of ribosomal precursor RNA in macronuclei of Tetrahymena thermophila.

We have characterized a 1.5 kb RNA species in T. thermophila macronuclei previously found in vivo and including intron sequences linked to the 3' exon. This IVS-3' exon RNA could be detected in gels as a discrete molecule only after denaturation of nuclear RNA. After addition of 32P-GTP, as splicing cofactor in a nuclear in vitro system, the IVS-3' exon RNA was labeled at its 5' terminus, as was the by-product of splicing, the excised IVS RNA. The time course of labeling indicates that the IVS-3' exon RNA acts like a reaction intermediate and specifically a kinetic precursor to IVS RNA. Partial nuclease digestions showed that the IVS-3' exon RNA and the IVS RNA have the same 5' terminal sequence. In addition the IVS-3' exon RNA can release the 15-mer oligonucleotide cleaved off during circularization of IVS RNA under conditions of high temperature. Taken together, the structural, functional, and kinetic properties of the IVS-3' exon RNA strongly suggest that it represents a previously postulated in vivo intermediate in the splicing pathway.

RNA as an Enzyme

The catalytic activity of ribonucleic acid is reviewed, with the intervening sequence (IVS) of the ribosomal RNA precursor of Tetrahymena serving as a major example. The IVS catalyzes its own excision from the precursor RNA and at the same time ligation of the flanking sequences, a reaction termed self-splicing. The excised IVS RNA can act as an enzyme to catalyze sequence-specific cleavage and ligation reactions on substrate RNA molecules. The RNA polymerization activity of the IVS supports the possibility that RNA catalysis could have been important in establishing a prebiotic self-replicating system. Other systems in which RNA catalysis has been found include related group I IVSs, group II IVSs, ribonuclease P, and certain plant infectious RNAs.

Cytological approach to the nucleolar functions detected by silver staining

The pattern of nucleolar silver staining was determined in the interphase and mitosis of plant cells using two cytological techniques, the selectivity of which was tested densitometrically. For complete accuracy, silver grains were removed and the sections were then observed under uranyl and lead staining. During interphase, the highest density of silver grains occurred in the portions of the dense fibrillar component nearest to the fibrillar centres; the latter were also stained except for the condensed chromatin inclusions typical of the heterogeneous type. During mitosis, not only the nucleolar organiser region (NOR) was stained, but also the prenucleolar material in anaphase and telophase. Two different proteins or sets of proteins are suggested to be responsible for this staining pattern; one of them is associated with NOR chromatin (which is not contained in the mitotic prenucleolar material) and the other with early ribosomal precursors (which have never been found in fibrillar centres). From these two proteins, the “Ag-NOR” reveals either transcriptionally active or potentially active chromatin, characterized by an uncoiled, extended structure, regardless of the state of transcription. This is the structural feature detected by Ag-NOR staining.

Localization of human histone gene transcripts predominantly in the nonmatrix nuclear fraction.

Using S1 nuclease protection assays, we have examined the representation of cell cycle-dependent H4 histone RNAs in the nuclear matrix and nonmatrix nuclear fractions of human cells. Cytoplasmic and nuclear fractions were prepared from exponentially growing HeLa S3 cells by double detergent (sodium deoxycholate and NP40) lysis. The nuclear matrix and nonmatrix nuclear fractions were then prepared by digestion of nuclei with RNase-free DNase I and subsequent high-salt [0.4 M (NH4)2SO4] extraction. Subcellular fractions were characterized by 1) DNA, RNA, and protein composition; 2) electrophoretic analysis of the proteins in each fraction; 3) the representation of 45S ribosomal RNA precursors and processed 18S and 28S ribosomal RNAs; and 4) the presence of mitochondrial RNAs. In contrast to ribosomal and messenger RNA precursors, which are largely associated with the nuclear matrix, the human H4 histone RNAs in the nucleus were found predominantly in the nonmatrix nuclear fraction. The presence of H4 histone RNA in the nonmatrix nuclear fraction appeared to be coupled to DNA replication, since inhibition of DNA synthesis by hydroxyurea resulted in a loss of histone RNA from the nucleus. Our results suggest either that the association of histone RNAs with the nuclear matrix is very transient or that posttranscriptional modifications of the rapidly processed histone gene transcripts do not involve the nuclear matrix.

Ribozyme inhibitors: deoxyguanosine and dideoxyguanosine are competitive inhibitors of self-splicing of the Tetrahymena ribosomal ribonucleic acid precursor.

The intervening sequence (IVS) of the Tetrahymena rRNA precursor catalyzes its own splicing. During splicing the 3'-hydroxyl of guanosine is ligated to the 5' terminus of the IVS. One catalytic strategy of the IVS RNA is to specifically bind its guanosine substrate. Deoxyguanosine (dG) and dideoxyguanosine (ddG) are found to be competitive inhibitors of self-splicing. Comparison of the kinetic parameters (Ki = 1.1 mM for dG; Ki = 5.4 mM for ddG; Km = 0.032 mM for guanosine) indicates that the ribose hydroxyls are necessary for optimal binding of guanosine to the RNA. dG is not a substrate for the reaction even at very high concentrations. Thus, in addition to aiding in binding, the 2'-hydroxyl is necessary for reaction of the 3'-hydroxyl. A second catalytic strategy of the IVS RNA is to enhance the reactivity of specific bonds. For example, the phosphodiester bond at the 3' splice site is extremely labile to hydrolysis. We find that dG and ddG, as well as 2'-O-methylguanosine and 3'-O-methylguanosine, reduce hydrolysis at the 3' splice site. These data are consistent with an RNA structure that brings the 5' and 3' splice sites proximal to the guanosine binding site.

Inactivation of a Single Eucaryotic Gene Irradiated in Vitro in Transcriptionally Active Chromatin Form.

The transcriptional activity of endogenous RNA polymerases has been used to study the inactivation of a single eucaryotic gene irradiated in chromatin form in aqueous solution. The gene codes for the ribosomal RNA precursor in Tetrahymena where it exists in multiple copies as free extrachromosomal molecules. The gene chromatin is isolated with intact RNA polymerases on the coding strand. Irradiation with 10-MeV electrons reduces the in vitro RNA synthesis on the isolated chromatin in a dose-dependent manner which allows the amount of DNA damage to be determined. Irradiation of the gene chromatin in dilute solution under various gas atmospheres demonstrates that OH radicals account for most of the inactivation. Under these conditions, $e_{{\rm aq}}^{-}$ is unimportant, while the results suggest that H may contribute to inactivation of gene transcription.

New reactions of the ribosomal RNA precursor of Tetrahymena and the mechanism of self-splicing

The availability of Tetrahymena pre-rRNA of discrete size, produced by transcription of recombinant plasmids with bacteriophage SP6 RNA polymerase, has permitted a more detailed investigation of the self-splicing reaction. The predicted splicing intermediate, the product of cleavage by guanosine at the 5′ splice site, was identified. This intermediate was tested in the intermolecular exon ligation reaction and found to be competent to undergo the second step of splicing. These results and others that evaluated the reactivity of the 5′ and 3′ splice sites independently show that splicing occurs in two separable steps. The 3′ splice site was found to be susceptible to site-specific hydrolysis leaving a 3′ hydroxyl terminus. This is interpreted as an indication that the 3′ splice site is activated for nucleophilic attack in general and for exon ligation in particular. Preliminary evidence for specific hydrolysis at the 5′ splice site was also obtained. All of the newly characterized intervening sequence RNA-mediated reactions as well as those found previously are divided into three categories: (1) transesterification by guanosine at sites following two or three pyrimidine nucleotides (and, as a minor reaction, at sites following other guanosine residues); (2) transesterification by oligopyrimidines or by the 5′ exon (which terminates with C-U-C-U-C-U OH) at the site following the 3′-terminal guanosine residue of the intervening sequence; and (3) specific hydrolysis at the splice sites. One of the products of the reactions at the 3′ splice site is a molecule that contains the 5′ exon still attached to the intervening sequence. It has a 3′-terminal G OH and undergoes cyclization both at the normal cyclization site within the intervening sequence and at the 5′ splice site. The finding that the splice site can act as a cyclization site, combined with the earlier observation that the normal cyclization site is subject to attack by guanosine mononucleotide, leads us to propose that all these reactions may be occurring in the same active site. Translocation (a conformational change) would then bring different oligopyrimidine sequences into the active site for attack by guanosine. On the basis of the experimental results, a model for the local structure at the active site is described. A key feature of the model is the interaction between the U at the end of the oligopyrimidine sequence, a G residue within the internal guide sequence in the intervening sequence, and another G residue that can be either the attacking group for transesterification or the 3′-terminal G of the intervening sequence.

The inhibitory effects of 5-fluorouracil on the metabolism of preribosomal and ribosomal RNA in L-1210 cells in vitro.

Addition of 5FU to the culture medium of mouse L-1210 cells resulted in inhibition of the maturation process of ribosomal RNA precursors in vitro. In the presence of 10(-6) M 5FU for 2 h, the 45S preribosomal RNA was processed to 32S preribosomal RNA, but 28S rRNA was not produced. The processing to 18S rRNA was intact at this drug concentration. Higher concentrations of 5FU for a longer incubation period affected the RNA processing more severely. At 10(-5) M of the drug for 24 h the processing to 28S rRNA and 32S preribosomal RNA. When the cells were labeled with 14C-UR for 2 h following 3H-5FU at 10(-6) M for 24 h, the radioactivities of newly synthesized RNA labeled with 14C-UR accumulated in the region of 45S and 32S preribosomal RNA, and no processing to 28S rRNA was observed. Radioactivity corresponding to 3H-5FU did not persist in the preribosomal RNA region, because further maturation proceeded in the condition of depletion of 5FU after the long incubation period. Thus, inhibition of the processing of preribosomal RNA to 28S rRNA was not brought about by the accumulation of 5FU-substituted 45S preribosomal RNA, but by some other, yet unknown, mechanism.

Splice-site selection by a self-splicing RNA of Tetrahymena

5′ Splice-site selection by the self-splicing Tetrahymena ribosomal RNA precursor requires the formation of a particular RNA helix by base pairing between an intron ‘guide sequence’ and bases surrounding the 5′ splice junction. Part of the guide sequence subsequently holds and positions the 3′ end of the cut 5′ exon to attack the 3′ splice junction.

Role of conserved sequence elements 9L and 2 in self-splicing of the Tetrahymena ribosomal RNA precursor

Oligonucleotide-directed mutagenesis has been used to alter highly conserved sequences within the intervening sequence (IVS) of the Tetrahymena large ribosomal RNA precursor. Mutations within either sequence element 9L or element 2 eliminate splicing activity under standard in vitro splicing conditions. A double mutant with compensatory base changes in elements 9L and 2 has accurate splicing activity restored. Thus, the targeted nucleotides of elements 9L and 2 base-pair with one another in the IVS RNA, and pairing is important for self-splicing. Mutant splicing activities are restored by increased magnesium ion concentrations, supporting the conclusion that the role of the targeted bases in splicing is primarily structural. Based on the temperature dependence, we propose that a conformational switch involving pairing and unpairing of elements 9L and 2 is required for splicing.

Evidence that the methylation inhibitor cycloleucine causes accumulation of a discrete ribosomal RNA precursor in hamster mitochondria.

A novel RNA fraction, 'Cy RNA,' that accumulates in mitochondria when hamster cells are treated with the methylation inhibitor cycloleucine, has been characterized by high resolution acrylamide gel electrophoresis and DNA-RNA hybridization. Cy RNA ran in gels as a discrete band, with an apparent chain length of 2 600. It hybridized specifically to restriction fragments containing genes for the mitochondrial ribosomal RNAs. We infer that Cy RNA is a discrete polycistronic ribosomal RNA precursor transcript, whose processing is dependent on normal methylation.

Reactions of the intervening sequence of the Tetrahymena ribosomal ribonucleic acid precursor: pH dependence of cyclization and site-specific hydrolysis.

During self-splicing of the Tetrahymena rRNA precursor, the intervening sequence (IVS) is excised as a unique linear molecule and subsequently cyclized. Cyclization involves formation of a phosphodiester bond between the 3' end and nucleotide 16 of the linear RNA, with release of an oligonucleotide containing the first 15 nucleotides. We find that the rate of cyclization is independent of pH in the range 4.7-9.0. A minor site of cyclization at nucleotide 20 is characterized. Cyclization to this site becomes more prominent at higher pHs, although under all conditions examined it is minor compared to cyclization at nucleotide 16. The circular IVS RNAs are unstable, undergoing hydrolysis at the phosphodiester bond that was formed during cyclization. We find that the rate of site-specific hydrolysis is first order with respect to hydroxide ion concentration, with a rate constant 10(3)-10(4)-fold greater than that of hydrolysis of strained cyclic phosphate esters. On the basis of these results, we propose that circular IVS RNA hydrolysis involves direct attack of OH- on the phosphate at the ligation junction, that particular phosphate being made particularly reactive by the folding of the RNA molecule. Cyclization, on the other hand, appears to occur by direct attack of the 3'-terminal hydroxyl group of the linear IVS RNA without prior deprotonation.

Oligomerization of Intervening Sequence RNA Molecules in the Absence of Proteins

The intervening sequence RNA excised from the ribosomal RNA precursor of Tetrahymena forms linear and circular oligomers when exposed to a heating-cooling treatment in vitro. The reactions require no protein or external energy source. Oligomerization is different from other self-catalyzed reactions of the intervening sequence RNA in that it involves intermolecular rather than intramolecular recombination, producing RNA molecules that are substantially larger than the original. The observation that RNA molecules can catalyze their own oligomerization has possible implications for the evolution of chromosomes and for the replicative cycle of plant viroids and virus-associated RNA's.

DNA-methylation in mouse embryo development : R. Balling, R. Kothary, J. Rossant, D. Huszar and A. Bernstein, Dept. of Biol. Sci., Brock Univ., St. Catharines, Ont., L253A1, and Dept. of Med. Biophys., U. of Toronto, Ont. M4X1

1. The minor nucleotide composition of the “ribosomal precursor” RNA of Escherichia coli has been compared with that of mature ribosomal RNA. When cells were treated with chloramphenicol, or with streptomycin, or when a “relaxed” auxotroph was starved of a required amino acid, the newly synthesized, “ribosomal precursor”, 16-S RNA (centrifugally purified) was found to be undermethylated by 70–80 %, and 23-S RNA by 40–45 %. Nucleotide analyses showed that the 23S precursor component was about equally deficient in all normal methylated nucleotides, except for an O′-ribose-methylated nucleotide, whose accumulation was more drastically affected. The 16S precursor component had virtually no methylated nucleotides, except for approximately one 5-methylcytidylic acid residue per molecule. These results are in accord with the idea that undermethylation causes previously observed centrifugal and chromatographic abnormalities of ribosomal precursor RNA.2. Chloramphenicol treatment was also found to inhibit the formation of pseudouridylic acid in newly synthesized ribosomal RNA.3. No differences in ribosomal RNA methylation patterns were noted among strains B, W, or K12, or between streptomycin-sensitive and streptomycin-resistant strains.4. A model is presented whereby certain “structural” ribosomal proteins also function as ribosomal RNA-modifying enzymes during ribosome assembly.

Coupling of Tetrahymena ribosomal RNA splicing to beta-galactosidase expression in Escherichia coli.

Splicing of the Tetrahymena ribosomal RNA precursor is mediated by the folded structure of the RNA molecule and therefore occurs in the absence of any protein in vitro. The Tetrahymena intervening sequence (IVS) has been inserted into the gene for the alpha-donor fragment of beta-galactosidase in a recombinant plasmid. Production of functional beta-galactosidase is dependent on RNA splicing in vivo in Escherichia coli. Thus RNA self-splicing can occur at a rate sufficient to support gene expression in a prokaryote, despite the likely presence of ribosomes on the nascent RNA. The beta-galactosidase messenger RNA splicing system provides a useful method for screening for splicing-defective mutations, several of which have been characterized.

Self-splicing of yeast mitochondrial ribosomal and messenger RNA precursors

We have previously shown linear and circular splicing intermediates resembling intermediates that result from self-splicing of ribosomal precursor RNA of Tetrahymena to be present in mitochondrial RNA. Here we show that splicing of yeast mitochondrial precursor RNA also occurs in vitro in the absence of mitochondrial proteins. The large ribosomal RNA gene, consisting of the intron and part of the flanking exon regions, was inserted behind the SP6 promoter in a recombinant plasmid and was transcribed in vitro. The resulting RNA shows self-catalyzed splicing via incorporation of GTP at the 5′-end of the excised intron, 5′- to 3′-axon ligation, and intron circularization. When purified mitochondrial RNA is incubated under similar conditions with α- 32P-GTP, the excised ribosomal intron RNA is also labeled, as well as several other RNA species. Some of these RNAs are derived from excised introns from the multiply split gene coding for cytochrome oxidase subunit I.

In situ ultrastructural localization of sugar‐binding sites in lizard granulosa cell nuclei

Mannose-binding sites were detected at the ultrastructural level in nuclei of lizard granulosa cells in situ by means of mannosylated ferritin. When ultrathin sections of material embedded in glycol methacrylate were incubated with mannosylated ferritin, a strong labelling was observed over nucleoli, chromatin and the external leaflet of the nuclear envelope, but no labelling was detected in the perinuclear space except for nuclear pores. This labelling was clearly inhibited when sections were incubated in a solution containing both mannosylated ferritin and a sugar-related neoglycoprotein (mannosylated serum albumin). These ultrastructural data supporting the existence of nuclear lectin-like components in reptilian cells are in agreement with our previous findings about such components in nuclei isolated from mammalian cells. Owing to the unique organization of the granular component of the nucleoli in specialized granulosa cells, we are able to show that some of the mannose-binding sites are associated with ribosomal precursors.

A search for nuclear RNA lost in azo dye-induced transplantable hepatoma.

Studies were conducted to characterize the nuclear RNA (nRNA) species that were present in rat liver but absent in the hepatoma. Nuclear RNA was compared between Donryu rat liver and AH136B hepatoma, an azo dye-induced transplantable cell line, by DNA-RNA competitive hybridization. The hepatoma lacked 13-14% of nRNA according to measurements of radioactivity of the hybridized 32P-labeled liver nRNA, and this loss was shown to be due to the failure to transcribe such RNA rather than to the deletion of the relevant DNA in the genome. Characterization of the lost RNA was first attempted by fractionating liver nRNA by density gradient sedimentation and polyacrylamide gel electrophoresis. A comparison of the additive effects of the fractionated RNA's in the competitive hybridization indicated that the pertinent RNA was present in the large RNA molecules (greater than 14S), not in the low molecular weight RNA's. Then poly(A) nRNA was found to show a strong additive effect in the competitive hybridization while nucleolar RNA showed little additive effect, indicating that the pertinent RNA was present in the heterogeneous nRNA, not in the ribosomal precursor. Further characterization was done by fractionating DNA with regard to the repetition in the genome. A comparison of the competitive hybridizations on the fractionated DNA's showed that the loss occurred mostly in RNA transcribed from highly repetitive DNA. In conclusion, the RNA species lost in the hepatoma were components of heterogeneous nRNA transcribed from highly repetitive DNA.

Nucleolar cycles during the fifth stadium in Manduca epidermis

The changing pattern of nucleolar structure in the epidermal cells of Manduca sexta has been correlated with hormonal changes taking place during the fifth stadium. The epidermal nucleoli show three cycles of development, the first and third of which occur at the beginnings of the intermoult and moult phases respectively and are related to larval and pupal syntheses. The second phase occurs in the middle of the stadium but prior to the onset of wandering and commitment to pupation. A phase of mitosis separates the second and third cycles. The three cycles thus correspond in time to those found in Calpodes. The three cycles of nucleolar change are superimposed over nuclear changes relating to the degree of ploidy. Each phase begins with an expansion of the condensed nucleoli to form lobed rings and then necklaces. In the first phase (day 0–3), the rings and necklaces progress to form threaded networks. Both rings and networks have many ribosomal precursor granules that are lacking in condensed nucleoli. The rings and networks are therefore presumed to be more active in rRNA synthesis than the condensed state. The first and third phases of nucleolar change occur after elevated titres of haemolymph ecdysteroid. Post-thoracic ligation of animals at ecdysis blocks nucleolar changes as well as the appearance of polyploid nuclei. Nucleolar changes may be a primary response of the epidermis to stimulation by ecdysone.

DNA replication in binucleate cells of the Malpighian tubules of Hemipteran insects

The cells of Malpighian tubules of the Hemipteran blood-sucking insect, Rhodnius prolixus, are binucleate. The cells grow without division and, at each larval moult, the DNA content of the nuclei doubles. At the final moult to the adult, however, the DNA content does not change, even though the tubules grow considerably thereafter. In contrast, the DNA content of the tubule nuclei of two other Heteropteran Hemipteran insects, Dysdercus and Oncopeltus, doubles at every moult, including the final one to the adult. If extra larval moults are induced in Rhodnius, by treatment with juvenile hormone, DNA doubling is induced at each such supernumerary larval moult. Shortly after the DNA content increases in Rhodnius tubules, the chromosomes can be seen in a condensed state; presumably, therefore, DNA replication is achieved by endomitosis. Both before this DNA doubling, and within a day after, multiple nucleoli are prominent and appear actively engaged in producing ribosomal precursors. In fed fourth stage Rhodnius, the DNA content of the tubule cell nuclei increases 5–6 days after the blood meal. Neither the rate of fluid secretion that can be induced by stimulation nor the rate of transport of p-aminohippuric acid show any change at the time of DNA replication nor in the remaining days before ecdysis to the fifth stage. Malpighian tubule cell growth without division is thus well adapted to providing, without interruption, for the excretory needs of the growing insect.