Ribosomal Precursor Research Articles

RNAs and ribonucleoproteins in recognition and catalysis

CONTENTS. 1. Did life begin with catalytic RNA?–2. Self‐splicing and self‐cleaving RNAs–2.1 Self‐splicing of group I introns – 2.2 Self‐splicing of group II introns – 2.3 Self‐cleaving RNAs–3. Splicing mediated by trans‐acting factors–3.1 Group III introns – 3.2 Splicing of nuclear pre‐mRNAs – 3.3 Trans‐splicing – 3.4 Is nuclear pre‐mRNA splicing evolutionarily related to group I and group II self‐splicing?– 3.5 Non‐RNA mediated splicing of tRNAs–4. Processing of ribosomal precursor RNAs–5. Processing of pre‐mRNA 3′ ends–5.1 Polyadenylation – 5.2 Histone pre‐mRNA 3′ processing–6. Other RNPs involved in metabolic mechanisms–6.1 5′ end processing of pre‐tRNAs by RNase P – 6.2 The signal recognition particle – 6.3 Telomerase – 6.4 RNA editing in trypanosomatid mitochondria–7. Why RNA?

The nucleolus.

Nucleoli are the sites of biosynthesis of the ribosomal precursors. They contain may copies of the genes for the main rRNAs (18S- and 28 S-rRNA) in the form of tandemly arranged repeats at the chromosomal nucleolar organizer regions (NORs). They also contain the small rRNA (5S-rRNA) that is synthesized outside the nucleolus, specific nucleolar proteins, among them the factors and enzymes necessary for transcription and transcript processing, and the precursor units of the ribosomes. In man as in may vertebrate species, three main components of nucleoli, besides chromatin, can be detected: fibrillar centres (FC), dense fibrillar component (DCF), and granular component (GC). Within a nucleolus the FCs are in many cases situated in its central region. The DFc forms a network of strands surrounding the FCs, but may sometimes reach for out towards the periphery of the nucleolus. The GC is usually situated in the peripheral regions of the nucleolus. In cells with a low level of ribosomal biosynthesis the nucleoli are small, usually with a single FC and little surrounding DFC and GC ("ring-shaped nucleolus"). In active cells the DFC forms a large network enclosing several, sometimes up to hundreds of FCs, and the GC covers a large area in the periphery ("compact nucleoli"). In cells at the onset of a new stimulation, the DFC is very prominent whereas the FCs are few and small, and the GC is also not very extensive ("reticulate nucleoli"). In some special cell types that are very active other arrangements of the structural components are found. In Sertoli cells, for instance, only one nucleolus is found, or occasionally two, each with a single large FC and a distinct area of GC, both areas being engulfed by DFC intermingled with some peripheral GC. Immunocytological and in situ hybridization studies to localize the rRNA genes within the nucleolus have so far led to divergent results. Both fibrillar components, the FCs and the DFC, have been claimed as the most probable candidates. Transcription of rDNA and the subsequent early steps of ribosome biosynthesis are localized in the DFC, whereas later steps (mature rRNA, preribosomes) are localized in the GC. The FCs may also serve as sites for the preparation of the rDNA for transcription, and as a store for certain nucleolar proteins. During mitosis, parts of the nucleolar proteins remain at the NORs. A direct contact between the nucleolus and the nuclear envelope is frequently observed but is not dependent on nucleolar activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Small nuclear RNAs in messenger RNA and ribosomal RNA processing.

Ribonucleoproteins (RNPs) play essential roles in many aspects of gene expression. Two families of nuclear RNPs are involved in the processing of primary transcripts made by RNA polymerases I and II (pol I and II), two of the three polymerases present in the nuclei of eukaryotic cells. Ribosomal RNA precursor transcription by pol I, subsequent processing of the precursor, and the initial steps of ribosome assembly all take place in the nucleolus. A group of nucleolar RNPs containing small RNAs (small nucleolar RNAs or snoRNAs) are involved in the posttranscriptional nucleolar events of ribosome biosynthesis. Six members of a related family of small nuclear RNAs (snRNAs) are required for the processing of mRNA precursors in the nucleoplasm. Five of these snRNAs (U1, U2, U4-6) participate in the removal of intervening sequences while the sixth (U7) plays an essential role in the 3' processing of a subset of mRNA precursors, the histone pre-mRNAs. This is a review of structural and functional aspects of the U1-U7 snRNAs and of snoRNAs.

Regulation of ribosomal RNA synthesis in sea urchin embryos

By the use of labeled DNA probes which selectively hybridize to non mature ribosomal RNA precursors and by the combined use of Northern blots and ofin vivo labeling experiments we provide evidence for the following facts: 1) the rate of rRNA synthesis per embryonic nucleus in the sea urchinP. lividus is very low in the pre-blastular stages and is markedly enhanced at blastula; 2) partially matured rRNA precursors of 2IS and 27S deriving from oogenesis are stored in the embryo at the pre-blastular stages. A negative regulatory role in the rRNA synthesis is hypotesized for these immature precursors.

The gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis contains a group I intron.

The nucleotide sequence of the gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis was determined. It revealed the presence of a group I intron with a length of 411 nucleotides. This is the third occurrence of such an intron discovered in a small subunit rRNA gene encoded by a eukaryotic nuclear genome. The other two occurrences are in Pneumocystis carinii, a fungus of uncertain taxonomic status, and Ankistrodesmus stipitatus, a green alga. The nucleotides of the conserved core structure of 101 group I intron sequences present in different genes and genome types were aligned and their evolutionary relatedness was examined. This revealed a cluster including all group I introns hitherto found in eukaryotic nuclear genes coding for small and large subunit rRNAs. A secondary structure model was designed for the area of the Ustilago maydis small ribosomal subunit RNA precursor where the intron is situated. It shows that the internal guide sequence pairing with the intron boundaries fits between two helices of the small subunit rRNA, and that minimal rearrangement of base pairs suffices to achieve the definitive secondary structure of the 18S rRNA upon splicing.

In vitro transcription in isolated nucleoli of Tetrahymena pyriformis.

An in vitro transcription system was established using extrachromosomal nucleoli from Tetrahymena pyriformis macronuclei as a template. Ribosomal precursor RNA (pre-rRNA) and nascent pre-rRNA chains were removed from isolated nucleoli by treatment with RNase T1 and Sarkosyl. Nucleoli were then incubated in a RNA synthetic cocktail containing a cellular extract from Tetrahymena thermophila. The transcription product was examined for the presence of transcripts from T. pyriformis ribosomal DNA (rDNA) by P1 nuclease protection mapping using a DNA probe from a T. pyriformis rDNA clone. A sequence difference between T. pyriformis and T. thermophila in the 5' region of their 35S pre-rRNAs permitted exclusive detection of T. pyriformis transcripts. The results showed that faithful transcription initiation occurred in vitro from the in vivo initiation site of the nucleolar template and that the nucleolar template had a much higher efficiency of transcription than that of the purified rDNA clone. This system may offer unique advantages for future studies of transcriptional control during development and differentiation.

Function of P11, a tertiary base pairing in self-splicing introns of subgroup IA

There is phylogenetic evidence for the existence of a new pairing in subgroup IA1 self-splicing introns. This tertiary interaction, called P11, which is extraneous to the catalytic centre of these ribozymes was modelled after a “pseudoknot” and grafted by computer modelling on the common core structure of group I introns that was recently proposed by Michel & Westhof. In order to probe the function of the P11 pairing, we mutated the P11 helix in the intron of the large ribosomal precursor of Saccharomyces cerevisiae mitochondria (Sc.LSU). Our experimental data show that the P11 pairing plays a role in stabilizing the overall fold of the RNA molecule. While P11 is not essential for self-splicing activity in vitro , mutants with disrupted P11 require higher concentration of MgCl 2 for self-splicing. By contrast, mutants with a reinforced P11 pairing ( via introduction of several G · C base-pairs) self-splice more efficiently than the wild-type at 55 °C. Based on this work, the possible engineering of new stable versions of the ribozyme is discussed.

Function of P11, a tertiary base pairing in self-splicing introns of subgroup IA

There is phylogenetic evidence for the existence of a new pairing in subgroup IA1 self-splicing introns. This tertiary interaction, called P11, which is extraneous to the catalytic centre of these ribozymes was modelled after a “pseudoknot” and grafted by computer modelling on the common core structure of group I introns that was recently proposed by Michel & Westhof. In order to probe the function of the P11 pairing, we mutated the P11 helix in the intron of the large ribosomal precursor of Saccharomyces cerevisiae mitochondria (Sc.LSU). Our experimental data show that the P11 pairing plays a role in stabilizing the overall fold of the RNA molecule. While P11 is not essential for self-splicing activity in vitro, mutants with disrupted P11 require higher concentration of MgCl 2 for self-splicing. By contrast, mutants with a reinforced P11 pairing ( via introduction of several G · C base-pairs) self-splice more efficiently than the wild-type at 55 °C. Based on this work, the possible engineering of new stable versions of the ribozyme is discussed.

Group I Intron Self-Splicing with Adenosine: Evidence for a Single Nucleoside-Binding Site

For self-splicing of Tetrahymena ribosomal RNA precursor, guanosine binding is required for 5' splice-site cleavage and exon ligation. Whether these two reactions use the same or different guanosine-binding sites has been debated. A double mutation in a previously identified guanosine-binding site within the intron resulted in preference for adenosine (or adenosine triphosphate) as the substrate for cleavage at the 5' splice site. However, splicing was blocked in the exon ligation step. Blockage was reversed by a change from guanine to adenine at the 3' splice site. These results indicate that a single determinant specifies nucleoside binding for both steps of splicing. Furthermore, it suggests that RNA could form an active site specific for adenosine triphosphate.

S. pombe pac1+, whose overexpression inhibits sexual development, encodes a ribonuclease III-like RNase.

The Schizosaccharomyces pombe pac1 gene is a multicopy suppressor of the pat1 temperature-sensitive mutation, which directs uncontrolled meiosis at the restrictive temperature. Overexpression of the pac1 gene had no apparent effect on vegetative growth but inhibited mating and sporulation in wild type S. pombe cells. In such cells, expression of certain genes required for mating or meiosis was inhibited. The pac1 gene is essential for vegetative cell growth. The deduced pac1 gene product has 363 amino acids. Its C-terminal 230 residues revealed 25% amino acid identity with ribonuclease III, an enzyme that digests double-stranded RNA and is involved in processing ribosomal RNA precursors and certain mRNAs in Escherichia coli. The pac1 gene product could degrade double-stranded RNA in vitro. These observations establish the presence of a RNase III homolog in eukaryotic cells. The pac1 gene product probably inhibits mating and meiosis by degrading a specific mRNA(s) required for sexual development. It is likely that mRNA processing is involved in the regulation of sexual development in fission yeast.

Control points in eucaryotic ribosome biogenesis

Ribosome biogenesis in eucaryotic cells involves the coordinated synthesis of four rRNA species, transcribed by RNA polymerase I (18S, 28S, 5.8S) and RNA polymerase III (5S), and approximately 80 ribosomal proteins translated from mRNAs synthesized by RNA polymerase II. Assembly of the ribosomal subunits in the nucleolus, the site of 45S rRNA precursor gene transcription, requires the movement of 5S rRNA and ribosomal proteins from the nucleoplasm and cytoplasm, respectively, to this structure. To integrate these events and ensure the balanced production of individual ribosomal components, different strategies have been developed by eucaryotic organisms in response to a variety of physiological changes. This review presents an overview of the mechanisms modulating the production of ribosomal precursor molecules and the rate of ribosome biogenesis in various biological systems.

Termination of transcription of ribosomal RNA in Saccharomyces cerevisiae

We have attempted to determine the site of termination of transcription of ribosomal RNA in the yeast, Saccharomyces cerevisiae. While a quantitative description of the termination sites of RNA polymerase I is not possible using presently available methods, we conclude that transcription of most molecules continues through a large portion of the adjacent enhancer region. There are two potential termination sites within the enhancer, one of which is near the binding site of the DNA binding protein REBI. In addition there is an apparently fail-safe termination site approximately 950 nucleotides beyond the 3' end of 35S ribosomal precursor RNA. Processing at the end of 35S RNA influences the choice of downstream termination site. Conversely downstream sequences also influence the site of termination.

Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA

The discovery of RNA enzymes has, for the first time, provided a single molecule that has both genetic and catalytic properties. We have devised techniques for the mutation, selection and amplification of catalytic RNA, all of which can be performed rapidly in vitro. Here we describe how these techniques can be integrated and performed repeatedly within a single reaction vessel. This allows evolution experiments to be carried out in response to artificially imposed selection constraints. We worked with the Tetrahymena ribozyme, a self-splicing group I intron derived from the large ribosomal RNA precursor of Tetrahymena thermophila that catalyses sequence-specific phosphoester transfer reactions involving RNA substrates. It consists of 413 nucleotides, and assumes a well-defined secondary and tertiary structure responsible for its catalytic activity. We selected for variant forms of the enzyme that could best react with a DNA substrate. This led to the recovery of a mutant form of the enzyme that cleaves DNA more efficiently than the wild-type enzyme. The selected molecule represents the discovery of the first RNA enzyme known to cleave single-stranded DNA specifically.

Core filaments of the nuclear matrix.

The nuclear matrix is concealed by a much larger mass of chromatin, which can be removed selectively by digesting nuclei with DNase I followed by elution of chromatin with 0.25 M ammonium sulfate. This mild procedure removes chromatin almost completely and preserves nuclear matrix morphology. The complete nuclear matrix consists of a nuclear lamina with an interior matrix composed of thick, polymorphic fibers and large masses that resemble remnant nucleoli. Further extraction of the nuclear matrices of HeLa or MCF-7 cells with 2 M sodium chloride uncovered a network of core filaments. A few dark masses remained enmeshed in the filament network and may be remnants of the nuclear matrix thick fibers and nucleoli. The highly branched core filaments had diameters of 9 and 13 nm measured relative to the intermediate filaments. They may serve as the core structure around which the matrix is constructed. The core filaments retained 70% of nuclear RNA. This RNA consisted both of ribosomal RNA precursors and of very high molecular weight hnRNA with a modal size of 20 kb. Treatment with RNase A removed the core filaments. When 2 M sodium chloride was used directly to remove chromatin after DNase I digestion without a preceding 0.25 M ammonium sulfate extraction, the core filaments were not revealed. Instead, the nuclear interior was filled with amorphous masses that may cover the filaments. This reflected a requirement for a stepwise increase in ionic strength because gradual addition of sodium chloride to a final concentration of 2 M without an 0.25 M ammonium sulfate extraction uncovered core filaments.

Juvenile hormone controls previtellogenic proliferation of ribosomal RNA in the mosquito fat body

During the previtellogenic development of mosquito fat body cells, the nucleolus, the organelle responsible for producing ribosomes, enlarges threefold, reaching maximal size between 2 and 3 days after eclosion. The granular component of the nucleolus containing ribosomal precursors increases considerably as well. These signs of nucleolar activation correlate with the synthetic rate and accumulation of poly(A) − RNA (predominantly ribosomal RNA) in the fat body cells. The amount of poly(A) − RNA in fat body cells increases during the first 2 days after eclosion and then declines gradually. The rate of RNA synthesis exhibits similar kinetics, but both the rise and the decline are sharper than for the accumulation of RNA. All the characteristics of nucleolar activation, its enlargement, accumulation of poly(A) − RNA, and the increased rate of RNA synthesis, are blocked by removal of the corpora allata (CA) in newly eclosed adult females but could be restored by either implantation of CA or topical application of juvenile hormone III or its analog, 7- S-methoprene, to allatectomized females. Thus, previtellogenic activation of fat body nucleoli for ribosomal RNA production is controlled by juvenile hormone from the corpora allata.

High resolution mapping of Xenopus laevis 5S and ribosomal RNA genes by EM in situ hybridization

We have developed a modification of in situ hybridization at the electron microscope level that permits simultaneous detection of at least two sequences. Probes are labelled with either biotin or AAF and detected with two distinct sizes of colloidal gold. This protocol has been applied to map the positions of Xenopus laevis oocyte-type 5S genes relative to ribosomal precursor genes in several independently derived cell lines. The results for the line TRXO, which expresses some oocyte 5S RNA, indicate that this inappropriate expression is not due to translocation from telomeric sites into the nucleolus organizer, as previously hypothesized. In addition we found that four other Xenopus cell lines, none of which express these genes, also contain distinct 5S oocyte translocations. These results suggest that an alteration in chromosome position is insufficient to result in gene activation and that sequences which are telomeric-proximal are exceptionally prone to translocation.

The 5′ end of U3 snRNA can be crosslinked in vivo to the external transcribed spacer of rat ribosomal RNA precursors

From previous work it was known that U3 RNA is hydrogen bonded to nucleolar 28 S to 35 S RNA and can be covalently crosslinked to RNA of >28 S by irradiation in vivo with long-wave ultraviolet light in the presence of 4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT psoralen). Here we use a novel sandwich blot technique to identify these large nucleolar RNA species as rRNA precursors and to map the site(s) of crosslinking in vivo. The crosslink occurs between one or more residues near the 5′ end of U3 RNA and a 380 nucleotide region of the rat rRNA external transcribed spacer (ETS1). We have sequenced this region of the rat ETS and we show that it includes an RNA-processing site analogous to those previously mapped to approximately 3.5 kb upstream from the 5′ end of mouse and human 18 S rRNAs.

U3, U8 and U13 comprise a new class of mammalian snRNPs localized in the cell nucleolus.

Using anti-(U3)RNP autoantibodies, we have isolated and characterized two additional small nucleolar RNAs from HeLa cells, which are less abundant than U3 RNA. Both RNAs possess a trimethylguanosine cap as judged by precipitation with anti-TMG antibody, but are not precipitated by either anti-Sm or anti-La antibodies. In addition, both RNAs are not precipitable by anti-Th serum, which recognizes another nucleolar RNP autoantigen. Sequence analysis revealed that one of these RNAs, 136 nucleotides long, is the human U8 homolog; while the other, 105 nucleotides long, represents a novel species which we designate U13. Both RNAs share with U3 two conserved sequences (boxes C and D). The role of one or both of these boxes in binding the common 34 kd antigenic protein, otherwise known as fibrillarin, is discussed. Fractionation of HeLa cells revealed that U8 and U13, like U3, reside in the nucleolus. In glycerol gradients both RNAs cosediment with larger structures possibly representing ribosomal precursors. We propose that U3, U8 and U13 comprise a new subset of mammalian snRNPs whose roles in ribosome biogenesis are discussed.

U3 small nuclear RNA can be psoralen-cross-linked in vivo to the 5' external transcribed spacer of pre-ribosomal-RNA.

U3 small nuclear RNA is hydrogen-bonded to high molecular weight nucleolar RNA and can be isolated from greater than 60S pre-ribosomal ribonucleoprotein particles, suggesting that it is involved in processing of ribosomal RNA precursors (pre-rRNA) or in ribosome biogenesis. Here we have used in vivo psoralen cross-linking to identify the region of pre-rRNA interacting with U3 RNA. Quantitative hybridization selection/depletion experiments with clones of rRNA-encoding DNA (rDNA) and cross-linked nuclear RNA showed that all of the cross-linked U3 RNA was associated with a region that includes the external transcribed spacer (ETS) at the 5' end of the human rRNA precursor. To further identify the site of interaction within the approximately 3.7-kilobase ETS, Southern blots of rDNA clones were sandwich-hybridized with cross-linked RNA and then probed for cross-linked U3 RNA. These experiments showed that U3 RNA was cross-linked to a 258-base sequence between nucleotides +438 and +695, just downstream of the ETS early cleavage site (+414). The localization of U3 to this region of the rRNA precursor was not expected from previous models for a base-paired U3-rRNA interaction and suggests that U3 plays a role in the initial pre-rRNA processing event.

Processing of transcripts of a dimeric tRNA gene in yeast uses the nuclease responsible for maturation of the 3′ termini upon 5 S and 37 S precursor rRNAs

The rna82 mutation of Saccharomyces cerevisiae inactivates an RNA processing activity responsible for maturation of 3′-terminal sequences upon 5 S and 37 S ribosomal RNA precursors. This study describes a difference in the processing of transcripts of an S. cerevisiae dimeric tRNA gene (tRNA Arg-tRNA Asp) in RNA polymerase III in vitro transcription extracts prepared from rna82 and wild-type cells. The mutant extract accumulated additional processing intermediates containing tRNA Arg sequences as compared to the extract from wild-type cells. The structure of these intermediates revealed a defect in removal of the 10 nucleotides left 3′ to the tRNA Arg sequence by the RNase P cleavage immediately 5′ to tRNA Asp. This is the first demonstration of a mutational defect affecting maturation of 3′ sequences upon a eukaryotic tRNA precursor.