Processing Of Ribosomal RNA Precursors Research Articles

Upon heat stress processing of ribosomal RNA precursors into mature rRNAs is compromised after cleavage at primary P site in Arabidopsis thaliana

ABSTRACT Transcription and processing of 45S rRNAs in the nucleolus are keystones of ribosome biogenesis. While these processes are severely impacted by stress conditions in multiple species, primarily upon heat exposure, we lack information about the molecular mechanisms allowing sessile organisms without a temperature-control system, like plants, to cope with such circumstances. We show that heat stress disturbs nucleolar structure, inhibits pre-rRNA processing and provokes imbalanced ribosome profiles in Arabidopsis thaliana plants. Notably, the accuracy of transcription initiation and cleavage at the primary P site in the 5’ETS (5’ External Transcribed Spacer) are not affected but the levels of primary 45S and 35S transcripts are, respectively, increased and reduced. In contrast, precursors of 18S, 5.8S and 25S RNAs are rapidly undetectable upon heat stress. Remarkably, nucleolar structure, pre-rRNAs from major ITS1 processing pathway and ribosome profiles are restored after returning to optimal conditions, shedding light on the extreme plasticity of nucleolar functions in plant cells. Further genetic and molecular analysis to identify molecular clues implicated in these nucleolar responses indicate that cleavage rate at P site and nucleolin protein expression can act as a checkpoint control towards a productive pre-rRNA processing pathway.

The enigmatic RNase MRP of kinetoplastids

ABSTRACT The ribonucleoprotein RNase MRP is responsible for the processing of ribosomal RNA precursors. It is found in virtually all eukaryotes that have been examined. In the Euglenozoa, including the genera Euglena, Diplonema and kinetoplastids, MRP RNA and protein subunits have so far escaped detection using bioinformatic methods. However, we now demonstrate that the RNA component is widespread among the Euglenozoa and that these RNAs have secondary structures that conform to the structure of all other phylogenetic groups. In Euglena, we identified the same set of P/MRP protein subunits as in many other protists. However, we failed to identify any of these proteins in the kinetoplastids. This finding poses interesting questions regarding the structure and function of RNase MRP in these species.

The HIV Tat protein affects processing of ribosomal RNA precursor

BackgroundInside the cell, the HIV Tat protein is mainly found in the nucleus and nucleolus. The nucleolus, the site of ribosome biogenesis, is a highly organized, non-membrane-bound sub-compartment where proteins with a high affinity for nucleolar components are found. While it is well known that Tat accumulates in the nucleolus via a specific nucleolar targeting sequence, its function in this compartment it still unknown.ResultsTo clarify the significance of the Tat nucleolar localization, we induced the expression of the protein during oogenesis in Drosophila melanogaster strain transgenic for HIV-tat gene. Here we show that Tat localizes in the nucleoli of Drosophila oocyte nurse cells, where it specifically co-localizes with fibrillarin. Tat expression is accompanied by a significant decrease of cytoplasmic ribosomes, which is apparently related to an impairment of ribosomal rRNA precursor processing. Such an event is accounted for by the interaction of Tat with fibrillarin and U3 snoRNA, which are both required for pre-rRNA maturation.ConclusionOur data contribute to understanding the function of Tat in the nucleolus, where ribosomal RNA synthesis and cell cycle control take place. The impairment of nucleolar pre-rRNA maturation through the interaction of Tat with fibrillarin-U3snoRNA complex suggests a process by which the virus modulates host response, thus contributing to apoptosis and protein shut-off in HIV-uninfected cells.

Molecular cloning and targeting of a fibrillarin homolog from Arabidopsis.

Fibrillarin is a nucleolar protein known to be involved in the processing of ribosomal RNA precursors. We isolated AtFbr1, a cDNA encoding a homolog of fibrillarin in Arabidopsis. The cDNA is 1.2 kb in size and encodes a polypeptide of 310 amino acid residues with a molecular mass of 33 kD. AtFbr1 is expressed at high levels in the flower and root tissue and at a slightly lower level in leaf tissue, whereas it was nearly undetectable in siliques. Expression of AtFbr1 was compared with that of the FLP (fibrillarin-like protein) gene identified by the Arabidopsis genome project. Abscisic acid treatment resulted in the down-regulation of the expression of both AtFbr1 and FLP genes in seedlings, although the degree of suppression was higher for FLP than for AtFbr1. In addition, the expression level of FLP decreased with the age of the seedlings, whereas AtFbr1 did not exhibit any detectable change. The subcellular localization of AtFbrl was studied with an in vivo targeting approach using a fusion protein, and was found to be correctly targeted to the nucleolus in protoplasts when expressed as a green fluorescent fusion protein (GFP). Deletion experiments showed that the N-terminal glycine- and arginine-rich region is necessary and sufficient to target AtFbr1 to the nucleolus.

Small nucleolar RNAs.

Many small RNA species associate with the nucleolar structure. Some of these small nucleolar RNAs (snoRNAs) are required for cleavage processing of ribosomal RNA precursors. There are many pseudouridine residues and methylated riboses in mature ribosomal RNA. For most, if not all, of these modifications, each site is selected by base pairing with a specific snoRNA species. Some snoRNAs are needed for the 2'-O-ribose methylation of at least one spliceosomal small nuclear RNA. Many snoRNAs, particularly in yeast, are generated from independent transcription units. Most vertebrate snoRNAs are produced by processing of introns from protein-coding transcripts. Some snoRNAs are made by processing of introns from non-protein-coding transcripts.

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.

Adaptation of Ribonucleic Acid Metabolism to Anoxia in Rice Embryos

Rice (Oryza sativa var. Cigalon) is a plant which can adapt to very stringent anoxic conditions. It has previously been shown that during the adaptation period the energy charge can be used as a marker for metabolic activity. We have studied RNA metabolism during this period and correlated it with changes in the energy charge. Uptake of labeled precursor, UTP-specific activity, and incorporation were measured. Immediately after transfer to anaerobic conditions, the UTP pool size is reduced and the overall rate of incorporation drops. During adaptation, the rate of incorporation increases and stabilizes at about half of its value in aerobic conditions. Analysis of RNA shows that rRNA and mRNA are synthesized and that the processing of ribosomal RNA precursor is altered. Polyribosomes are present throughout the adaptation period although their amount is reduced during the first hour of anoxia. Changes in poly(A) content were noticed, indicating that some mRNA are rapidly degraded. Taken together, the results show that the RNA metabolism can be modulated during adaptation to anoxia in a parallel manner with energy charge changes.

Synthesis of polyadenylated messenger RNA during the cell cycle of Saccharomyces cerevisiae

The rates of ribosomal and polyadenylated messenger RNA synthesis were measured through the cell cycle of the budding yeast Saccharomyces cerevisiae. An exponentially growing culture was pulse-labelled with [2- 3H]adenine for 0.1 generation time, then fractionated according to cell volume by zonal centrifugation. A control experiment with a synchronously dividing culture showed that zonal fractionation corresponded to a fractionation by stage in the cell cycle. Nucleic acids were extracted from each fraction of the cell cycle from the zonal rotor, and DNA, ribosomal and polyadenylated messenger RNAs were isolated. DNA synthesis occurred between 0.4 and 0.8 of the cell cycle as fractionated on the zonal rotor. The rate of ribosomal RNA synthesis per cell was constant during G 1‡ doubled early in the period of DNA synthesis, then remained constant at the new level for the remainder of the cell cycle. The rate of processing of ribosomal RNA precursors to mature ribosomal RNAs declined at the time of the increase in rate of ribosomal RNA synthesis, but recovered later in the cycle. This could mean that the pool of ribosomal proteins is small, and that the rate of ribosomal protein synthesis increases later in the cell cycle than the rate of ribosomal RNA synthesis. The rate of synthesis of polyadenylated messenger RNA was constant during G 1 and the earliest part of the period of DNA synthesis, then doubled over the remainder of the period of DNA synthesis. After DNA synthesis, the rate of polyadenylated messenger RNA synthesis remained steady for the remainder of the cell cycle. It is suggested that gene number controls the rates of ribosomal and messenger RNA synthesis. When the number of genes doubles during the DNA synthesis period, the amount of transcription also quickly doubles. Repeat experiments suggested that the genes coding for ribosomal RNA are reproducibly replicated earlier, and over a shorter proportion of the DNA replication period than the genes coding for messenger RNAs. It is calculated that a minimum of 625 genes coding for messenger RNA per haploid genome, transcribed at the maximum rate throughout the cell cycle, would be required to give the observed cell-cycle pattern of messenger RNA synthesis. The total content of polyadenylated messenger RNA of cells at different stages of the cell cycle was measured by the formation of ribonuclease-resistant complexes between polyadenylic acid sequences in total UNA from non-radioactive yeast and [ 3H]polyuridylic acid. The amount of polyadenylated messenger RNA per cell rose after the doubling of the rate of polyadenylated messenger RNA synthesis, and by the end of the cell cycle was twice the level at the beginning of the cycle. The cell-cycle patterns of messenger and ribosomal RNA synthesis are discussed in relation to the control of balanced growth and doubling of all cellular components during the cell cycle, and in relation to known patterns of enzyme activity during the cell cycle of budding yeast.

Studies on nucleic acids in lymphocytes of chronic lymphocytic leukaemia.

The resting lymphocytes of patients with chronic lymphocytic leukaemia (CLL) contain the same amount of RNA as do those of normal individuals, and in terms of different molecular species, as analyzed by polyacrylamide gels, there is very little to distinguish the leukaemic cells. However, a relatively large amount of low molecular weight RNA, similar to the SnRNA found in several other tissues, is present in the CLL cells. The products of transcription of the leukaemic cell nucleus have been studied by the incorporation of labelled uridine and methionine. The leukaemic lymphocytes show a build-up and apparent delay in processing of ribosomal RNA precursor, when compared to normals, but studies of methylation reveal that the production of mature ribosomal RNA occurs at a normal rate. The nature of the stable high molecular weight material produced is now being studied. The production of proteins on the ribosome of CLL cells seems likely to be faulty, as evidenced by the deficiency of active ribosomes in the leukaemic cells. Studies with selective inhibitors will show whether this is due to some fault in transcription of messenger RNA.

The effect of temperature on nucleotide pool formation in Tetrahymena pyriformis.

AbstractWarming of exponentially growing T. pyriformis to 34°C results in severe inhibition of nucleotide pool formation. The utilization of the pool for stable RNA synthesis is poorly affected at the high temperature. It thus appears that the synthesis and processing of ribosomal RNA precursors are not primarily impaired at 34°C.

A temperature-sensitive mutation affecting 28S ribosomal RNA production in mammalian cells.

We have characterized a temperature-sensitive (ts) mutant of the hamster cell line BHK 21 that appears to have a defect in the processing of ribosomal RNA precursors at 39 degrees . Mutant ts 422E grows at a normal rate at 33 degrees , but upon shift to 39 degrees growth stops after about one cell doubling. The appearance of 28S rRNA and large ribosomal subunits in the cytoplasm of ts 422E at 39 degrees is inhibited by about 95%, when compared to wild-type BHK cells. Production of 18S rRNA and small ribosomal subunits is unaffected. Shift-up experiments show that the defect in 28S rRNA production can be detected as early as 2-3 hr after the shift to 39 degrees . Synthesis of the larger rRNA precursor is normal at high temperature, but the processing appears to be arrested after the formation of 32S rRNA. 32S rRNA accumulates to some extent in the nucleoli of ts 422E. ts 422E cells appear to have a single mutation, directly affecting the conversion of 32S to 28S rRNA. The reduced amount of 28S rRNA in the cytoplasm of ts 422E cells at 39 degrees seems therefore responsible for their inability to grow at this temperature.

Destructive processing of the 45-S ribosomal precursor in the presence of 5-azacytidine

The base analogue 5-azacytidine causes aberrant processing of ribosomal RNA precursor. Radioactivity in the 45-S precursor molecule decreases, but the products of cleavage are found in greatly reduced quantities. It appears that most of the 45-S material is degraded.

Sites of synthesis and processing of ribosomal RNA presurosrs within the nucleolus of Urechis caupo eggs.

Nucleoli from unfertilized Urechis eggs, labeled with tritiated RNA precursors, have been isolated for simultaneous autoradiographic localization and biochemical analysis of labeled RNA. The production of the ribosomal RNA precursor (38S) and its first cleavage occur at the fibrillar core region of the nucleolus. The products, predominantly 30S RNA, are then rapidly transported and stored in the granular cortex of the nucleolus. The formation of the nucleolar cortex, therefore, seems to result from an accumulation of partially processed ribosomal RNA with its associated proteins.

Processing of ribosomal RNA precursor in isolated insect nuclei

In isolated nuclei, derived from developing silk moth wings pulse-labeled with H 3-uridine, progressive redistribution of label occurs among various classes of ribosomal RNA during a period of in , vitro culture in non-radioactive medium. The results indicate that the in , vivo labeled 39s precursor can be processed into smaller ribosomal RNA species in a cell-free system.

The fractionation of nuclei and the integrity of purified nucleoli in HeLa cells

The retention of structural and functional properties by nucleoli after purification has been investigated in HeLa cells. It is shown that: 1. 1. The partition of 60-S nuclear particles between the nucleolar and nucleoplasmic fractions depends on the salt concentration. 2. 2. The partition of nuclear heterodisperse RNA is also dependent on salt concentration. 3. 3. 15–20 % of the 28-S containing particles detected in the nucleoplasm at high salt concentration are in the form of very heavy clusters of 60-S particles. 4. 4. The nucleoli purified in high salt concentration conserve the capacity to carry on in vitro the initial stages of processing of ribosomal RNA precursors.