Processing Of Ribosomal Precursor RNA Research Articles

Uncovering a delicate balance between endonuclease RNase III and ribosomal protein S15 in E. coli ribosome assembly

The bacterial ribosomal protein S15 is located in the platform, a functional region of the 30S ribosomal subunit. While S15 is critical for in vitro formation of E. coli small subunits (SSUs), it is dispensable for in vivo biogenesis and growth. In this work, a novel synergistic interaction between rpsO, the gene that encodes S15, and rnc (the gene that encodes RNase III), was uncovered in E. coli. RNase III catalyzes processing of precursor ribosomal RNA (rRNA) transcripts and thus is involved in functional ribosome subunit maturation. Strains lacking S15 (ΔrpsO), RNase III (Δrnc) or both genes were examined to understand the relationship between these two factors and the impact of this double deletion on rRNA processing and SSU maturation. The double deletion of rpsO and rnc partially alleviates the observed cold sensitivity of ΔrpsO alone. A novel 16S rRNA precursor (17S∗ rRNA) that is detected in free 30S subunits of Δrnc is incorporated in 70S-like ribosomes in the double deletion. The stable accumulation of 17S∗ rRNA suggests that timing of processing events is closely coupled with SSU formation events in vivo. The double deletion has a suppressive effect on the cell elongation phenotype of ΔrpsO. The alteration of the phenotypes associated with S15 loss, due to the absence of RNase III, indicates that pre-rRNA processing and improvement of growth, relative to that observed for ΔrpsO, are connected. The characterization of the functional link between the two factors illustrates that there are redundancies and compensatory pathways for SSU maturation.

Cryo-EM reveals active site coordination within a multienzyme pre-rRNA processing complex

Ribosome assembly is a complex process reliant on the coordination of trans-acting enzymes to produce functional ribosomal subunits and secure the translational capacity of cells. The endoribonuclease (RNase) Las1 and the polynucleotide kinase (PNK) Grc3 assemble into a multienzyme complex, herein designated RNase PNK, to orchestrate processing of precursor ribosomal RNA (rRNA). RNase PNK belongs to the functionally diverse HEPN nuclease superfamily, whose members rely on distinct cues for nuclease activation. To establish how RNase PNK coordinates its dual enzymatic activities, we solved a series of cryo-EM structures of Chaetomium thermophilum RNase PNK in multiple conformational states. The structures reveal that RNase PNK adopts a butterfly-like architecture, harboring a composite HEPN nuclease active site flanked by discrete RNA kinase sites. We identify two molecular switches that coordinate nuclease and kinase function. Together, our structures and corresponding functional studies establish a new mechanism of HEPN nuclease activation essential for ribosome production.

Structural insight into precursor ribosomal RNA processing by ribonuclease MRP.

Ribonuclease (RNase) MRP is a conserved eukaryotic ribonucleoprotein complex that plays essential roles in precursor ribosomal RNA (pre-rRNA) processing and cell cycle regulation. In contrast to RNase P, which selectively cleaves transfer RNA-like substrates, it has remained a mystery how RNase MRP recognizes its diverse substrates. To address this question, we determined cryo-electron microscopy structures of Saccharomyces cerevisiae RNase MRP alone and in complex with a fragment of pre-rRNA. These structures and the results of biochemical studies reveal that coevolution of both protein and RNA subunits has transformed RNase MRP into a distinct ribonuclease that processes single-stranded RNAs by recognizing a short, loosely defined consensus sequence. This broad substrate specificity suggests that RNase MRP may have myriad yet unrecognized substrates that could play important roles in various cellular contexts.

Cryo-EM reveals active site coordination within a multienzyme pre-rRNA processing complex.

Ribosome assembly is a complex process reliant on the coordination of trans-acting enzymes to produce functional ribosomal subunits and secure the translational capacity of cells. The endoribonuclease (RNase) Las1 and the poly-nucleotide kinase (PNK) Grc3 assemble into a multienzyme complex, herein designated RNase PNK, to orchestrate processing of precursor ribosomal RNA. RNase PNK belongs to the functionally-diverse HEPN nuclease superfamily, whose members rely on distinct cues for nuclease activation. To establish how RNase PNK coordinates its dual enzymatic activities, we solved a series of cryo-electron microscopy structures of Chaetomium thermophilum RNase PNK in multiple conformational states. The structures reveal that RNase PNK adopts a butterfly-like architecture harboring a composite HEPN nuclease active site flanked by discrete RNA kinase sites. We identify two molecular switches that coordinate nuclease and kinase function. Together our structures and corresponding functional studies establish a new mechanism of HEPN nuclease activation essential for ribosome production.

Arabidopsis protein arginine methyltransferase 3 is required for ribosome biogenesis by affecting precursor ribosomal RNA processing.

Ribosome biogenesis is a fundamental and tightly regulated cellular process, including synthesis, processing, and assembly of rRNAs with ribosomal proteins. Protein arginine methyltransferases (PRMTs) have been implicated in many important biological processes, such as ribosome biogenesis. Two alternative precursor rRNA (pre-rRNA) processing pathways coexist in yeast and mammals; however, how PRMT affects ribosome biogenesis remains largely unknown. Here we show that Arabidopsis PRMT3 (AtPRMT3) is required for ribosome biogenesis by affecting pre-rRNA processing. Disruption of AtPRMT3 results in pleiotropic developmental defects, imbalanced polyribosome profiles, and aberrant pre-rRNA processing. We further identify an alternative pre-rRNA processing pathway in Arabidopsis and demonstrate that AtPRMT3 is required for the balance of these two pathways to promote normal growth and development. Our work uncovers a previously unidentified function of PRMT in posttranscriptional regulation of rRNA, revealing an extra layer of complexity in the regulation of ribosome biogenesis.

Active regulator of SIRT1 is required for ribosome biogenesis and function

Active regulator of SIRT1 (AROS) binds and upregulates SIRT1, an NAD+-dependent deacetylase. In addition, AROS binds RPS19, a structural ribosomal protein, which also functions in ribosome biogenesis and is implicated in multiple disease states. The significance of AROS in relation to ribosome biogenesis and function is unknown. Using human cells, we now show that AROS localizes to (i) the nucleolus and (ii) cytoplasmic ribosomes. Co-localization with nucleolar proteins was verified by confocal immunofluorescence of endogenous protein and confirmed by AROS depletion using RNAi. AROS association with cytoplasmic ribosomes was analysed by sucrose density fractionation and immunoprecipitation, revealing that AROS selectively associates with 40S ribosomal subunits and also with polysomes. RNAi-mediated depletion of AROS leads to deficient ribosome biogenesis with aberrant precursor ribosomal RNA processing, reduced 40S subunit ribosomal RNA and 40S ribosomal proteins (including RPS19). Together, this results in a reduction in 40S subunits and translating polysomes, correlating with reduced overall cellular protein synthesis. Interestingly, knockdown of AROS also results in a functionally significant increase in eIF2α phosphorylation. Overall, our results identify AROS as a factor with a role in both ribosome biogenesis and ribosomal function.

Downregulation of pre-rRNA processing gene Mamrd1 decreases growth, conidiation and virulence in the entomopathogenic fungus Metarhizium acridum

Mrd1 is one of the trans-acting proteins and plays an important role in precursor ribosomal RNA processing. Here we characterized the Mamrd1 gene from Metarhizium acridum and studied its function in growth, conidiation and virulence using RNA interference. The Mamrd1 gene was identified as participating in the processing of pre-rRNA in M. acridum and was highly upregulated during the infection process. A Mamrd1-RNAi strain exhibited an appearance of fluffy mycelia, a defective branching pattern and delayed conidiation compared to the wild-type strain. Downregulation of Mamrd1 in M. acridum suppressed growth both on artificial medium and inside the insect, and significantly reduced hyphal biomass, conidium production and virulence against Locusta migratoria manilensis. These results demonstrated that Mamrd1 plays an important role in growth, conidiation and virulence in M. acridum.

Molecular cloning and gene expression of a fibrillarin homolog of tobacco BY-2 cells

Fibrillarin is known to play an important role in precursor ribosomal RNA processing and ribosome assembly. The present study describes a fibrillarin homolog gene isolated from tobacco BY-2 cells and its expression during the cell cycle. The cDNA for a fibrillarin homolog, named NtFib1, was first cloned in Nicotiana tabacum with degenerate primers. It encodes 314 amino acids and the deduced amino acid sequence has some highly conserved functional domains, such as the glycine and arginine-rich (GAR) domain for nucleolar localization and the RNA-binding motif. The C-terminal region is highly conserved and has 7 beta-sheets and 7 alpha-helices which are peculiar to fibrillarin. Thus, it is suggested that the fibrillarin homolog of this plant species functions in the same way as the fibrillarin already known from human and yeast cells. Northern blot analysis of BY-2 cells synchronized with aphidicolin or a combination of aphidicolin and propyzamide showed that the histone H4 gene was specifically expressed in the S phase but NtFib1 mRNA remained at high levels during the cell cycle. Examination of the localization of NtFib1 protein tagged with green-fluorescent protein (GFP) suggested that some persisting in the mitotic apparatus was eventually incorporated into reconstructed nucleoli in late telophase. Newly synthesized GFP-tagged NtFib1 protein in the cytoplasm was added to the recycled protein in early mitosis. Highly concentrated actinomycin D completely inhibited the transcription of genes coding for rRNA (rDNA) but did not significantly suppress the amount of either NtFib1 mRNA or protein, although the NtFib1 protein was reversibly dislocated from nucleoli. Although hypoxic shock completely prohibited rDNA transcription, NtFib1 mRNA remained at the same level as in the control experiment, even after the 4 h treatment. These results indicate that the transcription of NtFib1 mRNA is not related to rDNA transcription and NtFib1 mRNA is resistant to disrupting factors during the cell cycle.

Nucleolin Responds to DNA Damage

Stimuli that damage DNA activate stress-signaling pathways that include the activation of the tumor suppressor p53. Daniely et al. report that ionizing radiation (IR) or the drug camptothecin produce a transient p53-dependent redistribution of nucleolin from nucleoli to the nucleoplasm. This redistribution did not occur in cells lacking p53 or in cells in which p53 expression was inhibited through a tetracycline-repressible system. Nucleolin and p53 were coprecipitable, and the formation of the complex was stimulated by IR or camptothecin. Not all stresses that stimulated phosphorylation of p53, such as ultraviolet radiation, also stimulated the formation of the nucleolin-p53 complex. Although p53 was required for the redistribution of nucleolin, analysis of cells expressing mutant versions of p53 showed that transcriptional activity of p53 was not required. However, cells expressing a COOH-terminally truncated version of p53 did not redistribute nucleolin in response to IR, nor was a complex detectable between the p53 COOH-terminal truncation mutant and nucleolin. Nucleolin when present in nucleoli is important for processing of precursor ribosomal RNA. Once released from the nucleoli into the nucleoplasm, nucleolin may inhibit DNA replication through interactions with replication protein A and may stimulate DNA annealing, which would promote DNA repair. Y. Daniely, D. D. Dimitrova, J. A. Borowiec, Stress-dependent nucleolin mobilization mediated by p53-nucleolin complex formation. Mol. Cell. Biol. 22 , 6014-6022 (2002). [Abstract] [Full Text]

In vivo release of mitotic silencing of ribosomal gene transcription does not give rise to precursor ribosomal RNA processing.

Nuclear RNA transcription is repressed when eukaryotic cells enter mitosis. Here, we found that the derepression of ribosomal gene (rDNA) transcription that normally takes place in telophase may be induced in prometaphase, metaphase, and anaphase mitotic HeLa cells, and therefore appears not to be dependent on completion of mitosis. We demonstrate for the first time that in vivo inhibition of the cdc2- cyclin B kinase activity is sufficient to give rise to okadaic acid-sensitive dephosphorylation of the mitotically phosphorylated forms of components of the rDNA transcription machinery, and consequently to restore rDNA transcription in mitotic cells. These results, showing that during mitosis the rDNA transcription machinery is maintained repressed by the cdc2-cyclin B kinase activity, provide an in vivo demonstration of the cell cycle-dependent regulation of rDNA transcription. Interestingly in mitotic cells, the newly synthesized 47S precursor ribosomal RNA (pre-rRNA) is not processed into the mature rRNAs, indicating that rDNA transcription and pre-rRNA processing may be uncoupled. Moreover this suggests that inhibition of the cdc2- cyclin B kinase is not sufficient to activate the 47S pre-rRNA processing machinery and/or to induce its relocalization at the level of newly synthesized 47S pre-rRNA. This in vivo approach provides new possibilities to investigate the correlation between pre-rRNA synthesis and pre-rRNA processing when the nucleolus reforms.

Processing of pre‐ribosomal RNA in Saccharomyces cerevisiae

Post-transcriptional processing of precursor-ribosomal RNA comprises a complex pathway of endonucleolytic cleavages, exonucleolytic digestion and covalent modifications. The general order of the various processing steps is well conserved in eukaryotic cells, but the underlying mechanisms are largely unknown. Recent analysis of pre-rRNA processing, mainly in the yeast Saccharomyces cerevisiae, has significantly improved our understanding of this important cellular activity. Here we will review the data that have led to our current picture of yeast pre-rRNA processing.

Molecular characterisation of plant U14 small nucleolar RNA genes: closely linked genes are transcribed as polycistronic U14 transcripts.

U14snoRNAs are highly conserved eukaryotic nucleolar small RNAs involved in precursor ribosomal RNA processing. In vertebrates, U14snoRNAs and a number of other snoRNAs are transcribed within introns of protein coding genes and are released by processing. We have isolated potato and maize genomic U14 clones using PCR-amplified plant U14 probes. Plant U14s show extensive homology to those from yeast and animals but contain plant-specific sequences. One of the isolated maize clones contains a cluster of four U14 genes in a region of only 761 bp, confirming the close linkage of U14 genes in maize, potato and barley as established by PCR. The absence of known plant promoter elements, the proximity of the genes and the detection of transcripts containing linked U14s by RT-PCR indicates that some plant U14snoRNAs are transcribed as precursor RNAs which are then processed to release individual U14s. Whether plant U14snoRNAs are intron-encoded or transcribed from novel promoter sequences, remains to be established.

Ribosomal RNA processing. Limited cleavages of mouse preribosomal RNA by a nucleolar endoribonuclease include the early +650 processing site.

A highly purified nucleolar associated endoribonuclease was tested for possible involvement in the processing of precursor ribosomal RNA at a primary cleavage site approximately 650 nucleotides downstream from the transcription initiation site. Preribosomal RNA sequences containing the +650 region were synthesized in vitro and subsequently digested over a range of concentrations of the nucleolar endoribonuclease. Cleavages generated by the nucleolar endoribonuclease were localized both by S1 nuclease protection analysis and primer extension analysis. A more precise determination of the specificity of cleavage was achieved by chemical cleavage DNA sequence analysis. These data demonstrated that the purified nucleolar endoribonuclease specifically cleaved the precursor ribosomal RNA transcript at the +650 site. Additional enzyme-dependent cleavages were observed upstream to the +650 site in a region which is rapidly degraded following processing at the +650 site in vivo. No major cleavages were observed for a distance of approximately 250 nucleotides downstream from the +650 site in a conserved region of sequence previously shown to be important in specifying processing at the +650 site. As a control, pancreatic ribonuclease, a single strand-specific endoribonuclease, was shown not to produce similar cleavages in the +650 region, indicating that cleavage by the nucleolar RNase was not simply due to accessibility of the RNA at the +650 site. Taken together, these results suggest that the nucleolar endoribonuclease may be necessary and sufficient to catalyze one of the initial endonucleolytic cleavages in preribosomal RNA processing.

Processing of precursor ribosomal RNA and the presence of a modified ribosome assembly scheme in Escherichia coli relaxed strain

An electrophoretic system capable of separating 25 S, 23 S, 17.5 S and 16 S ribosomal RNA (rRNA) species was used to study the synthesis and fate of rRNA during amino acid starvation and resupplementation of E. coli relaxed strain KL99. This E. coli relA1 strain responded to an amino acid starvation by increasing the rate of synthesis of 25 S and 17.5 S precursor rRNA. When the limiting amino acid was resupplemented, a previously observed 40-fold increase in the cellular guanosine 5'-diphosphate, 3'-diphosphate content [Mol. Gen. Genet. (1983) 192, 5-9] appeared to cause a reduction in new rRNA synthesis. Following amino acid resupplementation, the precursor 25 S and 17.5 S rRNA accumulated during the amino acid starvation were conserved and processed to 23 S and 16 S rRNA species, respectively. This suggests that a modified ribosome assembly scheme involving stable precursor rRNA exists in relA 1 bacteria during periods of amino acid limitation and resupplementation.

RNA synthesis and control of cell division in the yeast S. cerevisiae

Cells of the yeast Saccharomyces cerevisiae rapidly accumulated in the G1 phase of the cell cycle when exposed to the chelating agents o-phenanthroline (OP) or 8-hydroxyquinoline (HQ). Zinc salts fully reversed the growth-inhibitory effect of both OP and HQ. Cells treated with these chelating agents showed limited RNA accumulation and little RNA degradation. Rates of RNA synthesis were drastically reduced by low concentrations of these compounds, whereas rates of protein synthesis were essentially unaffected. Rates of synthesis of mRNA and tRNA were less affected than were rates of synthesis of high molecular weight RNA. Processing of ribosomal precursor RNA was altered. These results suggest that the primary effect of OP and HQ is on rRNA synthesis. RNA metabolism must therefore have a key role in the regulation of the cell cycle.

Ribosomal RNA transcription in a mutant of Saccharomyces cerevisiae defective in ribosomal protein synthesis

In Saccharomyces cerevisiae, the transcription of ribosomal precursor RNA is severely inhibited in the absence of protein synthesis. However, such transcription is not dependent on the synthesis of ribosomal proteins, nor on the synthesis of mRNA for ribosomal proteins, nor on the processing of ribosomal precursor RNA.

Early estrogen action: Stimulation of the synthesis of methylated ribosomal and transfer RNAs

The effects of estrogen on the rates of incorporation in vivo of radioactive uridine and Me-methionine, administered together, into RNA in the uterus of the ovariectomized adult rat have been measured. The ratio of incorporation of methionine to uridine during a 45-min labeling period was increased severalfold by hormone treatment. The increased rate of methylation was apparent in the uterus taken from the rat administered estrogen for 1 h, and the effect was more striking following 2 and 3 h of hormone treatment. This stimulation of methylation of RNA occurred in association with an increase in the whole-organ concentration of RNA. Analysis of the doubly-labeled uterine RNA on sucrose gradients revealed that the methylated species were mainly ribosomal and transfer RNA. These results show that very little methylation of RNA occurs in the atrophied uterus of the ovariectomized rat. During the first 3 h following estrogen administration to the ovariectomized animal, an increasing percentage of the newly synthesized RNA formed by the uterus is methylated ribosomal and transfer RNA. This result is discussed in light of recent studies of the efficiency of processing of ribosomal precursor RNA, as well as the synthesis of high-molecular-weight heterogeneous RNA in the early action of estrogen.

Defective rRNA synthesis in human leukaemic blast cells?

RECENT studies suggest that processing of ribosomal precursor RNA might be impaired in blast cells of acute leukaemia (AML)1,2. Following incubation with 3H-methyl-methionine blast cells of human acute leukaemias partially failed to methylate the 45S and 32S ribosomal precursors1. Using autoradiographic techniques, previous studies have also shown an increase in intranuclear RNA and decreased cytoplasmic RNA in these cells3. More recently, competitive hybridisation was performed with normal lymphocyte DNA and 35S–55S RNA fractions extracted from both acute leukaemia blast cells and normal PHA-stimulated lymphocytes. Using incubations with actinomycin D these experiments have led to the conclusion that leukaemic blast cells synthesise heterogeneous nuclear RNA molecules which are not transcribed in PHA-stimulated lymphocytes and it was also suggested—in contrast to the earlier data—that the amount of unmethylated unprocessed rRNA precursor was small in leukaemic blast cells4.

Processing of precursor ribosomal RNA in suspensions of higher plant cells

FEBS LettersVolume 28, Issue 2 p. 183-187 Full-length articleFree Access Processing of precursor ribosomal RNA in suspensions of higher plant cells J.P. Cecchini, J.P. Cecchini Laboratoire de Physiologie Cellulaire Végétale associé au C.N.R.S., Université d'Aix-Marseille, Centre de Luminy, 13288 Marseille, cedex 2, FranceSearch for more papers by this authorR. Miassod, R. Miassod Laboratoire de Physiologie Cellulaire Végétale associé au C.N.R.S., Université d'Aix-Marseille, Centre de Luminy, 13288 Marseille, cedex 2, FranceSearch for more papers by this authorJ. Ricard, J. Ricard Laboratoire de Physiologie Cellulaire Végétale associé au C.N.R.S., Université d'Aix-Marseille, Centre de Luminy, 13288 Marseille, cedex 2, FranceSearch for more papers by this author J.P. Cecchini, J.P. Cecchini Laboratoire de Physiologie Cellulaire Végétale associé au C.N.R.S., Université d'Aix-Marseille, Centre de Luminy, 13288 Marseille, cedex 2, FranceSearch for more papers by this authorR. Miassod, R. Miassod Laboratoire de Physiologie Cellulaire Végétale associé au C.N.R.S., Université d'Aix-Marseille, Centre de Luminy, 13288 Marseille, cedex 2, FranceSearch for more papers by this authorJ. Ricard, J. Ricard Laboratoire de Physiologie Cellulaire Végétale associé au C.N.R.S., Université d'Aix-Marseille, Centre de Luminy, 13288 Marseille, cedex 2, FranceSearch for more papers by this author First published: December 01, 1972 https://doi.org/10.1016/0014-5793(72)80707-5Citations: 15AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume28, Issue2December 01, 1972Pages 183-187 ReferencesRelatedInformation

Ribosome formation is blocked by camptothecin, a reversible inhibitor of RNA synthesis.

A new drug, camptothecin, has been used to study the regulation of ribosome synthesis in HeLa cells. 5 muM camptothecin inhibits the synthesis of heterogeneously sedimenting nuclear RNA by about 70%. Camptothecin also blocks a specific step in the processing of ribosomal precursor RNA, allowing the conversion of 45S RNA to 32S RNA, but inhibiting the conversion of 32S RNA to 28S RNA. The action of camptothecin, unlike that of actinomycin D, is rapidly reversible. Within 5 min after the removal of the drug, ribosomal precursor RNA synthesis and maturation resume at the normal rate. Ribosomal proteins made in the presence of camptothecin do not accumulate in the nucleolus, and are not subsequently used to form ribosomes if RNA synthesis is allowed to resume.