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1
Aquino, Gerald Ryan R., Nicolai Krogh, Philipp Hackert, Roman Martin, Jimena Davila Gallesio, Robert W. van Nues, Claudia Schneider, et al. "RNA helicase-mediated regulation of snoRNP dynamics on pre-ribosomes and rRNA 2′-O-methylation." Nucleic Acids Research 49, no. 7 (March 15, 2021): 4066–84. http://dx.doi.org/10.1093/nar/gkab159.
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Abstract RNA helicases play important roles in diverse aspects of RNA metabolism through their functions in remodelling ribonucleoprotein complexes (RNPs), such as pre-ribosomes. Here, we show that the DEAD box helicase Dbp3 is required for efficient processing of the U18 and U24 intron-encoded snoRNAs and 2′-O-methylation of various sites within the 25S ribosomal RNA (rRNA) sequence. Furthermore, numerous box C/D snoRNPs accumulate on pre-ribosomes in the absence of Dbp3. Many snoRNAs guiding Dbp3-dependent rRNA modifications have overlapping pre-rRNA basepairing sites and therefore form mutually exclusive interactions with pre-ribosomes. Analysis of the distribution of these snoRNAs between pre-ribosome-associated and ‘free’ pools demonstrated that many are almost exclusively associated with pre-ribosomal complexes. Our data suggest that retention of such snoRNPs on pre-ribosomes when Dbp3 is lacking may impede rRNA 2′-O-methylation by reducing the recycling efficiency of snoRNPs and by inhibiting snoRNP access to proximal target sites. The observation of substoichiometric rRNA modification at adjacent sites suggests that the snoRNPs guiding such modifications likely interact stochastically rather than hierarchically with their pre-rRNA target sites. Together, our data provide new insights into the dynamics of snoRNPs on pre-ribosomal complexes and the remodelling events occurring during the early stages of ribosome assembly.
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Shiao, Yih-Horng. "Promising Assays for Examining a Putative Role of Ribosomal Heterogeneity in COVID-19 Susceptibility and Severity." Life 12, no. 2 (January 28, 2022): 203. http://dx.doi.org/10.3390/life12020203.
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The heterogeneity of ribosomes, characterized by structural variations, arises from differences in types, numbers, and/or post-translational modifications of participating ribosomal proteins (RPs), ribosomal RNAs (rRNAs) sequence variants plus post-transcriptional modifications, and additional molecules essential for forming a translational machinery. The ribosomal heterogeneity within an individual organism or a single cell leads to preferential translations of selected messenger RNA (mRNA) transcripts over others, especially in response to environmental cues. The role of ribosomal heterogeneity in SARS-CoV-2 coronavirus infection, propagation, related symptoms, or vaccine responses is not known, and a technique to examine these has not yet been developed. Tools to detect ribosomal heterogeneity or to profile translating mRNAs independently cannot identify unique or specialized ribosome(s) along with corresponding mRNA substrate(s). Concurrent characterizations of RPs and/or rRNAs with mRNA substrate from a single ribosome would be critical to decipher the putative role of ribosomal heterogeneity in the COVID-19 disease, caused by the SARS-CoV-2, which hijacks the host ribosome to preferentially translate its RNA genome. Such a protocol should be able to provide a high-throughput screening of clinical samples in a large population that would reach a statistical power for determining the impact of a specialized ribosome to specific characteristics of the disease. These characteristics may include host susceptibility, viral infectivity and transmissibility, severity of symptoms, antiviral treatment responses, and vaccine immunogenicity including its side effect and efficacy. In this study, several state-of-the-art techniques, in particular, chemical probing of ribosomal components or rRNA structures, proximity ligation to generate rRNA-mRNA chimeras for sequencing, nanopore gating of individual ribosomes, nanopore RNA sequencing and/or structural analyses, single-ribosome mass spectrometry, and microfluidic droplets for separating ribosomes or indexing rRNAs/mRNAs, are discussed. The key elements for further improvement and proper integration of the above techniques to potentially arrive at a high-throughput protocol for examining individual ribosomes and their mRNA substrates in a clinical setting are also presented.
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Sleiman, Sophie, and Francois Dragon. "Recent Advances on the Structure and Function of RNA Acetyltransferase Kre33/NAT10." Cells 8, no. 9 (September 5, 2019): 1035. http://dx.doi.org/10.3390/cells8091035.
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Ribosome biogenesis is one of the most energy demanding processes in the cell. In eukaryotes, the main steps of this process occur in the nucleolus and include pre-ribosomal RNA (pre-rRNA) processing, post-transcriptional modifications, and assembly of many non-ribosomal factors and ribosomal proteins in order to form mature and functional ribosomes. In yeast and humans, the nucleolar RNA acetyltransferase Kre33/NAT10 participates in different maturation events, such as acetylation and processing of 18S rRNA, and assembly of the 40S ribosomal subunit. Here, we review the structural and functional features of Kre33/NAT10 RNA acetyltransferase, and we underscore the importance of this enzyme in ribosome biogenesis, as well as in acetylation of non-ribosomal targets. We also report on the role of human NAT10 in Hutchinson–Gilford progeria syndrome.
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Rivas, Mario, and George E. Fox. "Nonstandard RNA/RNA interactions likely enhance folding and stability of segmented ribosomes." RNA 28, no. 3 (December 7, 2021): 340–52. http://dx.doi.org/10.1261/rna.079006.121.
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The ribosome is the molecular factory that catalyzes all coded protein synthesis in extant organisms. Eukaryotic ribosomes are typically assembled out of four rRNAs; namely, 5S, 5.8S, 18S, and 28S. However, the 28S rRNA of some trypanosomatid organisms has been found to be segmented into six independent rRNAs of different sizes. The two largest segments have multiple sites where they jointly form stems comprised of standard base pairs that can hold them together. However, such regions of interaction are not observed among the four smaller RNAs. Early reports suggested that trypanosomatid segmented ribosome assembly was essentially achieved thanks to their association with rProteins. However, examination of cryo-EM ribosomal structures from Trypanosoma brucei, Leishmania donovani, and Trypanosoma cruzi reveals several long-range nonstandard RNA/RNA interactions. Most of these interactions are clusters of individual hydrogen bonds and so are not readily predictable. However, taken as a whole, they represent significant stabilizing energy that likely facilitates rRNA assembly and the overall stability of the segmented ribosomes. In the context of origin of life studies, the current results provide a better understanding of the true nature of RNA sequence space and what might be possible without an RNA replicase.
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Larson, D. E., P. Zahradka, and B. H. Sells. "Control points in eucaryotic ribosome biogenesis." Biochemistry and Cell Biology 69, no. 1 (January 1, 1991): 5–22. http://dx.doi.org/10.1139/o91-002.
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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.Key words: rRNA, ribosomal proteins, nucleolus, ribosome.
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O’Sullivan, Justin M., Dave A. Pai, Andrew G. Cridge, David R. Engelke, and Austen R. D. Ganley. "The nucleolus: a raft adrift in the nuclear sea or the keystone in nuclear structure?" BioMolecular Concepts 4, no. 3 (June 1, 2013): 277–86. http://dx.doi.org/10.1515/bmc-2012-0043.
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AbstractThe nucleolus is a prominent nuclear structure that is the site of ribosomal RNA (rRNA) transcription, and hence ribosome biogenesis. Cellular demand for ribosomes, and hence rRNA, is tightly linked to cell growth and the rRNA makes up the majority of all the RNA within a cell. To fulfill the cellular demand for rRNA, the ribosomal RNA (rDNA) genes are amplified to high copy number and transcribed at very high rates. As such, understanding the rDNA has profound consequences for our comprehension of genome and transcriptional organization in cells. In this review, we address the question of whether the nucleolus is a raft adrift the sea of nuclear DNA, or actively contributes to genome organization. We present evidence supporting the idea that the nucleolus, and the rDNA contained therein, play more roles in the biology of the cell than simply ribosome biogenesis. We propose that the nucleolus and the rDNA are central factors in the spatial organization of the genome, and that rapid alterations in nucleolar structure in response to changing conditions manifest themselves in altered genomic structures that have functional consequences. Finally, we discuss some predictions that result from the nucleolus having a central role in nuclear organization.
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Wang, Xiangxiang, Zhiyong Yue, Feifei Xu, Sufang Wang, Xin Hu, Junbiao Dai, and Guanghou Zhao. "Coevolution of ribosomal RNA expansion segment 7L and assembly factor Noc2p specializes the ribosome biogenesis pathway between Saccharomyces cerevisiae and Candida albicans." Nucleic Acids Research 49, no. 8 (April 6, 2021): 4655–67. http://dx.doi.org/10.1093/nar/gkab218.
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Abstract Ribosomes of different species share an evolutionarily conserved core, exhibiting flexible shells formed partially by the addition of species-specific ribosomal RNAs (rRNAs) with largely unexplored functions. In this study, we showed that by swapping the Saccharomyces cerevisiae 25S rRNA genes with non-S. cerevisiae homologs, species-specific rRNA variations caused moderate to severe pre-rRNA processing defects. Specifically, rRNA substitution by the Candida albicans caused severe growth defects and deficient pre-rRNA processing. We observed that such defects could be attributed primarily to variations in expansion segment 7L (ES7L) and could be restored by an assembly factor Noc2p mutant (Noc2p-K384R). We showed that swapping ES7L attenuated the incorporation of Noc2p and other proteins (Erb1p, Rrp1p, Rpl6p and Rpl7p) into pre-ribosomes, and this effect could be compensated for by Noc2p-K384R. Furthermore, replacement of Noc2p with ortholog from C. albicans could also enhance the incorporation of Noc2p and the above proteins into pre-ribosomes and consequently restore normal growth. Taken together, our findings help to elucidate the roles played by the species-specific rRNA variations in ribosomal biogenesis and further provide evidence that coevolution of rRNA expansion segments and cognate assembly factors specialized the ribosome biogenesis pathway, providing further insights into the function and evolution of ribosome.
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Lo, Amy C., Wangyi Liu, Doreen E. Culham, and Ross N. Nazar. "Effects of ribosome dissociation on the structure of the ribosome-associated 5.8S RNA." Biochemistry and Cell Biology 65, no. 6 (June 1, 1987): 536–42. http://dx.doi.org/10.1139/o87-069.
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Diethyl pyrocarbonate reactivity and thermal denaturation were used to probe potential ribosomal interactions between tRNA and the small 5.8S and 5S rRNAs. Puromycin, an analogue of the terminal aminoacyl-adenosine portion of aminoacyl-tRNA, was observed to increase the accessibility of the 5.8S rRNA, including the highly conserved GAACp sequences. EDTA which releases both tRNA and the 5S rRNA – protein complex resulted in an even greater accessibility in the 5.8S rRNA. The thermal dissociation of whole ribosomes resulted in the release of all three RNAs, with a striking similarity in the denaturation profiles. These results strongly suggest an interdependence in the ribosome-associated structures of the small rRNAs and provide in situ evidence for the various 5S rRNA, 5.8S rRNA, and tRNA containing ribonucleoprotein complexes previously reconstituted through affinity chromatography.
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Bogdanov, Alexey A., Olga A. Dontsova, Svetlana S. Dokudovskaya, and Inna N. Lavrik. "Structure and function of 5S rRNA in the ribosome." Biochemistry and Cell Biology 73, no. 11-12 (December 1, 1995): 869–76. http://dx.doi.org/10.1139/o95-094.
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5S rRNA is a small RNA molecule that is a component of a ribosome from almost all living organisms. In this review, we discuss the biogenesis of 5S rRNA and its properties as an independent structural domain of a ribosome as well as the current concepts concerning the higher order structure of 5S rRNA in free state and in its complexes with ribosomal proteins and its folding in the ribosome. Special attention is paid to recent experimental approaches that have been useful in 5S rRNA studies. Our own data on topography of 5S rRNA in the ribosomes are discussed in detail. The hypothesis describing the possible functional role of 5S rRNA for ribosome functioning is discussed.Key words: 5S rRNA, ribosomes, 23S rRNA, site-directed chemical cross-linking, RNA folding.
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Root-Bernstein, Robert, and Meredith Root-Bernstein. "The Ribosome as a Missing Link in Prebiotic Evolution III: Over-Representation of tRNA- and rRNA-Like Sequences and Plieofunctionality of Ribosome-Related Molecules Argues for the Evolution of Primitive Genomes from Ribosomal RNA Modules." International Journal of Molecular Sciences 20, no. 1 (January 2, 2019): 140. http://dx.doi.org/10.3390/ijms20010140.
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We propose that ribosomal RNA (rRNA) formed the basis of the first cellular genomes, and provide evidence from a review of relevant literature and proteonomic tests. We have proposed previously that the ribosome may represent the vestige of the first self-replicating entity in which rRNAs also functioned as genes that were transcribed into functional messenger RNAs (mRNAs) encoding ribosomal proteins. rRNAs also encoded polymerases to replicate itself and a full complement of the transfer RNAs (tRNAs) required to translate its genes. We explore here a further prediction of our “ribosome-first” theory: the ribosomal genome provided the basis for the first cellular genomes. Modern genomes should therefore contain an unexpectedly large percentage of tRNA- and rRNA-like modules derived from both sense and antisense reading frames, and these should encode non-ribosomal proteins, as well as ribosomal ones with key cell functions. Ribosomal proteins should also have been co-opted by cellular evolution to play extra-ribosomal functions. We review existing literature supporting these predictions. We provide additional, new data demonstrating that rRNA-like sequences occur at significantly higher frequencies than predicted on the basis of mRNA duplications or randomized RNA sequences. These data support our “ribosome-first” theory of cellular evolution.
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Bayas, Camille A., Jiarui Wang, Marissa K. Lee, Jared M. Schrader, Lucy Shapiro, and W. E. Moerner. "Spatial organization and dynamics of RNase E and ribosomes inCaulobacter crescentus." Proceedings of the National Academy of Sciences 115, no. 16 (April 2, 2018): E3712—E3721. http://dx.doi.org/10.1073/pnas.1721648115.
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We report the dynamic spatial organization ofCaulobacter crescentusRNase E (RNA degradosome) and ribosomal protein L1 (ribosome) using 3D single-particle tracking and superresolution microscopy. RNase E formed clusters along the central axis of the cell, while weak clusters of ribosomal protein L1 were deployed throughout the cytoplasm. These results contrast with RNase E and ribosome distribution inEscherichia coli, where RNase E colocalizes with the cytoplasmic membrane and ribosomes accumulate in polar nucleoid-free zones. For both RNase E and ribosomes inCaulobacter, we observed a decrease in confinement and clustering upon transcription inhibition and subsequent depletion of nascent RNA, suggesting that RNA substrate availability for processing, degradation, and translation facilitates confinement and clustering. Importantly, RNase E cluster positions correlated with the subcellular location of chromosomal loci of two highly transcribed rRNA genes, suggesting that RNase E’s function in rRNA processing occurs at the site of rRNA synthesis. Thus, components of the RNA degradosome and ribosome assembly are spatiotemporally organized inCaulobacter, with chromosomal readout serving as the template for this organization.
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Ramrath, David J. F., Moritz Niemann, Marc Leibundgut, Philipp Bieri, Céline Prange, Elke K. Horn, Alexander Leitner, Daniel Boehringer, André Schneider, and Nenad Ban. "Evolutionary shift toward protein-based architecture in trypanosomal mitochondrial ribosomes." Science 362, no. 6413 (September 13, 2018): eaau7735. http://dx.doi.org/10.1126/science.aau7735.
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Ribosomal RNA (rRNA) plays key functional and architectural roles in ribosomes. Using electron microscopy, we determined the atomic structure of a highly divergent ribosome found in mitochondria of Trypanosoma brucei, a unicellular parasite that causes sleeping sickness in humans. The trypanosomal mitoribosome features the smallest rRNAs and contains more proteins than all known ribosomes. The structure shows how the proteins have taken over the role of architectural scaffold from the rRNA: They form an autonomous outer shell that surrounds the entire particle and stabilizes and positions the functionally important regions of the rRNA. Our results also reveal the “minimal” set of conserved rRNA and protein components shared by all ribosomes that help us define the most essential functional elements.
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Scull, Catherine E., Guy Twa, Yinfeng Zhang, Naiheng J. Yang, Robert N. Hunter, Corinne E. Augelli-Szafran, and David A. Schneider. "Small Molecule RBI2 Disrupts Ribosome Biogenesis through Pre-rRNA Depletion." Cancers 15, no. 13 (June 23, 2023): 3303. http://dx.doi.org/10.3390/cancers15133303.
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Cancer cells are especially sensitive to perturbations in ribosome biogenesis as they rely on finely tuned protein homeostasis to facilitate their rapid growth and proliferation. While ribosome synthesis and cancer have a well-established relationship, ribosome biogenesis has only recently drawn interest as a cancer therapeutic target. In this study, we exploited the relationship between ribosome biogenesis and cancer cell proliferation by using a potent ribosome biogenesis inhibitor, RBI2 (Ribosome Biogenesis Inhibitor 2), to perturb cancer cell growth and viability. We demonstrate herein that RBI2 significantly decreases cell viability in malignant melanoma cells and breast cancer cell lines. Treatment with RBI2 dramatically and rapidly decreased ribosomal RNA (rRNA) synthesis, without affecting the occupancy of RNA polymerase I (Pol I) on the ribosomal DNA template. Next-generation RNA sequencing (RNA-seq) revealed that RBI2 and previously described ribosome biogenesis inhibitor CX-5461 induce distinct changes in the transcriptome. An investigation of the content of the pre-rRNAs through RT-qPCR revealed an increase in the polyadenylation of cellular rRNA after treatment with RBI2, constituting a known pathway by which rRNA degradation occurs. Northern blotting revealed that RBI2 does not appear to impair or alter rRNA processing. Collectively, these data suggest that RBI2 inhibits rRNA synthesis differently from other previously described ribosome biogenesis inhibitors, potentially acting through a novel pathway that upregulates the turnover of premature rRNAs.
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Maksimova, Elena, Olesya Kravchenko, Alexey Korepanov, and Elena Stolboushkina. "Protein Assistants of Small Ribosomal Subunit Biogenesis in Bacteria." Microorganisms 10, no. 4 (March 30, 2022): 747. http://dx.doi.org/10.3390/microorganisms10040747.
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Ribosome biogenesis is a fundamental and multistage process. The basic steps of ribosome assembly are the transcription, processing, folding, and modification of rRNA; the translation, folding, and modification of r-proteins; and consecutive binding of ribosomal proteins to rRNAs. Ribosome maturation is facilitated by biogenesis factors that include a broad spectrum of proteins: GTPases, RNA helicases, endonucleases, modification enzymes, molecular chaperones, etc. The ribosome assembly factors assist proper rRNA folding and protein–RNA interactions and may sense the checkpoints during the assembly to ensure correct order of this process. Inactivation of these factors is accompanied by severe growth phenotypes and accumulation of immature ribosomal subunits containing unprocessed rRNA, which reduces overall translation efficiency and causes translational errors. In this review, we focus on the structural and biochemical analysis of the 30S ribosomal subunit assembly factors RbfA, YjeQ (RsgA), Era, KsgA (RsmA), RimJ, RimM, RimP, and Hfq, which take part in the decoding-center folding.
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Stern, Seth, and Prakash Purohit. "An oligonucleotide analog approach to the decoding region of 16S rRNA." Biochemistry and Cell Biology 73, no. 11-12 (December 1, 1995): 899–905. http://dx.doi.org/10.1139/o95-097.
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Despite the passage of about 30 years since the discovery of the translational activities of ribosomes and the outlining of the roles of the large and small subunits, the actual molecular basis for the mRNA decoding activities of the small subunit has remained essentially obscure. In this paper, we describe a new approach using oligonucleotide analogs of 16S ribosomal RNA, in which the small ribosomal subunit is effectively deconstructed into a smaller more experimentally tractable form. Specifically, we review the results of experiments using an oligonucleotide analog of the decoding region of 16S ribosomal RNA, suggesting that the decoding region is the functional core of the small subunit, that it contacts both mRNA codons and tRNA anticodons, and that it mediates and probably enhances codon–anticodon base pairing, that is, decoding.Key words: translation, ribosome, 30S, 16S, RNA, decoding, antibiotic.
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Sergeeva, Olga, Philipp Sergeev, Pavel Melnikov, Tatiana Prikazchikova, Olga Dontsova, and Timofei Zatsepin. "Modification of Adenosine196 by Mettl3 Methyltransferase in the 5’-External Transcribed Spacer of 47S Pre-rRNA Affects rRNA Maturation." Cells 9, no. 4 (April 24, 2020): 1061. http://dx.doi.org/10.3390/cells9041061.
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Ribosome biogenesis is among the founding processes in the cell. During the first stages of ribosome biogenesis, polycistronic precursor of ribosomal RNA passes complex multistage maturation after transcription. Quality control of preribosomal RNA (pre-rRNA) processing is precisely regulated by non-ribosomal proteins and structural features of pre-rRNA molecules, including modified nucleotides. However, many participants of rRNA maturation are still unknown or poorly characterized. We report that RNA m6A methyltransferase Mettl3 interacts with the 5′ external transcribed spacer (5′ETS) of the 47S rRNA precursor and modifies adenosine 196. We demonstrated that Mettl3 knockdown results in the increase of pre-rRNA processing rates, while intracellular amounts of rRNA processing machinery components (U3, U8, U13, U14, and U17 small nucleolar RNA (snoRNA)and fibrillarin, nucleolin, Xrn2, and rrp9 proteins), rRNA degradation rates, and total amount of mature rRNA in the cell stay unchanged. Increased efficacy of pre-rRNA cleavage at A’ and A0 positions led to the decrease of 47S and 45S pre-rRNAs in the cell and increase of mature rRNA amount in the cytoplasm. The newly identified conserved motif DRACH sequence modified by Mettl3 in the 5′-ETS region is found and conserved only in primates, which may suggest participation of m6A196 in quality control of pre-rRNA processing at initial stages demanded by increased complexity of ribosome biogenesis.
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Björk, Petra, Göran Baurén, ShaoBo Jin, Yong-Guang Tong, Thomas R. Bürglin, Ulf Hellman, and Lars Wieslander. "A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis." Molecular Biology of the Cell 13, no. 10 (October 2002): 3683–95. http://dx.doi.org/10.1091/mbc.e02-03-0138.
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Synthesis of the ribosomal subunits from pre-rRNA requires a large number of trans-acting proteins and small nucleolar ribonucleoprotein particles to execute base modifications, RNA cleavages, and structural rearrangements. We have characterized a novel protein, RNA-binding domain-1 (RBD-1), that is involved in ribosome biogenesis. This protein contains six consensus RNA-binding domains and is conserved as to sequence, domain organization, and cellular location from yeast to human. RBD-1 is essential in Caenorhabditis elegans. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In cytoplasmic extracts, 20–30% of Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.
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Buyan, Andrey, Ivan Kulakovskiy, and Sergey Dmitriev. "Abstract P-22: Enhanced Crosslinking and Immunoprecipitation (Eclip) Data Reveal Interactions of RNA Binding Proteins with the Human Ribosome." International Journal of Biomedicine 11, Suppl_1 (June 1, 2021): S21. http://dx.doi.org/10.21103/ijbm.11.suppl_1.p22.
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Background: The ribosome is a protein-synthesizing molecular machine composed of four ribosomal RNAs (rRNAs) and dozens of ribosomal proteins. In mammals, the ribosome has a complicated structure with an additional outer layer of rRNA, including large tentacle-like extensions. A number of RNA binding proteins (RBPs) interact with this layer to assist ribosome biogenesis, nuclear export and decay, or to modulate translation. Plenty of methods have been developed in the last decade in order to study such protein-RNA interactions, including RNA pulldown and crosslinking-immunoprecipitation (CLIP) assays. Methods: In the current study, using publicly available data of the enhanced CLIP (eCLIP) experiments for 223 proteins studied in the ENCODE project, we found a number of RBPs that bind rRNAs in human cells. To locate their binding sites in rRNAs, we used a newly developed computational protocol for mapping and evaluation of the eCLIP data with the respect to the repetitive sequences. Results: For two proteins with known ribosomal localization, uS3/RPS3 and uS17/RPS11, the identified sites were in good agreement with structural data, thus validating our approach. Then, we identified rRNA contacts of overall 22 RBPs involved in rRNA processing and ribosome maturation (DDX21, DDX51, DDX52, NIP7, SBDS, UTP18, UTP3, WDR3, and WDR43), translational control during stress (SERBP1, G3BP1, SND1), IRES activity (PCBP1/hnRNPE1), and other translation-related functions. In many cases, the identified proteins interact with the rRNA expansion segments (ES) of the human ribosome pointing to their important role in protein synthesis. Conclusion: Our study identifies a number of RBPs as interacting partners of the human ribosome and sheds light on the role of rRNA expansion segments in translation.
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Laptev, Ivan, Olga Dontsova, and Petr Sergiev. "Epitranscriptomics of Mammalian Mitochondrial Ribosomal RNA." Cells 9, no. 10 (September 27, 2020): 2181. http://dx.doi.org/10.3390/cells9102181.
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Modified nucleotides are present in all ribosomal RNA molecules. Mitochondrial ribosomes are unique to have a set of methylated residues that includes universally conserved ones, those that could be found either in bacterial or in archaeal/eukaryotic cytosolic ribosomes and those that are present exclusively in mitochondria. A single pseudouridine within the mt-rRNA is located in the peptidyltransferase center at a position similar to that in bacteria. After recent completion of the list of enzymes responsible for the modification of mammalian mitochondrial rRNA it became possible to summarize an evolutionary history, functional role of mt-rRNA modification enzymes and an interplay of the mt-rRNA modification and mitoribosome assembly process, which is a goal of this review.
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Burger, Kaspar, Bastian Mühl, Michaela Rohrmoser, Britta Coordes, Martin Heidemann, Markus Kellner, Anita Gruber-Eber, Vigo Heissmeyer, Katja Strässer, and Dirk Eick. "Cyclin-dependent Kinase 9 Links RNA Polymerase II Transcription to Processing of Ribosomal RNA." Journal of Biological Chemistry 288, no. 29 (June 6, 2013): 21173–83. http://dx.doi.org/10.1074/jbc.m113.483719.
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Ribosome biogenesis is a process required for cellular growth and proliferation. Processing of ribosomal RNA (rRNA) is highly sensitive to flavopiridol, a specific inhibitor of cyclin-dependent kinase 9 (Cdk9). Cdk9 has been characterized as the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Here we studied the connection between RNAPII transcription and rRNA processing. We show that inhibition of RNAPII activity by α-amanitin specifically blocks processing of rRNA. The block is characterized by accumulation of 3′ extended unprocessed 47 S rRNAs and the entire inhibition of other 47 S rRNA-specific processing steps. The transcription rate of rRNA is moderately reduced after inhibition of Cdk9, suggesting that defective 3′ processing of rRNA negatively feeds back on RNAPI transcription. Knockdown of Cdk9 caused a strong reduction of the levels of RNAPII-transcribed U8 small nucleolar RNA, which is essential for 3′ rRNA processing in mammalian cells. Our data demonstrate a pivotal role of Cdk9 activity for coupling of RNAPII transcription with small nucleolar RNA production and rRNA processing.
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Choe, Donghui, Richard Szubin, Saugat Poudel, Anand Sastry, Yoseb Song, Yongjae Lee, Suhyung Cho, Bernhard Palsson, and Byung-Kwan Cho. "RiboRid: A low cost, advanced, and ultra-efficient method to remove ribosomal RNA for bacterial transcriptomics." PLOS Genetics 17, no. 9 (September 27, 2021): e1009821. http://dx.doi.org/10.1371/journal.pgen.1009821.
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RNA sequencing techniques have enabled the systematic elucidation of gene expression (RNA-Seq), transcription start sites (differential RNA-Seq), transcript 3′ ends (Term-Seq), and post-transcriptional processes (ribosome profiling). The main challenge of transcriptomic studies is to remove ribosomal RNAs (rRNAs), which comprise more than 90% of the total RNA in a cell. Here, we report a low-cost and robust bacterial rRNA depletion method, RiboRid, based on the enzymatic degradation of rRNA by thermostable RNase H. This method implemented experimental considerations to minimize nonspecific degradation of mRNA and is capable of depleting pre-rRNAs that often comprise a large portion of RNA, even after rRNA depletion. We demonstrated the highly efficient removal of rRNA up to a removal efficiency of 99.99% for various transcriptome studies, including RNA-Seq, Term-Seq, and ribosome profiling, with a cost of approximately $10 per sample. This method is expected to be a robust method for large-scale high-throughput bacterial transcriptomic studies.
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Szaflarski, Witold, Marta Leśniczak-Staszak, Mateusz Sowiński, Sandeep Ojha, Anaïs Aulas, Dhwani Dave, Sulochan Malla, Paul Anderson, Pavel Ivanov, and Shawn M. Lyons. "Early rRNA processing is a stress-dependent regulatory event whose inhibition maintains nucleolar integrity." Nucleic Acids Research 50, no. 2 (December 20, 2021): 1033–51. http://dx.doi.org/10.1093/nar/gkab1231.
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Abstract The production of ribosomes is an energy-intensive process owing to the intricacy of these massive macromolecular machines. Each human ribosome contains 80 ribosomal proteins and four non-coding RNAs. Accurate assembly requires precise regulation of protein and RNA subunits. In response to stress, the integrated stress response (ISR) rapidly inhibits global translation. How rRNA is coordinately regulated with the rapid inhibition of ribosomal protein synthesis is not known. Here, we show that stress specifically inhibits the first step of rRNA processing. Unprocessed rRNA is stored within the nucleolus, and when stress resolves, it re-enters the ribosome biogenesis pathway. Retention of unprocessed rRNA within the nucleolus aids in the maintenance of this organelle. This response is independent of the ISR or inhibition of cellular translation but is independently regulated. Failure to coordinately control ribosomal protein translation and rRNA production results in nucleolar fragmentation. Our study unveils how the rapid translational shut-off in response to stress coordinates with rRNA synthesis production to maintain nucleolar integrity.
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Maser, R. L., and J. P. Calvet. "U3 small nuclear RNA can be psoralen-cross-linked in vivo to the 5' external transcribed spacer of pre-ribosomal-RNA." Proceedings of the National Academy of Sciences 86, no. 17 (September 1989): 6523–27. http://dx.doi.org/10.1073/pnas.86.17.6523.
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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.
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Moraleva, Anastasia A., Alexander S. Deryabin, Yury P. Rubtsov, Maria P. Rubtsova, and Olga A. Dontsova. "Eukaryotic Ribosome Biogenesis: The 60S Subunit." Acta Naturae 14, no. 2 (July 21, 2022): 39–49. http://dx.doi.org/10.32607/actanaturae.11541.
Full textAbstract:
Ribosome biogenesis is consecutive coordinated maturation of ribosomal precursors in the nucleolus, nucleoplasm, and cytoplasm. The formation of mature ribosomal subunits involves hundreds of ribosomal biogenesis factors that ensure ribosomal RNA processing, tertiary structure, and interaction with ribosomal proteins. Although the main features and stages of ribosome biogenesis are conservative among different groups of eukaryotes, this process in human cells has become more complicated due to the larger size of the ribosomes and pre-ribosomes and intricate regulatory pathways affecting their assembly and function. Many of the factors involved in the biogenesis of human ribosomes have been identified using genome-wide screening based on RNA interference. A previous part of this review summarized recent data on the processing of the primary rRNA transcript and compared the maturation of the small 40S subunit in yeast and human cells. This part of the review focuses on the biogenesis of the large 60S subunit of eukaryotic ribosomes.
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Noller, Harry F., Rachel Green, Gabriele Heilek, Vernita Hoffarth, Alexander Hüttenhofer, Simpson Joseph, Inho Lee, et al. "Structure and function of ribosomal RNA." Biochemistry and Cell Biology 73, no. 11-12 (December 1, 1995): 997–1009. http://dx.doi.org/10.1139/o95-107.
Full textAbstract:
A refined model has been developed for the folding of 16S rRNA in the 30S subunit, based on additional constraints obtained from new experimental approaches. One set of constraints comes from hydroxyl radical footprinting of each of the individual 30S ribosomal proteins, using free Fe2+–EDTA complex. A second approach uses localized hydroxyl radical cleavage from a single Fe2+tethered to unique positions on the surface of single proteins in the 30S subunit. This has been carried out for one position on the surface of protein S4, two on S17, and three on S5. Nucleotides in 16S rRNA that are essential for P-site tRNA binding were identified by a modification interference strategy. Ribosomal subunits were partially inactivated by chemical modification at a low level. Active, partially modified subunits were separated from inactive ones by binding 3′-biotin-derivatized tRNA to the 30S subunits and captured with streptavidin beads. Essential bases are those that are unmodified in the active population but modified in the total population. The four essential bases, G926, 2mG966, G1338, and G1401 are a subset of those that are protected from modification by P-site tRNA. They are all located in the cleft of our 30S subunit model. The rRNA neighborhood of the acceptor end of tRNA was probed by hydroxyl radical probing from Fe2+tethered to the 5′ end of tRNA via an EDTA linker. Cleavage was detected in domains IV, V, and VI of 23S rRNA, but not in 5S or 16S rRNA. The sites were all found to be near bases that were protected from modification by the CCA end of tRNA in earlier experiments, except for a set of E-site cleavages in domain IV and a set of A-site cleavages in the α-sarcin loop of domain VI. In vitro genetics was used to demonstrate a base-pairing interaction between tRNA and 23S rRNA. Mutations were introduced at positions C74 and C75 of tRNA and positions 2252 and 2253 of 23S rRNA. Interaction of the CCA end of tRNA with mutant ribosomes was tested using chemical probing in conjunction with allele-specific primer extension. The interaction occurred only when there was a Watson–Crick pairing relationship between positions 74 of tRNA and 2252 of 23S rRNA. Using a novel chimeric in vitro reconstitution method, it was shown that the peptidyl transferase reaction depends on this same Watson–Crick base pair.Key words: rRNA, ribosome, tRNA, hydroxyl radical, ribosomal protein.
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Antony, Charles, Subin S. George, Justin Blum, Patrick Somers, Dexter Wu-corts, Maxim Pimkin, and Vikram R. Paralkar. "CEBPA Directly Binds Ribosomal DNA and Promotes Ribosomal RNA Transcription in Myeloid Progenitors." Blood 138, Supplement 1 (November 5, 2021): 3269. http://dx.doi.org/10.1182/blood-2021-148929.
Full textAbstract:
Abstract Hematopoietic stem cells (HSCs) form a hierarchy of lineage restricted progenitor cells to produce mature hematopoietic cells that vary in function, size, proliferation, and protein synthesis rates. Different hematopoietic cells also vary in the rate of ribosomal RNA (rRNA) transcription, the key rate-limiting step in ribosome biogenesis that occurs in the nucleolus. Leukemic blast cells have long been identified by their prominent nucleoli, indicating high ribosome biogenesis rates (Fig A). Ribosome biogenesis is an extremely energy intensive process begins with transcription of multi-copy rDNA genes by RNA polymerase I (Pol I) to produce 47S precursor rRNA (pre-rRNA) which further processed into the generation of mature 18S, 5.8S, and 28S rRNA and assembled with 5S rRNA and 80 different ribosomal proteins to form mature ribosomes (Fig B). This process is highly dynamic and regulated at the level of rRNA transcription. Despite cell-type and disease-specific variations, rRNA transcription has long been considered a housekeeping process. Hence, cell or tissue type-specific regulation of rRNA transcription has rarely been explored. To identify cell-type-specific regulators of rRNA transcription in hematopoiesis, we mapped 2200 publicly available ChIP-Seq datasets representing 249 hematopoietic transcription factors (TFs) and epigenetic factors to create an atlas of hematopoietic TF-rDNA binding. We identified CEBPA that shows consistent and abundant binding to rDNA at a conserved, previously unknown motif in both species (Fig C). CEBPA is a myeloid lineage specific TF whose knockout leads to complete loss of all myeloid lineage cells. It is also frequently mutated (10%) in AML patients. So we picked CEBPA to further characterize its role in rRNA transcription. Since CEBPA deletion causes loss of granulocyte-monocyte progenitors (GMPs), we used the mouse HoxA9-ER cell line (which closely resembles GMPs). To study the immediate consequences of CEBPA loss, We generated a stable degron cell line by biallelically fusing FKBP degron into endogenous loci of Cebpa, enabling to rapidly degrade endogenous CEBPA protein on treatment with dTagV ligand (Fig D, E). To precisely quantify the rate of rRNA transcription, we developed a novel assay called '47S-FISH-Flow' that involves hybridizing fluorescent oligos unique to 5' end of 47S pre-rRNA, which only marks newly synthesized nascent rRNA in the nucleolus, and quantify using flow cytometry (Fig F, G). We found that depleting CEBPA caused rapid decrease in 47S rRNA level and occupancy of Pol I on rDNA (Fig H, I). In summary, we found that myeloid lineage specific TF CEBPA abundantly binds to a conserved motif in rDNA and the depletion of CEBPA rapidly reduces nascent 47S rRNA, indicating that it directly promotes rRNA transcription. Our results, and the tools and experimental systems we have developed, shed light on an important and largely unexplored aspect of hematopoietic biology: the regulation of rRNA transcription by lineage-specific hematopoietic TFs. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Rabany, Ofri, and Daphna Nachmani. "Small Nucleolar (Sno)RNA: Therapy Lays in Translation." Non-Coding RNA 9, no. 3 (June 8, 2023): 35. http://dx.doi.org/10.3390/ncrna9030035.
Full textAbstract:
The ribosome is one of the largest complexes in the cell. Adding to its complexity are more than 200 RNA modification sites present on ribosomal RNAs (rRNAs) in a single human ribosome. These modifications occur in functionally important regions of the rRNA molecule, and they are vital for ribosome function and proper gene expression. Until recent technological advancements, the study of rRNA modifications and their profiles has been extremely laborious, leaving many questions unanswered. Small nucleolar RNAs (snoRNAs) are non-coding RNAs that facilitate and dictate the specificity of rRNA modification deposition, making them an attractive target for ribosome modulation. Here, we propose that through the mapping of rRNA modification profiles, we can identify cell-specific modifications with high therapeutic potential. We also describe the challenges of achieving the targeting specificity needed to implement snoRNAs as therapeutic targets in cancers.
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Conrad-Webb, H., and R. A. Butow. "A polymerase switch in the synthesis of rRNA in Saccharomyces cerevisiae." Molecular and Cellular Biology 15, no. 5 (May 1995): 2420–28. http://dx.doi.org/10.1128/mcb.15.5.2420.
Full textAbstract:
Transcription of ribosomal DNA by RNA polymerase I is believed to be the sole source of the 25S, 18S, and 5.8S rRNAs in wild-type cells of Saccharomyces cerevisiae. Here we present evidence for a switch from RNA polymerase I to RNA polymerase II in the synthesis of a substantial fraction of those rRNAs in respiratory-deficient (petite) cells. The templates for the RNA polymerase II transcripts are largely, if not exclusively, episomal copies of ribosomal DNA arising from homologous recombination events within the ribosomal DNA repeat on chromosome XII. Ribosomal DNA contains a cryptic RNA polymerase II promoter that is activated in petites; it overlaps the RNA polymerase I promoter and produces a transcript equivalent to the 35S precursor rRNA made by RNA polymerase I. Yeast cells that lack RNA polymerase I activity, because of a disruption of the RPA135 gene that encodes subunit II of the enzyme, can survive by using the RNA polymerase II promoter in ribosomal DNA to direct the synthesis of the 35S rRNA precursor. This polymerase switch could provide cells with a mechanism to synthesize rRNA independent of the controls of RNA polymerase I transcription.
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Beniac, Daniel R., Gregory J. Czarnota, Brenda L. Rutherford, F. Peter Ottensmeyer, and George Harauz. "Probing Ribosomal RNA By Electron Spectroscopic Imaging and Three-Dimensional Reconstruction." Microscopy Today 5, no. 1 (January 1997): 10–11. http://dx.doi.org/10.1017/s1551929500059940.
Full textAbstract:
The ribosome is the protein synthetic machinery in the cell. Knowledge of the structures of ribosomal RNA (rRNA) macromolecules in situ is essential to understanding their roles in ribosome mediated protein synthesis. We are using a microanalytical technique that identifies and maps elements directly, electron spectroscopic imaging, to determine the rRNA phosphorus distributions within Escherichia coli ribosomal subunits, and to combine the two-dimensional maps into a three-dimensional elemental distribution by iterative quaternion-assisted angular reconstitution of ribosomal particles at random orientations.
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Lundkvist, Pär, Sara Jupiter, Åsa Segerstolpe, Yvonne N. Osheim, Ann L. Beyer, and Lars Wieslander. "Mrd1p Is Required for Release of Base-Paired U3 snoRNA within the Preribosomal Complex." Molecular and Cellular Biology 29, no. 21 (August 24, 2009): 5763–74. http://dx.doi.org/10.1128/mcb.00428-09.
Full textAbstract:
ABSTRACT In eukaryotes, ribosomes are made from precursor rRNA (pre-rRNA) and ribosomal proteins in a maturation process that requires a large number of snoRNPs and processing factors. A fundamental problem is how the coordinated and productive folding of the pre-rRNA and assembly of successive pre-rRNA-protein complexes is achieved cotranscriptionally. The conserved protein Mrd1p, which contains five RNA binding domains (RBDs), is essential for processing events leading to small ribosomal subunit synthesis. We show that full function of Mrd1p requires all five RBDs and that the RBDs are functionally distinct and needed during different steps in processing. Mrd1p mutations trap U3 snoRNA in pre-rRNP complexes both in base-paired and non-base-paired interactions. A single essential RBD, RBD5, is involved in both types of interactions, but its conserved RNP1 motif is not needed for releasing the base-paired interactions. RBD5 is also required for the late pre-rRNP compaction preceding A2 cleavage. Our results suggest that Mrd1p modulates successive conformational rearrangements within the pre-rRNP that influence snoRNA-pre-rRNA contacts and couple U3 snoRNA-pre-rRNA remodeling and late steps in pre-rRNP compaction that are essential for cleavage at A0 to A2. Mrd1p therefore coordinates key events in biosynthesis of small ribosome subunits.
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Monaco, Piero, Virginie Marcel, Jean-Jacques Diaz, and Frédéric Catez. "2′-O-Methylation of Ribosomal RNA: Towards an Epitranscriptomic Control of Translation?" Biomolecules 8, no. 4 (October 3, 2018): 106. http://dx.doi.org/10.3390/biom8040106.
Full textAbstract:
Ribosomal RNA (rRNA) undergoes post-transcriptional modification of over 200 nucleotides, predominantly 2′-O-methylation (2′-O-Me). 2′-O-Methylation protects RNA from hydrolysis and modifies RNA strand flexibility but does not contribute to Watson-Crick base pairing. The contribution of 2′-O-Me to the translational capacity of ribosomes has been established. Yet, how 2′-O-Me participates in ribosome biogenesis and ribosome functioning remains unclear. The development of 2′-O-Me quantitative mapping methods has contributed to the demonstration that these modifications are not constitutive but rather provide heterogeneity to the ribosomal population. Moreover, recent advances in ribosome structure analysis and in vitro translation assays have proven, for the first time, that 2′-O-Me contributes to regulating protein synthesis. This review highlights the recent data exploring the impact of 2′-O-Me on ribosome structure and function, and the emerging idea that the rRNA epitranscriptome is involved in translational control.
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Piñol-Roma, Serafı́n. "Association of Nonribosomal Nucleolar Proteins in Ribonucleoprotein Complexes during Interphase and Mitosis." Molecular Biology of the Cell 10, no. 1 (January 1999): 77–90. http://dx.doi.org/10.1091/mbc.10.1.77.
Full textAbstract:
rRNA precursors are bound throughout their length by specific proteins, as the pre-rRNAs emerge from the transcription machinery. The association of pre-rRNA with proteins as ribonucleoprotein (RNP) complexes persists during maturation of 18S, 5.8S, and 28S rRNA, and through assembly of ribosomal subunits in the nucleolus. Preribosomal RNP complexes contain, in addition to ribosomal proteins, an unknown number of nonribosomal nucleolar proteins, as well as small nucleolar RNA-ribonucleoproteins (sno-RNPs). This report describes the use of a specific, rapid, and mild immunopurification approach to isolate and analyze human RNP complexes that contain nonribosomal nucleolar proteins, as well as ribosomal proteins and rRNA. Complexes immunopurified with antibodies to nucleolin—a major nucleolar RNA-binding protein—contain several distinct specific polypeptides that include, in addition to nucleolin, the previously identified nucleolar proteins B23 and fibrillarin, proteins with electrophoretic mobilities characteristic of ribosomal proteins including ribosomal protein S6, and a number of additional unidentified proteins. The physical association of these proteins with one another is mediated largely by RNA, in that the complexes dissociate upon digestion with RNase. Complexes isolated from M-phase cells are similar in protein composition to those isolated from interphase cell nuclear extracts. Therefore, the predominant proteins that associate with nucleolin in interphase remain in RNP complexes during mitosis, despite the cessation of rRNA synthesis and processing in M-phase. In addition, precursor rRNA, as well as processed 18S and 28S rRNA and candidate rRNA processing intermediates, is found associated with the immunopurified complexes. The characteristics of the rRNP complexes described here, therefore, indicate that they represent bona fide precursors of mature cytoplasmic ribosomal subunits.
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Grewal, Savraj S., Justin R. Evans, and Bruce A. Edgar. "Drosophila TIF-IA is required for ribosome synthesis and cell growth and is regulated by the TOR pathway." Journal of Cell Biology 179, no. 6 (December 17, 2007): 1105–13. http://dx.doi.org/10.1083/jcb.200709044.
Full textAbstract:
Synthesis of ribosomal RNA (rRNA) is a key step in ribosome biogenesis and is essential for cell growth. Few studies, however, have investigated rRNA synthesis regulation in vivo in multicellular organisms. Here, we present a genetic analysis of transcription initiation factor IA (TIF-IA), a conserved RNA polymerase I transcription factor. Drosophila melanogaster Tif-IA−/− mutants have reduced levels of rRNA synthesis and sustain a developmental arrest caused by a block in cellular growth. We find that the target of rapamycin (TOR) pathway regulates TIF-IA recruitment to rDNA. Furthermore, we show that the TOR pathway regulates rRNA synthesis in vivo and that TIF-IA overexpression can maintain rRNA transcription when TOR activity is reduced in developing larvae. We propose that TIF-IA acts in vivo as a downstream growth–regulatory target of the TOR pathway. Overexpression of TIF-IA also elevates levels of both 5S RNA and messenger RNAs encoding ribosomal proteins. Stimulation of rRNA synthesis by TIF-IA may therefore provide a feed-forward mechanism to coregulate the levels of other ribosome components.
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Erales, Jenny, Virginie Marchand, Baptiste Panthu, Sandra Gillot, Stéphane Belin, Sandra E. Ghayad, Maxime Garcia, et al. "Evidence for rRNA 2′-O-methylation plasticity: Control of intrinsic translational capabilities of human ribosomes." Proceedings of the National Academy of Sciences 114, no. 49 (November 20, 2017): 12934–39. http://dx.doi.org/10.1073/pnas.1707674114.
Full textAbstract:
Ribosomal RNAs (rRNAs) are main effectors of messenger RNA (mRNA) decoding, peptide-bond formation, and ribosome dynamics during translation. Ribose 2′-O-methylation (2′-O-Me) is the most abundant rRNA chemical modification, and displays a complex pattern in rRNA. 2′-O-Me was shown to be essential for accurate and efficient protein synthesis in eukaryotic cells. However, whether rRNA 2′-O-Me is an adjustable feature of the human ribosome and a means of regulating ribosome function remains to be determined. Here we challenged rRNA 2′-O-Me globally by inhibiting the rRNA methyl-transferase fibrillarin in human cells. Using RiboMethSeq, a nonbiased quantitative mapping of 2′-O-Me, we identified a repertoire of 2′-O-Me sites subjected to variation and demonstrate that functional domains of ribosomes are targets of 2′-O-Me plasticity. Using the cricket paralysis virus internal ribosome entry site element, coupled to in vitro translation, we show that the intrinsic capability of ribosomes to translate mRNAs is modulated through a 2′-O-Me pattern and not by nonribosomal actors of the translational machinery. Our data establish rRNA 2′-O-Me plasticity as a mechanism providing functional specificity to human ribosomes.
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Sharifi, Samim, and Holger Bierhoff. "Regulation of RNA Polymerase I Transcription in Development, Disease, and Aging." Annual Review of Biochemistry 87, no. 1 (June 20, 2018): 51–73. http://dx.doi.org/10.1146/annurev-biochem-062917-012612.
Full textAbstract:
Ribosome biogenesis is a complex and highly energy-demanding process that requires the concerted action of all three nuclear RNA polymerases (Pol I–III) in eukaryotes. The three largest ribosomal RNAs (rRNAs) originate from a precursor transcript (pre-rRNA) that is encoded by multicopy genes located in the nucleolus. Transcription of these rRNA genes (rDNA) by Pol I is the key regulation step in ribosome production and is tightly controlled by an intricate network of signaling pathways and epigenetic mechanisms. In this article, we give an overview of the composition of the basal Pol I machinery and rDNA chromatin. We discuss rRNA gene regulation in response to environmental signals and developmental cues and focus on perturbations occurring in diseases linked to either excessive or limited rRNA levels. Finally, we discuss the emerging view that rDNA integrity and activity may be involved in the aging process.
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Baudin-Baillieu, Agnès, and Olivier Namy. "Saccharomyces cerevisiae, a Powerful Model for Studying rRNA Modifications and Their Effects on Translation Fidelity." International Journal of Molecular Sciences 22, no. 14 (July 10, 2021): 7419. http://dx.doi.org/10.3390/ijms22147419.
Full textAbstract:
Ribosomal RNA is a major component of the ribosome. This RNA plays a crucial role in ribosome functioning by ensuring the formation of the peptide bond between amino acids and the accurate decoding of the genetic code. The rRNA carries many chemical modifications that participate in its maturation, the formation of the ribosome and its functioning. In this review, we present the different modifications and how they are deposited on the rRNA. We also describe the most recent results showing that the modified positions are not 100% modified, which creates a heterogeneous population of ribosomes. This gave rise to the concept of specialized ribosomes that we discuss. The knowledge accumulated in the yeast Saccharomyces cerevisiae is very helpful to better understand the role of rRNA modifications in humans, especially in ribosomopathies.
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Choi, Ilyeong, Young Jeon, Youngki Yoo, Hyun-Soo Cho, and Hyun-Sook Pai. "The in vivo functions of ARPF2 and ARRS1 in ribosomal RNA processing and ribosome biogenesis in Arabidopsis." Journal of Experimental Botany 71, no. 9 (April 10, 2020): 2596–611. http://dx.doi.org/10.1093/jxb/eraa019.
Full textAbstract:
Abstract Yeast Rpf2 plays a critical role in the incorporation of 5S rRNA into pre-ribosomes by forming a binary complex with Rrs1. The protein characteristics and overexpression phenotypes of Arabidopsis Ribosome Production Factor 2 (ARPF2) and Arabidopsis Regulator of Ribosome Synthesis 1 (ARRS1) have been previously studied. Here, we analyze loss-of-function phenotypes of ARPF2 and ARRS1 using virus-induced gene silencing to determine their functions in pre-rRNA processing and ribosome biogenesis. ARPF2 silencing in Arabidopsis led to pleiotropic developmental defects. RNA gel blot analysis and circular reverse transcription–PCR revealed that ARPF2 depletion delayed pre-rRNA processing, resulting in the accumulation of multiple processing intermediates. ARPF2 fractionated primarily with the 60S ribosomal subunit. Metabolic rRNA labeling and ribosome profiling suggested that ARPF2 deficiency mainly affected 25S rRNA synthesis and 60S ribosome biogenesis. ARPF2 and ARRS1 formed the complex that interacted with the 60S ribosomal proteins RPL5 and RPL11. ARRS1 silencing resulted in growth defects, accumulation of processing intermediates, and ribosome profiling similar to those of ARPF2-silenced plants. Moreover, depletion of ARPF2 and ARRS1 caused nucleolar stress. ARPF2-deficient plants excessively accumulated anthocyanin and reactive oxygen species. Collectively, these results suggest that the ARPF2–ARRS1 complex plays a crucial role in plant growth and development by modulating ribosome biogenesis.
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Phan, Tamara, Fatima Khalid, and Sebastian Iben. "Nucleolar and Ribosomal Dysfunction—A Common Pathomechanism in Childhood Progerias?" Cells 8, no. 6 (June 4, 2019): 534. http://dx.doi.org/10.3390/cells8060534.
Full textAbstract:
The nucleolus organizes around the sites of transcription by RNA polymerase I (RNA Pol I). rDNA transcription by this enzyme is the key step of ribosome biogenesis and most of the assembly and maturation processes of the ribosome occur co-transcriptionally. Therefore, disturbances in rRNA transcription and processing translate to ribosomal malfunction. Nucleolar malfunction has recently been described in the classical progeria of childhood, Hutchinson–Gilford syndrome (HGPS), which is characterized by severe signs of premature aging, including atherosclerosis, alopecia, and osteoporosis. A deregulated ribosomal biogenesis with enlarged nucleoli is not only characteristic for HGPS patients, but it is also found in the fibroblasts of “normal” aging individuals. Cockayne syndrome (CS) is also characterized by signs of premature aging, including the loss of subcutaneous fat, alopecia, and cataracts. It has been shown that all genes in which a mutation causes CS, are involved in rDNA transcription by RNA Pol I. A disturbed ribosomal biogenesis affects mitochondria and translates into ribosomes with a reduced translational fidelity that causes endoplasmic reticulum (ER) stress and apoptosis. Therefore, it is speculated that disease-causing disturbances in the process of ribosomal biogenesis may be more common than hitherto anticipated.
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Fox, Jesse M., Rebekah L. Rashford, and Lasse Lindahl. "Co-Assembly of 40S and 60S Ribosomal Proteins in Early Steps of Eukaryotic Ribosome Assembly." International Journal of Molecular Sciences 20, no. 11 (June 8, 2019): 2806. http://dx.doi.org/10.3390/ijms20112806.
Full textAbstract:
In eukaryotes three of the four ribosomal RNA (rRNA) molecules are transcribed as a long precursor that is processed into mature rRNAs concurrently with the assembly of ribosomal subunits. However, the relative timing of association of ribosomal proteins with the ribosomal precursor particles and the cleavage of the precursor rRNA into the subunit-specific moieties is not known. To address this question, we searched for ribosomal precursors containing components from both subunits. Particles containing specific ribosomal proteins were targeted by inducing synthesis of epitope-tagged ribosomal proteins followed by pull-down with antibodies targeting the tagged protein. By identifying other ribosomal proteins and internal rRNA transcribed spacers (ITS1 and ITS2) in the immuno-purified ribosomal particles, we showed that eS7/S7 and uL4/L4 bind to nascent ribosomes prior to the separation of 40S and 60S specific segments, while uS4/S9, uL22, and eL13/L13 are bound after, or simultaneously with, the separation. Thus, the incorporation of ribosomal proteins from the two subunits begins as a co-assembly with a single rRNA molecule, but is finished as an assembly onto separate precursors for the two subunits.
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Gaviraghi, Vivori, and Tonon. "How Cancer Exploits Ribosomal RNA Biogenesis: A Journey beyond the Boundaries of rRNA Transcription." Cells 8, no. 9 (September 17, 2019): 1098. http://dx.doi.org/10.3390/cells8091098.
Full textAbstract:
The generation of new ribosomes is a coordinated process essential to sustain cell growth. As such, it is tightly regulated according to cell needs. As cancer cells require intense protein translation to ensure their enhanced growth rate, they exploit various mechanisms to boost ribosome biogenesis. In this review, we will summarize how oncogenes and tumor suppressors modulate the biosynthesis of the RNA component of ribosomes, starting from the description of well-characterized pathways that converge on ribosomal RNA transcription while including novel insights that reveal unexpected regulatory networks hacked by cancer cells to unleash ribosome production.
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Sirri, Valentina, Pascal Roussel, and Danièle Hernandez-Verdun. "In Vivo Release of Mitotic Silencing of Ribosomal Gene Transcription Does Not Give Rise to Precursor Ribosomal RNA Processing." Journal of Cell Biology 148, no. 2 (January 24, 2000): 259–70. http://dx.doi.org/10.1083/jcb.148.2.259.
Full textAbstract:
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.
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Noller, Harry F., John Paul Donohue, and Robin R. Gutell. "The universally conserved nucleotides of the small subunit ribosomal RNAs." RNA 28, no. 5 (February 3, 2022): 623–44. http://dx.doi.org/10.1261/rna.079019.121.
Full textAbstract:
The ribosomal RNAs, along with their substrates the transfer RNAs, contain the most highly conserved nucleotides in all of biology. We have assembled a database containing structure-based alignments of sequences of the small-subunit rRNAs from organisms that span the entire phylogenetic spectrum, to identify the nucleotides that are universally conserved. In its simplest (bacterial and archaeal) forms, the small-subunit rRNA has ∼1500 nt, of which we identify 140 that are absolutely invariant among the 1961 species in our alignment. We examine the positions and detailed structural and functional interactions of these universal nucleotides in the context of a half century of biochemical and genetic studies and high-resolution structures of ribosome functional complexes. The vast majority of these nucleotides are exposed on the subunit interface surface of the small subunit, where the functional processes of the ribosome take place. However, only 40 of them have been directly implicated in specific ribosomal functions, such as contacting the tRNAs, mRNA, or translation factors. The roles of many other invariant nucleotides may serve to constrain the positions and orientations of those nucleotides that are directly involved in function. Yet others can be rationalized by participation in unusual noncanonical tertiary structures that may uniquely allow correct folding of the rRNA to form a functional ribosome. However, there remain at least 50 nt whose universal conservation is not obvious, serving as a metric for the incompleteness of our understanding of ribosome structure and function.
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Penev, Petar I., Sara Fakhretaha-Aval, Vaishnavi J. Patel, Jamie J. Cannone, Robin R. Gutell, Anton S. Petrov, Loren Dean Williams, and Jennifer B. Glass. "Supersized Ribosomal RNA Expansion Segments in Asgard Archaea." Genome Biology and Evolution 12, no. 10 (August 12, 2020): 1694–710. http://dx.doi.org/10.1093/gbe/evaa170.
Full textAbstract:
Abstract The ribosome’s common core, comprised of ribosomal RNA (rRNA) and universal ribosomal proteins, connects all life back to a common ancestor and serves as a window to relationships among organisms. The rRNA of the common core is similar to rRNA of extant bacteria. In eukaryotes, the rRNA of the common core is decorated by expansion segments (ESs) that vastly increase its size. Supersized ESs have not been observed previously in Archaea, and the origin of eukaryotic ESs remains enigmatic. We discovered that the large ribosomal subunit (LSU) rRNA of two Asgard phyla, Lokiarchaeota and Heimdallarchaeota, considered to be the closest modern archaeal cell lineages to Eukarya, bridge the gap in size between prokaryotic and eukaryotic LSU rRNAs. The elongated LSU rRNAs in Lokiarchaeota and Heimdallarchaeota stem from two supersized ESs, called ES9 and ES39. We applied chemical footprinting experiments to study the structure of Lokiarchaeota ES39. Furthermore, we used covariation and sequence analysis to study the evolution of Asgard ES39s and ES9s. By defining the common eukaryotic ES39 signature fold, we found that Asgard ES39s have more and longer helices than eukaryotic ES39s. Although Asgard ES39s have sequences and structures distinct from eukaryotic ES39s, we found overall conservation of a three-way junction across the Asgard species that matches eukaryotic ES39 topology, a result consistent with the accretion model of ribosomal evolution.
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Shu, Wen-Jie, Runfa Chen, Zhao-Hong Yin, Feng Li, Heng Zhang, and Hai-Ning Du. "Rph1 coordinates transcription of ribosomal protein genes and ribosomal RNAs to control cell growth under nutrient stress conditions." Nucleic Acids Research 48, no. 15 (July 3, 2020): 8360–73. http://dx.doi.org/10.1093/nar/gkaa558.
Full textAbstract:
Abstract Coordinated regulation of ribosomal RNA (rRNA) synthesis and ribosomal protein gene (RPG) transcription by eukaryotic RNA polymerases (RNAP) is a key requirement for growth control. Although evidence for balance between RNPI-dependent 35S rRNA production and RNAPII-mediated RPG transcription have been described, the molecular basis is still obscure. Here, we found that Rph1 modulates the transcription status of both rRNAs and RPGs in yeast. We show that Rph1 widely associates with RNAPI and RNAPII-transcribed genes. Deletion of RPH1 remarkably alleviates cell slow growth caused by TORC1 inhibition via derepression of rRNA and RPG transcription under nutrient stress conditions. Mechanistically, Rim15 kinase phosphorylates Rph1 upon rapamycin treatment. Phosphorylation-mimetic mutant of Rph1 exhibited more resistance to rapamycin treatment, decreased association with ribosome-related genes, and faster cell growth compared to the wild-type, indicating that Rph1 dissociation from chromatin ensures cell survival upon nutrient stress. Our results uncover the role of Rph1 in coordination of RNA polymerases-mediated transcription to control cell growth under nutrient stress conditions.
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Albanèse, Véronique, Stefanie Reissmann, and Judith Frydman. "A ribosome-anchored chaperone network that facilitates eukaryotic ribosome biogenesis." Journal of Cell Biology 189, no. 1 (April 5, 2010): 69–81. http://dx.doi.org/10.1083/jcb.201001054.
Full textAbstract:
Molecular chaperones assist cellular protein folding as well as oligomeric complex assembly. In eukaryotic cells, several chaperones termed chaperones linked to protein synthesis (CLIPS) are transcriptionally and physically linked to ribosomes and are implicated in protein biosynthesis. In this study, we show that a CLIPS network comprising two ribosome-anchored J-proteins, Jjj1 and Zuo1, function together with their partner Hsp70 proteins to mediate the biogenesis of ribosomes themselves. Jjj1 and Zuo1 have overlapping but distinct functions in this complex process involving the coordinated assembly and remodeling of dozens of proteins on the ribosomal RNA (rRNA). Both Jjj1 and Zuo1 associate with nuclear 60S ribosomal biogenesis intermediates and play an important role in nuclear rRNA processing, leading to mature 25S rRNA. In addition, Zuo1, acting together with its Hsp70 partner, SSB (stress 70 B), also participates in maturation of the 35S rRNA. Our results demonstrate that, in addition to their known cytoplasmic roles in de novo protein folding, some ribosome-anchored CLIPS chaperones play a critical role in nuclear steps of ribosome biogenesis.
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Rzhetsky, A. "Estimating substitution rates in ribosomal RNA genes." Genetics 141, no. 2 (October 1, 1995): 771–83. http://dx.doi.org/10.1093/genetics/141.2.771.
Full textAbstract:
Abstract A model is introduced describing nucleotide substitution in ribosomal RNA (rRNA) genes. In this model, substitution in the stem and loop regions of rRNA is modeled with 16- and four-state continuous time Markov chains, respectively. The mean substitution rates at nucleotide sites are assumed to follow gamma distributions that are different for the two types of regions. The simplest formulation of the model allows for explicit expressions for transition probabilities of the Markov processes to be found. These expressions were used to analyze several 16S-like rRNA genes from higher eukaryotes with the maximum likelihood method. Although the observed proportion of invariable sites was only slightly higher in the stem regions, the estimated average substitution rates in the stem regions were almost two times as high as in the loop regions. Therefore, the degree of site heterogeneity of substitution rates in the stem regions seems to be higher than in the loop regions of animal 16S-like rRNAs due to presence of a few rapidly evolving sites. The model appears to be helpful in understanding the regularities of nucleotide substitution in rRNAs and probably minimizing errors in recovering phylogeny for distantly related taxa from these genes.
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Dunn, Sianadh, Olivia Lombardi, and Victoria H. Cowling. "c-Myc co-ordinates mRNA cap methylation and ribosomal RNA production." Biochemical Journal 474, no. 3 (January 20, 2017): 377–84. http://dx.doi.org/10.1042/bcj20160930.
Full textAbstract:
The mRNA cap is a structure added to RNA pol II transcripts in eukaryotes, which recruits factors involved in RNA processing, nuclear export and translation initiation. RNA guanine-7 methyltransferase (RNMT)–RNA-activating miniprotein (RAM), the mRNA cap methyltransferase complex, completes the basic functional mRNA cap structure, cap 0, by methylating the cap guanosine. Here, we report that RNMT–RAM co-ordinates mRNA processing with ribosome production. Suppression of RNMT–RAM reduces synthesis of the 45S ribosomal RNA (rRNA) precursor. RNMT–RAM is required for c-Myc expression, a major regulator of RNA pol I, which synthesises 45S rRNA. Constitutive expression of c-Myc restores rRNA synthesis when RNMT–RAM is suppressed, indicating that RNMT–RAM controls rRNA production predominantly by controlling c-Myc expression. We report that RNMT–RAM is recruited to the ribosomal DNA locus, which may contribute to rRNA synthesis in certain contexts.
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Rodgers, Margaret L., Yunsheng Sun, and Sarah A. Woodson. "Ribosomal Protein S12 Hastens Nucleation of Co-Transcriptional Ribosome Assembly." Biomolecules 13, no. 6 (June 6, 2023): 951. http://dx.doi.org/10.3390/biom13060951.
Full textAbstract:
Ribosomal subunits begin assembly during transcription of the ribosomal RNA (rRNA), when the rRNA begins to fold and associate with ribosomal proteins (RPs). In bacteria, the first steps of ribosome assembly depend upon recognition of the properly folded rRNA by primary assembly proteins such as S4, which nucleates assembly of the 16S 5′ domain. Recent evidence, however, suggests that initial recognition by S4 is delayed due to variable folding of the rRNA during transcription. Here, using single-molecule colocalization co-transcriptional assembly (smCoCoA), we show that the late-binding RP S12 specifically promotes the association of S4 with the pre-16S rRNA during transcription, thereby accelerating nucleation of 30S ribosome assembly. Order of addition experiments suggest that S12 helps chaperone the rRNA during transcription, particularly near the S4 binding site. S12 interacts transiently with the rRNA during transcription and, consequently, a high concentration is required for its chaperone activity. These results support a model in which late-binding RPs moonlight as RNA chaperones during transcription in order to facilitate rapid assembly.
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Matzov, Donna, Masato Taoka, Yuko Nobe, Yoshio Yamauchi, Yehuda Halfon, Nofar Asis, Ella Zimermann, et al. "Cryo-EM structure of the highly atypical cytoplasmic ribosome of Euglena gracilis." Nucleic Acids Research 48, no. 20 (October 22, 2020): 11750–61. http://dx.doi.org/10.1093/nar/gkaa893.
Full textAbstract:
Abstract Ribosomal RNA is the central component of the ribosome, mediating its functional and architectural properties. Here, we report the cryo-EM structure of a highly divergent cytoplasmic ribosome from the single-celled eukaryotic alga Euglena gracilis. The Euglena large ribosomal subunit is distinct in that it contains 14 discrete rRNA fragments that are assembled non-covalently into the canonical ribosome structure. The rRNA is substantially enriched in post-transcriptional modifications that are spread far beyond the catalytic RNA core, contributing to the stabilization of this highly fragmented ribosome species. A unique cluster of five adenosine base methylations is found in an expansion segment adjacent to the protein exit tunnel, such that it is positioned for interaction with the nascent peptide. As well as featuring distinctive rRNA expansion segments, the Euglena ribosome contains four novel ribosomal proteins, localized to the ribosome surface, three of which do not have orthologs in other eukaryotes.
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Petrov, Anton S., Burak Gulen, Ashlyn M. Norris, Nicholas A. Kovacs, Chad R. Bernier, Kathryn A. Lanier, George E. Fox, et al. "History of the ribosome and the origin of translation." Proceedings of the National Academy of Sciences 112, no. 50 (November 30, 2015): 15396–401. http://dx.doi.org/10.1073/pnas.1509761112.
Full textAbstract:
We present a molecular-level model for the origin and evolution of the translation system, using a 3D comparative method. In this model, the ribosome evolved by accretion, recursively adding expansion segments, iteratively growing, subsuming, and freezing the rRNA. Functions of expansion segments in the ancestral ribosome are assigned by correspondence with their functions in the extant ribosome. The model explains the evolution of the large ribosomal subunit, the small ribosomal subunit, tRNA, and mRNA. Prokaryotic ribosomes evolved in six phases, sequentially acquiring capabilities for RNA folding, catalysis, subunit association, correlated evolution, decoding, energy-driven translocation, and surface proteinization. Two additional phases exclusive to eukaryotes led to tentacle-like rRNA expansions. In this model, ribosomal proteinization was a driving force for the broad adoption of proteins in other biological processes. The exit tunnel was clearly a central theme of all phases of ribosomal evolution and was continuously extended and rigidified. In the primitive noncoding ribosome, proto-mRNA and the small ribosomal subunit acted as cofactors, positioning the activated ends of tRNAs within the peptidyl transferase center. This association linked the evolution of the large and small ribosomal subunits, proto-mRNA, and tRNA.