Review

Authors: Takehiko Kobayashi

The ribosomal RNA gene (rDNA) repeats form a historically well-researched region in the chromosome. Their highly repetitive structure can be identified easily which has enabled studies on DNA replication, recombination, and transcription. The region is one of the most unstable regions in the genome because of deleterious recombination among the repeats. The ribosomal RNA gene repeats use a unique gene amplification system to restore the copy number after this has been reduced due to recombination. It has been shown that unstable features in the genome can accelerate cellular senescence that restricts the lifespan of a cell. Here, I will introduce a study by our group that shows how the stability of rDNA is maintained and affects lifespan. I propose that the ribosomal RNA gene repeats constitute a center from which the stability of the whole genome is regulated and the lifespan of the cell is controlled.

Topics: Animals; Cellular Senescence; DNA, Ribosomal; Genes, rRNA; Humans; RNA Stability; RNA, Ribosomal; Repetitive Sequences, Nucleic Acid

PubMed: 24727936
DOI: 10.2183/pjab.90.119

Authors: Alejandro A Schäffer, Richard McVeigh, Barbara Robbertse...

BACKGROUND

The DNA sequences encoding ribosomal RNA genes (rRNAs) are commonly used as markers to identify species, including in metagenomics samples that may combine many organismal communities. The 16S small subunit ribosomal RNA (SSU rRNA) gene is typically used to identify bacterial and archaeal species. The nuclear 18S SSU rRNA gene, and 28S large subunit (LSU) rRNA gene have been used as DNA barcodes and for phylogenetic studies in different eukaryote taxonomic groups. Because of their popularity, the National Center for Biotechnology Information (NCBI) receives a disproportionate number of rRNA sequence submissions and BLAST queries. These sequences vary in quality, length, origin (nuclear, mitochondria, plastid), and organism source and can represent any region of the ribosomal cistron.

RESULTS

To improve the timely verification of quality, origin and loci boundaries, we developed Ribovore, a software package for sequence analysis of rRNA sequences. The ribotyper and ribosensor programs are used to validate incoming sequences of bacterial and archaeal SSU rRNA. The ribodbmaker program is used to create high-quality datasets of rRNAs from different taxonomic groups. Key algorithmic steps include comparing candidate sequences against rRNA sequence profile hidden Markov models (HMMs) and covariance models of rRNA sequence and secondary-structure conservation, as well as other tests. Nine freely available blastn rRNA databases created and maintained with Ribovore are used for checking incoming GenBank submissions and used by the blastn browser interface at NCBI. Since 2018, Ribovore has been used to analyze more than 50 million prokaryotic SSU rRNA sequences submitted to GenBank, and to select at least 10,435 fungal rRNA RefSeq records from type material of 8350 taxa.

CONCLUSION

Ribovore combines single-sequence and profile-based methods to improve GenBank processing and analysis of rRNA sequences. It is a standalone, portable, and extensible software package for the alignment, classification and validation of rRNA sequences. Researchers planning on submitting SSU rRNA sequences to GenBank are encouraged to download and use Ribovore to analyze their sequences prior to submission to determine which sequences are likely to be automatically accepted into GenBank.

Topics: DNA, Ribosomal; Databases, Nucleic Acid; Phylogeny; RNA, Ribosomal; RNA, Ribosomal, 16S; RNA, Ribosomal, 18S; Sequence Analysis, RNA

PubMed: 34384346
DOI: 10.1186/s12859-021-04316-z

Review

Authors: Griffin A Welfer, Ryan A Brady, S Kundhavai Natchiar...

Since the framing of the Central Dogma, it has been speculated that physically distinct ribosomes within cells may influence gene expression and cellular physiology. While heterogeneity in ribosome composition has been reported in bacteria, protozoans, fungi, zebrafish, mice and humans, its functional implications remain actively debated. Here, we review recent evidence demonstrating that expression of conserved variant ribosomal DNA (rDNA) alleles in bacteria, mice and humans renders their actively translating ribosome pool intrinsically heterogeneous at the level of ribosomal RNA (rRNA). In this context, we discuss reports that nutrient limitation-induced stress in leads to changes in variant rRNA allele expression, programmatically altering transcription and cellular phenotype. We highlight that cells expressing ribosomes from distinct operons exhibit distinct drug sensitivities, which can be recapitulated and potentially rationalized by subtle perturbations in ribosome structure or in their dynamic properties. Finally, we discuss evidence that differential expression of variant rDNA alleles results in different populations of ribosome subtypes within mammalian tissues. These findings motivate further research into the impacts of rRNA heterogeneities on ribosomal function and predict that strategies targeting distinct ribosome subtypes may hold therapeutic potential.This article is part of the discussion meeting issue 'Ribosome diversity and its impact on protein synthesis, development and disease'.

Topics: RNA, Ribosomal; Animals; Ribosomes; Phenotype; Humans; Escherichia coli; Genetic Variation; Mice; Gene Expression

PubMed: 40045785
DOI: 10.1098/rstb.2023.0379

Authors: Maryam Hosseini, Poorna Roy, Marie Sissler...

Mammalian mitochondrial ribosomes evolved from bacterial ribosomes by reduction of ribosomal RNAs, increase of ribosomal protein content, and loss of guanine nucleotides. Guanine is the base most sensitive to oxidative damage. By systematically comparing high-quality, small ribosomal subunit RNA sequence alignments and solved 3D ribosome structures from mammalian mitochondria and bacteria, we deduce rules for folding a complex RNA with the remaining guanines shielded from solvent. Almost all conserved guanines in both bacterial and mammalian mitochondrial ribosomal RNA form guanine-specific, local or long-range, RNA-RNA or RNA-protein interactions. Many solvent-exposed guanines conserved in bacteria are replaced in mammalian mitochondria by bases less sensitive to oxidation. New guanines, conserved only in the mitochondrial alignment, are strategically positioned at solvent inaccessible sites to stabilize the ribosomal RNA structure. New mitochondrial proteins substitute for truncated RNA helices, maintain mutual spatial orientations of helices, compensate for lost RNA-RNA interactions, reduce solvent accessibility of bases, and replace guanines conserved in bacteria by forming specific amino acid-RNA interactions.

Topics: Animals; Base Sequence; Escherichia coli; Guanine; Mitochondria; Models, Molecular; Nucleic Acid Conformation; Protein Binding; RNA Folding; RNA, Bacterial; RNA, Mitochondrial; RNA, Ribosomal; Ribosomal Proteins; Ribosomes; Sus scrofa

PubMed: 30215760
DOI: 10.1093/nar/gky762

Authors: Weidong An, Yunxiao Yan, Keqiong Ye...

Ribosomal RNAs are processed in a complex pathway. We profiled rRNA processing intermediates in yeast at single-molecule and single-nucleotide levels with circularization, targeted amplification and deep sequencing (CircTA-seq), gaining significant mechanistic insights into rRNA processing and surveillance. The long form of the 5' end of 5.8S rRNA is converted to the short form and represents an intermediate of a unified processing pathway. The initial 3' end processing of 5.8S rRNA involves trimming by Rex1 and Rex2 and Trf4-mediated polyadenylation. The 3' end of 25S rRNA is formed by sequential digestion by four Rex proteins. Intermediates with an extended A1 site are generated during 5' degradation of aberrant 18S rRNA precursors. We determined precise polyadenylation profiles for pre-rRNAs and show that the degradation efficiency of polyadenylated 20S pre-rRNA critically depends on poly(A) lengths and degradation intermediates released from the exosome are often extensively re-polyadenylated.

Topics: RNA Processing, Post-Transcriptional; Saccharomyces cerevisiae; RNA, Ribosomal; RNA, Ribosomal, 5.8S; Saccharomyces cerevisiae Proteins; RNA Precursors; RNA, Ribosomal, 18S; Polyadenylation; RNA, Fungal; Exosome Multienzyme Ribonuclease Complex; High-Throughput Nucleotide Sequencing; RNA Stability

PubMed: 38994562
DOI: 10.1093/nar/gkae606

Review

Authors: Petr V Sergiev, Alexey A Bogdanov, Olga A Dontsova...

Five nearly universal methylated guanine-(N2) residues are present in bacterial rRNA in the ribosome. To date four out of five ribosomal RNA guanine-(N2)-methyltransferases are described. RsmC(YjjT) methylates G1207 of the 16S rRNA. RlmG(YgjO) and RlmL(YcbY) are responsible for the 23S rRNA m(2)G1835 and m(2)G2445 formation, correspondingly. RsmD(YhhF) is necessary for methylation of G966 residue of 16S rRNA. Structure of Escherichia coli RsmD(YhhF) methyltransferase and the structure of the Methanococcus jannaschii RsmC ortholog were determined. All ribosomal guanine-(N2)-methyltransferases have similar AdoMet-binding sites. In relation to the ribosomal substrate recognition, two enzymes that recognize assembled subunits are relatively small single domain proteins and two enzymes that recognize naked rRNA are larger proteins containing separate methyltransferase- and RNA-binding domains. The model for recognition of specific target nucleotide is proposed. The hypothetical role of the m(2)G residues in rRNA is discussed.

Topics: Bacteria; Guanine; Methyltransferases; RNA, Ribosomal; Substrate Specificity

PubMed: 17389639
DOI: 10.1093/nar/gkm104

Review

Authors: James Ofengand

Pseudouridines are found in virtually all ribosomal RNAs but their function is unknown. There are four to eight times more pseudouridines in eukaryotes than in eubacteria. Mapping 19 Haloarcula marismortui pseudouridines on the three-dimensional 50S subunit does not show clustering. In bacteria, specific enzymes choose the site of pseudouridine formation. In eukaryotes, and probably also in archaea, selection and modification is done by a guide RNA-protein complex. No unique specific role for ribosomal pseudouridines has been identified. We propose that pseudouridine's function is as a molecular glue to stabilize required RNA conformations that would otherwise be too flexible.

Topics: Animals; Archaea; Eubacterium; Eukaryotic Cells; Humans; Models, Molecular; Nucleic Acid Conformation; Pseudouridine; RNA, Ribosomal; RNA, Small Untranslated

PubMed: 11904174
DOI: 10.1016/s0014-5793(02)02305-0

Authors: Roman Martin, Philipp Hackert, Maike Ruprecht...

Ribosome biogenesis in yeast requires 75 small nucleolar RNAs (snoRNAs) and a myriad of cofactors for processing, modification, and folding of the ribosomal RNAs (rRNAs). For the 19 RNA helicases implicated in ribosome synthesis, their sites of action and molecular functions have largely remained unknown. Here, we have used UV cross-linking and analysis of cDNA (CRAC) to reveal the pre-rRNA binding sites of the RNA helicase Rok1, which is involved in early small subunit biogenesis. Several contact sites were identified in the 18S rRNA sequence, which interestingly all cluster in the "foot" region of the small ribosomal subunit. These include a major binding site in the eukaryotic expansion segment ES6, where Rok1 is required for release of the snR30 snoRNA. Rok1 directly contacts snR30 and other snoRNAs required for pre-rRNA processing. Using cross-linking, ligation and sequencing of hybrids (CLASH) we identified several novel pre-rRNA base-pairing sites for the snoRNAs snR30, snR10, U3, and U14, which cluster in the expansion segments of the 18S rRNA. Our data suggest that these snoRNAs bridge interactions between the expansion segments, thereby forming an extensive interaction network that likely promotes pre-rRNA maturation and folding in early pre-ribosomal complexes and establishes long-range rRNA interactions during ribosome synthesis.

Topics: Base Pairing; DEAD-box RNA Helicases; Nucleic Acid Conformation; Protein Binding; RNA Precursors; RNA, Ribosomal; RNA, Ribosomal, 18S; RNA, Small Nucleolar; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

PubMed: 24947498
DOI: 10.1261/rna.044669.114

Review

Authors: Maxime Aubert, Marie-Françoise O'Donohue, Simon Lebaron...

Ribosomal RNAs, the most abundant cellular RNA species, have evolved as the structural scaffold and the catalytic center of protein synthesis in every living organism. In eukaryotes, they are produced from a long primary transcript through an intricate sequence of processing steps that include RNA cleavage and folding and nucleotide modification. The mechanisms underlying this process in human cells have long been investigated, but technological advances have accelerated their study in the past decade. In addition, the association of congenital diseases to defects in ribosome synthesis has highlighted the central place of ribosomal RNA maturation in cell physiology regulation and broadened the interest in these mechanisms. Here, we give an overview of the current knowledge of pre-ribosomal RNA processing in human cells in light of recent progress and discuss how dysfunction of this pathway may contribute to the physiopathology of congenital diseases.

Topics: Disease; Humans; Nucleic Acid Conformation; RNA Processing, Post-Transcriptional; RNA, Ribosomal; Ribosomal Proteins; Ribosomes

PubMed: 30356013
DOI: 10.3390/biom8040123

Authors: Charles Antony, Patrick Somers, Erin M Gray...

FISH-Flow (fluorescence in situ hybridization-flow cytometry) involves hybridizing fluorescent oligos to RNA and quantifying fluorescence at a single-cell level using flow cytometry. Here, we present a FISH-Flow protocol to quantify nascent 47S and mature 18S and 28S rRNAs in mouse and human cells, including rRNA quantification across cell cycle stages using DNA staining. We describe steps for cell preparation, hybridization of fluorescent probes against rRNA, and DNA staining. We then detail procedures for flow cytometry and data analysis. For complete details on the use and execution of this protocol, please refer to Antony et al. (2022)..

Topics: Humans; Animals; Mice; RNA, Ribosomal; In Situ Hybridization, Fluorescence; RNA, Ribosomal, 28S; RNA; DNA

PubMed: 37481729
DOI: 10.1016/j.xpro.2023.102463