Profiling of 2'-O-Me in human rRNA reveals a subset of fractionally modified positions and provides evidence for ribosome heterogeneity
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE76393 | GEO | 2016/06/02
SECONDARY ACCESSION(S): PRJNA307191
REPOSITORIES: GEO
Similar Datasets
Project description:A sequencing-based profiling method (RiboMeth-seq) for ribose methylations was used to study methylation patterns in mouse adult tissues and during development. In contrast to previous reports based on studies of human cancer cell lines, almost all methylation sites were close to fully methylated in adult tissues. A subset of sites was differentially modified in developing tissues compared to their adult counterparts and showed clear developmental dynamics. This provides the first evidence for ribosome heterogeneity at the level of rRNA modifications during mouse development. In a prominent example, the expression levels of SNORD78 during development appeared to be regulated by alternative splicing of the Gas5 host-gene and to correlate with the methylation level of its target site at LSU-G4593. The results are discussed in the context of the specialized ribosome hypothesis.
2019-09-24 | GSE128947 | GEO
Project description:The major information-carrying macromolecules in the cell, DNA, RNA, and protein, carry an additional layer of information on top of their sequence in the form of modifications of residues. The modifications provide additional functional groups and impact the structure and function of the molecules. Cellular RNA molecules contain more than 100 different modifications and are found in all domains of life and in all major classes of RNA in eukaryotic organisms. Together these modifications constitute the epitranscriptome of which two-thirds are methylations with 2’-O methylation of the ribose moiety of the nucleotide as the most abundant. Many aspects of ribose methylation are underexplored because the existing methods for their detection are laborious and can only address a few modification sites at a time. Here, we introduce RiboMeth-Seq, a high-throughput sequencing based method and applies it to yeast ribosomal RNA. We detect all of the known as well as one new methylation site and provide evidence for hypomethylation at specific residues. Furthermore we demonstrate that many methylation events are interdependent and outline the timing of modifications during ribosome biogenesis. Our results demonstrate a novel and efficient approach to understanding of the role of modifications in ribosomal RNA folding and ribosome function. Recent evidence point to changes in ribose methylation patterns in cancer ribosomes and we anticipate that RiboMeth-Seq can be applied here as well as to other diseases in which ribosomes are affected, including the heritable ribosomopathies. Yeast RNA was degraded at denaturing conditions into small fragments, long enough to be mapped by sequence alignment to the reference genome (20-40 nucleotides). The fragments were ligated to RNA oligos using a tRNA ligase that was mutated to remove its kinase activity, reverse transcribed and the cDNA used as input for Ion semiconductor sequencing. The first and last nucleotides of the inserts were recorded using the full sequence for mapping and the read-ends plotted against the sequence. 2'-O-Methylated RNA positions are protected from cleavage, and read ends from such positions are therefore underrepresented compared to the surrounding positions.
2014-11-24 | E-GEOD-54655 | biostudies-arrayexpress
Project description:The major information-carrying macromolecules in the cell, DNA, RNA, and protein, carry an additional layer of information on top of their sequence in the form of modifications of residues. The modifications provide additional functional groups and impact the structure and function of the molecules. Cellular RNA molecules contain more than 100 different modifications and are found in all domains of life and in all major classes of RNA in eukaryotic organisms. Together these modifications constitute the epitranscriptome of which two-thirds are methylations with 2’-O methylation of the ribose moiety of the nucleotide as the most abundant. Many aspects of ribose methylation are underexplored because the existing methods for their detection are laborious and can only address a few modification sites at a time. Here, we introduce RiboMeth-Seq, a high-throughput sequencing based method and applies it to yeast ribosomal RNA. We detect all of the known as well as one new methylation site and provide evidence for hypomethylation at specific residues. Furthermore we demonstrate that many methylation events are interdependent and outline the timing of modifications during ribosome biogenesis. Our results demonstrate a novel and efficient approach to understanding of the role of modifications in ribosomal RNA folding and ribosome function. Recent evidence point to changes in ribose methylation patterns in cancer ribosomes and we anticipate that RiboMeth-Seq can be applied here as well as to other diseases in which ribosomes are affected, including the heritable ribosomopathies.
2014-11-24 | GSE54655 | GEO
Project description:Emerging evidence suggests that changes in ribosomal RNA 2’O-ribose methylation (rRNA 2’OMe) in the human ribosome play a key role in regulating translation thereby contributing in setting particular phenotypes such as hallmarks of cancer cells. Here, by analysing the rRNA 2’OMe in a large series of 195 human primary breast tumours using the RiboMETH-seq approach we have identified the positions within the rRNA molecules which can tolerate the absence of 2’OMe in humans. The frequency of rRNA 2’OMe at some positions is highly stable between individuals (termed “stable”) whereas it can vary substantially at other positions (termed “variant”). Differential association of rRNA 2’OMe with breast cancer features, evolution and structure/function characteristics suggest the stable positions showing a low inter-variability might be required to maintain the core structure and thus the function of the ribosome – representing compulsory rRNA 2’OMe positions. In contrast, the variant positions displaying high inter-variability might be the source of regulatory events – representing plastic rRNA 2’OMe positions. Therefore our data issued from rRNA 2’OMe profiling of a large-scale human sample series bring the first compelling evidence to resolve two divergent long lasting visions of the role of rRNA 2’O-Me and sustain the emerging role of the ribosome plasticity in regulating translation in cancer.
2020-12-22 | GSE143415 | GEO
Project description:Nucleotides in RNA and DNA are chemically modified by numerous enzymes that alter their function. Eukaryotic ribosomal RNA (rRNA) is modified at more than 100 locations, particularly at highly conserved and functionally important nucleotides. During ribosome biogenesis, modifications are added at various stages of assembly. The existence of differently modified classes of ribosomes in normal cells is unknown because no method exists to simultaneously evaluate the modification status at all sites, within a single rRNA molecule. Using a combination of yeast genetics and nanopore direct RNA sequencing, we developed a reliable method to track the modification status of single rRNA molecules at 37 sites in 18S rRNA and 73 sites in 25S rRNA. We use our method to characterize patterns of modification heterogeneity and identify concerted modification of nucleotides found near functional centers of the ribosome. Distinct undermodified subpopulations of rRNAs accumulate upon loss of Dbp3 or Prp43 RNA helicases, suggesting overlapping roles in ribosome biogenesis. Modification profiles are surprisingly resistant to change in response to many genetic and environmental conditions that affect translation, ribosome biogenesis, and pre-mRNA splicing. The ability to capture single-molecule RNA modification profiles provides new insights into the roles of nucleotide modifications in RNA function.
2021-12-03 | GSE186634 | GEO
Project description: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.
2021-03-18 | GSE155720 | GEO
Project description:In cell biology, ribosomal RNA (rRNA) 2'O-methyl (2'-O-Me) is the most prevalent post-transcriptional chemical modification contributing to ribosome heterogeneity. The modification involves a family of small nucleolar RNAs (snoRNAs) and is specified by box C/D snoRNAs (SNORDs). Given the importance of ribosome biogenesis for skeletal muscle growth, we asked if rRNA 2'-O-Me in nascent ribosomes synthesized in response to a growth stimulus is an unrecognized mode of ribosome heterogeneity in muscle. To determine the pattern and dynamics of 2'-O-Me rRNA, we used a sequencing-based profiling method called RiboMeth-seq. We applied this method to tissue-derived rRNA of skeletal muscle and rRNA specifically from the muscle fiber using an inducible myofiber-specific RiboTag mouse in sedentary and mechanically overloaded conditions. These analyses were complemented by myonuclear-specific small RNA sequencing to profile SNORDs and link the rRNA epitranscriptome to known regulatory elements generated within the muscle fiber. We demonstrate for the first time that mechanical overload of skeletal muscle 1) induces decreased 2'-O-Me at a subset of skeletal muscle rRNAand 2) alters the SNORD profile in isolated myonuclei. These findings point to a transient diversification of the ribosome pool via 2'-O-Me during growth and adaptation in skeletal muscle. These findings suggest changes in ribosome heterogeneity at the 2'-O-Me level during muscle hypertrophy and lay the foundation for studies investigating the functional implications of these newly identified "growth-induced" ribosomes.
2024-07-24 | GSE273053 | GEO
Project description:Ribosomal RNAs (rRNAs) are main effectors of 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 display 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 (FBL) in human cells. Using RiboMethSeq, a non-biased 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 (CrPV) IRES element, as a model, coupled to in vitro translation, we show that the intrinsic capability of ribosomes to translate mRNAs is modulated through 2'-O-Me pattern and not by non-ribosomal actors of the translational machinery. Our results establish rRNA 2'-O-methylation plasticity as a mechanism providing functional specificity to human ribosomes.
2018-01-19 | GSE105248 | GEO
Project description:RNase E, an essential endoribonuclease in Escherichia coli, is the key component of the multienzyme RNA degradosome, which is localized to the inner cytoplasmic membrane. Until now, the reason for membrane localization of RNase E was not known. We have analyzed ribosome assembly in the rneΔMTS strain, which expresses a cytoplasmic variant of RNase E (cRNase E) resulting in a cytoplasmic RNA degradosome. In this mutant strain, there is a mild slowdown in the rates of growth and mRNA degradation. Here we document the striking accumulation of intermediates in ribosome assembly in the rneΔMTS strain in which precursors of 16S and 23S rRNA are cleaved by cRNase E. In vitro, we show that ribosomes partially unfolded in low ionic strength buffer are cleaved by RNase E. Mapping of in vivo and in vitro cleavage sites shows that they overlap and that their consensus sequence matches previously mapped RNase E cleavage sites. In vivo, fragments of 16S and 23S rRNA as well as a precursor of 5S rRNA are degraded in a pathway involving polyadenylation and 3’ exonucleases. Since the pathway for rRNA degradation is the same as the pathway for mRNA degradation, the slowdown of mRNA degradation in the rneΔMTS strain could be due to competition by rRNA degradation. Our results strongly suggest that the RNA degradosome participates in the quality control of ribosome assembly and that localization on the inner cytoplasmic membrane protects newly synthesized intermediates from wasteful degradation. Ribosome synthesis is costly. Since growth rate correlates with ribosome synthesis rate, slow growth rate of the rneΔMTS strain is due to the degradation of a proportion of newly synthesized ribosomes. Avoiding wasteful degradation of intermediates in ribosome assembly likely explains why localization of RNase E homologues to the inner cytoplasmic membrane is conserved throughout the γ-Proteobacteria.
2023-01-16 | PXD038317 | Pride
Project description:NOL12 5'-3' exoribonucleases, conserved among eukaryotes, play important roles in pre-rRNA processing, ribosome assembly and export. The most well-described yeast counterpart, Rrp17, is required for maturation of 5.8 and 25S rRNAs, whereas human hNOL12 is crucial for the separation of the large (LSU) and small (SSU) ribosome subunit rRNA precursors. In this study we demonstrate that plant AtNOL12 is also involved in rRNA biogenesis, specifically in the processing of the LSU rRNA precursor, 27S pre-rRNA. Importantly, the absence of AtNOL12 alters the expression of many ribosomal protein and ribosome biogenesis genes. These changes could potentially exacerbate rRNA biogenesis defects, or, conversely, they might stem from the disturbed ribosome assembly caused by delayed pre-rRNA processing. Moreover, exposure of the nol12 mutant to stress factors, including heat and pathogen Pseudomonas syringae, enhances the observed molecular phenotypes, linking pre-rRNA processing to stress response pathways. The aberrant rRNA processing, dependent on AtNOL12, could impact ribosome function, as suggested by improved mutant resistance to ribosome-targeting antibiotics. Despite extensive studies, the pre-rRNA processing pathway in plants remains insufficiently characterized. Our investigation reveals the involvement of AtNOL12 in the maturation of rRNA precursors, correlating this process to stress response in Arabidopsis. These findings contribute to a more comprehensive understanding of plant ribosome biogenesis.
2023-11-07 | GSE232067 | GEO