Concerted modification of nucleotides at functional centers of the ribosome revealed by single-molecule RNA modification profiling

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.

18S rRNA variability maps reveal three highly divergent, conserved motifs within Rotifera

Background 18S rRNA is a major component of the small subunit of the eukaryotic ribosome and an important phylogenetic marker for many groups, often to the point of being the only marker available for some. A core structure across eukaryotes exists for this molecule that can help to inform about its evolution in different groups. Using an alignment of 18S rDNA for Rotifera as traditionally recognized (=Bdelloidea, Monogononta, and Seisonacea, but not Acanthocephala), I fitted sequences for three exemplar species (Adineta vaga, Brachionus plicatilis, and Seison nebaliae, respectively) to the core structure and used these maps to reveal patterns of evolution for the remainder of this diverse group of microscopic animals. Results The obtained variability maps of the 18S rRNA molecule revealed a pattern of high diversity among the three major rotifer clades coupled with strong conservation within each of bdelloids and monogononts. A majority of individual sites (ca. 60%) were constant even across rotifers as a whole with variable sites showing only intermediate rates of evolution. Although the three structural maps each showed good agreement with the inferred core structure for eukaryotic 18S rRNA and so were highly similar to one another at the secondary and tertiary levels, the overall pattern is of three highly distinct, but conserved motifs within the group at the primary sequence level. A novel finding was that of a variably expressed deletion at the 3' end of the V3 hypervariable region among some bdelloid species that occasionally extended into and included the pseudoknot structure following this region as well as the central “square” of the 18S rRNA molecule. Compared to other groups, levels of variation and rates of evolution for 18S rRNA in Rotifera roughly matched those for Gastropoda and Acanthocephala, despite increasing evidence for the latter being a clade within Rotifera. Conclusions The lack of comparative data for comparable groups makes interpretation of the results (i.e., very low variation within each of the three major rotifer clades, but high variation between them) and their potential novelty difficult. However, these findings in combination with the high morphological diversity within rotifers potentially help to explain why no clear consensus has been reached to date with regard to the phylogenetic relationships among the major groups.

Co-Assembly of 40S and 60S Ribosomal Proteins in Early Steps of Eukaryotic Ribosome Assembly

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.

Simultaneous Bayesian inference of phylogeny and molecular coevolution

Patterns of molecular coevolution can reveal structural and functional constraints within or among organic molecules. These patterns are better understood when considering the underlying evolutionary process, which enables us to disentangle the signal of the dependent evolution of sites (coevolution) from the effects of shared ancestry of genes. Conversely, disregarding the dependent evolution of sites when studying the history of genes negatively impacts the accuracy of the inferred phylogenetic trees. Although molecular coevolution and phylogenetic history are interdependent, analyses of the two processes are conducted separately, a choice dictated by computational convenience, but at the expense of accuracy. We present a Bayesian method and associated software to infer how many and which sites of an alignment evolve according to an independent or a pairwise dependent evolutionary process, and to simultaneously estimate the phylogenetic relationships among sequences. We validate our method on synthetic datasets and challenge our predictions of coevolution on the 16S rRNA molecule by comparing them with its known molecular structure. Finally, we assess the accuracy of phylogenetic trees inferred under the assumption of independence among sites using synthetic datasets, the 16S rRNA molecule and 10 additional alignments of protein-coding genes of eukaryotes. Our results demonstrate that inferring phylogenetic trees while accounting for dependent site evolution significantly impacts the estimates of the phylogeny and the evolutionary process.

A non-cytotoxic but ribonucleolytically specific ribotoxin variant: implication of tryptophan residues in the cytotoxicity of hirsutellin A

Ribotoxins are a family of toxic proteins that exert a highly specific cleavage at the universally conserved sarcin/ricin loop (SRL) of the larger rRNA molecule. Before this ribonucleolytic action, passage through the cell membrane is a necessary step for ribotoxin internalization and the limiting factor for cytotoxicity. Although extensive knowledge of their ribonucleolytic activity and substrate recognition has been accumulated, little is known about the mechanisms of cell entry of ribotoxins. Hirsutellin A (HtA) is a recently described member of this family, which accommodates the main abilities of previously characterized ribotoxins into a shorter sequence, but exhibits some differences regarding membrane interaction properties. This work investigates the contribution of tryptophan (Trp) residues 71 and 78 to both endoribonucleolytic activity and cellular toxicity of this ribotoxin. Substitution mutants W71F and W78F, as well as the double mutant W71/78F, were obtained and assayed against isolated ribosomes, synthetic SRL, and human tumor cells. The results provide evidence that cell membrane passage and internalization, as well as substrate-specific recognition, require the participation of the region involving both Trp 71 and Trp 78. Additionally, the mutant W71/78F is the first non-cytotoxic but specific ribosome-cleaving ribotoxin mutant obtained to date.

Feasibility of Transferring Fluorescent In Situ Hybridization Probes to an 18S rRNA Gene Phylochip and Mapping of Signal Intensities

ABSTRACT DNA microarray technology offers the possibility to analyze microbial communities without cultivation, thus benefiting biodiversity studies. We developed a DNA phylochip to assess phytoplankton diversity and transferred 18S rRNA probes from dot blot or fluorescent in situ hybridization (FISH) analyses to a microarray format. Similar studies with 16S rRNA probes have been done determined that in order to achieve a signal on the microarray, the 16S rRNA molecule had to be fragmented, or PCR amplicons had to be <150 bp in length to minimize the formation of a secondary structure in the molecule so that the probe could bind to the target site. We found different results with the 18S rRNA molecule. Four out of 12 FISH probes exhibited false-negative signals on the microarray; eight exhibited strong but variable signals using full-length 18S RNA molecules. A systematic investigation of the probe's accessibility to the 18S rRNA gene was made using Prymenisum parvum as the target. Fourteen additional probes identical to this target covered the regions not tested with existing FISH probes. Probes with a binding site in the first 900 bp of the gene generated positive signals. Six out of nine probes binding in the last 900 bp of the gene produced no signal. Our results suggest that although secondary structure affected probe binding, the effect is not the same for the 18S rRNA gene and the 16S rRNA gene. For the 16S rRNA gene, the secondary structure is stronger in the first half of the molecule, whereas in the 18S rRNA gene, the last half of the molecule is critical. Probe-binding sites within 18S rRNA gene molecules are important for the probe design for DNA phylochips because signal intensity appears to be correlated with the secondary structure at the binding site in this molecule. If probes are designed from the first half of the 18S rRNA molecule, then full-length 18S rRNA molecules can be used in the hybridization on the chip, avoiding the fragmentation and the necessity for the short PCR amplicons that are associated with using the 16S rRNA molecule. Thus, the 18S rRNA molecule is a more attractive molecule for use in environmental studies where some level of quantification is desired. Target size was a minor problem, whereas for 16S rRNA molecules target size rather than probe site was important.

Novel Plasmid-Mediated 16S rRNA m1A1408 Methyltransferase, NpmA, Found in a Clinically Isolated Escherichia coli Strain Resistant to Structurally Diverse Aminoglycosides

ABSTRACT We have isolated a multiple-aminoglycoside-resistant Escherichia coli strain, strain ARS3, and have been the first to identify a novel plasmid-mediated 16S rRNA methyltransferase, NpmA. This new enzyme shared a relatively low level of identity (30%) to the chromosomally encoded 16S rRNA methyltransferase (KamA) of Streptomyces tenjimariensis, an actinomycete aminoglycoside producer. The introduction of a recombinant plasmid carrying npmA could confer on E. coli consistent resistance to both 4,6-disubstituted 2-deoxystreptamines, such as amikacin and gentamicin, and 4,5-disubstituted 2-deoxystreptamines, including neomycin and ribostamycin. The histidine-tagged NpmA elucidated methyltransferase activity against 30S ribosomal subunits but not against 50S subunits and the naked 16S rRNA molecule in vitro. We further confirmed that NpmA is an adenine N-1 methyltransferase specific for the A1408 position at the A site of 16S rRNA. Drug footprinting data indicated that binding of aminoglycosides to the target site was apparently interrupted by methylation at the A1408 position. These observations demonstrate that NpmA is a novel plasmid-mediated 16S rRNA methyltransferase that provides a panaminoglycoside-resistant nature through interference with the binding of aminoglycosides toward the A site of 16S rRNA through N-1 methylation at position A1408.

Temperature-based variation of rRNA secondary structure models: a case study in the insect Drosophila simulans, the land snail Isabellaria adriani, and the crustacean Daphnia pulex

The influence of a temperature default on ribosomal RNA (rRNA) secondary structure models was studied with the "Mfold" energy-optimization program. Folding models of the internal transcribed spacer (ITS) 1 rRNA for both Drosophila simulans (Insecta) and Isabellaria adriani (Gastropoda) were generated at two different temperatures. The folding models are compared with the models previously shown for the ITS-1 of D. melanogaster Oregon R strain and I. adriani. A search for phylogenetically informative ITS-1 folding motifs was conducted for D. simulans. In I. adriani, a new approach for ITS-1 secondary structure analyses is suggested. The paper also elucidates results inferred from three energy-optimizing programs (Mfold, GeneBee, and STAR). These three folding programs give different information on the structure and free energy of a ITS-1 rRNA molecule. Furthermore, secondary-structure models of the small subunit (ssu) rRNA of Daphnia pulex (Crustacea: Cladocera) were investigated. The ssu rRNA molecule is usually folded according to alignment information. Here, ssu folding patterns are computed with Mfold using two temperature conditions. The two Mfold models are compared with the alignment model previously suggested for D. pulex. Three cladoceran-specific motifs and a short stem motif within the ssu rRNA of eukaryotes are discussed with respect to structure and phylogenetic information.

The phylogeny of bacteria from a modern Antarctic refuge

The 16S rRNAs of nine new species of prokaryotes, that had been isolated from four lakes of the Vestfold Hills, have been sequenced. These sequences were compared with those of their closest taxonomic relatives available from publicly available databases. The Antarctic species were of wide diversity with representatives from the domains Archaea and Bacteria (sensu Woese). Generally, they were most closely related to organisms from marine environments. The sequence dissimilarity between the rRNA sequences of the Antarctic strains and their nearest known relatives suggest they diverged from each other much earlier than the establishment of their modern Antarctic habitat. The conserved nature of the 16S rRNA molecule suggests it may not be as useful for detecting evolutionary change in Antarctic prokaryotes as distinct from non-Antarctic prokaryotes. Although the optimal temperature for growth of each species is well above the temperature of its environment, each has a reduced optimal temperature for growth when compared with its taxonomic counterpart from non-Antarctic environments. The vast majority of Antarctic prokaryotes remains to be described.