rRNA Molecules Research Articles

Comparative Gut Microbiota of 59 Neotropical Bird Species.

The gut microbiota of vertebrates are essential to host health. Most non-model vertebrates, however, lack even a basic description of natural gut microbiota biodiversity. Here, we sampled 116 intestines from 59 Neotropical bird species and used the V6 region of the 16S rRNA molecule as a microbial fingerprint (average coverage per bird ~80,000 reads). A core microbiota of Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria was identified, as well as several gut-associated genera. We tested 18 categorical variables associated with each bird for significant correlation to the gut microbiota; host taxonomic categories were most frequently significant and explained the most variation. Ecological variables (e.g., diet, foraging stratum) were also frequently significant but explained less variation. Little evidence was found for a significant influence of geographic space. Finally, we suggest that microbial sampling during field collection of organisms would propel biological understanding of evolutionary history and ecological significance of host-associated microbiota.

C/D box sRNA-guided 2'-O-methylation patterns of archaeal rRNA molecules.

BackgroundIn archaea and eukaryotes, ribonucleoprotein complexes containing small C/D box s(no)RNAs use base pair complementarity to target specific sites within ribosomal RNA for 2'-O-ribose methylation. These modifications aid in the folding and stabilization of nascent rRNA molecules and their assembly into ribosomal particles. The genomes of hyperthermophilic archaea encode large numbers of C/D box sRNA genes, suggesting an increased necessity for rRNA stabilization at extreme growth temperatures.ResultsWe have identified the complete sets of C/D box sRNAs from seven archaea using RNA-Seq methodology. In total, 489 C/D box sRNAs were identified, each containing two guide regions. A combination of computational and manual analyses predicts 719 guide interactions with 16S and 23S rRNA molecules. This first pan-archaeal description of guide sequences identifies (i) modified rRNA nucleotides that are frequently conserved between species and (ii) regions within rRNA that are hotspots for 2'-O-methylation. Gene duplication, rearrangement, mutational drift and convergent evolution of sRNA genes and guide sequences were observed. In addition, several C/D box sRNAs were identified that use their two guides to target locations distant in the rRNA sequence but close in the secondary and tertiary structure. We propose that they act as RNA chaperones and facilitate complex folding events between distant sequences.ConclusionsThis pan-archaeal analysis of C/D box sRNA guide regions identified conserved patterns of rRNA 2'-O-methylation in archaea. The interaction between the sRNP complexes and the nascent rRNA facilitates proper folding and the methyl modifications stabilize higher order rRNA structure within the assembled ribosome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1839-z) contains supplementary material, which is available to authorized users.

Sensitive and rapid RT-qPCR quantification of pathogenic Candida species in human blood

For accurate diagnosis and appropriate treatment of candidiasis, we developed a highly sensitive quantitative RT-PCR (RT-qPCR) system for five Candida species that have been reported to be the major causes of bloodstream fungal infection (Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei), together with a system for all pathogenic Candida species. Cells of each fungal species spiked into human peripheral blood (PB) were specifically detected at a lower detection limit of 100cell/1mL PB by this system using the newly developed specific primer sets targeting 18S or 26S rRNA of the five Candida species, together with the existing group primer set. The total count of the five Candida spp. as the sum of those obtained by using the five species primer sets was equivalent to the count obtained by using the group primer set, indicating that the group set covered the major five Candida spp. in human blood with the same degree of accuracy as the species primer sets. The RT-qPCR counts of the Candida species were in good agreement with CFU counts obtained by their culture on CHROMagar™, with a lower detection limit of 100cell/mL of PB. Candida rRNA molecules were stably stored for at least 7days at 4°C by keeping the blood specimens in an RNA stabilizing reagent. These results strongly suggest that this sensitive system is useful for accurate and rapid diagnosis of Candida bloodstream infections.

5.8S rRNA sequence and secondary structure in Monotropa hypopitys and related Ericaceae species.

This is the first study to investigate the secondary structure of 5.8S rRNA in M. hypopitys and related Ericaceae species. The identified nucleotide substitutions are localized mostly at the 3' and 5' ends of the gene, in the region of the third hairpin, and do not significantly affect the topology of the secondary structure of the 5.8S rRNA molecule.

A new method for efficient detection of Cryptosporidium RNA by real-time reverse transcription-PCR with surfactants

Cryptosporidium is one of the most common causes of waterborne diseases worldwide. Its oocysts possess a robust wall that is extremely resistant to the chlorine used for potable water disinfection. The current procedures of nucleic acid extraction and purification, such as the freeze–thaw (F/T) method and the commercial kits, are time consuming and expensive. To this end, a surfactant extraction treatment (SET) was developed as a method to extract nucleic acids from Cryptosporidium using only surfactants. The use of 18S rRNA improves the sensitivity of Cryptosporidium detection for real-time polymerase chain reaction (PCR), because 18S rRNA molecules are constitutively present in high copy numbers. Therefore, we applied SET to the detection of Cryptosporidium 18S rRNA using reverse transcription (RT)-PCR for the first time. RT-PCR was inhibited by 0.01% of the anionic surfactant sodium dodecyl sulfate (SDS), whereas the inhibition did not occur with 5% of the nonionic surfactants Tween 20, Triton X-100, Tween 80, and Triton X-114. However, the nonionic surfactants could not completely suppress the inhibition induced by 0.1% SDS. We successfully extracted 18S rRNA genes from oocysts by SET without the F/T method and detected them by real-time RT-PCR.

Highly divergent 18S rRNA gene paralogs in a Cryptosporidium genotype from eastern chipmunks (Tamias striatus)

Cryptosporidium is an apicomplexan parasite that causes the disease cryptosporidiosis in humans, livestock, and other vertebrates. Much of the knowledge on Cryptosporidium diversity is derived from 18S rRNA gene (18S rDNA) phylogenies. Eukaryote genomes generally have multiple 18S rDNA copies that evolve in concert, which is necessary for the accurate inference of phylogenetic relationships. However, 18S rDNA copies in some genomes evolve by a birth-and-death process that can result in sequence divergence among copies. Most notably, divergent 18S rDNA paralogs in the apicomplexan Plasmodium share only 89–95% sequence similarity, encode structurally distinct rRNA molecules, and are expressed at different life cycle stages. In the present study, Cryptosporidium 18S rDNA was amplified from 28/72 (38.9%) eastern chipmunks (Tamias striatus). Phylogenetic analyses showed the co-occurrence of two 18S rDNA types, Type A and Type B, in 26 chipmunks, and Type B clustered with a sequence previously identified as Cryptosporidium chipmunk genotype II. Types A and B had a sister group relationship but shared less than 93% sequence similarity. In contrast, actin and heat shock protein 70 gene sequences were homogeneous in samples with both Types A and B present. It was therefore concluded that Types A and B are divergent 18S rDNA paralogs in Cryptosporidium chipmunk genotype II. Substitution patterns in Types A and B were consistent with functionally constrained evolution; however, Type B evolved more rapidly than Type A and had a higher G+C content (46.3% versus 41.0%). Oocysts of Cryptosporidium chipmunk genotype II measured 4.17μm (3.73–5.04μm)×3.94μm (3.50–4.98μm) with a length-to-width ratio of 1.06±0.06μm, and infection occurred naturally in the jejunum, cecum, and colon of eastern chipmunks. The findings of this study have implications for the use of 18S rDNA sequences to infer phylogenetic relationships.

Out of sight, out of mind.

Out of sight, out of mind.

Riboswitches as Potential Targets for Aminoglycosides Compared with rRNA Molecules: In Silico Study

Riboswitches are cis acting riboregulaters in non-coding region of the mRNAs. Their possible contribution in antibiotic targeting especially for FMN, TPP and lysine riboswitches in bacteria has been revealed since a decade ago. Regarding some studies on the possibility of the interaction between aminoglycosides and the artificial riboswitches, in this study we attempted to evaluate the binding potential of different types of aminoglycosides including gentamicin, amikacin, kanamycin, neomycin, tobramaycin, sisomicin and paromomycin with various classes of riboswitches using computational methods. Applying Auto Dock vina, it was shown that the binding energy of each kind of riboswitches with different types of aminoglycosides (riboswitches/aminoglycosides) is almost similar or sometimes more than the binding energy of the aminoglycoside with the corresponding binding cage of “16S rRNA A site” (16S rRNA A site/aminoglycosides) as aminoglycosides’ target site. The affinity between riboswitches and aminoglycosides is almost the same or higher than the affinity of riboswitches/natural ligands. In this study ampicillin was used as the negative control antibiotic and 5S rRNA was employed as the negative control RNA. Results showed that the binding energies of riboswitches/ampicillin and 5S rRNA/aminoglycosides are usually lower than the energy of riboswitches/aminoglycosides. Accordingly, lysine, glycine and SAM-I riboswitches were recognized as the best RNA targets for all of the aminoglycosides because of their higher binding energy. In the next step, docking results were further validated by rDock program. Furthermore, it was shown that hydrogen binding makes a key role in the binding energy between aminoglycosides and riboswitches. Moreover, MD simulation studies on lysine riboswitch/paromomycin complex confirmed the stability of the docked structure in the solvent containing magnesium and chloride ions.

The role of RNase R in trans-translation and ribosomal quality control.

Gene expression not only depends on the rate of transcription but is also largely controlled at the post-transcriptional level. Translation rate and mRNA decay greatly influence the final protein levels. Surveillance mechanisms are essential to ensure the quality of the RNA and proteins produced. Trans-translation is one of the most important systems in the quality control of bacterial translation. This process guarantees the destruction of abnormal proteins and also leads to degradation of the respective defective RNAs through the action of Ribonuclease R (RNase R). This exoribonuclease hydrolyzes RNAs starting from their 3' end. Besides its involvement in trans-translation, RNase R also participates in the quality control of rRNA molecules involved in ribosomal biogenesis. RNase R is thus emerging as a key factor in ensuring translation accuracy. This review focuses on issues related to the quality control of translation, with special emphasis on the role of RNase R.

Direct activation of ribosome-associated double-stranded RNA-dependent protein kinase (PKR) by deoxynivalenol, anisomycin and ricin: a new model for ribotoxic stress response induction.

Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is a critical upstream mediator of the ribotoxic stress response (RSR) to the trichothecene deoxynivalenol (DON) and other translational inhibitors. Here, we employed HeLa cell lysates to: (1) characterize PKR’s interactions with the ribosome and ribosomal RNA (rRNA); (2) demonstrate cell-free activation of ribosomal-associated PKR and (3) integrate these findings in a unified model for RSR. Robust PKR-dependent RSR was initially confirmed in intact cells. PKR basally associated with 40S, 60S, 80S and polysome fractions at molar ratios of 7, 2, 23 and 3, respectively. Treatment of ATP-containing HeLa lysates with DON or the ribotoxins anisomycin and ricin concentration-dependently elicited phosphorylation of PKR and its substrate eIF2α. These phosphorylations could be blocked by PKR inhibitors. rRNA immunoprecipitation (RNA-IP) of HeLa lysates with PKR-specific antibody and sequencing revealed that in the presence of DON or not, the kinase associated with numerous discrete sites on both the 18S and 28S rRNA molecules, a number of which contained double-stranded hairpins. These findings are consistent with a sentinel model whereby multiple PKR molecules basally associate with the ribosome positioning them to respond to ribotoxin-induced alterations in rRNA structure by dimerizing, autoactivating and, ultimately, evoking RSR.

Analysis of the sequences of internal transcribed spacers ITS1, ITS2 and the 5.8S ribosomal gene of species of the Amaranthus genus

Analysis of the sequence ITS1-5.8S-ITS2 in 11 samples of the amaranth species (Amaranthus caudatus, A. cruentus, A. hybridus, A. tricolor, A. paniculatus, A. hypohondriacus) was performed. It has been shown that the variability of the sequences of the intergenic spacers ITS1, ITS2 and 5.8S rRNA gene of the amaranth species analyzed is extremely low. A possible secondary structure of the 5.8S rRNA molecule was determined for the first time; three conservative motifs were identified. A single nucleotide substitution found in A. hybridus did not change the loop topology. In the sample of Celosia cristata taken as an external group, a four-nucleotide insertion in the 5'-end of the gene and a one-nucleotide deletion in the fourth hairpin not affecting the general topology of the 5.8S rRNA molecule were found.

Variation in secondary structure of the 16S rRNA molecule in cyanobacteria with implications for phylogenetic analysis.

The analysis of the phylogenetic signal in the sequence of the 16S rRNA gene in cyanobacteria and bacteria has been primarily limited to use of unweighted primary sequence in various cladistic analyses. Such analyses ignore the significance of secondary structure in the 16S rRNA molecule. Structure of this molecule is directly tied to its function, and consequently changes in primary sequence that lead to changes in the secondary structure theoretically have greater evolutionary significance than those changes that do not lead to structural changes. Very little analysis of the impact of primary sequence data on the secondary structure of the small subunit rRNA molecule in cyanobacteria has been reported. Herein is reported a secondary structure model of the full 16S rRNA sequence for Nostoc commune Vaucher ex Bornet et Flahault. We also assess the variability in secondary structure of six variable helices in 180 OTUs of cyanobacteria. Variations were minor in most instances, and the observed motifs were not only similar among these cyanobacteria but also were similar to Escherichia coli, a well-studied, representative non-photosynthetic bacterium. Helix 17 was the most variable helix in terms of structure and length of primary sequence. In order to maintain the motifs, we assumed the presence of non-canonical base pairings in the helices. Phylogenies using primary sequence data alone and using primary sequence data in conjunction with coding for secondary structure were very similar, although secondary structural considerations produced trees that more closely reflected the current ultrastructure-based modern taxonomy in the group.

Mechanisms of rDNA silencing and the Nucleolar Remodelling Complex (NoRC)

Protein synthesis in living cells requires functional ribosomes which are composed of ribosomal proteins and ribosomal RNA (rRNA) molecules. rRNA is transcribed from tandemly repeated ribosomal DNA (rDNA) which is organised into a nuclear compartment termed the nucleolus in S-phase cells. It is essential that rDNA transcription is properly regulated in order to meet the cell's requirements for ribosomes and hence protein synthesis without wasting metabolic energy. In the last twenty years many proteins involved in regulating this process have been identified, suggesting that most organisms contain multiple protein complexes that regulate rDNA packaging and transcription. Importantly, it has become clear that errors in the function of these proteins can permit aberrant cellular growth, including in several classes of cancer. In this review, I discuss the history of how protein complexes such as the Nucleolar Remodelling Complex (NoRC) were discovered, using examples from humans and from model research organisms from different biological groups. I will discuss recent discoveries of the critical roles of rDNA-binding complexes in nucleolar assembly, the widespread occurrence of regulatory non-coding RNAs which interact with these complexes, and the pathways which regulate rDNA transcription in response to cellular energy status. Finally, I will review the growing evidence that misregulation of rDNA transcription not only allows the growth of cancerous cells, but can trigger oncogenesis itself.

Indications for algae-degrading benthic microbial communities in deep-sea sediments along the Antarctic Polar Front

Phytoplankton blooms in surface waters of the oceans are known to influence the food web and impact microbial as well as zooplankton communities. Numerous studies have investigated the fate of phytoplankton-derived organic matter in surface waters and shelf sediments, however, little is known about the effect of sinking algal biomass on microbial communities in deep-sea sediments. Here, we analyzed sediments of four regions in the Southern Atlantic Ocean along the Antarctic Polar Front that had different exposures to phytoplankton bloom derived organic matter. We investigated the microbial communities in these sediments using high-throughput sequencing of 16S rRNA molecules to determine microorganisms that were active and catalyzed reporter deposition fluorescence in situ hybridization to infer their abundance and distribution. The sediments along the Antarctic Polar Front harbored microbial communities that were highly diverse and contained microbial clades that seem to preferably occur in regions of high primary productivity. We showed that organisms affiliated with the gammaproteobacterial clade NOR5/OM60, which is known from surface waters and coastal sediments, thrive in the deep-sea. Benthic deep-sea NOR5 were abundant, diverse, distinct from pelagic NOR5 and likely specialized on the degradation of phytoplankton-derived organic matter, occupying a similar niche as their pelagic relatives. Algal detritus seemed to not only fuel the benthic microbial communities of large areas in the deep-sea, but also to influence communities locally, as we found a peak in Flavobacteriaceae-related clades that also include degraders of algal biomass. The results strongly suggest that phytoplankton-derived organic matter was rapidly exported to the deep-sea, nourished distinct benthic microbial communities and seemed to be the main energy source for microbial life in the seafloor of vast abyssal regions along the Antarctic Polar Front.

Ribosomal Gene Polymorphism in Small Genomes: Analysis of Different 16S rRNA Sequences Expressed in the Honeybee Parasite Nosema ceranae (Microsporidia)

To date, few organisms have been shown to possess variable ribosomal RNA, otherwise considered a classic example of uniformity by concerted evolution. The polymorphism for the 16S rRNA in Nosema ceranae analysed here is striking as Microsporidia are intracellular parasites which have suffered a strong reduction in their genomes and cellular organization. Moreover, N. ceranae infects the honeybee Apis mellifera, and has been associated with the colony-loss phenomenon during the last decade. The variants of 16S rRNA include single nucleotide substitutions, one base insertion-deletion, plus a tetranucleotide indel. We show that different gene variants are expressed. The polymorphic sites tend to be located in particular regions of the rRNA molecule, and the comparison to the Escherichia coli 16S rRNA secondary structure indicates that most variations probably do not preclude ribosomal activity. The fact that the polymorphisms in such a minimal organism as N. ceranae are maintained in samples collected worldwide suggest that the existence of differently expressed rRNA may play an adaptive role in the microsporidian.

Indigenous and acquired modifications in the aminoglycoside binding sites of Pseudomonas aeruginosa rRNAs

Aminoglycoside antibiotics remain the drugs of choice for treatment of Pseudomonas aeruginosa infections, particularly for respiratory complications in cystic-fibrosis patients. Previous studies on other bacteria have shown that aminoglycosides have their primary target within the decoding region of 16S rRNA helix 44 with a secondary target in 23S rRNA helix 69. Here, we have mapped P. aeruginosa rRNAs using MALDI mass spectrometry and reverse transcriptase primer extension to identify nucleotide modifications that could influence aminoglycoside interactions. Helices 44 and 45 contain indigenous (housekeeping) modifications at m4Cm1402, m3U1498, m2G1516, m62A1518, and m62A1519; helix 69 is modified at m3Ψ1915, with m5U1939 and m5C1962 modification in adjacent sequences. All modifications were close to stoichiometric, with the exception of m3Ψ1915, where about 80% of rRNA molecules were methylated. The modification status of a virulent clinical strain expressing the acquired methyltransferase RmtD was altered in two important respects: RmtD stoichiometrically modified m7G1405 conferring high resistance to the aminoglycoside tobramycin and, in doing so, impeded one of the methylation reactions at C1402. Mapping the nucleotide methylations in P. aeruginosa rRNAs is an essential step toward understanding the architecture of the aminoglycoside binding sites and the rational design of improved drugs against this bacterial pathogen.

Transcriptome-Wide Mapping of 5-methylcytidine RNA Modifications in Bacteria, Archaea, and Yeast Reveals m5C within Archaeal mRNAs

The presence of 5-methylcytidine (m5C) in tRNA and rRNA molecules of a wide variety of organisms was first observed more than 40 years ago. However, detection of this modification was limited to specific, abundant, RNA species, due to the usage of low-throughput methods. To obtain a high resolution, systematic, and comprehensive transcriptome-wide overview of m5C across the three domains of life, we used bisulfite treatment on total RNA from both gram positive (B. subtilis) and gram negative (E. coli) bacteria, an archaeon (S. solfataricus) and a eukaryote (S. cerevisiae), followed by massively parallel sequencing. We were able to recover most previously documented m5C sites on rRNA in the four organisms, and identified several novel sites in yeast and archaeal rRNAs. Our analyses also allowed quantification of methylated m5C positions in 64 tRNAs in yeast and archaea, revealing stoichiometric differences between the methylation patterns of these organisms. Molecules of tRNAs in which m5C was absent were also discovered. Intriguingly, we detected m5C sites within archaeal mRNAs, and identified a consensus motif of AUCGANGU that directs methylation in S. solfataricus. Our results, which were validated using m5C-specific RNA immunoprecipitation, provide the first evidence for mRNA modifications in archaea, suggesting that this mode of post-transcriptional regulation extends beyond the eukaryotic domain.

Note: steps taken to optimise probe specificity and signal intensity prior to field validation of the MIDTAL (Microarray for the Detection of Toxic Algae)

A microarray for the detection of toxic algal species was developed in the European Union 7th Framework project MIDTAL. We initially tested all available fluorescence in situ hybridisation probes for toxic algae, which are normally designed to a length of 18 nt, and found that in most cases the signal was rather weak or all probes designed from the second half of the molecule were inaccessible in a microarray format because of secondary structure of the ribosomal RNA molecule We modified the length of the probes, the fragmentation of the rRNA, the stringency of the washing buffers and the length of the spacer molecules linking the probes to the glass surface of the microarray. Because of the secondary structure of the rRNA molecule, regions of the molecule can be difficult to access by the probes. Each of these modifications has improved probe accessibility and probe specificity to reduce false positives.

A New Generation of Food-Borne Pathogen Detection Based on Ribosomal RNA

Listeria and Salmonella detection assays for food and environmental surfaces that target ribosomal RNA (rRNA) have been developed. The large number of rRNA molecules in bacteria enabled the development of molecular assays that use enrichment times as short as 12 hours for Salmonella and 24 hours for Listeria. These assays run on a fully automated molecular pathogen detection system, which provides walk-away capability and produces 300 assay results in eight hours.

Autophagy Induction Is a Tor- and Tp53-Independent Cell Survival Response in a Zebrafish Model of Disrupted Ribosome Biogenesis

Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (ttis450), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome. The biochemical impacts of this lesion are decreased production of mature 18S rRNA molecules, activation of Tp53, and impaired ribosome biogenesis. In ttis450, the growth of the endodermal organs, eyes, brain, and craniofacial structures is severely arrested and autophagy is up-regulated, allowing intestinal epithelial cells to evade cell death. Inhibiting autophagy in ttis450 larvae markedly reduces their lifespan. Somewhat surprisingly, autophagy induction in ttis450 larvae is independent of the state of the Tor pathway and proceeds unabated in Tp53-mutant larvae. These data demonstrate that autophagy is a survival mechanism invoked in response to ribosomal stress. This response may be of relevance to therapeutic strategies aimed at killing cancer cells by targeting ribosome biogenesis. In certain contexts, these treatments may promote autophagy and contribute to cancer cells evading cell death.