rRNA Fragments Research Articles

El efecto de una solución superoxidada sobre el uroepitelio de la vejiga de ratas

Aim: The present study aimed to identify the changes on the bladder mucosa in rats after intravesical infusion of Super-oxidized solution (SOS) (Mycrodacyn60® Mexico).
 Design: Eighteen Wistar Albino rats weighing 250-300 g were randomly categorized into three groups. Rats in Group 1 were infused with a single dose of 1.5 ml of 0.9% saline solution (SS) whereas those in group 2 were administered a single dose of 1.5 ml pH neutral SOS. Control rats in Group 3 did not undergo any procedure.
 Results: The absence of bacteria was verified before and after the administration of the solution, performing urine cultures and amplifying the 16S rRNA fragment by PCR and histopathological examinations for each bladder. The absence of bacterial DNA in the uroepithelium was demonstrated, histopathological examination revealed, that irrigation with SOS, in histological observation, no structural alterations were identified in the urothelial mucosa.
 Limitations: It was not possible to analyze the effect of SOS absorption indamage to the uroepithelium secondary to traumatic infection or injury. The effect of irrigation at different times and volumes with SOS was also not analyzed.
 Originality: It is known that SOS is useful as disinfectant in skin and serosa bacterial infections, lacking toxicity and it is effective against Escherichia coli. The use of superoxide solutions in cavities such as the bladder, has not been investigated yet.
 Conclusion: The pH-neutral SOS infused transurethral in the bladder of rats has no adverse effects on the uroepithelium, which was supported by histopathological evidence.

The effect of a super-oxidized solution on the bladder uroepithelium of rats

Aim: The present study aimed to identify the changes on the bladder mucosa in rats after intravesical infusion of Super-oxidized solution (SOS) (Mycrodacyn60® Mexico). Design: Eighteen Wistar Albino rats weighing 250-300 g were randomly categorized into three groups. Rats in Group 1 were infused with a single dose of 1.5 ml of 0.9% saline solution (SS) whereas those in group 2 were administered a single dose of 1.5 ml pH neutral SOS. Control rats in Group 3 did not undergo any procedure. Results: The absence of bacteria was verified before and after the administration of the solution, performing urine cultures and amplifying the 16S rRNA fragment by PCR and histopathological examinations for each bladder. The absence of bacterial DNA in the uroepithelium was demonstrated, histopathological examination revealed, that irrigation with SOS, in histological observation, no structural alterations were identified in the urothelial mucosa. Limitations: It was not possible to analyze the effect of SOS absorption indamage to the uroepithelium secondary to traumatic infection or injury. The effect of irrigation at different times and volumes with SOS was also not analyzed. Originality: It is known that SOS is useful as disinfectant in skin and serosa bacterial infections, lacking toxicity and it is effective against Escherichia coli. The use of superoxide solutions in cavities such as the bladder, has not been investigated yet. Conclusion: The pH-neutral SOS infused transurethral in the bladder of rats has no adverse effects on the uroepithelium, which was supported by histopathological evidence.

A Practical Approach to Identifying Processed White Meat of Guinea Fowl, Rabbit, and Selected Fish Species Using End-Point PCR.

Among the foodstuff, most often adulterated are white meat and meat products as well as fish and fish products. For this reason, we evaluated in practice the possibilities of identifying selected species of white meat, i.e., guinea fowl and rabbit as well as four fish species, namely, pollock, hake, sole, and panga, in thermally treated samples. The aim was to check whether the previously published in the scientific literature species-specific primers allows for the identification of processed meat using the end-point PCR technique. To identify the six species, the short sequence fragments (from 130 to 255 bp) of 12S rRNA, COX3, mitochondrial ATP synthase Fo subunit 6 (ATP6) gene, pantophysin (Pan I) gene, 5S rRNA gene, and microsatellite markers (locus: Phy01-KUL) were selected. Stability and specificity of the six pair primers were evaluated on cooked and autoclaved meat, and commercially processed food samples such as rabbit and guinea pâtés, ready-made baby food, and breaded, fried, and deep-frozen fish products. The method proved to be useful for the authentication of severely processed food products against fraudulent species substitution and mislabelling and this approach may be an alternative to more advanced and more expensive PCR techniques.

Molecular phylogeny and revised classification of the Buccinoidea (Neogastropoda)

AbstractThe superfamily Buccinoidea is distributed across the oceans of the world from the Arctic Ocean to the Antarctic and from intertidal to abyssal depths. It encompasses 3351 recent species in 337 genera. The latest taxonomic account recognized eight full families. For the first time, the monophyly of the superfamily and the relationships among the families are tested with molecular data supplemented by anatomical and radula data. Five genetic markers were used: fragments of mitochondrial COI, 16S rRNA, 12S rRNA and nuclear Histone 3 (H3) and 28S rRNA genes (for 225 species of 117 genera). Our analysis recovered Buccinoidea monophyletic in Bayesian analyses. The relationships between the formerly recognized families and subfamilies are drastically revised and a new classification of the superfamily is here proposed, now including 20 taxa of family rank and 23 subfamilies. Five new families (Chauvetiidae, Dolicholatiridae, Eosiphonidae, Prodotiidae and Retimohniidae) and one subfamily of Nassariidae (Tomliniinae) are described. Austrosiphonidae and Tudiclidae are resurrected from synonymy and employed in a new taxonomical extension. All but 40 recent genera are reclassified. Our results demonstrate that anatomy is rather uniform within the superfamily. With exceptions, the rather uniform radular morphology alone does not allow the allocation of genera to a particular family without additional molecular data.

First Report of 16SrII-V Phytoplasma Associated with Green Manure Soybean (Glycine max L.) in Taiwan.

QING PI DOU, a local variety of soybean (Glycine max (L.) Merrill) with small seed size, is primarily cultivated in the southern region of Taiwan. Due to the advantage of high germination rate, fast growth and high nitrogen fixation capacity, QING PI DOU has widely used as green manure in rotation with rice to increase soil fertility in Taiwan. In the summer of 2020, phytoplasma-induced disease symptoms were observed in QING PI DOU with 23% (18/78) disease incidence in Yunlin County, Taiwan. These plants exhibited severe disease symptoms such as little leaf, yellowing, phyllody, virescence, and witches' broom compared to healthy plants. Leaf samples of the symptomatic plants were subsequently collected and examined through transmission electron microscopy (TEM), PCR, and western blotting analyses. The ultrathin sections of the diseased QING PI DOU were double-stained with uranyl acetate and lead citrate. The typical phytoplasma-like pleomorphic bodies were observed in sieve elements of leaf veins by TEM. To investigate the association of phytoplasma with the diseased QING PI DOU, total DNA extracted by the Plant Genomic DNA Purification Kit (DP022, Genemark, Taiwan) was examined by nested PCR using the phytoplasma universal primer pair P1/P7 followed by R16F2n/R16R2 (Lee et al. 1993). The 1.2 kb PCR product specific for 16S ribosomal RNA (16S rRNA) gene was only amplified from symptomatic plants but not from healthy plants. BLAST analysis demonstrated that the sequence (accession no. MW393690) of amplified DNA fragment of 16S rRNA is identical to that of GenBank accession no. NZ_AMWZ01000008 (complement [31109 to 32640]) of peanut witches' broom (PnWB) phytoplasma, a 'Candidatus phytoplasma aurantifolia'-related strain (Firrao et al. 2004). Further analysis on the virtual RFLP pattern of MW393690 generated by iPhyClassifier confirmed that the phytoplasma identified in the diseased QING PI DOU can be classified into the 16SrII-V subgroup. Samples examined by nested PCR were further selected for total cell extracts preparation and characterized by western blotting using the polyclonal antibody raised against the immunodominant membrane protein (Imp) of PnWB phytoplasma (Chien et al. 2020). An expected signal of 19 kDa specific for Imp was only detected in symptomatic plants but not in healthy plants. Moreover, the PCR products encoding SAP11 and phyllogen, the virulence factors responsible for phytoplasma-induced witches' broom and phyllody symptoms (Namba 2019), were also amplified from symptomatic QING PI DOU by PCR using the primer pairs 5'-ATGGCTCCCGAAAAAAATGATAAAGG-3'/5'-TTTTTTAGAATCATCAGGCTTTTTAG-3' (0.28 kb) and 5'-ATGGATCCAAAACTTCCAGAAACT-3'/5'-GTTTTTTTCATCATTTAAATCAT-3' (0.27 kb), respectively. Further analysis by BLAST revealed that SAP11 and phyllogen identified in symptomatic QING PI DOU are identical with those of PnWB phytoplasma. To the best of our knowledge, this report is the first to describe phytoplasma-associated soybean (Glycine max L.) witches' broom disease in green manure soybean in Taiwan.

Novel rRNA-depletion methods for total RNA sequencing and ribosome profiling developed for avian species

Deep sequencing of RNAs has greatly aided the study of the transcriptome, enabling comprehensive gene expression profiling and the identification of novel transcripts. While messenger RNAs (mRNAs) are of the greatest interest in gene expression studies as they encode for proteins, mRNAs make up only 3 to 5% of total RNAs, with the majority comprising ribosomal RNAs (rRNAs). Therefore, applications of deep sequencing to RNA face the challenge of how to efficiently enrich mRNA species prior to library construction. Traditional methods extract mRNAs using oligo-dT primers targeting the poly-A tail on mRNAs; however, this approach is not comprehensive as it does not capture mRNAs lacking the poly-A tail or other long non-coding RNAs that we may be interested in. Alternative mRNA enrichment methods deplete rRNAs, but such approaches require species-specific probes and the commercially available kits are costly and have only been developed for a limited number of model organisms. Here, we describe a quick, cost-effective method for depleting rRNAs using custom-designed oligos, using chickens as an example species for probe design. With this optimized protocol, we have not only removed the rRNAs from total RNAs for RNA-seq library construction but also depleted rRNA fragments from ribosome-protected fragments for ribosome profiling. Currently, this is the only rRNA depletion-based method for avian species; this method thus provides a valuable resource for both the scientific community and the poultry industry.

Angiostrongylus vasorum in foxes (Vulpes vulpes) and wolves (Canis lupus italicus) from Abruzzo region, Italy

In Europe wildlife animals such as the red fox (Vulpes vulpes) are considered the main reservoir for Angiostrongylus vasorum as well as a potential threat for domestic dog infection. Though this parasite is endemic in fox populations, data on A. vasorum infection in wolves (Canis lupus italicus) are still scant, having only recently been described in Northwestern Spain, in Italy, in Croatia and in Slovakia.Based on the rising number of cases of canine lungworm infection in Central Italy (Abruzzo region), the aim of the present study was to investigate the infection by A. vasorum in fox and wolf populations sharing the same geographical area of dogs.From October 2008 to November 2019, A. vasorum specimens were collected, through routine post-mortem examination, from 56 carcasses (44 foxes and 12 wolves). Adult parasites were searched for in the right side of the heart and in pulmonary artery of all carcasses. First stage of larvae (L1) was searched in faeces using the Baermann technique and in lungs by tissue impressions. Overall, 230 adult specimens were collected and identified on a morphological basis. To confirm the morphological identification, 4 adult specimens (n = 3 from fox, n = 1 from wolf) were molecularly identified as A. vasorum by amplification of partial fragment of nuclear 18S rRNA (~1700 bp) genes.The anatomo-pathological and parasitological examinations indicated the presence of A. vasorum in 33 foxes (75%) and in 8 wolves (66.7%). The level of prevalence of infested wolves was higher than the previous one reported in other European countries. Interestingly, the prevalence of infection in foxes herein recorded was higher than that described in dogs (8.9%) living in the same geographical area. This result may confirm the hypothesis that the spread of canine angiostrongylosis is linked to fox populations infection.

HDL-small RNA Export, Transport, and Functional Delivery in Atherosclerosis.

This review highlights recent advances on the mechanisms and impact of HDL-small non-coding RNAs (sRNA) on intercellular communication in atherosclerosis. Studies demonstrate that HDL-microRNAs (miRNA) are significantly altered in atherosclerotic cardiovascular disease (ASCVD), and are responsive to diet, obesity, and diabetes. Immune cells, pancreatic beta cells, and neurons are shown to export miRNAs to HDL. In turn, HDL can deliver functional miRNAs to recipient hepatocytes and endothelial cells regulating adhesion molecule expression, cytokines, and angiogenesis. With high-throughput sRNA sequencing, we now appreciate the full sRNA signature on circulating HDL, including the transport of rRNA and tRNA-derived fragments. Strikingly, HDL were highly enriched with exogenous microbial sRNAs. HDL transport a diverse set of host and non-host sRNAs that are altered in cardiometabolic diseases. Given the bioactivity of these sRNAs, they likely contribute to cellular communication within atherosclerotic lesions, and are potential disease biomarkers and therapeutic targets.

A novel fragmented mitochondrial genome in the protist pathogen Toxoplasma gondii and related tissue coccidia.

Mitochondrial genome content and structure vary widely across the eukaryotic tree of life, with protists displaying extreme examples. Apicomplexan and dinoflagellate protists have evolved highly reduced mitochondrial genome sequences, mtDNA, consisting of only three cytochrome genes and fragmented rRNA genes. Here, we report the independent evolution of fragmented cytochrome genes in Toxoplasma and related tissue coccidia and evolution of a novel genome architecture consisting minimally of 21 sequence blocks (SBs) totaling 5.9 kb that exist as nonrandom concatemers. Single-molecule Nanopore reads consisting entirely of SBs ranging from 0.1 to 23.6 kb reveal both whole and fragmented cytochrome genes. Full-length cytochrome transcripts including a divergent coxIII are detected. The topology of the mitochondrial genome remains an enigma. Analysis of a cob point mutation reveals that homoplasmy of SBs is maintained. Tissue coccidia are important pathogens of man and animals, and the mitochondrion represents an important therapeutic target. The mtDNA sequence has been elucidated, but a definitive genome architecture remains elusive.

Metazoan diversity and seasonality through eDNA metabarcoding at a Mediterranean long-term ecological research site

Abstract Metabarcoding of environmental DNA has provided striking insights into marine microbial diversity. With this approach, we assessed the diversity of metazoan assemblages and their temporal variations at the Long-Term Ecological Research site MareChiara (LTER-MC) in the Gulf of Naples (Mediterranean Sea). The Illumina sequencing of the V4-18S rRNA fragment from 48 surface samples collected from 2011 to 2013 produced a total of 5 011 047 marine metazoan reads. The normalized dataset was generally dominated by copepods (60.3%), followed by annelids (34.7%) mostly represented by the invasive benthic polychaete Hydroides elegans. Non-copepod holoplankton was mainly represented by siphonophores, rotifers, and appendicularians, with occasional mass occurrences of jellyfish. The rest of meroplankton (mainly molluscs, annelids, and anthozoans) showed a high diversity, covering all 11 zooplankton phyla, from Porifera to Craniata. A high number of copepod genera were identified, with seasonal recurrence matching patterns observed in 30 years of studies in the Gulf of Naples. Despite limitations related to the molecular marker resolution and reference dataset, the study provided valuable insights into diversity and seasonal patterns of the whole metazoan assemblage, expanding the knowledge on rare or hardly identifiable taxa and confirming DNA metabarcoding as a powerful approach to be integrated at LTER sites.

Molecular detection of Rickettsia sp. cf. Rickettsia monacensis in Ixodes sp. cf. Ixodes affinis collected from white-tailed deer in Campeche, Mexico.

Deer encompass a group of large-sized vertebrates that serve as hosts for a wide variety of ectoparasites, mainly ticks. In Mexico, ticks have relevance as vectors of pathogenic microorganisms, and 20 species of hard ticks are associated with four species of deer, although only a single study has been conducted to detect bacterial agents associated with ticks from deer in the country. In February, 2019 three white-tailed deers (Odocoileus virginianus) were hunted from the locality of Chiná from the municipality of Campeche, Mexico. The sampled deers were parasitized by 26 ticks belonged to three species: Amblyomma mixtum (5♀, 1♂), Amblyomma ovale (2♀, 1♂), and Ixodes sp. cf. Ixodes affinis (15♀, 2♂). Specimens were screened individually for Anaplasma, Borrelia, Ehrlichia, and Rickettsia DNA by the amplification of several fragments of 16S rRNA, gltA, 17-kDa, and flaB genes. This study report for the first time the presence of Rickettsia sp. cf. Rickettsia monacensis in Ixodes sp. cf. Ixodes affinis in Mexico.

Molecular-genetic identification of microorganism strains isolated from activated sludge based on an analysis of nucleotide sequences of the 16s rRNA gene

A molecular-genetic identification of four bacterial strains isolated from activated sludge of urban wastewater treatment plants (Ulan-Ude) and the industrial enterprise OJSC “Selenginsky Pulp and Paper Mill” (Selenginsk) was carried out. Bacterial strains were identified by a capillary sequencer ABI 3130XL Genetic Analyzer (Applied Biosystems) using 16S primers 27F and 1492R at the Genomics Collective Use Center of the Institute of Chemical Biology and Fundamental Medicine, SB RAS, Novosibirsk. The results were obtained using the method of determination of the direct nucleotide sequence of a 16S rRNA fragment followed by comparison of the nucleotide identity with the sequences deposited in the international database GenBank. Bacterial strains isolated from activated sludge were identified according to the GenBank database: strain B 1.1 corresponds to Paenibacillus dendritiformis strain P411 (similarity 99.93%), strains B 1.2 and B 1.3 correspond to Bacillus licheniformis strain PB399 (similarity 86 and 100%, respectively), strain P 1.1 corresponds to the Paenibacillus polymyxa strain ISSDS-85 (similarity 99.86%). The biochemical properties of the identified strains were determined: amylolytic, proteolytic and lipolytic activity; the ability to ferment carbohydrates in Hiss’ nutrient medium; the ability to form ammonia, urea and nitrate reduction. The bacterial strains isolated from activated sludge may be promising for the destruction of wastewater pollutants. On their basis, it is planned to create a consortium of microorganisms for the destruction of protein and fatty pollutants in wastewater.

Sticky problems: extraction of nucleic acids from molluscs

Traditional molecular methods and omics-techniques across molluscan taxonomy increasingly inform biology of Mollusca. Recovery of DNA and RNA for such studies is challenged by common biological properties of the highly diverse molluscs. Molluscan biomineralization, adhesive structures and mucus involve polyphenolic proteins and mucopolysaccharides that hinder DNA extraction or copurify to inhibit enzyme-catalysed molecular procedures. DNA extraction methods that employ the detergent hexadecyltrimethylammoniumbromide (CTAB) to remove these contaminants importantly facilitate molecular-level study of molluscs. Molluscan pigments may stain DNA samples and interfere with spectrophotometry, necessitating gel electrophoresis or fluorometry for accurate quantification. RNA can reliably be extracted but the ‘hidden break’ in 28S rRNA of molluscs (like most protostomes) causes 18S and 28S rRNA fragments to co-migrate electrophoretically. This challenges the standard quality control based on the ratio of 18S and 28S rRNA, developed for deuterostome animals. High-AT content in molluscan rRNA prevents the effective purification of polyadenylated mRNA. Awareness of these matters aids the continuous expansion of molecular malacology, enabling work also with museum specimens and next-generation sequencing, with the latter imposing unprecedented demands on DNA quality. Alternative methods to extract nucleic acids from molluscs are available from literature and, importantly, from communications with others who study the molecular biology of molluscs. This article is part of the Theo Murphy meeting issue ‘Molluscan genomics: broad insights and future directions for a neglected phylum’.

Biodiversity of prokaryotic microorganisms in whey tofu from Lembang, Sumedang and Garut West Java

Tofu is one of the products processed from soybeans that contain nutrients for the body. Tofu from Lembang, Tofu from Sumedang and Tofu from Garut are three types tofu that are produced in different places. Types of microorganisms found in whey can influence the flavor of tofu. This research was conducted to find out the type of microorganisms contained in whey Tofu from Lembang, Sumedang and Garut through metagenom approach. Through this approach, the microbial species in an environment can be known, even without the need to cultivate the microorganism. Whey samples filtered by cellulose acetate membrane of 0.2 µm. Total DNA was isolated by the Klijn method, and used as a template to obtain a fragment of 16S rRNA with ± 400 bp long using touchdown PCR. Amplicons obtained then separated again by DGGE analysis. Optimization of DGGE was made with 8% gel and a denaturant range of 30%-80%. The microorganisms contained in whey Tofu from Lembang have closer homology to the Bacterium EM, microorganisms contained in Tofu from Sumedang were closer with Acinobacter sp. and Uncultured Bacterium, while microorganisms contained in Tofu from Garut were closer to Uncultured bacterium.

Computational meta-analysis of ribosomal RNA fragments: potential targets and interaction mechanisms.

The most abundant cellular RNA species, ribosomal RNA (rRNA), appears to be a source of massive amounts of non-randomly generated fragments. We found rRNA fragments (rRFs) in immunoprecipitated Argonaute (Ago-IP) complexes in human and mouse cells and in small RNA sequencing datasets. In human Ago1-IP, guanine-rich rRFs were preferentially cut in single-stranded regions of mature rRNAs between pyrimidines and adenosine, and non-randomly paired with cellular transcripts in crosslinked chimeras. Numerous identical rRFs were found in the cytoplasm and nucleus in mouse Ago2-IP. We report specific interaction motifs enriched in rRF-target pairs. Locations of such motifs on rRFs were compatible with the Ago structural features and patterns of the Ago-RNA crosslinking in both species. Strikingly, many of these motifs may bind to double-stranded regions on target RNAs, suggesting a potential pathway for regulating translation by unwinding mRNAs. Occurring on either end of rRFs and matching intronic, untranslated or coding regions in targets, such interaction sites extend the concept of microRNA seed regions. Targeting both borders of certain short introns, rRFs may be involved in their biogenesis or function, facilitated by Ago. Frequently dismissed as noise, rRFs are poised to greatly enrich the known functional spectrum of small RNA regulation.

Spiroplasma Isolated From Third-Generation Laboratory Colony Ixodes persulcatus Ticks.

Spiroplasma are vertically-transmitted endosymbionts of ticks and other arthropods. Field-collected Ixodes persulcatus have been reported to harbour Spiroplasma, but nothing is known about their persistence during laboratory colonisation of this tick species. We successfully isolated Spiroplasma from internal organs of 6/10 unfed adult ticks, belonging to the third generation of an I. persulcatus laboratory colony, into tick cell culture. We screened a further 51 adult male and female ticks from the same colony for presence of Spiroplasma by genus-specific PCR amplification of fragments of the 16S rRNA and rpoB genes; 100% of these ticks were infected and the 16S rRNA sequence showed 99.8% similarity to that of a previously-published Spiroplasma isolated from field-collected I. persulcatus. Our study shows that Spiroplasma endosymbionts persist at high prevalence in colonised I. persulcatus through at least three generations, and confirms the usefulness of tick cell lines for isolation and cultivation of this bacterium.

Impact of DNA extraction methods on 16S rRNA-based profiling of bacterial communities in cheese

Numerous factors associated with sample preparation, DNA extraction, primer choice, sequencing platform and data analysis can affect the accuracy of 16S rRNA sequencing results. The DNA extraction method is considered critical for the success of sequencing as it can be the source of considerable variations in the analysis of the microbiome. In this study, the impact of various DNA extraction methods on the results of analysis of bacterial communities in cheese was evaluated. DNA was isolated from Mozzarella as a model cheese using optimized bead-based homogenization followed by different extraction procedures. Five commercial kits and two open-formula DNA extraction protocols were evaluated for amplicon sequencing of a 16S rRNA fragment of ~1460 bp. In addition, model cheese samples artificially contaminated by defined concentrations of Listeria monocytogenes and Escherichia coli, as representatives of Gram positive and Gram negative bacteria, were analysed. Six out of seven DNA extraction procedures were found to be able to provide amplifiable bacterial DNA suitable for 16S rRNA sequence analysis, but individual extraction procedures led to variable results. In particular, lysis supported with bead-beating led to a higher proportion of G+ bacteria in relative abundance profiles, probably because of the more efficient cell wall disruption. Artificially added bacterial species were reliably detected with a quantitative response. The results demonstrated a risk in comparing the data on bacterial communities in cheese when different DNA extraction protocols are used and highlighted the need to choose a standardized approach when comparison across multiple sequencing runs is required.

Ribo-ODDR: oligo design pipeline for experiment-specific rRNA depletion in Ribo-seq.

MotivationRibosome Profiling (Ribo-seq) has revolutionized the study of RNA translation by providing information on ribosome positions across all translated RNAs with nucleotide-resolution. Yet several technical limitations restrict the sequencing depth of such experiments, the most common of which is the overabundance of rRNA fragments. Various strategies can be employed to tackle this issue, including the use of commercial rRNA depletion kits. However, as they are designed for more standardized RNAseq experiments, they may perform suboptimally in Ribo-seq. In order to overcome this, it is possible to use custom biotinylated oligos complementary to the most abundant rRNA fragments, however currently no computational framework exists to aid the design of optimal oligos.ResultsHere, we first show that a major confounding issue is that the rRNA fragments generated via Ribo-seq vary significantly with differing experimental conditions, suggesting that a ‘one-size-fits-all’ approach may be inefficient. Therefore we developed Ribo-ODDR, an oligo design pipeline integrated with a user-friendly interface that assists in oligo selection for efficient experiment-specific rRNA depletion. Ribo-ODDR uses preliminary data to identify the most abundant rRNA fragments, and calculates the rRNA depletion efficiency of potential oligos. We experimentally show that Ribo-ODDR designed oligos outperform commercially available kits and lead to a significant increase in rRNA depletion in Ribo-seq.Availability and implementationRibo-ODDR is freely accessible at https://github.com/fallerlab/Ribo-ODDR.Supplementary informationSupplementary data are available at Bioinformatics online.

Recognition of non-CpG repeats in Alu and ribosomal RNAs by the Z-RNA binding domain of ADAR1 induces A-Z junctions

Adenosine-to-inosine (A-to-I) editing of eukaryotic cellular RNAs is essential for protection against auto-immune disorders. Editing is carried out by ADAR1, whose innate immune response-specific cytoplasmic isoform possesses a Z-DNA binding domain (Zα) of unknown function. Zα also binds to CpG repeats in RNA, which are a hallmark of Z-RNA formation. Unexpectedly, Zα has been predicted — and in some cases even shown — to bind to specific regions within mRNA and rRNA devoid of such repeats. Here, we use NMR, circular dichroism, and other biophysical approaches to demonstrate and characterize the binding of Zα to mRNA and rRNA fragments. Our results reveal a broad range of RNA sequences that bind to Zα and adopt Z-RNA conformations. Binding is accompanied by destabilization of neighboring A-form regions which is similar in character to what has been observed for B-Z-DNA junctions. The binding of Zα to non-CpG sequences is specific, cooperative and occurs with an affinity in the low micromolar range. This work allows us to propose a model for how Zα could influence the RNA binding specificity of ADAR1.

Reduced rDNA transcription diminishes skeletal muscle ribosomal capacity and protein synthesis in cancer cachexia.

Muscle wasting in cancer is associated with deficits in protein synthesis, yet, the mechanisms underlying this anabolic impairment remain poorly understood. The capacity for protein synthesis is mainly determined by the abundance of muscle ribosomes, which is in turn regulated by transcription of the ribosomal (r)RNA genes (rDNA). In this study, we investigated whether muscle loss in a preclinical model of ovarian cancer is associated with a reduction in ribosomal capacity and was a consequence of impaired rDNA transcription. Tumor bearing resulted in a significant loss in gastrocnemius muscle weight and protein synthesis capacity, and was consistent with a significant reduction in rDNA transcription and ribosomal capacity. Despite the induction of the ribophagy receptor NUFIP1 mRNA and the loss of NUFIP1 protein, in vitro studies revealed that while inhibition of autophagy rescued NUFIP1, it did not prevent the loss of rRNA. Electrophoretic analysis of rRNA fragmentation from both in vivo and in vitro models showed no evidence of endonucleolytic cleavage, suggesting that rRNA degradation may not play a major role in modulating muscle ribosome abundance. Our results indicate that in this model of ovarian cancer-induced cachexia, the ability of skeletal muscle to synthesize protein is compromised by a reduction in rDNA transcription and consequently a lower ribosomal capacity. Thus, impaired ribosomal production appears to play a key role in the anabolic deficits associated with muscle wasting in cancer cachexia.