Primer extension coupled with fragment analysis for rapid and quantitative evaluation of 5.8S rRNA isoforms

The ribosomal RNA 5.8S is one of the four rRNAs that constitute ribosomes. In human cells, like in all eukaryotes, it derives from the extensive processing of a long precursor containing the sequence of 18S, 5.8S and 28S rRNAs. It has been confirmed also in human cells the presence of three isoforms of 5.8S rRNA: one more abundant called 5.8S short, one called 5.8S long bearing 5 extra-nucleotides at its 5’ end and one 10 nucleotide shorter called 5.8S cropped. So far, little is known about 5.8S long specific role in cell biology and its function in human pathology. The lack of studies on the three 5.8S isoforms could be due to the techniques usually applied to study ribosome biogenesis, such as Northern blot with radioactively labelled probes, that require strict protective measures, and abundant and high-quality samples. To overcome this issue, we optimized a method that combines primer extension with a fluorescently labeled reverse primer designed on the 3’ of 5.8S rRNA sequence and fragment analysis. The resulting electropherogram shows the peaks corresponding to the three isoforms of 5.8S rRNA. The estimation of the area underneath the peaks allows to directly quantify the isoforms and to express their relative abundance. The relative abundance of 5.8S long and 5.8S short remains constant using scalar dilution of RNA and in samples subjected to partial degradation. 5.8S cropped abundance varies significantly in lower concentrate RNA samples. This method allows to analyze rapidly and safely the abundance of 5.8S rRNA isoforms in samples that have been so far considered not suitable such as poorly concentrated samples, RNA derived from frozen tissue or unique samples.

USE OF SEQUENCING METHODS FOR SPECIES IDENTIFICATION EXEMPLIFIED BY PHYLOGENETIC RELATIONSHIPS WITHIN GENUS HEDYSARUM L.

At the moment, a relevant objective in pharmacognosy, is the use of all kinds of the DNA analysis methods for identifying plant materials, detecting counterfeits, genetically modified crops and products.The aim of the research is to study the possibility of using molecular genetic research methods in the analysis of the genus Hedysarum L., for the identification of medicinal plant materials. This article presents the results of the application of molecular genetic research methods in the analysis of the genus Hedysarum L. in the flora of the North Caucasus.Materials and methods. The study material was the samples of the genus Hedysarum L. species collected in the North Caucasus: Hedysarum caucasicum M. Bieb. (in the fruiting phase in the territory of the Karachay-Cherkess Republic); Hedysarum grandiflorum Pall. (in the fruiting phase in the Volgograd region); Hedysarum daghestanicum Rupr. ex Boiss. (in the flowering phase in the Republic of Dagestan). Sequencing of the ITS1-5.8S-ITS2 marker region of gene 5.8S by the RNA ribosome was carried out according to the Sanger method on the AbiPrism 3130 genetic analyzer at the laboratory of biosystematics and cytology of Komarov Botanical Institute of the Russian Academy of Sciences.Results. Based on a comparative study of the marker region of the nuclear ribosomal gene 5.8S rRNA, marker nucleotide substitutions of Hedysarum caucasicum M. Bieb., Hedysarum daghestanicum Rupr. ex Boiss., Hedysarum grandiflorum Pall, have been identified. The most probable secondary structure of 5.8S rRNA has been constructed. It has been shown that based on the analysis performed, it is possible to predict additional raw material sources of mangiferin and other groups of xanthones using the molecular data exemplified by the Obscura section.Conclusion. Based on the data obtained, it can be concluded that the morphological classification of the genus Hedysarum L. can be confirmed within the Obscura section.

Role of Ribosomal RNA Released from Red Cells in Blood Coagulation in Zebrafish and Humans

Hemolytic disorders are characterized by hemolysis and are prone to thrombosis. Previously, it has been shown that the RNA released from damaged blood cells activates clotting. However, the nature of RNA released from hemolysis is still elusive. We found that after hemolysis, RBCs from both zebrafish and humans released 5.8S rRNA. This RNA activated coagulation in zebrafish and human plasmas. Using both natural and synthetic 5.8S rRNA and its truncated fragments, we found that the 3'-end 26 nucleotide-long RNA (3'-26 RNA) and its stem-loop secondary structure were necessary and sufficient for clotting activity. Corn trypsin inhibitor (CTI), a coagulation factor XII (FXII) inhibitor blocked 3'-26 RNA-mediated coagulation activation of both zebrafish and human plasma. CTI also inhibited zebrafish coagulation in vivo. 5.8S rRNA monoclonal antibody inhibited both 5.8S rRNA- and 3'-26 RNA-mediated zebrafish coagulation activity. Both 5.8S rRNA and 3'-26 RNA activates normal human plasma but did not activate FXII-deficient human plasma. Taken together, these results suggested that the activation of zebrafish plasma is via FXII-like protein. Since zebrafish has no FXII and hepatocyte growth factor activator (Hgfac) has sequence similarities to FXII, we knocked down the hgfac in adult zebrafish. We found that plasma from this knockdown fish does not respond to 3'-26 RNA. In conclusion, we identified 5.8S rRNA released in hemolysis activates clotting in human and zebrafish plasma. Only 3'-end 26 nucleotides of the 5.8S rRNA is needed for the clotting activity. Furthermore, we showed that fish Hgfac plays a role in 5.8S rRNA-mediated activation of coagulation.

Genomic constitution of diploid species of the genus Avena L. with A-genome according to the data of the next-generation sequencing (NGS)

For diploid (2x) species with the A-genome, as well as for hexaploid (6x) from the genus Avena, a locus-specific next-generation sequencing (NGS) of the sequence of the region of the internal transcribed spacer ITS1 and the beginning of the 5.8S rRNA gene was carried out on the Illumina platform. The high diversity and heterogeneity of the genomes of diploid species are shown. It was revealed that the genomes of modern diploid oat species are relatively far removed from the hexaploid species. It was found that A. canariensis occupies an isolated position among other diploid species, and also takes only an insignificant role in the formation of hexaploid genomes.

Molecular phylogenetic study of rare weed-field species of the genus Avena L.

A molecular phylogenetic study of weed-field species of the genus Avena L. using marker sequences ITS1–5.8S rRNA gene–ITS2 was undertaken. In addition, next-generation sequencing (NGS) was performed on the Illuminaplatform for the ITS1 sequence and the beginning of the gene 5.8S rRNA. Sanger sequencing results revealed the separateclade of microspecies with a good level of support and small level of difference between themselves. According to NGSsequencing data, the two most abundant subgenomes in terms of the number of sequences were identified. Among thecommon sequences of hexaploids, those associated with the C-genome were not found. The presence of unique ribotypeswas shown for A. persica and A. georgica.

Detection and Identification of Novel Genes from Fungi Isolated from Wastewater

The environment in Mosul city is very rich, containing a wide variety of microorganisms which have not been recognised for a long time. Five new fungal genes were identified and registered for the first time in the gene bank. These included Fusarium falciforme 2020-06-MIK-F1 genes for 5.8S rRNA with Accession no. LC555741, Nectriaceae sp. 2020-06-MIK-F2 genes for ITS1 with Accession no. LC555742, Trichoderma asperellum MIK3 genes for 5.8S rRNA with Accession no. LC575020, Penecillum sp. MIK4 genes for 5.8S rRNA with Accession no. LC575021, and Neurospora crassa MIK5 genes for 5.8S rRNA with Accession no. LC575022. These fungal genes were isolated from wastewater of Khosr river in Mosul city/ Iraq, which has many contamination sources.

Comparison of Two Real-Time PCR Assays Targeting Ribosomal Sequences for the Identification of Cystoisospora belli in Human Stool Samples

Cystoisospora (C.) belli is a coccidian parasite associated with acute or chronic gastroenteritis in immunocompromised patients. Dissatisfactory sensitivity of microscopy as the diagnostic standard approach has been described. Here, we comparatively evaluated two real-time PCRs targeting ribosomal RNA gene sequences of C. belli in stool in a test comparison without a reference standard applying latent class analysis. Therefore, 1000 stool samples from Ghanaian HIV (human immunodeficiency virus) patients (n = 905) as well as military returnees from the tropics (n = 95) were assessed by both assays in parallel. After the exclusion of 33 samples showing PCR inhibition, 29 and 33 positive results were recorded with the 5.8S rRNA gene/ITS-2 sequence PCR and the ITS-2 sequence PCR, respectively, resulting in an accuracy-adjusted prevalence of 3.2%. Nearly perfect agreement between both assays was indicated by Fleiss’ kappa of 0.933 with sensitivity and specificity of 92.8% and 100% as well as 100% and 99.8% for the 5.8S rRNA gene/ITS-2 sequence PCR and the ITS-2 sequence PCR, respectively. Both assays proved to be suitable for the diagnosis of C. belli in human stool samples with slightly better sensitivity of the ITS-2 sequence assay, while the 5.8S rRNA gene/ITS-2 sequence PCR may be considered for confirmatory testing.

Phosphorylation of PUF-A/PUM3 on Y259 modulates PUF-A stability and cell proliferation

Human PUF-A/PUM3 is a RNA and DNA binding protein participating in the nucleolar processing of 7S to 5.8S rRNA. The nucleolar localization of PUF-A redistributes to the nucleoplasm upon the exposure to genotoxic agents in cells. However, little is known regarding the roles of PUF-A in tumor progression. Phosphoprotein database analysis revealed that Y259 phosphorylation of PUF-A is the most prevalent residue modified. Here, we reported the importance of PUF-A’s phosphorylation on Y259 in tumorigenesis. PUF-A gene was knocked out by the Crispr/Cas9 method in human cervix epithelial HeLa cells. Loss of PUF-A in HeLa cells resulted in reduced clonogenic and lower transwell invasion capacity. Introduction of PUF-AY259F to PUF-A deficient HeLa cells was unable to restore colony formation. In addition, the unphosphorylated mutant of PUF-A, PUF-AY259F, attenuated PUF-A protein stability. Our results suggest the important role of Y259 phosphorylation of PUF-A in cell proliferation.

Acanthoparyphium shinanense n. sp. (Digenea: Echinostomatidae) from Experimental Chicks Infected with Metacercariae Encysted in Brackish Water Clams in the Republic of Korea

Acanthoparyphium shinanense n. sp. (Digenea: Echinostomatidae) is described from chicks experimentally infected with the metacercariae encysted in 2 brackish water clam species, Ruditapes philippinarum and Coecella chinensis, in the Republic of Korea. The metacercariae were round to oval, armed with 23 collar spines, and 0.216 (0.203-0.226) mm in diameter. From 5 chicks experimentally infected each with 200 metacercariae, 34 juvenile (5-day-old worms) and 104 adult flukes (7-day-old worms) were harvested from their small intestines, with the average worm recovery rate of 13.8%. The adult flukes were 3.18 (2.89-3.55) mm long and 0.68 (0.61-0.85) mm wide, with an elongated, posteriorly tapering body, and a prominent head collar armed with 23 collar spines arranged in a single uninterrupted row. The posterior testis of A. shinanense was longitudinally elongated, which is similar to Acanthoparyphium spinulosum Johnston, 1917 but unique from the other closely related species, including Acanthoparyphium tyosenense Yamaguti, 1939, Acanthoparyphium kurogamo Yamaguti, 1939, and Acanthoparyphium marilae Yamaguti, 1934. The eggs of A. shinanense were larger than those of A. spinulosum, and the anterior extent of 2 lateral groups of vitellaria was slightly more limited in A. shinanense than in A. spinulosum. Molecular analysis of nuclear and mitochondrial genes revealed low homology with A. spinulosum from USA (96.1% in 5.8S rRNA) and Ukraine (97.9% in 28S rRNA), Acanthoparyphium n. sp. from USA (98.0% in 28S rRNA), and Acanthoparyphium sp. from Australia, Kuwait, and New Zealand. Biological characteristics, including its first intermediate host and natural definitive hosts, as well as its zoonotic capability, should be elucidated.

A Novel Model for the RNase MRP-Induced Switch between the Formation of Different Forms of 5.8S rRNA

Processing of the RNA polymerase I pre-rRNA transcript into the mature 18S, 5.8S, and 25S rRNAs requires removing the “spacer” sequences. The canonical pathway for the removal of the ITS1 spacer involves cleavages at the 3′ end of 18S rRNA and at two sites inside ITS1. The process can generate either a long or a short 5.8S rRNA that differs in the number of ITS1 nucleotides retained at the 5.8S 5′ end. Here we document a novel pathway to the long 5.8S, which bypasses cleavage within ITS1. Instead, the entire ITS1 is degraded from its 5′ end by exonuclease Xrn1. Mutations in RNase MRP increase the accumulation of long relative to short 5.8S rRNA. Traditionally this is attributed to a decreased rate of RNase MRP cleavage at its target in ITS1, called A3. However, results from this work show that the MRP-induced switch between long and short 5.8S rRNA formation occurs even when the A3 site is deleted. Based on this and our published data, we propose that the link between RNase MRP and 5.8S 5′ end formation involves RNase MRP cleavage at unknown sites elsewhere in pre-rRNA or in RNA molecules other than pre-rRNA.