Nuclear RNA Research Articles

Hepatitis B virus RNAs co-opt ELAVL1 for stabilization and CRM1-dependent nuclear export.

Hepatitis B virus (HBV) chronically infects 296 million people worldwide, posing a major global health threat. Export of HBV RNAs from the nucleus to the cytoplasm is indispensable for viral protein translation and genome replication, however the mechanisms regulating this critical process remain largely elusive. Here, we identify a key host factor embryonic lethal, abnormal vision, Drosophila-like 1 (ELAVL1) that binds HBV RNAs and controls their nuclear export. Using an unbiased quantitative proteomics screen, we demonstrate direct binding of ELAVL1 to the HBV pregenomic RNA (pgRNA). ELAVL1 knockdown inhibits HBV RNAs posttranscriptional regulation and suppresses viral replication. Further mechanistic studies reveal ELAVL1 recruits the nuclear export receptor CRM1 through ANP32A and ANP32B to transport HBV RNAs to the cytoplasm via specific AU-rich elements, which can be targeted by a compound CMLD-2. Moreover, ELAVL1 protects HBV RNAs from DIS3+RRP6+ RNA exosome mediated nuclear RNA degradation. Notably, we find HBV core protein is dispensable for HBV RNA-CRM1 interaction and nuclear export. Our results unveil ELAVL1 as a crucial host factor that regulates HBV RNAs stability and trafficking. By orchestrating viral RNA nuclear export, ELAVL1 is indispensable for the HBV life cycle. Our study highlights a virus-host interaction that may be exploited as a new therapeutic target against chronic hepatitis B.

HRDE-2 drives small RNA specificity for the nuclear Argonaute protein HRDE-1

RNA interference (RNAi) is a conserved gene silencing process that exists in diverse organisms to protect genome integrity and regulate gene expression. In C. elegans, the majority of RNAi pathway proteins localize to perinuclear, phase-separated germ granules, which are comprised of sub-domains referred to as P granules, Mutator foci, Z granules, and SIMR foci. However, the protein components and function of the newly discovered SIMR foci are unknown. Here we demonstrate that HRDE-2 localizes to SIMR foci and interacts with the germline nuclear Argonaute HRDE-1 in its small RNA unbound state. In the absence of HRDE-2, HRDE-1 exclusively loads CSR-class 22G-RNAs rather than WAGO-class 22G-RNAs, resulting in inappropriate H3K9me3 deposition on CSR-target genes. Thus, our study demonstrates that the recruitment of unloaded HRDE-1 to germ granules, mediated by HRDE-2, is critical to ensure that the correct small RNAs are used to guide nuclear RNA silencing in the C. elegans germline.

USP4 regulates TUT1 ubiquitination status in concert with SART3

The ubiquitin system plays pivotal roles in diverse cellular processes, including signal transduction, transcription and translation, organelle quality control, and protein degradation. Recent investigations have revealed the regulatory influence of ubiquitin systems on RNA metabolism. Previously, we reported that the deubiquitinating enzyme, ubiquitin specific peptidase 15 (USP15), promotes deubiquitination of terminal uridylyl transferase 1 (TUT1), a key regulator within the U4/U6 spliceosome, thereby instigating significant alterations in global RNA splicing [1]. In this study, we report that ubiquitin specific peptidase 4 (USP4), a homologous protein to USP15, also exerts control over the ubiquitination status of TUT1. Analogous to USP15, the expression of USP4 results in a reduction of TUT1 ubiquitination. Furthermore, squamous cell carcinoma antigen recognized by T-cells 3 (SART3) collaborates in enhancing the deubiquitinating activity of USP4 towards TUT1. A crucial revelation is that USP4 orchestrates the subnuclear relocation of TUT1 from the nucleolus to the nucleoplasm and facilitates the stability of U6 small nuclear RNA (snRNA). Notably, USP4 has a more profound effect on TUT1 redistribution compared to USP15. Our findings suggest that USP4 intricately modulates the ubiquitination status of TUT1, thereby exerting pronounced effects on the spliceosome functions.

Frontiers and Challenges of Computing ncRNAs Biogenesis, Function and Modulation.

Non-coding RNAs (ncRNAs), generated from nonprotein coding DNA sequences, constitute 98-99% of the human genome. Non-coding RNAs encompass diverse functional classes, including microRNAs, small interfering RNAs, PIWI-interacting RNAs, small nuclear RNAs, small nucleolar RNAs, and long non-coding RNAs. With critical involvement in gene expression and regulation across various biological and physiopathological contexts, such as neuronal disorders, immune responses, cardiovascular diseases, and cancer, non-coding RNAs are emerging as disease biomarkers and therapeutic targets. In this review, after providing an overview of non-coding RNAs' role in cell homeostasis, we illustrate the potential and the challenges of state-of-the-art computational methods exploited to study non-coding RNAs biogenesis, function, and modulation. This can be done by directly targeting them with small molecules or by altering their expression by targeting the cellular engines underlying their biosynthesis. Drawing from applications, also taken from our work, we showcase the significance and role of computer simulations in uncovering fundamental facets of ncRNA mechanisms and modulation. This information may set the basis to advance gene modulation tools and therapeutic strategies to address unmet medical needs.

Nuclear and cytoplasmic specific RNA binding proteome enrichment and its changes upon ferroptosis induction

The key role of RNA-binding proteins (RBPs) in posttranscriptional regulation of gene expression is intimately tied to their subcellular localization. Here, we show a subcellular-specific RNA labeling method for efficient enrichment and deep profiling of nuclear and cytoplasmic RBPs. A total of 1221 nuclear RBPs and 1333 cytoplasmic RBPs were enriched and identified using nuclear/cytoplasm targeting enrichment probes, representing an increase of 54.4% and 85.7% compared with previous reports. The probes were further applied in the omics-level investigation of subcellular-specific RBP-RNA interactions upon ferroptosis induction. Interestingly, large-scale RBPs display enhanced interaction with RNAs in nucleus but reduced association with RNAs in cytoplasm during ferroptosis process. Furthermore, we discovered dozens of nucleoplasmic translocation candidate RBPs upon ferroptosis induction and validated representative ones by immunofluorescence imaging. The enrichment of Tricarboxylic acid cycle in the translocation candidate RBPs may provide insights for investigating their possible roles in ferroptosis induced metabolism dysregulation.

Light controls mesophyll-specific post-transcriptional splicing of photoregulatory genes by AtPRMT5

Light plays a central role in plant growth and development, providing an energy source and governing various aspects of plant morphology. Previous study showed that many polyadenylated full-length RNA molecules within the nucleus contain unspliced introns (post-transcriptionally spliced introns, PTS introns), which may play a role in rapidly responding to changes in environmental signals. However, the mechanism underlying post-transcriptional regulation during initial light exposure of young, etiolated seedlings remains elusive. In this study, we used FLEP-seq2, a Nanopore-based sequencing technique, to analyze nuclear RNAs in Arabidopsis (Arabidopsis thaliana) seedlings under different light conditions and found numerous light-responsive PTS introns. We also used single-nucleus RNA sequencing (snRNA-seq) to profile transcripts in single nucleus and investigate the distribution of light-responsive PTS introns across distinct cell types. We established that light-induced PTS introns are predominant in mesophyll cells during seedling de-etiolation following exposure of etiolated seedlings to light. We further demonstrated the involvement of the splicing-related factor A. thaliana PROTEIN ARGININE METHYLTRANSFERASE 5 (AtPRMT5), working in concert with the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a critical repressor of light signaling pathways. We showed that these two proteins orchestrate light-induced PTS events in mesophyll cells and facilitate chloroplast development, photosynthesis, and morphogenesis in response to ever-changing light conditions. These findings provide crucial insights into the intricate mechanisms underlying plant acclimation to light at the cell-type level.

Parasaturnius maurepasi n. gen. et n. comb. (Digena: Bunocotylidae) from the stomach of the silver mullet, Mugil curema (Perciformes: Mugilidae) in coastal lagoons of northern Yucatán, Mexico

Bunocotylid trematodes represent a group of 149 species with a rather complex taxonomic history. The current concept of the subfamily only includes three genera, Bunocotyle, Saturnius, and Robinia. Specimens of a bunocotylid were collected from the silver mullet, M. curema, from a coastal lagoon of Yucatán and identified as belonging to Saturnius. Further detailed morphological study revealed they corresponded to S. maurepasi, a species previously reported from the stripped mullet, Mugil cephalus in Mississippi, USA. Specimens were sequenced for the LSU of nuclear ribosomal RNA gene (28S) to test their phylogenetic position. We discovered that they do not belong in Saturnius since they nest as an independent lineage which is the sister taxa of a clade formed by Robinia, and Saturnius + Bunocotyle; additionally, the new genus exhibits high genetic divergence (10-12%) with respect to species allocated in the other bunocotylid genera. The species S. maurepasi was then transferred to the new genus as Parasaturnius maurepasin. gen., n. comb. that was created to accommodate it, and was redescribed based on newly sampled specimens.

Clinical value assessment for serum hsa_tsr013526 in the diagnosis of gastric carcinoma.

Gastric carcinoma (GC) is a malignant tumor that is detrimental to human health. Transfer RNA-derived small RNAs are a newly identified class of noncoding small RNAs with specific biological functions that are aberrantly expressed in cancer. The aim of this study was to investigate the potential of hsa_tsr013526 as a biomarker for GC. Quantitative real-time fluorescence polymerase chain reaction was used to detect the expression level of hsa_tsr013526. The molecular characteristics of hsa_tsr013526 were verified by agarose gel electrophoresis, Sanger sequencing, and separation of nuclear and cytoplasmic RNA fractions. By testing the receiver operating characteristic (ROC) curves, the diagnostic efficiency of GC using hsa_tsr013526 was determined. Finally, we predicted the downstream of hsa_tsr013526 using functional assays and bioinformatics analysis. Serum expression of hsa_tsr013526 was higher in GC patients than in healthy donors. Serum expression showed differential changes in GC patients, gastritis patients, and healthy donors. Chi-squared tests showed that high expression of hsa_tsr013526 was significantly correlated with T stage, lymphatic metastasis, and tumor node metastasis stage. ROC curve analysis indicated that GC patients could be discriminated from healthy donors or gastritis patients based on their serum levels of hsa_tsr013526. Furthermore, hsa_tsr013526 expression was significantly reduced in postoperative GC patients (p = .0016). High expression of hsa_tsr013526 promotes gastric cancer cell proliferation, invasion, and migration. Serum hsa_tsr013526 was stable and specific, and could be used for dynamic monitoring of GC patients. Therefore, hsa_tsr013526 may be a new biomarker for the diagnosis and postoperative monitoring of GC patients.

Human milk exosome-derived circDNAJB6 improves bronchopulmonary dysplasia model by promoting DNAJB6 gene transcription.

To verify the protective effect of circDNAJB6 on Bronchopulmonary dysplasia (BPD) cell and animal models and to explore the possible mechanism of its protective effect. The function of circDNAJB6 was investigated at the cell and animal levels. Nuclear and Cytoplasmic RNA extraction kits and fluorescence in situ hybridization (FISH) were used to explore the distribution of circDNAJB6 in cells, and the potential mechanism of circDNAJB6 was verified by q-PCR, luciferase assays and rescue experiments.CircDNAJB6 is abundant in breast milk exosomes. Overexpression of circDNAJB6 can ameliorate damage in BPD models caused by hyperoxia exposure in vivo and in vitro. Mechanistically, circDNAJB6 can target the downstream DNAJB6 gene and promote the transcription of DNAJB6, exertive a protective effect on the experimental BPD model. Our results showed that circDNAJB6 alleviated damage and inhibited the proliferation of alveolar epithelial cells in the BPD model by promoting transcription of parent gene DNAJB6. Human milk exosome-derived circDNAJB6 provides new directions for preventing and treating BPD.

Insights into the genome of the ‘Loco’ Concholepas concholepas (Gastropoda: Muricidae) from low-coverage short-read sequencing: genome size, ploidy, transposable elements, nuclear RNA gene operon, mitochondrial genome, and phylogenetic placement in the family Muricidae

BackgroundThe Peruvian ‘chanque’ or Chilean ‘loco’ Concholepas concholepas is an economically, ecologically, and culturally important muricid gastropod heavily exploited by artisanal fisheries in the temperate southeastern Pacific Ocean. In this study, we have profited from a set of bioinformatics tools to recover important biological information of C. concholepas from low-coverage short-read NGS datasets. Specifically, we calculated the size of the nuclear genome, ploidy, and estimated transposable elements content using an in silico k-mer approach, we discovered, annotated, and quantified those transposable elements, we assembled and annotated the 45S rDNA RNA operon and mitochondrial genome, and we confirmed the phylogenetic position of C. concholepas within the muricid subfamily Rapaninae based on translated protein coding genes.ResultsUsing a k-mer approach, the haploid genome size estimated for the predicted diploid genome of C. concholepas varied between 1.83 Gbp (with kmer = 24) and 2.32 Gbp (with kmer = 36). Between half and two thirds of the nuclear genome of C. concholepas was composed of transposable elements. The most common transposable elements were classified as Long Interspersed Nuclear Elements and Short Interspersed Nuclear Elements, which were more abundant than DNA transposons, simple repeats, and Long Terminal Repeats. Less abundant repeat elements included Helitron mobile elements, 45S rRNA DNA, and Satellite DNA, among a few others.The 45S rRNA DNA operon of C. concholepas that encodes for the ssrRNA, 5.8S rRNA, and lsrRNA genes was assembled into a single contig 8,090 bp long. The assembled mitochondrial genome of C. concholepas is 15,449 bp long and encodes 13 protein coding genes, two ribosomal genes, and 22 transfer RNAs.ConclusionThe information gained by this study will inform the assembly of a high quality nuclear genome for C. concholepas and will support bioprospecting and biomonitoring using environmental DNA to advance development of conservation and management plans in this overexploited marine snail.

The putative RNA helicase DDX1 associates with the nuclear RNA exosome and modulates RNA/DNA hybrids (R-loops)

The RNA exosome is a ribonuclease complex that mediates both RNA processing and degradation. This complex is evolutionarily conserved, ubiquitously expressed, and required for fundamental cellular functions, including rRNA processing. The RNA exosome plays roles in regulating gene expression and protecting the genome, including modulating the accumulation of RNA-DNA hybrids (R-loops). The function of the RNA exosome is facilitated by cofactors, such as the RNA helicase MTR4, which binds/remodels RNAs. Recently, missense mutations in RNA exosome subunit genes have been linked to neurological diseases. One possibility to explain why missense mutations in genes encoding RNA exosome subunits lead to neurological diseases is that the complex may interact with cell- or tissue-specific cofactors that are impacted by these changes. To begin addressing this question, we performed immunoprecipitation of the RNA exosome subunit, EXOSC3, in a neuronal cell line (N2A) followed by proteomic analyses to identify novel interactors. We identified the putative RNA helicase, DDX1, as an interactor. DDX1 plays roles in double-strand break repair, rRNA processing, and R-loop modulation. To explore the functional connections between EXOSC3 and DDX1, we examined the interaction following double-strand breaks, and analyzed changes in R-loops in N2A cells depleted for EXOSC3 or DDX1 by DNA/RNA immunoprecipitation followed by sequencing (DRIP-Seq). We find that EXOSC3 interaction with DDX1 is decreased in the presence of DNA damage and that loss of EXOSC3 or DDX1 alters R-loops. These results suggest EXOSC3 and DDX1 interact during events of cellular homeostasis and potentially suppress unscrupulous expression of genes promoting neuronal projection.

Resettlement of Eurytemora velox (Crustacea: Copepoda) in Europe, the Urals and Western Siberia

The recent distribution of the Ponto-Caspian calanoid copepod, Eurytemora velox, in Western and Eastern Europe has been well-documented; however, there are no studies on the genetic diversity of the recently discovered Western Siberian species population. To contribute towards filling this gap, genetic diversity and distribution were investigated for E. velox collected in the Urals and Western Siberia of the Russian Federation to compare with the European populations. In this study, 44 specimens were dissected for the morphological studies, and 22 specimens of E. velox from different geographical points were analyzed for mitochondrial cytochrome oxidase I (CO1), nuclear ITS1-ITS2 (nITS) and nuclear ribosomal RNA (18SrRNA) gene fragments. Analysis of the CO1 gene region showed that the studied populations from the Urals and Western Siberia differ significantly from European ones and represent a separate genetic line. However, the nuclear nITS and 18SrRNA genes, as well as the results of morphological analysis, did not show such isolation of the Trans-Ural populations from those in Europe. As in many studies on E. velox, we found atypical structural features of the fifth prosomal leg among females in the Ob Bay. Their share was 36% of the number of studied individuals. The CO1 shows that the divergence of genetic lines occurred approximately in the Middle Pleistocene, and the species itself is Paleogene–Neogene by origin. Based on these data, a different scenario of E. velox distribution or possible refuge survival is discussed.

PABPN1 loss-of-function causes APA-shift in oculopharyngeal muscular dystrophy

Alternative polyadenylation (APA) at the 3' UTR of transcripts contributes to the cell transcriptome. APA is suppressed by the nuclear RNA-binding protein PABPN1. Aging-associated reduced PABPN1 levels in skeletal muscles lead to muscle wasting. Muscle weakness in oculopharyngeal muscular dystrophy (OPMD) is caused by short alanine expansion in PABPN1 exon1. The expanded PABPN1 forms nuclear aggregates, an OPMD hallmark. Whether the expanded PABPN1 affects APA and how it contributes to muscle pathology is unresolved. To investigate these questions, we developed a procedure including RNA library preparation and a simple pipeline calculating the APA-shift ratio as a readout for PABPN1 activity. Comparing APA-shift results to previously published PAS utilization and APA-shift results, we validated this procedure. The procedure was then applied on the OPMD cell model and on RNA from OPMD muscles. APA-shift was genome-wide in the mouse OPMD model, primarily affecting muscle transcripts. In OPMD individuals, APA-shift was enriched with muscle transcripts. In an OPMD cell model APA-shift was not significant. APA-shift correlated with reduced expression levels of a subset of PABPN1 isoforms, whereas the expression of the expanded PABPN1 did not correlate with APA-shift. PABPN1 activity is not affected by the expression of expanded PABPN1, but rather by reduced PABPN1 expression levels. In muscles, PABPN1 activity initially affects muscle transcripts. We suggest that muscle weakness in OPMD is caused by PABPN1 loss-of-function leading to APA-shift that primarily affects in muscle transcripts.

7SK small nuclear RNA (Rn7SK) induces apoptosis through intrinsic and extrinsic pathways in human embryonic kidney cell line.

Rn7SK, a highly conserved small nuclear non-coding RNA, controls Polymerase II transcription machinery by activating of the Positive Transcriptional Elongation Factor b (P-TEFb). Apart from its role in transcriptional regulation, the potential functions of Rn7SK in cell apoptosis are poorly understood. In a previous study, we demonstrated that overexpression of 7SK induces apoptosis in HEK cells. However, it remains unclear whether 7SK-mediated apoptosis induction is exerted through the intrinsic or extrinsic pathways. Rn7SK was overexpressed in HEK 293T cell line using Lipofectamine 2000 reagent to investigate its potential apoptotic functions. The overexpression of Rn7SK resulted in reduced cell viability through the induction of apoptosis, as evidenced by MTT assay and Annexin V/PI staining. Concurrently, alterations in the expression levels of key apoptosis-related genes were observed, as determined by quantitative RT-PCR. Furthermore, Rn7SK overexpression led to a decrease in cell proliferation, as assessed by colony formation assay and growth curve analysis. This reduction was associated with downregulated expression of key proliferative-related genes. Additionally, the migration and invasion capabilities of cells were significantly inhibited upon upregulation of Rn7SK, as demonstrated by transwell assays. This study suggests the apoptotic role of 7SK through both intrinsic and extrinsic pathways, necessitating further investigation into its underlying mechanisms.

Sm complex assembly and 5' cap trimethylation promote selective processing of snRNAs by the 3' exonuclease TOE1.

Competing exonucleases that promote 3' end maturation or degradation direct quality control of small non-coding RNAs, but how these enzymes distinguish normal from aberrant RNAs is poorly understood. The Pontocerebellar Hypoplasia 7 (PCH7)-associated 3' exonuclease TOE1 promotes maturation of canonical small nuclear RNAs (snRNAs). Here, we demonstrate that TOE1 achieves specificity toward canonical snRNAs through their Sm complex assembly and cap trimethylation, two features that distinguish snRNAs undergoing correct biogenesis from other small non-coding RNAs. Indeed, disruption of Sm complex assembly via snRNA mutations or protein depletions obstructs snRNA processing by TOE1, and in vitro snRNA processing by TOE1 is stimulated by a trimethylated cap. An unstable snRNA variant that normally fails to undergo maturation becomes fully processed by TOE1 when its degenerate Sm binding motif is converted into a canonical one. Our findings uncover the molecular basis for how TOE1 distinguishes snRNAs from other small non-coding RNAs and explain how TOE1 promotes maturation specifically of canonical snRNAs undergoing proper processing.

Pleiotropic effects of different exonic nucleotide changes at the same position contribute to hemophilia B phenotypic variation

BackgroundThe disease-causing effects of genetic variations often depend on their location within a gene. Exonic changes generally lead to alterations in protein production, secretion, activity, or clearance. However, owing to the overlap between proteins and splicing codes, missense variants can also affect messenger RNA splicing, thus adding a layer of complexity and influencing disease phenotypes. ObjectivesTo extensively characterize a panel of 13 exonic variants in the F9 gene occurring at 6 different factor IX positions and associated with varying severities of hemophilia B (HB). MethodsComputational predictions, splicing analysis, and recombinant factor IX assays were exploited to characterize F9 variants. ResultsWe demonstrated that 5 (38%) of 13 selected F9 exonic variants have pleiotropic effects. Although bioinformatic approaches accurately classified effects, extensive experimental assays were required to elucidate and deepen the molecular mechanisms underlying the pleiotropic effects. Importantly, their characterization was instrumental in developing tailored RNA therapeutics based on engineered U7 small nuclear RNA to mask cryptic splice sites and compensatory U1 small nuclear RNA to enhance exon definition. ConclusionOverall, albeit a multitool bioinformatic approach suggested the molecular effects of multiple HB variants, the deep investigation of molecular mechanisms revealed insights into the HB phenotype–genotype relationship, enabling accurate classification of HB variants. Importantly, knowledge of molecular mechanisms allowed the development of tailored RNA therapeutics, which can also be translated to other genetic diseases.

Prognostic effect of programmed cell death ligand 1/programmed cell death 1 expression in cancer stem cells of human oral squamous cell carcinoma.

The relationship between cancer stem cells (CSCs) in oral squamous cell carcinoma (OSCC) and programmed cell death ligand 1 (PD-L1)/programmed cell death 1 (PD-1) remains unclear. Therefore, the present study aimed to clarify the association between the CD44v3high/CD24low immunophenotype of CSCs in OSCC and PD-L1/PD-1 co-expression, and to assess the prognostic effect of CSCs in terms of immune checkpoint molecules. Formalin-fixed, paraffin-embedded tissue samples and clinicopathological data from 168 patients with OSCC were retrospectively retrieved. Immunohistochemical staining and reverse transcription quantitative polymerase chain reaction were applied to a tissue microarray of the invasive front of each case. Semi-automated cell counting was used to assess CD44v3, CD24, PD-L1 and PD-1 expression by immunohistochemistry (IHC) using a digital image analysis program. Associations between immunological markers and clinicopathological variables were estimated. Patients with the CSC immunophenotype CD44v3high/CD24low, and patients with a high PD-L1/PD-1-positive cell density in the tumor parenchyma and stroma had significantly lower survival rates. Furthermore, patients with the CSC immunophenotype (CD44v3high/CD24low) and high PD-L1/PD-1 co-expression had even lower survival rates (P<0.01, log-rank test). Notably, there was a positive correlation between CD44v3 and PD-L1 expression (τ=0.1096, P=0.0366, Kendall rank correlation coefficient) and a negative correlation between CD24 and PD-1 expression (τ=-0.1387, P=0.0089, Kendall rank correlation coefficient). Additionally, the high CD44v3 expression group, as determined by IHC, exhibited significantly decreased expression of U2 small nuclear RNA auxiliary factor 1 (U2AF1) at the mRNA level compared with that in the low CD44v3 expression group (P<0.001, Mann-Whitney U test), and U2AF1 and PD-L1 mRNA expression exhibited a significant negative correlation (τ=-0.3948, P<0.001, Kendall rank correlation coefficient). In conclusion, CSCs in OSCC may evade host immune mechanisms and maintain CSC stemness via PD-L1/PD-1 co-expression, resulting in unfavorable clinical outcomes.

A New Species of Ganoderma (Ganodermataceae, Polyporales) from Southern China and Optimum Condition for Mycelia Production

The present study sought to propose Ganoderma guixiense sp. nov. as a new species based on phenotypic and genotypic evidence. Phylogenetic analyses were carried out based on the internal transcribed spacer (ITS), the large subunit of nuclear ribosomal RNA gene (nLSU), and the second subunit of RNA polymerase II (RPB2) sequence data. G. guixiense has been characterized by pileate basidiomata, long stipe, in addition to reddish-black zonate pileal surface. Basidiospores are broadly ellipsoid with one end tapering at maturity, and measuring 9–12.8 × 6.5–9.3 μm. Basidia are oval to subglobose. This study marks the first exploration of the biological characteristics of G. guixiense. The result indicated that the optimal medium of mycelial growth was observed on malt extract agar (MEA) and yeast extract peptone dextrose agar (YPD) while the optimal temperature was found to be 25–30 °C with pH range of 6–7.

Morphological and molecular characterization of the rust fungus, Phakopsora apoda causing leaf rust on Brachiaria grass in Rwanda

Brachiaria grass is one of the major forages that plays a key role in push pull technology. Different diseases including leaf rust caused by rust fungus, Phakopsora apoda have recently become an important challenge resulting in yield loss of Brachiaria grass in different countries including Rwanda. The objective of this study was to provide morphological and molecular characteristics of Phakopsora apoda causing Brachiaria leaf rust. Spores isolated from rusted Brachiaria leaves collected in five districts of Rwanda were analyzed. Samples were put in paper bags and left at room temperature for two days. Thereafter, spores were removed using brushes and put in eppendorf tubes, stored at 4oC in darkness for further characterization. For morphological characteristics, colour and measurements of spores were recorded using Optika B-350 microscope with installed camera and calibrated micrometer. Multigene analysis using rust specific primers and large subunit (LSU) of nuclear ribosomal RNA was performed for molecular characterization. The results indicated that the colour of spores was yellowish or brownish while the shape was ellipsoidal or circular with average measurements of 25.1µm and 16.8µm for length and width respectively. Primers amplified the size of DNA between 1291bp and 1381bp and the size between 874bp to 882bp for rust specific primers and LSU respectively. The identity of the sequence was 96% with e-value of 0, sequence coverage of 60% for rust primer and the sequence identity was 94.97% to 95.37%, sequence coverage of 99% to 100% and e-value of 0 for LSU primer. All sequences recovered from five isolates of leaf rust matched to the genebank accession number MG461668.1 for both rust specific and LSU sequences. The results of this study provide useful information to develop effective options for leaf rust disease management in Rwanda. Key words: Internal transcribed spacer, large subunit, nuclear ribosomal RNA, Phakopsora apoda, sequence, spore.

The Functions of N6-Methyladenosine in Nuclear RNAs.

N6-methyladenosine (m6A) is one of the most common modifications in both eukaryotic and prokaryotic mRNAs. It has been experimentally confirmed that m6A methylation is involved in the regulation of stability and translation of various mRNAs. Until recently, the majority of m6A-related studies have been focused on the cytoplasmic functions of this modification. Here, we review new data on the role of m6A in several key biological processes taking place in the cell nucleus, such as transcription, chromatin organization, splicing, nuclear-cytoplasmic transport, and R-loop metabolism. Based on analysis of these data, we suggest that m6A methylation of nuclear RNAs is another level of gene expression regulation which, together with DNA methylation and histone modifications, controls chromatin structure and functioning in various biological contexts.