RNA Primer Research Articles

DNA primase large subunit is an essential plant gene for geminiviruses, putatively priming viral ss-DNA replication.

The family of Geminiviridae consists of more than 500 circular single-stranded (ss) DNA viral species that can infect numerous dicot and monocot plants. Geminiviruses replicate their genome in the nucleus of a plant cell, taking advantage of the host's DNA replication machinery. For converting their DNA into double-stranded DNA, and subsequent replication, these viruses rely on host DNA polymerases. However, the priming of the very first step of this process, i.e. the conversion of incoming circular ssDNA into a dsDNA molecule, has remained elusive for almost 30 years. In this study, sequencing of melon (Cucumis melo) accession K18 carrying the Tomato leaf curl New Delhi virus (ToLCNDV) recessive resistance quantitative trait locus (QTL) in chromosome 11, and analyses of DNA sequence data from 100 melon genomes, showed a conservation of a shared mutation in the DNA Primase Large subunit (PRiL) of all accessions that exhibited resistance upon a challenge with ToLCNDV. Silencing of (native) Nicotiana benthamiana PriL and subsequent challenging with three different geminiviruses showed a severe reduction in titers of all three viruses, altogether emphasizing an important role of PRiL in geminiviral replication. A model is presented explaining the role of PriL during initiation of geminiviral DNA replication, i.e. as a regulatory subunit of primase that generates an RNA primer at the onset of DNA replication in analogy to DNA Primase-mediated initiation of DNA replication in all living organisms.

Identification of potential biomarkers for colorectal cancer by clinical database analysis and Kaplan-Meier curves analysis.

This study aimed to explore critical genes as potential biomarkers for the diagnosis and prognosis of colorectal cancer (CRC) for clinical utility. To identify and screen candidate genes involved in CRC carcinogenesis and disease progression, we downloaded microarray datasets GSE89076, GSE73360, and GSE32323 from the GEO database identified differentially expressed genes (DEGs), and performed a functional enrichment analysis. A protein-protein interaction network was constructed, and correlated module analysis was performed using STRING and Cytoscape. The Kaplan-Meier survival curve shows the survival of the hub genes. The expression of cyclin-dependent kinase (CDK1), cyclin B1 (CCNB1), and PCNA in tissues and changes in tumor grade were analyzed. A total of 329 DEGs were identified, including 264 upregulated and 65 downregulated genes. The functions and pathways of DEGs include the mitotic cell cycle, poly(A) RNA binding replication, ATP binding, DNA replication, ribosome biogenesis in eukaryotes, and RNA transport. Forty-seven Hub genes were identified, and biological process analysis showed that these genes were mainly enriched in cell cycle and DNA replication. Patients with mutations in CDK1, PCNA, and CCNB1 had poorer survival rates. CDK1, PCNA, and CCNB1 were significantly overexpressed in the tumor tissues. The expression of CDK1 and CCNB1 gradually decreased with increasing tumor grade. CDK1, CCNB1, and PCNA can be used as potential markers for the diagnosis and prognosis of CRC. These genes are overexpressed in colon cancer tissues and are associated with low survival rates in CRC patients.

Single-molecule dynamics of ribonuclease H enzymes in Bacillus subtilis.

Transcription and replication are fundamental processes for gene expression during which RNA-DNA hybrids (such as R-loops, single-ribonucleotide errors, RNA primers, ribopatches, and complementary RNA:DNA) may form. When RNA-DNA hybrids are not resolved, replication fork stress can occur leading to compromised genome integrity and an increase in cell mutation rates. Ribonuclease (RNase) H family enzymes contribute to genome maintenance by cleaving the RNA portion of RNA-DNA hybrid structures. Recent work has deepened our understanding of how RNA-DNA hybrids form and what could occur if they are not resolved. However, how these enzymes are recruited to the sites of RNA-DNA hybrids and the individual contributions of each enzyme remain unclear. Here, we use live-cell single-molecule imaging and super-resolution microscopy techniques to study the dynamics of the two RNase H enzymes in Bacillus subtilis. We observe changes in the single-molecule dynamics and localization patterns of each RNase H enzyme upon arresting transcription and inhibiting replication. Our results show that inhibition of transcription and replication both affect the fraction of enzymes existing in a functional binding state, as well as the localization of the enzymes at the replication fork. Our work provides insight into the specific functions of RNase H enzymes for different types of RNA-DNA hybrids in B. subtilis.

Enzymatic Synthesis of High-Density RNA Microarrays.

Oligonucleotide microarrays are used to investigate the interactome of nucleic acids. DNA microarrays are commercially available, whereas equivalent RNA microarrays are not. This protocol describes a method to convert DNA microarrays of any density and complexity into RNA microarrays using only readily available materials and reagents. This simple conversion protocol will facilitate the accessibility of RNA microarrays to a wide range of researchers. In addition to general considerations for the design of a template DNA microarray, this procedure describes the experimental steps of hybridization of an RNA primer to the immobilized DNA, followed by its covalent attachment via psoralen-mediated photocrosslinking. The subsequent enzymatic processing steps comprise the extension of the primer with T7 RNA polymerase to generate complementary RNA, and finally the removal of the DNA template with TURBO DNase. Beyond the conversion process, we also describe approaches to detect the RNA product either by internal labeling with fluorescently labeled NTPs or via hybridization to the product strand, a step that can then be complemented by an RNase H assay to confirm the nature of the product. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Conversion of a DNA microarray to an RNA microarray Alternate Protocol: Detection of RNA via incorporation of Cy3-UTP Support Protocol 1: Detection of RNA via hybridization Support Protocol 2: RNase H assay.

Cystic neutrophilic granulomatous mastitis: sensitivity and specificity of 16s rRNA and Sanger sequencing for Corynebacterium spp

AimsCystic neutrophilic granulomatous mastitis (CNGM) is a subtype of granulomatous mastitis (GM) associated with Corynebacterium spp infection. We aimed to analyse the prevalence of Corynebacteria in CNGM and non-CNGM cases.MethodsBreast...

Transcriptomics Analysis of the Toxicological Impact of Enrofloxacin in an Aquatic Environment on the Chinese Mitten Crab (Eriocheir sinensis).

Enrofloxacin is an important antimicrobial drug that is widely used in aquaculture. Enrofloxacin residues can have negative effects on aquatic environments and animals. The toxicological effects of different concentrations of enrofloxacin residues in cultured water on Chinese mitten crabs (Eriocheir sinensis) were compared. A histological analysis of the E. sinensis hepatopancreas demonstrated that the hepatopancreas was damaged by the different enrofloxacin residue concentrations. The hepatopancreas transcriptome results revealed that 1245 genes were upregulated and that 1298 genes were downregulated in the low-concentration enrofloxacin residue group. In the high-concentration enrofloxacin residue group, 380 genes were upregulated, and 529 genes were downregulated. The enrofloxacin residues led to differentially expressed genes related to the immune system and metabolic processes in the hepatopancreas of the Chinese mitten crab, such as the genes for alkaline phosphatase, NF-kappa B inhibitor alpha, alpha-amylase, and beta-galactosidase-like. The gene ontology terms "biological process" and "molecular function" were enriched in the carboxylic acid metabolic process, DNA replication, the synthesis of RNA primers, the transmembrane transporter activity, the hydrolase activity, and the oxidoreductase activity. A Kyoto Encyclopedia of Genes and Genomes pathway analysis determined that the immune and metabolic signal transduction pathways were significantly enriched. Furthermore, the nonspecific immune enzyme (alkaline phosphatase) and the metabolic enzyme system played a role in the enrofloxacin metabolism in the E. sinensis hepatopancreas. These findings helped us to further understand the basis of the toxicological effects of enrofloxacin residues on river crabs and provided valuable information for the better utilization of enrofloxacin in aquatic water environments.

Fork-Remodeling Helicase Rad5 Preferentially Reverses Replication Forks with Gaps in the Leading Strand

DNA damage bypass pathways promote the replication of damaged DNA when replication forks stall at sites of DNA damage. Template switching is a DNA damage bypass pathway in which fork-reversal helicases convert stalled replication forks into four-way DNA junctions called chicken foot intermediates, which are subsequently extended by replicative DNA polymerases. In yeast, fork-reversal is carried out by the Rad5 helicase using an unknown mechanism. To better understand the mechanism of Rad5 and its specificity for different fork DNA substrates, we used a FRET-based assay to observe fork reversal in real time. We examined the ability of Rad5 to bind and catalyze the reversal of various fork DNA substrates in the presence of short gaps in the leading or lagging strand as well as in the presence or absence of RPA and RNA primers in the lagging strand. We found that Rad5 preferentially reverses fork DNA substrates with short gaps (10 to 30 nt.) in the leading strand. Thus, Rad5 preferentially reverses fork DNA substrates that form chicken foot intermediates with 5′ overhangs that can be extended by replicative DNA polymerases during the subsequent steps of template switching.

Flap Endonuclease 1 Endonucleolytically Processes RNA to Resolve R-Loops through DNA Base Excision Repair.

Flap endonuclease 1 (FEN1) is an essential enzyme that removes RNA primers and base lesions during DNA lagging strand maturation and long-patch base excision repair (BER). It plays a crucial role in maintaining genome stability and integrity. FEN1 is also implicated in RNA processing and biogenesis. A recent study from our group has shown that FEN1 is involved in trinucleotide repeat deletion by processing the RNA strand in R-loops through BER, further suggesting that the enzyme can modulate genome stability by facilitating the resolution of R-loops. However, it remains unknown how FEN1 can process RNA to resolve an R-loop. In this study, we examined the FEN1 cleavage activity on the RNA:DNA hybrid intermediates generated during DNA lagging strand processing and BER in R-loops. We found that both human and yeast FEN1 efficiently cleaved an RNA flap in the intermediates using its endonuclease activity. We further demonstrated that FEN1 was recruited to R-loops in normal human fibroblasts and senataxin-deficient (AOA2) fibroblasts, and its R-loop recruitment was significantly increased by oxidative DNA damage. We showed that FEN1 specifically employed its endonucleolytic cleavage activity to remove the RNA strand in an R-loop during BER. We found that FEN1 coordinated its DNA and RNA endonucleolytic cleavage activity with the 3'-5' exonuclease of APE1 to resolve the R-loop. Our results further suggest that FEN1 employed its unique tracking mechanism to endonucleolytically cleave the RNA strand in an R-loop by coordinating with other BER enzymes and cofactors during BER. Our study provides the first evidence that FEN1 endonucleolytic cleavage can result in the resolution of R-loops via the BER pathway, thereby maintaining genome integrity.

A Nested-PCR assay for detection of Cryptosporidium spp. in cattle in Sulawesi Island, Indonesia

Cryptosporidium spp. is a protozoan parasite that cause cryptosporidiosis, a gastroenteric disease in wide range of animals and humans. Cryptosporidiosis affects significant economic loss in livestock production due to poor management practices, leading to faecal contamination. The parasite can be transmitted by ingestion of oocysts which have high survival ability in various type of environment and resistant to water chlorination treatment. In this study, we aimed to investigate the presence of Cryptosporidium spp. in various faecal samples of cattle (n=24, aged 1-2 years old) obtained from several farms in Sulawesi. Following faecal DNA extraction, we performed a nested-PCR assay using validated-Cryptosporidium 18S ribosomal RNA (18S rRNA) primers, resulting a size of amplified DNA approximately 770 bps. A total four samples (16.7%) were regarded as positive for Cryptosporidium spp., including samples obtained from farms in South Sulawesi (n=2), Central Sulawesi (n=1) and Gorontalo (n=1). This result confirms the risk of cryptosporidiosis not only among cattle in Sulawesi but also potential transfer of this parasite to humans especially from contaminated water and food. Therefore, early detection of the spread of oocysts is also crucial for monitoring animal health including cattle as production animals, human health, and environment.

Human DNA polymerase α has a strong mutagenic potential at the initial steps of DNA synthesis.

DNA polymerase α (Polα) is essential for DNA replication initiation and makes a notable contribution to genome mutagenesis. The activity and fidelity of Polα during the early steps of DNA replication have not been well studied. Here we show that at the beginning of DNA synthesis, when extending the RNA primer received from primase, Polα is more mutagenic than during the later DNA elongation steps. Kinetic and binding studies revealed substantially higher activity and affinity to the template:primer when Polα interacts with ribonucleotides of a chimeric RNA-DNA primer. Polα activity greatly varies during first six steps of DNA synthesis, and the bias in the rates of correct and incorrect dNTP incorporation leads to impaired fidelity, especially upon the second step of RNA primer extension. Furthermore, increased activity and stability of Polα/template:primer complexes containing RNA-DNA primers result in higher efficiency of mismatch extension.

Trypanosoma brucei Mitochondrial DNA Polymerase POLIB Contains a Novel Polymerase Domain Insertion That Confers Dominant Exonuclease Activity.

Trypanosoma brucei and related parasites contain an unusual catenated mitochondrial genome known as kinetoplast DNA (kDNA) composed of maxicircles and minicircles. The kDNA structure and replication mechanism are divergent and essential for parasite survival. POLIB is one of three Family A DNA polymerases independently essential to maintain the kDNA network. However, the division of labor among the paralogs, particularly which might be a replicative, proofreading enzyme, remains enigmatic. De novo modeling of POLIB suggested a structure that is divergent from all other Family A polymerases, in which the thumb subdomain contains a 369 amino acid insertion with homology to DEDDh DnaQ family 3'-5' exonucleases. Here we demonstrate recombinant POLIB 3'-5' exonuclease prefers DNA vs RNA substrates and degrades single- and double-stranded DNA nonprocessively. Exonuclease activity prevails over polymerase activity on DNA substrates at pH 8.0, while DNA primer extension is favored at pH 6.0. Mutations that ablate POLIB polymerase activity slow the exonuclease rate suggesting crosstalk between the domains. We show that POLIB extends an RNA primer more efficiently than a DNA primer in the presence of dNTPs but does not incorporate rNTPs efficiently using either primer. Immunoprecipitation of Pol I-like paralogs from T. brucei corroborates the pH selectivity and RNA primer preferences of POLIB and revealed that the other paralogs efficiently extend a DNA primer. The enzymatic properties of POLIB suggest this paralog is not a replicative kDNA polymerase, and the noncanonical polymerase domain provides another example of exquisite diversity among DNA polymerases for specialized function.

Mechanistic investigation of human maturation of Okazaki fragments reveals slow kinetics

The final steps of lagging strand synthesis induce maturation of Okazaki fragments via removal of the RNA primers and ligation. Iterative cycles between Polymerase δ (Polδ) and Flap endonuclease-1 (FEN1) remove the primer, with an intermediary nick structure generated for each cycle. Here, we show that human Polδ is inefficient in releasing the nick product from FEN1, resulting in non-processive and remarkably slow RNA removal. Ligase 1 (Lig1) can release the nick from FEN1 and actively drive the reaction toward ligation. These mechanisms are coordinated by PCNA, which encircles DNA, and dynamically recruits Polδ, FEN1, and Lig1 to compete for their substrates. Our findings call for investigating additional pathways that may accelerate RNA removal in human cells, such as RNA pre-removal by RNase Hs, which, as demonstrated herein, enhances the maturation rate ~10-fold. They also suggest that FEN1 may attenuate the various activities of Polδ during DNA repair and recombination.

Hyperbaric Oxygen Treatment in Spinal Cord Injury Recovery: Profiling Long Noncoding RNAs.

A functional, transcriptome, and long noncoding RNAs (lncRNAs) expression analysis in the spinal cord of mice after hyperbaric oxygen (HBO) treatment. We aimed to explore the mechanism by which HBO treats spinal cord injury (SCI) at the level of lncRNAs. Immense amounts of research have established that HBO treatment promotes the recovery of neurological function after SCI. The mechanism of action remains to be clarified. High-throughput RNA sequencing, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were used to profile lncRNA expression and analyze biological function in the spinal cords of mice from sham-operated, SCI, and HBO-treated groups. The differential expression of lncRNA between the groups was assessed using real-time quantitative polymerase chain reaction. Differential expression across 577 lncRNAs was identified among the three groups. GO analysis showed that free ubiquitin chain polymerization, ubiquitin homeostasis, DNA replication, synthesis of RNA primer, single-stranded telomeric DNA binding, and alpha-amylase activity were significantly enriched. Kyoto Encyclopedia of Genes and Genomes enrichment analysis displayed that vitamin B6 metabolism, one carbon pool by folate, DNA replication, lysine degradation, beta-alanine metabolism, fanconi anemia pathway, and Notch signal pathway were the main pathways with enrichment significance. LncRNAs NONMMUT 092674.1, NONMMUT042986.2, and NONMMUT018850.2 showed significantly different expression between the SCI and the other two groups (P<0.05, <0.01). This study is the first to determine the expression profiles of lncRNAs in the injured spinal cord after HBO treatment. We identified several important dysregulated lncRNAs in this setting. These results help us better understand the mechanism by which HBO treats SCI and provide new potential therapeutic targets for SCI.

CRISPR/LbCas12a-mediated targeted mutation of Gracilariopsis lemaneiformis (Rhodophyta).

CRISPR/LbCas12a-mediated targeted mutation of Gracilariopsis lemaneiformis (Rhodophyta).

Translocation pause of remdesivir-containing primer/template RNA duplex within SARS-CoV-2's RNA polymerase complexes.

The mechanism of remdesivir incorporation into the RNA primer by the RNA-dependent RNA polymerase (RdRp) of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains to be fully established at the molecular level. Here, we compare molecular dynamics (MD) simulations after incorporation of either remdesivir monophosphate (RMP) or adenosine monophosphate (AMP). We find that the Mg2+-pyrophosphate (PPi) binds more tightly to the polymerase when the added RMP is at the third primer position than in the AMP added complex. The increased affinity of Mg2+-PPi to the RMP-added primer/template (P/T) RNA duplex complex introduces a new hydrogen bond of a substituted cyano group in RMP with the K593 sidechain. The new interactions disrupt a switching mechanism of a hydrogen bond network that is essential for translocation of the P/T duplex product and for opening of a vacant NTP-binding site necessary for next primer extension. Furthermore, steric interactions between the sidechain of S861 and the 1'-cyano group of RMP at position i+3 hinders translocation of RMP to the i + 4 position, where i labels the insertion site. These findings are particularly valuable to guide the design of more effective inhibitors of SARS-CoV-2 RNA polymerase.

Selective RNA Labeling by RNA-Compatible Type II Restriction Endonuclease and RNA-Extending DNA Polymerase.

RNAs not only offer valuable information regarding our bodies but also regulate cellular functions, allowing for their specific manipulations to be extensively explored for many different biological and clinical applications. In particular, rather than temporary hybridization, permanent labeling is often required to introduce functional tags to target RNAs; however, direct RNA labeling has been revealed to be challenging, as native RNAs possess unmodifiable chemical moieties or indefinable dummy sequences at the ends of their strands. In this work, we demonstrate the combinatorial use of RNA-compatible restriction endonucleases (REs) and RNA-extending polymerases for sequence-specific RNA cleavage and subsequent RNA functionalization. Upon the introduction of complementary DNAs to target RNAs, Type II REs, such as AvrII and AvaII, could precisely cut the recognition site in the RNA-DNA heteroduplexes with exceptionally high efficiency. Subsequently, the 3′ ends of the cleaved RNAs were selectively and effectively modified when Therminator DNA polymerase template-dependently extended the RNA primers with a variety of modified nucleotides. Based on this two-step RNA labeling, only the target RNA could be chemically labeled with the desired moieties, such as bioconjugation tags or fluorophores, even in a mixture of various RNAs, demonstrating the potential for efficient and direct RNA modifications.

Molecular detection of disease-causing agent infecting Malvastrum coromandelianum L. in Bahawalpur District Punjab Pakistan

Weeds are common ancestors of modern crops. They are critical source of genes for resistance to diseases, pests and stresses such as drought and extreme environmental conditions. The wild relatives led to improve resistance to wheat curl mite, late blight in potato and to grassy stunt disease in rice. Agricultural pathogens are infecting economically important cash crops and weeds as well. Malvastrum coromandelianum is highly invasive weed specie of family Malvaceae. It is distributed all over the world in all climatic condition of tropical, sub-tropical and temperate region. The Malvaceae family is economically important as cotton (Gossipium hirsutem) belongs to it. M. coromandelianum has ethnobotanical importance and is extensively applied in the treatment of different diseases in south Punjab of Pakistan. It has been observed that there is a specific disease that affects the leaves of M. coromandelianum. In this study, we investigated the disease-causing agents (viruses and bacteria) at molecular level. Molecular investigation includes DNA isolation of unknown pathogens, verification through agarose gel electrophoresis, for identification of bacterial pathogens, 16s RNA primers were applied, and for viral pathogen using specific primers and RCA (rolling circle amplification) product as template. PCR was carried out for the amplification of full-length genomes. Manifestation of 1.4 kb bands showed the presence of the alpha and beta satellite of begomoviruse. Two types of begomoviruses, Malvestrum yellow vain younnan virus (MaYVYV) and Malvastrum yellow vain virus (MaYVV) was confirmed in this weed. The intermediate host Bemisia tabaci is the most common for the begomovirus transmission in crops and weeds. Intercrops and trap-crops techniques decrease the rate of whitefly population. Cultural methods, use of chemicals and biocontrol are useful practices to eliminate whitefly from field.

Quantification of Circular RNAs Using Digital Droplet PCR

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 water-in-oil droplets, and the PCR occurs in the individual droplets. The dd-PCR has several advantages over conventional real-time qPCR, including increased accuracy in detecting low-abundance targets, omitting reference genes for quantification, eliminating technical replicates for samples, and showing high resilience to inhibitors in the samples. Recently, dd-PCR has become one of the most popular methods for accurately quantifying target DNA or RNA for gene expression analysis and diagnostics. Circular RNAs (circRNAs) are a large family of recently discovered covalently closed RNA molecules lacking 5' and 3' ends. They have been shown to regulate gene expression by acting as sponges for RNA-binding proteins and microRNAs. Furthermore, circRNAs are secreted into body fluids, and their resistance to exonucleases makes them serve as biomarkers for disease diagnosis. This article aims to show how to perform divergent primer design, RNA extraction, cDNA synthesis, and dd-PCR analysis to accurately quantify specific circular RNA (circRNA) levels in cells. In conclusion, we demonstrate the precise quantification of circRNAs using dd-PCR.

Quantification of Circular RNAs Using Digital Droplet PCR.

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 water-in-oil droplets, and the PCR occurs in the individual droplets. The dd-PCR has several advantages over conventional real-time qPCR, including increased accuracy in detecting low-abundance targets, omitting reference genes for quantification, eliminating technical replicates for samples, and showing high resilience to inhibitors in the samples. Recently, dd-PCR has become one of the most popular methods for accurately quantifying target DNA or RNA for gene expression analysis and diagnostics. Circular RNAs (circRNAs) are a large family of recently discovered covalently closed RNA molecules lacking 5' and 3' ends. They have been shown to regulate gene expression by acting as sponges for RNA-binding proteins and microRNAs. Furthermore, circRNAs are secreted into body fluids, and their resistance to exonucleases makes them serve as biomarkers for disease diagnosis. This article aims to show how to perform divergent primer design, RNA extraction, cDNA synthesis, and dd-PCR analysis to accurately quantify specific circular RNA (circRNA) levels in cells. In conclusion, we demonstrate the precise quantification of circRNAs using dd-PCR.

Multiscale characterization reveals oligomerization dependent phase separation of primer-independent RNA polymerase nsp8 from SARS-CoV-2

RNA replication and transcription machinery is an important drug target for fighting against coronavirus. Non-structure protein nsp8 was proposed harboring primase activity. However, the RNA primer synthesis mechanism of nsp8 is still largely unknown. Here, we purified dimer and tetramer forms of SARS-CoV-2 nsp8. Combined with dynamic light scattering, small-angle neutron scattering and thermo-stability analysis, we found that both dimer and tetramer become loosened and destabilized with decreasing salt concentration, and the dimer form is more stable than the tetramer form. Further investigation showed that nsp8 dimer and tetramer can undergo phase separation but exhibit different phase separation behaviors. Nsp8 dimer can form liquid-like droplets in the buffer with a low concentration of NaCl; phase separation of nsp8 tetramer depends on the assistance of RNA. Our findings on different phase separation behaviors of nsp8 dimer and tetramer may provide insight into the functional studies of nsp8 in coronavirus.