CRISPR/Cas12a Collateral Cleavage Activity for Sensitive 3'-5' Exonuclease Assay.

This study presents a technique for detecting 3'-5' exonuclease activity through the use of CRISPR/Cas12a. These enzymes, including 3'-5' exonuclease (Exo III), perform crucial roles in various cellular processes and are associated with life expectancy. However, imbalances in their expression can increase susceptibility to diseases such as cancer, particularly under prolonged stress. In this study, an activator sequence of CRISPR/Cas12a was constructed on the 5'-end of a hairpin probe (HP), forming a blunt end. When the 3'-end of the HP was hydrolyzed with Exo III activity, the activator sequence of Cas12a was exposed, which led to collateral cleavage of the DNA signal probe and generated a fluorescent signal, allowing sensitive and highly specific Exo III detection. This detection principle relied on the fact that Exo III exclusively cleaves the 3'-end mononucleotide of dsDNA and does not affect ssDNA. Based on this strategy, Exo III activity was successfully assayed at 0.0073 U/mL, demonstrating high sensitivity. In addition, this technique was used to screen candidate inhibitors of Exo III activity.

Identification of a novel family B DNA polymerase from Enterococcus phage IME199 and its overproduction in Escherichia coli BL21(DE3).

BACKGROUND: Identification and characterization of novel, faithful and processive DNA polymerases is a driving force in the development of DNA amplification methods. Purification of proteins from natural phages is often time-consuming, cumbersome and low yielding. Escherichia coli is a host bacterium widely used for the production of recombinant proteins, is the cell factory of choice for in vitro studies of phage protein function. RESULTS: We expressed the gene encoding Enterococcus faecium phage IME199 DNA polymerase (IME199 DNAP) in Escherichia coli BL21(DE3), and characterized protein function. IME199 DNAP has 3'-5' exonuclease activity, but does not have 5'-3' exonuclease activity. In addition, IME199 DNAP has dNTP-dependent 5'-3' polymerase activity and can amplify DNA at 15-35 °C and a pH range of 5.5-9.5. The amino acid residues Asp30, Glu32, Asp112 and Asp251 are the 3'-5' exonuclease active sites of IME199 DNAP, while residues Asp596 and Tyr639 are essential for DNA synthesis by IME199 DNAP. More importantly, the IME199 DNAP has strand displacement and processive synthesis capabilities, and can perform rolling circle amplification and multiple displacement amplification with very low error rates (approximately 3.67 × 10-6). CONCLUSIONS: A novel family B DNA polymerase was successfully overproduced in Escherichia coli BL21(DE3). Based on the characterized properties, IME199 DNAP is expected to be developed as a high-fidelity polymerase for DNA amplification at room temperature.

Bacillus subtilis NrnB is expressed during sporulation and acts as a unique 3'-5' exonuclease.

All cells employ a combination of endo- and exoribonucleases to degrade long RNA polymers to fragments 2-5 nucleotides in length. These short RNA fragments are processed to monoribonucleotides by nanoRNases. Genetic depletion of nanoRNases has been shown to increase abundance of short RNAs. This deleteriously affects viability, virulence, and fitness, indicating that short RNAs are a metabolic burden. Previously, we provided evidence that NrnA is the housekeeping nanoRNase for Bacillus subtilis. Herein, we investigate the biological and biochemical functions of the evolutionarily related protein, B. subtilis NrnB (NrnBBs). These experiments show that NrnB is surprisingly different from NrnA. While NrnA acts at the 5' terminus of RNA substrates, NrnB acts at the 3' terminus. Additionally, NrnA is expressed constitutively under standard growth conditions, yet NrnB is selectively expressed during endospore formation. Furthermore, NrnA processes only short RNAs, while NrnB unexpectedly processes both short RNAs and longer RNAs. Indeed, inducible expression of NrnB can even complement the loss of the known global 3'-5' exoribonucleases, indicating that it acts as a general exonuclease. Together, these data demonstrate that NrnB proteins, which are widely found in Firmicutes, Epsilonproteobacteria and Archaea, are fundamentally different than NrnA proteins and may be used for specialized purposes.

RecJ3/4-aRNase J form a Ubl-associated nuclease complex functioning in survival against DNA damage in Haloferax volcanii.

Nucleases are strictly regulated and often localized in the cell to avoid the uncontrolled degradation of DNA and RNA. Here, a new type of nuclease complex, composed of RecJ3, RecJ4, and aRNase J, was identified through its ATP-dependent association with the ubiquitin-like SAMP1 and AAA-ATPase Cdc48a. The complex was discovered in Haloferax volcanii, an archaeon lacking an RNA exosome. Genetic analysis revealed aRNase J to be essential and RecJ3, RecJ4, and Cdc48a to function in the recovery from DNA damage including genotoxic agents that generate double-strand breaks. The RecJ3:RecJ4:aRNase J complex (isolated in 2:2:1 stoichiometry) functioned primarily as a 3'-5' exonuclease in hydrolyzing RNA and ssDNA, with the mechanism non-processive for ssDNA. aRNase J could also be purified as a homodimer that catalyzed endoribonuclease activity and, thus, was not restricted to the 5'-3' exonuclease activity typical of aRNase J homologs. Moreover, RecJ3 and RecJ4 could be purified as a 560-kDa subcomplex in equimolar subunit ratio with nuclease activities mirroring the full RecJ3/4-aRNase J complex. These findings prompted reconstitution assays that suggested RecJ3/4 could suppress, alter, and/or outcompete the nuclease activities of aRNase J. Based on the phenotypic results, this control mechanism of aRNase J by RecJ3/4 is not necessary for cell growth but instead appears important for DNA repair. IMPORTANCE Nucleases are critical for various cellular processes including DNA replication and repair. Here, a dynamic type of nuclease complex is newly identified in the archaeon Haloferax volcanii, which is missing the canonical RNA exosome. The complex, composed of RecJ3, RecJ4, and aRNase J, functions primarily as a 3'-5' exonuclease and was discovered through its ATP-dependent association with the ubiquitin-like SAMP1 and Cdc48a. aRNase J alone forms a homodimer that has endonuclease function and, thus, is not restricted to 5'-3' exonuclease activity typical of other aRNase J enzymes. RecJ3/4 appears to suppress, alter, and/or outcompete the nuclease activities of aRNase J. While aRNase J is essential for growth, RecJ3/4, Cdc48a, and SAMPs are important for recovery against DNA damage. These biological distinctions may correlate with the regulated nuclease activity of aRNase J in the RecJ3/4-aRNaseJ complex.

The evolution and structure of snake venom phosphodiesterase (svPDE) highlight its importance in venom actions.

For decades, studies of snake venoms focused on the venom-ome-specific toxins (VSTs). VSTs are dominant soluble proteins believed to contribute to the main venomous effects and emerged into gene clusters for fast adaptation and diversification of snake venoms. However, the conserved minor venom components, such as snake venom phosphodiesterase (svPDE), remain largely unexplored. Here, we focus on svPDE by genomic and transcriptomic analysis across snake clades and demonstrate that soluble svPDE is co-opted from the ancestral membrane-attached ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase 3) gene by replacing the original 5' exon with the exon encoding a signal peptide. Notably, the exons, promoters, and transcription/translation starts have been replaced multiple times during snake evolution, suggesting the evolutionary necessity of svPDE. The structural and biochemical analyses also show that svPDE shares the similar functions with ENPP family, suggesting its perturbation to the purinergic signaling and insulin transduction in venomous effects.

Identification of a Rho-Dependent Termination Site In Vivo Using Synthetic Small RNA.

Rho promotes Rho-dependent termination (RDT) at the Rho-dependent terminator, producing a variable-length region without secondary structure at the 3' end of mRNA. Determining the exact RDT site in vivo is challenging, because the 3' end of mRNA is rapidly removed after RDT by 3'-to-5' exonuclease processing. Here, we applied synthetic small RNA (sysRNA) to identify the RDT region in vivo by exploiting its complementary base-pairing ability to target mRNA. Through the combined analyses of rapid amplification of cDNA 3' ends, primer extension, and capillary electrophoresis, we could precisely map and quantify mRNA 3' ends. We found that complementary double-stranded RNA (dsRNA) formed between sysRNA and mRNA was efficiently cleaved by RNase III in the middle of the dsRNA region. The formation of dsRNA appeared to protect the cleaved RNA 3' ends from rapid degradation by 3'-to-5' exonuclease, thereby stabilizing the mRNA 3' end. We further verified that the signal intensity at the 3' end was positively correlated with the amount of mRNA. By constructing a series of sysRNAs with close target sites and comparing the difference in signal intensity at the 3' end of wild-type and Rho-impaired strains, we finally identified a region of increased mRNA expression within the 21-bp range, which was determined as the RDT region. Our results demonstrated the ability to use sysRNA as a novel tool to identify RDT regions in vivo and expand the range of applications of sysRNA. IMPORTANCE sysRNA, which was formerly widely employed, has steadily lost popularity as more novel techniques for suppressing gene expression come into existence because of issues such as unstable inhibition effect and low inhibition efficiency. However, it remains an interesting topic as a regulatory tool due to its ease of design and low metabolic burden on cells. Here, for the first time, we discovered a new method to identify RDT regions in vivo using sysRNA. This new feature is important because since the discovery of the Rho protein in 1969, specific identification of RDT sites in vivo has been difficult due to the rapid processing of RNA 3' ends by exonucleases, and sysRNA might provide a new approach to address this challenge.

Manganese-dependent microRNA trimming by 3'→5' exonucleases generates 14-nucleotide or shorter tiny RNAs.

MicroRNAs (miRNAs) are about 22-nucleotide (nt) noncoding RNAs forming the effector complexes with Argonaute (AGO) proteins to repress gene expression. Although tiny RNAs (tyRNAs) shorter than 19 nt have been found to bind to plant and vertebrate AGOs, their biogenesis remains a long-standing question. Here, our in vivo and in vitro studies show several 3'→5' exonucleases, such as interferon-stimulated gene 20 kDa (ISG20), three prime repair exonuclease 1 (TREX1), and ERI1 (enhanced RNAi, also known as 3'hExo), capable of trimming AGO-associated full-length miRNAs to 14-nt or shorter tyRNAs. Their guide trimming occurs in a manganese-dependent manner but independently of the guide sequence and the loaded four human AGO paralogs. We also show that ISG20-mediated guide trimming makes Argonaute3 (AGO3) a slicer. Given the high Mn2+ concentrations in stressed cells, virus-infected cells, and neurodegeneration, our study sheds light on the roles of the Mn2+-dependent exonucleases in remodeling gene silencing.

Crystal structure of DNA polymerase I from Thermus phage G20c.

This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3'-5' exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5'-3' exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SßαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SßαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3'-5' exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SßαR motif, was first determined to 2.19 Šresolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Šresolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearothermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.

Research on Werner Syndrome: Trends from Past to Present and Future Prospects.

A rare and autosomal recessive premature aging disorder, Werner syndrome (WS) is characterized by the early onset of aging-associated diseases, including shortening stature, alopecia, bilateral cataracts, skin ulcers, diabetes, osteoporosis, arteriosclerosis, and chromosomal instability, as well as cancer predisposition. WRN, the gene responsible for WS, encodes DNA helicase with a 3' to 5' exonuclease activity, and numerous studies have revealed that WRN helicase is involved in the maintenance of chromosome stability through actions in DNA, e.g., DNA replication, repair, recombination, and epigenetic regulation via interaction with DNA repair factors, telomere-binding proteins, histone modification enzymes, and other DNA metabolic factors. However, although these efforts have elucidated the cellular functions of the helicase in cell lines, they have not been linked to the treatment of the disease. Life expectancy has improved for WS patients over the past three decades, and it is hoped that a fundamental treatment for the disease will be developed. Disease-specific induced pluripotent stem (iPS) cells have been established, and these are expected to be used in drug discovery and regenerative medicine for WS patients. In this article, we review trends in research to date and present some perspectives on WS research with regard to the application of pluripotent stem cells. Furthermore, the elucidation of disease mechanisms and drug discovery utilizing the vast amount of scientific data accumulated to date will be discussed.

The 5'-phosphate enhances the DNA-binding and exonuclease activities of human mitochondrial genome maintenance exonuclease 1 (MGME1).

In higher eukaryotes, mitochondria play multiple roles in energy production, signaling, and biosynthesis. Mitochondria possess multiple copies of mitochondrial DNA (mtDNA), which encodes 37 genes that are essential for mitochondrial and cellular function. When mtDNA is challenged by endogenous and exogenous factors, mtDNA undergoes repair, degradation, and compensatory synthesis. mtDNA degradation is an emerging pathway in mtDNA damage response and maintenance. A key factor involved is the human mitochondrial genome maintenance exonuclease 1 (MGME1). Despite previous biochemical and functional studies, controversies exist regarding the polarity of MGME1-mediated DNA cleavage. Also, how DNA sequence may affect the activities of MGME1 remains elusive. Such information is not only fundamental to the understanding of MGME1 but critical for deciphering the mechanism of mtDNA degradation. Herein, we use quantitative assays to examine the effects of substrate structure and sequence on the DNA-binding and enzymatic activities of MGME1. We demonstrate that MGME1 binds to and cleaves from the 5'-end of single-stranded DNA substrates, especially in the presence of 5'-phosphate, which plays an important role in DNA binding and optimal cleavage by MGME1. In addition, MGME1 tolerates certain modifications at the terminal end, such as a 5'-deoxyribosephosphate intermediate formed in base excision repair. We show that MGME1 processes different sequences with varying efficiencies, with dT and dC sequences being the most and least efficiently digested, respectively. Our results provide insights into the enzymatic properties of MGME1 and a rationale for the coordination of MGME1 with the 3'-5' exonuclease activity of DNA polymerase γ in mtDNA degradation.

Molecular Characterization of a DNA Polymerase from Thermus thermophilus MAT72 Phage vB_Tt72: A Novel Type-A Family Enzyme with Strong Proofreading Activity.

We present a structural and functional analysis of the DNA polymerase of thermophilic Thermus thermophilus MAT72 phage vB_Tt72. The enzyme shows low sequence identity (<30%) to the members of the type-A family of DNA polymerases, except for two yet uncharacterized DNA polymerases of T. thermophilus phages: φYS40 (91%) and φTMA (90%). The Tt72 polA gene does not complement the Escherichia colipolA− mutant in replicating polA-dependent plasmid replicons. It encodes a 703-aa protein with a predicted molecular weight of 80,490 and an isoelectric point of 5.49. The enzyme contains a nucleotidyltransferase domain and a 3'-5' exonuclease domain that is engaged in proofreading. Recombinant enzyme with His-tag at the N-terminus was overproduced in E. coli, subsequently purified by immobilized metal affinity chromatography, and biochemically characterized. The enzyme exists in solution in monomeric form and shows optimum activity at pH 8.5, 25 mM KCl, and 0.5 mM Mg2+. Site-directed analysis proved that highly-conserved residues D15, E17, D78, D180, and D184 in 3'-5' exonuclease and D384 and D615 in the nucleotidyltransferase domain are critical for the enzyme's activity. Despite the source of origin, the Tt72 DNA polymerase has not proven to be highly thermoresistant, with a temperature optimum at 55 °C. Above 60 °C, the rapid loss of function follows with no activity > 75 °C. However, during heat treatment (10 min at 75 °C), trehalose, trimethylamine N-oxide, and betaine protected the enzyme against thermal inactivation. A midpoint of thermal denaturation at Tm = 74.6 °C (ΔHcal = 2.05 × 104 cal mol−1) and circular dichroism spectra > 60 °C indicate the enzyme's moderate thermal stability.

Structure and activity of a bacterial defense-associated 3'-5' exonuclease.

The widespread CBASS (cyclic oligonucleotide-based anti-phage signaling system) immune systems in bacteria protect their hosts from bacteriophage infection by triggering programmed cell death. CBASS systems all encode a cyclic oligonucleotide synthase related to eukaryotic cGAS but use diverse regulators and effector proteins including nucleases, phospholipases, and membrane-disrupting proteins to effect cell death. Cap18 is a predicted 3'-5' exonuclease associated with hundreds of CBASS systems, whose structure, biochemical activities, and biological roles remain unknown. Here we show that Cap18 is a DEDDh-family exonuclease related to the bacterial exonucleases RNase T and Orn and has nonspecific 3'-5' DNA exonuclease activity. Cap18 is commonly found in CBASS systems with associated CapW or CapH+CapP transcription factors, suggesting that it may coordinate with these proteins to regulate CBASS transcription in response to DNA damage. These data expand the repertoire of enzymatic activities associated with bacterial CBASS systems and provide new insights into the regulation of these important bacterial immune systems.

Effect of DNA Methylation on the 3'→5' Exonuclease Activity of Major Human Abasic Site Endonuclease APEX1.

Apurinic/apyrimidinic (AP) endonucleases are the key enzymes in the DNA base excision repair, as they hydrolyze the phosphodiester bond in the AP site formed after removal of the damaged base. Major human AP endonuclease APEX1 also possesses the 3'-phosphodiesterase and 3'→5' exonuclease activities. The biological role of the latter has not been established yet; it is assumed that it corrects DNA synthesis errors during DNA repair. If DNA is damaged at the 3'-side of 5-methylcytosine (mC) residue, the 3'→5' exonuclease activity can change the epigenetic methylation status of the CpG dinucleotide. It remains unclear whether the 3'→5' exonuclease activity of APEX1 contributes to the active epigenetic demethylation or, on the contrary, is limited in the case of methylated CpG dinucleotides in order to preserve the epigenetic status upon repair of accidental DNA damage. Here, we report the results of the first systematic study on the efficiency of removal of 3'-terminal nucleotides from the substrates modeling DNA repair intermediates in the CpG dinucleotides. The best substrates for the 3'→5' exonuclease activity of APEX1 were oligonucleotides with the 3'-terminal bases non-complementary to the template, while the worst substrates contained mC. The presence of mC in the complementary strand significantly reduced the reaction rate even for the non-complementary 3'-ends. Therefore, the efficiency of the 3'→5' exonuclease reaction catalyzed by APEX1 is limited in the case of the methylated CpG dinucleotides, which likely reflects the need to preserve the epigenetic status during DNA repair.

A single point mutation in the Plasmodium falciparum 3'-5' exonuclease does not alter piperaquine susceptibility.

BACKGROUND: The rise in Plasmodium falciparum resistance to dihydroartemisinin-piperaquine (DHA-PPQ) treatment has been documented in the Greater Mekong Subregion with associations with mutations in the P. falciparum chloroquine resistance transporter (pfcrt) and plasmepsin 2 (pfpm2) genes. However, it is unclear whether other genes also play a role with PPQ resistance, such as the E415G mutation in the exonuclease (pfexo) gene. The aim of this study was to investigate the role of this mutation in PPQ resistance by generating transgenic parasites expressing the pfexo-E415G mutant allele. METHODS: Transgenic parasite clones carrying the E415G mutation in PfEXO of the B5 isolate were derived by CRISPR-Cas9 gene editing and verified using PCR and gene sequencing. Polymorphisms of pfkelch-13, pfcrt, and pfexo were examined by PCR while the copy number variations of pfpm2 were examined by both relative quantitative real-time PCR and the duplication breakpoint assay. Drug sensitivity against a panel of antimalarials, the ring-stage survival assay (RSA), the PPQ survival assay (PSA), and bimodal dose-response curves were used to evaluate antimalarial susceptibility. RESULTS: The transgenic line, B5-rexo-E415G-B8, was successfully generated. The PPQ-IC90, %PPQ survival, and the bimodal dose-response clearly showed that E415G mutation in PfEXO of B5 isolate remained fully susceptible to PPQ. Furthermore, growth assays demonstrated that the engineered parasites grew slightly faster than the unmodified parental isolates whereas P. falciparum isolates harbouring pfkelch-13, pfcrt, and pfexo mutations with multiple copies of pfpm2 grew much more slowly. CONCLUSIONS: Insertion of the E415G mutation in PfEXO did not lead to increased PPQ-IC90 and %PPQ survival, suggesting that this mutation alone may not be associated with PPQ resistance, but could still be an important marker if used in conjunction with other markers for monitoring PPQ-resistant parasites. The results also highlight the importance of monitoring and evaluating suspected genetic mutations with regard to parasite fitness and resistance.

Mechanistic decoupling of exonuclease III multifunctionality into AP endonuclease and exonuclease activities at the single-residue level.

Bacterial exonuclease III (ExoIII) is a multifunctional enzyme that uses a single active site to perform two conspicuous activities: (i) apurinic/apyrimidinic (AP)-endonuclease and (ii) 3'→5' exonuclease activities. The AP endonuclease activity results in AP site incision, while the exonuclease activity results in the continuous excision of 3' terminal nucleobases to generate a partial duplex for recruiting the downstream DNA polymerase during the base excision repair process (BER). The key determinants of functional selection between the two activities are poorly understood. Here, we use a series of mutational analyses and single-molecule imaging to unravel the pivotal rules governing these endo- and exonuclease activities at the single amino acid level. An aromatic residue, either W212 or F213, recognizes AP sites to allow for the AP endonuclease activity, and the F213 residue also participates in the stabilization of the melted state of the 3' terminal nucleobases, leading to the catalytically competent state that activates the 3'→5' exonuclease activity. During exonucleolytic cleavage, the DNA substrate must be maintained as a B-form helix through a series of phosphate-stabilizing residues (R90, Y109, K121 and N153). Our work decouples the AP endonuclease and exonuclease activities of ExoIII and provides insights into how this multifunctional enzyme controls each function at the amino acid level.

Optimal intervention time of ADSCs for hepatic ischemia-reperfusion combined with partial resection injury in rats.

AIMS: Hepatic ischemia reperfusion injury (HIRI) is a complication of liver surgery and liver transplantation. Adipose-derived stem cells (ADSCs) can inhibit oxidative stress and inflammation through a paracrine effect. This study aimed to determine the optimal time window of ADSCs transplantation to restore liver function after HIRI. MAIN METHODS: A rat model of hepatic ischemia reperfusion combined with partial hepatectomy (HIR/PH) was established. The animals were injected intravenously with 2 × 106 rat ADSCs 2 h before, immediately after, or 6 h after surgery. Liver tissues and blood samples were collected for routine histological and biochemical assays. The molecular changes were analyzed by qRT-PCR and western blotting. KEY FINDINGS: ADSCs significantly improved liver tissue structure and decreased the levels of AST, ALT and ALP, which was indicative of functional recovery. In addition, transplantation of ADSCs immediately after operation decreased the levels of inflammation-related cytokines such as TNF-α, IL-1ß and IL-6, and significantly increased the activity of antioxidant enzymes. At the same time, the expression of MDA was decreased. Mechanistically, ADSCs activated the Keap1/Nrf2 pathway in the injured liver. Transplantation of ADSCs pre- and 6 h post-operation did not significantly affect some indices such as mRNA and protein expression of HO-1, and protein expression of NQO1. SIGNIFICANCE: Transplanting ADSCs immediately after surgery accelerated tissue repair and functional recovery of the liver by activating the Keap1/Nrf2 pathway, which inhibited hepatic inflammation and oxidative stress, and restored the hepatic microenvironment.

Micro-flow hydrophilic interaction liquid chromatography coupled with triple quadrupole mass spectrometry detects modified nucleosides in the transfer RNA pool of cyanobacteria.

Post-transcriptional modification of nucleosides is observed in almost all elements of RNA. Modified nucleosides finely tune the structure of RNA molecules and affect vital functions, such as the modified wobble position 34 of transfer RNAs expanding the reading preference of anticodons to codons. Recent investigations have revealed that the modification species and their frequencies in an RNA element are not stable but vary with specific cellular factors including metabolites and particular proteins (writers, readers, and erasers). To understand the link between dynamic RNA modifications and biological processes, sensitive and reliable methods for determining modified nucleosides are required. In this study, micro-flow (8 µL/min) hydrophilic interaction liquid chromatography was coupled with triple quadrupole mass spectrometry for the simultaneous determination of adenosine, uridine, cytidine, guanosine, and 20 modified nucleosides. The method was calibrated using 0.1-1000 nM standards (∼0.03-300 ng/mL) and successfully applied to the determination of transfer RNA modifications in the model cyanobacterium Synechococcus elongatus PCC 7942. A protocol for the isolation of a clean transfer RNA pool was optimized, requiring only 25 ng for the identification and quantification of transfer RNA modifications. This micro-flow liquid chromatography-tandem mass spectrometry method constitutes the first step toward monitoring dynamic ribonucleoside modifications in a limited RNA sample.

Role of phosphodiesterase 1 in the pathophysiology of diseases and potential therapeutic opportunities.

Cyclic nucleotide phosphodiesterases (PDEs) are superfamily of enzymes that regulate the spatial and temporal relationship of second messenger signaling in the cellular system. Among the 11 different families of PDEs, phosphodiesterase 1 (PDE1) sub-family of enzymes hydrolyze both 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) in a mutually competitive manner. The catalytic activity of PDE1 is stimulated by their binding to Ca2+/calmodulin (CaM), resulting in the integration of Ca2+ and cyclic nucleotide-mediated signaling in various diseases. The PDE1 family includes three subtypes, PDE1A, PDE1B and PDE1C, which differ for their relative affinities for cAMP and cGMP. These isoforms are differentially expressed throughout the body, including the cardiovascular, central nervous system and other organs. Thus, PDE1 enzymes play a critical role in the pathophysiology of diseases through the fundamental regulation of cAMP and cGMP signaling. This comprehensive review provides the current research on PDE1 and its potential utility as a therapeutic target in diseases including the cardiovascular, pulmonary, metabolic, neurocognitive, renal, cancers and possibly others.

Quantitative analysis of RNA by HPLC and evaluation of RT-dPCR for coronavirus RNA quantification.

Nucleic acid detection and quantification have been known to be important at various fields, from genetically modified organisms and gene expression to virus detection. For DNA molecules, digital PCR has been developed as an absolute quantification method which is not dependent on external calibrators. While when it comes to RNA molecules, reverse transcription (RT) step must be taken before PCR amplification to obtain cDNA. With different kinds of reverse transcriptase (RTase) and RT reaction conditions being used in laboratory assays, the efficiency of RT process differs a lot which led variety in quantification results of RNA molecules. In this study, we developed HPLC method combined with enzymatic digestion of RNA to nucleotides for quantification of RNA without RT process. This method was metrologically traceable to four nuceloside monophosphate (NMP) Certification Reference Materials of National Institute of Metrology, China (NIMC) for insurance of accuracy. The established method was used to evaluate the reverse transcription digital polymerase chain reaction (RT-dPCR) of three target genes of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) RNA, including open reading frame 1ab (ORF1ab), nucleocapsid protein (N) and envelope protein (E) gene. Three available RT kits had been evaluated and disparities were observed for the RT efficiency varied from 9% to 182%. It is thus demonstrated that HPLC combined with enzymatic digestion could be a useful method to quantify RNA molecules and evaluate RT efficiency. It is suggested that RT process should be optimized and identified in RNA quantification assays.

A cell-based ribozyme reporter system employing a chromosomally-integrated 5' exonuclease gene.

BACKGROUND: Bioinformatic genome surveys indicate that self-cleaving ribonucleic acids (ribozymes) appear to be widespread among all domains of life, although the functions of only a small number have been validated by biochemical methods. Alternatively, cell-based reporter gene assays can be used to validate ribozyme function. However, reporter activity can be confounded by phenomena unrelated to ribozyme-mediated cleavage of RNA. RESULTS: We established a ribozyme reporter system in Escherichia coli in which a significant reduction of reporter activity is manifest when an active ribozyme sequence is fused to the reporter gene and the expression of a foreign Bacillus subtilis RNaseJ1 5' exonuclease is induced from a chromosomally-integrated gene in the same cell. CONCLUSIONS: The reporter system could be useful for validating ribozyme function in candidate sequences identified from bioinformatics.