Phosphate End Research Articles

Kinetic and structural insights into the requirement of fungal tRNA ligase for a 2'-phosphate end.

Fungal RNA ligase (LIG) is an essential tRNA splicing enzyme that joins 3'-OH,2'-PO4 and 5'-PO4 RNA ends to form a 2'-PO4,3'-5' phosphodiester splice junction. Sealing entails three divalent cation-dependent adenylate transfer steps. First, LIG reacts with ATP to form a covalent ligase-(lysyl-Nζ)-AMP intermediate and displace pyrophosphate. Second, LIG transfers AMP to the 5'-PO4 RNA terminus to form an RNA-adenylate intermediate (A5'pp5'RNA). Third, LIG directs the attack of an RNA 3'-OH on AppRNA to form the splice junction and displace AMP. A defining feature of fungal LIG vis-à-vis canonical polynucleotide ligases is the requirement for a 2'-PO4 to synthesize a 3'-5' phosphodiester bond. Fungal LIG consists of an N-terminal adenylyltransferase domain and a unique C-terminal domain. The C-domain of Chaetomium thermophilum LIG (CthLIG) engages a sulfate anion thought to be a mimetic of the terminal 2'-PO4 Here, we interrogated the contributions of the C-domain and the conserved sulfate ligands (His227, Arg334, Arg337) to ligation of a pRNA2'p substrate. We find that the C-domain is essential for end-joining but dispensable for ligase adenylylation. Mutations H227A, R334A, and R337A slowed the rate of step 2 RNA adenylation by 420-fold, 120-fold, and 60-fold, respectively, vis-à-vis wild-type CthLIG. An R334A-R337A double-mutation slowed step 2 by 580-fold. These results fortify the case for the strictly conserved His-Arg-Arg triad as the enforcer of the 2'-PO4 end-specificity of fungal tRNA ligases and as a target for small molecule interdiction of fungal tRNA splicing.

Enhanced chemical recovery of phosphorus from residues of recirculating aquaculture systems (RAS)

In recirculating aquaculture systems, cultivated fish cannot incorporate all the phosphate contained in the feed. Excess phosphate ends up in the culture water and in the sludge produced. If the sludge cannot be valorised directly in agriculture, a chemical recovery as concentrated phosphate is an interesting option to help closing the phosphorus cycle. This study investigates the extent to which accumulated phosphorus can be recovered by acid re-dissolution with subsequent precipitation on sludge from two different recirculating aquaculture systems cultivating African Catfish (Clarias gariepinus). Acid treatment could increase the ratio of dissolved phosphorus available for subsequent precipitation by 53% for extensive and by 61% for intensive fish farming. With the consecutive precipitation in total up to 86% of the phosphorus from the sludge could be recovered. Phosphorus re-dissolution with citric acid requires the highest amount of acid per g dry matter as well as of sodium hydroxide for subsequent precipitation. Sulfuric and nitric acids have comparably lower demands.

Anaerobic Digestion of Olive Mill Wastewater and Process Derivatives—Biomethane Potential, Operation of a Continuous Fixed Bed Digester, and Germination Index

Olive mill wastewater (OMW) management is an economic and environmental challenge for olive oil-producing countries. The recovery of components with high added value, such as antioxidants, is a highly researched approach that could help refinance performant wastewater treatment systems. Anaerobic (co-)digestion is a suitable process to valorize the energetic and nutritional content of OMW and OMW-derived waste streams from resource recovery processes. Issues of process stability, operation, and yields discourage industrial application. Deepening the understanding of biomethane potential, continuous anaerobic digester operational parameters, and co-substrates is key to large-scale implementation. The biomethane potential of different OMW-derived samples and organic solid market waste as co-substrate was 106–350 NL methane per kg volatile solids (VS). The highest yields were obtained with the co-substrate and depolyphenolized OMW mixed with retentate from an ultrafiltration pretreatment. Over 150 days, an anaerobic fixed-bed 300 L digester was operated with different OMW-derived substrates, including OMW with selectively reduced polyphenol concentrations. Different combinations of organic loading rate and hydraulic retention time were set. The biogas yields ranged from 0.97 to 0.99 L of biogas per g of volatile solids (VS) eliminated, with an average methane content in the produced biogas of 64%. Potential inhibition of the process due to high polyphenol concentrations or over-acidification through volatile fatty acids was avoided in the continuous process through process and substrate manipulation. High concentrations of potassium and low concentrations of nitrogen and phosphate end up in the digestate. Sulfate reduction results in high H2S concentrations in the biogas. The digestate was tested for phytotoxic properties via the germination index. Diluted digestate samples improved germination by up to 50%.

Phosphate toxicity and SERCA2a dysfunction in sudden cardiac arrest.

Almost half of the people who die from sudden cardiac arrest have no detectable heart disease. Among children and young adults, the cause of approximately one-third of deaths from sudden cardiac arrest remains unexplained after thorough examination. Sudden cardiac arrest and related sudden cardiac death are attributed to dysfunctional cardiac ion-channels. The present perspective paper proposes a pathophysiological mechanism by which phosphate toxicity from cellular accumulation of dysregulated inorganic phosphate interferes with normal calcium handling in the heart, leading to sudden cardiac arrest. During cardiac muscle relaxation following contraction, SERCA2a pumps actively transport calcium ions into the sarcoplasmic reticulum, powered by ATP hydrolysis that produces ADP and inorganic phosphate end products. Reviewed evidence supports the proposal that end-product inhibition of SERCA2a occurs as increasing levels of inorganic phosphate drive up phosphate toxicity and bring cardiac function to a sudden and unexpected halt. The paper concludes that end-product inhibition from ATP hydrolysis is the mediating factor in the association of sudden cardiac arrest with phosphate toxicity. However, current technology lacks the ability to directly measure this pathophysiological mechanism in active myocardium, and further research is needed to confirm phosphate toxicity as a risk factor in individuals with sudden cardiac arrest. Moreover, phosphate toxicity may be reduced through modification of dietary phosphate intake, with potential for employing low-phosphate dietary interventions to reduce the risk of sudden cardiac arrest.

DNA Binding and Cleavage, Stopped-Flow Kinetic, Mechanistic, and Molecular Docking Studies of Cationic Ruthenium(II) Nitrosyl Complexes Containing "NS4" Core.

This work aimed to evaluate in vitro DNA binding mechanistically of cationic nitrosyl ruthenium complex [RuNOTSP]+ and its ligand (TSPH2) in detail, correlate the findings with cleavage activity, and draw conclusions about the impact of the metal center. Theoretical studies were performed for [RuNOTSP]+, TSPH2, and its anion TSP-2 using DFT/B3LYP theory to calculate optimized energy, binding energy, and chemical reactivity. Since nearly all medications function by attaching to a particular protein or DNA, the in vitro calf thymus DNA (ctDNA) binding studies of [RuNOTSP]+ and TSPH2 with ctDNA were examined mechanistically using a variety of biophysical techniques. Fluorescence experiments showed that both compounds effectively bind to ctDNA through intercalative/electrostatic interactions via the DNA helix's phosphate backbone. The intrinsic binding constants (Kb), (2.4 ± 0.2) × 105 M-1 ([RuNOTSP]+) and (1.9 ± 0.3) × 105 M-1 (TSPH2), as well as the enhancement dynamic constants (KD), (3.3 ± 0.3) × 104 M-1 ([RuNOTSP]+) and (2.6 ± 0.2) × 104 M-1 (TSPH2), reveal that [RuNOTSP]+ has a greater binding propensity for DNA compared to TSPH2. Stopped-flow investigations showed that both [RuNOTSP]+ and TSPH2 bind through two reversible steps: a fast second-order binding, followed by a slow first-order isomerization reaction via a static quenching mechanism. For the first and second steps of [RuNOTSP]+ and TSPH2, the detailed binding parameters were established. The total binding constants for [RuNOTSP]+ (Ka = 43.7 M-1, Kd = 2.3 × 10-2 M-1, ΔG0 = -36.6 kJ mol-1) and TSPH2 (Ka = 15.1 M-1, Kd = 66 × 10-2 M, ΔG0 = -19 kJ mol-1) revealed that the relative reactivity is approximately ([RuNOTSP]+)/(TSPH2) = 3/1. The significantly negative ΔG0 values are consistent with a spontaneous binding reaction to both [RuNOTSP]+ and TSPH2, with the former being very favorable. The findings showed that the Ru(II) center had an effect on the reaction rate but not on the mechanism and that the cationic [RuNOTSP]+ was a more highly effective DNA binder than the ligand TSPH2 via strong electrostatic interaction with the phosphate end of DNA. Because of its higher DNA binding affinity, cationic [RuNOTSP]+ demonstrated higher cleavage efficiency towards the minor groove of pBR322 DNA via the hydrolytic pathway than TSPH2, revealing the synergy effect of TSPH2 in the form of the complex. Furthermore, the mode of interaction of both compounds with ctDNA has also been supported by molecular docking.

In Vitro Self-Assembly of a Modified Diphenylalanine Peptide to Nanofibers Induced by the Eye Absent Enzyme and Alkaline Phosphatase and Its Activity against Breast Cancer Cell Proliferation.

Drug-resistant breast cancers such as Triple negative breast cancer (TNBC) do not respond successfully to chemotherapy treatments because they lack the expression of receptor targets. Drug-resistant anti-cancer treatments require innovative approaches to target these cells without relying on the receptors. Intracellular self-assembly of small molecules induced by enzymes is a nanotechnology approach for inhibiting cancer cell growth. In this approach, enzymes will induce the self-assembly of small molecules to nanofibers, which leads to cell death. Here, we investigate the self-assembly of a modified small peptide induced by two different phosphatases: alkaline phosphatase (ALP) and eye absent tyrosine phosphatase (EYA). ALPs are expressed in many adult human tissues and are critical for many cellular functions. EYAs are embryonic enzymes that are over-expressed in drug-resistant breast cancers. We synthesized a small diphenylalanine-based peptide with a tyrosine phosphate end group as the substrate of phosphatase enzymes. Peptides were synthesized with solid phase techniques and were characterized by HPLC and MALDI-TOF. To characterize the self-assembly of peptides exposed to enzymes, different techniques were used such as scattering light intensity, microscopes, and phosphate detection kit. We then determined the toxicity effect of the peptide against normal breast cancer cells, MCF-7, and drug-resistant breast cancer cells, MDA-MB-231. The results showed that the EYA enzyme is able to initiate self-assembly at lower peptide concentration with higher self-assembling intensity compared to ALP. A significant decrease in the TNBC cell number was observed even with a low peptide concentration of 60 μM. These results collectively support the exploration of enzyme self-assembly to treat TNBC.

Biotechnological production of polyphosphate from industrial wash water.

Phosphate is mined from phosphate rock, which is a limited resource on a human time scale. For a sustainable phosphate supply, strategies for efficient use and recycling of phosphate must be developed. A German chemical company produces annually wash water containing phosphate and other inorganic substances (e.g., sodium, potassium, sulfate, and chloride) at a ton scale. Chemical precipitation is mostly used for phosphate removal. In this study, a biotechnological process utilizing Saccharomyces cerevisiae to upcycle phosphate-containing wastewater into pure sodium polyphosphate in powder form was developed. The process comprises fermentation and downstream processing (polyphosphate yields: 25% and 36%, respectively). The polyphosphate quality was independent of the wash water composition. Polyphosphate with a purity of 23% molar ratio Na to Na, K, and Mg of > 90%, and with an average chain length of 12.5 phosphate subunits was produced. The upcycled polyphosphate can be reused compared to phosphate fertilizer in many different applications. Overall, the here developed process can contribute to truly slowing down phosphate mining and finally enable a sustainable utilization of phosphate. Thereby, the benefit of the process is the cascade use of phosphate, reducing the need for phosphate rock before the phosphate ends up in the soil and ultimately in the sea.

Making Invisible RNA Visible: Discriminative Sequencing Methods for RNA Molecules with Specific Terminal Formations.

Next generation sequencing of RNA molecules (RNA-seq) has become a common tool to characterize the expression profiles of RNAs and their regulations in normal physiological processes and diseases. Although increasingly accumulating RNA-seq data are widely available through publicly accessible sites, most of the data for short non-coding RNAs (sncRNAs) have been obtained for microRNA (miRNA) analyses by standard RNA-seq, which only capture the sncRNAs with 5′-phosphate (5′-P) and 3′-hydroxyl (3′-OH) ends. The sncRNAs with other terminal formations such as those with a 5′-hydroxyl end (5′-OH), a 3′-phosphate (3′-P) end, or a 2′,3′-cyclic phosphate end (2′,3′-cP) cannot be efficiently amplified and sequenced by standard RNA-seq. Due to the invisibility in standard RNA-seq data, these non-miRNA-sncRNAs have been a hidden component in the transcriptome. However, as the functional significances of these sncRNAs have become increasingly apparent, specific RNA-seq methods compatible with various terminal formations of sncRNAs have been developed and started shedding light on the previously unrecognized sncRNAs that lack 5′-P/3′-OH ends. In this review, we summarize the expanding world of sncRNAs with various terminal formations and the strategic approaches of specific RNA-seq methods to distinctively characterize their expression profiles.

5′-Phosphonate modified oligoadenylates as potent activators of human RNase L

The oligoadenylate synthetase-ribonuclease L pathway is a major player in the interferon-induced antiviral defense mechanism of cells. Upon sensing viral dsRNA, 5′-phosphorylated 2′,5′-oligoadenylates are synthesized, and subsequently activate latent RNase L. To determine the influence of 5′-phosphate end on the activation of human RNase L, four sets of 5′-phosphonate modified oligoadenylates were prepared on solid-phase. The ability of these 5′-modified oligoadenylates bearing shortened, isosteric and prolonged phosphonate linkages to activate RNase L was explored. We found that isosteric linkages and linkages prolonged by one atom were in general well tolerated by the enzyme with the EC50 values comparable to that of the natural activator. In contrast, linkages shortened by one atom or prolonged by two atoms exhibited decrease in the activity.

Structure of 3'-PO4/5'-OH RNA ligase RtcB in complex with a 5'-OH oligonucleotide.

RtcB enzymes comprise a widely distributed family of manganese- and GTP-dependent RNA repair enzymes that join 2′,3′-cyclic phosphate ends to 5′-OH ends via RtcB-(histidinyl-N)–GMP, RNA 3′-phosphate, and RNA3′pp5′G intermediates. RtcB can ligate either 5′-OH RNA or 5′-OH DNA strands in vitro. The nucleic acid contacts of RtcB are uncharted. Here we report a 2.7 Å crystal structure of Pyrococcus horikoshii RtcB in complex with a 6-mer 5′-OH DNA oligonucleotide HOA1pT2pG3pT4pC5pC6, which reveals enzymic contacts of Asn202 to the terminal 5′-OH nucleophile; Arg238 to the A1pT2 and T2pG3 phosphates; Arg190 and Gln194 to the T2pG3 phosphate; and an Arg190 π-cation interaction with the G3 nucleobase. The structural insights affirm functional studies of E. coli RtcB that implicated the conserved counterpart of Arg238 in engagement of the 5′-OH strand for ligation. The essential active site Cys98 that coordinates two manganese ions is oxidized to cysteine sulfonic acid in our structure, raising the prospect that RtcB activity might be sensitive to modulation during oxidative stress.

An RNA Repair Operon Regulated by Damaged tRNAs

SUMMARYMany bacteria contain an RNA repair operon, encoding the RtcB RNA ligase and the RtcA RNA cyclase, that is regulated by the RtcR transcriptional activator. Although RtcR contains a divergent version of the CARF (CRISPR-associated Rossman fold) oligonucleotide-binding regulatory domain, both the specific signal that regulates operon expression and the substrates of the encoded enzymes are unknown. We report that tRNA fragments activate operon expression. Using a genetic screen in Salmonella enterica serovar Typhimurium, we find that the operon is expressed in the presence of mutations that cause tRNA fragments to accumulate. RtcA, which converts RNA phosphate ends to 2′, 3′-cyclic phosphate, is also required. Operon expression and tRNA fragment accumulation also occur upon DNA damage. The CARF domain binds 5′ tRNA fragments ending in cyclic phosphate, and RtcR oligomerizes upon binding these ligands, a prerequisite for operon activation. Our studies reveal a signaling pathway involving broken tRNAs and implicate the operon in tRNA repair.

Molecular basis for the distinct cellular functions of the Lsm1-7 and Lsm2-8 complexes.

Eukaryotes possess eight highly conserved Lsm (like Sm) proteins that assemble into circular, heteroheptameric complexes, bind RNA, and direct a diverse range of biological processes. Among the many essential functions of Lsm proteins, the cytoplasmic Lsm1–7 complex initiates mRNA decay, while the nuclear Lsm2–8 complex acts as a chaperone for U6 spliceosomal RNA. It has been unclear how these complexes perform their distinct functions while differing by only one out of seven subunits. Here, we elucidate the molecular basis for Lsm-RNA recognition and present four high-resolution structures of Lsm complexes bound to RNAs. The structures of Lsm2–8 bound to RNA identify the unique 2′,3′ cyclic phosphate end of U6 as a prime determinant of specificity. In contrast, the Lsm1–7 complex strongly discriminates against cyclic phosphates and tightly binds to oligouridylate tracts with terminal purines. Lsm5 uniquely recognizes purine bases, explaining its divergent sequence relative to other Lsm subunits. Lsm1–7 loads onto RNA from the 3′ end and removal of the Lsm1 carboxy-terminal region allows Lsm1–7 to scan along RNA, suggesting a gated mechanism for accessing internal binding sites. These data reveal the molecular basis for RNA binding by Lsm proteins, a fundamental step in the formation of molecular assemblies that are central to eukaryotic mRNA metabolism.

A streamlined protocol for the detection of mRNA-sRNA interactions using AMT-crosslinking in vitro.

Until recently, RNA-RNA interactions were mainly identified by crosslinking RNAs with interacting proteins, RNA proximity ligation and deep sequencing. Recently, AMT-based direct RNA crosslinking was established. Yet, several steps of these procedures are rather inefficient, reducing the output of identified interaction partners. To increase the local concentration of RNA ends, interacting RNAs are often fragmented. However, the resulting 2',3'-cyclic phosphate and 5'-OH ends are not accepted by T4 RNA ligase and have to be converted to 3'-OH and 5'-phosphate ends. Using an artificial mRNA/sRNA pair, we optimized the workflow downstream of the crosslinking reaction in vitro. The use of a tRNA ligase allows direct fusion of 2',3'-cyclic phosphate and 5'-OH RNA ends.

Utilization of 31P NMR and ITC to Study the Binding of ATP, ADP, and AMP with Metal Ions

Magnesium is known to be an important cofactor in many enzymatic processes involving adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP). The binding of magnesium to the phosphate end of the nucleotide phosphate is well known and crucial to the catalytic activity of many enzymatic reactions. There is high selectivity for magnesium and other metals are not found to participate in the binding and enzymatic catalysis of nucleotide phosphates. We aim to study the binding of various metals in addition to magnesium to see if it can be determined why other metals are not as crucial to enzymatic binding. To answer this question we assessed metal ion binding to ATP, ADP, and AMP in pH 6.9 and 8.0 HEPES buffer using 31P Nuclear Magnetic Resonance (NMR) as well as Isothermal Titration Calorimetry (ITC). These measurements will correlate the conformational changes seen in the 31P NMR to the binding energy measured in ITC. With magnesium we see increasing conformational changes in the 31P NMR that correlate with a large binding energy seen in ITC. With other metal ions we do see a conformational change in the NMR but the binding energy is not as large as that found for magnesium.Support or Funding InformationInstrumentation support was provided by the Carver Foundation and the R.W. and Mary Nelson FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Capture of Ribonucleotides in Yeast Genomic DNA Using Ribose-Seq.

Experiments conducted in yeast cells have recently shown abundant presence of ribonucleotides (rNMPs) embedded both in nuclear and mitochondrial DNA. Indeed, rNMPs are the most frequent, nonstandard nucleotides found in cellular DNA. rNMPs have a highly reactive 2'-hydroxyl group in the ribose sugar that gives rise to genome instability by altering the structure, function, and properties of DNA. In order to profile rNMPs embedded in yeast genomic DNA, as well as any other genomic DNA of interest, we developed "ribose-seq." Ribose-seq utilizes Arabidopsis thaliana tRNA ligase (AtRNL), which enables ligation of 2'-phosphate termini of DNA molecules terminating with an rNMP to the 5'-phosphate end of the same DNA molecules. Thus, a unique feature of ribose-seq is its capacity to specifically and directly capture the rNMPs present in DNA. Here we describe how ribose-seq is applied to yeast Saccharomyces cerevisiae DNA to capture rNMPs that are incorporated in the yeast genome and build libraries of rNMP incorporation for high-throughput sequencing. We also provide the advancements over our original ribose-seq protocol at the end of Subheading 1, and the specific details are provided in the methods part of this chapter.

Generation of 2′,3′-Cyclic Phosphate-Containing RNAs as a Hidden Layer of the Transcriptome

Cellular RNA molecules contain phosphate or hydroxyl ends. A 2′,3′-cyclic phosphate (cP) is one of the 3′-terminal forms of RNAs mainly generated from RNA cleavage by ribonucleases. Although transcriptome profiling using RNA-seq has become a ubiquitous tool in biological and medical research, cP-containing RNAs (cP-RNAs) form a hidden transcriptome layer, which is infrequently recognized and characterized, because standard RNA-seq is unable to capture them. Despite cP-RNAs’ invisibility in RNA-seq data, increasing evidence indicates that they are not accumulated simply as non-functional degradation products; rather, they have physiological roles in various biological processes, designating them as noteworthy functional molecules. This review summarizes our current knowledge of cP-RNA biogenesis pathways and their catalytic enzymatic activities, discusses how the cP-RNA generation affects biological processes, and explores future directions to further investigate cP-RNA biology.

Microbial Community Level Physiological Profiles of Active Mud Volcano Soils in Andaman and Nicobar Islands

The aim of this work was to determine if any differences exist in the community-level physiological profiles (CLPP) in different regions of mud volcano. The microbial functional diversity in the four soils sampled in the vicinity of a mud volcano, Baratang, Andaman and Nicobar Islands, India, was determined using Biolog EcoPlates. Comparisons of the patterns of substrate use showed differences in the community composition, reflecting the heterogeneous distribution of microorganisms in this environment. The results showed statistically significant differences among different volcanic environments both by total colony-forming unit and by average well colour development. On the basis of similarity percentage analysis, it was found that i-erythritol was the most commonly used substrate, but substrates such as dl-glycerol phosphate (end mud volcano × point mud volcano), l-asparagine (end mud volcano × dried mud volcano) and ketobutyric acid (outside mud volcano × point mud volcano) were found in only one environment. The metabolic diversity varied depending on the environments compared, indicating specific utilization of compounds’ effect. Microbial response in terms of the overall metabolic activity in outside mud volcano was higher than in other inside mud volcano soils. To the best of our knowledge, this is the first attempt to use CLPP study in volcanic environments in the Andaman and Nicobar Islands.

The Impact of Uremic Toxins on Vascular Smooth Muscle Cell Function.

Chronic kidney disease (CKD) is associated with profound vascular remodeling, which accelerates the progression of cardiovascular disease. This remodeling is characterized by intimal hyperplasia, accelerated atherosclerosis, excessive vascular calcification, and vascular stiffness. Vascular smooth muscle cell (VSMC) dysfunction has a key role in the remodeling process. Under uremic conditions, VSMCs can switch from a contractile phenotype to a synthetic phenotype, and undergo abnormal proliferation, migration, senescence, apoptosis, and calcification. A growing body of data from experiments in vitro and animal models suggests that uremic toxins (such as inorganic phosphate, indoxyl sulfate and advanced-glycation end products) may directly impact the VSMCs’ physiological functions. Chronic, low-grade inflammation and oxidative stress—hallmarks of CKD—are also strong inducers of VSMC dysfunction. Here, we review current knowledge about the impact of uremic toxins on VSMC function in CKD, and the consequences for pathological vascular remodeling.

Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay.

Tudor staphylococcal nuclease (TSN) is an evolutionarily conserved ribonuclease in eukaryotes that is composed of five staphylococcal nuclease-like domains (SN1–SN5) and a Tudor domain. TSN degrades hyper-edited double-stranded RNA, including primary miRNA precursors containing multiple I•U and U•I pairs, and mature miRNA during miRNA decay. However, how TSN binds and degrades its RNA substrates remains unclear. Here, we show that the C. elegans TSN (cTSN) is a monomeric Ca2+-dependent ribonuclease, cleaving RNA chains at the 5′-side of the phosphodiester linkage to produce degraded fragments with 5′-hydroxyl and 3′-phosphate ends. cTSN degrades single-stranded RNA and double-stranded RNA containing mismatched base pairs, but is not restricted to those containing multiple I•U and U•I pairs. cTSN has at least two catalytic active sites located in the SN1 and SN3 domains, since mutations of the putative Ca2+-binding residues in these two domains strongly impaired its ribonuclease activity. We further show by small-angle X-ray scattering that rice osTSN has a flexible two-lobed structure with open to closed conformations, indicating that TSN may change its conformation upon RNA binding. We conclude that TSN is a structure-specific ribonuclease targeting not only single-stranded RNA, but also unstructured regions of double-stranded RNA. This study provides the molecular basis for how TSN cooperates with RNA editing to eliminate duplex RNA in cell defense, and how TSN selects and degrades RNA during microRNA decay.

Mitochondrial tyrosyl-DNA phosphodiesterase 2 and its TDP2S short isoform.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2S) expressed from an alternative transcription start site. TDP2S contains a mitochondrial targeting sequence, contributing to its enrichment in the mitochondria and cytosol, while full-length TDP2 contains a nuclear localization signal and the ubiquitin-associated domain in the N-terminus. Our study reveals that both TDP2 isoforms are present and active in the mitochondria. Comparison of isogenic wild-type (WT) and TDP2 knockout (TDP2-/-/-) DT40 cells shows that TDP2-/-/- cells are hypersensitive to mitochondrial-targeted doxorubicin (mtDox), and that complementing TDP2-/-/- cells with human TDP2 restores resistance to mtDox. Furthermore, mtDox selectively depletes mitochondrial DNA in TDP2-/-/- cells. Using CRISPR-engineered human cells expressing only the TDP2S isoform, we show that TDP2S also protects human cells against mtDox. Finally, lack of TDP2 in the mitochondria reduces the mitochondria transcription levels in two different human cell lines. In addition to identifying a novel TDP2S isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria.