The tRNAVal half: A strong endogenous Toll-like receptor 7 ligand with a 5'-terminal universal sequence signature.

Toll-like receptors (TLRs) are crucial components of the innate immune system. Endosomal TLR7 recognizes single-stranded RNAs, yet its endogenous ssRNA ligands are not fully understood. We previously showed that extracellular (ex-) 5'-half molecules of tRNAHisGUG (the 5'-tRNAHisGUG half) in extracellular vesicles (EVs) of human macrophages activate TLR7 when delivered into endosomes of recipient macrophages. Here, we fully explored immunostimulatory ex-5'-tRNA half molecules and identified the 5'-tRNAValCAC/AAC half, the most abundant tRNA-derived RNA in macrophage EVs, as another 5'-tRNA half molecule with strong TLR7 activation capacity. Levels of the ex-5'-tRNAValCAC/AAC half were highly up-regulated in macrophage EVs upon exposure to lipopolysaccharide and in the plasma of patients infected with Mycobacterium tuberculosis. The 5'-tRNAValCAC/AAC half-mediated activation of TLR7 effectively eradicated bacteria infected in macrophages. Mutation analyses of the 5'-tRNAValCAC/AAC half identified the terminal GUUU sequence as a determinant for TLR7 activation. We confirmed that GUUU is the optimal ratio of guanosine and uridine for TLR7 activation; microRNAs or other RNAs with the terminal GUUU motif can indeed stimulate TLR7, establishing the motif as a universal signature for TLR7 activation. These results advance our understanding of endogenous ssRNA ligands of TLR7 and offer insights into diverse TLR7-involved pathologies and their therapeutic strategies.

ArcS from Thermococcus kodakarensis transfers L-lysine to preQ0 nucleoside derivatives as minimum substrate RNAs.

Archaeosine (G+) is an archaea-specific tRNA modification synthesized via multiple steps. In the first step, archaeosine tRNA guanine transglucosylase (ArcTGT) exchanges the G15 base in tRNA with 7-cyano-7-deazaguanine (preQ0). In Euryarchaea, preQ015 in tRNA is further modified by archaeosine synthase (ArcS). Thermococcus kodakarensis ArcS catalyzes a lysine-transfer reaction to produce preQ0-lysine (preQ0-Lys) as an intermediate. The resulting preQ0-Lys15 in tRNA is converted to G+15 by a radical S-adenosyl-L-methionine enzyme for archaeosine formation (RaSEA), which forms a complex with ArcS. Here, we focus on the substrate tRNA recognition mechanism of ArcS. Kinetic parameters of ArcS for lysine and tRNA-preQ0 were determined using a purified enzyme. RNA fragments containing preQ0 were prepared from Saccharomyces cerevisiae tRNAPhe-preQ015. ArcS transferred 14C-labeled lysine to RNA fragments. Furthermore, ArcS transferred lysine to preQ0 nucleoside and preQ0 nucleoside 5'-monophosphate. Thus, the L-shaped structure and the sequence of tRNA are not essential for the lysine-transfer reaction by ArcS. However, the presence of D-arm structure accelerates the lysine-transfer reaction. Because ArcTGT from thermophilic archaea recognizes the common D-arm structure, we expected the combination of T. kodakarensis ArcTGT and ArcS and RaSEA complex would result in the formation of preQ0-Lys15 in all tRNAs. This hypothesis was confirmed using 46 T. kodakarensis tRNA transcripts and three Haloferax volcanii tRNA transcripts. In addition, ArcTGT did not exchange the preQ0-Lys15 in tRNA with guanine or preQ0 base, showing that formation of tRNA-preQ0-Lys by ArcS plays a role in preventing the reverse reaction in G+ biosynthesis.

In vitro production and multiplex quantification of 2',3'-cyclic phosphate-containing 5'-tRNA half molecules.

RNA cleavages by many ribonucleases generate RNA molecules that contain a 2',3'-cyclic phosphate (cP) at their 3'-termini, and many cP-containing RNAs (cP-RNAs) are expressed as functional molecules in cells and tissues. 5'-tRNA half molecules are representative examples of functional cP-RNAs, playing important roles in various biological processes. We here show in vitro production of cP-containing 5'-tRNA half molecules that is able to prepare abundant synthetic cP-RNAs enough for functional analyses. Furthermore, we report a multiplex TaqMan RT-qPCR method which can simultaneously quantify multiple cP-containing 5'-tRNA half species. The method enabled us to efficiently quantify 5'-tRNA halves using samples with limited amounts, such as human plasma samples, revealing drastic enhancement of 5'-tRNA half levels at approximately 1,000-fold in patients infected with Mycobacterium tuberculosis. These in vitro production and multiplex quantification methods can be applied to any cP-RNAs, and they provide cost-effective, in-house techniques to accelerate expressional and functional characterizations of 5'-tRNA halves and other cP-RNAs.

Genome-wide identification of short 2',3'-cyclic phosphate-containing RNAs and their regulation in aging.

RNA molecules generated by ribonuclease cleavage sometimes harbor a 2',3'-cyclic phosphate (cP) at their 3'-ends. Those cP-containing RNAs (cP-RNAs) form a hidden layer of transcriptome because standard RNA-seq cannot capture them as a result of cP's prevention of an adapter ligation reaction. Here we provide genome-wide analyses of short cP-RNA transcriptome across multiple mouse tissues. Using cP-RNA-seq that can exclusively sequence cP-RNAs, we identified numerous novel cP-RNA species which are mainly derived from cytoplasmic tRNAs, mRNAs, and rRNAs. Determination of the processing sites of substrate RNAs for cP-RNA generation revealed highly-specific RNA cleavage events between cytidine and adenosine in cP-RNA biogenesis. cP-RNAs were not evenly derived from the overall region of substrate RNAs but rather from specific sites, implying that cP-RNAs are not from random degradation but are produced through a regulated biogenesis pathway. The identified cP-RNAs were abundantly accumulated in mouse tissues, and the expression levels of cP-RNAs showed age-dependent reduction. These analyses of cP-RNA transcriptome unravel a novel, abundant class of non-coding RNAs whose expression could have physiological roles.

Tadalafil Treatment of Mice with Fetal Growth Restriction and Preeclampsia Improves Placental mTOR Signaling.

Fetal growth restriction (FGR) is a major cause of poor perinatal outcomes. Although several studies have been conducted to improve the prognosis of FGR in infants, no effective intrauterine treatment method has been established. This study aimed to use tadalafil, a phosphodiesterase 5 inhibitor (PDE5) inhibitor, as a novel intrauterine treatment and conducted several basic and clinical studies. The study investigated the effects of tadalafil on placental mTOR signaling. Tadalafil was administered to mice with L-NG-nitroarginine methyl ester (L-NAME)-induced FGR and associated preeclampsia (PE). Placental phosphorylated mTOR (p-mTOR) signaling was assessed by fluorescent immunohistochemical staining and Western blotting. The expression of p-mTOR was significantly decreased in mice with FGR on 13 days post coitum (d.p.c.) but recovered to the same level as that of the control on 17 d.p.c. following tadalafil treatment. The results were similar for 4E-binding protein 1 (4E-BP1) and S6 ribosomal (S6R) protein, which act downstream in the mTOR signaling pathway. We demonstrate that the tadalafil treatment of FGR in mice improved placental mTOR signaling to facilitate fetal growth. Our study provides the key mechanistic detail about the mode of action of tadalafil and thus would be helpful for future clinical studies on FGR.

Identification of a radical SAM enzyme involved in the synthesis of archaeosine.

Archaeosine (G+), 7-formamidino-7-deazaguanosine, is an archaea-specific modified nucleoside found at the 15th position of tRNAs. In Euryarchaeota, 7-cyano-7-deazaguanine (preQ0)-containing tRNA (q0N-tRNA), synthesized by archaeal tRNA-guanine transglycosylase (ArcTGT), has been believed to be converted to G+-containing tRNA (G+-tRNA) by the paralog of ArcTGT, ArcS. However, we found that several euryarchaeal ArcSs have lysine transfer activity to q0N-tRNA to form q0kN-tRNA, which has a preQ0 lysine adduct as a base. Through comparative genomics and biochemical experiments, we found that ArcS forms a robust complex with a radical S-adenosylmethionine (SAM) enzyme named RaSEA. The ArcS-RaSEA complex anaerobically converted q0N-tRNA to G+-tRNA in the presence of SAM and lysine via q0kN-tRNA. We propose that ArcS and RaSEA should be considered an archaeosine synthase α-subunit (lysine transferase) and ß-subunit (q0kN-tRNA lyase), respectively.

The biogenesis pathway of tRNA-derived piRNAs in Bombyx germ cells.

Transfer RNAs (tRNAs) function in translational machinery and further serves as a source of short non-coding RNAs (ncRNAs). tRNA-derived ncRNAs show differential expression profiles and play roles in many biological processes beyond translation. Molecular mechanisms that shape and regulate their expression profiles are largely unknown. Here, we report the mechanism of biogenesis for tRNA-derived Piwi-interacting RNAs (td-piRNAs) expressed in Bombyx BmN4 cells. In the cells, two cytoplasmic tRNA species, tRNAAspGUC and tRNAHisGUG, served as major sources for td-piRNAs, which were derived from the 5'-part of the respective tRNAs. cP-RNA-seq identified the two tRNAs as major substrates for the 5'-tRNA halves as well, suggesting a previously uncharacterized link between 5'-tRNA halves and td-piRNAs. An increase in levels of the 5'-tRNA halves, induced by BmNSun2 knockdown, enhanced the td-piRNA expression levels without quantitative change in mature tRNAs, indicating that 5'-tRNA halves, not mature tRNAs, are the direct precursors for td-piRNAs. For the generation of tRNAHisGUG-derived piRNAs, BmThg1l-mediated nucleotide addition to -1 position of tRNAHisGUG was required, revealing an important function of BmThg1l in piRNA biogenesis. Our study advances the understanding of biogenesis mechanisms and the genesis of specific expression profiles for tRNA-derived ncRNAs.

Multisite-specific archaeosine tRNA-guanine transglycosylase (ArcTGT) from Thermoplasma acidophilum, a thermo-acidophilic archaeon.

Archaeosine (G(+)), which is found only at position 15 in many archaeal tRNA, is formed by two steps, the replacement of the guanine base with preQ0 by archaeosine tRNA-guanine transglycosylase (ArcTGT) and the subsequent modification of preQ0 to G(+) by archaeosine synthase. However, tRNA(Leu) from Thermoplasma acidophilum, a thermo-acidophilic archaeon, exceptionally has two G(+)13 and G(+)15 modifications. In this study, we focused on the biosynthesis mechanism of G(+)13 and G(+)15 modifications in this tRNA(Leu). Purified ArcTGT from Pyrococcus horikoshii, for which the tRNA recognition mechanism and structure were previously characterized, exchanged only the G15 base in a tRNA(Leu) transcript with (14)C-guanine. In contrast, T. acidophilum cell extract exchanged both G13 and G15 bases. Because T. acidophilum ArcTGT could not be expressed as a soluble protein in Escherichia coli, we employed an expression system using another thermophilic archaeon, Thermococcus kodakarensis. The arcTGT gene in T. kodakarensis was disrupted, complemented with the T. acidophilum arcTGT gene, and tRNA(Leu) variants were expressed. Mass spectrometry analysis of purified tRNA(Leu) variants revealed the modifications of G(+)13 and G(+)15 in the wild-type tRNA(Leu). Thus, T. acidophilum ArcTGT has a multisite specificity and is responsible for the formation of both G(+)13 and G(+)15 modifications.

Development of a Quenchbody for the Detection and Imaging of the Cancer-Related Tight-Junction-Associated Membrane Protein Claudin.

Claudins (CLs) are membrane proteins found in tight junctions and play a major role in establishing the intercellular barrier. However, some CLs are abnormally overexpressed on tumor cells and are valid clinical biomarkers for cancer diagnosis. Here, we constructed antibody Fab fragment-based Quenchbodies (Q-bodies) as effective and reliable fluorescent sensors for detecting and visualizing CLs on live tumor cells. The variable region genes for anti-CL1 and anti-CL4 antibodies were used to express recombinant Fab fragments, and clones recognizing CL4 with high affinity were selected for making Q-bodies. When two fluorescent dyes were conjugated to the N-terminal tags attached to the Fab, the fluorescent signal was significantly increased after adding nanomolar-levels of purified CL4. Moreover, addition of the Q-body to CL4-expressing cells including CL4-positive cancer cells led to a clear fluorescence signal with low background, even without washing steps. Our findings suggested that such Q-bodies would serve as a potent tool for specifically illuminating membrane targets expressed on cancer cells, both in vitro and in vivo.

Comparing Papanicolaou test results obtained during pregnancy and post-partum.

AIM: Cervical cancer onset initially occurs during youth. Papanicolaou tests performed in early pregnancy can detect cervical cancer; however, Papanicolaou tests during pregnancy have been noted to be inaccurate, reflecting changes associated with pregnancy. Therefore, we assessed the effect of pregnancy on Papanicolaou test results. METHODS: Of 1351 pregnant women who delivered at Ise Red Cross Hospital between January 2010 and December 2014, 1213 underwent Papanicolaou tests at early pregnancy and post-partum. We compared the Papanicolaou test results. RESULTS: The results of the Papanicolaou test were different in 32 patients. Of the 1191 patients negative for intraepithelial lesions or malignancy in early pregnancy, 16 had other cytological abnormalities post-partum. We performed therapeutic conization post-partum in four patients. The Papanicolaou test results in early pregnancy of the four patients were negative for intraepithelial lesions or malignancy in one patient, atypical squamous cells of undetermined significance in one and high-grade squamous intraepithelial lesion in two. CONCLUSION: The results of the Papanicolaou test during pregnancy may not be accurate because of the influence of hormones associated with pregnancy. Taking advantage of the one-month post-partum screening visit can lead to early detection and treatment of cervical cancer in young people.

The structure of metallo-DNA with consecutive thymine-HgII-thymine base pairs explains positive entropy for the metallo base pair formation.

We have determined the three-dimensional (3D) structure of DNA duplex that includes tandem Hg(II)-mediated T-T base pairs (thymine-Hg(II)-thymine, T-Hg(II)-T) with NMR spectroscopy in solution. This is the first 3D structure of metallo-DNA (covalently metallated DNA) composed exclusively of 'NATURAL' bases. The T-Hg(II)-T base pairs whose chemical structure was determined with the (15)N NMR spectroscopy were well accommodated in a B-form double helix, mimicking normal Watson-Crick base pairs. The Hg atoms aligned along DNA helical axis were shielded from the bulk water. The complete dehydration of Hg atoms inside DNA explained the positive reaction entropy (ΔS) for the T-Hg(II)-T base pair formation. The positive ΔS value arises owing to the Hg(II) dehydration, which was approved with the 3D structure. The 3D structure explained extraordinary affinity of thymine towards Hg(II) and revealed arrangement of T-Hg(II)-T base pairs in metallo-DNA.

X-ray structure of the fourth type of archaeal tRNA splicing endonuclease: insights into the evolution of a novel three-unit composition and a unique loop involved in broad substrate specificity.

Cleavage of introns from precursor transfer RNAs (tRNAs) by tRNA splicing endonuclease (EndA) is essential for tRNA maturation in Archaea and Eukarya. In the past, archaeal EndAs were classified into three types (α'2, α4 and α2ß2) according to subunit composition. Recently, we have identified a fourth type of archaeal EndA from an uncultivated archaeon Candidatus Micrarchaeum acidiphilum, referred to as ARMAN-2, which is deeply branched within Euryarchaea. The ARMAN-2 EndA forms an ε2 homodimer and has broad substrate specificity like the α2ß2 type EndAs found in Crenarchaea and Nanoarchaea. However, the precise architecture of ARMAN-2 EndA was unknown. Here, we report the crystal structure of the ε2 homodimer of ARMAN-2 EndA. The structure reveals that the ε protomer is separated into three novel units (αN, α and ßC) fused by two distinct linkers, although the overall structure of ARMAN-2 EndA is similar to those of the other three types of archaeal EndAs. Structural comparison and mutational analyses reveal that an ARMAN-2 type-specific loop (ASL) is involved in the broad substrate specificity and that K161 in the ASL functions as the RNA recognition site. These findings suggest that the broad substrate specificities of ε2 and α2ß2 EndAs were separately acquired through different evolutionary processes.

Raman spectroscopic detection of the T-Hg II-T base pair and the ionic characteristics of mercury.

Developing applications for metal-mediated base pairs (metallo-base-pair) has recently become a high-priority area in nucleic acid research, and physicochemical analyses are important for designing and fine-tuning molecular devices using metallo-base-pairs. In this study, we characterized the Hg(II)-mediated T-T (T-Hg(II)-T) base pair by Raman spectroscopy, which revealed the unique physical and chemical properties of Hg(II). A characteristic Raman marker band at 1586 cm(-1) was observed and assigned to the C4=O4 stretching mode. We confirmed the assignment by the isotopic shift ((18)O-labeling at O4) and density functional theory (DFT) calculations. The unusually low wavenumber of the C4=O4 stretching suggested that the bond order of the C4=O4 bond reduced from its canonical value. This reduction of the bond order can be explained if the enolate-like structure (N3=C4-O4(-)) is involved as a resonance contributor in the thymine ring of the T-Hg(II)-T pair. This resonance includes the N-Hg(II)-bonded state (Hg(II)-N3-C4=O4) and the N-Hg(II)-dissociated state (Hg(II+) N3=C4-O4(-)), and the latter contributor reduced the bond order of N-Hg(II). Consequently, the Hg(II) nucleus in the T-Hg(II)-T pair exhibited a cationic character. Natural bond orbital (NBO) analysis supports the interpretations of the Raman experiments.

Hg2+-trapping beads: Hg2+-specific recognition through thymine-Hg(II)-thymine base pairing.

Mercury pollution poses a severe threat to human health. To remove Hg(2+) from contaminated water, we synthesized Hg(2+)-trapping beads that include oligo-thymidine functionalities that can form thymine-Hg(II)-thymine base pairs on the solid support. The beads can selectively trap Hg(2+) even in the presence of other metal cations. More interestingly, Hg(2+)-trapping efficiency was higher in the presence of the co-existing cations. Thus, the developed Hg(2+)-trapping beads can capture Hg(2+) without affecting the mineral balance of water so much. The Hg(2+)-trapping beads presented here show promise for removing Hg(2+) from environmental water.

Transfer RNA methyltransferases from Thermoplasma acidophilum, a thermoacidophilic archaeon.

We investigated tRNA methyltransferase activities in crude cell extracts from the thermoacidophilic archaeon Thermoplasma acidophilum. We analyzed the modified nucleosides in native initiator and elongator tRNAMet, predicted the candidate genes for the tRNA methyltransferases on the basis of the tRNAMet and tRNALeu sequences, and characterized Trm5, Trm1 and Trm56 by purifying recombinant proteins. We found that the Ta0997, Ta0931, and Ta0836 genes of T. acidophilum encode Trm1, Trm56 and Trm5, respectively. Initiator tRNAMet from T. acidophilum strain HO-62 contained G+, m1I, and m22G, which were not reported previously in this tRNA, and the m2G26 and m22G26 were formed by Trm1. In the case of elongator tRNAMet, our analysis showed that the previously unidentified G modification at position 26 was a mixture of m2G and m22G, and that they were also generated by Trm1. Furthermore, purified Trm1 and Trm56 could methylate the precursor of elongator tRNAMet, which has an intron at the canonical position. However, the speed of methyl-transfer by Trm56 to the precursor RNA was considerably slower than that to the mature transcript, which suggests that Trm56 acts mainly on the transcript after the intron has been removed. Moreover, cellular arrangements of the tRNA methyltransferases in T. acidophilum are discussed.

Immunoactive signatures of circulating tRNA- and rRNA-derived RNAs in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is the most prevalent lung disease, and macrophages play a central role in the inflammatory response in COPD. We here report a comprehensive characterization of circulating short non-coding RNAs (sncRNAs) in plasma from patients with COPD. While circulating sncRNAs are increasingly recognized for their regulatory roles and biomarker potential in various diseases, the conventional RNA sequencing (RNA-seq) method cannot fully capture these circulating sncRNAs due to their heterogeneous terminal structures. By pre-treating the plasma RNAs with T4 polynucleotide kinase, which converts all RNAs to those with RNA-seq susceptible ends (5'-phosphate and 3'-hydroxyl), we comprehensively sequenced a wide variety of non-microRNA sncRNAs, such as 5'-tRNA halves containing a 2',3'-cyclic phosphate. We discovered a remarkable accumulation of the 5'-half derived from tRNAValCAC in plasma from COPD patients, whereas the 5'-tRNAGlyGCC half is predominant in healthy donors. Further, the 5'-tRNAValCAC half activates human macrophages via Toll-like receptor 7 and induces cytokine production. Additionally, we identified circulating rRNA-derived fragments that were upregulated in COPD patients and demonstrated their ability to induce cytokine production in macrophages. Our findings provide evidence of circulating, immune-active sncRNAs in patients with COPD, suggesting that they serve as inflammatory mediators in the pathogenesis of COPD.