NMR Studies of Genomic RNA in 3' Untranslated Region Unveil Pseudoknot Structure that Initiates Viral RNA Replication in SARS-CoV-2.

In the 3' untranslated region of the SARS-CoV-2 virus RNA genome, genomic RNA replication is initiated in the highly conserved region called 3'PK, containing three stem structures (P1pk, P2, and P5). According to one proposed mechanism, P1pk and distal P2 stems switch their structure to a pseudoknot through base-pairing, thereby initiating transcription by recruiting RNA-dependent RNA polymerase complexed with nonstructural proteins (nsp)7 and nsp8. However, experimental evidence of pseudoknot formation or structural switching is unavailable. Using SARS-CoV-2 3'PK fragments, we show that 3'PK adopted stem-loop and pseudoknot forms in a mutually exclusive manner. When P1pk and P2 formed a pseudoknot, the P5 stem, which includes a sequence at the 3' end, exited from the stem-loop structure and opened up. Interaction with the nsp7/nsp8 complex destabilized the stem-loop form but did not alter the pseudoknot form. These results suggest that the interaction between the pseudoknot and nsp7/nsp8 complex transformed the 3' end of viral genomic RNA into single-stranded RNA ready for synthesis, presenting the unique pseudoknot structure as a potential pharmacological target.

An NMR-based approach reveals the core structure of the functional domain of SINEUP lncRNAs.

Long non-coding RNAs (lncRNAs) are attracting widespread attention for their emerging regulatory, transcriptional, epigenetic, structural and various other functions. Comprehensive transcriptome analysis has revealed that retrotransposon elements (REs) are transcribed and enriched in lncRNA sequences. However, the functions of lncRNAs and the molecular roles of the embedded REs are largely unknown. The secondary and tertiary structures of lncRNAs and their embedded REs are likely to have essential functional roles, but experimental determination and reliable computational prediction of large RNA structures have been extremely challenging. We report here the nuclear magnetic resonance (NMR)-based secondary structure determination of the 167-nt inverted short interspersed nuclear element (SINE) B2, which is embedded in antisense Uchl1 lncRNA and upregulates the translation of sense Uchl1 mRNAs. By using NMR 'fingerprints' as a sensitive probe in the domain survey, we successfully divided the full-length inverted SINE B2 into minimal units made of two discrete structured domains and one dynamic domain without altering their original structures after careful boundary adjustments. This approach allowed us to identify a structured domain in nucleotides 31-119 of the inverted SINE B2. This approach will be applicable to determining the structures of other regulatory lncRNAs.

Structures and Catalytic Activities of Complexes between Heme and All Parallel-Stranded Monomeric G-Quadruplex DNAs.

Heme in its ferrous and ferric states [heme(Fe2+) and heme(Fe3+), respectively] binds selectively to the 3'-terminal G-quartet of all parallel-stranded monomeric G-quadruplex DNAs formed from inosine(I)-containing sequences, i.e., d(TAGGGTGGGTTGGGTGIG) DNA(18mer) and d(TAGGGTGGGTTGGGTGIGA) DNA(18mer/A), through a π-π stacking interaction between the porphyrin moiety of the heme and the G-quartet, to form 1:1 complexes [heme-DNA(18mer) and heme-DNA(18mer/A) complexes, respectively]. These complexes exhibited enhanced peroxidase activities, compared with that of heme(Fe3+) alone, and the activity of the heme(Fe3+)-DNA(18mer/A) complex was greater than that of the heme(Fe3+)-DNA(18mer) one, indicating that the 3'-terminal A of the DNA sequence acts as an acid-base catalyst that promotes the catalytic reaction. In the complexes, a water molecule (H2O) at the interface between the heme and G-quartet is coordinated to the heme Fe atom as an axial ligand and possibly acts as an electron-donating ligand that promotes heterolytic peroxide bond cleavage of hydrogen peroxide bound to the heme Fe atom, trans to the H2O, for the generation of an active species. The intermolecular nuclear Overhauser effects observed among heme, DNA, and Fe-bound H2O indicated that the H2O rotates about the H2O-Fe coordination bond with respect to both the heme and DNA in the complex. Thus, the H2O in the complex is unique in terms of not only its electronic properties but also its dynamic ones. These findings provide novel insights into the design of heme-deoxyribozymes and -ribozymes.

Identification of functional features of synthetic SINEUPs, antisense lncRNAs that specifically enhance protein translation.

SINEUPs are antisense long noncoding RNAs, in which an embedded SINE B2 element UP-regulates translation of partially overlapping target sense mRNAs. SINEUPs contain two functional domains. First, the binding domain (BD) is located in the region antisense to the target, providing specific targeting to the overlapping mRNA. Second, the inverted SINE B2 represents the effector domain (ED) and enhances translation. To adapt SINEUP technology to a broader number of targets, we took advantage of a high-throughput, semi-automated imaging system to optimize synthetic SINEUP BD and ED design in HEK293T cell lines. Using SINEUP-GFP as a model SINEUP, we extensively screened variants of the BD to map features needed for optimal design. We found that most active SINEUPs overlap an AUG-Kozak sequence. Moreover, we report our screening of the inverted SINE B2 sequence to identify active sub-domains and map the length of the minimal active ED. Our synthetic SINEUP-GFP screening of both BDs and EDs constitutes a broad test with flexible applications to any target gene of interest.

High Glucose Stimulates Mineralocorticoid Receptor Transcriptional Activity Through the Protein Kinase C ß Signaling.

Activation of mineralocorticoid receptor (MR) is shown in resistant hypertension including diabetes mellitus. Although protein kinase C (PKC) signaling is involved in the pathogenesis of diabetic complications, an association between PKC and MR is not known. Activation of PKCα and PKCß by TPA (12-O-Tetradecanoylphorbol 13-acetate) increased MR proteins and its transcriptional activities in HEK293-MR cells. In contrast, a high glucose condition resulted in PKCß but not PKCα activation, which is associated with elevation of MR protein levels and MR transcriptional activities. Reduction of endogenous PKCß by siRNA decreased those levels. Interestingly, high glucose did not affect MR mRNA levels, but rather decreased ubiquitination of MR proteins. In db/db mice kidneys, levels of phosphorylated PKCß2, MR and Sgk-1 proteins were elevated, and the administration of PKC inhibitor reversed these changes compared to db/+ mice. These data suggest that high glucose stimulates PKCß signaling, which leads to MR stabilization and its transcriptional activities.

Structure of Musashi1 in a complex with target RNA: the role of aromatic stacking interactions.

Mammalian Musashi1 (Msi1) is an RNA-binding protein that regulates the translation of target mRNAs, and participates in the maintenance of cell 'stemness' and tumorigenesis. Msi1 reportedly binds to the 3'-untranslated region of mRNA of Numb, which encodes Notch inhibitor, and impedes initiation of its translation by competing with eIF4G for PABP binding, resulting in triggering of Notch signaling. Here, the mechanism by which Msi1 recognizes the target RNA sequence using its Ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), RBD1 and RBD2 has been revealed on identification of the minimal binding RNA for each RBD and determination of the three-dimensional structure of the RBD1:RNA complex. Unique interactions were found for the recognition of the target sequence by Msi1 RBD1: adenine is sandwiched by two phenylalanines and guanine is stacked on the tryptophan in the loop between ß1 and α1. The minimal recognition sequences that we have defined for Msi1 RBD1 and RBD2 have actually been found in many Msi1 target mRNAs reported to date. The present study provides molecular clues for understanding the biology involving Musashi family proteins.

Prediction of recovery time from hypoglycemia in patients with insulin overdose.

Insulin overdose results in prolonged hypoglycemia. We hypothesized that if a huge amount of insulin is subcutaneously injected, the duration of hypoglycemia depends on the dose of insulin rather than the type of insulin. We conducted a literature review of insulin overdose and 33 cases were included in this study. We assessed the correlation between recovery time from hypoglycemia and insulin dose. As a result, there was a significant correlation between recovery time from hypoglycemia and insulin dose (r=0.88, p<0.0001) and this correlation was expressed as y=0.045x; where y is time (h) and x is insulin dose (U), corresponding to that if 1000 U insulin is injected, hypoglycemia will persist for ~45 h. This equation may be useful to predict the duration of glucose supplementation for treatment of insulin overdose.

Structural analysis of Musashi-RNA complex on the basis of long-range structural information.

Musashi protein is supposed to be involved in the regulation of differentiation of neural stem cells. Musashi binds to 3' untranslated region of target mRNA and represses the translation of mRNA. Musashi has two tandem RNA-binding domains (RBDs), RBD1 and RBD2. Both RBDs cooperatively bind to the target mRNA. Here, we determined the structure of RBD1-RBD2 in complex with target RNA. First, the structures of two RBDs in the complex were determined on the basis of short-range distance restrains derived from NOEs. However, the relative position of two RBDs was not determined due to the lack of long-range distance restraints across two RBDs. In order to overcome the situation, we introduced the paramagnetic center into Musashi by attaching MTSL carrying the NO radical. The long-range distance restraints (ca. 20-40 A) between two RBDs were derived from paramagnetic relaxation enhancement (PRE) caused by the paramagnetic center. The relative position of two RBDs was successfully determined on the basis of these long-range distance restraints. The change in the relative position of two RBDs on binding to the target RNA was also detected by PRE. The determined structure of RBD1-RBD2 in the complex has suggested how Musashi recognizes its target mRNA.

Unexpected A-form formation of 4'-thioDNA in solution, revealed by NMR, and the implications as to the mechanism of nuclease resistance.

Fully modified 4'-thioDNA, an oligonucleotide only comprising 2'-deoxy-4'-thionucleosides, exhibited resistance to an endonuclease, in addition to preferable hybridization with RNA. Therefore, 4'-thioDNA is promising for application as a functional oligonucleotide. Fully modified 4'-thioDNA was found to behave like an RNA molecule, but no details of its structure beyond the results of circular dichroism analysis are available. Here, we have determined the structure of fully modified 4'-thioDNA with the sequence of d(CGCGAATTCGCG) by NMR. Most sugars take on the C3'-endo conformation. The major groove is narrow and deep, while the minor groove is wide and shallow. Thus, fully modified 4'-thioDNA takes on the A-form characteristic of RNA, both locally and globally. The only structure reported for 4'-thioDNA showed that partially modified 4'-thioDNA that contained some 2'-deoxy-4'-thionucleosides took on the B-form in the crystalline form. We have determined the structure of 4'-thioDNA in solution for the first time, and demonstrated unexpected differences between the two structures. The origin of the formation of the A-form is discussed. The remarkable biochemical properties reported for fully modified 4'-thioDNA, including nuclease-resistance, are rationalized in the light of the elucidated structure.

Interactions with RNA/DNA of proteins involved in the regulation of transcription, translation and telomere elongation.

Interactions with DNA and RNA of three different proteins involved in the regulation of (1) transcription, (2) translation, and (3) telomere elongation were examined by NMR. In the first case, the combination of structural determination, dynamical analysis on the basis of relaxation data and identification of interactive surface for wild and phosphorylation-mimicking mutant proteins has given the insight on the increase of DNA-binding affinity through phosphorylation of the protein. In the second case, the arrangement of two tandem domains interacting with RNA has been determined with residual dipolar couplings and paramagnetic relaxation enhancement, which has given the idea on how the two tandem domains recognize the target RNA. In the third case, simultaneous binding of the other two tandem domains to both DNA and RNA has been analyzed with chemical shift perturbation analysis. The result has suggested that the protein composed of two tandem domains can recruit telomerase to telomere DNA.

Structure of 4'-thioDNA which exhibits endonuclease resistance.

4'-thio DNA consisting of 2'-deoxy-4'-thionucleosides exhibits resistance to both endonuclease and 3'-exonuclease cleavages. Interestingly, we found that 4'-thioDNA duplex behaved like RNA molecules in hybridization properties and structural aspects. Here, we have determined the structure of 4'-thioDNA duplex in solution by NMR. Most residues take on C3'-endosugar puckering, which is characteristic to A-form. The major groove of 4'-thioDNA duplex is narrow and deep, while the minor groove is wide and shallow. These features are also characteristic to A-form. Thus, although DNA duplex usually takes on B-form in solution, 4'-thioDNA takes on A-form, like RNA molecules. A-form-like groove features of 4'-thioDNA can account for the fact that 4'-thioDNA duplex interacts with an RNA major groove binder, but not with DNA groove binder. Interaction of 4'-thioDNA with RNase V1 and resistance to endonuclease may also be accounted for in the same context.

Structural analysis of DNA containing (6-4) photoproduct in complex with distamycin A by NMR.

We reported that distamycin A can bind to DNA containing the (6-4) photoproduct, one of the major UV lesions in DNA, by means of CD spectroscopy. Here, we have analyzed the structure of DNA containing the (6-4) photoproduct in complex with distamycin A by NMR. Broadening of NMR resonances and large chemical shift perturbation have been observed for residues located close to the (6-4) photoproduct in DNA, indicating the binding to this region. Intermolecular NOEs between DNA and distamycin A have been identified for some of these residues, suggesting the formation of the relatively tight complex. On the basis of these NOEs, structure determination of the complex is now in progress.

Formation of a complex of 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin with G-quadruplex DNA.

A water-soluble cationic porphyrin, 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin (TmPyP4), has been studied extensively because of its unique physicochemical properties that lead to interactions with nucleic acids, as well as its therapeutic application. Formation of a complex between TmPyP4 and parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d(TTAGGG), has been characterized in an effort to elucidate the mode of molecular recognition between TmPyP4 and the DNA. The study demonstrated that TmPyP4 intercalates into the A3pG4 step of [d(TTAGGG)]4 with an association constant of 6.2 x 10(6) M(-1) and a stoichiometric ratio of 1:1. The binding of TmPyP4 to the A3pG4 step of [d(TTAGGG)]4 was found to be stabilized by the pi-pi stacking interaction of the porphyrin ring of TmPyP4 with the G4 quartet as well as the A3 bases of the G-quadruplex DNA. These findings provide novel insights for the design of porphyrin derivatives that bind to DNA with high affinity and specificity.

Dynamics and thermodynamics of dimerization of parallel G-quadruplexed DNA formed from d(TTAGn) (n=3-5).

Parallel G-quadruplexes formed from oligonucleotide sequences, d(TTAGn), where n = 3-5, have been shown to form a dimer through end-to-end stacking of 3'-terminal G-tetrads. The monomers and dimers of the G-quadruplexes are in dynamic equilibrium with an exchange rate of approximately 1 s-1. A thermodynamic study demonstrated that the dimerization of the G-quadruplexes is largely enthalpic in origin.

Structural and functional characterization of novel G-quadruplexed DNA-heme coordination complex.

We analyzed the coordination structure of G-quadruplexed DNA-heme complex which exhibits remarkably similar spectroscopic characters to hemoprotein such as myoglobin. We found that some exogenous ligands can be accommodated at the 6th coordination site of heme in the G-quadruplexed DNA-heme complex with a DNA base coordinated to the 5th site. The G-quadruplexed DNA-hemin complex is expected to play a role as a novel DNAzyme. These findings provide novel insights into molecular design for creating artificial heme enzymes using spontaneous assembly between G-quadruplexed DNA and heme.

Dynamics and thermodynamics of dimerization of parallel G-quadruplexed DNA through pi-pi stacking interaction.

DNA fragments of d(TTAG(n)) (n = 3-5) form all-parallel G-quadruplexed structure in the presence of K+. We found that G-quadruplexed d(TTAG(n)) forms dimer through end-to-end stacking of the 3'-terminal G-tetrads. In this study, we report the structure of the dimer, and the dynamics and thermodynamics of the dimerization.

Binding of 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin to an AT-rich region of a duplex DNA.

Characterization of the interaction between DNA and small organic compounds is of considerable importance for gaining insights into the mechanism underlying molecular recognition, which could be highly relevant to drug design. In the present study, the interaction of a water-soluble cationic porphyrin, 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin, with a self-complementary duplex DNA, d(GCTTAAGC)2, has been investigated by means of absorption, circular dichroism, and NMR spectroscopies. The optical studies indicated that TMPyP binds to the TTAA region of d(GCTTAAGC)2 with a binding constant of 2.5 x 10(6) M(-1) and a stoichiometric ratio of 1:1. The observation of intermolecular nuclear Overhauser effect connectivities demonstrated that TMPyP binds in the major groove of d(GCTTAAGC)2. A model for the binding of TMPyP in the major groove of the AT-rich region of d(GCTTAAGC)2 is proposed.

Study on the complexation between DNA and cationic porphyrin derivatives.

A water-soluble cationic porphyrin, 5,10,15,20-Tetra(N-methylpyridinium-4-yl)-21H,23H-porphyrin has been shown to intercalate selectively into the A3-G4 gap of C-quadruplexed DNA d(TTAGGG)4.

Study on interaction of a cationic porphyrin with DNA.

5,10,15,20-Tetra(N-methyl-pyridinium-4-yl)-21H,23H-porphyrin has shown to bind to the major groove of AT-rich DNA predominantly through electrostatic interaction between positively charged N-methyl pyridinium moieties of the porphyrin and negatively charged phosphodiester backbone. Solution structure of the complex between the porphyrin and a double-stranded DNA fragment has been inferred from the measurements of the mixing time-dependent intermolecular nuclear Overhauser effects (NOEs).

Coordination complex between haemin and parallel-quadruplexed d(TTAGGG).

Haemin, iron(III)-protoporphyrin IX complex, and parallel-quadruplexed d(TTAGGG) have been shown to form a stable coordination complex which exhibits spectroscopic properties remarkably similar to those of haemoproteins.