Deficiency of m 6 A RNA methylation promotes ZBP1-mediated cell death.

m 6 A RNA methylation suppresses the immunostimulatory potential of endogenous RNA. Deficiency of m 6 A provokes inflammatory responses and cell death, but the underlying mechanisms remain elusive. Here we showed that the noncoding RNA 7SK gains immunostimulatory potential upon m 6 A depletion and subsequently activates the RIG-I/MAVS axis to spark interferon (IFN) signaling cascades. Concomitant excess of IFN and m 6 A deficiency synergistically facilitate the formation of RNA G-quadruplexes (rG4) to promote ZBP1-mediated necroptotic cell death. Collectively, our findings delineate a hitherto uncharacterized mechanism that links m 6 A dysregulation with ZBP1 activity in triggering inflammatory cell death.

Structural analysis of the regulation of blue-light receptors by GIGANTEA.

In Arabidopsis, GIGANTEA (GI), together with the blue-light receptors ZTL, LKP2, and FKF1, regulates degradation of the core clock protein TOC1 and the flowering repressor CDFs, thereby controlling circadian oscillation and flowering. Despite the significance of GI in diverse plant physiology, its molecular function is not much understood because of technical problems in protein preparation and a lack of structural information. Here, we report the purification of the GI monomer and the crystal structure of the GI/LKP2 complex. The crystal structure reveals that residues 1-813 of GI possess an elongated rigid structure formed by stacking hydrophobic α-helices and that the LOV domain of LKP2 binds to the middle region of the GI (residues 563-789). Interaction analysis further shows that LOV homodimers are converted to monomers by GI binding. Our results provide structural insights into the regulation of the circadian clock and photoperiodic flowering by GI and ZTL/LKP2/FKF1.

Crystal structure of a tandem B-box domain from Arabidopsis CONSTANS.

CONSTANS is a central protein in the regulation of photoperiodic flowering, which is expressed in response to day length and promotes the expression of Flowering Locus T. The tandem B-box domain in CONSTANS mediates interactions with various proteins to regulate the expression of Flowering Locus T. Although most plants, including Arabidopsis, have multiple B-box proteins, their B-box structures have not been elucidated. Here, we report the crystal structure of a tandem B-box domain from Arabidopsis CONSTANS. The crystal structure shows that each B-box adopts a canonical B-box fold and coordinates two zinc atoms. Furthermore, the crystal structure reveals that the B-box domain has a unique structure that distinguishes it from animal B-boxes at the monomer and dimer level.

The CAGE-MiR-181b-5p-S1PR1 Axis Regulates Anticancer Drug Resistance and Autophagy in Gastric Cancer Cells.

Cancer-associated gene (CAGE), a cancer/testis antigen, has been known to promote anticancer drug resistance. Since the underlying mechanisms of CAGE-promoted anticancer drug resistance are poorly understood, we established Anticancer drug-resistant gastric cancer cells (AGS R ) to better elucidate possible mechanisms. AGS R showed an increased expression level of CAGE and autophagic flux compared with anticancer drug-sensitive parental gastric cancer cells (AGS cells). AGS R cells showed higher invasion potential, growth rate, tumor spheroid formation, and angiogenic potential than AGS cells. CAGE exerted effects on the response to anticancer drugs and autophagic flux. CAGE was shown to bind to Beclin1, a mediator of autophagy. Overexpression of CAGE increased autophagic flux and invasion potential but inhibited the cleavage of PARP in response to anticancer drugs in CAGE CRISPR-Cas9 cell lines. TargetScan analysis was utilized to predict the binding of miR-302b-5p to the promoter sequences of CAGE, and the results show that miR-302b-5p directly regulated CAGE expression as illustrated by luciferase activity. MiR-302b-5p regulated autophagic flux and the response to anticancer drugs. CAGE was shown to bind the promoter sequences of miR-302b-5p. The culture medium of AGS R cells increased CAGE expression and autophagic flux in AGS cells. ImmunoEM showed CAGE was present in the exosomes of AGS R cells; exosomes of AGS R cells and human recombinant CAGE protein increased CAGE expression, autophagic flux, and resistance to anticancer drugs in AGS cells. MicroRNA array revealed miR-181b-5p as a potential negative regulator of CAGE. MiR-181b-5p inhibitor increased the expression of CAGE and autophagic flux in addition to preventing anticancer drugs from cleaving poly(ADP-ribose) polymerase (PARP) in AGS cells. TargetScan analysis predicted sphingosine 1-phosphate receptor 1 (SIPR1) as a potential target for miR-181b-5p. CAGE showed binding to the promoter sequences of S1PR1. The downregulation or inhibition of S1PR1 led to decreased autophagic flux but enhanced the sensitivity to anticancer drugs in AGS R cells. This study presents a novel role of the CAGE-miR-181b-5p-S1PR1 axis in anticancer drug resistance and autophagy.

Structural insights into the regulation of SigB activity by RsbV and RsbW.

Bacillus subtilis SigB is an alternative sigma factor that initiates the transcription of stress-responsive genes. The anti-sigma factor RsbW tightly binds SigB to suppress its activity under normal growth conditions and releases it when nonphosphorylated RsbV binds to RsbW in response to stress signals. To understand the regulation of SigB activity by RsbV and RsbW based on structural features, crystal structures and a small-angle X-ray scattering (SAXS) envelope structure of the RsbV-RsbW complex were determined. The crystal structures showed that RsbV and RsbW form a heterotetramer in a similar manner to a SpoIIAA-SpoIIAB tetramer. Multi-angle light scattering and SAXS revealed that the RsbV-RsbW complex is an octamer in solution. Superimposition of the crystal structure on the SAXS envelope structure showed that the unique dimeric interface of RsbW mediates the formation of an RsbV-RsbW octamer and does not prevent RsbV and SigB from binding to RsbW. These results provide structural insights into the molecular assembly of the RsbV-RsbW complex and the regulation of SigB activity.

Selective interactions between mimivirus uracil-DNA glycosylase and inhibitory proteins determined by a single amino acid.

Uracil-N-glycosylase (UNG) is found in most organisms as well as in large DNA viruses. Its inhibitory proteins, including uracil glycosylase inhibitor (UGI) and p56, tightly bind to the active site of UNG by mimicking the DNA substrates. As the binding motifs are conserved in UNG family proteins, the inhibitory proteins bind to various UNG proteins across species. However, the intercalation residue that penetrates the DNA minor groove during uracil excision is not conserved among UNG proteins. To understand the role of the intercalation residue in their binding to the inhibitory proteins, we prepared mutants of mimivirus UNG, measured the binding affinity between the UNG mutants and inhibitory proteins, and analyzed the interactions based on the crystal structures of mimivirus UNG mutants complexed with UGI. The results show that mimivirus UNG, which harbors Tyr as an intercalation residue, did not interact with the inhibitory proteins intrinsically, whereas mutations of the intercalation residue to Phe or Leu resulted in tight interactions with UGI and p56; mutation to Met resulted in tight interactions only with p56. The crystal structures revealed that Phe and Leu stabilize the interactions by fitting into the hydrophobic pocket of UGI. These results show that differences in size and hydrophobicity of the intercalation residues determine the interactions between UNG family proteins and the inhibitory proteins, UGI and p56.

Structural insights into stressosome assembly.

The stressosome transduces environmental stress signals to SigB to upregulate SigB-dependent transcription, which is required for bacterial viability. The stressosome core is composed of RsbS and at least one of the RsbR paralogs. A previous cryo-electron microscopy (cryo-EM) structure of the RsbRA-RsbS complex determined under a D2 symmetry restraint showed that the stressosome core forms a pseudo-icosahedron consisting of 60 STAS domains of RsbRA and RsbS. However, it is still unclear how RsbS and one of the RsbR paralogs assemble into the stressosome. Here, an assembly model of the stressosome is presented based on the crystal structure of the RsbS icosahedron and cryo-EM structures of the RsbRA-RsbS complex determined under diverse symmetry restraints (nonsymmetric C1, dihedral D2 and icosahedral I envelopes). 60 monomers of the crystal structure of RsbS fitted well into the I-restrained cryo-EM structure determined at 4.1 Šresolution, even though the STAS domains in the I envelope were averaged. This indicates that RsbS and RsbRA share a highly conserved STAS fold. 22 protrusions observed in the C1 envelope, corresponding to dimers of the RsbRA N-domain, allowed the STAS domains of RsbRA and RsbS to be distinguished in the stressosome core. Based on these, the model of the stressosome core was reconstructed. The mutation of RsbRA residues at the binding interface in the model (R189A/Q191A) significantly reduced the interaction between RsbRA and RsbS. These results suggest that nonconserved residues in the conserved STAS folds between RsbS and RsbR paralogs determine stressosome assembly.

Structural analysis of the recognition of the -35 promoter element by SigW from Bacillus subtilis.

Sigma factors are key proteins that mediate the recruitment of RNA polymerase to the promoter regions of genes, for the initiation of bacterial transcription. Multiple sigma factors in a bacterium selectively recognize their cognate promoter sequences, thereby inducing the expression of their own regulons. In this paper, we report the crystal structure of the σ4 domain of Bacillus subtilis SigW bound to the -35 promoter element. Purine-specific hydrogen bonds of the -35 promoter element with the recognition helix α9 of the σ4 domain occurs at three nucleotides of the consensus sequence (G-35, A-34, and G'-31 in G-35A-34A-33A-32C-31C-30T-29). The hydrogen bonds of the backbone with the α7 and α8 of the σ4 domain occurs at G'-30. These results elucidate the structural basis of the selective recognition of the promoter by SigW. In addition, comparison of SigW structures complexed with the -35 promoter element or with anti-sigma RsiW reveals that DNA recognition and anti-sigma factor binding of SigW are mutually exclusive.

Crystal structure of mimivirus uracil-DNA glycosylase.

Cytosine deamination induced by stresses or enzymatic catalysis converts deoxycytidine into deoxyuridine, thereby introducing a G to A mutation after DNA replication. Base-excision repair to correct uracil to cytosine is initiated by uracil-DNA glycosylase (UDG), which recognizes and eliminates uracil from DNA. Mimivirus, one of the largest known viruses, also encodes a distinctive UDG gene containing a long N-terminal domain (N-domain; residues 1-130) and a motif-I (residues 327-343), in addition to the canonical catalytic domain of family I UDGs (also called UNGs). To understand the structural and functional features of the additional segments, we have determined the crystal structure of UNG from Acanthamoeba polyphaga mimivirus (mvUNG). In the crystal structure of mvUNG, residues 95-130 in the N-domain bind to a hydrophobic groove in the catalytic domain, and motif-I forms a short ß-sheet with a positively charged surface near the active site. Circular dichroism spectra showed that residues 1-94 are in a random coil conformation. Deletion of the three additional fragments reduced the activity and thermal stability, compared to full-length mvUNG. The results suggested that the mvUNG N-domain and motif-I are required for its structural and functional integrity.