The transcription factor NFB is activated by phosphorylation and acetylation and plays important roles in inflammatory and immune responses in the cell. Additionally, posttranslational modification of the NFB p65 subunit by O-linked N-acetylglucosamine (OGlcNAc) has been reported, but the modification site of O-GlcNAc on NFB p65 and its exact function have not been elucidated. In this work, we show that O-GlcNAcylation of NFB p65 decreases binding to IB␣ and increases transcriptional activity under hyperglycemic conditions. Also, we demonstrate that both Thr-322 and Thr-352 of NFB p65 can be modified with O-GlcNAc, but modification on Thr-352, not Thr-322, is important for transcriptional activation. Our findings suggest that site-specific O-GlcNAcylation may be a reason why NFB activity increases continuously under hyperglycemic conditions.T ranscription factor NFB plays important roles in inflammatory, immune, and antiapoptotic responses (1-3). In mammals, NFB is present as a dimer composed of various combinations of Rel proteins such as p65 (RelA), RelB, c-Rel, p50/p105, and p52/p100. In most cell types, NFB is composed of p65 and p50 and is localized in the cytosol where it binds inhibitor (IB). Treatment with NFB-activating agents such as tumor necrosis factor ␣ (TNF␣) activates IB kinase (IKK) complexes, inducing phosphorylation in the N terminus of IB. The phosphorylation event induces IB degradation via a ubiquitin-dependent proteolysis. Free NFB translocates to the nucleus and activates the expression of target genes (1, 2).Posttranslational modifications such as phosphorylation (4-9) and acetylation (10, 11) regulate the transcriptional activity of NFB. The activity of NFB is influenced also by the hexosamine biosynthetic pathway, which produces a substrate of O-GlcNAcylation, UDP-GlcNAc (12). Many nucleocytoplasmic proteins are known to be dynamically modified with O-GlcNAc. This modification is modulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) (13-18). O-GlcNAcylation levels play an important role in transcription, translation, nuclear transport, protein stability, and protein-protein interactions and can be increased under hyperglycemic conditions caused by diabetes (16 -18). Although it is known that O-GlcNAcylation of NFB is involved in hyperglycemia-induced NFB activation (12) and is required for lymphocyte activation (19), the specific sites and the function of O-GlcNAcylation on NFB are not well understood.In this work, we show that OGA overexpression downregulates O-GlcNAcylation and inhibits hyperglycemia-induced NFB activation in rat vascular smooth muscle cells (VSMCs). In contrast, up-regulation of O-GlcNAcylation after OGT overexpression and treatment of cells with the O-GlcNAcase inhibitors streptozotocin (STZ) (20) and O-(2-acetamido-2-deoxy-Dglucopyranosylidene)amino-N-phenylcarbamate (PUGNAc)(21) increase NFB transcriptional activity. Additionally, we identify Thr-322 and Thr-352 as O-GlcNAcylation sites in a mutation study and with mass spectrometry analysis. Our data sho...

Protein O-phosphorylation often occurs reciprocally with O-GlcNAc modification and represents a regulatory principle for proteins. O-phosphorylation of serine by glycogen synthase kinase-3b on Snail1, a transcriptional repressor of E-cadherin and a key regulator of the epithelial-mesenchymal transition (EMT) programme, results in its proteasomal degradation. We show that by suppressing O-phosphorylation-mediated degradation, O-GlcNAc at serine112 stabilizes Snail1 and thus increases its repressor function, which in turn attenuates E-cadherin mRNA expression. Hyperglycaemic condition enhances O-GlcNAc modification and initiates EMT by transcriptional suppression of E-cadherin through Snail1. Thus, dynamic reciprocal O-phosphorylation and OGlcNAc modification of Snail1 constitute a molecular link between cellular glucose metabolism and the control of EMT.

Cytoplasmic male sterility (CMS) in plants is known to be associated with novel open reading frames (ORFs) that result from recombination events in the mitochondrial genome. In this study Southern and Northern blot analyses using several mitochondrial DNA probes were conducted to detect the presence of differing band patterns between male fertile and CMS lines of chili pepper (Capsicum annuum L.). In the CMS pepper, a novel ORF, termed orf456, was found at the 3'-end of the coxll gene. Western blot analysis revealed the expression of an approximately 17-kDa product in the CMS line, and the intensity of expression of this protein was severely reduced in the restorer pepper line. To investigate the functional role of the ORF456 protein in plant mitochondria, we carried out two independent experiments to transform Arabidopsis with a mitochondrion-targeted orf456 gene construct by Agrobacterium-mediated transformation. About 45 % of the T1 transgenic population showed the male-sterile phenotype and no seed set. Pollen grains from semi-sterile T1 plants were observed to have defects on the exine layer and vacuolated pollen phenotypes. It is concluded that this newly discovered orf456 may represent a strong candidate gene--from among the many CMS-associated mitochondrial genes--for determining the male-sterile phenotype of CMS in chili pepper.

Genome editing has been harnessed through the development of CRISPR system, and the CRISPR from Prevotella and Francisella 1 (Cpf1) system has emerged as a promising alternative to CRISPR-Cas9 for use in various circumstances. Despite the inherent multiple advantages of Cpf1 over Cas9, the adoption of Cpf1 has been unsatisfactory because of target-dependent insufficient indel efficiencies. Here, we report an engineered CRISPR RNA (crRNA) for highly efficient genome editing by Cpf1, which includes a 20-base target-complementary sequence and a uridinylate-rich 3′-overhang. When the crRNA is transcriptionally produced, crRNA with a 20-base target-complementary sequence plus a U4AU4 3′-overhang is the optimal configuration. U-rich crRNA also maximizes the utility of the AsCpf1 mutants and multiplexing genome editing using mRNA as the source of multiple crRNAs. Furthermore, U-rich crRNA enables a highly safe and specific genome editing using Cpf1 in human cells, contributing to the enhancement of a genome-editing toolbox.