TFIIB Research Articles

A critical role of a plant-specific TFIIB-related protein, BRP1, in salicylic acid-mediated immune response.

An integral part of plant immunity is transcription reprogramming by concerted action of specific transcription factors that activate or repress genes through recruitment or release of RNA polymerase II (Pol II). Pol II is assembled into Pol II holoenzyme at the promoters through association with a group of general transcription factors including transcription factor IIB (TFIIB) to activate transcription. Unlike other eukaryotic organisms, plants have a large family of TFIIB-related proteins with 15 members in Arabidopsis including several plant-specific TFIIB-related proteins (BRPs). Molecular genetic analysis has revealed important roles of some BRPs in plant reproductive processes. In this study, we report that Arabidopsis knockout mutants for BRP1, the founding member of the BRP protein family, were normal in growth and development, but were hypersusceptible to the bacterial pathogen Psuedomonas syringae. The enhanced susceptibility of the brp1 mutants was associated with reduced expression of salicylic acid (SA) biosynthetic gene ISOCHORISMATE SYNTHASE 1 (ICS1) and SA-responsive PATHOGENESIS-RELATED (PR) genes. Pathogen-induced SA accumulation was reduced in the brp1 mutants and exogenous SA rescued the brp1 mutants for resistance to the bacterial pathogen. In uninfected plants, BRP1 was primarily associated with the plastids but pathogen infection induced its accumulation in the nucleus. BRP1 acted as a transcription activator in plant cells and binded to the promoter of ICS1. These results collectively indicate that BRP1 is a functionally specialized transcription factor that increasingly accumulates in the nucleus in response to pathogen infection to promote defense gene expression.

TFIIB-Related Protein BRP5/PTF2 Is Required for Both Male and Female Gametogenesis and for Grain Formation in Rice.

Transcription factor IIB (TFIIB) is a general transcription factor for RNA polymerase II, exerting its influence across various biological contexts. In the majority of eukaryotes, TFIIB typically has two homologs, serving as general transcription factors for RNA polymerase I and III. In plants, however, the TFIIB-related protein family has expanded greatly, with 14 and 9 members in Arabidopsis and rice, respectively. BRP5/pollen-expressed transcription factor 2 (PTF2) proteins belong to a subfamily of TFIIB-related proteins found only in plants and algae. The prior analysis of an Arabidopsis atbrp5 mutant, characterized by a T-DNA insertion at the 5' untranslated region, demonstrated the essential role of BRP5/PTF2 during the process of pollen germination and embryogenesis in Arabidopsis. Using a rice transformation system based on CRISPR/Cas9 technology, we have generated transgenic rice plants containing loss-of-function frameshift mutations in the BRP5/PTF2 gene. Unlike in the Arabidopsis atbrp5 mutant, the brp5/ptf2 frameshift mutations were not transmitted to progeny in rice, indicating an essential role of BRP5/PTF2 in both male and female gamete development or viability. The silencing of rice BRP5/PTF2 expression through RNA interference (RNAi) had little effect on vegetative growth and panicle formation but strongly affected pollen development and grain formation. Genetic analysis revealed that strong RNAi silencing of rice BRP5/PTF2 was still transmissible to progeny almost exclusively through female gametes, as found in the Arabidopsis atbrp5 knockdown mutant. Thus, reduced rice BRP5/PTF2 expression impacted pollen preferentially by interfering with male gamete development or viability. Drawing upon these findings, we posit that BRP5/PTF2 assumes a distinct and imperative function in the realm of plant sexual reproduction.

A bioinformatics screen reveals hox and chromatin remodeling factors at the Drosophila histone locus

BackgroundCells orchestrate histone biogenesis with strict temporal and quantitative control. To efficiently regulate histone biogenesis, the repetitive Drosophila melanogaster replication-dependent histone genes are arrayed and clustered at a single locus. Regulatory factors concentrate in a nuclear body known as the histone locus body (HLB), which forms around the locus. Historically, HLB factors are largely discovered by chance, and few are known to interact directly with DNA. It is therefore unclear how the histone genes are specifically targeted for unique and coordinated regulation.ResultsTo expand the list of known HLB factors, we performed a candidate-based screen by mapping 30 publicly available ChIP datasets of 27 unique factors to the Drosophila histone gene array. We identified novel transcription factor candidates, including the Drosophila Hox proteins Ultrabithorax (Ubx), Abdominal-A (Abd-A), and Abdominal-B (Abd-B), suggesting a new pathway for these factors in influencing body plan morphogenesis. Additionally, we identified six other factors that target the histone gene array: JIL-1, hormone-like receptor 78 (Hr78), the long isoform of female sterile homeotic (1) (fs(1)h) as well as the general transcription factors TBP associated factor 1 (TAF-1), Transcription Factor IIB (TFIIB), and Transcription Factor IIF (TFIIF).ConclusionsOur foundational screen provides several candidates for future studies into factors that may influence histone biogenesis. Further, our study emphasizes the powerful reservoir of publicly available datasets, which can be mined as a primary screening technique.

Targeting BRF2: insights from in silico screening and molecular dynamic simulations

The Transcription factor II B (TFIIB)‑related factor 2 (BRF2) containing TFIIIB complex recruits RNA polymerase III multi-subunit complex to selective gene promoters that altogether are responsible for synthesizing a variety of small non-coding RNAs, including a special type of selenocysteine tRNA (tRNASec), micro-RNA (miRNA), and other regulatory RNAs. BRF2 has been identified as a potential oncogene that promotes cancer cell survival under oxidative stress through its genetic activation. The structure of the BRF2 protein was modeled using the Robetta server, refined, and validated using the Ramachandran plot. A virtual approach utilizing molecular docking was used to screen a natural compound library to determine potential compounds that can interact with the molecular pin motif of the BRF2 protein using Maestro (Schrodinger). Subsequent molecular dynamics simulation studies of the top four ligands that exhibited low glide scores were performed using GROMACS. The findings derived from the simulations, in conjunction with the exploration of hydrogen bonding patterns, evaluation of the free energy landscape, and thorough analysis of residue decomposition, collectively converged to emphasize the robust interaction characteristics exhibited by Ligand 366 (Deacetyl lanatoside C) and ligand 336 (Neogitogenin)—with the BRF2 protein. These natural compounds may be potential inhibitors of BRF2, which could modulate the regulation of selenoprotein synthesis in cancer cells. Targeting BRF2 using these promising compounds may offer a new therapeutic approach to sensitize cancer cells to ferroptosis and apoptosis. Communicated by Ramaswamy H. Sarma

Transcription factor GTF2B regulates AIP protein expression in growth hormone-secreting pituitary adenomas and influences tumor phenotypes.

Clinically, the low expression of wild-type aryl hydrocarbon receptor-interacting protein (AIP) in patients with sporadic growth hormone (GH)-secreting pituitary adenoma (GHPA) is associated with a more aggressive phenotype. However, the mechanism by which AIP expression is regulated in GHPA remains unclear. Herein, we investigated a transcription factor that regulates AIP expression and explored its role in tumor phenotypes. General transcription factor IIB (GTF2B) was predicted by several bioinformatic tools to regulate AIP expression transcriptionally. Regulation by GTF2B was evaluated using chromatin immunoprecipitation (ChIP), reverse transcription PCR, luciferase reporter, and western blot experiments in SH-SY5Y cells. Furthermore, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, transwell invasive assay, ELISA, western blot, immunohistochemical staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling were performed to investigate the effects of GTF2B and AIP on tumor cell proliferation, apoptosis, growth hormone secretion, and invasiveness in GH3 cells and mouse xenograft models. Moreover, correlations between GTF2B and AIP expression were explored in GHPA cases. ChIP and luciferase reporter studies demonstrated that the regulation of AIP expression by GTF2B was dependent on the intergenic-5' untranslated region element of AIP and the initial residual S65 of GTF2B. In vitro and in vivo experiments indicated that GTF2B regulated AIP expression to impact the GHPA phenotype; this was confirmed by data from 33 GHPA cases. We determined the regulation by GTF2B of AIP transcription in GHPA and its impact on tumor phenotype. Our findings suggest that GTF2B may be a potential therapeutic target for GHPA with low AIP expression.

Critical Involvement of TFIIB in Viral Pathogenesis.

Viral infections and the harm they cause to their host are a perpetual threat to living organisms. Pathogenesis and subsequent spread of infection requires replication of the viral genome and expression of structural and non-structural proteins of the virus. Generally, viruses use transcription and translation machinery of the host cell to achieve this objective. The viral genome encodes transcriptional regulators that alter the expression of viral and host genes by manipulating initiation and termination steps of transcription. The regulation of the initiation step is often through interactions of viral factors with gene specific factors as well as general transcription factors (GTFs). Among the GTFs, TFIIB (Transcription Factor IIB) is a frequent target during viral pathogenesis. TFIIB is utilized by a plethora of viruses including human immunodeficiency virus, herpes simplex virus, vaccinia virus, Thogoto virus, hepatitis virus, Epstein-Barr virus and gammaherpesviruses to alter gene expression. A number of viral transcriptional regulators exhibit a direct interaction with host TFIIB in order to accomplish expression of their genes and to repress host transcription. Some viruses have evolved proteins with a three-dimensional structure very similar to TFIIB, demonstrating the importance of TFIIB for viral persistence. Upon viral infection, host transcription is selectively altered with viral transcription benefitting. The nature of viral utilization of TFIIB for expression of its own genes, along with selective repression of host antiviral genes and downregulation of general host transcription, makes TFIIB a potential candidate for antiviral therapies.

Expansion and Functional Diversification of TFIIB-Like Factors in Plants.

As sessile organisms, plants have evolved unique patterns of growth and development, elaborate metabolism and special perception and signaling mechanisms to environmental cues. Likewise, plants have complex and highly special programs for transcriptional control of gene expression. A case study for the special transcription control in plants is the expansion of general transcription factors, particularly the family of Transcription Factor IIB (TFIIB)-like factors with 15 members in Arabidopsis. For more than a decade, molecular and genetic analysis has revealed important functions of these TFIIB-like factors in specific biological processes including gametogenesis, pollen tube growth guidance, embryogenesis, endosperm development, and plant-microbe interactions. The redundant, specialized, and diversified roles of these TFIIB-like factors challenge the traditional definition of general transcription factors established in other eukaryotes. In this review, we discuss general transcription factors in plants with a focus on the expansion and functional analysis of plant TFIIB-like proteins to highlight unique aspects of plant transcription programs that can be highly valuable for understanding the molecular basis of plant growth, development and responses to stress conditions.

Exosomes exist in nipple discharge of breast cancer.

Exosomes exist in nipple discharge of breast cancer.

Silencing of BRF2 inhibits the growth and metastasis of lung cancer cells.

Transcription factor II B (TFIIB)-related factor 2 (BRF2) is involved in the development of cancer, but its role in lung cancer is underreported. The present study aimed to explore the role of BRF2 in the regulation of lung cancer cells. Immunofluorescence staining and immunohistochemistry were performed to detect BRF2 protein expression in human lung cancer cells and tissues. Following cell transfection with small interfering RNA for silencing BRF2, the cell proliferation was examined by Cell Counting Kit-8 and MTT assays. Cell apoptosis, migration and invasion were determined by flow cytometry, wound-healing and Transwell assay. The expression levels of Akt, phosphorylated (p)-Akt, Bax, E-cadherin, Bcl-2, N-cadherin, Snail and epidermal growth factor receptor (EGFR) in human lung cancer A549 cells were detected by western blotting. The results demonstrated that BRF2 expression was increased in human lung cancer cells and tissues, and that silencing of BRF2 promoted cell apoptosis but inhibited cell proliferation and migration. The protein expression levels of Akt, E-cadherin, p-Akt, Bcl-2, N-cadherin, Snail and EGFR in A549 cells were inhibited by silencing of BRF2, while expression levels of Bax and E-cadherin were increased by silencing BRF2. In conclusion, BRF2 demonstrates high expression in lung cancer and silencing of BRF2 inhibits the growth and metastasis of lung cancer cells. The current findings provide a novel approach for the treatment of lung cancer.

Role of betaine in inhibiting the induction of RNA Pol III gene transcription and cell growth caused by alcohol

Alcohol has been classified as carcinogenic to humans by the International Agency for Research on Cancer (IARC). Studies have demonstrated that alcohol intake increases the risk of breast cancer, and alcohol also stimulates breast cancer cell growth. Deregulation of Pol III genes is tightly associated with tumour development. Transcription factor II-B (TFIIB)-related factor 1 (Brf1) is a transcription factor that specifically regulates Pol III gene transcription. Our in vivo and in vitro studies have indicated that alcohol enhances the transcription of Pol III genes to cause an alteration of cellular phenotypes, which is closely related with human breast cancer. Betaine is a vegetable alkaloid and has antitumor functions. Most reports about betaine show that the consumption level of betaine is inversely associated with a risk of breast cancer. Although different mechanisms of betaine against tumour have been investigated, nothing has been reported on the effect of betaine on the deregulation of Brf1 and Pol III genes. In this study, we determine the role of betaine in breast cancer cell growth and colony formation and explore its mechanism. Our results indicate that alcohol increases the rates of growth and colony formation of breast cancer cells, whereas betaine is able to significantly inhibit the effects of alcohol on these cell phenotypes. Betaine decreases the induction of Brf1 expression and Pol III gene transcription caused by ethanol to reduce the rates of cell growth and colony formation. Together, these studies provide novel insights into the role of betaine in alcohol-caused breast cancer cell growth and deregulation of Brf1 and Pol III genes. These results suggest that betaine consumption is able to prevent alcohol-associated human cancer development.

Plasma Induced Modification of Biomolecules (PLIMB) for Epitope Mapping

Epitope mapping is the process of identifying the site(s) of an antigen where an antibody binds. Knowledge of epitopes facilitates drug design and patent applications; hence it is a key element of vaccine and drug development. Hydroxyl radical protein footprinting coupled with mass spectrometry offers opportunities to map antibody-binding regions of antigens. We report here the use of hydroxyl radical protein footprinting via Plasma Induced Modification of Biomolecules (PLIMB) to characterize the epitopes of antithrombin bound to the serine-protease thrombin, and monoclonal antibody (mAb) IIB8 to human transcription factor II B (TFIIB). The protein samples were exposed to plasma for hydroxyl radical labeling and the labeled sites were identified using mass spectrometry. The data was analyzed using Byos (Protein Metrics). The PLIMB data is compared to previously published results that determined the epitope of thrombin and TFIIB using other techniques.

Analysis of MUTYH alternative transcript expression, promoter function, and the effect of human genetic variants.

The human DNA repair gene MUTYH, whose mutational loss causes a colorectal polyposis and cancer predisposition, contains three alternative first exons. In order to analyze alternative transcription and the effect of genetic alterations found in humans, we established a cell-based minigene experimental model supporting transcription and splicing and thoroughly verified its functionality. We identified highly conserved promoter areas and inactivated them in the minigene, and also introduced six human variants. Moreover, the potential contribution of CpG island methylation and specific transcription factors on MUTYH transcription was addressed. The findings allowed to attribute regulatory roles to three conserved motifs in the promoter: an M4 motif, a transcription factor IIB recognition element, and a GC box. Moreover, the data showed that three patient variants compromised MUTYH expression and therefore have the potential to cause pathogenic effects. We did not find evidence for a biologically relevant contribution of CpG island methylation or a direct transcriptional activation by DNA damage. Besides insight into the regulation of MUTYH transcription, the work therefore provides a functional MUTYH minigene experimental system suitable as a diagnostic tool for analyzing patient variants, and a functional map of the promotor that also can facilitate pathogenicity classifications of human variants.

CanTF, a Novel Transcription Factor in Pepper, Is Involved in Resistance to Phytophthora capsici as well as Abiotic Stresses

Stress is usually considered an important factor resulting in plant injury. In this study, we identified a novel transcription factor gene, CanTF (Capsicum annuum transcription factor IIB), and characterized its role in the response to biotic and abiotic stresses. The full-length CanTF cDNA consists of 1488 bp, with a 1281-nucleotide open reading frame (ORF), and encodes a protein containing 426 amino acids with a theoretical molecular weight of 46.84 kDa. Real-time quantitative PCR revealed that CanTF is a stress-induced gene, with increased expression levels under both biotic and abiotic stresses. The expression of CanTF in pepper organs, especially roots, was highly induced by inoculation with an avirulent strain of Phytophthora capsici. Additionally, the differential expression of CanTF was observed under abiotic stresses, i.e., earlier expression was detected after cold, drought, and SA treatments than after salt and H2O2 treatment, suggesting its role in responses to various abiotic stresses. Furthermore, the silencing of CanTF by virus-induced gene silencing reduced the expression of defense-related genes (CaPR1, CaDEF1, and CaSAR82) under P. capsici inoculation. POD and root activity levels were lower after gene silencing than in controls, demonstrating the positive regulatory effect of CanTF against P. capsici. These results suggested that CanTF is a stress-induced gene involved in strengthening the pepper defense against biotic and abiotic stresses.

Isolation and characterization of Aquaporin 1 (AQP1), sodium/potassium-transporting ATPase subunit alpha-1 (Na/K-ATPase α1), Heat Shock Protein 90 (HSP90), Heat Shock Cognate 71 (HSC71), Osmotic Stress Transcription Factor 1 (OSTF1) and Transcription Factor II B (TFIIB) genes from a euryhaline fish, Etroplus suratensis.

The present study reports the complete sequences of Aquaporin 1 (AQP1) gene and partial sequences of genes, Sodium/potassium-transporting ATPase subunit alpha-1 (Na/K-ATPase α1 subunit), Osmotic Stress Transcription Factor 1 (OSTF1), Transcription Factor II B (TFIIB), Heat Shock Cognate 71 (HSC71) and Heat Shock Protein 90 (HSP90) obtained from mRNA and genomic DNA of Etroplus suratensis. They are candidate genes involved in stress responses of fishes. AQP1 gene was 2163bp long. Its mRNA sequence has 55bp 5' UTR, 783bp open reading frame (ORF), 119bp 3' UTR, three intronic regions and 90% identity with AQP1 of Oreochromis niloticus. The partial Na/K-ATPase α1subunit gene obtained 5998bp length with an ORF of 2213bp and 12 intronic regions. The partial OSTF1, TF IIB, HSC71 and HSP90 mRNA sequences obtained were 1473bp, 587bp, 1708bp and 151bp in length respectively. All the genes showed a high sequence similarity with respective genes reported from fishes. Comparison of AQP1 and Na/K-ATPase α1 genomic DNA sequence of E. suratensis collected from different water system showed two type of AQP1 with one synonymous mutation in exon-1 and higher sequence difference in intronic regions (including addition, deletion, transition and transversion mutations) with few synonymous and non-synonymous mutations in the exons of Na/K-ATPase α1. The sequence information of these major candidate genes involved in stress responses will help in further studies on population genetics, adaptive variations and genetic improvement programs of this cichlid species having aquaculture, ornamental and evolutionary importance.

The vitamin D hormone and its nuclear receptor: molecular actions and disease states.

Vitamin D plays a major role in bone mineral homeostasis by promoting the transport of calcium and phosphate to ensure that the blood levels of these ions are sufficient for the normal mineralization of type I collagen matrix in the skeleton. In contrast to classic vitamin D-deficiency rickets, a number of vitamin D-resistant rachitic syndromes are caused by acquired and hereditary defects in the metabolic activation of the vitamin to its hormonal form, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), or in the subsequent functions of the hormone in target cells. The actions of 1,25(OH)2D3 are mediated by the nuclear vitamin D receptor (VDR), a phosphoprotein which binds the hormone with-high affinity and regulates the expression of genes via zinc finger-mediated DNA binding and protein-protein interactions. In hereditary hypocalcemic vitamin D-resistant rickets (HVDRR), natural mutations in human VDR that confer patients with tissue insensitivity to 1,25(OH)2D3 are particularly instructive in revealing VDR structure function relationships. These mutations fall into three categories: (i) DNA binding/nuclear localization, (ii) hormone binding and (iii) heterodimerization with retinoid X receptors (RXRs). That all three classes of VDR mutations generate the HVDRR phenotype is consistent with a basic model of the active receptor as a DNA-bound, 1,25(OH)2D3-liganded heterodimer of VDR and RXR. Vitamin D responsive elements (VDREs) consisting of direct hexanucleotide repeats with a spacer of three nucleotides have been identified in the promoter regions of positively controlled genes expressed in bone, such as osteocalcin, osteopontin, beta 3-integrin and vitamin D 24-OHase. The 1,25(OH)2D3 ligand promotes VDR-RXR heterodimerization and specific, high affinity VDRE binding, whereas the ligand for RXR, 9-cis retinoic acid (9-cis RA), is capable of suppressing 1,25(OH)2D3-stimulated transcription by diverting RXR to form homodimers. However, initial 1,25(OH)2D3 liganding of a VDR monomer renders it competent not only to recruit RXR into a heterodimer but also to conformationally silence the ability of its RXR partner to bind 9-cis RA and dissociate the heterodimer. Additional probing of protein-protein interactions has revealed that VDR also binds to basal transcription factor IIB (TFIIB) and, in the presence of 1,25(OH)2D3, an RXR-VDR-TFIIB ternary complex can be created in solution. Moreover, for transcriptional activation by 1,25(OH)2D3, both VDR and RXR require an intact short amphipathic alpha-helix, known as AF-2, positioned at their extreme C-termini. Because the AF-2 domains participate neither in VDR-RXR heterodimerization nor in TFIIB association, it is hypothesized that they contact, in a ligand-dependent fashion, transcriptional coactivators such as those of the steroid receptor coactivator family, constituting yet a third protein-protein interaction for VDR. Therefore, in VDR-mediated transcriptional activation, 1,25(OH)2D3 binding to VDR alters the conformation of the ligand binding domain such that it: (i) engages in strong heterodimerization with RXR to facilitate VDRE binding, (ii) influences the RXR ligand binding domain such that it is resistant to the binding of 9-cis RA but active in recruiting coactivator to its AF-2 and (iii) presents the AF-2 region in VDR for coactivator association. The above events, including bridging by coactivators to the TATA binding protein and associated factors, may position VDR such that it is able to attract TFIIB and the balance of the RNA polymerase II transcription machinery, culminating in repeated transcriptional initiation of VDRE-containing, vitamin D target genes. Such a model would explain the action of 1,25(OH)2D3 to elicit bone remodeling by stimulating osteoblast and osteoclast precursor gene expression, while concomitantly triggering the termination of its hormonal signal by inducing the 24-OHase catabolizing enzyme.

Structural dissection of an interaction between transcription initiation and termination factors implicated in promoter-terminator cross-talk

Functional cross-talk between the promoter and terminator of a gene has long been noted. Promoters and terminators are juxtaposed to form gene loops in several organisms, and gene looping is thought to be involved in transcriptional regulation. The general transcription factor IIB (TFIIB) and the C-terminal domain phosphatase Ssu72, essential factors of the transcription preinitiation complex and the mRNA processing and polyadenylation complex, respectively, are important for gene loop formation. TFIIB and Ssu72 interact both genetically and physically, but the molecular basis of this interaction is not known. Here we present a crystal structure of the core domain of TFIIB in two new conformations that differ in the relative distance and orientation of the two cyclin-like domains. The observed extraordinary conformational plasticity may underlie the binding of TFIIB to multiple transcription factors and promoter DNAs that occurs in distinct stages of transcription, including initiation, reinitiation, and gene looping. We mapped the binding interface of the TFIIB-Ssu72 complex using a series of systematic, structure-guided in vitro binding and site-specific photocross-linking assays. Our results indicate that Ssu72 competes with acidic activators for TFIIB binding and that Ssu72 disrupts an intramolecular TFIIB complex known to impede transcription initiation. We also show that the TFIIB-binding site on Ssu72 overlaps with the binding site of symplekin, a component of the mRNA processing and polyadenylation complex. We propose a hand-off model in which Ssu72 mediates a conformational transition in TFIIB, accounting for the role of Ssu72 in transcription reinitiation, gene looping, and promoter-terminator cross-talk.

Leading role of TBP in the Establishment of Complexity in Eukaryotic Transcription Initiation Systems

While both archaeal and eukaryotic transcription initiation systems utilize TBP (TATA box-binding protein) and TFIIB (transcription factor IIB), eukaryotic systems include larger numbers of initiation factors. It remains uncertain how eukaryotic transcription initiation systems have evolved. Here, we investigate the evolutionary development of TBP and TFIIB, each of which has an intramolecular direct repeat, using two evolutionary indicators. Inter-repeat sequence dissimilarity (dDR, distance between direct repeats) indicates that the asymmetry of two repeats in TBP and TFIIB has gradually increased during evolution. Interspecies sequence diversity (PD, phylogenetic diversity) indicates that the resultant asymmetric structure, which is related to the ability to interact with multiple factors, diverged in archaeal TBP and archaeal/eukaryotic TFIIB during evolution. Our findings suggest that eukaryotic TBP initially acquired multiple Eukarya-specific interactors through asymmetric evolution of the two repeats. After the asymmetric TBP generated the complexity of the eukaryotic transcription initiation systems, its diversification halted and its asymmetric structure spread throughout eukaryotic species.

Structural Basis for the Persistence of Homing Endonucleases in Transcription Factor IIB Inteins

Inteins are mobile genetic elements that are spliced out of proteins after translation. Some inteins contain a homing endonuclease (HEN) responsible for their propagation. Hedgehog/INTein (HINT) domains catalyzing protein splicing and their nested HEN domains are thought to be functionally independent because of the existence of functional mini-inteins without HEN domains. Despite the lack of obvious mutualism between HEN and HINT domains, HEN domains are persistently found at one specific site in inteins, indicating their potential functional role in protein splicing. Here we report crystal structures of inactive and active mini-inteins derived from inteins residing in the transcription factor IIB of Methanococcus jannaschii and Methanocaldococcus vulcanius, revealing a novel modified HINT fold that might provide new insights into the mutualism between the HEN and HINT domains. We propose an evolutionary model of inteins and a functional role of HEN domains in inteins.

Analysis of Senescence-Related Differentiation Potentials and Gene Expression Profiles in Human Dental Pulp Stem Cells

Introduction: Dental pulp stem cell (DPSC)-mediated dental pulp regeneration is considered a promising method for the treatment of deep caries with pulpitis. However, mesenchymal stem cell (MSC) senescence is an adverse factor from the perspective of cell-based therapies. In this study, we investigated the characteristics and expression profiles of DPSCs from young and old donors. Methods: DPSCs from young and old donors were cultured in differentiation medium, and their differentiation potentials were assessed. Long noncoding RNA (LncRNA) microarray assays and a bioinformatic analysis were performed to investigate differences in LncRNA and mRNA expression profiles between DPSCs from young and old donors. Results: We found that DPSCs from young donors exhibited more powerful proliferation ability and greater osteogenic and adipogenic differentiation potentials than DPSCs from old donors. In DPSCs from young donors, numerous LncRNAs were significantly up- (n = 389) or down-regulated (n = 172) compared to DPSCs from old donors. Furthermore, 304 mRNAs were differentially expressed, including 247 up-regulated genes and 57 down-regulated genes in DPSCs from young donors. The bioinformatic analysis identified that several pathways may be associated with DPSC characteristics, such as those involved in the cell cycle and RNA transport, and revealed nuclear transcription factor Y subunit β, general transcription factor IIB, and nuclear receptor subfamily 3 group C member 1 as core regulatory factors and FR249114, FR299091, and ENST00000450004 as core LncRNAs. Conclusions: Our results indicated that senescence impaired the proliferation and differentiation potentials of DPSCs and that donor age is an important factor that affects their use for tooth regeneration. We also provide insight into the mechanisms responsible for senescence in DPSCs.

GTPase Activating Protein (Sh3 Domain) Binding Protein 1 Regulates the Processing of MicroRNA-1 during Cardiac Hypertrophy

BackgroundMicroRNAs (miR) are small, posttranscriptional regulators, expressed as part of a longer primary transcript, following which they undergo nuclear and cytoplasmic processing by Drosha and Dicer, respectively, to form the functional mature ~20mer that gets incorporated into the silencing complex. Others and we have shown that mature miR-1 levels decrease with pressure-induced cardiac hypertrophy, however, there is little or no change in the primary transcript encompassing miR-1 stem-loop, suggesting critical regulatory step in microRNA processing. The objective of this study was to investigate the underlying mechanisms regulating miR-1 expression in cardiomyocytes.ResultsHere we report that GTPase–activating protein (SH3 domain) binding protein 1 (G3bp1), an endoribonuclease regulates miR-1 processing in cardiomyocytes. G3bp1 is upregulated during cardiac hypertrophy and restricts miR-1 processing by binding to its consensus sequence in the pre-miR-1-2 stem-loop. In accordance, exogenous G3bp1 is sufficient to reduce miR-1 levels, along with derepression of miR-1 targets; General transcription factor IIB (Gtf2b), cyclin dependent factor 9 (Cdk9) and eukaryotic initiation factor 4E (Eif4e). While Cdk9 and Gtf2b are essential for transcription, Eif4e is required for translation. Thus, downregulation of miR-1 is necessary for increase in these molecules. Similar to miR-1 knockdown, G3bp1 overexpression is not sufficient for development of cardiac hypertrophy. Conversely, knockdown of G3bp1 in hypertrophying cardiomyocytes inhibited downregulation of miR-1 and upregulation of its targets along with restricted hypertrophy, suggesting that G3bp1 is necessary for development of cardiac hypertrophy. These results indicate that G3bp1-mediated inhibition of miR-1 processing with growth stimulation results in decrease in mature miR-1 and, thereby, an increase of its targets, which play fundamental roles in the development of hypertrophy.ConclusionG3bp1 posttranscriptionally regulates miRNA-1 processing in the heart, and G3bp1 mediated downregulation of mature miRNA-1 levels is required for the derepression of its targets and increase in gene expression during cardiac hypertrophy.