Brain organoids: A revolutionary tool for modeling neurological disorders and development of therapeutics.

Brain organoids are self-organized, three-dimensional (3D) aggregates derived from pluripotent stem cells that have cell types and cellular architectures resembling those of the developing human brain. The current understanding of human brain developmental processes and neurological disorders has advanced significantly with the introduction of this in vitro model. Brain organoids serve as a translational link between two-dimensional (2D) cultures and in vivo models which imitate the neural tube formation at the early and late stages and the differentiation of neuroepithelium with whole-brain regionalization. In addition, the generation of region-specific brain organoids made it possible to investigate the pathogenic and etiological aspects of acquired and inherited brain disease along with drug discovery and drug toxicity testing. In this review article, we first summarize an overview of the existing methods and platforms used for generating brain organoids and their limitations and then discuss the recent advancement in brain organoid technology. In addition, we discuss how brain organoids have been used to model aspects of neurodevelopmental and neurodegenerative diseases, including autism spectrum disorder (ASD), Rett syndrome, Zika virus-related microcephaly, Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD).

CRISPR/Cas9-mediated mutations in both a cAMP response element and an ETS-binding site suppress FLT1 gene expression.

The Fms-like tyrosine kinase-1 (FLT1) gene is expressed in various types of cells, including vascular endothelial cells and placental trophoblasts, and regulates angiogenesis, inflammation, and pregnancy. However, the basal transcriptional machinery of FLT1 is still not well understood. In this study, we first examined FLT1 promoter activity in three different types of cells, that is, trophoblast-derived cells, vascular endothelial-related cells, and HEK293 cells, using plasmid-based luciferase reporter assays, and showed that a cAMP-response element (CRE) and an ETS-binding site (EBS) are important for FLT1 expression in all cell types. To further examine the importance of these sites at the chromosomal level using HEK293 cells, we introduced CRISPR/Cas9-mediated mutations in these sites on the genomic DNA. HEK293 cells carrying these mutations clearly showed a significant decrease in endogenous FLT1 gene expression. These results suggest that CRE and EBS transcription regulatory elements are crucial for FLT1 gene expression in human tissues.

Triazole fungicides disrupt embryonic stem cell differentiation: Potential modulatory role of the retinoic acid signaling pathway.

The developmental toxicity and human health risks of triazole fungicides (TFs) have attracted worldwide attention due to the ability to enter the human body in a variety of ways. Nevertheless, the specific mechanism by which TFs exert remains incompletely understood. Given that retinoic acid (RA) signaling pathway are closely related to development, this study aimed to screen and identify developmentally disabled chemicals in commonly used TFs and to reveal the potential effects of TFs on developmental retardation through the RA signaling pathway in mouse embryonic stem cells (mESCs). Specifically, six typical TFs (myclobutanil, tebuconazole, hexaconazole, propiconazole, difenoconazole, and flusilazole) were exposed through the construction of an embryoid bodies (EBs)-based in vitro global differentiation models. Our results clarified that various TFs disturbed lineage commitment during early embryonic development. Crucially, the activation of RA signaling pathway, which alters the expression of key genes and interferes the transport and metabolism of retinol, may be responsible for this effect. Furthermore, molecular docking, molecular dynamics simulations, and experiments using a retinoic acid receptor α inhibitor provide evidence supporting the potential modulatory role of the retinoic acid signaling pathway in developmental injury. The current study offers new insights into the TFs involved in the RA signaling pathway that interfere with the differentiation process of mESCs, which is crucial for understanding the impact of TFs on pregnancy and early development.

Autologous hGMSC-Derived iPS: A New Proposal for Tissue Regeneration.

The high mortality in the global population due to chronic diseases highlights the urgency to identify effective alternative therapies. Regenerative medicine provides promising new approaches for this purpose, particularly in the use of induced pluripotent stem cells (iPSCs). The aim of the work is to establish a new pluripotency cell line obtained for the first time by reprogramming human gingival mesenchymal stem cells (hGMSCs) by a non-integrating method. The hGMSC-derived iPS line characterization is performed through morphological analysis with optical and electron scanning microscopy and through the pluripotency markers expression evaluation in cytofluorimetry, immunofluorescence, and RT-PCR. To confirm the pluripotency of new hGMSC-derived iPS, the formation of embryoid bodies (EBs), as an alternative to the teratoma formation test, is studied in morphological analysis and through three germ layers' markers' expression in immunofluorescence and RT-PCR. At the end, a comparative study between parental hGMSCs and derived iPS cells is performed also for the extracellular vesicles (EVs) and their miRNA content. The new hGMSC-derived iPS line demonstrated to be pluripotent in all aspects, thus representing an innovative dynamic platform for personalized tissue regeneration.

A cell-based drug discovery assay identifies inhibition of cell stress responses as a new approach to treatment of epidermolysis bullosa simplex.

In the skin fragility disorder epidermolysis bullosa simplex (EBS), mutations in keratin 14 (K14, also known as KRT14) or keratin 5 (K5, also known as KRT5) lead to keratinocyte rupture and skin blistering. Severe forms of EBS are associated with cytoplasmic protein aggregates, with elevated kinase activation of ERK1 and ERK2 (ERK1/2; also known as MAPK3 and MAPK1, respectively), suggesting intrinsic stress caused by misfolded keratin protein. Human keratinocyte EBS reporter cells stably expressing GFP-tagged EBS-mimetic mutant K14 were used to optimize a semi-automated system to quantify the effects of test compounds on keratin aggregates. Screening of a protein kinase inhibitor library identified several candidates that reduced aggregates and impacted on epidermal growth factor receptor (EGFR) signalling. EGF ligand exposure induced keratin aggregates in EBS reporter keratinocytes, which was reversible by EGFR inhibition. EBS keratinocytes treated with a known EGFR inhibitor, afatinib, were driven out of activation and towards quiescence with minimal cell death. Aggregate reduction was accompanied by denser keratin filament networks with enhanced intercellular cohesion and resilience, which when extrapolated to a whole tissue context would predict reduced epidermal fragility in EBS patients. This assay system provides a powerful tool for discovery and development of new pathway intervention therapeutic avenues for EBS.

Beam-induced redox chemistry in iron oxide nanoparticle dispersions at ESRF-EBS.

The storage ring upgrade of the European Synchrotron Radiation Facility makes ESRF-EBS the most brilliant high-energy fourth-generation light source, enabling in situ studies with unprecedented time resolution. While radiation damage is commonly associated with degradation of organic matter such as ionic liquids or polymers in the synchrotron beam, this study clearly shows that highly brilliant X-ray beams readily induce structural changes and beam damage in inorganic matter, too. Here, the reduction of Fe3+ to Fe2+ in iron oxide nanoparticles by radicals in the brilliant ESRF-EBS beam, not observed before the upgrade, is reported. Radicals are created due to radiolysis of an EtOH-H2O mixture with low EtOH concentration (∼6 vol%). In light of extended irradiation times during insitu experiments in, for example, battery and catalysis research, beam-induced redox chemistry needs to be understood for proper interpretation of insitu data.

STUDYING ANXIETY AS A PREDICTOR IN STUDENTS TO PREDICT THE DEVELOPMENT OF BURNOUT.

OBJECTIVE: The aim: Research of a subjectively significant personal social-psychological and individual-psychological characteristic - anxiety in students, which can be a predictor of the occurrence of EBS. To identify the scale and prevalence of the specified predictor in the student environment. PATIENTS AND METHODS: Materials and methods: A survey was conducted among 556 respondents. The survey was conducted according to the Spielberg-Hanin Anxiety Scale, which is available online with automatic scoring and retrieval of results. The test involves determining the levels of situational (reactive) and personal anxiety. To achieve the goal of the research, a set of methods was used: a systematic approach, a sociological method, and a medical-statistical method. The data are provided in the form of relative values with errors. RESULTS: Results: Almost half of the students who participated in the study feel the influence of the anxiety factor, which increases the likelihood of emotional burnout. The "tension" phase - nervous (anxious tension) is a harbinger and "trigger" mechanism in the formation of emotional burnout. According to the results of the study, up to 50% of respondents are in the first phase of emotional burnout or have already passed it. This determines the need for preventive work with students, among whom the survey was conducted, to prevent the occurrence of emotional and, subsequently, professional burnout. The low level of anxiety, which was 8.49% ± 1.18% among respondents, needs further study, as it may indicate the suppression of experiences by consciousness and hidden anxiety, which is an even more provoking factor for emotional burnout than a high level of anxiety. CONCLUSION: Conclusions: Empirical research has established a significant prevalence among students of a high and medium level of personal characteristic - anxiety, which has signs of a negative internal factor and can be a predictor of the development of EBS.

Clinical heterogeneity in epidermolysis bullosa simplex with plectin (PLEC) mutations-A study of six unrelated families from India.

Epidermolysis bullosa simplex (EBS) with plectin mutations is a very rare subtype of EB usually associated with pyloric atresia (PA) or muscular dystrophy (MD). We report six unrelated children between ages 4 and 14 years from India with varied clinical manifestations. Only one had PA, and none has developed MD to date. All except the one with PA presented with early onset blistering along with laryngeal involvement in the form of hoarseness of voice and nail involvement. Patient with PA presented with aplasia cutis and died in the first week. Two patients had predominantly respiratory and gastrointestinal involvement with varying severity while two had features of myasthenic syndrome but no limb-girdle involvement and one patient phenocopied laryngo-onycho-cutaneous (LOC) syndrome. Using whole-exome sequencing, we identified novel mutations in PLEC. Histopathological analysis (Immunofluorescence antigen mapping) showed absence of staining to plectin antibodies. Our observations propose to append a phenotype of EBS, hoarseness of voice and nail dystrophy or LOC-like phenotype with plectin mutations. Long-term follow up is necessary to monitor for the development of muscular dystrophy.

The Role of SBI2/ALG12/EBS4 in the Regulation of Endoplasmic Reticulum-Associated Degradation (ERAD) Studied by a Null Allele.

Redundancy and lethality is a long-standing problem in genetics but generating minimal and lethal phenotypes in the knockouts of the same gene by different approaches drives this problem to a new high. In Asn (N)-linked glycosylation, a complex and ubiquitous cotranslational and post-translational protein modification required for the transfer of correctly folded proteins and endoplasmic reticulum-associated degradation (ERAD) of misfolded proteins, ALG12 (EBS4) is an α 1, 6-mannosyltransferase catalyzing a mannose into Glc3Man9GlcNAc2. In Arabidopsis, T-DNA knockout alg12-T is lethal while likely ebs4 null mutants isolated by forward genetics are most healthy as weak alleles, perplexing researchers and demanding further investigations. Here, we isolated a true null allele, sbi2, with the W258Stop mutation in ALG12/EBS4. sbi2 restored the sensitivity of brassinosteroid receptor mutants bri1-5, bri1-9, and bri1-235 with ER-trapped BRI1 to brassinosteroids. Furthermore, sbi2 maturated earlier than the wild-type. Moreover, concomitant with impaired and misfolded proteins accumulated in the ER, sbi2 had higher sensitivity to tunicamycin and salt than the wild-type. Our findings thus clarify the role of SBI2/ALG12/EBS4 in the regulation of the ERAD of misfolded glycoproteins, and plant growth and stress response. Further, our study advocates the necessity and importance of using multiple approaches to validate genetics study.

STUDY OF NEUROTICISM AND EXTRAVERSION AS PREDICTORS OF THE SYNDROME OF EMOTIONAL BURNOUT (EBS) IN STUDENTS.

OBJECTIVE: The aim: To investigate internal factors - neuroticism and extroversion, which can be predictors of emotional burnout syndrome and their prevalence among student youth. PATIENTS AND METHODS: Materials and methods: Used a complex of methods: theoretical - theoretical analysis, synthesis, systematization; sociological method of questioning; empirical: observation, testing-Eysenck's methods for assessing the impact of neuroticism and externality; statistical. The object of the research is the process of influence of personal characteristics (individual factors) on the development and spread of EBS among students. Subject - personal characteristics (individual factors) of students - extraversion and neuroticism. The number of respondents was 610 students. RESULTS: Results: A study of the personal characteristics of neuroticism and extraversion, which can be predictors in the genesis of EBS, was conducted. It has been established that several negative internal factors influence students. The significant prevalence of internal risk factors of EBS indicates the need for a comprehensive approach to its prevention and the need for the development and application of modern adequate methods, forms, and methods of prevention. CONCLUSION: Conclusions: The presence and influence of internal factors on the development of EBS have been theoretically proven. It has been established that there is a significant propagation of personal characteristics that may have signs of negative internal factors. The need for preventive measures for the development of EBS among students has been revealed.

An S-ribonuclease binding protein EBS1 and brassinolide signaling are specifically required for Arabidopsis tolerance to bicarbonate.

Bicarbonate (NaHCO3) present in soils is usually considered to be a mixed stress for plants, with salts and high pH. NaHCO3-specific signaling in plants has rarely been reported. In this study, transcriptome analyses were conducted in order to identify NaHCO3-specific signaling in Arabidopsis. Weighted correlation network analysis was performed to isolate NaHCO3-specific modules in comparison with acetate treatment. The genes in the NaHCO3-root-specific module, which exhibited opposite expression to that in sodium acetate treatments, were further examined with their corresponding knock-out mutants. The gene Exclusively Bicarbonate Sensitive 1 (EBS1) encoding an S-ribonuclease binding protein, was identified to be specifically involved in plant tolerance to NaHCO3, but not to the other two alkaline salts, acetate and phosphate. We also identified the genes that are commonly regulated by bicarbonate, acetate and phosphate. Multiple brassinosteroid-associated gene ontology terms were enriched in these genes. Genetic assays showed that brassinosteroid signaling positively regulated plant tolerance to NaHCO3 stress, but negatively regulated tolerance to acetate and phosphate. Overall, our data identified bicarbonate-specific genes, and confirmed that alkaline stress is mainly dependent on the specificities of the weak acid ions, rather than high pH.

An analysis of the prevalence of osteoporosis and osteopenia in patients with epidermolysis bullosa: A cross-sectional study.

BACKGROUND: Epidermolysis bullosa (EB) patients have multiple risk factors for osteoporosis. There is limited literature describing the prevalence of bone health in EB, particularly in adults and less severe EB types. OBJECTIVES: To investigate the prevalence of osteopenia or osteoporosis in EB patients from the Australasian Epidermolysis Bullosa Registry (AEBR). METHODS: Of 417 AEBR patients, 72 underwent a dual energy X-ray absorptiometry scan. Bone mineral density (BMD) T and Z-scores, EB Disease Activity and Scarring Index (EBDASI), and Quality of Life in EB (QOLEB) scores were obtained. RESULTS: T-scores of RDEB patients were significantly lower than the diagnostic cut-off value for osteopenia. EBDASI and QOLEB scores were inversely correlated with Z-scores. The prevalence of osteoporosis in adults was 75% in severe EB types (RDEB and JEB). In adults with less severe types (EBS and DDEB), the prevalence of osteopenia was 50% and 33%, respectively. CONCLUSIONS: This is the largest study of osteoporosis in EB to date and the first to include adult patients with EBS. The high prevalence of osteoporosis and osteopenia identified in these patients warrants larger, collaborative international studies. Nevertheless, EB patients with high disease severity and QOL scores, irrespective of type, should receive early osteoporosis screening and prophylaxis.

Early prediction of the differentiation potential during the formation of human iPSC-derived embryoid bodies.

Induced pluripotent stem cells (iPSCs) show huge variations in their differentiation potential, even in the same condition. However, methods for predicting these differentiation tendencies, especially in the early stage of differentiation, are still scarce. This study aimed to establish a simple and practical system to predict the differentiation tendency of iPSC lines using embryoid bodies (EBs) with identified parameters in the early stage. We compared four human iPSC lines in terms of the morphology and maintenance of EBs and their gene expression levels of specific markers for three germ-layers. Furthermore, the differentiation potentials of these iPSC lines into melanocytes, which are ectoderm-derived cells, were also compared and correlated with the above parameters. The results showed that iPSC lines forming regular, smooth, and not cystic EBs, which could be maintained in culture for a relatively longer time, also expressed higher levels of ectoderm-specific markers and lower levels of mesoderm/endoderm markers. Additionally, these iPSC lines showed greater potential in melanocyte differentiation using EB-based protocol, and the induced melanocytes expressed melanocytic markers and presented characteristics that were similar to those of normal human melanocytes. By contrast, iPSC lines that formed cystic EBs with bright or dark cavities and expressed relatively lower levels of ectoderm-specific markers failed in the melanocyte differentiation. Collectively, the differentiation tendency of human iPSC lines may be predicted by specific parameters in the EB stage. The formation and maintenance of optimal EBs and the expression of germ layer-specific markers are particularly important and practical for the prediction assay in the early stage.

An optimized protocol for generating labeled and transplantable photoreceptor precursors from human embryonic stem cells.

Cell replacement therapy is a promising approach for treatment of retinal degenerative diseases. Several protocols for the generation of photoreceptor precursors (PRP) from human embryonic stem cells (hESC) have been reported with variable efficiency. Herein, we show the advantages of use of size-controlled embryoid bodies in the ESC differentiation process using two differentiation protocols. We further explored cell-labeling methods for following the survival of PRP transplanted subretinally in rat eyes. Size-controlled embryoid bodies (EBs) generated using microwell dishes and non-size-controlled EBs generated using V-shaped 96-well plates were differentiated into PRP using two differentiation protocols. The differentiation protocols utilized two different combinations of growth factors. The first, Dkk1, Noggin, and IGF1, and the second protocol used IWR1e, SAG, and CHIR99021. Differentiation efficiency to PRP was analyzed by qPCR, immunocytochemistry, and fluorescence-assisted cell sorting (FACS). Size-controlled IWR1e yielded a significantly higher percent (86.4%) of PRP cells expressing CRX, compared with non-size-controlled IWR1e (51.4%, P = 0.026) or the size-controlled DKK1 protocol (70.5%, p = 0.007). In addition, the IWR1e differentiated cells exhibited a significantly higher fluorescence intensity of CRX immunostaining, compared with the DKK1 protocol, consistent with higher protein expression levels. The IWR1e cells exhibited higher maturation levels, as manifested by lower early neuronal marker PAX6 and pluripotency marker OCT4 levels compared with the DKK1 protocol. The expression of other late photoreceptor markers (NRL, recoverin) were similar among the differentiation groups. PRP cells were labeled by using hESC constitutively expressing EGFP or by AAV-GFP transduction. Finally, we transplanted the cells in the subretinal space of wild-type rats and monitored their survival over several weeks. The AAV2 serotype efficiently transduced the PRP cells, whereas other serotypes yielded low or no transduction. Following subretinal transplantation of GFP-labeled PRP, 63% of the cells were detected at 4 weeks post-transplantation. In conclusion, we show here that the IWR1e protocol using size-controlled EBs efficiently generated of PRP that could be labeled and followed in-vivo for weeks. The data from this study is an advance toward the goal of PRP transplantation therapy for retinal degenerative diseases.

A target-group-change couple with mass defect filtering strategy to identify the metabolites of "Dogel ebs" in rats plasma, urine and bile.

"Dogel ebs" was known as Sophora flavescens Ait., a classical traditional Chinese Mongolian herbal medicine, which had the effects on damp-heat dysentery, scrofula, and syndrome of accumulated dampness toxicity. Although the chemical constituents have been clarified by our previous studies, the metabolic transformation of "Dogel ebs" in vivo was still unclear. To explore the mechanism of "Dogel ebs," the metabolites in plasma, bile, and urine samples were investigated. A fast positive and negative ion switching technology was used for the simultaneous determination of flavonoids and alkaloids in "Dogel ebs" in a single run. And a target-group-change coupled with mass defect filtering strategy was utilized to analyze the collected data. 89 parent compounds and 82 metabolites were characterized by high-performance liquid chromatography with quadrupole exactive Orbitrap mass spectrometry. Both phase I and phase II metabolites were observed and the metabolic pathways involved in oxidation, demethylation, acetylation, and glucuronidation. 69 metabolites of "Dogel ebs," including three hydroxyls bonding xanthohumol, formononetin-7-O-glucuronide, 2'-hydroxyl-isoxanthohumol decarboxylation metabolite, oxysophocarpine dehydrogen, 9α-hydroxysophoramine-O-glucuronide, etc. were reported for the first time.

Keratins and epidermolysis bullosa simplex.

Keratin intermediate filaments play an important role in maintaining the integrity of the skin structure. Understanding the importance of this subject is possible with the investigation of keratin defects in epidermolysis bullosa simplex (EBS). Nowadays, in addition to clinical criteria, new molecular diagnostic methods, such as next generation sequencing, can help to distinguish the subgroups of EBS more precisely. Because the most important and most commonly occurring molecular defects in these patients are the defects of keratins 5 and14 (KRT5 and KRT14), comprehending the nature structure of these proteins and their involved processes can be very effective in understanding the pathophysiology of this disease and providing new and effective therapeutic platforms to treat it. Here, we summarized the various aspects of the presence of KRT5 and KRT14 in the epidermis, their relation to the incidence and severity of EBS phenotypes, and the processes with which these proteins can affect them.

Exploring the effect of end-binding proteins and microtubule targeting chemotherapy drugs on microtubule dynamic instability.

Microtubules (MTs) play a key role in normal cell development and are a primary target for many cancer chemotherapy MT targeting agents (MTAs). As such, understanding MT dynamics in the presence of such agents, as well as other proteins that alter MT dynamics, is extremely important. In general, MTs grow relatively slowly and shorten very fast (almost instantaneously), an event referred to as a catastrophe. These dynamics, referred to as dynamic instability, have been studied in both experimental and theoretical settings. In the presence of MTAs, it is well known that such agents work by suppressing MT dynamics, either by promoting MT polymerization or promoting MT depolymerization. However, recent in vitro experiments show that in the presence of end-binding proteins (EBs), low doses of MTAs can increase MT dynamic instability, rather than suppress it. Here, we develop a novel mathematical model, to describe MT and EB dynamics, something which has not been done in a theoretical setting. Our MT model is based on previous modeling efforts, and consists of a pair of partial differential equations to describe length distributions for growing and shortening MT populations, and an ordinary differential equation (ODE) system to describe the time evolution for concentrations of GTP- and GDP-bound tubulin. A new extension of our approach is the use of an integral term, rather than an advection term, to describe very fast MT shortening events. Further, we introduce an ODE system to describe the binding and unbinding of EBs with MTs. To compare simulation results with experiment, we define novel mathematical expressions for time- and distance-based catastrophe frequencies. These quantities help to define MT dynamics in in vivo and in vitro settings. Simulation results show that increasing concentrations of EBs work to increase time-based catastrophe while distance-based catastrophe is less affected by changes in EB concentration, a result that is consistent with experiment. We further describe how EBs and MTAs alter MT dynamics. In the context of this modeling framework, we show that it is likely that MTAs and EBs do not work independently from one another. Thus, we propose a mechanism for how EBs can work synergistically with MTAs to promote MT dynamic instability at low MTA dose.

Imprinted expression in cystic embryoid bodies shows an embryonic and not an extra-embryonic pattern.

A large subset of mammalian imprinted genes show extra-embryonic lineage (EXEL) specific imprinted expression that is restricted to placental trophectoderm lineages and to visceral yolk sac endoderm (ysE). Isolated ysE provides a homogenous in vivo model of a mid-gestation extra-embryonic tissue to examine the mechanism of EXEL-specific imprinted gene silencing, but an in vitro model of ysE to facilitate more rapid and cost-effective experiments is not available. Reports indicate that ES cells differentiated into cystic embryoid bodies (EBs) contain ysE, so here we investigate if cystic EBs model ysE imprinted expression. The imprinted expression pattern of cystic EBs is shown to resemble fetal liver and not ysE. To investigate the reason for this we characterized the methylome and transcriptome of cystic EBs in comparison to fetal liver and ysE, by whole genome bisulphite sequencing and RNA-seq. Cystic EBs show a fetal liver pattern of global hypermethylation and low expression of repeats, while ysE shows global hypomethylation and high expression of IAPEz retroviral repeats, as reported for placenta. Transcriptome analysis confirmed that cystic EBs are more similar to fetal liver than ysE and express markers of early embryonic endoderm. Genome-wide analysis shows that ysE shares epigenetic and repeat expression features with placenta. Contrary to previous reports, we show that cystic EBs do not contain ysE, but are more similar to the embryonic endoderm of fetal liver. This explains why cystic EBs reproduce the imprinted expression seen in the embryo but not that seen in the ysE.

Role of helical constraints of the EBS1-IBS1 duplex of a group II intron on demarcation of the 5' splice site.

Recognition of the 5' splice site by group II introns involves pairing between an exon binding sequence (EBS) 1 within the ID3 stem-loop of domain 1 and a complementary sequence at the 3' end of exon 1 (IBS1). To identify the molecular basis for splice site definition of a group IIB ai5γ intron, we probed the solution structure of the ID3 stem-loop alone and upon binding of its IBS1 target by solution NMR. The ID3 stem was structured. The base of the ID3 loop was stacked but displayed a highly flexible EBS1 region. The flexibility of EBS1 appears to be a general feature of the ai5γ and the smaller Oceanobacillus iheyensis (O.i.) intron and may help in effective search of conformational space and prevent errors in splicing as a result of fortuitous base-pairing. Binding of IBS1 results in formation of a structured seven base pair duplex that terminates at the 5' splice site in spite of the potential for additional A-U and G•U pairs. Comparison of these data with conformational features of EBS1-IBS1 duplexes extracted from published structures suggests that termination of the duplex and definition of the splice site are governed by constraints of the helical geometry within the ID3 loop. This feature and flexibility of the uncomplexed ID3 loop appear to be common for both the ai5γ and O.i. introns and may help to fine-tune elements of recognition in group II introns.

NMR structure of the 5' splice site in the group IIB intron Sc.ai5γ--conformational requirements for exon-intron recognition.

A crucial step of the self-splicing reaction of group II intron ribozymes is the recognition of the 5' exon by the intron. This recognition is achieved by two regions in domain 1 of the intron, the exon-binding sites EBS1 and EBS2 forming base pairs with the intron-binding sites IBS1 and IBS2 located at the end of the 5' exon. The complementarity of the EBS1•IBS1 contact is most important for ensuring site-specific cleavage of the phosphodiester bond between the 5' exon and the intron. Here, we present the NMR solution structures of the d3' hairpin including EBS1 free in solution and bound to the IBS1 7-mer. In the unbound state, EBS1 is part of a flexible 11-nucleotide (nt) loop. Binding of IBS1 restructures and freezes the entire loop region. Mg(2+) ions are bound near the termini of the EBS1•IBS1 helix, stabilizing the interaction. Formation of the 7-bp EBS1•IBS1 helix within a loop of only 11 nt forces the loop backbone to form a sharp turn opposite of the splice site, thereby presenting the scissile phosphate in a position that is structurally unique.