Modeling splicing outcome by combining 5'ss strength and splicing regulatory elements.

Correct pre-mRNA processing in higher eukaryotes vastly depends on splice site recognition. Beyond conserved 5'ss and 3'ss motifs, splicing regulatory elements (SREs) play a pivotal role in this recognition process. Here, we present in silico designed sequences with arbitrary a priori prescribed splicing regulatory HEXplorer properties that can be concatenated to arbitrary length without changing their regulatory properties. We experimentally validated in silico predictions in a massively parallel splicing reporter assay on more than 3000 sequences and exemplarily identified some SRE binding proteins. Aiming at a unified 'functional splice site strength' encompassing both U1 snRNA complementarity and impact from neighboring SREs, we developed a novel RNA-seq based 5'ss usage landscape, mapping the competition of pairs of high confidence 5'ss and neighboring exonic GT sites along HBond and HEXplorer score coordinate axes on human fibroblast and endothelium transcriptome datasets. These RNA-seq data served as basis for a logistic 5'ss usage prediction model, which greatly improved discrimination between strong but unused exonic GT sites and annotated highly used 5'ss. Our 5'ss usage landscape offers a unified view on 5'ss and SRE neighborhood impact on splice site recognition, and may contribute to improved mutation assessment in human genetics.

Ranking noncanonical 5' splice site usage by genome-wide RNA-seq analysis and splicing reporter assays.

Most human pathogenic mutations in 5' splice sites affect the canonical GT in positions +1 and +2, leading to noncanonical dinucleotides. On the other hand, noncanonical dinucleotides are observed under physiological conditions in ∼1% of all human 5'ss. It is therefore a challenging task to understand the pathogenic mutation mechanisms underlying the conditions under which noncanonical 5'ss are used. In this work, we systematically examined noncanonical 5' splice site selection, both experimentally using splicing competition reporters and by analyzing a large RNA-seq data set of 54 fibroblast samples from 27 subjects containing a total of 2.4 billion gapped reads covering 269,375 exon junctions. From both approaches, we consistently derived a noncanonical 5'ss usage ranking GC > TT > AT > GA > GG > CT. In our competition splicing reporter assay, noncanonical splicing was strictly dependent on the presence of upstream or downstream splicing regulatory elements (SREs), and changes in SREs could be compensated by variation of U1 snRNA complementarity in the competing 5'ss. In particular, we could confirm splicing at different positions (i.e., -1, +1, +5) of a splice site for all noncanonical dinucleotides "weaker" than GC. In our comprehensive RNA-seq data set analysis, noncanonical 5'ss were preferentially detected in weakly used exon junctions of highly expressed genes. Among high-confidence splice sites, they were 10-fold overrepresented in clusters with a neighboring, more frequently used 5'ss. Conversely, these more frequently used neighbors contained only the dinucleotides GT, GC, and TT, in accordance with the above ranking.

Effect fingerprints of antipsychotic drugs on neural networks in vitro.

We compared the acute effect of typical (haloperidol) and atypical (aripiprazole, clozapine, olanzapine) antipsychotic drugs (APDs) on spontaneous electrophysiological activity of in vitro neuronal networks cultured on microelectrode arrays (MEAs). Network burst analysis revealed a "regularizing" effect of all APDs at therapeutic concentrations, i.e., an increase of network-wide temporal synchronization. At supratherapeutic concentrations, all APDs but olanzapine mediated a decrease of burst and spike rates, burst duration, number of spikes in bursts, and network synchrony. The rank order of potency of APDs was: haloperidol > aripiprazole > clozapine > olanzapine (no suppression). Disruption of network function was not due to enhanced cell death as assessed by trypan blue staining. APDs promoted distinct concentration-dependent alterations yielding acute effect fingerprints of the tested compounds. These effects were rather characteristic for individual compounds than distinctive for typical vs. atypical APDs. Thus, this dichotomy may be of value in distinguishing clinical features but has no apparent basis on the network or local circuitry level.

Succession of splicing regulatory elements determines cryptic 5΄ss functionality.

A critical step in exon definition is the recognition of a proper splice donor (5΄ss) by the 5' end of U1 snRNA. In the selection of appropriate 5΄ss, cis-acting splicing regulatory elements (SREs) are indispensable. As a model for 5΄ss recognition, we investigated cryptic 5΄ss selection within the human fibrinogen Bß-chain gene (FGB) exon 7, where we identified several exonic SREs that simultaneously acted on up- and downstream cryptic 5΄ss. In the FGB exon 7 model system, 5΄ss selection iteratively proceeded along an alternating sequence of U1 snRNA binding sites and interleaved SREs which in principle supported different 3' exon ends. Like in a relay race, SREs either suppressed a potential 5΄ss and passed the splicing baton on or splicing actually occurred. From RNA-Seq data, we systematically selected 19 genes containing exons with silent U1 snRNA binding sites competing with nearby highly used 5΄ss. Extensive SRE analysis by different algorithms found authentic 5΄ss significantly more supported by SREs than silent U1 snRNA binding sites, indicating that our concept may permit generalization to a model for 5΄ss selection and 3' exon end definition.

Analysis of Competing HIV-1 Splice Donor Sites Uncovers a Tight Cluster of Splicing Regulatory Elements within Exon 2/2b.

The HIV-1 accessory protein Vif is essential for viral replication by counteracting the host restriction factor APOBEC3G (A3G), and balanced levels of both proteins are required for efficient viral replication. Noncoding exons 2/2b contain the Vif start codon between their alternatively used splice donors 2 and 2b (D2 and D2b). For vif mRNA, intron 1 must be removed while intron 2 must be retained. Thus, splice acceptor 1 (A1) must be activated by U1 snRNP binding to either D2 or D2b, while splicing at D2 or D2b must be prevented. Here, we unravel the complex interactions between previously known and novel components of the splicing regulatory network regulating HIV-1 exon 2/2b inclusion in viral mRNAs. In particular, using RNA pulldown experiments and mass spectrometry analysis, we found members of the heterogeneous nuclear ribonucleoparticle (hnRNP) A/B family binding to a novel splicing regulatory element (SRE), the exonic splicing silencer ESS2b, and the splicing regulatory proteins Tra2/SRSF10 binding to the nearby exonic splicing enhancer ESE2b. Using a minigene reporter, we performed bioinformatics HEXplorer-guided mutational analysis to narrow down SRE motifs affecting splice site selection between D2 and D2b. Eventually, the impacts of these SREs on the viral splicing pattern and protein expression were exhaustively analyzed in viral particle production and replication experiments. Masking of these protein binding sites by use of locked nucleic acids (LNAs) impaired Vif expression and viral replication.IMPORTANCE Based on our results, we propose a model in which a dense network of SREs regulates vif mRNA and protein expression, crucial to maintain viral replication within host cells with varying A3G levels and at different stages of infection. This regulation is maintained by several serine/arginine-rich splicing factors (SRSF) and hnRNPs binding to those elements. Targeting this cluster of SREs with LNAs may lead to the development of novel effective therapeutic strategies.

Genomic HEXploring allows landscaping of novel potential splicing regulatory elements.

Effective splice site selection is critically controlled by flanking splicing regulatory elements (SREs) that can enhance or repress splice site use. Although several computational algorithms currently identify a multitude of potential SRE motifs, their predictive power with respect to mutation effects is limited. Following a RESCUE-type approach, we defined a hexamer-based 'HEXplorer score' as average Z-score of all six hexamers overlapping with a given nucleotide in an arbitrary genomic sequence. Plotted along genomic regions, HEXplorer score profiles varied slowly in the vicinity of splice sites. They reflected the respective splice enhancing and silencing properties of splice site neighborhoods beyond the identification of single dedicated SRE motifs. In particular, HEXplorer score differences between mutant and reference sequences faithfully represented exonic mutation effects on splice site usage. Using the HIV-1 pre-mRNA as a model system highly dependent on SREs, we found an excellent correlation in 29 mutations between splicing activity and HEXplorer score. We successfully predicted and confirmed five novel SREs and optimized mutations inactivating a known silencer. The HEXplorer score allowed landscaping of splicing regulatory regions, provided a quantitative measure of mutation effects on splice enhancing and silencing properties and permitted calculation of the mutationally most effective nucleotide.

Impaired action potential initiation in GABAergic interneurons causes hyperexcitable networks in an epileptic mouse model carrying a human Na(V)1.1 mutation.

Mutations in SCN1A and other ion channel genes can cause different epileptic phenotypes, but the precise mechanisms underlying the development of hyperexcitable networks are largely unknown. Here, we present a multisystem analysis of an SCN1A mouse model carrying the NaV1.1-R1648H mutation, which causes febrile seizures and epilepsy in humans. We found a ubiquitous hypoexcitability of interneurons in thalamus, cortex, and hippocampus, without detectable changes in excitatory neurons. Interestingly, somatic Na(+) channels in interneurons and persistent Na(+) currents were not significantly changed. Instead, the key mechanism of interneuron dysfunction was a deficit of action potential initiation at the axon initial segment that was identified by analyzing action potential firing. This deficit increased with the duration of firing periods, suggesting that increased slow inactivation, as recorded for recombinant mutated channels, could play an important role. The deficit in interneuron firing caused reduced action potential-driven inhibition of excitatory neurons as revealed by less frequent spontaneous but not miniature IPSCs. Multiple approaches indicated increased spontaneous thalamocortical and hippocampal network activity in mutant mice, as follows: (1) more synchronous and higher-frequency firing was recorded in primary neuronal cultures plated on multielectrode arrays; (2) thalamocortical slices examined by field potential recordings revealed spontaneous activities and pathological high-frequency oscillations; and (3) multineuron Ca(2+) imaging in hippocampal slices showed increased spontaneous neuronal activity. Thus, an interneuron-specific generalized defect in action potential initiation causes multisystem disinhibition and network hyperexcitability, which can well explain the occurrence of seizures in the studied mouse model and in patients carrying this mutation.

Balanced splicing at the Tat-specific HIV-1 3'ss A3 is critical for HIV-1 replication.

BACKGROUND: The viral regulatory protein Tat is essential for establishing a productive transcription from the 5'-LTR promoter during the early phase of viral gene expression. Formation of the Tat-encoding mRNAs requires splicing at the viral 3'ss A3, which has previously been shown to be both negatively and positively regulated by the downstream splicing regulatory elements (SREs) ESS2p and ESE2/ESS2. However, using the novel RESCUE-type computational HEXplorer algorithm, we were recently able to identify another splicing enhancer (ESE(5807-5838), henceforth referred to as ESE tat ) located between ESS2p and ESE2/ESS2. Here we show that ESE tat has a great impact on viral tat-mRNA splicing and that it is fundamental for regulated 3'ss A3 usage. RESULTS: Mutational inactivation or locked nucleic acid (LNA)-directed masking of the ESE tat sequence in the context of a replication-competent virus was associated with a failure (i) to activate viral 3'ss A3 and (ii) to accumulate Tat-encoding mRNA species. Consequently, due to insufficient amounts of Tat protein efficient viral replication was drastically impaired. RNA in vitro binding assays revealed SRSF2 and SRSF6 as candidate splicing factors acting through ESE tat and ESE2 for 3'ss A3 activation. This notion was supported by coexpression experiments, in which wild-type, but not ESE tat -negative provirus responded to higher levels of SRSF2 and SRSF6 proteins with higher levels of tat-mRNA splicing. Remarkably, we could also find that SRSF6 overexpression established an antiviral state within provirus-transfected cells, efficiently blocking virus particle production. For the anti-HIV-1 activity the arginine-serine (RS)-rich domain of the splicing factor was dispensable. CONCLUSIONS: Based on our results, we propose that splicing at 3'ss A3 is dependent on binding of the enhancing SR proteins SRSF2 and SRSF6 to the ESE tat and ESE2 sequence. Mutational inactivation or interference specifically with ESE tat activity by LNA-directed masking seem to account for an early stage defect in viral gene expression, probably by cutting off the supply line of Tat that HIV needs to efficiently transcribe its genome.

Tra2-mediated recognition of HIV-1 5' splice site D3 as a key factor in the processing of vpr mRNA.

Small noncoding HIV-1 leader exon 3 is defined by its splice sites A2 and D3. While 3' splice site (3'ss) A2 needs to be activated for vpr mRNA formation, the location of the vpr start codon within downstream intron 3 requires silencing of splicing at 5'ss D3. Here we show that the inclusion of both HIV-1 exon 3 and vpr mRNA processing is promoted by an exonic splicing enhancer (ESE(vpr)) localized between exonic splicing silencer ESSV and 5'ss D3. The ESE(vpr) sequence was found to be bound by members of the Transformer 2 (Tra2) protein family. Coexpression of these proteins in provirus-transfected cells led to an increase in the levels of exon 3 inclusion, confirming that they act through ESE(vpr). Further analyses revealed that ESE(vpr) supports the binding of U1 snRNA at 5'ss D3, allowing bridging interactions across the upstream exon with 3'ss A2. In line with this, an increase or decrease in the complementarity of 5'ss D3 to the 5' end of U1 snRNA was accompanied by a higher or lower vpr expression level. Activation of 3'ss A2 through the proposed bridging interactions, however, was not dependent on the splicing competence of 5'ss D3 because rendering it splicing defective but still competent for efficient U1 snRNA binding maintained the enhancing function of D3. Therefore, we propose that splicing at 3'ss A2 occurs temporally between the binding of U1 snRNA and splicing at D3.

Correct mRNA processing at a mutant TT splice donor in FANCC ameliorates the clinical phenotype in patients and is enhanced by delivery of suppressor U1 snRNAs.

The U1 small nuclear RNA (U1 snRNA) as a component of the major U2-dependent spliceosome recognizes 5' splice sites (5'ss) containing GT as the canonical dinucleotide in the intronic positions +1 and +2. The c.165+1G>T germline mutation in the 5'ss of exon 2 of the Fanconi anemia C (FANCC) gene commonly predicted to prevent correct splicing was identified in nine FA patients from three pedigrees. RT-PCR analysis of the endogenous FANCC mRNA splicing pattern of patient-derived fibroblasts revealed aberrant mRNA processing, but surprisingly also correct splicing at the TT dinucleotide, albeit with lower efficiency. This consequently resulted in low levels of correctly spliced transcript and minute levels of normal posttranslationally processed FANCD2 protein, indicating that this naturally occurring TT splicing might contribute to the milder clinical manifestations of the disease in these patients. Functional analysis of this FANCC 5'ss within splicing reporters revealed that both the noncanonical TT dinucleotide and the genomic context of FANCC were required for the residual correct splicing at this mutant 5'ss. Finally, use of lentiviral vectors as a delivery system to introduce expression cassettes for TT-adapted U1 snRNAs into primary FANCC patient fibroblasts allowed the correction of the DNA-damage-induced G2 cell-cycle arrest in these cells, thus representing an alternative transcript-targeting approach for genetic therapy of inherited splice-site mutations.

Using routine data for quality assessment in NeuroNet telestroke care.

BACKGROUND: Systematic clinical trials are often unavailable to evaluate and optimize operational telestroke networks. In a complementary approach, readily available routine clinical data were analyzed in this study to evaluate the effect of a telestroke network over a 4-year period. METHODS: Routine clinical data from the HELIOS hospital information system were compared before and after implementation of the NeuroNet concept, including neurologic acute stroke teleconsultations, standard operating procedures, and peer review quality management in 3 hospital cohorts: 5 comprehensive stroke centers, 5 NeuroNet hospitals, and 5 matched control hospitals. RESULTS: During the study period, the rate of thrombolytic therapy increased by 4.8% in NeuroNet hospitals, while ischemic stroke in-hospital mortality decreased (relative risk reduction ~29% in NeuroNet and control hospitals). The odds ratio for thrombolytic therapy in comprehensive stroke centers compared to NeuroNet hospitals was reduced from 3.7 to 1.3 between 2006 and 2009. Comprehensive stroke care coding according to German Diagnosis Related Groups definitions increased by 45% in NeuroNet (P < .0001) and by 18% in control hospitals. CONCLUSIONS: Routine clinical data on in-hospital mortality, the rate of thrombolytic therapy, and comprehensive stroke care coding reflect different aspects of acute stroke care improvement related to the implementation of the telemedical NeuroNet concept and unified quality management (standard operating procedure teaching concept, peer review process). Similar evaluation processes could contribute to quality monitoring in other telestroke networks.

Modifying splice site usage with ModCon: Maintaining the genetic code while changing the underlying mRNP code.

Codon degeneracy of amino acid sequences permits an additional "mRNP code" layer underlying the genetic code that is related to RNA processing. In pre-mRNA splicing, splice site usage is determined by both intrinsic strength and sequence context providing RNA binding sites for splicing regulatory proteins. In this study, we systematically examined modification of splicing regulatory properties in the neighborhood of a GT site, i.e. potential splice site, without altering the encoded amino acids. We quantified the splicing regulatory properties of the neighborhood around a potential splice site by its Splice Site HEXplorer Weight (SSHW) based on the HEXplorer score algorithm. To systematically modify GT site neighborhoods, either minimizing or maximizing their SSHW, we designed the novel stochastic optimization algorithm ModCon that applies a genetic algorithm with stochastic crossover, insertion and random mutation elements supplemented by a heuristic sliding window approach. To assess the achievable range in SSHW in human splice donors without altering the encoded amino acids, we applied ModCon to a set of 1000 randomly selected Ensembl annotated human splice donor sites, achieving substantial and accurate changes in SSHW. Using ModCon optimization, we successfully switched splice donor usage in a splice site competition reporter containing coding sequences from FANCA, FANCB or BRCA2, while retaining their amino acid coding information. The ModCon algorithm and its R package implementation can assist in reporter design by either introducing novel splice sites, silencing accidental, undesired splice sites, and by generally modifying the entire mRNP code while maintaining the genetic code.

Differential acute impact of therapeutically effective and overdose concentrations of lithium on human neuronal single cell and network function.

Lithium salts are used as mood-balancing medication prescribed to patients suffering from neuropsychiatric disorders, such as bipolar disorder and major depressive disorder. Lithium salts cross the blood-brain barrier and reach the brain parenchyma within few hours after oral application, however, how lithium influences directly human neuronal function is unknown. We applied patch-clamp and microelectrode array technology on human induced pluripotent stem cell (iPSC)-derived cortical neurons acutely exposed to therapeutic (<1 mM) and overdose concentrations (>1 mM) of lithium chloride (LiCl) to assess how therapeutically effective and overdose concentrations of LiCl directly influence human neuronal electrophysiological function at the synapse, single-cell, and neuronal network level. We describe that human iPSC-cortical neurons exposed to lithium showed an increased neuronal activity under all tested concentrations. Furthermore, we reveal a lithium-induced, concentration-dependent, transition of regular synchronous neuronal network activity using therapeutically effective concentration (<1 mM LiCl) to epileptiform-like neuronal discharges using overdose concentration (>1 mM LiCl). The overdose concentration lithium-induced epileptiform-like activity was similar to the epileptiform-like activity caused by the GABAA-receptor antagonist. Patch-clamp recordings reveal that lithium reduces action potential threshold at all concentrations, however, only overdose concentration causes increased frequency of spontaneous AMPA-receptor mediated transmission. By applying the AMPA-receptor antagonist and anti-epileptic drug Perampanel, we demonstrate that Perampanel suppresses lithium-induced epileptiform-like activity in human cortical neurons. We provide insights in how therapeutically effective and overdose concentration of lithium directly influences human neuronal function at synapse, a single neuron, and neuronal network levels. Furthermore, we provide evidence that Perampanel suppresses pathological neuronal discharges caused by overdose concentrations of lithium in human neurons.

Cerebrospinal fluid of brain trauma patients inhibits in vitro neuronal network function via NMDA receptors.

Neurological diseases frequently induce pathological changes of cerebrospinal fluid (CSF) that might secondarily influence brain activity, as the CSF-brain barrier is partially permeable. However, functional effects of CSF on neuronal network activity have not been specified to date. Here, we report that CSF specimens from patients with reduced Glasgow Coma Scale values caused by severe traumatic brain injury suppress synchronous activity of in vitro-generated neuronal networks in comparison with controls. We present evidence that underlying mechanisms include increased N-methyl-D-aspartate receptor activity mediated by a CSF fraction containing elevated amino acid concentrations. These proof-of-principle data suggest that determining effects of CSF specimens on neuronal network activity might be of diagnostic value.

VarCon: An R Package for Retrieving Neighboring Nucleotides of an SNV.

Reporting of a single nucleotide variant (SNV) follows the Sequence Variant Nomenclature (http://varnomen.hgvs.org/), using an unambiguous numbering scheme specific for coding and noncoding DNA. However, the corresponding sequence neighborhood of a given SNV, which is required to assess its impact on splicing regulation, is not easily accessible from this nomenclature. Providing fast and easy access to this neighborhood just from a given SNV reference, the novel tool VarCon combines information of the Ensembl human reference genome and the corresponding transcript table for accurate retrieval. VarCon also displays splice site scores (HBond and MaxEnt scores) and HEXplorer profiles of an SNV neighborhood, reflecting position-dependent splice enhancing and silencing properties.

Human Cerebrospinal Fluid Promotes Neuronal Circuit Maturation of Human Induced Pluripotent Stem Cell-Derived 3D Neural Aggregates.

Human induced pluripotent stem cell (hiPSC)-derived in vitro neural and organoid models resemble fetal, rather than adult brain properties, indicating that currently applied cultivation media and supplements are insufficient to achieve neural maturation beyond the fetal stage. In vivo, cerebrospinal fluid molecules are regulating the transition of the immature fetal human brain into a mature adult brain. By culturing hiPSC-3D neural aggregates in human cerebrospinal fluid (hCSF) obtained from healthy adult individuals, we demonstrate that hCSF rapidly triggers neurogenesis, gliogenesis, synapse formation, neurite outgrowth, suppresses proliferation of residing neural stem cells, and results in the formation of synchronously active neuronal circuits in vitro within 3 days. Thus, a physiologically relevant and adult brain-like milieu triggers maturation of hiPSC-3D neural aggregates into highly functional neuronal circuits in vitro. The approach presented here opens a new avenue to identify novel physiological factors for the improvement of hiPSC neural in vitro models.

TGF-ß1 Suppresses Proliferation and Induces Differentiation in Human iPSC Neural in vitro Models.

Persistent neural stem cell (NSC) proliferation is, among others, a hallmark of immaturity in human induced pluripotent stem cell (hiPSC)-based neural models. TGF-ß1 is known to regulate NSCs in vivo during embryonic development in rodents. Here we examined the role of TGF-ß1 as a potential candidate to promote in vitro differentiation of hiPSCs-derived NSCs and maturation of neuronal progenies. We present that TGF-ß1 is specifically present in early phases of human fetal brain development. We applied confocal imaging and electrophysiological assessment in hiPSC-NSC and 3D neural in vitro models and demonstrate that TGF-ß1 is a signaling protein, which specifically suppresses proliferation, enhances neuronal and glial differentiation, without effecting neuronal maturation. Moreover, we demonstrate that TGF-ß1 is equally efficient in enhancing neuronal differentiation of human NSCs as an artificial synthetic small molecule. The presented approach provides a proof-of-concept to replace artificial small molecules with more physiological signaling factors, which paves the way to improve the physiological relevance of human neural developmental in vitro models.

Characterization and application of electrically active neuronal networks established from human induced pluripotent stem cell-derived neural progenitor cells for neurotoxicity evaluation.

Neurotoxicity is mediated by a variety of modes-of-actions leading to disturbance of neuronal function. In order to screen larger numbers of compounds for their neurotoxic potential, in vitro functional neuronal networks (NN) might be helpful tools. We established and characterized human NN (hNN) from hiPSC-derived neural progenitor cells by comparing hNN formation with two different differentiation media: in presence (CINDA) and absence (neural differentiation medium (NDM)) of maturation-supporting factors. As a NN control we included differentiating rat NN (rNN) in the study. Gene/protein expression and electrical activity from in vitro developing NN were assessed at multiple time points. Transcriptomes of 5, 14 and 28 days in vitro CINDA-grown hNN were compared to gene expression profiles of in vivo human developing brains. Molecular expression analyses as well as measures of electrical activity indicate that NN mature into neurons of different subtypes and astrocytes over time. In contrast to rNN, hNN are less electrically active within the same period of differentiation time, yet hNN grown in CINDA medium develop higher firing rates than hNN without supplements. Challenge of NN with neuronal receptor stimulators and inhibitors demonstrate presence of inhibitory, GABAergic neurons, whereas glutamatergic responses are limited. hiPSC-derived GABAergic hNN grown in CINDA medium might be a useful tool as part of an in vitro battery for assessing neurotoxicity.

Niche-dependent development of functional neuronal networks from embryonic stem cell-derived neural populations.

BACKGROUND: The present work was performed to investigate the ability of two different embryonic stem (ES) cell-derived neural precursor populations to generate functional neuronal networks in vitro. The first ES cell-derived neural precursor population was cultivated as free-floating neural aggregates which are known to form a developmental niche comprising different types of neural cells, including neural precursor cells (NPCs), progenitor cells and even further matured cells. This niche provides by itself a variety of different growth factors and extracellular matrix proteins that influence the proliferation and differentiation of neural precursor and progenitor cells. The second population was cultivated adherently in monolayer cultures to control most stringently the extracellular environment. This population comprises highly homogeneous NPCs which are supposed to represent an attractive way to provide well-defined neuronal progeny. However, the ability of these different ES cell-derived immature neural cell populations to generate functional neuronal networks has not been assessed so far. RESULTS: While both precursor populations were shown to differentiate into sufficient quantities of mature NeuN+ neurons that also express GABA or vesicular-glutamate-transporter-2 (vGlut2), only aggregate-derived neuronal populations exhibited a synchronously oscillating network activity 24 weeks after initiating the differentiation as detected by the microelectrode array technology. Neurons derived from homogeneous NPCs within monolayer cultures did merely show uncorrelated spiking activity even when differentiated for up to 12 weeks. We demonstrated that these neurons exhibited sparsely ramified neurites and an embryonic vGlut2 distribution suggesting an inhibited terminal neuronal maturation. In comparison, neurons derived from heterogeneous populations within neural aggregates appeared as fully mature with a dense neurite network and punctuated vGlut2 expression within presynaptic vesicles. Also those NPCs that had migrated away from adherent neural aggregates maintained their ability to generate a synchronously oscillating neuronal network, even if they were separated from adherent aggregates, dissociated and re-plated. CONCLUSION: These findings suggest that the complex environment within niches and aggregates of heterogeneous neural cell populations support the generation of fully mature neurons and functional neuronal networks from ES cell-derived neural cells. In contrast, homogeneous ES cell-derived NPCs within monolayer cultures exhibited an impaired functional neuronal maturation.

Context matters: Regulation of splice donor usage.

Elaborate research on splicing, starting in the late seventies, evolved from the discovery that 5' splice sites are recognized by their complementarity to U1 snRNA towards the realization that RNA duplex formation cannot be the sole basis for 5'ss selection. Rather, their recognition is highly influenced by a number of context factors including transcript architecture as well as splicing regulatory elements (SREs) in the splice site neighborhood. In particular, proximal binding of splicing regulatory proteins highly influences splicing outcome. The importance of SRE integrity especially becomes evident in the light of human pathogenic mutations where single nucleotide changes in SREs can severely affect the resulting transcripts. Bioinformatics tools nowadays greatly assist in the computational evaluation of 5'ss, their neighborhood and the impact of pathogenic mutations. Although predictions are already quite robust, computational evaluation of the splicing regulatory landscape still faces challenges to increase future reliability. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.