Splice site proximity influences alternative exon definition.

Alternative splicing enables higher eukaryotes to expand mRNA diversity from a finite number of genes through highly combinatorial splice site selection mechanisms that are influenced by the sequence of competing splice sites, cis-regulatory elements binding trans-acting factors, the length of exons and introns harbouring alternative splice sites and RNA secondary structures at putative splice junctions. To test the hypothesis that the intron definition or exon definition modes of splice site recognition direct the selection of alternative splice patterns, we created a database of alternative splice site usage (ALTssDB). When alternative splice sites are embedded within short introns (intron definition), the 5' and 3' splice sites closest to each other across the intron preferentially pair, consistent with previous observations. However, when alternative splice sites are embedded within large flanking introns (exon definition), the 5' and 3' splice sites closest to each other across the exon are preferentially selected. Thus, alternative splicing decisions are influenced by the intron and exon definition modes of splice site recognition. The results demonstrate that the spliceosome pairs splice sites that are closest in proximity within the unit of initial splice site selection.

The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes.

Metabolic labeling is a widely used tool to investigate different aspects of pre-mRNA splicing and RNA turnover. The labeling technology takes advantage of native cellular machineries where a nucleotide analog is readily taken up and incorporated into nascent RNA. One such analog is 4-thiouridine (4sU). Previous studies demonstrated that the uptake of 4sU at elevated concentrations (>50µM) and extended exposure led to inhibition of rRNA synthesis and processing, presumably induced by changes in RNA secondary structure. Thus, it is possible that 4sU incorporation may also interfere with splicing efficiency. To test this hypothesis, we carried out splicing analyses of pre-mRNA substrates with varying levels of 4sU incorporation (0-100%). We demonstrate that increased incorporation of 4sU into pre-mRNAs decreased splicing efficiency. The overall impact of 4sU labeling on pre-mRNA splicing efficiency negatively correlates with the strength of splice site signals such as the 3' and the 5' splice sites. Introns with weaker splice sites are more affected by the presence of 4sU. We also show that transcription by T7 polymerase and pre-mRNA degradation kinetics were impacted at the highest levels of 4sU incorporation. Increased incorporation of 4sU caused elevated levels of abortive transcripts, and fully labeled pre-mRNA is more stable than its uridine-only counterpart. Cell culture experiments show that a small number of alternative splicing events were modestly, but statistically significantly influenced by metabolic labeling with 4sU at concentrations considered to be tolerable (40 µM). We conclude that at high 4sU incorporation rates small, but noticeable changes in pre-mRNA splicing can be detected when splice sites deviate from consensus. Given these potential 4sU artifacts, we suggest that appropriate controls for metabolic labeling experiments need to be included in future labeling experiments.

Advantages and limitations of various treatment chamber designs for reversible and irreversible electroporation in life sciences.

The fundamental mechanisms of pulsed electric fields on biological cells are not yet fully elucidated, though it is apparent that membrane electroporation plays a crucial role. Little is known about treatment-chamber-specific effects, and systematic studies are scarce. Thus, the present study evaluates the (dis-)advantages of various treatment chamber designs for liquid applications at differing scales. Three chambers, namely parallel plate microfluidic (V̇: 0.1 ml/min; titanium electrodes), co-linear meso (V̇: 5.0 ml/min; stainless steel electrodes), and co-linear macro (V̇: 83.3 ml/min; stainless steel electrodes) chambers, were studied. Electroporation effects on Escherichia coli in media with 0.1-10.0 mS/cm were evaluated by plate counts and flow cytometry at 8, 16, and 20 kV/cm. For the microfluidic chamber, predominantly irreversible electroporation (2.5 logs10 reductions) was seen at 0.1 mS/cm, while high irreversible electroporation (4.2 logs10 reductions) at 10.0 mS/cm was observed for the macro chamber. The meso chamber indicated a similar trend towards increased conductivity, even though only low inactivation levels were present. Variation in conductivity and electrode configuration or area likely induces effects resulting in distinct electroporation levels, as observed for the micro and macro chamber. Suitable application scenarios, depending on targeted electroporation effects, were suggested.

Publisher Correction: Transcriptomic and proteomic signatures of stemness and differentiation in the colon crypt.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transcriptomic and proteomic signatures of stemness and differentiation in the colon crypt.

Intestinal stem cells are non-quiescent, dividing epithelial cells that rapidly differentiate into progenitor cells of the absorptive and secretory cell lineages. The kinetics of this process is rapid such that the epithelium is replaced weekly. To determine how the transcriptome and proteome keep pace with rapid differentiation, we developed a new cell sorting method to purify mouse colon epithelial cells. Here we show that alternative mRNA splicing and polyadenylation dominate changes in the transcriptome as stem cells differentiate into progenitors. In contrast, as progenitors differentiate into mature cell types, changes in mRNA levels dominate the transcriptome. RNA processing targets regulators of cell cycle, RNA, cell adhesion, SUMOylation, and Wnt and Notch signaling. Additionally, global proteome profiling detected >2,800 proteins and revealed RNA:protein patterns of abundance and correlation. Paired together, these data highlight new potentials for autocrine and feedback regulation and provide new insights into cell state transitions in the crypt.

Allosteric regulation of U1 snRNP by splicing regulatory proteins controls spliceosomal assembly.

Alternative splicing is responsible for much of the transcriptomic and proteomic diversity observed in eukaryotes and involves combinatorial regulation by many cis-acting elements and trans-acting factors. SR and hnRNP splicing regulatory proteins often have opposing effects on splicing efficiency depending on where they bind the pre-mRNA relative to the splice site. Position-dependent splicing repression occurs at spliceosomal E-complex, suggesting that U1 snRNP binds but cannot facilitate higher order spliceosomal assembly. To test the hypothesis that the structure of U1 snRNA changes during activation or repression, we developed a method to structure-probe native U1 snRNP in enriched conformations that mimic activated or repressed spliceosomal E-complexes. While the core of U1 snRNA is highly structured, the 5' end of U1 snRNA shows different SHAPE reactivities and psoralen crosslinking efficiencies depending on where splicing regulatory elements are located relative to the 5' splice site. A motif within the 5' splice site binding region of U1 snRNA is more reactive toward SHAPE electrophiles when repressors are bound, suggesting U1 snRNA is bound, but less base-paired. These observations demonstrate that splicing regulators modulate splice site selection allosterically.

Capacitive coupling increases the accuracy of cell-specific tumour disruption by electric fields.

Irreversible electroporation holds great potential for cell-specific lysis due to the size-dependent susceptibility of cells to externally imposed electric fields. Previous attempts at selective cell lysis lead to significant overlap between affected populations and struggle with inconsistent biological outcome. We propose that charge transfer at the electrode-liquid interface is responsible by inducing multifactorial effects originating from both the electric field and electrochemical reactions. A promising remedy is the coating of electrodes with a high-k dielectric layer. The resulting capacitive coupling restores the selective potential of electric field mediated lysis in a microfluidic setup. Initial experiments show the consistent depletion of erythrocytes from whole blood while leaving leukocytes intact. The same is true for the reproducible and selective depletion of Jurkat and MCF-7 cells in a mixture with leukocytes. Unexpectedly, the observed order of lysis cannot be correlated with cell size. This implies that the cellular response to capacitive coupling features a selective characteristic that is different from conventional lysis configurations.

Controllable cell manipulation in a microfluidic pipette-tip design using capacitive coupling of electric fields.

Systems designed toward cell manipulation by electric fields are inherently challenged by energy dissipation along the electrode-electrolyte interface. A promising remedy is the introduction of high-k electrode passivation, enabling efficient capacitive coupling of electric fields into biological samples. We present the implementation of this strategy in a reusable pipette tip design featuring a 10 µl chamber volume for life science applications. Prototype validation and comparison to conductive gold-coated electrodes reveal a consistent and controllable biological effect that significantly increases the reproducibility of lysis events. The system provides precise descriptions of HEK-293 lysis dependency to variables such as field strength, frequency, and conductivity. Over 80% of cells were reversibly electroporated with minimal electrical lysis over a broad range of field settings. Successful transfection requires exponential decay pulses and showcases how modulating capacitive coupling can advance our understanding of fundamental mechanics in the field of electroporation.

Ultra-deep sequencing reveals pre-mRNA splicing as a sequence driven high-fidelity process.

Alternative splicing diversifies mRNA transcripts in human cells. While the spliceosome pairs exons with a high degree of accuracy, the rates of rare aberrant and non-canonical pre-mRNA splicing have not been evaluated at the nucleotide level to determine the quantity and identity of these events across splice junctions. Using ultra-deep sequencing the frequency of aberrant and non-canonical splicing events for three splice junctions flanking exon 7 of SMN1 were determined at single nucleotide resolution. After correction for background noise introduced by PCR amplification and sequencing steps, pre-mRNA splicing was shown to maintain a low overall rate of aberrant and non-canonically spliced events. Several previously unannotated splicing events across 3 exon|intron junctions in SMN1 were identified. Mutations within SMN exon 7 were shown to affect splicing fidelity by modulating RNA secondary structures, by altering the binding site of regulatory proteins and by changing the 5' splice site strength. Mutations also create a truncated SMN1 exon 7 through the introduction of a de novo non-canonical 5' splice site. The results from the ultra-deep sequencing approach highlight the impressive fidelity of pre-mRNA splicing and demonstrate that the immediate sequence context around splice sites is the main driving force behind non-canonical splice site pairing.

Exon size and sequence conservation improves identification of splice-altering nucleotides.

Pre-mRNA splicing is regulated through multiple trans-acting splicing factors. These regulators interact with the pre-mRNA at intronic and exonic positions. Given that most exons are protein coding, the evolution of exons must be modulated by a combination of selective coding and splicing pressures. It has previously been demonstrated that selective splicing pressures are more easily deconvoluted when phylogenetic comparisons are made for exons of identical size, suggesting that exon size-filtered sequence alignments may improve identification of nucleotides evolved to mediate efficient exon ligation. To test this hypothesis, an exon size database was created, filtering 76 vertebrate sequence alignments based on exon size conservation. In addition to other genomic parameters, such as splice-site strength, gene position, or flanking intron length, this database permits the identification of exons that are size- and/or sequence-conserved. Highly size-conserved exons are always sequence-conserved. However, sequence conservation does not necessitate exon size conservation. Our analysis identified evolutionarily young exons and demonstrated that length conservation is a strong predictor of alternative splicing. A published data set of approximately 5000 exonic SNPs associated with disease was analyzed to test the hypothesis that exon size-filtered sequence comparisons increase detection of splice-altering nucleotides. Improved splice predictions could be achieved when mutations occur at the third codon position, especially when a mutation decreases exon inclusion efficiency. The results demonstrate that coding pressures dominate nucleotide composition at invariable codon positions and that exon size-filtered sequence alignments permit identification of splice-altering nucleotides at wobble positions.

Neurotoxic potential of reactive astrocytes in canine distemper demyelinating leukoencephalitis.

Canine distemper virus (CDV) causes a fatal demyelinating leukoencephalitis in young dogs resembling human multiple sclerosis. Astrocytes are the main cellular target of CDV and undergo reactive changes already in pre-demyelinating brain lesions. Based on their broad range of beneficial and detrimental effects in the injured brain reactive astrogliosis is in need of intensive investigation. The aim of the study was to characterize astrocyte plasticity during the course of CDV-induced demyelinating leukoencephalitis by the aid of immunohistochemistry, immunofluorescence and gene expression analysis. Immunohistochemistry revealed the presence of reactive glial fibrillary acidic protein (GFAP)+ astrocytes with increased survivin and reduced aquaporin 4, and glutamine synthetase protein levels, indicating disturbed blood brain barrier function, glutamate homeostasis and astrocyte maladaptation, respectively. Gene expression analysis revealed 81 differentially expressed astrocyte-related genes with a dominance of genes associated with neurotoxic A1-polarized astrocytes. Accordingly, acyl-coA synthetase long-chain family member 5+/GFAP+, and serglycin+/GFAP+ cells, characteristic of A1-astrocytes, were found in demyelinating lesions by immunofluorescence. In addition, gene expression revealed a dysregulation of astrocytic function including disturbed glutamate homeostasis and altered immune function. Observed findings indicate an astrocyte polarization towards a neurotoxic phenotype likely contributing to lesion initiation and progression in canine distemper leukoencephalitis.

Combinatorial regulation of alternative splicing.

The generation of protein coding mRNAs from pre-mRNA is a fundamental biological process that is required for gene expression. Alternative pre-mRNA splicing is responsible for much of the transcriptomic and proteomic diversity observed in higher order eukaryotes. Aberrations that disrupt regular alternative splicing patterns are known to cause human diseases, including various cancers. Alternative splicing is a combinatorial process, meaning many factors affect which two splice sites are ligated together. The features that dictate exon inclusion are comprised of splice site strength, intron-exon architecture, RNA secondary structure, splicing regulatory elements, promoter use and transcription speed by RNA polymerase and the presence of post-transcriptional nucleotide modifications. A comprehensive view of all of the factors that influence alternative splicing decisions is necessary to predict splicing outcomes and to understand the molecular basis of disease. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.

Pancreatic pericytes originate from the embryonic pancreatic mesenchyme.

The embryonic origin of pericytes is heterogeneous, both between and within organs. While pericytes of coelomic organs were proposed to differentiate from the mesothelium, a single-layer squamous epithelium, the embryonic origin of pancreatic pericytes has yet to be reported. Here, we show that adult pancreatic pericytes originate from the embryonic pancreatic mesenchyme. Our analysis indicates that pericytes of the adult mouse pancreas originate from cells expressing the transcription factor Nkx3.2. In the embryonic pancreas, Nkx3.2-expressing cells constitute the multilayered mesenchyme, which surrounds the pancreatic epithelium and supports multiple events in its development. Thus, we traced the fate of the pancreatic mesenchyme. Our analysis reveals that pancreatic mesenchymal cells acquire various pericyte characteristics, including gene expression, typical morphology, and periendothelial location, during embryogenesis. Importantly, we show that the vast majority of pancreatic mesenchymal cells differentiate into pericytes already at embryonic day 13.5 and progressively acquires a more mature pericyte phenotype during later stages of pancreas organogenesis. Thus, our study indicates the embryonic pancreatic mesenchyme as the primary origin to adult pancreatic pericytes. As pericytes of other coelomic organs were suggested to differentiate from the mesothelium, our findings point to a distinct origin of these cells in the pancreas. Thus, our study proposes a complex ontogeny of pericytes of coelomic organs.

Association of Varroa destructor females in multiply infested cells of the honeybee Apis mellifera.

The genetic diversity of Varroa destructor (Anderson & Trueman) is limited outside its natural range due to population bottlenecks and its propensity to inbreed. In light of the arms race between V. destructor and its honeybee (Apis mellifera L.) host, any mechanism enhancing population admixture of the mite may be favored. One way that admixture can occur is when two genetically dissimilar mites coinvade a brood cell, with the progeny of the foundresses admixing. We determined the relatedness of 393 pairs of V. destructor foundresses, each pair collected from a single bee brood cell (n = five colonies). We used six microsatellites to identify the genotypes of mites coinvading a cell and calculated the frequency of pairs with different or the same genotypes. We found no deviation from random coinvasion, but the frequency of cells infested by mites with different genotypes was high. This rate of recombination, coupled with a high transmission rate of mites, homogenized the allelic pool of mites within the apiary.

Molecular Mechanisms for CFIm-Mediated Regulation of mRNA Alternative Polyadenylation.

Alternative mRNA processing is a critical mechanism for proteome expansion and gene regulation in higher eukaryotes. The SR family proteins play important roles in splicing regulation. Intriguingly, mammalian genomes encode many poorly characterized SR-like proteins, including subunits of the mRNA 3'-processing factor CFIm, CFIm68 and CFIm59. Here we demonstrate that CFIm functions as an enhancer-dependent activator of mRNA 3' processing. CFIm regulates global alternative polyadenylation (APA) by specifically binding and activating enhancer-containing poly(A) sites (PASs). Importantly, the CFIm activator functions are mediated by the arginine-serine repeat (RS) domains of CFIm68/59, which bind specifically to an RS-like region in the CPSF subunit Fip1, and this interaction is inhibited by CFIm68/59 hyper-phosphorylation. The remarkable functional similarities between CFIm and SR proteins suggest that interactions between RS-like domains in regulatory and core factors may provide a common activation mechanism for mRNA 3' processing, splicing, and potentially other steps in RNA metabolism.

Preparation of Splicing Competent Nuclear Extract from Mammalian Cells and In Vitro Pre-mRNA Splicing Assay.

The ability to perform in vitro splicing assays has paved the way for in-depth studies of the mechanisms and machinery involved in the process of splicing. The in vitro splicing assay is a valuable experimental approach that combines the complexity of the spliceosome and regulatory systems with the flexibility of performing endless splicing and alternative splicing reactions. Through the use of crude nuclear extract and radiolabeled pre-mRNA, spliced mRNAs can be visualized using autoradiography for downstream analysis. This chapter describes the necessary steps to perform an in vitro splicing reaction, including the generation of the key components necessary for the splicing reaction; nuclear extract.

Isolation of Newly Transcribed RNA Using the Metabolic Label 4-Thiouridine.

Isolation of newly transcribed RNA is an invaluable approach that can be used to study the dynamic life of RNA in cellulo. Traditional methods of whole-cell RNA extraction limit subsequent gene expression analyses to the steady-state levels of RNA abundance, which often masks changes in RNA synthesis and processing. This chapter describes a methodology with low cytotoxicity that permits the labeling and isolation of nascent pre-mRNA in cell culture. The resulting isolate is suitable for use in a series of downstream applications aimed at studying changes in RNA synthesis, processing, or stability.

Seasonal cycle of inbreeding and recombination of the parasitic mite Varroa destructor in honeybee colonies and its implications for the selection of acaricide resistance.

Varroa destructor is the most devastating parasite of the Western honeybee, Apis mellifera. In the light of the arm race opposing the host and its parasite, the population dynamics and genetic diversity of these organisms are key parameters. However, the life cycle of V. destructor is characterized by extreme inbreeding due to full sibling mating in the host brood cells. We here present an equation reflecting the evolution of inbreeding in such a clonal system, and compare our predictions with empirical data based on the analysis of seven microsatellite markers. This comparison revealed that the mites perform essentially incestuous mating in the beginning of the brood season. However, this pattern changes with the development of mite infestation. Despite the fact that the overall level of genetic diversity of the mites remained low through the season, multiple inbred lineages were identified in the mites we sampled in June. As a response to the decrease of brood availability and the increase of the parasite population in parallel in the colonies, these lineages recombined towards the end of the season as mites co-infest brood cells. Our results suggest that the ratio of the number of mite per brood cell in the colony determines the genetic structure of the populations of V. destructor. This intracolonial population dynamics has great relevance for the selection of acaricide resistance in V. destructor. If chemical treatments occur before the recombination phase, inbreeding will greatly enhance the fixation of resistance alleles at the colony level.

Dual-component collagenous peptide/reactive oligomer hydrogels as potential nerve guidance materials - from characterization to functionalization.

Toward a new generation of improved nerve guidance conduits (NGCs), novel biomaterials are required to address pressing clinical shortcomings in peripheral nerve regeneration (PNR) and to promote biological performance. A dual-component hydrogel system formed by cross-linking reaction between maleic anhydride groups in an oligomeric building block for cross-linking of free amine functionalities in partially hydrolyzed collagen is formulated for continuous processing and NGC fabrication. The influence of the gelation base is optimized for processing from a double syringe delivery system with a static mixer. A hydrophilic low-concentrated base was introduced to control network formation and to utilize highly reactive macromers for gelation. Cross-linking extent and building block conversion were improved and homogenous monoliths were fabricated. Chemically derivatized hydrogels were obtained by conversion of a fraction of anhydride groups in the oligomeric precursor with monovalent primary amine-containing grafting molecules prior to gelation. Network stability in functionalized hydrogels was maintained and cationic moieties were implement to the gel that promoted in vitro cell attachment and spreading irrespective of mechanical stiffness. A molding strategy was introduced that allowed for fabrication of flexible tubular conduits in tunable dimensions and with chemically patterned structures. These hydrogel-based conduits hold promise for the next generation NGCs with integrated chemical cues for PNR.