Proteomic identification of heterogeneous nuclear ribonucleoprotein L as a novel component of SLM/Sam68 nuclear bodies

Background: Active pre-mRNA splicing occurs co-transcriptionally, and takes place throughout the nucleoplasm of eukaryotic cells. Splicing decisions are controlled by networks of nuclear RNA-binding proteins and their target sequences, sometimes in response to signalling pathways. Sam68 (Src-associated in mitosis 68 kDa) is the prototypic member of the STAR (Signal Transduction and Activation of RNA) family of RNA-binding proteins, which regulate splicing in response to signalling cascades. Nuclear Sam68 protein is concentrated within subnuclear organelles called SLM/Sam68 Nuclear Bodies (SNBs), which also contain some other splicing regulators, signalling components and nucleic acids. Results: We used proteomics to search for the major interacting protein partners of nuclear Sam68. In addition to Sam68 itself and known Sam68-associated proteins (heterogeneous nuclear ribonucleoproteins hnRNP A1, A2/B1 and G), we identified hnRNP L as a novel Sam68-interacting protein partner. hnRNP L protein was predominantly present within small nuclear protein complexes approximating to the expected size of monomers and dimers, and was quantitatively associated with nucleic acids. hnRNP L spatially co-localised with Sam68 as a novel component of SNBs and was also observed within the general nucleoplasm. Localisation within SNBs was highly specific to hnRNP L and was not shared by the closely-related hnRNP LL protein, nor any of the other Sam68-interacting proteins we identified by proteomics. The interaction between Sam68 and hnRNP L proteins was observed in a cell line which exhibits low frequency of SNBs suggesting that this association also takes place outside SNBs. Although ectopic expression of hnRNP L and Sam68 proteins independently affected splicing of CD44 variable exon v5 and TJP1 exon 20 minigenes, these proteins did not, however, co-operate with each other in splicing regulation of these target exons. Conclusion: Here we identify hnRNP L as a novel SNB component. We show that, compared with other identified Sam68-associated hnRNP proteins and hnRNP LL, this co-localisation within SNBs is specific to hnRNP L. Our data suggest that the novel Sam68-hnRNP L protein interaction may have a distinct role within SNBs

Can Enzymatic Treatment of Sugar Beet Pectins Reduce Coalescence Effects in High-Pressure Processes?

While sugar beet pectins (SBPs) are well known for effectively stabilizing fine oil droplets in low-fat food and beverages, e.g., low-fat dressings and soft drinks, it often fails in products of higher oil contents. The aim of this study was to improve the emulsifying properties of SBPs and, consequently, their ability to reduce coalescence during high pressure homogenization of products with increased oil content. Therefore, the molecular size of SBPs was reduced by partial cleavage of the homogalacturonan backbone using the enzymes exo- and endo-polygalacturonanase and varying incubation times. The sizes of SBPs were compared based on the molecular size distribution and hydrodynamic diameter. In addition, to obtain information on the interfacial activity and adsorption rate of SBPs, the dynamic interfacial tension was measured by drop profile analysis tensiometry. The (non)modified SBPs were used as emulsifying agents in 30 wt% mct oil–water emulsions stabilized with 0.5 wt% SBP at pH 3, prepared by high-pressure homogenization (400–1000 bar). By analyzing the droplet size distributions, conclusions could be drawn about the coalescence that occurred after droplet breakup. It could be shown that SBPs modified by exo-polygalacturonanase stabilized the oil–water interface more rapidly, resulting in less coalescence and the smallest oil droplets. In contrast, SBPs modified with endo-polygalacturonanase resulted in poorer emulsification properties, and thus larger oil droplets with increasing incubation time. The differences could be attributed to the different cleavage pattern of the enzymes used. The results suggest that a minimum molecular size is required for the stabilization of fine oil droplets with SBPs as emulsifiers

Effect of Conformation of Sugar Beet Pectin on the Interfacial and Emulsifying Properties

The influence of the conformation of sugar beet pectin (SBP) on the interfacial and emulsifying properties was investigated. The colloidal properties of SBP, such as zeta potential and hydrodynamic diameter, were characterized at different pH levels. Furthermore, pendant drop tensiometry and quartz crystal microgravimetry were used to study adsorption behavior (adsorbed mass and adsorption rate) and stabilizing mechanism (layer thickness and interfacial tension). A more compact conformation resulted in a faster reduction of interfacial tension, higher adsorbed mass, and a thicker adsorption layer. In addition, emulsions were prepared at varying conditions (pH 3–5) and formulations (1–30 wt% MCT oil, 0.1–2 wt% SBP), and their droplet size distributions were measured. The smallest oil droplets could be stabilized at pH 3. However, significantly more pectin was required at pH 3 compared to pH 4 or 5 to sufficiently stabilize the oil droplets. Both phenomena were attributed to the more compact conformation of SBP at pH < pK$_{a}$: On the one hand, pectins adsorbed faster and in greater quantity, forming a thicker interfacial layer. On the other hand, they covered less interfacial area per SBP molecule. Therefore, the SBP concentration must be chosen appropriately depending on the conformation

Optimization of glycolipid synthesis in hydrophilic deep eutectic solvents

Glycolipids are considered an alternative to petrochemically based surfactants because they are non-toxic, biodegradable, and less harmful to the environment while having comparable surface-active properties. They can be produced chemically or enzymatically in organic solvents or in deep eutectic solvents (DES) from renewable resources. DES are non-flammable, non-volatile, biodegradable, and almost non-toxic. Unlike organic solvents, sugars are easily soluble in hydrophilic DES. However, DES are highly viscous systems and restricted mass transfer is likely to be a major limiting factor for their application. Limiting factors for glycolipid synthesis in DES are not generally well understood. Therefore, the influence of external mass transfer, fatty acid concentration, and distribution on initial reaction velocity in two hydrophilic DES (choline:urea and choline:glucose) was investigated. At agitation speeds of and higher than 60 rpm, the viscosity of both DES did not limit external mass transfer. Fatty acid concentration of 0.5 M resulted in highest initial reaction velocity while higher concentrations had negative effects. Fatty acid accessibility was identified as a limiting factor for glycolipid synthesis in hydrophilic DES. Mean droplet sizes of fatty acid-DES emulsions can be significantly decreased by ultrasonic pretreatment resulting in significantly increased initial reaction velocity and yield (from 0.15 ± 0.03 μmol glucose monodecanoate/g DES to 0.57 ± 0.03 μmol/g) in the choline: urea DES. The study clearly indicates that fatty acid accessibility is a limiting factor in enzymatic glycolipid synthesis in DES. Furthermore, it was shown that physical pretreatment of fatty acid-DES emulsions is mandatory to improve the availability of fatty acids

Impact of Global Climate Change on the European Barley Market Requires Novel Multi-Method Approaches to Preserve Crop Quality and Authenticity

Most recently in 2018 and 2019, large parts of Europe were affected by periods of massive drought. Resulting losses in cereal yield pose a major risk to the global supply of barley, as more than 60% of global production is based in Europe. Despite the arising price fluctuations on the cereal market, authenticity of the crop must be ensured, which includes correct declaration of harvest years. Here, we show a novel approach that allows such differentiation for spring barley samples, which takes advantage of the chemical changes caused by the extreme drought. Samples from 2018 were successfully differentiated from those of 2017 by analysis of changes in near-infrared spectra, enrichment in the isotope (13)C, and strong accumulation of the plant-physiological marker betaine. We demonstrate that through consideration of multiple modern analysis techniques, not only can fraudulent labelling be prevented, but indispensable knowledge on the drought tolerance of crops can be obtained

SMN-assisted assembly of snRNP-specific Sm cores in trypanosomes.

Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring (U1, U5, spliced leader snRNPs), or variant Sm cores with snRNA-specific Sm subunits (U2, U4 snRNPs). Searching for specificity factors, we identified SMN and Gemin2 proteins that are highly divergent from known orthologs. SMN is splicing-essential in trypanosomes and nuclear-localized, suggesting that Sm core assembly in trypanosomes is nuclear. We demonstrate in vitro that SMN is sufficient to confer specificity of canonical Sm core assembly and to discriminate against binding to nonspecific RNA and to U2 and U4 snRNAs. SMN interacts transiently with the SmD3B subcomplex, contacting specifically SmB. SMN remains associated throughout the assembly of the Sm heteroheptamer and dissociates only when a functional Sm site is incorporated. These data establish a novel role of SMN, mediating snRNP specificity in Sm core assembly, and yield new biochemical insight into the mechanism of SMN activity

2 Terbium(III) Footprinting as a Probe of RNA Structure and Metal Binding Sites

Introduction Cations play a pivotal role in RNA structure and function. A functional RNA tertiary structure is stabilized by metal ions that neutralize and, in the case of multivalent ions, bridge the negatively charged phosphoribose backbon

HnRNP L and L-like cooperate in multiple-exon regulation of CD45 alternative splicing

CD45 encodes a trans-membrane protein-tyrosine phosphatase expressed in diverse cells of the immune system. By combinatorial use of three variable exons 4–6, isoforms are generated that differ in their extracellular domain, thereby modulating phosphatase activity and immune response. Alternative splicing of these CD45 exons involves two heterogeneous ribonucleoproteins, hnRNP L and its cell-type specific paralog hnRNP L-like (LL). To address the complex combinatorial splicing of exons 4–6, we investigated hnRNP L/LL protein expression in human B-cells in relation to CD45 splicing patterns, applying RNA-Seq. In addition, mutational and RNA-binding analyses were carried out in HeLa cells. We conclude that hnRNP LL functions as the major CD45 splicing repressor, with two CA elements in exon 6 as its primary target. In exon 4, one element is targeted by both hnRNP L and LL. In contrast, exon 5 was never repressed on its own and only co-regulated with exons 4 and 6. Stable L/LL interaction requires CD45 RNA, specifically exons 4 and 6. We propose a novel model of combinatorial alternative splicing: HnRNP L and LL cooperate on the CD45 pre-mRNA, bridging exons 4 and 6 and looping out exon 5, thereby achieving full repression of the three variable exons