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It remains a formidable challenge to characterize the diverse complexes of RNA binding proteins and their targets. While crosslink and immunoprecipitation (CLIP) methods are powerful techniques that identify RNA targets on a global scale, the resolution and consistency of these methods is a matter of debate. Here we present a comparative analysis of LIN28-pre-let-7 UV-induced crosslinking using a tandem mass spectrometry (MS/MS) and deep sequencing interrogation of in vitro crosslinked complexes. Interestingly, analyses by the two methods diverge in their identification of crosslinked nucleotide identity – whereas bioinformatics and sequencing analyses suggest guanine in mammalian cells, MS/MS identifies uridine. This work suggests the need for comprehensive analysis and validation of crosslinking methodologies.

Origins of life hypotheses often invoke a transitional phase of nonenzymatic template-directed RNA replication prior to the emergence of ribozyme-catalyzed copying of genetic information. Here, using NMR and ITC, we interrogate the binding affinity of guanosine 5′-monophosphate (GMP) for primer–template complexes when either another GMP, or a helper oligonucleotide, can bind downstream. Binding of GMP to a primer–template complex cannot be significantly enhanced by the possibility of downstream monomer binding, because the affinity of the downstream monomer is weaker than that of the first monomer. Strikingly, GMP binding affinity can be enhanced by ca. 2 orders of magnitude when a helper oligonucleotide is stably bound downstream of the monomer binding site. We compare these thermodynamic parameters to those previously reported for T7 RNA polymerase-mediated replication to help address questions of binding affinity in related nonenzymatic processes.

Early protocells are likely to have arisen from the self-assembly of RNA, peptide, and lipid molecules that were generated and concentrated within geologically favorable environments on the early Earth. The reactivity of these components in a prebiotic environment that supplied sources of chemical energy could have produced additional species with properties favorable to the emergence of protocells. The geochemically plausible activation of amino acids by carbonyl sulfide has been shown to generate short peptides via the formation of cyclic amino acid N-carboxyanhydrides (NCAs). Here, we show that the polymerization of valine-NCA in the presence of fatty acids yields acylated amino acids and peptides via a mixed anhydride intermediate. Notably, Nα-oleoylarginine, a product of the reaction between arginine and oleic acid in the presence of valine-NCA, partitions spontaneously into vesicle membranes and mediates the association of RNA with the vesicles. Our results suggest a potential mechanism by which activated amino acids could diversify the chemical functionality of fatty acid membranes and colocalize RNA with vesicles during the formation of early protocells.

High affinity RNA-protein interactions are critical to cellular function, but directly identifying the determinants of binding within these complexes is often difficult. Here, we introduce a stable isotope mass labeling technique to assign specific interacting nucleotides in an oligonucleotide-protein complex by photo-cross-linking. The method relies on generating site-specific oxygen-18-labeled phosphodiester linkages in oligonucleotides, such that covalent peptide-oligonucleotide cross-link sites arising from ultraviolet irradiation can be assigned to specific sequence positions in both RNA and protein simultaneously by mass spectrometry. Using Lin28A and a let-7 pre-element RNA, we demonstrate that mass labeling permits unambiguous identification of the cross-linked sequence positions in the RNA-protein complex.

Some ceramides have been shown to displace cholesterol from liquid ordered domains rich in sphingomyelin or saturated phosphatidylcholine in model membranes. The addition of ceramide will stabilize the formed ceramide/sphingomyelin domains and the subsequent partitioning of cholesterol to the disordered phase will also increase order and stabilization in this phase. The displacement of cholesterol may be due to more favorable interactions of ceramide with the ordered phase lipids, mainly sphingomyelin, compared to cholesterol. The ceramide could also interact less favorably with the disordered phase lipids compared to cholesterol, driving it into the ordered domains. It would also be of interest to better understand how cholesterol and ceramide can interact with each other in bilayer membranes. To study these questions, we have prepared a cholesterol analog with a phosphocholine head group on the 3-oxygen. We have studied the distribution and behavior of chol-PC and ceramides in bilayer systems on their own, and together with saturated and monounsaturated phospholipids. Chol-PC together with ceramide was able to form stable bilayer phases (extruded LUVs, electroformed GUVs). Phosphatidylcholine acyl chain order measurements showed that chol-PC was less efficient in inducing acyl chain order compared to cholesterol. Fluorescence quenching measurements in POPC/ceramide/sterol mixtures further showed a decrease in ordered domain stability of saturated ceramide in the presence of cholesterol or chol-PC. We have also examined the possibility to use chol-PC:ceramide mixtures to load ceramide into cultured cells. Proliferation experiments on HeLa and FRTL-5 cells indicates that chol-PC:ceramide bilayer mixtures are more efficient in inducing apoptosis than DMSO:ceramide mixtures. Our results suggest that chol-PC and ceramide interact with each other under specific conditions, and that the binary bilayers could constitute an interesting formulation for providing cells and tissues with solvent-free ceramides.

BackgroundBirth by caesarean section is linked to an increased risk of developing asthma, but the underlying mechanisms are unclear.ObjectiveTo elucidate the link between birth by caesarean section and asthma using newborn metabolomic profiles and integrating early-life gut microbiome data and cord blood immunology.MethodsWe investigated the influence of caesarean section on liquid chromatography mass spectrometry metabolomic profiles of dried blood spots from newborns of the two independent Copenhagen Prospective Studies on Asthma in Childhood cohorts, i.e. COPSAC2010 (n=677) and COPSAC2000 (n=387). We assessed the associations between the caesarean section metabolic profile, gut microbiome data and frequency of cord blood regulatory T-cells (Tregs) at 1 week of age.ResultsIn COPSAC2010, a partial least square discriminant analysis model showed that children born by caesarean section versus natural delivery had different metabolic profiles (area under the curve (AUC)=0.77, p=2.2×10−16), which was replicated in COPSAC2000 (AUC=0.66, p=1.2×10−5). The metabolic profile of caesarean section was significantly associated with an increased risk of asthma at school age in both COPSAC2010 (p=0.03) and COPSAC2000 (p=0.005). Caesarean section was associated with lower abundance of tryptophan, bile acid and phenylalanine metabolites, indicative of a perturbed gut microbiota. Furthermore, gut bacteria dominating after natural delivery, i.e. Bifidobacterium and Bacteroides were correlated with caesarean section-discriminative microbial metabolites, suggesting maternal microbial transmission during birth regulating the newborn's metabolism. Finally, the caesarean section metabolic profile was associated with frequency of cord blood Tregs.ConclusionsThese findings propose that caesarean section programmes the risk of childhood asthma through perturbed immune responses and gut microbial colonisation patterns reflected in the blood metabolome at birth.

The polymorphic phase behavior of different phospholipids depends strongly on fine details of their head group and backbone structures. Studies of phospholipid head group modification offers an attractive strategy in elucidating how structural elements of the head group effect membrane properties. We are investigating phosphatidyl alcohols with saturated fatty acyl chains and their interactions with phospholipids and sterols. Molecules of interest are dipalmitoyl glycerophospholipids with methanol, ethanol, propanol and butanol as different headgroups. These molecules can be formed from e.g., corresponding phosphatidylcholines due to the transphosphatidylation activity of phospholipase D. Phosphatidyl ethanol is of physiological relevance since it is increasingly formed in cells of chronic alcoholics. Thermodynamic properties such as phase transition temperatures, heat capacity of transitions and transition cooperativity have been studied with high sensitivity differential scanning calorimetry (DSC). The miscibility and effects of cholesterol on mixtures of these negatively charged phospholipids with high Tm and low Tm phospholipids have been determined using DSC, and with fluorescence spectroscopy using domain-selective lipid probes. The relative acyl-chain order in the bilayer membranes has been assessed using steady-state DPH anisotropy. We will report on the phase behavior of these molecules in simple and more complex bilayer systems. The formation, by the dipalmitoyl phosphatidyl alcohols, of sterol-enriched or ordered domains in fluid bilayer membranes will also be determined. Comparison of the properties of these phosphatidyl alcohols with molecules having closely related structures will help in understanding the relationships between the chemical structures, physical properties and functional properties of the phospholipid molecules in model as well as biological membranes.