2'-19F labelling of ribose in RNAs: a tool to analyse RNA/protein interactions by NMR in physiological conditions.

Protein-RNA interactions are central to numerous cellular processes. In this work, we present an easy and straightforward NMR-based approach to determine the RNA binding site of RNA binding proteins and to evaluate the binding of pairs of proteins to a single-stranded RNA (ssRNA) under physiological conditions, in this case in nuclear extracts. By incorporation of a 19F atom on the ribose of different nucleotides along the ssRNA sequence, we show that, upon addition of an RNA binding protein, the intensity of the 19F NMR signal changes when the 19F atom is located near the protein binding site. Furthermore, we show that the addition of pairs of proteins to a ssRNA containing two 19F atoms at two different locations informs on their concurrent binding or competition. We demonstrate that such studies can be done in a nuclear extract that mimics the physiological environment in which these protein-ssRNA interactions occur. Finally, we demonstrate that a trifluoromethoxy group (-OCF3) incorporated in the 2'ribose position of ssRNA sequences increases the sensitivity of the NMR signal, leading to decreased measurement times, and reduces the issue of RNA degradation in cellular extracts.

FluoroTensor: Identification and tracking of colocalised molecules and their stoichiometries in multi-colour single molecule imaging via deep learning.

The identification of photobleaching steps in single molecule fluorescence imaging is a well-established procedure for analysing the stoichiometries of molecular complexes. Nonetheless, the method is challenging with protein fluorophores because of the high levels of noise, rapid bleaching and highly variable signal intensities, all of which complicate methods based on statistical analyses of intensities to identify bleaching steps. It has recently been shown that deep learning by convolutional neural networks can yield an accurate analysis with a relatively short computational time. We describe here an improved use of such an approach that detects bleaching events even in the first time point of observation, and we have included this within an integrated software package incorporating fluorescence spot detection, colocalisation, tracking, FRET and photobleaching step analyses of single molecules or complexes. This package, known as FluoroTensor, is written in Python with a self-explanatory user interface.

Structural insights into the complex of oncogenic KRas4BG12V and Rgl2, a RalA/B activator.

About a quarter of total human cancers carry mutations in Ras isoforms. Accumulating evidence suggests that small GTPases, RalA, and RalB, and their activators, Ral guanine nucleotide exchange factors (RalGEFs), play an essential role in oncogenic Ras-induced signalling. We studied the interaction between human KRas4B and the Ras association (RA) domain of Rgl2 (Rgl2RA), one of the RA-containing RalGEFs. We show that the G12V oncogenic KRas4B mutation changes the interaction kinetics with Rgl2RA The crystal structure of the KRas4BG12V: Rgl2RA complex shows a 2:2 heterotetramer where the switch I and switch II regions of each KRasG12V interact with both Rgl2RA molecules. This structural arrangement is highly similar to the HRasE31K:RALGDSRA crystal structure and is distinct from the well-characterised Ras:Raf complex. Interestingly, the G12V mutation was found at the dimer interface of KRas4BG12V with its partner. Our study reveals a potentially distinct mode of Ras:effector complex formation by RalGEFs and offers a possible mechanistic explanation for how the oncogenic KRas4BG12V hyperactivates the RalA/B pathway.

Contemporary biophysical approaches for studying 14-3-3 protein-protein interactions.

14-3-3 proteins are a family of regulatory hubs that function through a vast network of protein-protein interactions. Their dysfunction or dysregulation is implicated in a wide range of diseases, and thus they are attractive drug targets, especially for molecular glues that promote protein-protein interactions for therapeutic intervention. However, an incomplete understanding of the molecular mechanisms that underpin 14-3-3 function hampers progress in drug design and development. Biophysical methodologies are an essential element of the 14-3-3 analytical toolbox, but in many cases have not been fully exploited. Here, we present a contemporary review of the predominant biophysical techniques used to study 14-3-3 protein-protein interactions, with a focus on examples that address key questions and challenges in the 14-3-3 field.

Surface Passivation with a Perfluoroalkane Brush Improves the Precision of Single-Molecule Measurements.

Single-molecule imaging is invaluable for investigating the heterogeneous behavior and interactions of biological molecules. However, an impediment to precise sampling of single molecules is the irreversible adsorption of components onto the surfaces of cover glasses. This causes continuous changes in the concentrations of different molecules dissolved or suspended in the aqueous phase from the moment a sample is dispensed, which will shift, over time, the position of chemical equilibria between monomeric and multimeric components. Interferometric scattering microscopy (iSCAT) is a technique in the single-molecule toolkit that has the capability to detect unlabeled proteins and protein complexes both as they adsorb onto and desorb from a glass surface. Here, we examine the reversible and irreversible interactions between a number of different proteins and glass via analysis of the adsorption and desorption of protein at the single-molecule level. Furthermore, we present a method for surface passivation that virtually eliminates irreversible adsorption while still ensuring the residence time of molecules on surfaces is sufficient for detection of adsorption by iSCAT. By grafting high-density perfluoroalkane brushes on cover-glass surfaces, we observe approximately equal numbers of adsorption and desorption events for proteins at the measurement surface (±1%). The fluorous-aqueous interface also prevents the kinetic trapping of protein complexes and assists in establishing a thermodynamic equilibrium between monomeric and multimeric components. This surface passivation approach is valuable for in vitro single-molecule experiments using iSCAT microscopy because it allows for continuous monitoring of adsorption and desorption of protein without either a decline in detection events or a change in sample composition due to the irreversible binding of protein to surfaces.

Biophysical characterisation of the Bcl-x pre-mRNA and binding specificity of the ellipticine derivative GQC-05: Implication for alternative splicing regulation.

The BCL2L1 gene expresses two isoforms of Bcl-x protein via the use of either of two alternative 5' splice sites (5'ss) in exon 2. These proteins have antagonistic actions, Bcl-XL being anti-apoptotic and Bcl-XS pro-apoptotic. In a number of cancers the Bcl-XL isoform is over-expressed, resulting in cancer cell survival and growth, so switching splicing to the Xs isoform could have therapeutic benefits. We have previously proposed that a putative G-quadruplex (G4) exists downstream of the XS 5'ss and shown that the ellipticine derivative GQC-05, a previously identified DNA G4-specific ligand, induces an increase in the XS/XL ratio both in vitro and in cells. Here, we demonstrate that this G4 forms in vitro and that the structure is stabilised in the presence of GQC-05. We also show that GQC-05 binds RNA non-specifically in buffer conditions, but selectively to the Bcl-x G4 in the presence of nuclear extract, highlighting the limitations of biophysical measurements taken outside of a functional environment. We also demonstrate that GQC-05 is able to shift the equilibrium between competing G4 and duplex structures towards the G4 conformation, leading to an increase in accessibility of the XS 5'ss, supporting our previous model on the mechanism of action of GQC-05.

Exon-independent recruitment of SRSF1 is mediated by U1 snRNP stem-loop 3.

SRSF1 protein and U1 snRNPs are closely connected splicing factors. They both stimulate exon inclusion, SRSF1 by binding to exonic splicing enhancer sequences (ESEs) and U1 snRNPs by binding to the downstream 5' splice site (SS), and both factors affect 5' SS selection. The binding of U1 snRNPs initiates spliceosome assembly, but SR proteins such as SRSF1 can in some cases substitute for it. The mechanistic basis of this relationship is poorly understood. We show here by single-molecule methods that a single molecule of SRSF1 can be recruited by a U1 snRNP. This reaction is independent of exon sequences and separate from the U1-independent process of binding to an ESE. Structural analysis and cross-linking data show that SRSF1 contacts U1 snRNA stem-loop 3, which is required for splicing. We suggest that the recruitment of SRSF1 to a U1 snRNP at a 5'SS is the basis for exon definition by U1 snRNP and might be one of the principal functions of U1 snRNPs in the core reactions of splicing in mammals.

Understanding the Logistics for the Distribution of Heme in Cells.

Heme is essential for the survival of virtually all living systems-from bacteria, fungi, and yeast, through plants to animals. No eukaryote has been identified that can survive without heme. There are thousands of different proteins that require heme in order to function properly, and these are responsible for processes such as oxygen transport, electron transfer, oxidative stress response, respiration, and catalysis. Further to this, in the past few years, heme has been shown to have an important regulatory role in cells, in processes such as transcription, regulation of the circadian clock, and the gating of ion channels. To act in a regulatory capacity, heme needs to move from its place of synthesis (in mitochondria) to other locations in cells. But while there is detailed information on how the heme lifecycle begins (heme synthesis), and how it ends (heme degradation), what happens in between is largely a mystery. Here we summarize recent information on the quantification of heme in cells, and we present a discussion of a mechanistic framework that could meet the logistical challenge of heme distribution.

Unravelling the mechanisms controlling heme supply and demand.

In addition to heme's role as the prosthetic group buried inside many different proteins that are ubiquitous in biology, there is new evidence that heme has substantive roles in cellular signaling and regulation. This means that heme must be available in locations distant from its place of synthesis (mitochondria) in response to transient cellular demands. A longstanding question has been to establish the mechanisms that control the supply and demand for cellular heme. By fusing a monomeric heme-binding peroxidase (ascorbate peroxidase, mAPX) to a monomeric form of green-fluorescent protein (mEGFP), we have developed a heme sensor (mAPXmEGFP) that can respond to heme availability. By means of fluorescence lifetime imaging, this heme sensor can be used to quantify heme concentrations; values of the mean fluorescence lifetime (τMean) for mAPX-mEGFP are shown to be responsive to changes in free (unbound) heme concentration in cells. The results demonstrate that concentrations are typically limited to one molecule or less within cellular compartments. These miniscule amounts of free heme are consistent with a system that sequesters the heme and is able to buffer changes in heme availability while retaining the capability to mobilize heme when and where it is needed. We propose that this exchangeable supply of heme can operate using mechanisms for heme transfer that are analogous to classical ligand-exchange mechanisms. This exquisite control, in which heme is made available for transfer one molecule at a time, protects the cell against the toxic effect of excess heme and offers a simple mechanism for heme-dependent regulation in single-molecule steps.

Formation of pre-pore complexes of pneumolysin is accompanied by a decrease in short-range order of lipid molecules throughout vesicle bilayers.

Oligomers of pneumolysin form transmembrane channels in cholesterol-containing lipid bilayers. The mechanism of pore formation involves a multistage process in which the protein, at first, assembles into a ring-shaped complex on the outer-bilayer leaflet. In a subsequent step, the complex inserts into the membrane. Contrary to most investigations of pore formation that have focussed on protein changes, we have deduced how the lipid-packing order is altered in different stages of the pore-forming mechanism. An optical tweezing apparatus was used, in combination with microfluidics, to isolate large-unilamellar vesicles and control exposure of the bilayer to pneumolysin. By monitoring Raman-scattered light from a single-trapped liposome, the effect of the protein on short-range order and rotational diffusion of lipids could be inferred from changes in the envelope of the C-H stretch. A significant change in the lipid-packing order takes place during assembly of pre-pore oligomers. We were not able to detect a change in the lipid-packing order during the initial stage of protein binding, or any further change during the insertion of oligomers. Pre-pore complexes induce a transformation in which a bilayer, resembling a liquid-ordered phase is changed into a bilayer resembling a fluid-liquid-disordered phase surrounding ordered microdomains enriched in cholesterol and protein complexes.

Patterns of conservation of spliceosomal intron structures and spliceosome divergence in representatives of the diplomonad and parabasalid lineages.

BACKGROUND: Two spliceosomal intron types co-exist in eukaryotic precursor mRNAs and are excised by distinct U2-dependent and U12-dependent spliceosomes. In the diplomonad Giardia lamblia, small nuclear (sn) RNAs show hybrid characteristics of U2- and U12-dependent spliceosomal snRNAs and 5 of 11 identified remaining spliceosomal introns are trans-spliced. It is unknown whether unusual intron and spliceosome features are conserved in other diplomonads. RESULTS: We have identified spliceosomal introns, snRNAs and proteins from two additional diplomonads for which genome information is currently available, Spironucleus vortens and Spironucleus salmonicida, as well as relatives, including 6 verified cis-spliceosomal introns in S. vortens. Intron splicing signals are mostly conserved between the Spironucleus species and G. lamblia. Similar to 'long' G. lamblia introns, RNA secondary structural potential is evident for 'long' (> 50 nt) Spironucleus introns as well as introns identified in the parabasalid Trichomonas vaginalis. Base pairing within these introns is predicted to constrain spatial distances between splice junctions to similar distances seen in the shorter and uniformly-sized introns in these organisms. We find that several remaining Spironucleus spliceosomal introns are ancient. We identified a candidate U2 snRNA from S. vortens, and U2 and U5 snRNAs in S. salmonicida; cumulatively, illustrating significant snRNA differences within some diplomonads. Finally, we studied spliceosomal protein complements and find protein sets in Giardia, Spironucleus and Trepomonas sp. PC1 highly- reduced but well conserved across the clade, with between 44 and 62 out of 174 studied spliceosomal proteins detectable. Comparison with more distant relatives revealed a highly nested pattern, with the more intron-rich fornicate Kipferlia bialata retaining 87 total proteins including nearly all those observed in the diplomonad representatives, and the oxymonad Monocercomonoides retaining 115 total proteins including nearly all those observed in K. bialata. CONCLUSIONS: Comparisons in diplomonad representatives and species of other closely-related metamonad groups indicates similar patterns of intron structural conservation and spliceosomal protein composition but significant divergence of snRNA structure in genomically-reduced species. Relative to other eukaryotes, loss of evolutionarily-conserved snRNA domains and common sets of spliceosomal proteins point to a more streamlined splicing mechanism, where intron sequences and structures may be functionally compensating for the minimalization of spliceosome components.

Rapid metagenomics analysis of EMS vehicles for monitoring pathogen load using nanopore DNA sequencing.

Pathogen monitoring, detection and removal are essential to public health and outbreak management. Systems are in place for monitoring the microbial load of hospitals and public health facilities with strategies to mitigate pathogen spread. However, no such strategies are in place for ambulances, which are tasked with transporting at-risk individuals in immunocompromised states. As standard culturing techniques require a laboratory setting, and are time consuming and labour intensive, our approach was designed to be portable, inexpensive and easy to use based on the MinION third-generation sequencing platform from Oxford Nanopore Technologies. We developed a transferable sampling-to-analysis pipeline to characterize the microbial community in emergency medical service vehicles. Our approach identified over sixty-eight organisms in ambulances to the genera level, with a proportion of these being connected with health-care associated infections, such as Clostridium spp. and Staphylococcus spp. We also monitored the microbiome of different locations across three ambulances over time, and examined the dynamic community of microorganisms found in emergency medical service vehicles. Observed differences identified hot spots, which may require heightened monitoring and extensive cleaning. Through metagenomics analysis it is also possible to identify how microorganisms spread between patients and colonize an ambulance over time. The sequencing results aid in the development of practices to mitigate disease spread, while also providing a useful tool for outbreak prediction through ongoing analysis of the ambulance microbiome to identify new and emerging pathogens. Overall, this pipeline allows for the tracking and monitoring of pathogenic microorganisms of epidemiological interest, including those related to health-care associated infections.

Precise determination of heme binding affinity in proteins.

Accumulating evidence suggests a new role for cellular heme as a signalling molecule, in which interactions with target proteins are more transient than found with traditionally-defined hemoproteins. To study this role, a precise method is needed for determining the heme-binding affinity (or dissociation constant, Kd). Estimates of Kd are commonly made following a spectrophotometric titration of an apo-protein with hemin. An impediment to precise determination is, however, the challenge in discriminating between the Soret absorbance for the product (holo-protein) and that for the titrant (hemin). An altogether different approach has been used in this paper to separate contributions made by these components to absorbance values. The pure component spectra and concentration profiles are estimated by a multivariate curve-resolution (MCR) algorithm. This approach has significant advantages over existing methods. First, a more precise determination of Kd can be made as concentration profiles for all three components (apo-protein/holo-protein/hemin) are determined and can be simultaneously fitted to a theoretical-binding model. Second, an absorption spectrum for the holo-protein is calculated. This is a unique advantage of MCR and attractive for investigating proteins in which the nature of heme binding has not, hitherto, been characterised because the holo-protein spectrum provides information on the interaction.

Rapid generation of sequence-diverse terminator libraries and their parameterization using quantitative Term-Seq.

Synthetic biology and the rational design and construction of biological devices require vast numbers of characterized biological parts, as well as reliable design tools to build increasingly complex, multigene architectures. Design principles for intrinsic terminators have been established; however, additional sequence-structure studies are needed to refine parameters for termination-based genetic devices. We report a rapid single-pot method to generate libraries of thousands of randomized bidirectional intrinsic terminators and a modified quantitative Term-Seq (qTerm-Seq) method to simultaneously identify terminator sequences and measure their termination efficiencies (TEs). Using qTerm-Seq, we characterize hundreds of additional strong terminators (TE > 90%) with some terminators reducing transcription read-through by up to 1000-fold in Escherichia coli. Our terminator library and qTerm-Seq pipeline constitute a flexible platform enabling identification of terminator parts that can achieve transcription termination not only over a desired range but also to investigate their sequence-structure features, including for specific genetic and application contexts beyond the common in vivo systems such as E. coli.

RNA-Seq employing a novel rRNA depletion strategy reveals a rich repertoire of snoRNAs in Euglena gracilis including box C/D and Ψ-guide RNAs targeting the modification of rRNA extremities.

Previous mRNA transcriptome studies of Euglena gracilis have shown that this organism possesses a large and diverse complement of protein coding genes; however, the study of non-coding RNA classes has been limited. The natural extensive fragmentation of the E. gracilis large subunit ribosomal RNA presents additional barriers to the identification of non-coding RNAs as size-selected small RNA libraries will be dominated by rRNA sequences. In this study we have developed a strategy to significantly reduce rRNA amplification prior to RNA-Seq analysis thereby producing a ncRNA library allowing for the identification of many new E. gracilis small RNAs. Library analysis reveals 113 unique new small nucleolar (sno) RNAs and a large collection of snoRNA isoforms, as well as the first significant collection of nuclear tRNAs in this organism. A 3' end AGAUGN consensus motif and conserved structural features can now be defined for E. gracilis pseudouridine guide RNAs. snoRNAs of both classes were identified that target modification of the 3' extremities of rRNAs utilizing predicted base-pairing interactions with internally transcribed spacers (ITS), providing insight into the timing of steps in rRNA maturation. Cumulatively, this represents the most comprehensive analysis of small ncRNAs in Euglena gracilis to date.

The mechanisms of a mammalian splicing enhancer.

Exonic splicing enhancer (ESE) sequences are bound by serine & arginine-rich (SR) proteins, which in turn enhance the recruitment of splicing factors. It was inferred from measurements of splicing around twenty years ago that Drosophila doublesex ESEs are bound stably by SR proteins, and that the bound proteins interact directly but with low probability with their targets. However, it has not been possible with conventional methods to demonstrate whether mammalian ESEs behave likewise. Using single molecule multi-colour colocalization methods to study SRSF1-dependent ESEs, we have found that that the proportion of RNA molecules bound by SRSF1 increases with the number of ESE repeats, but only a single molecule of SRSF1 is bound. We conclude that initial interactions between SRSF1 and an ESE are weak and transient, and that these limit the activity of a mammalian ESE. We tested whether the activation step involves the propagation of proteins along the RNA or direct interactions with 3' splice site components by inserting hexaethylene glycol or abasic RNA between the ESE and the target 3' splice site. These insertions did not block activation, and we conclude that the activation step involves direct interactions. These results support a model in which regulatory proteins bind transiently and in dynamic competition, with the result that each ESE in an exon contributes independently to the probability that an activator protein is bound and in close proximity to a splice site.

The Emergency Medical Service Microbiome.

Emergency medical services (EMS) personnel are an integral component of the health care framework and function to transport patients from various locations to and between care facilities. In addition to physical injury, EMS personnel are expected to be at high risk to acquire and transmit health care-associated infections (HAIs) in the workplace. However, currently, little is known about EMS biosafety risk factors and the epidemiological contribution of EMS to pathogen transmission within and outside the health care sector. Health care facility microbiomes contain diverse bacterial, fungal, and viral pathogens that cause over 1.7 million HAIs each year in the United States alone. While hospital microbiomes have been relatively well studied, there is scant information about EMS infrastructure and equipment microbiomes or the role(s) they play in HAI transmission between health care facilities. We review recent literature investigating the microbiome of ambulances and other EMS service facilities which consistently identify antibiotic-resistant pathogens causing HAIs, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus, and Klebsiella pneumoniae Our review provides evidence that EMS microbiomes are dynamic and important pathogen reservoirs, and it underscores the need for more widespread and in-depth microbiome studies to elucidate patterns of pathogen transmission. We discuss emerging DNA sequencing technologies and other methods that can be applied to characterize and mitigate EMS biosafety risks in the future. Understanding the complex interplay between EMS and hospital microbiomes will provide key insights into pathogen transmission mechanisms and identify strategies to minimize HAIs and community infection.

Light Scattering By Optically-Trapped Vesicles Affords Unprecedented Temporal Resolution Of Lipid-Raft Dynamics.

A spectroscopic technique is presented that is able to identify rapid changes in the bending modulus and fluidity of vesicle lipid bilayers on the micrometer scale, and distinguish between the presence and absence of heterogeneities in lipid-packing order. Individual unilamellar vesicles have been isolated using laser tweezers and, by measuring the intensity modulation of elastic back-scattered light, changes in the biophysical properties of lipid bilayers were revealed. Our approach offers unprecedented temporal resolution and, uniquely, physical transformations of lipid bilayers can be monitored on a length scale of micrometers. As an example, the deformation of a membrane bilayer following the gel-to-fluid phase transition in a pure phospholipid vesicle was observed to take place across an interval of 54 ± 5 ms corresponding to an estimated full-width of only ~1 m°C. Dynamic heterogeneities in packing order were detected in mixed-lipid bilayers. Using a ternary mixture of lipids, the modulated-intensity profile of elastic back-scattered light from an optically-trapped vesicle revealed an abrupt change in the bending modulus of the bilayer which could be associated with the dissolution of ordered microdomains (i.e., lipid rafts). This occurred across an interval of 30 ± 5 ms (equivalent to ~1 m°C).

Stoichiometries of U2AF35, U2AF65 and U2 snRNP reveal new early spliceosome assembly pathways.

The selection of 3΄ splice sites (3΄ss) is an essential early step in mammalian RNA splicing reactions, but the processes involved are unknown. We have used single molecule methods to test whether the major components implicated in selection, the proteins U2AF35 and U2AF65 and the U2 snRNP, are able to recognize alternative candidate sites or are restricted to one pre-specified site. In the presence of adenosine triphosphate (ATP), all three components bind in a 1:1 stoichiometry with a 3΄ss. Pre-mRNA molecules with two alternative 3΄ss can be bound concurrently by two molecules of U2AF or two U2 snRNPs, so none of the components are restricted. However, concurrent occupancy inhibits splicing. Stoichiometric binding requires conditions consistent with coalescence of the 5΄ and 3΄ sites in a complex (I, initial), but if this cannot form the components show unrestricted and stochastic association. In the absence of ATP, when complex E forms, U2 snRNP association is unrestricted. However, if protein dephosphorylation is prevented, an I-like complex forms with stoichiometric association of U2 snRNPs and the U2 snRNA is base-paired to the pre-mRNA. Complex I differs from complex A in that the formation of complex A is associated with the loss of U2AF65 and 35.

Correction: Resonance Raman spectroscopy as an in situ probe for monitoring catalytic events in a Ru-porphyrin mediated amination reaction.

Correction for 'Resonance Raman spectroscopy as an in situ probe for monitoring catalytic events in a Ru-porphyrin mediated amination reaction' by Paolo Zardi et al., Analyst, 2016, 141, 3050-3058.