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The critical roles of RNA-binding proteins (RBPs) in all aspects of RNA biology fostered the development of methods utilizing ultraviolet (UV) crosslinking and method-specific RNA enrichment steps for proteome-wide identification and assessment of RBP function. Despite the substantial contributions of these UV-based RNA-centric methods to our understanding of RNA-protein interaction networks, their utility is constrained by biases in RBP recovery and significant noise contributions, which can confound meaningful interpretation. To overcome these issues, we recently developed a method termed Liquid Emulsion-Assisted Purification of RNA-Bound Protein (LEAP-RBP) and introduced quantitative signal-to-noise (S:N)-based metrics for the proteome-wide identification of RNA interactomes and accurate assessment of global RBP occupancy dynamics. Compared to existing methodologies, LEAP-RBP provides significant advantages in speed, cost, efficiency, and selectivity for RNA-bound proteins. In this work, we provide a step-by-step guide for the successful application of the LEAP-RBP method for both small- and large-scale investigations of RNA-bound proteomes. Key features • Unbiased and efficient isolation of total RNA-bound protein, RNA, and protein from biological samples. • Cost-effective identification of proteome-wide RNA interactomes and validation of direct RNA-binding protein functionality. • Robust and accurate assessment of context- and/or condition-dependent RBP occupancy state dynamics.

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AT-rich interacting domain (ARID)-containing proteins, Arids, are a heterogeneous DNA-binding protein family involved in transcription regulation and chromatin processing. For the member Arid5a, no exact DNA-binding preference has been experimentally defined so far. Additionally, the protein binds to mRNA motifs for transcript stabilization, supposedly through the DNA-binding ARID domain. To date, however, no unbiased RNA motif definition and clear dissection of nucleic acid-binding through the ARID domain have been undertaken. Using NMR-centered biochemistry, we here define the Arid5a DNA preference. Further, high-throughput in vitro binding reveals a consensus RNA-binding motif engaged by the core ARID domain. Finally, transcriptome-wide binding (iCLIP2) reveals that Arid5a has a weak preference for (A)U-rich regions in pre-mRNA transcripts of factors related to RNA processing. We find that the intrinsically disordered regions flanking the ARID domain modulate the specificity and affinity of DNA binding, while they appear crucial for RNA interactions. Ultimately, our data suggest that Arid5a uses its extended ARID domain for bifunctional gene regulation and that the involvement of IDR extensions is a more general feature of Arids in interacting with different nucleic acids at the chromatin-mRNA interface.

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In this work, a water-soluble (hydrophilic) polymer was used to form a hydrophobic coating on silicon substrates (Si) in a two-step process comprising (i) the transformation of the polymer into an insoluble material and (ii) the structuring of this coating at nanometric and micrometric scales to achieve the desired hydrophobic behavior. Polyvinylpyrrolidone (PVP), a water-soluble commodity polymer, was crosslinked using benzophenone and UV irradiation to produce a water-insoluble PVP coating. The nanometric scale roughness of the coating was achieved by the addition of silica nanoparticles (NPs) in the coating. The micrometric scale roughness was achieved by forming vertical pillars of PVP/NP coating. To prepare these pillars, a perforated polystyrene (PS) template was filled with a PVP/NP suspension. Micrometer scale vertical pillars of PVP/silica NPs were produced by this method, which allowed us to tune the wettability of the surface, by combining the micrometric scale roughness of the pillars to the nanometric scale roughness provided by the nanoparticles at the surface. By adjusting the various experimental parameters, a hydrophobic PVP coating was prepared with a water contact angle of 110°, resulting in an improvement of more than 80% compared to the bare flat film with an equal amount of nanoparticles. This study paves the way for the development of a more simplified experimental approach, relying on a blend of polymers containing PVP and NPs, to form the micro/nano-structured PVP pillars directly after the deposition step and the selective etching of the sacrificial major phase.

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UV-crosslinking has proven to be an invaluable tool for the identification of RNA-protein interactomes. The paucity of methods for distinguishing background from bona fide RNA-protein interactions, however, makes attribution of RNA-binding function on UV-crosslinking alone challenging. To address this need, we previously reported an RNA-binding protein (RBP) confidence scoring metric (RCS), incorporating both signal-to-noise (S:N) and protein abundance determinations to distinguish high- and low-confidence candidate RBPs. Although RCS has utility, we sought a direct metric for quantification and comparative evaluation of protein-RNA interactions. Here we propose the use of protein-specific UV-crosslinking efficiency (%CL), representing the molar fraction of a protein that is crosslinked to RNA, for functional evaluation of candidate RBPs. Application to the HeLa RNA interactome yielded %CL values for 1097 proteins. Remarkably, %CL values span over five orders of magnitude. For the HeLa RNA interactome, %CL values comprise a range from high efficiency, high specificity interactions, e.g., the Elav protein HuR and the Pumilio homolog Pum2, with %CL values of 45.9 and 24.2, respectively, to very low efficiency and specificity interactions, for example, the metabolic enzymes glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, and alpha-enolase, with %CL values of 0.0016, 0.006, and 0.008, respectively. We further extend the utility of %CL through prediction of protein domains and classes with known RNA-binding functions, thus establishing it as a useful metric for RNA interactome analysis. We anticipate that this approach will benefit efforts to establish functional RNA interactomes and support the development of more predictive computational approaches for RBP identification.

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Electrospun nanofibrous membranes are of great interest for tissue engineering, active material delivery, and wound dressing. These nanofibers possess unique three-dimensional (3D) interconnected porous structures that result in a higher surface-area-to-volume ratio and porosity. This study was carried out to prepare nanofibrous membranes by electrospinning a blend of PVA/chitosan polymeric solution functionalized with different ratios of copper oxide. Chitosan-stabilized CuO nanoparticles (CH-CuO NPs) were biosynthesized successfully utilizing chitosan as the capping and reducing agent. XRD analysis confirmed the monoclinic structure of CH-CuO NPs. In addition, the electrospun nanofibrous membranes were UV-crosslinked for a definite time. The membranes containing CH-CuO NPs were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy, ultraviolet-visible (UV-vis) spectrophotometry, and dynamic light scattering (DLS). SEM results showed the nanosize of the fiber diameter in the range of 147-207 nm. The FTIR spectroscopy results indicated the successful incorporation of CH-CuO NPs into the PVA/chitosan nanofibrous membranes. DSC analysis proved the enhanced thermal stability of the nanofibrous membranes due to UV-crosslinking. Swelling and degradation tests were carried out to ensure membrane stability. Greater antimicrobial activity was observed in the nanoparticle-loaded membrane. An in vitro release study of Cu2+ ions from the membrane was carried out for 24 h. The cytotoxicity of CH-CuO NP-incorporated membranes was investigated to estimate the safe dose of nanoparticles. An in vivo test using the CH-CuO NP-loaded PVA/chitosan membrane was conducted on a mice model, in which wound healing occurred in approximately 12 days. These results confirmed that the biocompatible, nontoxic nanofibrous membranes are ideal for wound-dressing applications.

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Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen accounting for high mortality rates among infected patients. Transcriptomic regulation by small RNAs (sRNAs) has been shown to regulate networks promoting antibiotic resistance and virulence in S. aureus. Yet, the biological role of most sRNAs during MRSA host infection remains unknown. To fill this gap, in collaboration with the lab of Jai Tree, we performed comprehensive RNA-RNA interactome analyses in MRSA using CLASH under conditions that mimic the host environment. Here we present a detailed version of this optimized CLASH (cross-linking, ligation, and sequencing of hybrids) protocol we recently developed, which has been tailored to explore the RNA interactome in S. aureus as well as other Gram-positive bacteria. Alongside, we introduce a compilation of helpful Python functions for analyzing folding energies of putative RNA-RNA interactions and streamlining sRNA and mRNA seed discovery in CLASH data. In the accompanying computational demonstration, we aim to establish a standardized strategy to evaluate the likelihood that observed chimeras arise from true RNA-RNA interactions.

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RNA-binding proteins (RBPs) are at the heart of many biological processes and are therefore essential for cellular life. Following identification of single RBPs by classical genetics and molecular biology methods, approaches for RBP discovery on a systems level have recently emerged. For instance, RNA interactome capture (RIC) enables the global purification of RBPs cross-linked to polyadenylated RNA using oligo(dT) probes. RIC was originally developed for eukaryotic organisms but was recently established for capturing RBPs in bacteria. In this chapter, we provide a detailed step-by-step protocol for performing RIC in bacteria. The protocol is based on its application to Escherichia coli but should be amenable for charting other genetically tractable bacterial species.

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Multifunctional bio-adhesives with tunable mechanical properties are obtained by controlling the orientation of anisotropic particles in a blend of fast-curing hydrogel with an imposed capillary flow. The suspensions' microstructural evolution was monitored by the small-angle light scattering (SALS) method during flow up to the critical Péclet number (Pe≈1) necessary for particle orientation and hydrogel crosslinking. The multifunctional bio-adhesives were obtained by combining flow and UV light exposure for rapid photo-curing of PEGDA medium and freezing titania rods' ordered microstructures. Blending the low- and high-molecular weight of PEGDA polymer improved the mechanical properties of the final hydrogel. All the hydrogel samples were non-cytotoxic up to 72 h after cell culturing. The system shows rapid blood hemostasis and promotes adhesive and cohesive strength matching targeted tissue properties with an applicating methodology compatible with surgical conditions. The developed SALS approach to optimize nanoparticles' microstructures in bio-adhesive applies to virtually any optically transparent nanocomposite and any type of anisotropic nanoparticles. As such, this method enables rational design of bio-adhesives with enhanced anisotropic mechanical properties which can be tailored to potentially any type of tissue.

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Spent coffee grounds (SCGs) have numerous applications and are often blended with polymers to create composites. However, SCGs are physically trapped within the polymer matrix, lacking strong chemical bonding. Therefore, this study has developed a new method for UV crosslinking composites using phenyl azide to address the issue of SCG leakage and limited durability of the composites. The main approach involves grafting phenyl azide onto chitosan, which is then combined with SCGs. When exposed to UV light, the SCGs become covalently linked to the chitosan chains. This method not only resolves the problem of chitosan's porous material fragility but also prevents SCG detachment, surpassing the performance of glutaraldehyde-crosslinked composites. Regarding applications, CS/SCG composites exhibit rapid heating and photothermal stability, making them suitable for use as thermal pads in evaporative water purification, enabling for the collection of pure water from contaminated sources. Furthermore, SCGs have the ability to adsorb metal ions, significantly enhancing the Cu2+ adsorption capacity of CS/SCG composites compared to pure CS, with an increase of more than twofold. This research not only presents a practical solution for stabilizing fillers within polymer matrices but also demonstrates the reusability of SCGs.

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The UV cross-linking immunoprecipitation (CLIP) technique was first established in 2003. Sequences of target RNAs and binding sites of specific RNA-binding proteins (RBPs) were identified within the entire transcriptome by UV cross-linking, immunoprecipitation, reverse transcription, and subsequent high-throughput sequencing. Over the last 20 years, CLIP has been continuously modified and improved. Advanced operability and accuracy have extended its application category. Currently, the widely used CLIP technologies include high-throughput sequencing with crosslinking-immunoprecipitation (HITS-CLIP), photoactivatable-ribonucleoside-enhanced CLIP (PAR-CLIP), individual nucleotide resolution CLIP (iCLIP), enhanced CLIP (eCLIP), infrared-CLIP (irCLIP), etc. HITS-CLIP combines high-throughput sequencing with UV cross-linking immunoprecipitation. The 254 nm UV cross-linking and RNAase digestion steps allow the technology to capture transient intracellular RBP-RNA interactions. However, there are limitations in the efficiency of UV cross-linking, with low resolution and high intrinsic background noise. For PAR-CLIP, photoactivatable ribonucleoside was incorporated into RNA molecules, and RBP cross-linked with RNA by 365 nm UV light to improve cross-linking efficiency and resolution. Cross-linking mediated single-base mutations provide more accurate binding site information and reduce interference from background sequences. Long-term alternative nucleotide incorporation, on the other hand, can be cytotoxic and may skew experimental results. iCLIP can identify RBP-RNA cross-linking sites at the single nucleotide level through cDNA circularization and subsequent re-linearization steps, but it has more experimental procedures, and partial cDNAs lost in the circularization step are inevitable. eCLIP discards the radioisotope labeling procedure and reduces RNA loss by ligating adaptors in two separate steps, greatly improving the library-building efficiency, and reducing bias associated with PCR amplification; however, the efficiency of immunoprecipitation cannot be visually assessed at the early stage of the experiment. The irCLIP technique replaces radioisotopes with infrared dyes and greatly reduces the initial number of cells required for the experiment; however, an infrared imaging scanner is essential for the irCLIP application. To address more particular scientific issues, derivative CLIP-related techniques such as PAPERCLIP, cTag-PAPERCLIP, hiCLIP, and tiCLIP have also been developed in recent years. In practice, the aforementioned CLIP approaches have their advantages and disadvantages. When deciding on a technical strategy, we should take into account our experimental objectives and conditions, such as whether we need to precisely define the RNA site for binding to RBP; whether we have the necessary experimental conditions for working with radioisotopes or performing infrared imaging; the amount of initial sample size, and so on. In addition, the CLIP technique has a relatively large number of procedures and can be divided into several successive experimental modules. We can try to combine modules from different mainstream CLIP technologies to meet our experimental requirements, which also gives us more opportunities to improve and refine them and to build more targeted derivative CLIP technologies according to our research objectives.

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After the development of polymer coatings and films based on renewable resources, there remains a challenge of combining the advantages of water-borne acrylic latexes with the excellent physical properties of cross-linked solvent-borne coatings. After polymerization, the renewable 4-oxocyclopentenyl acrylate (4CPA) is capable of undergoing photocyclodimerization under UV light, yielding a cross-linked polyacrylate. In this work, we investigate the polymerization-induced self-assembly (PISA) of 4CPA with several renewable acrylic monomers in the presence of a macro-RAFT agent. The produced latexes have a small particle size, good colloidal stability, and are free of volatile organic compounds. After film formation and UV curing, flexible to rigid films can be obtained depending on the monomer composition and UV irradiation time. The cross-linked films show promise as oil and water barriers in paper coating applications. This work outlines the development and application of renewable and functional cross-linkable latexes synthesized by PISA.

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Regulatory small RNA (sRNA) have been extensively studied in model Gram-negative bacteria, but the functional characterisation of these post-transcriptional gene regulators in Gram-positives remains a major challenge. Our previous work in enterohaemorrhagic E. coli utilised the proximity-dependant ligation technique termed CLASH (UV-crosslinking, ligation, and sequencing of hybrids) for direct high-throughput sequencing of the regulatory sRNA-RNA interactions within the cell. Recently, we adapted the CLASH technique and demonstrated that UV-crosslinking and RNA proximity-dependant ligation can be applied to Staphylococcus aureus, which uncovered the first RNA-RNA interaction network in a Gram-positive bacterium. In this chapter, we describe modifications to the CLASH technique that were developed to capture the RNA interactome associated with the double-stranded endoribonuclease RNase III in two clinical isolates of S. aureus. To briefly summarise our CLASH methodology, regulatory RNA-RNA interactions were first UV-crosslinked in vivo to the RNase III protein and protein-RNA complexes were affinity-purified using the His6-TEV-FLAG tags. Linkers were ligated to RNase III-bound RNA during library preparation and duplexed RNA-RNA species were ligated together to form a single contiguous RNA 'hybrid'. The RNase III-RNA binding sites and RNA-RNA interactions occurring on RNase III (RNA hybrids) were then identified by paired-end sequencing technology. RNase III-CLASH represents a step towards a systems-level understanding of regulatory RNA in Gram-positive bacteria.

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Introduction: Poly(1,3-trimethylene carbonate) (PTMC) is a flexible amorphous polymer with good degradability and biocompatibility. The degradation of PTMC is critical for its application as a degradable polymer, more convenient and easy-to-control cross-linking strategies for preparing PTMC are required. Methods: The blends of poly(trimethylene carbonate) (PTMC) and cross-linked poly(ethylene glycol) diacrylate (PEGDA) were prepared by mixing photoactive PEGDA and PTMC and subsequently photopolymerizing the mixture with uv light. The physical properties and in vitro enzymatic degradation of the resultant PTMC/cross-linked PEGDA blends were investigated. Results: The results showed that the gel fraction of PTMC/cross-linked PEGDA blends increased while the swelling degree decreased with the content of PEGDA dosage. The results of in vitro enzymatic degradation confirmed that the degradation of PTMC/cross-linked PEGDA blends in the lipase solution occurred under the surface erosion mechanism, and the introduction of the uv cross-linked PEGDA significantly improved the resistance to lipase erosion of PTMC; the higher the cross-linking degree, the lower the mass loss. Discussion: The results indicated that the blends/cross-linking via PEGDA is a simple and effective strategy to tailor the degradation rate of PTMC.

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Individual nucleotide resolution UV cross-linking and immunoprecipitation followed by high-throughput sequencing (iCLIP-seq) is a powerful technique that is used to identify RNA-binding proteins' (RBP) binding sites on target RNAs and to characterize the molecular basis of posttranscriptional regulatory pathways. Several variants of CLIP have been developed to improve its efficiency and simplify the protocol [e.g., iCLIP2 and enhanced CLIP (eCLIP)]. We have recently reported that transcription factor SP1 functions in the regulation of alternative cleavage and polyadenylation through direct RNA binding. We utilized a modified iCLIP method to identify RNA-binding sites for SP1 and several of the cleavage and polyadenylation complex subunits, including CFIm25, CPSF7, CPSF100, CPSF2, and Fip1. Our revised protocol takes advantage of several features of the eCLIP procedure and also improves on certain steps of the original iCLIP method, including optimization of circularization of cDNA. Herein, we describe a step-by-step procedure for our revised iCLIP-seq protocol, that we designate as iCLIP-1.5, and provide alternative approaches for certain difficult-to-CLIP proteins. Key features Identification of RNA-binding sites of RNA-binding proteins (RBPs) at nucleotide resolution. iCLIP-seq provides precise positional and quantitative information on the RNA-binding sites of RBPs in living cells. iCLIP facilitates the identification of sequence motifs recognized by RBPs. Allows quantitative analysis of genome-wide changes in protein-RNA interactions. Revised iCLIP-1.5 protocol is more efficient and highly robust; it provides higher coverage even for low-input samples. Graphical overview.

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RNA-protein interactions regulate a myriad of biological functions through formation of ribonucleoprotein complexes. These complexes may consist of one or more RNA-protein interaction network(s) providing additional layers of regulatory potential to the RNA. Moreover, since the protein-binding also regulates local and global structure of the RNA by structurally remodeling the latter, it is important to correlate RNA nucleotide flexibility with the site of protein-binding. We have discussed methods for chemical probing of structure of the RNA in the protein-free and protein-bound states in the preceding chapters. In this chapter, we describe a ribonucleoprotein mutational profiling (RNP-MaP) method for probing RNA-protein interaction networks.

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RNA-protein interactions are important in development and disease, but identification of novel RNA-protein interactions remains challenging. Here, we describe an updated capture method to identify direct and specific RNA-protein interactions. First, RNA and protein are covalently cross-linked in living cells by treatment with UV light at 254 nanometers wavelength. The antisense purification approach is dependent upon nucleic acid hybridization between biotinylated DNA probes and a target RNA. Target protein:RNA:DNA complexes are enriched by capture on streptavidin magnetic beads and purified through several denaturing washes that remove nonspecific protein and nucleic acid interactors. Mass spectrometry is used to identify proteins that are specifically enriched in the target RNA capture. This method has been applied to discover the protein interactions of noncoding RNAs but can be used to capture any RNA where the target sequence is known.

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RNA-binding proteins (RBPs) govern the lifespan of nearly all transcripts and play key roles in adaptive responses in microbes. A robust approach to examine protein-RNA interactions involves irradiating cells with UV light to form covalent adducts between RBPs and their cognate RNAs. Combined with RNA or protein purification, these procedures can provide global RBP censuses or transcriptomic maps for all target sequences of a single protein in living cells. The recent development of novel methods has quickly populated the RBP landscape in microorganisms. Here, we provide an overview of prominent UV cross-linking techniques which have been applied to investigate RNA interactomes in microbes. By assessing their advantages and caveats, this technical evaluation intends to guide the selection of appropriate methods and experimental design as well as to encourage the use of complementary UV-dependent techniques to inspect RNA-binding activity.

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Trichomonas vaginalis is one of the most common sexually transmitted parasites in humans. This protozoan has high iron requirements for growth, metabolism, and virulence. However, iron concentrations also differentially modulate T. vaginalis gene expression as in the genes encoding cysteine proteinases TvCP4 and TvCP12. Our goal was to identify the regulatory mechanism mediating the upregulation of tvcp12 under iron-restricted (IR) conditions. Here, we showed by RT-PCR, Western blot, and immunocytochemistry assays that IR conditions increase mRNA stability and amount of TvCP12. RNA electrophoretic mobility shift assay (REMSA), UV cross-linking, and competition assays demonstrated that a non-canonical iron-responsive element (IRE)-like structure at the 3'-untranslated region of the tvcp12 transcript (IRE-tvcp12) specifically binds to human iron regulatory proteins (IRPs) and to atypical RNA-binding cytoplasmic proteins from IR trichomonads, such as HSP70 and α-Actinin 3. These data were confirmed by REMSA supershift and Northwestern blot assays. Thus, our findings show that a positive gene expression regulation under IR conditions occurs at the posttranscriptional level possibly through RNA-protein interactions between atypical RNA-binding proteins and non-canonical IRE-like structures at the 3'-UTR of the transcript by a parallel mechanism to the mammalian IRE/IRP system that can be applied to other iron-regulated genes of T. vaginalis.

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The fungal cell wall provides protection and structure and is an important target for antifungal compounds. A mitogen-activated protein (MAP) kinase cascade termed the cell wall integrity (CWI) pathway regulates transcriptional responses to cell wall damage. Here, we describe a posttranscriptional pathway that plays an important complementary role. We report that the RNA-binding proteins (RBPs) Mrn1 and Nab6 specifically target the 3' UTRs of a largely overlapping set of cell wall-related mRNAs. These mRNAs are downregulated in the absence of Nab6, indicating a function in target mRNA stabilization. Nab6 acts in parallel to CWI signaling to maintain appropriate expression of cell wall genes during stress. Cells lacking both pathways are hypersensitive to antifungal compounds targeting the cell wall. Deletion of MRN1 partially alleviates growth defects associated with Δnab6, and Mrn1 has an opposing function in mRNA destabilization. Our results uncover a posttranscriptional pathway that mediates cellular resistance to antifungal compounds.

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Hemocompatibility tuning was adopted to explore and refine an innovative, GA-free preparation strategy combining decellularization, riboflavin/UV crosslinking, and low-energy electron irradiation (SULEEI) procedure. A SULEEI-protocol was established to avoid GA-dependent deterioration that results in insufficient long-term aortic valve bioprosthesis durability. Final SULEEI-pericardium, intermediate steps and GA-fixed reference pericardium were exposed in vitro to fresh human whole blood to elucidate effects of preparation parameters on coagulation and inflammation activation and tissue histology. The riboflavin/UV crosslinking step showed to be less efficient in inactivating extracellular matrix (ECM) protein activity than the GA fixation, leading to tissue-factor mediated blood clotting. Intensifying the riboflavin/UV crosslinking with elevated riboflavin concentration and dextran caused an enhanced activation of the complement system. Yet activation processes induced by the previous protocol steps were quenched with the final electron beam treatment step. An optimized SULEEI protocol was developed using an intense and extended, trypsin-containing decellularization step to inactivate tissue factor and a dextran-free, low riboflavin, high UV crosslinking step. The innovative and improved GA-free SULEEI-preparation protocol results in low coagulant and low inflammatory bovine pericardium for surgical application.

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