U1A Protein Research Articles

High Energy Channeling and Malleable Transition States: Molecular Dynamics Simulations and Free Energy Landscapes for the Thermal Unfolding of Protein U1A and 13 Mutants.

The spliceosome protein U1A is a prototype case of the RNA recognition motif (RRM) ubiquitous in biological systems. The in vitro kinetics of the chemical denaturation of U1A indicate that the unfolding of U1A is a two-state process but takes place via high energy channeling and a malleable transition state, an interesting variation of typical two-state behavior. Molecular dynamics (MD) simulations have been applied extensively to the study of two-state unfolding and folding of proteins and provide an opportunity to obtain a theoretical account of the experimental results and a molecular model for the transition state ensemble. We describe herein all-atom MD studies including explicit solvent of up to 100 ns on the thermal unfolding (UF) of U1A and 13 mutants. Multiple MD UF trajectories are carried out to ensure accuracy and reproducibility. A vector representation of the MD unfolding process in RMSD space is obtained and used to calculate a free energy landscape for U1A unfolding. A corresponding MD simulation and free energy landscape for the protein CI2, well known to follow a simple two state folding/unfolding model, is provided as a control. The results indicate that the unfolding pathway on the MD calculated free energy landscape of U1A shows a markedly extended transition state compared with that of CI2. The MD results support the interpretation of the observed chevron plots for U1A in terms of a high energy, channel-like transition state. Analysis of the MDUF structures shows that the transition state ensemble involves microstates with most of the RRM secondary structure intact but expanded by ~14% with respect to the radius of gyration. Comparison with results on a prototype system indicates that the transition state involves an ensemble of molten globule structures and extends over the region of ~1–35 ns in the trajectories. Additional MDUF simulations were carried out for 13 U1A mutants, and the calculated φ-values show close accord with observed results and serve to validate our methodology.

Spliceosomal SL1 RNA binding to U1-70K: gaurdian under stress

Spliceosome is a very dynamic machine in the eukaryotic cell which splices pre-mRNA into mRNA. Its components assemble and disassemble routinely and are transported among different cell compartments and membraneless organelles during function. The U1 small nuclear ribonucleoprotein particle (snRNP) consists of a single RNA folded into four hairpin loops, with loop SL1 binding to the protein U1-70K RNA recognition motif (RRM), loop SL2 binding to the protein U1A. SL1 RNA binds to the region surrounding the RRM of protein U1-70K as strongly as it does to the whole U1-70K protein.

Obtaining Crystals of Nucleic Acids in Complex with the Protein U1A Using the Soaking Method.

X-ray crystallography is one of the most prominent techniques for determining high-resolution structures of nucleic acids. The major challenges are to obtain well-diffracting single crystals and to solve the phase problem. The absence of structural information impedes the elucidation of the molecular details of biological processes. A particularly intriguing example is the RNA-cleavage catalyzed by the 10-23 deoxyribozyme (DNAzyme). This DNAzyme consists of a catalytic core that is flanked by two substrate binding arms, which can be designed to bind any RNA of interest. Structure elucidation of the 10-23 DNAzyme in a biologically relevant conformation faces three major challenges: (1) stabilization of the RNA substrate to capture the DNA:RNA complex in the pre-catalytic conformation, (2) prevention of the formation of an artificial duplex conformation due to a self-complementary sequence in the catalytic core of the DNAzyme, and (3) the crystallization of nucleic acids with their uniform surfaces. Here, we provide a protocol for an innovative strategy facilitating the crystallization of protein:nucleic acid complexes using a soaking approach and discuss on how to apply this protocol for the structure elucidation of the 10-23 DNAzyme. For this purpose, we describe the purification procedure of an optimized variant of the RNA-binding protein U1A, the crystallization of this specific U1A variant, the soaking process with its specific RNA hairpin loop, and finally suggest a strategy for applying this procedure on the 10-23 DNAzyme in complex with its specific RNA target.

Cellular Sticking Can Strongly Reduce Complex Binding by Speeding Dissociation.

While extensive studies have been carried out to determine protein-RNA binding affinities, mechanisms, and dynamics in vitro, such studies do not take into consideration the effect of the many weak nonspecific interactions in a cell filled with potential binding partners. Here we experimentally tested the role of the cellular environment on affinity and binding dynamics between a protein and RNA in living U-2 OS cells. Our model system is the spliceosomal protein U1A and its binding partner SL2 of the U1 snRNA. The binding equilibrium was perturbed by a laser-induced temperature jump and monitored by Förster resonance energy transfer. The apparent binding affinity in live cells was reduced by up to 2 orders of magnitude compared to in vitro. The measured in-cell dissociation rate coefficients were up to 2 orders of magnitude larger, whereas no change in the measured association rate coefficient was observed. The latter is not what would be anticipated due to macromolecular crowding or nonspecific sticking of the uncomplexed U1A and SL2 in the cell. A quantitative model fits our experimental results, with the major cellular effect being that U1A and SL2 sticking to cellular components are capable of binding, just not as strongly as the free complex. This observation suggests that high binding affinities measured or designed in vitro are necessary for proper binding in vivo, where competition with many nonspecific interactions exists, especially for strongly interacting species with high charge or large hydrophobic surface areas.

A study on allosteric communication in U1A-snRNA binding interactions: network analysis combined with molecular dynamics data

The allosteric regulation during the binding interactions between small nuclear RNAs (snRNAs) and the associated protein factors is critical to the function of spliceosomes in alternative RNA splicing. Although network models combined with molecular dynamics simulations have shown to be powerful tools for the analysis of protein allostery, the atomic-level simulations are, however, too expensive and with limited accuracy for the large-size systems. In this work, we use a residual network model combined with a coarse-grained Gaussian network model (GNM) to investigate the binding interactions between the snRNA and the human U1A protein which is a major component of the spliceosomal U1 small nuclear ribonucleoprotein particle, and to identify the residues that play an important role in the allosteric communication in U1A during this process. We also utilize the Girvan-Newman method to detect the structural organization in U1A-snRNA recognition and interactions. Our results reveal that: (Ι) not only the residues at the binding sites that are traditionally considered to play a major role in U1A-snRNA association, but those residues that are far away from the RNA binding interface participate in the U1A's allosteric signal transmission induced by the RNA binding; (Π) the structure of U1A protein is well organized with different communities acting different roles for its RNA binding and allosteric regulation. The study demonstrates that the combination of the residual network and elastic network models is an effective and efficient method which can be readily extended to the investigation of the allosteric communication for other macromolecular interaction systems.

Expanding crystallization tools for nucleic acid complexes using U1A protein variants

The major bottlenecks in structure elucidation of nucleic acids are crystallization and phasing. Co-crystallization with proteins is a straight forward approach to overcome these challenges. The human RNA-binding protein U1A has previously been established as crystallization module, however, the absence of UV-active residues and the predetermined architecture in the asymmetric unit constitute clear limitations of the U1A system. Here, we report three crystal structures of tryptophan-containing U1A variants, which expand the crystallization toolbox for nucleic acids. Analysis of the structures complemented by SAXS, NMR spectroscopy, and optical spectroscopy allow for insights into the potential of the U1A variants to serve as crystallization modules for nucleic acids. In addition, we report a fast and efficient protocol for crystallization of RNA by soaking and present a fluorescence-based approach for detecting RNA-binding in crystallo. Our results provide a new tool set for the crystallization of RNA and RNA:DNA complexes.

Overexpression system for recombinant RNA in Corynebacterium glutamicum using a strong promoter derived from corynephage BFK20

In recent years, it has been shown that recombinant RNA molecules have a great potential in mRNA therapy and as novel agricultural pesticides. We developed a fundamental system for efficient production of target RNA molecules in Corynebacterium glutamicum, composed of a strong promoter named F1 and a terminator derived from corynephage BFK20 in a high-copy number plasmid vector. As a target model RNA for overexpression, we designed and used an RNA molecule [designated U1A*-RNA, ∼160 nucleotides (nt) long] containing a stem/loop II (SL-II, hairpin-II) structure from U1 small nuclear RNA (snRNA), which binds to U1A protein, forming a U1 sn-ribonucleoprotein, which is essential in the pre-mRNA splicing process. C.glutamicum strains harboring the U1A*-RNA expression plasmid were cultured and the total RNA was analyzed. We observed prominent expression of RNA corresponding to the U1A*-RNA transcript along with lower expression of a 3'-region-truncated form of the transcript (∼110nt) in an rnc (encoding RNase III)-deficient strain. We also found that the produced U1A*-RNA bound to the U1A RNA-binding domain protein, which was separately prepared with C.glutamicum. In a batch cultivation using a fermentor, the total accumulated amount of the target RNA reached about 300mg/L by 24h. Thus, our results indicated that our system can serve as an efficient platform for large-scale preparation of an RNA of interest.

Interpreting the Dynamics of Binding Interactions of snRNA and U1A Using a Coarse-Grained Model

The binding interactions of small nuclear RNAs (snRNA) and the associated protein factors are critical to the function of spliceosomes in alternatively splicing primary RNA transcripts. Although molecular dynamics simulations are a powerful tool to interpret the mechanism of biological processes, the atomic-level simulations are, however, too expensive and with limited accuracy for the large-size systems, such as snRNA-protein complexes. We extend the coarse-grained Gaussian network model, which models the RNA-protein complexes as a harmonic chain of Cα, P, and O4′ atoms, to investigating the impact of the snRNA-binding interaction on the dynamic stability of the human U1A protein, which is a major component of the spliceosomal U1 small nuclear ribonucleoprotein particle. The results reveal that the first and third loops and the C-terminal helix regions of the U1A domain undergo a significant loss of flexibility upon the RNA binding due to the forming of mostly electrostatic and hydrogen bond interactions with RNA 5′ stem and loop. By examining the residues whose mutations significantly change the binding free energy between U1A and snRNA, the Gaussian network model-based calculations show that not only the residues at the binding sites that are traditionally considered to play a major role in U1A-RNA association but also those residues that are far away from the RNA-binding interface can participate in the long-range allosteric signal transmission; these calculations are quantitatively consistent with the data observed in the recent snRNA binding experiments. The study demonstrates a useful avenue to utilize the simplified elastic network model to investigate the dynamics characteristics of the biologically important macromolecular interactions.

Spliceosomal protein U1A is involved in alternative splicing and salt stress tolerance in Arabidopsis thaliana.

Soil salinity is a significant threat to sustainable agricultural production worldwide. Plants must adjust their developmental and physiological processes to cope with salt stress. Although the capacity for adaptation ultimately depends on the genome, the exceptional versatility in gene regulation provided by the spliceosome-mediated alternative splicing (AS) is essential in these adaptive processes. However, the functions of the spliceosome in plant stress responses are poorly understood. Here, we report the in-depth characterization of a U1 spliceosomal protein, AtU1A, in controlling AS of pre-mRNAs under salt stress and salt stress tolerance in Arabidopsis thaliana. The atu1a mutant was hypersensitive to salt stress and accumulated more reactive oxygen species (ROS) than the wild-type under salt stress. RNA-seq analysis revealed that AtU1A regulates AS of many genes, presumably through modulating recognition of 5′ splice sites. We showed that AtU1A is associated with the pre-mRNA of the ROS detoxification-related gene ACO1 and is necessary for the regulation of ACO1 AS. ACO1 is important for salt tolerance because ectopic expression of ACO1 in the atu1a mutant can partially rescue its salt hypersensitive phenotype. Our findings highlight the critical role of AtU1A as a regulator of pre-mRNA processing and salt tolerance in plants.

Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells.

Synthetic biology has great potential for future therapeutic applications including autonomous cell programming through the detection of protein signals and the production of desired outputs. Synthetic RNA devices are promising for this purpose. However, the number of available devices is limited due to the difficulty in the detection of endogenous proteins within a cell. Here, we show a strategy to construct synthetic mRNA devices that detect endogenous proteins in living cells, control translation and distinguish cell types. We engineered protein-binding aptamers that have increased stability in the secondary structures of their active conformation. The designed devices can efficiently respond to target proteins including human LIN28A and U1A proteins, while the original aptamers failed to do so. Moreover, mRNA delivery of an LIN28A-responsive device into human induced pluripotent stem cells (hiPSCs) revealed that we can distinguish living hiPSCs and differentiated cells by quantifying endogenous LIN28A protein expression level. Thus, our endogenous protein-driven RNA devices determine live-cell states and program mammalian cells based on intracellular protein information.

Spliceosomal U1A Protein-SL2 RNA Binding Affinity Decreases in Cells

While extensive biochemical and biophysical studies have been carried out to elucidate protein-RNA binding mechanisms and dynamics in vitro, most of these studies do not take into consideration the effect of the cellular environment. Here we have experimentally tested the role of the cellular environment on protein transport and binding affinity in one of the most widely studied RNA recognition motifs, the spliceosomal protein U1A, and its binding partner, stem loop 2 (SL2) of the U1 small nuclear RNA. U1A-SL2 localization, stability and binding kinetics were monitored in live U2OS cells by fast relaxation imaging (FreI), which combines a temperature jump with fluorescence microscopy of the FRET (Fluorescence resonance energy transfer)-labeled RNA and protein. U1A protein alone was found to diffuse across the cell, whereas SL2 RNA and the U1A-SL2 complex localized in the nucleus. This demonstrates that SL2 RNA mediates transport of the U1A protein to the nucleus. The binding affinity in live cells was reduced compared to in vitro. The dissociation rate was unchanged in cells; however, the association rate was an order of magnitude lower, resulting in a two order of magnitude decrease in the dissociation constant. Introduction of a macromolecular crowder, Ficoll 70, in vitro further stabilized the complex. This suggests that differences between binding affinities as measured in vitro and in live cells cannot be explained by crowding alone. Instead, high binding affinities as measured in vitro may be necessary for selectivity in vivo, where competition exists between multiple binding partners.

Integrating Omics and Alternative Splicing Reveals Insights into Grape Response to High Temperature.

Heat stress is one of the primary abiotic stresses that limit crop production. Grape (Vitis vinifera) is a cultivated fruit with high economic value throughout the world, with its growth and development often influenced by high temperature. Alternative splicing (AS) is a widespread phenomenon increasing transcriptome and proteome diversity. We conducted high-temperature treatments (35°C, 40°C, and 45°C) on grapevines and assessed transcriptomic (especially AS) and proteomic changes in leaves. We found that nearly 70% of the genes were alternatively spliced under high temperature. Intron retention (IR), exon skipping, and alternative donor/acceptor sites were markedly induced under different high temperatures. Among all differential AS events, IR was the most abundant up- and down-regulated event. Moreover, the occurrence frequency of IR events at 40°C and 45°C was far higher than at 35°C. These results indicated that AS, especially IR, is an important posttranscriptional regulatory event during grape leaf responses to high temperature. Proteomic analysis showed that protein levels of the RNA-binding proteins SR45, SR30, and SR34 and the nuclear ribonucleic protein U1A gradually rose as ambient temperature increased, which revealed a reason why AS events occurred more frequently under high temperature. After integrating transcriptomic and proteomic data, we found that heat shock proteins and some important transcription factors such as MULTIPROTEIN BRIDGING FACTOR1c and HEAT SHOCK TRANSCRIPTION FACTOR A2 were involved mainly in heat tolerance in grape through up-regulating transcriptional (especially modulated by AS) and translational levels. To our knowledge, these results provide the first evidence for grape leaf responses to high temperature at simultaneous transcriptional, posttranscriptional, and translational levels.

Therapeutic activity of modified U1 core spliceosomal particles.

Modified U1 snRNAs bound to intronic sequences downstream of the 5′ splice site correct exon skipping caused by different types of mutations. Here we evaluate the therapeutic activity and structural requirements of these exon-specific U1 snRNA (ExSpeU1) particles. In a severe spinal muscular atrophy, mouse model, ExSpeU1, introduced by germline transgenesis, increases SMN2 exon 7 inclusion, SMN protein production and extends life span. In vitro, RNA mutant analysis and silencing experiments show that while U1A protein is dispensable, the 70K and stem loop IV elements mediate most of the splicing rescue activity through improvement of exon and intron definition. Our findings indicate that precise engineering of the U1 core spliceosomal RNA particle has therapeutic potential in pathologies associated with exon-skipping mutations.

Use of the U1A Protein to Facilitate Crystallization and Structure Determination of Large RNAs.

The preparation of well-ordered crystals of RNAs with complex three-dimensional architecture can be facilitated by engineering a binding site for the spliceosomal protein U1A into a functionally and structurally dispensable stem-loop of the RNA of interest. Once suitable crystals are obtained, the U1A protein, of known structure, can be employed to facilitate preparation of heavy atom or anomalously scattering atom derivatives, or as a source of partial model phases for the molecular replacement method. Here, we describe the methods for making U1A preparations suitable for cocrystallization with RNA. As an example, the cocrystallization of the tetracycline aptamer with U1A is also described.

An integrated, peptide-based approach to site-specific protein immobilization for detection of biomolecular interactions.

We have developed an integrated solution for the site-specific immobilization of proteins on a biosensor surface, which may be widely applicable for high throughput analytical purposes. The gold surface of a biosensor was coated with an anti-fouling layer of zwitterionic peptide molecules from which leucine zipper peptides protrude. Proteins of interest, the autoantigenic proteins La and U1A, were immobilized via a simple incubation procedure by using the complementary leucine zipper sequence as a genetically fused binding tag. This tag forms a strong coiled-coil interaction that is stable during multiple consecutive measurements and under common regeneration conditions. Visualization of the immobilized proteins of interest via antibody binding with multiplex surface plasmon resonance imaging demonstrated 2.5 times higher binding responses than when these proteins were randomly attached to the surface via the commonly applied activated ester-mediated coupling. The proteins could also be immobilized in a leucine zipper-dependent manner directly from complex mixtures like bacterial lysates, eliminating the need for laborious purification steps. This method allows the production of uniform functional protein arrays by control over immobilized protein orientation and geometry and is compatible with high-throughput procedures.

Mechanisms of autoantibody production in systemic lupus erythematosus.

Autoantibodies against a panoply of self-antigens are seen in systemic lupus erythematosus, but only a few (anti-Sm/RNP, anti-Ro/La, anti-dsDNA) are common. The common lupus autoantigens are nucleic acid complexes and levels of autoantibodies can be extraordinarily high. We explore why that is the case. Lupus is associated with impaired central or peripheral B-cell tolerance and increased circulating autoreactive B cells. However, terminal differentiation is necessary for autoantibody production. Nucleic acid components of the major lupus autoantigens are immunostimulatory ligands for toll-like receptor (TLR)7 or TLR9 that promote plasma cell differentiation. We show that the levels of autoantibodies against the U1A protein (part of a ribonucleoprotein) are markedly higher than autoantibodies against other antigens, including dsDNA and the non-nucleic acid-associated autoantigens insulin and thyroglobulin. In addition to driving autoantibody production, TLR7 engagement is likely to contribute to the pathogenesis of inflammatory disease in lupus.

In the beginning was the U1A protein: a personal reflection.

In the beginning was the U1A protein: a personal reflection.

Native Conformational Dynamics of the Spliceosomal U1A Protein

The complex of spliceosomal U1A protein and its cognate SL2 RNA is a prototype system for protein-RNA binding studies. A major question is whether U1A protein alone is capable of undergoing conformational dynamics similar to structural rearrangements upon RNA binding. Using a fast temperature jump and tryptophan fluorescence detection, we uncover a ∼20 μs conformational transition for the Lys22Gln/Phe56Trp-only mutant of U1A, yet a Phe56Trp-only control mutant does not show the transition. To explain this observation, we performed extensive molecular dynamics (MD) simulations. The simulations explain why only the Lys22Gln/Phe56Trp-only mutant shows a fluorescence signal: in the other mutant, the tryptophan probe is not quenched upon structural rearrangement. The simulations support helix C movement as the underlying structural rearrangement, although the simulated time scale is faster than experimentally detected. On the basis of our MD results, we propose a reversible two-pathway three-state transition for the helix C movement and assign T-jump kinetics to a closed to semi-closed transition of the helix. Our result provides a specific example of how alternative protein conformations on the native side of the folding barrier can be functionally important, for example in conformational selection by a binding partner.

Electrostatic Interaction Effects on the Binding of Spliceosomal U1A Protein-SL2 RNA Hairpin

The U1A protein is a component of the U1 small nuclear ribonucleoprotein particle, which forms part of the spliceosome. Since U1A protein is highly charged, electrostatic interactions have been suggested to be of importance in the association of the protein with RNA and the stability of the complex. To understand this effect, positively charged residues of the protein have been mutated in three positions (K20, K22 and K50) that do not have direct interactions with the SL2 RNA.We performed extensive molecule dynamics simulations on the mutant complexes and characterized the changes in the networks of interactions. To reveal more global variations within the mutant complexes, we also determined the changes on the subsections of the proteins-RNA (called communities) that move in concert together. These communities have been calculated based on generalized correlations to account for the collective atomic fluctuations within the protein-RNA complexes. These results are in agreement with the experimental complex dissociation binding studies. We then determined a measure based on the collective atomic fluctuations of different mutant complexes that have a linear correlation with the experimentally measured dissociation constants. This correlation can be used to further predict the effect of a mutation on the dissociation constant of the U1A-SL2 RNA complexes.

Combined mechanism of conformational selection and induced fit in U1A-RNA molecular recognition.

In this study, we demonstrate that U1A-RNA molecular recognition is mediated by a combined mechanism of conformational selection and induced fit. The binding of U1A to RNA has been discussed in the context of induced fit that involves the reorientation of the α-helix in the C-terminal region (Helix-C) of U1A to permit RNA access only when U1A correctly recognizes RNA. However, according to our molecular dynamics simulations, even in the absence of RNA, Helix-C spontaneously reoriented to permit RNA access. Nonetheless, such a conformational change was still incomplete. Helix-C was often partially or even fully unfolded and in an infrequent RNA-accessible conformation, which can be detected using state-of-the-art nuclear magnetic resonance methodology. These results suggest that the formation of an energetically stabilized complex is promoted by specific interactions between U1A and RNA. In conclusion, in the recognition of RNA by U1A protein, we propose a combined mechanism that requires the reorientation of Helix-C and the subsequent contact with RNA through conformational selection, although the stabilization of the U1A-RNA complex is caused by induced fit. We further propose a modification to the conventional assumption regarding the mechanism of U1A-RNA molecular recognition.