The N-terminal RNA recognition motif domain (RRM1) of polypyrimidine tract binding protein (PTB) forms an additional C-terminal helix α3, which docks to one edge of the β-sheet upon binding to a stem-loop RNA containing a UCUUU pentaloop. Importantly, α3 does not contact the RNA. The α3 helix therefore represents an allosteric means to regulate the conformation of adjacent domains in PTB upon binding structured RNAs. Here we investigate the process of dynamic adaptation by stem-loop RNA and RRM1 using NMR and MD in order to obtain mechanistic insights on how this allostery is achieved. Relaxation data and NMR structure determination of the free protein show that α3 is partially ordered and interacts with the domain transiently. Stem-loop RNA binding quenches fast time scale dynamics and α3 becomes ordered, however microsecond dynamics at the protein-RNA interface is observed. MD shows how RRM1 binding to the stem-loop RNA is coupled to the stabilization of the C-terminal helix and helps to transduce differences in RNA loop sequence into changes in α3 length and order. IRES assays of full length PTB and a mutant with altered dynamics in the α3 region show that this dynamic allostery influences PTB function in cultured HEK293T cells.

• MeSH
• Allosteric Regulation MeSH
• Nucleic Acid Conformation MeSH
• Humans MeSH
• RNA Recognition Motif MeSH
• Polypyrimidine Tract-Binding Protein * metabolism   chemistry   MeSH
• Protein Domains MeSH
• RNA * chemistry   metabolism   MeSH
• Protein Folding MeSH
• Molecular Dynamics Simulation MeSH
• Protein Binding * MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Polypyrimidine Tract-Binding Protein * MeSH
• RNA * MeSH

The conserved protein Hfq is a key factor in the RNA-mediated control of gene expression in most known bacteria. The transient intermediates Hfq forms with RNA support intricate and robust regulatory networks. In Pseudomonas, Hfq recognizes repeats of adenine-purine-any nucleotide (ARN) in target mRNAs via its distal binding side, and together with the catabolite repression control (Crc) protein, assembles into a translation-repression complex. Earlier experiments yielded static, ensemble-averaged structures of the complex, but details of its interface dynamics and assembly pathway remained elusive. Using explicit solvent atomistic molecular dynamics simulations, we modeled the extensive dynamics of the Hfq-RNA interface and found implications for the assembly of the complex. We predict that syn/anti flips of the adenine nucleotides in each ARN repeat contribute to a dynamic recognition mechanism between the Hfq distal side and mRNA targets. We identify a previously unknown binding pocket that can accept any nucleotide and propose that it may serve as a 'status quo' staging point, providing nonspecific binding affinity, until Crc engages the Hfq-RNA binary complex. The dynamical components of the Hfq-RNA recognition can speed up screening of the pool of the surrounding RNAs, participate in rapid accommodation of the RNA on the protein surface, and facilitate competition among different RNAs. The register of Crc in the ternary assembly could be defined by the recognition of a guanine-specific base-phosphate interaction between the first and last ARN repeats of the bound RNA. This dynamic substrate recognition provides structural rationale for the stepwise assembly of multicomponent ribonucleoprotein complexes nucleated by Hfq-RNA binding.

• Keywords
• ARN repeats, Crc protein, Hfq protein, RNA metabolism, RNA-binding protein, dynamic recognition, molecular dynamics, protein–nucleic acid interaction,
• MeSH
• RNA, Bacterial chemistry   genetics   metabolism   MeSH
• Nucleic Acid Conformation MeSH
• Protein Conformation MeSH
• Nucleotide Motifs * MeSH
• Host Factor 1 Protein chemistry   genetics   metabolism   MeSH
• Pseudomonas aeruginosa genetics   metabolism   MeSH
• Gene Expression Regulation, Bacterial * MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Bacterial MeSH
• Host Factor 1 Protein MeSH

The human prototypical SR protein SRSF1 is an oncoprotein that contains two RRMs and plays a pivotal role in RNA metabolism. We determined the structure of the RRM1 bound to RNA and found that the domain binds preferentially to a CN motif (N is for any nucleotide). Based on this solution structure, we engineered a protein containing a single glutamate to asparagine mutation (E87N), which gains the ability to bind to uridines and thereby activates SMN exon7 inclusion, a strategy that is used to cure spinal muscular atrophy. Finally, we revealed that the flexible inter-RRM linker of SRSF1 allows RRM1 to bind RNA on both sides of RRM2 binding site. Besides revealing an unexpected bimodal mode of interaction of SRSF1 with RNA, which will be of interest to design new therapeutic strategies, this study brings a new perspective on the mode of action of SRSF1 in cells.

• MeSH
• Asparagine genetics   MeSH
• Exons genetics   MeSH
• HEK293 Cells MeSH
• Glutamic Acid genetics   MeSH
• Humans MeSH
• RNA Splice Sites genetics   MeSH
• RNA Recognition Motif genetics   MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Survival of Motor Neuron 1 Protein genetics   MeSH
• Protein Engineering MeSH
• Recombinant Proteins genetics   isolation & purification   metabolism   ultrastructure   MeSH
• Serine-Arginine Splicing Factors genetics   isolation & purification   metabolism   ultrastructure   MeSH
• RNA Splicing * MeSH
• Molecular Dynamics Simulation MeSH
• Muscular Atrophy, Spinal genetics   therapy   MeSH
• Amino Acid Substitution MeSH
• Uridine metabolism   MeSH
• Computational Biology MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Asparagine MeSH
• Glutamic Acid MeSH
• RNA Splice Sites MeSH
• Survival of Motor Neuron 1 Protein MeSH
• Recombinant Proteins MeSH
• Serine-Arginine Splicing Factors MeSH
• SMN1 protein, human MeSH Browser
• SRSF1 protein, human MeSH Browser
• Uridine MeSH

The RNA recognition motif (RRM) is the most common RNA binding domain across eukaryotic proteins. It is therefore of great value to engineer its specificity to target RNAs of arbitrary sequence. This was recently achieved for the RRM in Rbfox protein, where four mutations R118D, E147R, N151S, and E152T were designed to target the precursor to the oncogenic miRNA 21. Here, we used a variety of molecular dynamics-based approaches to predict specific interactions at the binding interface. Overall, we have run approximately 50 microseconds of enhanced sampling and plain molecular dynamics simulations on the engineered complex as well as on the wild-type Rbfox·pre-miRNA 20b from which the mutated systems were designed. Comparison with the available NMR data on the wild type molecules (protein, RNA, and their complex) served to establish the accuracy of the calculations. Free energy calculations suggest that further improvements in affinity and selectivity are achieved by the S151T replacement.

• MeSH
• Nucleic Acid Conformation MeSH
• Humans MeSH
• MicroRNAs chemistry   genetics   metabolism   MeSH
• Models, Molecular MeSH
• RNA Recognition Motif * genetics   MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Protein Engineering MeSH
• RNA-Binding Proteins chemistry   genetics   metabolism   MeSH
• RNA chemistry   metabolism   MeSH
• Amino Acid Sequence MeSH
• Molecular Dynamics Simulation MeSH
• RNA Stability MeSH
• Protein Binding MeSH
• Binding Sites genetics   MeSH
• Computational Biology MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• MicroRNAs MeSH
• MIRN20b microRNA, human MeSH Browser
• MIRN21 microRNA, human MeSH Browser
• RNA-Binding Proteins MeSH
• RNA MeSH

The neomycin sensing riboswitch is the smallest biologically functional RNA riboswitch, forming a hairpin capped with a U-turn loop-a well-known RNA motif containing a conserved uracil. It was shown previously that a U→C substitution of the eponymous conserved uracil does not alter the riboswitch structure due to C protonation at N3. Furthermore, cytosine is evolutionary permitted to replace uracil in other U-turns. Here, we use molecular dynamics simulations to study the molecular basis of this substitution in the neomycin sensing riboswitch and show that a structure-stabilizing monovalent cation-binding site in the wild-type RNA is the main reason for its negligible structural effect. We then use NMR spectroscopy to confirm the existence of this cation-binding site and to demonstrate its effects on RNA stability. Lastly, using quantum chemical calculations, we show that the cation-binding site is altering the electronic environment of the wild-type U-turn so that it is more similar to the cytosine mutant. The study reveals an amazingly complex and delicate interplay between various energy contributions shaping up the 3D structure and evolution of nucleic acids.

• MeSH
• Cytosine chemistry   MeSH
• Potassium MeSH
• Magnesium MeSH
• Ions chemistry   MeSH
• Cations chemistry   MeSH
• Nucleic Acid Conformation MeSH
• Ligands MeSH
• Mutation MeSH
• Neomycin MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Base Pairing MeSH
• Riboswitch * MeSH
• Molecular Dynamics Simulation MeSH
• Uracil chemistry   MeSH
• Binding Sites MeSH
• Hydrogen Bonding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytosine MeSH
• Potassium MeSH
• Magnesium MeSH
• Ions MeSH
• Cations MeSH
• Ligands MeSH
• Neomycin MeSH
• Riboswitch * MeSH
• Uracil MeSH

With both catalytic and genetic functions, ribonucleic acid (RNA) is perhaps the most pluripotent chemical species in molecular biology, and its functions are intimately linked to its structure and dynamics. Computer simulations, and in particular atomistic molecular dynamics (MD), allow structural dynamics of biomolecular systems to be investigated with unprecedented temporal and spatial resolution. We here provide a comprehensive overview of the fast-developing field of MD simulations of RNA molecules. We begin with an in-depth, evaluatory coverage of the most fundamental methodological challenges that set the basis for the future development of the field, in particular, the current developments and inherent physical limitations of the atomistic force fields and the recent advances in a broad spectrum of enhanced sampling methods. We also survey the closely related field of coarse-grained modeling of RNA systems. After dealing with the methodological aspects, we provide an exhaustive overview of the available RNA simulation literature, ranging from studies of the smallest RNA oligonucleotides to investigations of the entire ribosome. Our review encompasses tetranucleotides, tetraloops, a number of small RNA motifs, A-helix RNA, kissing-loop complexes, the TAR RNA element, the decoding center and other important regions of the ribosome, as well as assorted others systems. Extended sections are devoted to RNA-ion interactions, ribozymes, riboswitches, and protein/RNA complexes. Our overview is written for as broad of an audience as possible, aiming to provide a much-needed interdisciplinary bridge between computation and experiment, together with a perspective on the future of the field.

• MeSH
• DNA chemistry   MeSH
• Catalysis MeSH
• Nucleic Acid Conformation * MeSH
• Computer Simulation MeSH
• RNA chemistry   MeSH
• Molecular Dynamics Simulation * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• DNA MeSH
• RNA MeSH

The cyclooxygenase-2 is a pro-inflammatory and cancer marker, whose mRNA stability and translation is regulated by the CUG-binding protein 2 interacting with AU-rich sequences in the 3' untranslated region. Here, we present the solution NMR structure of CUG-binding protein 2 RRM3 in complex with 5'-UUUAA-3' originating from the COX-2 3'-UTR. We show that RRM3 uses the same binding surface and protein moieties to interact with AU- and UG-rich RNA motifs, binding with low and high affinity, respectively. Using NMR spectroscopy, isothermal titration calorimetry and molecular dynamics simulations, we demonstrate that distinct sub-states characterized by different aromatic side-chain conformations at the RNA-binding surface allow for high- or low-affinity binding with functional implications. This study highlights a mechanism for RNA discrimination possibly common to multiple RRMs as several prominent members display a similar rearrangement of aromatic residues upon binding their targets.The RNA Recognition Motif (RRM) is the most ubiquitous RNA binding domain. Here the authors combined NMR and molecular dynamics simulations and show that the RRM RNA binding surface exists in different states and that a conformational switch of aromatic side-chains fine-tunes sequence specific binding affinities.

• MeSH
• 3' Untranslated Regions MeSH
• Amino Acid Motifs MeSH
• CELF Proteins chemistry   genetics   metabolism   MeSH
• Cyclooxygenase 2 genetics   MeSH
• Phenylalanine chemistry   metabolism   MeSH
• Protein Conformation MeSH
• Magnetic Resonance Spectroscopy MeSH
• RNA, Messenger chemistry   metabolism   MeSH
• Nerve Tissue Proteins chemistry   genetics   metabolism   MeSH
• Molecular Dynamics Simulation MeSH
• Amino Acid Substitution MeSH
• AU Rich Elements MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 3' Untranslated Regions MeSH
• CELF Proteins MeSH
• CELF2 protein, human MeSH Browser
• Cyclooxygenase 2 MeSH
• Phenylalanine MeSH
• RNA, Messenger MeSH
• Nerve Tissue Proteins MeSH
• PTGS2 protein, human MeSH Browser

The Fox-1 RNA recognition motif (RRM) domain is an important member of the RRM protein family. We report a 1.8 Å X-ray structure of the free Fox-1 containing six distinct monomers. We use this and the nuclear magnetic resonance (NMR) structure of the Fox-1 protein/RNA complex for molecular dynamics (MD) analyses of the structured hydration. The individual monomers of the X-ray structure show diverse hydration patterns, however, MD excellently reproduces the most occupied hydration sites. Simulations of the protein/RNA complex show hydration consistent with the isolated protein complemented by hydration sites specific to the protein/RNA interface. MD predicts intricate hydration sites with water-binding times extending up to hundreds of nanoseconds. We characterize two of them using NMR spectroscopy, RNA binding with switchSENSE and free-energy calculations of mutant proteins. Both hydration sites are experimentally confirmed and their abolishment reduces the binding free-energy. A quantitative agreement between theory and experiment is achieved for the S155A substitution but not for the S122A mutant. The S155 hydration site is evolutionarily conserved within the RRM domains. In conclusion, MD is an effective tool for predicting and interpreting the hydration patterns of protein/RNA complexes. Hydration is not easily detectable in NMR experiments but can affect stability of protein/RNA complexes.

• MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• RNA Recognition Motif genetics   MeSH
• Mutagenesis, Site-Directed MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• RNA metabolism   MeSH
• RNA Splicing Factors chemistry   genetics   metabolism   MeSH
• Molecular Dynamics Simulation MeSH
• Amino Acid Substitution MeSH
• Binding Sites MeSH
• Water chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• RBFOX1 protein, human MeSH Browser
• Recombinant Proteins MeSH
• RNA MeSH
• RNA Splicing Factors MeSH
• Water MeSH