Recognition of single-stranded RNA (ssRNA) by RNA recognition motif (RRM) domains is an important class of protein-RNA interactions. Many such complexes were characterized using nuclear magnetic resonance (NMR) and/or X-ray crystallography techniques, revealing ensemble-averaged pictures of the bound states. However, it is becoming widely accepted that better understanding of protein-RNA interactions would be obtained from ensemble descriptions. Indeed, earlier molecular dynamics simulations of bound states indicated visible dynamics at the RNA-RRM interfaces. Here, we report the first atomistic simulation study of spontaneous binding of short RNA sequences to RRM domains of HuR and SRSF1 proteins. Using a millisecond-scale aggregate ensemble of unbiased simulations, we were able to observe a few dozen binding events. HuR RRM3 utilizes a pre-binding state to navigate the RNA sequence to its partially disordered bound state and then to dynamically scan its different binding registers. SRSF1 RRM2 binding is more straightforward but still multiple-pathway. The present study necessitated development of a goal-specific force field modification, scaling down the intramolecular van der Waals interactions of the RNA which also improves description of the RNA-RRM bound state. Our study opens up a new avenue for large-scale atomistic investigations of binding landscapes of protein-RNA complexes, and future perspectives of such research are discussed.

• MeSH
• HuR protein metabolismus   MeSH
• motiv rozpoznávající RNA genetika   MeSH
• proteiny vázající RNA * metabolismus   MeSH
• RNA * chemie   MeSH
• RRM proteiny metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HuR protein MeSH
• proteiny vázající RNA * MeSH
• RNA * MeSH
• RRM proteiny 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
• asparagin genetika   MeSH
• exony genetika   MeSH
• HEK293 buňky MeSH
• kyselina glutamová genetika   MeSH
• lidé MeSH
• místa sestřihu RNA genetika   MeSH
• motiv rozpoznávající RNA genetika   MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• protein přežití motorických neuronů 1 genetika   MeSH
• proteinové inženýrství MeSH
• rekombinantní proteiny genetika   izolace a purifikace   metabolismus   ultrastruktura   MeSH
• serin-arginin sestřihové faktory genetika   izolace a purifikace   metabolismus   ultrastruktura   MeSH
• sestřih RNA * MeSH
• simulace molekulární dynamiky MeSH
• spinální svalová atrofie genetika   terapie   MeSH
• substituce aminokyselin MeSH
• uridin metabolismus   MeSH
• výpočetní biologie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• asparagin MeSH
• kyselina glutamová MeSH
• místa sestřihu RNA MeSH
• protein přežití motorických neuronů 1 MeSH
• rekombinantní proteiny MeSH
• serin-arginin sestřihové faktory MeSH
• SMN1 protein, human MeSH Prohlížeč
• SRSF1 protein, human MeSH Prohlížeč
• uridin MeSH

Human antigen R (HuR) is a key regulator of cellular mRNAs containing adenylate/uridylate-rich elements (AU-rich elements; AREs). These are a major class of cis elements within 3' untranslated regions, targeting these mRNAs for rapid degradation. HuR contains three RNA recognition motifs (RRMs): a tandem RRM1 and 2, followed by a flexible linker and a C-terminal RRM3. While RRM1 and 2 are structurally characterized, little is known about RRM3. Here we present a 1.9-Å-resolution crystal structure of RRM3 bound to different ARE motifs. This structure together with biophysical methods and cell-culture assays revealed the mechanism of RRM3 ARE recognition and dimerization. While multiple RNA motifs can be bound, recognition of the canonical AUUUA pentameric motif is possible by binding to two registers. Additionally, RRM3 forms homodimers to increase its RNA binding affinity. Finally, although HuR stabilizes ARE-containing RNAs, we found that RRM3 counteracts this effect, as shown in a cell-based ARE reporter assay and by qPCR with native HuR mRNA targets containing multiple AUUUA motifs, possibly by competing with RRM12.

• Klíčová slova
• NMR spectroscopy, RNA-binding protein, crystal structure, dimerization, multiple register,
• MeSH
• 3' nepřekládaná oblast MeSH
• dimerizace MeSH
• encefalomyelitida paraneoplastická - Hu antigeny chemie   MeSH
• HuR protein chemie   genetika   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• motiv rozpoznávající RNA genetika   MeSH
• nádorové supresorové proteiny chemie   MeSH
• proteiny vázající RNA chemie   genetika   MeSH
• ribonukleosiddifosfátreduktasa chemie   MeSH
• úseky bohaté na AU genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3' nepřekládaná oblast MeSH
• encefalomyelitida paraneoplastická - Hu antigeny MeSH
• HuR protein MeSH
• nádorové supresorové proteiny MeSH
• proteiny vázající RNA MeSH
• ribonucleotide reductase M2 MeSH Prohlížeč
• ribonukleosiddifosfátreduktasa MeSH
• RRM1 protein, human MeSH Prohlížeč

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
• konformace nukleové kyseliny MeSH
• lidé MeSH
• mikro RNA chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• motiv rozpoznávající RNA * genetika   MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• proteinové inženýrství MeSH
• proteiny vázající RNA chemie   genetika   metabolismus   MeSH
• RNA chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• simulace molekulární dynamiky MeSH
• stabilita RNA MeSH
• vazba proteinů MeSH
• vazebná místa genetika   MeSH
• výpočetní biologie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mikro RNA MeSH
• MIRN20b microRNA, human MeSH Prohlížeč
• MIRN21 microRNA, human MeSH Prohlížeč
• proteiny vázající RNA MeSH
• RNA MeSH

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
• krystalografie rentgenová MeSH
• lidé MeSH
• motiv rozpoznávající RNA genetika   MeSH
• mutageneze cílená MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• RNA metabolismus   MeSH
• sestřihové faktory chemie   genetika   metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• substituce aminokyselin MeSH
• vazebná místa MeSH
• voda chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• RBFOX1 protein, human MeSH Prohlížeč
• rekombinantní proteiny MeSH
• RNA MeSH
• sestřihové faktory MeSH
• voda MeSH

RNA recognition motif (RRM) proteins represent an abundant class of proteins playing key roles in RNA biology. We present a joint atomistic molecular dynamics (MD) and experimental study of two RRM-containing proteins bound with their single-stranded target RNAs, namely the Fox-1 and SRSF1 complexes. The simulations are used in conjunction with NMR spectroscopy to interpret and expand the available structural data. We accumulate more than 50 μs of simulations and show that the MD method is robust enough to reliably describe the structural dynamics of the RRM-RNA complexes. The simulations predict unanticipated specific participation of Arg142 at the protein-RNA interface of the SRFS1 complex, which is subsequently confirmed by NMR and ITC measurements. Several segments of the protein-RNA interface may involve competition between dynamical local substates rather than firmly formed interactions, which is indirectly consistent with the primary NMR data. We demonstrate that the simulations can be used to interpret the NMR atomistic models and can provide qualified predictions. Finally, we propose a protocol for 'MD-adapted structure ensemble' as a way to integrate the simulation predictions and expand upon the deposited NMR structures. Unbiased μs-scale atomistic MD could become a technique routinely complementing the NMR measurements of protein-RNA complexes.

• MeSH
• konformace proteinů MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• molekulární modely MeSH
• motiv rozpoznávající RNA genetika   MeSH
• multiproteinové komplexy chemie   genetika   MeSH
• RNA chemie   genetika   MeSH
• sekvence aminokyselin genetika   MeSH
• serin-arginin sestřihové faktory chemie   genetika   MeSH
• sestřihové faktory chemie   genetika   MeSH
• simulace molekulární dynamiky MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• multiproteinové komplexy MeSH
• RBFOX1 protein, human MeSH Prohlížeč
• RNA MeSH
• serin-arginin sestřihové faktory MeSH
• sestřihové faktory MeSH
• SRSF1 protein, human MeSH Prohlížeč