In the bacterial Csr/Rsm system, non-coding RNAs activate mRNA translation by removing homodimeric Csr/Rsm proteins from the ribosome-binding sites of mRNAs. In Pseudomonas protegens, each RsmZ ncRNA sequesters up to five RsmE dimers sequentially and specifically within a narrow affinity range, functioning as a 'protein sponge'. Although the RsmE binding cascade is cooperative, binding of the highest affinity stem-loop RNA in RsmZ (SL2) reduces RNA binding affinity at the second site by 10- to 30-fold. This unusual negative cooperativity may facilitate RsmE release from tightly bound mRNA for handover to the non-coding RNA, yet the underlying mechanisms remain unclear. Using Isothermal Titration Calorimetry, NMR spectroscopy and Molecular Dynamic simulations, we show that the initial binding event increases conformational entropy at the empty site, partially unfolding the C-terminal helix. Moreover, we reveal an allosteric mechanism coupling RNA binding at the first site to conformational changes at the second site, explaining the reduced affinity of the second binding event. The anti-parallel β-sheets in the RsmE dimer facilitate communication between sites, with H-bond constriction at the bound site and relaxation at the empty site, resembling a Newton's cradle.

• Publikační typ
• časopisecké články MeSH
• preprinty MeSH

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
• alosterická regulace MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• motiv rozpoznávající RNA MeSH
• protein vázající polypyrimidinové úseky RNA * metabolismus   chemie   MeSH
• proteinové domény MeSH
• RNA * chemie   metabolismus   MeSH
• sbalování proteinů MeSH
• simulace molekulární dynamiky MeSH
• vazba proteinů * MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protein vázající polypyrimidinové úseky RNA * MeSH
• RNA * MeSH

The YTH domain of YTHDC1 belongs to a class of protein "readers", recognizing the N6-methyladenosine (m6A) chemical modification in mRNA. Static ensemble-averaged structures revealed details of N6-methyl recognition via a conserved aromatic cage. Here, we performed molecular dynamics (MD) simulations along with nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) to examine how dynamics and solvent interactions contribute to the m6A recognition and negative selectivity toward an unmethylated substrate. The structured water molecules surrounding the bound RNA and the methylated substrate's ability to exclude bulk water molecules contribute to the YTH domain's preference for m6A. Intrusions of bulk water deep into the binding pocket disrupt binding of unmethylated adenosine. The YTHDC1's preference for the 5'-Gm6A-3' motif is partially facilitated by a network of water-mediated interactions between the 2-amino group of the guanosine and residues in the m6A binding pocket. The 5'-Im6A-3' (where I is inosine) motif can be recognized too, but disruption of the water network lowers affinity. The D479A mutant also disrupts the water network and destabilizes m6A binding. Our interdisciplinary study of the YTHDC1 protein-RNA complex reveals an unusual physical mechanism by which solvent interactions contribute toward m6A recognition.

• MeSH
• adenosin analogy a deriváty   MeSH
• magnetická rezonanční spektroskopie MeSH
• proteiny nervové tkáně metabolismus   MeSH
• proteiny vázající RNA * genetika   metabolismus   MeSH
• sestřihové faktory metabolismus   MeSH
• simulace molekulární dynamiky * MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosin MeSH
• N-methyladenosine MeSH Prohlížeč
• proteiny nervové tkáně MeSH
• proteiny vázající RNA * MeSH
• sestřihové faktory 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

Understanding the RNA binding specificity of protein is of primary interest to decipher their function in the cell. Here, we review the methodology used to solve the structures of protein-RNA complexes using solution-state NMR spectroscopy: from sample preparation to structure calculation procedures. We also describe how molecular dynamics simulations can help providing additional information on the role of key amino acid side chains and of water molecules in protein-RNA recognition.

• Klíčová slova
• MD simulation, Protein–RNA interactions, Solution-state NMR, Structures,
• MeSH
• CELF proteiny chemie   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• konformace nukleové kyseliny MeSH
• konformace proteinů, alfa-helix MeSH
• konformace proteinů, beta-řetězec MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie metody   MeSH
• RNA chemie   genetika   metabolismus   MeSH
• sestřihové faktory chemie   metabolismus   MeSH
• simulace molekulární dynamiky * MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• vodíková vazba MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CELF proteiny MeSH
• RBFOX1 protein, human MeSH Prohlížeč
• RNA MeSH
• sestřihové faktory 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 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' nepřekládaná oblast MeSH
• aminokyselinové motivy MeSH
• CELF proteiny chemie   genetika   metabolismus   MeSH
• cyklooxygenasa 2 genetika   MeSH
• fenylalanin chemie   metabolismus   MeSH
• konformace proteinů MeSH
• magnetická rezonanční spektroskopie MeSH
• messenger RNA chemie   metabolismus   MeSH
• proteiny nervové tkáně chemie   genetika   metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• substituce aminokyselin MeSH
• úseky bohaté na AU MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3' nepřekládaná oblast MeSH
• CELF proteiny MeSH
• CELF2 protein, human MeSH Prohlížeč
• cyklooxygenasa 2 MeSH
• fenylalanin MeSH
• messenger RNA MeSH
• proteiny nervové tkáně MeSH
• PTGS2 protein, human MeSH Prohlížeč

TDP-43 encodes an alternative-splicing regulator with tandem RNA-recognition motifs (RRMs). The protein regulates cystic fibrosis transmembrane regulator (CFTR) exon 9 splicing through binding to long UG-rich RNA sequences and is found in cytoplasmic inclusions of several neurodegenerative diseases. We solved the solution structure of the TDP-43 RRMs in complex with UG-rich RNA. Ten nucleotides are bound by both RRMs, and six are recognized sequence specifically. Among these, a central G interacts with both RRMs and stabilizes a new tandem RRM arrangement. Mutations that eliminate recognition of this key nucleotide or crucial inter-RRM interactions disrupt RNA binding and TDP-43-dependent splicing regulation. In contrast, point mutations that affect base-specific recognition in either RRM have weaker effects. Our findings reveal not only how TDP-43 recognizes UG repeats but also how RNA binding-dependent inter-RRM interactions are crucial for TDP-43 function.

• MeSH
• DNA vazebné proteiny chemie   metabolismus   fyziologie   MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• protein CFTR genetika   metabolismus   MeSH
• proteiny vázající RNA chemie   metabolismus   fyziologie   MeSH
• sekvence aminokyselin MeSH
• sestřih RNA fyziologie   MeSH
• terciární struktura proteinů MeSH
• vazebná místa MeSH
• zastoupení bazí MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CFTR protein, human MeSH Prohlížeč
• DNA vazebné proteiny MeSH
• protein CFTR MeSH
• proteiny vázající RNA MeSH