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
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.
- 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
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.
- 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
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
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