Frédéric H.-T. Allain

The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression

The RNA recognition motif (RRM), also known as RNA‐binding domain (RBD) or ribonucleoprotein domain (RNP) is one of the most abundant protein domains in eukaryotes. Based on the comparison of more than 40 structures including 15 complexes (RRM–RNA or RRM–protein), we reviewed the structure–function relationships of this domain. We identified and classified the different structural elements of the RRM that are important for binding a multitude of RNA sequences and proteins. Common structural aspects were extracted that allowed us to define a structural leitmotif of the RRM–nucleic acid interface with its variations. Outside of the two conserved RNP motifs that lie in the center of the RRM β‐sheet, the two external β‐strands, the loops, the C‐ and N‐termini, or even a second RRM domain allow high RNA‐binding affinity and specific recognition. Protein–RRM interactions that have been found in several structures reinforce the notion of an extreme structural versatility of this domain supporting the numerous biological functions of the RRM‐containing proteins.

The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression.

The RNA recognition motif (RRM), also known as RNA‐binding domain (RBD) or ribonucleoprotein domain (RNP) is one of the most abundant protein domains in eukaryotes. Based on the comparison of more than 40 structures including 15 complexes (RRM–RNA or RRM–protein), we reviewed the structure–function relationships of this domain. We identified and classified the different structural elements of the RRM that are important for binding a multitude of RNA sequences and proteins. Common structural aspects were extracted that allowed us to define a structural leitmotif of the RRM–nucleic acid interface with its variations. Outside of the two conserved RNP motifs that lie in the center of the RRM β‐sheet, the two external β‐strands, the loops, the C‐ and N‐termini, or even a second RRM domain allow high RNA‐binding affinity and specific recognition. Protein–RRM interactions that have been found in several structures reinforce the notion of an extreme structural versatility of this domain supporting the numerous biological functions of the RRM‐containing proteins.

Gac/Rsm signal transduction pathway of γ‐proteobacteria: from RNA recognition to regulation of social behaviour

In many γ‐proteobacteria, the conserved GacS/GacA (BarA/UvrY) two‐component system positively controls the expression of one to five genes specifying small RNAs (sRNAs) that are characterized by repeated unpaired GGA motifs but otherwise appear to belong to several independent families. The GGA motifs are essential for binding small, dimeric RNA‐binding proteins of a single conserved family designated RsmA (CsrA). These proteins, which also occur in bacterial species outside the γ‐proteobacteria, act as translational repressors of certain mRNAs when these contain an RsmA/CsrA binding site at or near the Shine‐Dalgarno sequence plus additional binding sites located in the 5′ untranslated leader mRNA. Recent structural data have established that the RsmA‐like protein RsmE of Pseudomonas fluorescens makes specific contacts with an RNA consensus sequence 5′‐A/UCANGGANGU/A‐3′ (where N is any nucleotide). Interaction with an RsmA/CsrA protein promotes the formation of a short stem supporting an ANGGAN loop. This conformation hinders access of 30S ribosomal subunits and hence translation initiation. The output of the Gac/Rsm cascade varies widely in different bacterial species and typically involves management of carbon storage and expression of virulence or biocontrol factors. Unidentified signal molecules co‐ordinate the activity of the Gac/Rsm cascade in a cell population density‐dependent manner.

RNA recognition motifs: boring? Not quite.

The RNA recognition motif (RRM) is one of the most abundant protein domains in eukaryotes. While the structure of this domain is well characterized by the packing of two alpha-helices on a four-stranded beta-sheet, the mode of protein and RNA recognition by RRMs is not clear owing to the high variability of these interactions. Here we report recent structural data on RRM-RNA and RRM-protein interactions showing the ability of this domain to modulate its binding affinity and specificity using each of its constitutive elements (beta-strands, loops, alpha-helices). The extreme structural versatility of the RRM interactions explains why RRM-containing proteins have so diverse biological functions.

Sequence-specific binding of single-stranded RNA: is there a code for recognition?

A code predicting the RNA sequence that will be bound by a certain protein based on its amino acid sequence or its structure would provide a useful tool for the design of RNA binders with desired sequence-specificity. Such de novo designed RNA binders could be of extraordinary use in both medical and basic research applications. Furthermore, a code could help to predict the cellular functions of RNA-binding proteins that have not yet been extensively studied. A comparative analysis of Pumilio homology domains, zinc-containing RNA binders, hnRNP K homology domains and RNA recognition motifs is performed in this review. Based on this, a set of binding rules is proposed that hints towards a code for RNA recognition by these domains. Furthermore, we discuss the intermolecular interactions that are important for RNA binding and summarize their importance in providing affinity and specificity.

Molecular basis of RNA recognition by the human alternative splicing factor Fox‐1

The Fox‐1 protein regulates alternative splicing of tissue‐specific exons by binding to GCAUG elements. Here, we report the solution structure of the Fox‐1 RNA binding domain (RBD) in complex with UGCAUGU. The last three nucleotides, UGU, are recognized in a canonical way by the four‐stranded β‐sheet of the RBD. In contrast, the first four nucleotides, UGCA, are bound by two loops of the protein in an unprecedented manner. Nucleotides U1, G2, and C3 are wrapped around a single phenylalanine, while G2 and A4 form a base‐pair. This novel RNA binding site is independent from the β‐sheet binding interface. Surface plasmon resonance analyses were used to quantify the energetic contributions of electrostatic and hydrogen bond interactions to complex formation and support our structural findings. These results demonstrate the unusual molecular mechanism of sequence‐specific RNA recognition by Fox‐1, which is exceptional in its high affinity for a defined but short sequence element.

SRSF2 Mutations Contribute to Myelodysplasia by Mutant-Specific Effects on Exon Recognition

Mutations affecting spliceosomal proteins are the most common mutations in patients with myelodysplastic syndromes (MDS), but their role in MDS pathogenesis has not been delineated. Here we report that mutations affecting the splicing factor SRSF2 directly impair hematopoietic differentiation in vivo, which is not due to SRSF2 loss of function. By contrast, SRSF2 mutations alter SRSF2s normal sequence-specific RNA binding activity, thereby altering the recognition of specific exonic splicing enhancer motifs to drive recurrent mis-splicing of key hematopoietic regulators. This includes SRSF2 mutation-dependent splicing of EZH2, which triggers nonsense-mediated decay, which, in turn, results in impaired hematopoietic differentiation. These data provide a mechanistic link between a mutant spliceosomal protein, alterations in the splicing of key regulators, and impaired hematopoiesis.

The Solution Structure of the ADAR2 dsRBM-RNA Complex Reveals a Sequence-Specific Readout of the Minor Groove.

Sequence-dependent recognition of dsDNA-binding proteins is well understood, yet sequence-specific recognition of dsRNA by proteins remains largely unknown, despite their importance in RNA maturation pathways. Adenosine deaminases that act on RNA (ADARs) recode genomic information by the site-selective deamination of adenosine. Here, we report the solution structure of the ADAR2 double-stranded RNA-binding motifs (dsRBMs) bound to a stem-loop pre-mRNA encoding the R/G editing site of GluR-2. The structure provides a molecular basis for how dsRBMs recognize the shape, and also more surprisingly, the sequence of the dsRNA. The unexpected direct readout of the RNA primary sequence by dsRBMs is achieved via the minor groove of the dsRNA and this recognition is critical for both editing and binding affinity at the R/G site of GluR-2. More generally, our findings suggest a solution to the sequence-specific paradox faced by many dsRBM-containing proteins that are involved in post-transcriptional regulation of gene expression.

Molecular basis of messenger RNA recognition by the specific bacterial repressing clamp RsmA/CsrA

Proteins of the RsmA/CsrA family are global translational regulators in many bacterial species. We have determined the solution structure of a complex formed between the RsmE protein, a member of this family from Pseudomonas fluorescens, and a target RNA encompassing the ribosome-binding site of the hcnA gene. The RsmE homodimer with its two RNA-binding sites makes optimal contact with an 5′-A/UCANGGANGU/A-3′ sequence in the mRNA. When tightly gripped by RsmE, the ANGGAN core folds into a loop, favoring the formation of a 3-base-pair stem by flanking nucleotides. We validated these findings by in vivo and in vitro mutational analyses. The structure of the complex explains well how, by sequestering the Shine-Dalgarno sequence, the RsmA/CsrA proteins repress translation.

Molecular basis for temperature sensing by an RNA thermometer

Regulatory RNA elements, like riboswitches, respond to intracellular signals by three‐dimensional (3D) conformational changes. RNA thermometers employ a similar strategy to sense temperature changes in the cell and regulate the translational machinery. We present here the first 3D NMR structure of the functional domain of a highly conserved bacterial RNA thermometer containing the ribosome binding site that remains occluded at normal temperatures (30°C). We identified a region adjacent to the Shine–Dalgarno sequence that has a network of weak hydrogen bonds within the RNA helix. With the onset of heat shock at 42°C, destabilisation of the RNA structure initiates at this region and favours the release of the ribosome binding site and of the start codon. Deletion of a highly conserved G residue leads to the formation of a stable regular RNA helix that loses thermosensing ability. Our results indicate that RNA thermometers are able to sense temperature changes without the aid of accessory factors.