Antoine Cléry

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.

RRM-RNA recognition: NMR or crystallography…and new findings.

To characterize protein-RNA recognition at the molecular level, structural biology has turned out to be an indispensable approach. Detailed and direct insights into the mechanism of RNA binding and specificity have emerged from protein-RNA structures, especially from the most abundant RNA recognition motif (RRM). Although this protein domain has a very conserved α-β fold, it can recognize a large number of different RNA sequences and shapes and can be involved in a multitude of biological processes. Here, we report on recent single and multiple RRM-RNA structures and point out those features that provide novel insights into the mechanism of RNA recognition by RRMs. We further outline inherent problems to both NMR spectroscopy and X-ray crystallography methods and review recent strategies that emphasize the need to use both methodologies for more rapid and accurate structure determinations.

Molecular basis of purine-rich RNA recognition by the human SR-like protein Tra2-β1

Tra2-β1 is a unique splicing factor as its single RNA recognition motif (RRM) is located between two RS (arginine-serine) domains. To understand how this protein recognizes its RNA target, we solved the structure of Tra2-β1 RRM in complex with RNA. The central 5′-AGAA-3′ motif is specifically recognized by residues from the β-sheet of the RRM and by residues from both extremities flanking the RRM. The structure suggests that RNA binding by Tra2-β1 induces positioning of the two RS domains relative to one another. By testing the effect of Tra2-β1 and RNA mutations on the splicing of SMN2 exon 7, we validated the importance of the RNA-protein contacts observed in the structure for the function of Tra2-β1 and determined the functional sequence of Tra2-β1 in SMN2 exon 7. Finally, we propose a model for the assembly of multiple RNA binding proteins on this exon.

A syn – anti conformational difference allows SRSF2 to recognize guanines and cytosines equally well

SRSF2 (SC35) is a key player in the regulation of alternative splicing events and binds degenerated RNA sequences with similar affinity in nanomolar range. We have determined the solution structure of the SRSF2 RRM bound to the 5′‐UCCAGU‐3′ and 5′‐UGGAGU‐3′ RNA, both identified as SRSF2 binding sites in the HIV‐1 tat exon 2. RNA recognition is achieved through a novel sandwich‐like structure with both termini forming a positively charged cavity to accommodate the four central nucleotides. To bind both RNA sequences equally well, SRSF2 forms a nearly identical network of intermolecular interactions by simply flipping the bases of the two consecutive C or G nucleotides into either anti or syn conformation. We validate this unusual mode of RNA recognition functionally by in‐vitro and in‐vivo splicing assays and propose a 5′‐SSNG‐3′ (S=C/G) high‐affinity binding consensus sequence for SRSF2. In conclusion, in addition to describe for the first time the RNA recognition mode of SRSF2, we provide the precise consensus sequence to identify new putative binding sites for this splicing factor.

Isolated pseudo–RNA-recognition motifs of SR proteins can regulate splicing using a noncanonical mode of RNA recognition

Significance Serine/arginine (SR) proteins are key regulators of eukaryotic gene expression and have been associated with multiple human diseases including cancers. Several members of this protein family contain a noncanonical RNA recognition motif (RRM), the pseudo-RRM, for which the mode of RNA recognition is unknown. Here, we solved the structure of SRSF1 pseudo-RRM bound to RNA. It reveals the RNA motif recognized and a very unusual mode of interaction, which is conserved for all the SR proteins containing pseudo-RRMs. Finally, we show that the pseudo-RRM in isolation often is sufficient to regulate splicing, and we reveal its mechanism of action. Serine/arginine (SR) proteins, one of the major families of alternative-splicing regulators in Eukarya, have two types of RNA-recognition motifs (RRMs): a canonical RRM and a pseudo-RRM. Although pseudo-RRMs are crucial for activity of SR proteins, their mode of action was unknown. By solving the structure of the human SRSF1 pseudo-RRM bound to RNA, we discovered a very unusual and sequence-specific RNA-binding mode that is centered on one α-helix and does not involve the β-sheet surface, which typically mediates RNA binding by RRMs. Remarkably, this mode of binding is conserved in all pseudo-RRMs tested. Furthermore, the isolated pseudo-RRM is sufficient to regulate splicing of about half of the SRSF1 target genes tested, and the bound α-helix is a pivotal element for this function. Our results strongly suggest that SR proteins with a pseudo-RRM frequently regulate splicing by competing with, rather than recruiting, spliceosome components, using solely this unusual RRM.

One, Two, Three, Four! How Multiple RRMs Read the Genome Sequence.

RRM-containing proteins are involved in most of the RNA metabolism steps. Their functions are closely related to their mode of RNA recognition, which has been studied by structural biologists for more than 20 years. In this chapter, we report on high-resolution structures of single and multi RRM-RNA complexes to explain the numerous strategies used by these domains to interact specifically with a large repertoire of RNA sequences. We show that multiple variations of their canonical fold can be used to adapt to different single-stranded sequences with a large range of affinities. Furthermore, we describe the consequences on RNA binding of the different structural arrangements found in tandem RRMs and higher order RNPs. Importantly, these structures also reveal with very high accuracy the RNA motifs bound specifically by RRM-containing proteins, which correspond very often to consensus sequences identified with genome-wide approaches. Finally, we show how structural and cellular biology can benefit from each other and pave a way for understanding, defining, and predicting a code of RNA recognition by the RRMs.

Characterization of the RNA recognition mode of hnRNP G extends its role in SMN2 splicing regulation

Regulation of SMN2 exon 7 splicing is crucial for the production of active SMN protein and the survival of Spinal Muscular Atrophy (SMA) patients. One of the most efficient activators of exon 7 inclusion is hnRNP G, which is recruited to the exon by Tra2-β1. We report that in addition to the C-terminal region of hnRNP G, the RNA Recognition Motif (RRM) and the middle part of the protein containing the Arg–Gly–Gly (RGG) box are important for this function. To better understand the mode of action of hnRNP G in this context we determined the structure of its RRM bound to an SMN2 derived RNA. The RRM interacts with a 5′-AAN-3′ motif and specifically recognizes the two consecutive adenines. By testing the effect of mutations in hnRNP G RRM and in its putative binding sites on the splicing of SMN2 exon 7, we show that it specifically binds to exon 7. This interaction is required for hnRNP G splicing activity and we propose its recruitment to a polyA tract located upstream of the Tra2-β1 binding site. Finally, our data suggest that hnRNP G plays a major role in the recruitment of the Tra2-β1/hnRNP G/SRSF9 trimeric complex to SMN2 exon 7.

Synergy between NMR measurements and MD simulations of protein/RNA complexes: application to the RRMs, the most common RNA recognition motifs.

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.

Binding to SMN2 pre-mRNA-protein complex elicits specificity for small molecule splicing modifiers

Small molecule splicing modifiers have been previously described that target the general splicing machinery and thus have low specificity for individual genes. Several potent molecules correcting the splicing deficit of the SMN2 (survival of motor neuron 2) gene have been identified and these molecules are moving towards a potential therapy for spinal muscular atrophy (SMA). Here by using a combination of RNA splicing, transcription, and protein chemistry techniques, we show that these molecules directly bind to two distinct sites of the SMN2 pre-mRNA, thereby stabilizing a yet unidentified ribonucleoprotein (RNP) complex that is critical to the specificity of these small molecules for SMN2 over other genes. In addition to the therapeutic potential of these molecules for treatment of SMA, our work has wide-ranging implications in understanding how small molecules can interact with specific quaternary RNA structures.Small molecules correcting the splicing deficit of the survival of motor neuron 2 (SMN2) gene have been identified as having therapeutic potential. Here, the authors provide evidence that SMN2 mRNA forms a ribonucleoprotein complex that can be specifically targeted by these small molecules.

NMR spectroscopy: an excellent tool to understand RNA and carbohydrate recognition by proteins.

Structural biology plays a key role in understanding how networks of protein interactions with their partners are organized at the atomic level. In this review, we show that NMR is a very efficient method to solve 3D structures of protein - RNA and protein-carbohydrate complexes of high quality. We explain the importance of studying such interactions and describe the main steps that are required to determine structures of these types of complexes by NMR. Finally, we show that X-ray crystallography and NMR are complementary methods and briefly report on advantages and disadvantages of each approach.