Christophe Maris

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.

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 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.

A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage

Structural information on RNA, emerging more and more as a major regulator in gene expression, dramatically lags behind compared with information on proteins. Although NMR spectroscopy has proven to be an excellent tool to solve RNA structures, it is hampered by the severe spectral resonances overlap found in RNA, limiting its use for large RNA molecules. Segmental isotope labeling of RNA or ligation of a chemically synthesized RNA containing modified nucleotides with an unmodified RNA fragment have proven to have high potential in overcoming current limitations in obtaining structural information on RNA. However, low yields, cumbersome preparations and sequence requirements have limited its broader application in structural biology. Here we present a fast and efficient approach to generate multiple segmentally labeled RNAs with virtually no sequence requirements with very high yields (up to 10-fold higher than previously reported). We expect this approach to open new avenues in structural biology of RNA.

Automated and assisted RNA resonance assignment using NMR chemical shift statistics

The three-dimensional structure determination of RNAs by NMR spectroscopy relies on chemical shift assignment, which still constitutes a bottleneck. In order to develop more efficient assignment strategies, we analysed relationships between sequence and 1H and 13C chemical shifts. Statistics of resonances from regularly Watson–Crick base-paired RNA revealed highly characteristic chemical shift clusters. We developed two approaches using these statistics for chemical shift assignment of double-stranded RNA (dsRNA): a manual approach that yields starting points for resonance assignment and simplifies decision trees and an automated approach based on the recently introduced automated resonance assignment algorithm FLYA. Both strategies require only unlabeled RNAs and three 2D spectra for assigning the H2/C2, H5/C5, H6/C6, H8/C8 and H1′/C1′ chemical shifts. The manual approach proved to be efficient and robust when applied to the experimental data of RNAs with a size between 20 nt and 42 nt. The more advanced automated assignment approach was successfully applied to four stem-loop RNAs and a 42 nt siRNA, assigning 92–100% of the resonances from dsRNA regions correctly. This is the first automated approach for chemical shift assignment of non-exchangeable protons of RNA and their corresponding 13C resonances, which provides an important step toward automated structure determination of RNAs.

The Signature of the Five-Stranded vRRM Fold Defined by Functional, Structural and Computational Analysis of the hnRNP L Protein

The RNA recognition motif (RRM) is the far most abundant RNA binding domain. In addition to the typical β1α1β2β3α2β4 fold, various sub-structural elements have been described and reportedly contribute to the high functional versatility of RRMs. The heterogeneous nuclear ribonucleoprotein L (hnRNP L) is a highly abundant protein of 64 kDa comprising four RRM domains. Involved in many aspects of RNA metabolism, hnRNP L specifically binds to RNAs containing CA repeats or CA-rich clusters. However, a comprehensive structural description of hnRNP L including its sub-structural elements is missing. Here, we present the structural characterization of the RRM domains of hnRNP L and demonstrate their function in repressing exon 4 of SLC2A2. By comparison of the sub-structural elements between the two highly similar paralog families of hnRNP L and PTB, we defined signatures underlying interacting C-terminal coils (ICCs), the RRM34 domain interaction and RRMs with a C-terminal fifth β-strand, a variation we denoted vRRMs. Furthermore, computational analysis revealed new putative ICC-containing RRM families and allowed us to propose an evolutionary scenario explaining the origins of the ICC and fifth β-strand sub-structural extensions. Our studies provide insights of domain requirements in alternative splicing mediated by hnRNP L and molecular descriptions for the sub-structural elements. In addition, the analysis presented may help to classify other abundant RRM extensions and to predict structure-function relationships.

Evidence for cooperative tandem binding of hnRNP C RRMs in mRNA processing.

The human hnRNP C is a ubiquitous cellular protein involved in mRNA maturation. Recently, we have shown that this protein specifically recognizes uridine (U) pentamers through its single RNA recognition motif (RRM). However, a large fraction of natural RNA targets of hnRNP C consists of much longer contiguous uridine stretches. To understand how these extended sites are recognized, we studied the binding of the RRM to U-tracts of 8-11 bases. In vivo investigation of internal translation activation of unr (upstream of N-ras) mRNA indicates that the conservation of the entire hnRNP C binding site, UC(U)8, is required for hnRNP C-dependent IRES activation. The assays further suggest a synergistic interplay between hnRNP C monomers, dependent on the proteins ability to oligomerize. In vitro spectroscopic and thermodynamic analyses show that isolated RRMs bind to (U)11 oligomers as dimers. Structural modeling of a ternary double-RRM/RNA complex indicates additionally that two RRM copies can be accommodated on the canonical sequence UC(U)8. The proposed tandem RRM binding is in very good agreement with the transcriptome-wide recognition of extended U-tracts by full-length hnRNP C, which displays a cross-linking pattern consistent with a positively cooperative RRM dimer binding model.

Structural basis of siRNA recognition by TRBP double‐stranded RNA binding domains

The accurate cleavage of pre‐micro(mi)RNAs by Dicer and mi/siRNA guide strand selection are important steps in forming the RNA‐induced silencing complex (RISC). The role of Dicer binding partner TRBP in these processes remains poorly understood. Here, we solved the solution structure of the two N‐terminal dsRNA binding domains (dsRBDs) of TRBP in complex with a functionally asymmetric siRNA using NMR, EPR, and single‐molecule spectroscopy. We find that siRNA recognition by the dsRBDs is not sequence‐specific but rather depends on the RNA shape. The two dsRBDs can swap their binding sites, giving rise to two equally populated, pseudo‐symmetrical complexes, showing that TRBP is not a primary sensor of siRNA asymmetry. Using our structure to model a Dicer‐TRBP‐siRNA ternary complex, we show that TRBPs dsRBDs and Dicers RNase III domains bind a canonical 19 base pair siRNA on opposite sides, supporting a mechanism whereby TRBP influences Dicer‐mediated cleavage accuracy by binding the dsRNA region of the pre‐miRNA during Dicer cleavage.