Tandem RNA Recognition Motif Domains Research Articles

Analysis of the inter-domain orientation of tandem RRM domains with diverse linkers: connecting experimental with AlphaFold2 predicted models.

The RNA recognition motif (RRM) is the most prevalent RNA binding domain in eukaryotes and is involved in most RNA metabolism processes. Single RRM domains have a limited RNA specificity and affinity and tend to be accompanied by other RNA binding domains, frequently additional RRMs that contribute to an avidity effect. Within multi-RRM proteins, the most common arrangement are tandem RRMs, with two domains connected by a variable linker. Despite their prevalence, little is known about the features that lead to specific arrangements, and especially the role of the connecting linker. In this work, we present a novel and robust way to investigate the relative domain orientation in multi-domain proteins using inter-domain vectors referenced to a stable secondary structure element. We apply this method to tandem RRM domains and cluster experimental tandem RRM structures according to their inter-domain and linker-domain contacts, and report how this correlates with their orientation. By extending our analysis to AlphaFold2 predicted structures, with particular attention to the inter-domain predicted aligned error, we identify new orientations not reported experimentally. Our analysis provides novel insights across a range of tandem RRM orientations that may help for the design of proteins with a specific RNA binding mode.

VdNop12, containing two tandem RNA recognition motif domains, is a crucial factor for pathogenicity and cold adaption in Verticillium dahliae.

Previous studies have reported the ability of fungi to overwinter in soil or on crop debris under different environmental conditions, but how fungi adapt to chilling is still largely unknown. In this study, we have identified and characterized the RNA binding protein (RBP) (VdNop12) by screening an Agrobacterium tumefaciens-mediated transformation-mediated insertional mutational library of Verticillium dahliae. We determined that this protein was essential to the pathogen for virulence on cotton plants. VdNop12 contains two tandem RNA recognition motif domains, and its orthologs are widely distributed in filamentous fungi. Mutants produced by disruption of VdNop12 showed defects in vegetative growth, conidiation and cell wall integrity. The mutant also showed an increase in sensitivity to low temperature, as compared to the wildtype and complementation strains. Yeast complementation assay showed that VdNop12 could functionally restore the growth phenotype of ΔScNop12 mutant of Saccharomyces cerevisiae at 15°C. We demonstrated that the VdNop12 is localized in the nucleus, and its loss resulted in the downregulated expression of several genes related to cAMP-PKA and MAPK pathways in V. dahliae. Our results demonstrated a crucial role of RBPs in the regulation of morphology, cold adaption, and pathogenic development in V. dahliae.

Early Metastable Assembly during the Stress-Induced Formation of Worm-like Amyloid Fibrils of Nucleic Acid Binding Domains of TDP-43.

TDP-43 protein travels between the cytosol and the nucleus to perform its nucleic acid binding functions through its two tandem RNA recognition motif domains (TDP-43tRRM). When exposed to various environmental stresses, it forms abnormal aggregates in the cytosol of neurons, which are the hallmarks of amyotrophic lateral sclerosis and other TDP-43 proteinopathies. However, the nature of early structural changes upon stress sensing and the consequent steps during the course of aggregation are not well understood. In this study, we show that under low-pH conditions, mimicking starvation stress, TDP-43tRRM undergoes a conformational opening reaction linked to the protonation of buried ionizable residues and grows into a metastable oligomeric assembly (called the "low-pH form" or the "L form"). In the L form, the protein molecules have disrupted tertiary structure, solvent-exposed hydrophobic patches, and mobile side chains but the native-like secondary structure remains intact. The L form structure is held by weak interactions and has a steep dependence on ionic strength. In the presence of as little as 15 mM KCl, it fully misfolds and further oligomerizes to form a β-sheet rich "β form" in at least two distinct steps. The β form has an ordered, stable structure that resembles worm-like amyloid fibrils. The unstructured regions of the protein gain structure during L ⇌ β conversion. Our results suggest that TDP-43tRRM could function as a stress sensor and support a recent model in which stress sensing during neurodegeneration occurs by assembly of proteins into metastable assemblies that are precursors to the solid aggregates.

ATP is a cryptic binder of TDP-43 RRM domains to enhance stability and inhibit ALS/AD-associated fibrillation

ATP is the universal energy currency for all cells but has cellular concentrations of 2–12 mM, much higher than required for its classic functions. RNA-recognition motif (RRM) constitutes one of the most abundant domains in eukaryotes and most heterogeneous nuclear ribonucleoproteins (hnRNP) contain RRM domains which not only mediate direct interactions with nucleic acids, but whose aggregation/fibrillation is the pathological hallmark of various human diseases. Here, by NMR and molecular docking, ATP has been decoded to bind TDP-43 two tandem RRM domains with distinctive types of interactions, thus resulting in diverse affinities. Most strikingly, the binding of ATP enhances thermodynamic stability of TDP-43 RRM domains and inhibits ALS-/AD-associated fibrillation. Together, ATP is a cryptic binder of RRM-containing proteins which generally safeguards functional phase separation from transforming into pathological aggregation/fibrillation associated with various diseases and ageing. Our study thus reveals a mechanism of ATP to control protein homeostasis by specific binding.

The Folding and Aggregation Energy Landscapes of Tethered RRM Domains of Human TDP-43 Are Coupled via a Metastable Molten Globule-like Oligomer.

Stress-induced misfolding and intraneuronal aggregation of the highly conserved nucleic acid binding protein TDP-43 (transactive response DNA binding protein 43 kDa) and its fragments have been implicated in amyotrophic lateral sclerosis and several other neurodegenerative diseases. However, the physicochemical mechanism of its misfolding from the functional folded state is poorly understood. TDP-43 is a four-domain protein and performs the essential nucleic acid binding function with the help of its two tandem RNA recognition motif domains naturally tethered by a linker (called here the tethered RRM domain of TDP-43 or TDP-43tRRM). Here, we show that the monomeric native form of TDP-43tRRM remains in a pH-dependent and reversible thermodynamic equilibrium with a protonated, nanosized, 40-meric form (the A form). Under the stress-like low-pH condition, the A form becomes predominantly populated. In the A form, protein molecules have restricted dynamics of surface side-chain residues but native-like secondary structure. This self-assembled form possesses a loosely packed core in which the intrinsically disordered and aggregation-prone regions are in the proximity. The A form is metastable and swiftly aggregates into a highly stable amyloid-like protofibrillar form (β form) mediated by the disorder-to-order transition of intrinsically disordered regions upon small environmental perturbations. Interestingly, the A form and the β form are not formed when TDP-43tRRM is bound to DNA, indicating that the nucleic acid binding regions of the protein participate in their formation. Our results reveal how the energy landscapes of folding and aggregation of TDP-43tRRM are coupled by a metastable molten-globule like oligomeric form and modulated by stress-like conditions.

Recognition of the 3′ splice site RNA by the U2AF heterodimer involves a dynamic population shift

An essential early step in the assembly of human spliceosomes onto pre-mRNA involves the recognition of regulatory RNA cis elements in the 3' splice site by the U2 auxiliary factor (U2AF). The large (U2AF65) and small (U2AF35) subunits of the U2AF heterodimer contact the polypyrimidine tract (Py-tract) and the AG-dinucleotide, respectively. The tandem RNA recognition motif domains (RRM1,2) of U2AF65 adopt closed/inactive and open/active conformations in the free form and when bound to bona fide Py-tract RNA ligands. To investigate the molecular mechanism and dynamics of 3' splice site recognition by U2AF65 and the role of U2AF35 in the U2AF heterodimer, we have combined single-pair FRET and NMR experiments. In the absence of RNA, the RRM1,2 domain arrangement is highly dynamic on a submillisecond time scale, switching between closed and open conformations. The addition of Py-tract RNA ligands with increasing binding affinity (strength) gradually shifts the equilibrium toward an open conformation. Notably, the protein-RNA complex is rigid in the presence of a strong Py-tract but exhibits internal motion with weak Py-tracts. Surprisingly, the presence of U2AF35, whose UHM domain interacts with U2AF65 RRM1, increases the population of the open arrangement of U2AF65 RRM1,2 in the absence and presence of a weak Py-tract. These data indicate that the U2AF heterodimer promotes spliceosome assembly by a dynamic population shift toward the open conformation of U2AF65 to facilitate the recognition of weak Py-tracts at the 3' splice site. The structure and RNA binding of the heterodimer was unaffected by cancer-linked myelodysplastic syndrome mutants.

RNA recognition and self-association of CPEB4 is mediated by its tandem RRM domains.

Cytoplasmic polyadenylation is regulated by the interaction of the cytoplasmic polyadenylation element binding proteins (CPEB) with cytoplasmic polyadenylation element (CPE) containing mRNAs. The CPEB family comprises four paralogs, CPEB1–4, each composed of a variable N-terminal region, two RNA recognition motif (RRM) and a C-terminal ZZ-domain. We have characterized the RRM domains of CPEB4 and their binding properties using a combination of biochemical, biophysical and NMR techniques. Isothermal titration calorimetry, NMR and electrophoretic mobility shift assay experiments demonstrate that both the RRM domains are required for an optimal CPE interaction and the presence of either one or two adenosines in the two most commonly used consensus CPE motifs has little effect on the affinity of the interaction. Both the single RRM1 and the tandem RRM1–RRM2 have the ability to dimerize, although representing a minor population. Self-association does not affect the proteins’ ability to interact with RNA as demonstrated by ion mobility–mass spectrometry. Chemical shift effects measured by NMR of the apo forms of the RRM1–RRM2 samples indicate that the two domains are orientated toward each other. NMR titration experiments show that residues on the β-sheet surface on RRM1 and at the C-terminus of RRM2 are affected upon RNA binding. We propose a model of the CPEB4 RRM1–RRM2–CPE complex that illustrates the experimental data.

The structure of the ARE-binding domains of Hu antigen R (HuR) undergoes conformational changes during RNA binding

Human RNA-binding protein (HuR), a ubiquitously expressed member of the Hu protein family, plays an important role in mRNA degradation and has been implicated as a key post-transcriptional regulator. HuR contains three RNA-recognition motif (RRM) domains. The two N-terminal tandem RRM domains can selectively bind AU-rich elements (AREs), while the third RRM domain (RRM3) contributes to interactions with the poly-A tail of target mRNA and other ligands. Here, the X-ray structure of two methylated tandem RRM domains (RRM1/2) of HuR in their RNA-free form was solved at 2.9 Å resolution. The crystal structure of RRM1/2 complexed with target mRNA was also solved at 2.0 Å resolution; comparisons of the two structures show that HuR RRM1/2 undergoes conformational changes upon RNA binding. Fluorescence polarization assays (FPA) were used to study the protein-RNA interactions. Both the structure and the FPA analysis indicated that RRM1 is the primary ARE-binding domain in HuR and that the conformational changes induce subsequent contacts of the RNA substrate with the inter-domain linker and RRM2 which greatly improve the RNA-binding affinity of HuR.

Promiscuity in protein‐RNA interactions: Conformational ensembles facilitate molecular recognition in the spliceosome

Here I discuss findings that suggest a universal mechanism for proteins (and RNA) to recognize and interact with various binding partners by selectively binding to different conformations that pre-exist in the free protein's conformational ensemble. The tandem RNA recognition motif domains of splicing factor U2AF⁶⁵ fluctuate in solution between a predominately closed conformation in which the RNA binding site of one of the domains is blocked, and a lowly populated open conformation in which both RNA binding pockets are accessible. RNA binding to U2AF⁶⁵ may thus occur through the weakly populated open conformation, and the binding interaction stabilizes the open conformation. The conformational diversity observed in U2AF⁶⁵ might also facilitate binding to diverse RNA sequences as found in the polypyrimidine tracts that help define 3' splice sites. Similar binding pathways in other systems have important consequences in biological regulation, molecular evolution, and information storage.

Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF

Many cellular functions involve multi-domain proteins, which are composed of structurally independent modules connected by flexible linkers. Although it is often well understood how a given domain recognizes a cognate oligonucleotide or peptide motif, the dynamic interaction of multiple domains in the recognition of these ligands remains to be characterized. Here we have studied the molecular mechanisms of the recognition of the 3'-splice-site-associated polypyrimidine tract RNA by the large subunit of the human U2 snRNP auxiliary factor (U2AF65) as a key early step in pre-mRNA splicing. We show that the tandem RNA recognition motif domains of U2AF65 adopt two remarkably distinct domain arrangements in the absence or presence of a strong (that is, high affinity) polypyrimidine tract. Recognition of sequence variations in the polypyrimidine tract RNA involves a population shift between these closed and open conformations. The equilibrium between the two conformations functions as a molecular rheostat that quantitatively correlates the natural variations in polypyrimidine tract nucleotide composition, length and functional strength to the efficiency to recruit U2 snRNP to the intron during spliceosome assembly. Mutations that shift the conformational equilibrium without directly affecting RNA binding modulate splicing activity accordingly. Similar mechanisms of cooperative multi-domain conformational selection may operate more generally in the recognition of degenerate nucleotide or amino acid motifs by multi-domain proteins.

Preliminary crystallographic analysis of the RNA-binding domain of HuR and its poly(U)-binding properties

Human antigen R (HuR), a ubiquitously expressed member of the Hu protein family, is an important post-transcriptional regulator which has three RNA-recognition motif (RRM) domains. The two tandem N-terminal RRM domains can selectively bind to the AU-rich element (ARE), while the third one interacts with the poly(A) tail and other proteins. Here, the recombinant ARE-binding region of HuR (residues 18-186) was crystallized in space group P2(1)2(1)2, with unit-cell parameters a = 41.2, b = 133.1, c = 31.4 Å. X-ray diffraction data were collected to a resolution of 2.8 Å. Mutagenesis analysis and SPR assays revealed its poly(U)-binding properties.

Crystal structure of the two N-terminal RRM domains of Pub1 and the poly(U)-binding properties of Pub1

Yeast poly(U)-binding protein (Pub1) is a major nuclear and cytoplasmic protein that contains three RNA recognition motif (RRM) domains (termed Pub1RRM1, Pub1RRM2 and Pub1RRM3). Pub1 has been implicated as a regulator of cellular mRNA decay. Nearly 10% of all yeast mRNA decay occurs in a Pub1-dependent manner. Pub1 binds to and stabilizes AU-rich element (ARE) and ARE-like sequence-containing transcripts by protecting them from degradation through the deadenylation-dependent pathway, and also binds to and stabilizes stabilizer element (STE)-containing transcripts by preventing their degradation via the nonsense-mediated decay (NMD) pathway. RNA-binding analyses showed that Pub1 binds to poly(U) in vitro. Here we show the crystal structures of Pub1RRM2 and the first two tandem RRM domains (Pub1RRM12). Crystallography showed that the structure of Pub1RRM12 is a domain-swapped dimer. Size exclusion chromatography assay and analytical ultracentrifugation (AUC) showed that Pub1RRM12 is a monomer in solution. Kinetic analysis showed that all three individual RRM domains can bind to poly(U) with similar affinities and Pub1RRM12 binds to a long poly(U) segment with higher affinity. Mutagenesis analysis revealed that residues on the beta-sheets of Pub1RRM1 and Pub1RRM2 are critical for poly(U) binding.

Identification of the Polypyrimidine Tract Binding Protein-associated Splicing Factor·p54(nrb) Complex as a Candidate DNA Double-strand Break Rejoining Factor

The biological effects of ionizing radiation are attributable, in large part, to induction of DNA double-strand breaks. We report here the identification of a new protein factor that reconstitutes efficient double-strand break rejoining when it is added to a reaction containing the five other polypeptides known to participate in the human nonhomologous end-joining pathway. The factor is a stable heteromeric complex of polypyrimidine tract-binding protein-associated splicing factor (PSF) and a 54-kDa nuclear RNA-binding protein (p54(nrb)). These polypeptides, to which a variety of functions have previously been attributed, share extensive homology, including tandem RNA recognition motif domains. The PSF.p54(nrb) complex cooperates with Ku protein to form a functional preligation complex with substrate DNA. Based on structural comparison with related proteins, we propose a model where the four RNA recognition motif domains in the heteromeric PSF.p54(nrb) complex cooperate to align separate DNA molecules.