Search

Dead End (DND1) is an RNA-binding protein essential for germline development through its role in post-transcriptional gene regulation. The molecular mechanisms behind selection and regulation of its targets are unknown. Here, we present the solution structure of DND1's tandem RNA Recognition Motifs (RRMs) bound to AU-rich RNA. The structure reveals how an NYAYUNN element is specifically recognized, reconciling seemingly contradictory sequence motifs discovered in recent genome-wide studies. RRM1 acts as a main binding platform, including atypical extensions to the canonical RRM fold. RRM2 acts cooperatively with RRM1, capping the RNA using an unusual binding pocket, leading to an unusual mode of tandem RRM-RNA recognition. We show that the consensus motif is sufficient to mediate upregulation of a reporter gene in human cells and that this process depends not only on RNA binding by the RRMs, but also on DND1's double-stranded RNA binding domain (dsRBD), which is dispensable for binding of a subset of targets in cellulo. Our results point to a model where DND1 target selection is mediated by a non-canonical mode of AU-rich RNA recognition by the tandem RRMs and a role for the dsRBD in the recruitment of effector complexes responsible for target regulation., (© 2022. The Author(s).)

Stress granules are non-membrane bound RNA-protein granules essential for survival during acute cellular stress. TIA-1 is a key protein in the formation of stress granules that undergoes liquid-liquid phase separation by association with specific RNAs and protein-protein interactions. However, the fundamental properties of the TIA-1 protein that enable phase-separation also render TIA-1 susceptible to the formation of irreversible fibrillar aggregates. Despite this, within physiological stress granules, TIA-1 is not present as fibrils, pointing to additional factors within the cell that prevent TIA-1 aggregation. Here we show that heterotypic interactions with stress granule co-factors Zn 2+ and RGG-rich regions from FUS each act together with nucleic acid to induce the liquid-liquid phase separation of TIA-1. In contrast, these co-factors do not enhance nucleic acid induced fibril formation of TIA-1, but rather robustly inhibit the process. NMR titration experiments revealed specific interactions between Zn 2+ and H94 and H96 in RRM2 of TIA-1. Strikingly, this interaction promotes multimerization of TIA-1 independently of the prion-like domain. Thus, through different molecular mechanisms, these stress granule co-factors promote TIA-1 liquid-liquid phase separation and suppress fibrillar aggregates, potentially contributing to the dynamic nature of stress granules and the cellular protection that they provide., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 West, Loughlin, Rivero-Rodríguez, Vankadari, Velázquez-Cruz, Corrales-Guerrero, Díaz-Moreno and Wilce.)

TIA-1 is an RNA-binding protein that sequesters target RNA into stress granules under conditions of cellular stress. Promotion of stress granule formation by TIA-1 depends upon self-association of its prion-like domain that facilitates liquid-liquid phase separation and is thought to be enhanced via RNA binding. However, the mechanisms underlying the influence of RNA on TIA-1 self-association have not been previously demonstrated. Here we have investigated the self-associating properties of full-length TIA-1 in the presence of designed and native TIA-1 nucleic acid binding sites in vitro, monitoring phase separation, fibril formation and shape. We show that single stranded RNA and DNA induce liquid-liquid phase separation of TIA-1 in a multisite, sequence-specific manner and also efficiently promote formation of amyloid-like fibrils. Although RNA binding to a single site induces a small conformational change in TIA-1, this alone does not enhance phase separation of TIA-1. Tandem binding sites are required to enhance phase separation of TIA-1 and this is finely tuned by the protein:binding site stoichiometry rather than nucleic acid length. Native tandem TIA-1 binding sites within the 3' UTR of p53 mRNA also efficiently enhance phase separation of TIA-1 and thus may potentially act as potent nucleation sites for stress granule assembly., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mutations in the RNA-binding protein Fused in Sarcoma (FUS) cause early-onset amyotrophic lateral sclerosis (ALS). However, a detailed understanding of central RNA targets of FUS and their implications for disease remain elusive. Here, we use a unique blend of crosslinking and immunoprecipitation (CLIP) and NMR spectroscopy to identify and characterise physiological and pathological RNA targets of FUS. We find that U1 snRNA is the primary RNA target of FUS via its interaction with stem-loop 3 and provide atomic details of this RNA-mediated mode of interaction with the U1 snRNP. Furthermore, we show that ALS-associated FUS aberrantly contacts U1 snRNA at the Sm site with its zinc finger and traps snRNP biogenesis intermediates in human and murine motor neurons. Altogether, we present molecular insights into a FUS toxic gain-of-function involving direct and aberrant RNA-binding and strengthen the link between two motor neuron diseases, ALS and spinal muscular atrophy (SMA).

RNA-binding proteins TDP-43 and FUS play essential roles in pre-mRNA splicing, localization, granule formation and other aspects of RNA metabolism. Both proteins are implicated in neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Despite their apparent similarities, each protein has unique structural characteristics. Here we present the current structural understanding of RNA-binding and self-association mechanisms. Both globular and intrinsically disordered domains contribute to RNA binding, each with different specificities, affinities and kinetics. Self-associating Prion-like domains in each protein form multivalent interactions and labile cross-β structures. These interactions are modulated by distinctive additional domains including a globular oligomerization domain in TDP-43 and synergistic interactions with intrinsically disordered Arginine-Glycine rich domains in FUS. These insights contribute to a better understanding of native biological functions of TDP-43 and FUS and potential molecular pathways in neurodegenerative diseases., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

Fused in sarcoma (FUS) is an RNA binding protein involved in regulating many aspects of RNA processing and linked to several neurodegenerative diseases. Transcriptomics studies indicate that FUS binds a large variety of RNA motifs, suggesting that FUS RNA binding might be quite complex. Here, we present solution structures of FUS zinc finger (ZnF) and RNA recognition motif (RRM) domains bound to RNA. These structures show a bipartite binding mode of FUS comprising of sequence-specific recognition of a NGGU motif via the ZnF and an unusual shape recognition of a stem-loop RNA via the RRM. In addition, sequence-independent interactions via the RGG repeats significantly increase binding affinity and promote destabilization of structured RNA conformation, enabling additional binding. We further show that disruption of the RRM and ZnF domains abolishes FUS function in splicing. Altogether, our results rationalize why deciphering the RNA binding mode of FUS has been so challenging., (Copyright © 2018 Elsevier Inc. All rights reserved.)

N-terminal gluconoylation is a moderately widespread modification in recombinant proteins expressed in Escherichia coli, in particular in proteins bearing an N-terminal histidine-tag. This post-translational modification has been investigated mainly by mass spectrometry. Although its NMR signals must have been observed earlier in spectra of 13 C/ 15 N labeled proteins, their chemical shifts were not yet reported. Here we present the complete 1 H and 13 C chemical shift assignment of the N-terminal gluconoyl post-translational modification, based on a selection of His-tagged protein constructs (CCL2, hnRNP A1 and Lin28) starting with Met-Gly-...-(His) 6 . In addition, we show that the modification can hydrolyze over time, resulting in a free N-terminus and gluconate. This leads to the disappearance of the gluconoyl signals and the appearance of gluconate signals during the NMR measurements. The chemical shifts presented here can now be used as a reference for the identification of gluconoylation in recombinant proteins, in particular when isotopically labeled.

TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Å resolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expression., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In recent years, evidence has emerged for the existence of many diverse types of RNA, which play roles in a wide range of biological processes in all kingdoms of life. These molecules generally do not, however, act in isolation, and identifying which proteins partner with RNA is a major challenge. Many methods, in vivo and in vitro, have been used to address this question, including combinatorial or high-throughput approaches, such as systematic evolution of ligands, cross-linking and immunoprecipitation and RNA immunoprecipitation combined with deep sequencing. However, most of these methods are not trivial to pursue and often require substantial optimization before results can be achieved. Here, we demonstrate a simple technique that allows one to screen proteins for RNA-binding properties in a gel-shift experiment and can be easily implemented in any laboratory. This assay should be a useful first-pass tool for assessing whether a protein has RNA- or DNA-binding properties, prior to committing resources to more complex procedures.

Lin28 inhibits the biogenesis of let-7 miRNAs through a direct interaction with the terminal loop of pre-let-7. This interaction requires the zinc-knuckle domains of Lin28. We show that the zinc knuckle domains of Lin28 are sufficient to provide binding selectivity for pre-let-7 miRNAs and present the NMR structure of human Lin28 zinc knuckles bound to the short sequence 5'-AGGAGAU-3'. The structure reveals that each zinc knuckle recognizes an AG dinucleotide separated by a single nucleotide spacer. This defines a new 5'-NGNNG-3' consensus motif that explains how Lin28 selectively recognizes pre-let-7 family members. Binding assays in cell lysates and functional assays in cultured cells demonstrate that the interactions observed in the solution structure also occur between the full-length protein and members of the pre-let-7 family. The consensus sequence explains several seemingly disparate previously published observations on the binding properties of Lin28.

The recognition of single-stranded RNA (ssRNA) is an important aspect of gene regulation, and a number of different classes of protein domains that recognize ssRNA in a sequence-specific manner have been identified. Recently, we demonstrated that the RanBP2-type zinc finger (ZnF) domains from the human splicing factor ZnF Ran binding domain-containing protein 2 (ZRANB2) can bind to a sequence containing the consensus AGGUAA. Six other human proteins, namely, Ewing's sarcoma (EWS), translocated in liposarcoma (TLS)/FUS, RNA-binding protein 56 (RBP56), RNA-binding motif 5 (RBM5), RNA-binding motif 10 (RBM10) and testis-expressed sequence 13A (TEX13A), each contains a single ZnF with homology to the ZRANB2 ZnFs, and several of these proteins have been implicated in the regulation of mRNA processing. Here, we show that all of these ZnFs are able to bind with micromolar affinities to ssRNA containing a GGU motif. NMR titration data reveal that binding is mediated by the corresponding surfaces on each ZnF, and we also show that sequence selectivity is largely limited to the GGU core motif and that substitution of the three flanking adenines that were selected in our original selection experiment has a minimal effect on binding affinity. These data establish a subset of RanBP2-type ZnFs as a new family of ssRNA-binding motifs., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The alternative splicing of mRNA is a critical process in higher eukaryotes that generates substantial proteomic diversity. Many of the proteins that are essential to this process contain arginine/serine-rich (RS) domains. ZRANB2 is a widely-expressed and highly-conserved RS-domain protein that can regulate alternative splicing but lacks canonical RNA-binding domains. Instead, it contains 2 RanBP2-type zinc finger (ZnF) domains. We demonstrate that these ZnFs recognize ssRNA with high affinity and specificity. Each ZnF binds to a single AGGUAA motif and the 2 domains combine to recognize AGGUAA(N(x))AGGUAA double sites, suggesting that ZRANB2 regulates alternative splicing via a direct interaction with pre-mRNA at sites that resemble the consensus 5' splice site. We show using X-ray crystallography that recognition of an AGGUAA motif by a single ZnF is dominated by side-chain hydrogen bonds to the bases and formation of a guanine-tryptophan-guanine "ladder." A number of other human proteins that function in RNA processing also contain RanBP2 ZnFs in which the RNA-binding residues of ZRANB2 are conserved. The ZnFs of ZRANB2 therefore define another class of RNA-binding domain, advancing our understanding of RNA recognition and emphasizing the versatility of ZnF domains in molecular recognition.

ZRANB2 is a zinc-finger protein that has been shown to influence alternative splice-site selection. The protein comprises a C-terminal arginine/serine-rich domain that interacts with spliceosomal proteins and two N-terminal RanBP2-type zinc fingers that have been implicated in RNA recognition. The second zinc finger bound to a six-nucleotide single-stranded RNA target sequence crystallized in the hexagonal space group P6(5)22 or P6(1)22, with unit-cell parameters a = 54.52, b = 54.52, c = 48.07 A; the crystal contains one monomeric complex per asymmetric unit. This crystal form has a solvent content of 39% and diffracted to 1.4 A resolution using synchrotron radiation.

The design of metal-binding functionality in proteins is expanding into many different areas with a wide range of practical and research applications. Here we review several developing areas of metal-related protein design, including the use of metals to induce protein-protein interactions or facilitate the assembly of extended nanostructures; the design of metallopeptides that bind metal and other inorganic surfaces, an area with potential in diverse applications ranging from nanoelectronics and photonics to biotechnology and biomedicine; and, the creation of sensitive and selective metal sensors for use both in vivo and in vitro.

The gene-for-gene mechanism of plant disease resistance involves direct or indirect recognition of pathogen avirulence (Avr) proteins by plant resistance (R) proteins. Flax rust (Melampsora lini) AvrL567 avirulence proteins and the corresponding flax (Linum usitatissimum) L5, L6, and L7 resistance proteins interact directly. We determined the three-dimensional structures of two members of the AvrL567 family, AvrL567-A and AvrL567-D, at 1.4- and 2.3-A resolution, respectively. The structures of both proteins are very similar and reveal a beta-sandwich fold with no close known structural homologs. The polymorphic residues in the AvrL567 family map to the surface of the protein, and polymorphisms in residues associated with recognition differences for the R proteins lead to significant changes in surface chemical properties. Analysis of single amino acid substitutions in AvrL567 proteins confirm the role of individual residues in conferring differences in recognition and suggest that the specificity results from the cumulative effects of multiple amino acid contacts. The structures also provide insights into possible pathogen-associated functions of AvrL567 proteins, with nucleic acid binding activity demonstrated in vitro. Our studies provide some of the first structural information on avirulence proteins that bind directly to the corresponding resistance proteins, allowing an examination of the molecular basis of the interaction with the resistance proteins as a step toward designing new resistance specificities.

Zinc-fingers (ZnFs) are extremely abundant in higher eukaryotes. Once considered to function exclusively as sequence-specific DNA-binding motifs, ZnFs are now known to have additional activities such as the recognition of RNA and other proteins. Here we discuss recent advances in our understanding of ZnFs as specific modules for protein recognition. Structural studies of ZnF complexes reveal considerable diversity in terms of protein partners, binding modes and affinities, and highlight the often underestimated versatility of ZnF structure and function. An appreciation of the structural features of ZnF-protein interactions will contribute to our ability to engineer and to use ZnFs with tailored protein-binding properties.

Homeodomain-only protein (HOP) is an 8-kDa transcriptional corepressor that is essential for the normal development of the mammalian heart. Previous studies have shown that HOP, which consists entirely of a putative homeodomain, acts downstream of Nkx2.5 and associates with the serum response factor (SRF), repressing transcription from SRF-responsive genes. HOP is also able to recruit histone deacetylase (HDAC) activity, consistent with its ability to repress transcription. Unlike other classic homeodomain proteins, HOP does not appear to interact with DNA, although it has been unclear if this is because of an overall divergent structure or because of specific amino acid differences between HOP and other homeodomains. To work toward an understanding of HOP function, we have determined the 3D structure of full-length HOP and used a range of biochemical assays to define the parts of the protein that are functionally important for its repression activity. We show that HOP forms a classical homeodomain fold but that it cannot recognize double stranded DNA, a result that emphasizes the importance of caution in predicting protein function from sequence homology alone. We also demonstrate that two distinct regions on the surface of HOP are required for its ability to repress an SRF-driven reporter gene, and it is likely that these motifs direct interactions between HOP and partner proteins such as SRF- and HDAC-containing complexes. Our results demonstrate that the homeodomain fold has been co-opted during evolution for functions other than sequence-specific DNA binding and suggest that HOP functions as an adaptor protein to mediate transcriptional repression.

GATA-1 and friend of GATA (FOG) are zinc-finger transcription factors that physically interact to play essential roles in erythroid and megakaryocytic development. Several naturally occurring mutations in the GATA-1 gene that alter the FOG-binding domain have been reported. The mutations are associated with familial anemias and thrombocytopenias of differing severity. To elucidate the molecular basis for the GATA-1/FOG interaction, we have determined the three-dimensional structure of a complex comprising the interaction domains of these proteins. The structure reveals how zinc fingers can act as protein recognition motifs. Details of the architecture of the contact domains and their physical properties provide a molecular explanation for how the GATA-1 mutations contribute to distinct but related genetic diseases.