Selective RNA Binding Research Articles

The glycine-rich domain of GRP7 plays a crucial role in binding long RNAs and facilitating phase separation

Microscale thermophoresis (MST) is a well-established method to quantify protein-RNA interactions. In this study, we employed MST to analyze the RNA binding properties of glycine-rich RNA binding protein 7 (GRP7), which is known to have multiple biological functions related to its ability to bind different types of RNA. However, the exact mechanism of GRP7’s RNA binding is not fully understood. While the RNA-recognition motif of GRP7 is known to be involved in RNA binding, the glycine-rich region (known as arginine-glycine-glycine-domain or RGG-domain) also influences this interaction. To investigate to which extend the RGG-domain of GRP7 is involved in RNA binding, mutation studies on putative RNA interacting or modulating sites were performed. In addition to MST experiments, we examined liquid–liquid phase separation of GRP7 and its mutants, both with and without RNA. Furthermore, we systemically investigated factors that might affect RNA binding selectivity of GRP7 by testing RNAs of different sizes, structures, and modifications. Consequently, our study revealed that GRP7 exhibits a high affinity for a variety of RNAs, indicating a lack of pronounced selectivity. Moreover, we established that the RGG-domain plays a crucial role in binding longer RNAs and promoting phase separation.

The RNA-binding Selectivity of the RGG/RG Motifs of hnRNP U is Abolished by Elements Within the C-terminal Intrinsically Disordered Region

The abundant nuclear protein hnRNP U interacts with a broad array of RNAs along with DNA and protein to regulate nuclear chromatin architecture. The RNA-binding activity is achieved via a disordered ∼100 residue C-terminal RNA-binding domain (RBD) containing two distinct RGG/RG motifs. Although the RNA-binding capabilities of RGG/RG motifs have been widely reported, less is known about hnRNP U’s RNA-binding selectivity. Furthermore, while it is well established that hnRNP U binds numerous nuclear RNAs, it remains unknown whether it selectively recognizes sequence or structural motifs in target RNAs. To address this question, we performed equilibrium binding assays using fluorescence anisotropy (FA) and electrophoretic mobility shift assays (EMSAs) to quantitatively assess the ability of human hnRNP U RBD to interact with segments of cellular RNAs identified from eCLIP data. These RNAs often, but not exclusively, contain poly-uridine or 5′-AGGGAG sequence motifs. Detailed binding analysis of several target RNAs reveal that the hnRNP U RBD binds RNA in a promiscuous manner with high affinity for a broad range of structured RNAs, but with little preference for any distinct sequence motif. In contrast, the isolated RGG/RG of hnRNP U motif exhibits a strong preference for G-quadruplexes, similar to that observed for other RGG motif bearing peptides. These data reveal that the hnRNP U RBD attenuates the RNA binding selectivity of its core RGG motifs to achieve an extensive RNA interactome. We propose that a critical role of RGG/RG motifs in RNA biology is to alter binding affinity or selectivity of adjacent RNA-binding domains.

Cleaving Folded RNA by Multifunctional DNAzyme Nanomachines.

Both tight and specific binding of folded biological mRNA is required for gene silencing by oligonucleotide gene therapy agents. However, this is fundamentally impossible using the conventional oligonucleotide probes according to the affinity/specificity dilemma. This study addresses this problem for cleaving folded RNA by using multicomponent agents (dubbed 'DNA nanomachine' or DNM). DNMs bind RNA by four short RNA binding arms, which ensure tight and highly selective RNA binding. Along with the improved affinity, DNM maintain the high sequence selectivity of the conventional DNAzymes. DNM enabled up to 3-fold improvement in DNAzymes catalytic efficiency (kcat/Km) by facilitating both RNA substrate binding and product release steps of the catalytic cycle. This study demonstrates that multicomponent probes organized in sophisticated structures can help to achieve the balance between affinity and selectivity in recognizing folded RNA and thus creates a foundation for applying complex DNA nanostructures derived by DNA nanotechnology in gene therapy.

Translational enhancement of target endogenous mRNA in mammalian cells using programmable RNA-binding pentatricopeptide repeat proteins

Programmable protein scaffolds are invaluable in the development of genome engineering tools. The pentatricopeptide repeat (PPR) protein is an attractive platform for RNA manipulation because of its programmable RNA-binding selectivity, which is determined by the combination of amino acid species at three specific sites in the PPR motif. Translation is a key RNA regulatory step that determines the final gene expression level and is involved in various human diseases. In this study, designer PPR protein was used to develop a translational enhancement technique by fusion with the translation initiation factor eIF4G. The results showed that the PPR-eIF4G fusion protein could activate the translation of endogenous c-Myc and p53 mRNAs and control cell fate, indicating that PPR-based translational enhancement is a versatile technique applicable to various endogenous mRNAs in mammalian cells. In addition, the translational enhancement was dependent on both the target position and presence of eIF4G, suggesting the presence of an unknown translation activation mechanism.

LIPRNAseq: a method to discover lipid interacting RNAs by sequencing.

Current biological research extensively describes the interactions of molecules such as RNA with other nucleic acids or proteins. However, the relatively recent discovery of nuclear phospholipids playing biologically relevant processes outside membranes, as well as, RNA-lipid interactions shows the need for new methods to explore the identity of these RNAs. In this study, we describe the method for LIPID-RNA isolation followed by sequencing and analysis of the RNA that has the ability to interact with the selected lipids. Here we utilized specific phospholipid coated beads for selective RNA binding. We tested RNA from organisms belonging to different realms (human, plant, and yeast), and tested their ability to bind a specific lipid. The results show several RNAs differentially enriched in the pull-down of phosphatidyl Inositol 4,5 bisphosphate coated beads. This method is helpful to screen lipid-binding RNA, which may have relevant biological functions. The method can be used with different lipids and comparison of pull-downs and can narrow the selection of RNAs that interact with a particular lipid for further studies.

The RIG-I receptor adopts two different conformations for distinguishing host from viral RNA ligands

RIG-I is an essential innate immune receptor for detecting and responding to infection by RNA viruses. RIG-I specifically recognizes the unique molecular features of viral RNA molecules and selectively distinguishes them from closely related RNAs abundant in host cells. The physical basis for this exquisite selectivity is revealed through a series of high-resolution cryo-EM structures of RIG-I in complex with host and viral RNA ligands. These studies demonstrate that RIG-I actively samples double-stranded RNAs in the cytoplasm and distinguishes them by adopting two different types of protein folds. Upon binding viral RNA, RIG-I adopts a high-affinity conformation that is conducive to signaling, while host RNA induces an autoinhibited conformation that stimulates RNA release. By coupling protein folding with RNA binding selectivity, RIG-I distinguishes RNA molecules that differ by as little as one phosphate group, thereby explaining the molecular basis for selective antiviral sensing and the induction of autoimmunity upon RIG-I dysregulation.

Structural basis for Gemin5 decamer-mediated mRNA binding

Gemin5 in the Survival Motor Neuron (SMN) complex serves as the RNA-binding protein to deliver small nuclear RNAs (snRNAs) to the small nuclear ribonucleoprotein Sm complex via its N-terminal WD40 domain. Additionally, the C-terminal region plays an important role in regulating RNA translation by directly binding to viral RNAs and cellular mRNAs. Here, we present the three-dimensional structure of the Gemin5 C-terminal region, which adopts a homodecamer architecture comprised of a dimer of pentamers. By structural analysis, mutagenesis, and RNA-binding assays, we find that the intact pentamer/decamer is critical for the Gemin5 C-terminal region to bind cognate RNA ligands and to regulate mRNA translation. The Gemin5 high-order architecture is assembled via pentamerization, allowing binding to RNA ligands in a coordinated manner. We propose a model depicting the regulatory role of Gemin5 in selective RNA binding and translation. Therefore, our work provides insights into the SMN complex-independent function of Gemin5.

Chemical Synthesis of Modified Oligonucleotides Containing 5'-Amino-5'-Deoxy-5'-Hydroxymethylthymidine Residues.

Introduction of cationic modifications into an oligonucleotide can increase its nuclease resistance and duplex- or triplex-forming abilities. In a recent study, we found that the nuclease resistance and RNA binding selectivity of an oligonucleotide containing a 5'-(R)-amino-5'-deoxy-5'-(R)-hydroxymethylthymidine residue were greater than those of the unmodified oligonucleotide. In this article, we describe the synthesis of 5'-amino-5'-deoxy-5'-hydroxymethylthymidine via dihydroxylation of the 5'-alkene derivative using either of two commercial AD (asymmetric dehydroxylation) mixes or via epoxidation and ring opening. We also provide detailed protocols for the syntheses of oligonucleotides containing 5'-amino-5'-deoxy-5'-hydroxymethylthymidine residues. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 5'-amino-5'-deoxy-5'-hydroxymethylthymidine phosphoramidites 9a and 9b Basic Protocol 2: Synthesis of oligonucleotides 1 and 2 containing 5'-amino-5'-deoxy-5'-hydoxymethylthymidine residues (R T and S T).

Photoswitches for controllable RNA binding: a future approach in the RNA-targeting therapy.

RNA is an emerging drug target that opens new perspectives in the treatment of viral and bacterial infections, cancer and a range of so far incurable genetic diseases. Among the various strategies towards the design and development of selective and efficient ligands for targeting and detection of therapeutically relevant RNA, photoswitchable RNA binders represent a very promising approach due to the possibility to control the ligand-RNA and protein-RNA interactions by light with high spatiotemporal resolution. However, the field of photoswitchable RNA binders still remains underexplored due to challenging design of lead structures that should combine high RNA binding selectivity with efficient photochemical performance. The aim of this highlight article is to describe the development of photoswitchable noncovalent RNA binders and to outline the current situation and perspectives of this emerging interdisciplinary field.

Two-step mechanism for selective incorporation of lncRNA into a chromatin modifier

The MLE DExH helicase and the roX lncRNAs are essential components of the chromatin modifying Dosage Compensation Complex (DCC) in Drosophila. To explore the mechanism of ribonucleoprotein complex assembly, we developed vitRIP, an unbiased, transcriptome-wide in vitro assay that reveals RNA binding specificity. We found that MLE has intrinsic specificity for U-/A-rich sequences and tandem stem-loop structures and binds many RNAs beyond roX in vitro. The selectivity of the helicase for physiological substrates is further enhanced by the core DCC. Unwinding of roX2 by MLE induces a highly selective RNA binding surface in the unstructured C-terminus of the MSL2 subunit and triggers-specific association of MLE and roX2 with the core DCC. The exquisite selectivity of roX2 incorporation into the DCC thus originates from intimate cooperation between the helicase and the core DCC involving two distinct RNA selection principles and their mutual refinement.

The minimal meningococcal ProQ protein has an intrinsic capacity for structure-based global RNA recognition

FinO-domain proteins are a widespread family of bacterial RNA-binding proteins with regulatory functions. Their target spectrum ranges from a single RNA pair, in the case of plasmid-encoded FinO, to global RNA regulons, as with enterobacterial ProQ. To assess whether the FinO domain itself is intrinsically selective or promiscuous, we determine in vivo targets of Neisseria meningitidis, which consists of solely a FinO domain. UV-CLIP-seq identifies associations with 16 small non-coding sRNAs and 166 mRNAs. Meningococcal ProQ predominantly binds to highly structured regions and generally acts to stabilize its RNA targets. Loss of ProQ alters transcript levels of >250 genes, demonstrating that this minimal ProQ protein impacts gene expression globally. Phenotypic analyses indicate that ProQ promotes oxidative stress resistance and DNA damage repair. We conclude that FinO domain proteins recognize some abundant type of RNA shape and evolve RNA binding selectivity through acquisition of additional regions that constrain target recognition.

Deciphering the Nucleotide and RNA Binding Selectivity of the Mayaro Virus Macro Domain

Mayaro virus (MAYV) is a member of Togaviridae family, which also includes Chikungunya virus as a notorious member. MAYV recently emerged in urban areas of the Americas, and this emergence emphasized the current paucity of knowledge about its replication cycle. The macro domain (MD) of MAYV belongs to the N-terminal region of its non-structural protein 3, part of the replication complex. Here, we report the first structural and dynamical characterization of a previously unexplored Alphavirus MD investigated through high-resolution NMR spectroscopy, along with data on its ligand selectivity and binding properties. The structural analysis of MAYV MD reveals a typical “macro” (ββαββαβαβα) fold for this polypeptide, while NMR-driven interaction studies provide in-depth insights into MAYV MD–ligand adducts. NMR data in concert with thermodynamics and biochemical studies provide convincing experimental evidence for preferential binding of adenosine diphosphate ribose (ADP‐r) and adenine-rich RNAs to MAYV MD, thus shedding light on the structure–function relationship of a previously unexplored viral MD. The emerging differences with any other related MD are expected to enlighten distinct functions.

Microprocessor depends on hemin to recognize the apical loop of primary microRNA.

Microprocessor, which consists of a ribonuclease III DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) maturation by cleaving primary miRNA transcripts (pri-miRNAs). We recently demonstrated that the DGCR8 dimer recognizes the apical elements of pri-miRNAs, including the UGU motif, to accurately locate and orient Microprocessor on pri-miRNAs. However, the mechanism underlying the selective RNA binding remains unknown. In this study, we find that hemin, a ferric ion-containing porphyrin, enhances the specific interaction between the apical UGU motif and the DGCR8 dimer, allowing Microprocessor to achieve high efficiency and fidelity of pri-miRNA processing in vitro. Furthermore, by generating a DGCR8 mutant cell line and carrying out rescue experiments, we discover that hemin preferentially stimulates the expression of miRNAs possessing the UGU motif, thereby conferring differential regulation of miRNA maturation. Our findings reveal the molecular action mechanism of hemin in pri-miRNA processing and establish a novel function of hemin in inducing specific RNA-protein interaction.

An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription.

Potent and selective recognition and modulation of disease-relevant RNAs remain a daunting challenge. We previously examined the utility of the U1A N-terminal RNA recognition motif as a scaffold for tailoring new RNA hairpin recognition and showed that as few as one or two mutations can result in moderate affinity (low μM dissociation constant) for the human immunodeficiency virus (HIV) trans-activation response element (TAR) RNA, an RNA hairpin controlling transcription of the human immunodeficiency virus (HIV) genome. Here, we use yeast display and saturation mutagenesis of established RNA-binding regions in U1A to identify new synthetic proteins that potently and selectively bind TAR RNA. Our best candidate has truly altered, not simply broadened, RNA-binding selectivity; it binds TAR with subnanomolar affinity (apparent dissociation constant of ∼0.5 nM) but does not appreciably bind the original U1A RNA target (U1hpII). It specifically recognizes the TAR RNA hairpin in the context of the HIV-1 5'-untranslated region, inhibits the interaction between TAR RNA and an HIV trans-activator of transcription (Tat)-derived peptide, and suppresses Tat/TAR-dependent transcription. Proteins described in this work are among the tightest TAR RNA-binding reagents-small molecule, nucleic acid, or protein-reported to date and thus have potential utility as therapeutics and basic research tools. Moreover, our findings demonstrate how a naturally occurring RNA recognition motif can be dramatically resurfaced through mutation, leading to potent and selective recognition-and modulation-of disease-relevant RNA.

A new selective ‘turn-on’ small fluorescent cationic probe for recognition of RNA in cells

Fluorescent imaging probes have revolutionised cell biology by monitoring cellular objects. However, the lack of fluorescent probes with high selectivity for RNA has been a drawback. Thus, selective RNA binding for fluorescent sensors is essential. Here, we report the selective fluorescence enhancement upon addition of RNA. By exploiting a selective recognition of small tetra-cationic probe 1 for RNA, we also explain the possible binding mode for RNA. As a membrane-permeant fluorescence probe, 1 provides selective imaging of RNA not only in human neuroblastoma tumour SH-SY5Y cell line used for Parkinson's disease but also in the unicellular green alga cells. Further exploitation could open new opportunities in neurotoxin and cancer biology.

Human FMRP contains an integral tandem Agenet (Tudor) and KH motif in the amino terminal domain.

Fragile X syndrome, a common cause of intellectual disability and autism, is due to mutational silencing of the FMR1 gene leading to the absence of its gene product, fragile X mental retardation protein (FMRP). FMRP is a selective RNA binding protein owing to two central K-homology domains and a C-terminal arginine-glycine-glycine (RGG) box. However, several properties of the FMRP amino terminus are unresolved. It has been documented for over a decade that the amino terminus has the ability to bind RNA despite having no recognizable functional motifs. Moreover, the amino terminus has recently been shown to bind chromatin and influence the DNA damage response as well as function in the presynaptic space, modulating action potential duration. We report here the amino terminal crystal structures of wild-type FMRP, and a mutant (R138Q) that disrupts the amino terminus function, containing an integral tandem Agenet and discover a novel KH motif.

Myosin Va is required for the transport of fragile X mental retardation protein (FMRP) granules

Fragile X mental retardation protein (FMRP) is a selective RNA binding protein that functions as a translational inhibitor. It also plays a role in directing the transport of a subset of mRNAs to their site of translation and several recent reports have implicated microtubule motor proteins in the transport of FMRP-messenger ribonucleoprotein (mRNP) granules in neurons. Earlier work reported the association of the actin-based motor protein myosin Va with FMRP granules. Here, we follow up on this finding and confirm that myosin Va does in fact associate with FMRP and is required for its correct intracellular localisation. FMRP is concentrated in the perinuclear region of myosin Va-null mouse melanoma cells which contrasts starkly with the evenly distributed punctate pattern observed in wild-type cells. Similarly, overexpression of a dominant-negative mutant of myosin Va results in the accumulation of FMRP in large aggregate-like structures. FRAP experiments demonstrate that FMRP is largely immobile in the absence of myosin Va. Combining these data, we propose a model in which myosin Va and kinesin play key roles in the assembly and subsequent transport of FMRP granules along microtubules to the periphery of the cell. Myosin Va captures the complex onto peripheral actin structures and mediates the local delivery of the FMRP granule to the site of mRNA translation.

Cadmium coordination to the zinc binding domains of the non-classical zinc finger protein Tristetraprolin affects RNA binding selectivity

Tristetraprolin [(TTP), also known as NUP475 and TIS11] is a non-classical zinc finger protein that regulates inflammatory response via a protein-RNA interaction. Specifically, TTP recognizes AU-rich RNA sequences located on the 3′ untranslated region of messenger RNA associated with cytokines. Recently, TTP was shown to be upregulated when cells were exposed to cadmium. Other types of zinc finger proteins have been shown to bind Cd(II), thus the Cd(II) binding properties of TTP were pursued. Metal binding titrations using Co(II) as a spectroscopic probe for Cd(II) were performed. Cd(II) was found to coordinate to the two Cys3His structural zinc sites of TTP (TTP-2D) with an upper-limit dissociation constant, Kd, of 3.5±0.1nM. Upon reconstitution of TTP-2D with Cd(II), the protein recognized target RNA, UUUAUUUAUUU, with a dissociation constant, Kd, of 2.4±0.2nM. The Cd(II)TTP-2D/RNA binding event was more sensitive to base mutations than the Zn(II)TTP-2D/RNA binding event. A single base mutation within the UUUAUUUAUUU oligomer decreased the Cd(II)TTP-2D binding affinity 50-fold and a double mutation decreased the affinity 1000–2000 fold. In comparison, only 2-fold and 15–25 fold changes for Zn(II)TTP-2D binding to the identical RNA sequences are measured.

Probing Binding Preferences of DNA and RNA: Backbone Chirality of Thioacetamido-Linked Nucleic Acids and iso-Thioacetamido-Linked Nucleic Acids to Differentiate DNA versus RNA Selective Binding

Subtle differences in RNA and DNA duplex geometry could be sensed by the changed stereochemistry at 3'-amino function in the 5-atom thioacetamido linker of thioacetamido-linked nucleic acids and iso-thioacetamido-linked nucleic acids modified oligomers. In contrast to the preferred N-type sugar conformations for either 3'- ribo- or xylo amino nucleosides, predominant S-type sugar conformations were found in the dimers. Although the CD spectral differences for the dimer blocks were found to be identical for those found in phosphodiester linked ribo/xylo dimers, the 5-atom thioactamido linker could reverse the RNA binding selectivity to DNA binding selectivity by the change in configuration at the 3'-amino-substituted sugar.

RNA extraction method for the PCR detection of hepatitis A virus in shellfish

Viruses are the leading cause of foodborne illness associated with the consumption of raw or slightly-cooked contaminated shellfish. This study evaluated the E.Z.N.A. Mollusc RNA extraction and purification kit for the detection of HAV in shellfish. The E.Z.N.A. method, based on the cationic detergent, cetyltrimethyl ammonium bromide, in conjunction with a selective RNA binding silica matrix, efficiently isolated viral RNA with a detection limit of 1 TCID 50/ml by hemi-nested PCR. This method proved to be faster and less expensive than PEG precipitation-based procedures. It is also technically undemanding, requiring no extensive processing steps or excess manipulation, minimizing RNA degradation and ensuring the absence of PCR inhibitors. The E.Z.N.A. method applied to HAV screening of shellfish samples from the Apulian region, revealed a high level of contamination. These results confirm that conventional faecal indicators are unreliable for demonstrating the presence or absence of viruses.