On the covalent nature of lysine polyphosphorylation.

Post-translational modifications of proteins (PTMs) introduce an extra layer of complexity to cellular regulation. Although phosphorylation of serine, threonine, and tyrosine residues is well-known as PTMs, lysine is, in fact, the most heavily modified amino acid, with over 30 types of PTMs on lysine having been characterized. One of the most recently discovered PTMs on lysine residues is polyphosphorylation, which sees linear chains of inorganic polyphosphates (polyP) attached to lysine residues. The labile nature of phosphoramidate bonds raises the question of whether this modification is covalent in nature. Here, we used buffers with very high ionic strength, which would disrupt any non-covalent interactions, and confirmed that lysine polyphosphorylation occurs covalently on proteins containing PASK domains (polyacidic, serine-, and lysine-rich), such as the budding yeast protein nuclear signal recognition 1 (Nsr1) and the mammalian protein nucleolin. This Matters Arising Response paper addresses the Neville et al. (2024) Matters Arising paper, published concurrently in Molecular Cell.

UPF1 helicase orchestrates mutually exclusive interactions with the SMG6 endonuclease and UPF2.

Nonsense-mediated mRNA decay (NMD) is a conserved co-translational mRNA surveillance and turnover pathway across eukaryotes. NMD has a central role in degrading defective mRNAs and also regulates the stability of a significant portion of the transcriptome. The pathway is organized around UPF1, an RNA helicase that can interact with several NMD-specific factors. In human cells, degradation of the targeted mRNAs begins with a cleavage event that requires the recruitment of the SMG6 endonuclease to UPF1. Previous studies have identified functional links between SMG6 and UPF1, but the underlying molecular mechanisms have remained elusive. Here, we used mass spectrometry, structural biology and biochemical approaches to identify and characterize a conserved short linear motif in SMG6 that interacts with the cysteine/histidine-rich (CH) domain of UPF1. Unexpectedly, we found that the UPF1-SMG6 interaction is precluded when the UPF1 CH domain is engaged with another NMD factor, UPF2. Based on cryo-EM data, we propose that the formation of distinct SMG6-containing and UPF2-containing NMD complexes may be dictated by different conformational states connected to the RNA-binding status of UPF1. Our findings rationalize a key event in metazoan NMD and advance our understanding of mechanisms regulating activity and guiding substrate recognition by the SMG6 endonuclease.

Structural basis of pri-let-7 recognition by human pseudouridine synthase TruB1.

Let-7 was one of the first microRNAs (miRNAs) to be discovered and its expression promotes differentiation during development and function as tumor suppressors in various cancers. The maturation process of let-7 miRNA is tightly regulated by multiple RNA-binding proteins. For example, LIN28 binds to the terminal loops of the precursors of let-7 family and block their processing into mature miRNAs. Trim25 promotes the uridylation-mediated degradation of pre-let-7 modified by LIN28/TUT4. Recently, human pseudouridine synthase TruB1 has been reported to facilitate let-7 maturation by directly binding to pri-let-7 and recruiting Drosha-DGCR8 microprocessor. Through biochemical assay and structural investigation, we show that human TruB1 binds specifically the terminal loop of pri-let-7a1 at nucleotides 31-41, which folds as a small stem-loop architecture. Although TruB1 recognizes the terminal loop of pri-let-7a1 in a way similar to how E. coli TruB interacts with tRNA, a conserved KRKK motif in human and other higher eukaryotes adds an extra binding interface and strengthens the recognition of TruB1 for pri-let-7a1 through electrostatic interactions. These findings reveal the structural basis of TruB1-pri-let-7 interaction which may assists the elucidation of precise role of TruB1 in biogenesis of let-7.

Simultaneous enhancement in phosphorescence and its lifetime of PtOEP-peptide assembly triggered by protein interaction.

We fabricated hybrid nanoparticles consisting of organic semiconducting material with peptide sequence to reflect the target protein interaction. A phosphorescent OLED material, platinum octaethylporphyrin (PtOEP) was self-assembled by reprecipitation with the A17 peptide (YCAYYSPRHKTTF) selected as a probe ligand in order to recognize heat shock protein 70 (HSP70). The phosphorescence intensity of the PtOEP-A17 assembly was enhanced by 125 % after treatment with HSP70. The specificity of the protein interaction was confirmed in both solution and solid states of the PtOEP-A17 assembly against to BSA and nucleolin. We figured out that the phosphorescence lifetime of PtOEP-A17 assembly after exposed to HSP70 increased significantly to 153 ns from initial 115 ns. These simultaneous enhancements in phosphorescence and lifetime triggered by the specific protein interaction would open new applications of PtOEP, a representative material of light-emitting device fields.

Selection of bifunctional RNAs with specificity for arginine and lipid membranes.

The molecular mechanisms of selective RNA loading into exosomes and other extracellular vesicles are not yet completely understood. In order to show that a pool of RNA sequences binds both the amino acid arginine and lipid membranes, we constructed a bifunctional RNA 10Arg aptamer specific for arginine and lipid vesicles. The preference of RNA 10Arg for lipid rafts was visualized and confirmed using FRET microscopy in neuroblastoma cells. The selection-amplification (SELEX) method using a doped (with the other three nucleotides) pool of RNA 10Arg sequences yielded several RNA 10Arg(D) sequences, and the affinities of these RNAs both to arginine and liposomes are improved in comparison to pre-doped RNA. Generation of these bispecific aptamers supports the hypothesis that an RNA molecule can bind both to RNA-binding proteins (RBPs) through arginine within the RBP-binding site and to membrane lipid rafts, thus facilitating RNA loading into exosomes and other extracellular vesicles.

Molecular basis of A. thaliana KEOPS complex in biosynthesizing tRNA t6A.

In archaea and eukaryotes, the evolutionarily conserved KEOPS is composed of four core subunits-Kae1, Bud32, Cgi121 and Pcc1, and a fifth Gon7/Pcc2 that is found in fungi and metazoa. KEOPS cooperates with Sua5/YRDC to catalyze the biosynthesis of tRNA N6-threonylcarbamoyladenosine (t6A), an essential modification needed for fitness of cellular organisms. Biochemical and structural characterizations of KEOPSs from archaea, yeast and humans have determined a t6A-catalytic role for Kae1 and auxiliary roles for other subunits. However, the precise molecular workings of KEOPSs still remain poorly understood. Here, we investigated the biochemical functions of A. thaliana KEOPS and determined a cryo-EM structure of A. thaliana KEOPS dimer. We show that A. thaliana KEOPS is composed of KAE1, BUD32, CGI121 and PCC1, which adopts a conserved overall arrangement. PCC1 dimerization leads to a KEOPS dimer that is needed for an active t6A-catalytic KEOPS-tRNA assembly. BUD32 participates in direct binding of tRNA to KEOPS and modulates the t6A-catalytic activity of KEOPS via its C-terminal tail and ATP to ADP hydrolysis. CGI121 promotes the binding of tRNA to KEOPS and potentiates the t6A-catalytic activity of KEOPS. These data and findings provide insights into mechanistic understanding of KEOPS machineries.

Ewing Sarcoma Related protein 1 recognizes R-loops by binding DNA forks.

EWSR1 (Ewing Sarcoma Related protein 1) is an RNA binding protein that is ubiquitously expressed across cell lines and involved in multiple parts of RNA processing, such as transcription, splicing, and mRNA transport. EWSR1 has also been implicated in cellular mechanisms to control formation of R-loops, a three-stranded nucleic acid structure consisting of a DNA:RNA hybrid and a displaced single-stranded DNA strand. Unscheduled R-loops result in genomic and transcription stress. Loss of function of EWSR1 functions commonly found in Ewing Sarcoma correlates with high abundance of R-loops. In this study, we investigated the mechanism for EWSR1 to recognize an R-loop structure specifically. Using electrophoretic mobility shift assays (EMSA), we detected the high affinity binding of EWSR1 to substrates representing components found in R-loops. EWSR1 specificity could be isolated to the DNA fork region, which transitions between double- and single-stranded DNA. Our data suggests that the Zinc-finger domain (ZnF) with flanking arginine and glycine rich (RGG) domains provide high affinity binding, while the RNA recognition motif (RRM) with its RGG domains offer improved specificity. This model offers a rational for EWSR1 specificity to encompass a wide range in contexts due to the DNA forks always found with R-loops.

Pliability in the m6A-Binding Region Extends Druggability of YTH Domains.

Epitranscriptomic mRNA modifications affect gene expression, with their altered balance detected in various cancers. YTHDF proteins contain the YTH reader domain recognizing the m6A mark on mRNA and represent valuable drug targets. Crystallographic structures have been determined for all three family members; however, discrepancies are present in the organization of the m6A-binding pocket. Here, we present new crystallographic structures of the YTH domain of YTHDF1, accompanied by computational studies, showing that this domain can exist in different stable conformations separated by a significant energetic barrier. During the transition, additional conformations are explored, with peculiar druggable pockets appearing and offering new opportunities for the design of YTH-interfering small molecules.

Development of stapled NONO-associated peptides reveals unexpected cell permeability and nuclear localisation.

The non-POU domain-containing octamer-binding protein (NONO) is a nucleic acid-binding protein with diverse functions that has been identified as a potential cancer target in cell biology studies. Little is known about structural motifs that mediate binding to NONO apart from its ability to form homodimers, as well as heterodimers and oligomers with related homologues. We report a stapling approach to macrocyclise helical peptides derived from the insulin-like growth factor binding protein (IGFBP-3) that NONO interacts with, and also from the dimerisation domain of NONO itself. Using a range of chemistries including Pd-catalysed cross-coupling, cysteine arylation and cysteine alkylation, we successfully improved the helicity and observed modest peptide binding to the NONO dimer, although binding could not be saturated at micromolar concentrations. Unexpectedly, we observed cell permeability and preferential nuclear localisation of various dye-labelled peptides in live confocal microscopy, indicating the potential for developing peptide-based tools to study NONO in a cellular context.

Assessing the Binding Mode of a Splicing Modulator Stimulating Pre-mRNA Binding to the Plastic U2AF2 Splicing Factor.

RNA recognition motifs (RRMs) play a pivotal role in RNA metabolism and the regulation of gene expression. Owing to their plasticity and fuzziness, targeting RRM/RNA interfaces with small molecules is a daunting challenge for drug discovery campaigns. The U2AF2 splicing factor, which recognizes the polypyrimidine (polyPy) sequence of premature messenger (pre-m)RNA, exhibits a dynamic architecture consisting of two RRMs joined by a disordered linker. An inhibitor, NSC-194308, was shown to enhance the binding of pre-mRNA to U2AF2, selectively triggering cell death in leukemia cell lines containing spliceosome mutations. The NSC-194308 binding mode remains elusive; yet, unraveling its knowledge may offer intriguing insights for effectively targeting U2AF2 and other flexible protein/protein/RNA interfaces with small molecules. To infer plausible NSC-194308 binding poses to U2AF2, here, we applied and benchmarked the performance of static and dynamic docking approaches, elucidating the molecular basis of the NSC-194308-induced pre-mRNA stabilization on U2AF2. We demonstrate that introducing dynamic effects is mandatory to assess the binding mode of the inhibitors when they target plastic and modular architectures, such as those formed by interacting RRMs. The latter are widespread across RNA binding proteins; therefore, this mechanism may be broadly applicable to discover new therapeutics aimed at selectively modulating the RNA function by targeting protein/protein/RNA interfaces.

Helicases in R-loop Formation and Resolution.

With the development and wide usage of CRISPR technology, the presence of R-loop structures, which consist of an RNA-DNA hybrid and a displaced single-strand (ss) DNA, has become well accepted. R-loop structures have been implicated in a variety of circumstances and play critical roles in the metabolism of nucleic acid and relevant biological processes, including transcription, DNA repair, and telomere maintenance. Helicases are enzymes that use an ATP-driven motor force to unwind double-strand (ds) DNA, dsRNA, or RNA-DNA hybrids. Additionally, certain helicases have strand-annealing activity. Thus, helicases possess unique positions for R-loop biogenesis: they utilize their strand-annealing activity to promote the hybridization of RNA to DNA, leading to the formation of R-loops; conversely, they utilize their unwinding activity to separate RNA-DNA hybrids and resolve R-loops. Indeed, numerous helicases such as senataxin (SETX), Aquarius (AQR), WRN, BLM, RTEL1, PIF1, FANCM, ATRX (alpha-thalassemia/mental retardation, X-linked), CasDinG, and several DEAD/H-box proteins are reported to resolve R-loops; while other helicases, such as Cas3 and UPF1, are reported to stimulate R-loop formation. Moreover, helicases like DDX1, DDX17, and DHX9 have been identified in both R-loop formation and resolution. In this review, we will summarize the latest understandings regarding the roles of helicases in R-loop metabolism. Additionally, we will highlight challenges associated with drug discovery in the context of targeting these R-loop helicases.

Spirocyclic Chromenopyrazole Inhibitors Disrupting the Interaction between the RNA-Binding Protein LIN28 and Let-7.

Small-molecule inhibitors of the RNA-binding and regulating protein LIN28 have the potential to be developed as chemical probes for biological perturbation and as therapeutic candidates. Reported small molecules disrupting the interaction between LIN28 and let-7 miRNA suffer from moderate to weak inhibitory activity and flat structure-activity relationship, which hindered the development of next-generation LIN28 inhibitors that warrant further evaluations. We report herein the identification of new LIN28 inhibitors utilizing a spirocyclization strategy based on a chromenopyrazole scaffold. Representative compounds 2-5 showed potent in vitro inhibitory activity against LIN28-let-7 interaction and single-digit micromolar potency in inhibiting the proliferation of LIN28-expressing JAR cancer cells. The spirocyclic compound 5 incorporated a position that is amenable for functional group appendage and further structural modifications. The binding mode of compound 5 with the LIN28 cold shock domain was rationalized via a molecular docking analysis.

Remodeling oncogenic transcriptomes by small molecules targeting NONO.

Much of the human proteome is involved in mRNA homeostasis, but most RNA-binding proteins lack chemical probes. Here we identify electrophilic small molecules that rapidly and stereoselectively decrease the expression of transcripts encoding the androgen receptor and its splice variants in prostate cancer cells. We show by chemical proteomics that the compounds engage C145 of the RNA-binding protein NONO. Broader profiling revealed that covalent NONO ligands suppress an array of cancer-relevant genes and impair cancer cell proliferation. Surprisingly, these effects were not observed in cells genetically disrupted for NONO, which were instead resistant to NONO ligands. Reintroduction of wild-type NONO, but not a C145S mutant, restored ligand sensitivity in NONO-disrupted cells. The ligands promoted NONO accumulation in nuclear foci and stabilized NONO-RNA interactions, supporting a trapping mechanism that may prevent compensatory action of paralog proteins PSPC1 and SFPQ. These findings show that NONO can be co-opted by covalent small molecules to suppress protumorigenic transcriptional networks.

Iron-dependent post transcriptional control of mitochondrial aconitase expression.

Iron regulatory proteins (IRPs) control the translation of animal cell mRNAs encoding proteins with diverse roles. This includes the iron storage protein ferritin and the tricarboxylic cycle (TCA) enzyme mitochondrial aconitase (ACO2) through iron-dependent binding of IRP to the iron responsive element (IRE) in the 5' untranslated region (UTR). To further elucidate the mechanisms allowing IRPs to control translation of 5' IRE-containing mRNA differentially, we focused on Aco2 mRNA, which is weakly controlled versus the ferritins. Rat liver contains two classes of Aco2 mRNAs, with and without an IRE, due to alterations in the transcription start site. Structural analysis showed that the Aco2 IRE adopts the canonical IRE structure but lacks the dynamic internal loop/bulge five base pairs 5' of the CAGUG(U/C) terminal loop in the ferritin IREs. Unlike ferritin mRNAs, the Aco2 IRE lacks an extensive base-paired flanking region. Using a full-length Aco2 mRNA expression construct, iron controlled ACO2 expression in an IRE-dependent and IRE-independent manner, the latter of which was eliminated with the ACO23C3S mutant that cannot bind the FeS cluster. Iron regulation of ACO23C3S encoded by the full-length mRNA was completely IRE-dependent. Replacement of the Aco23C3S 5' UTR with the Fth1 IRE with base-paired flanking sequences substantially improved iron responsiveness, as did fusing of the Fth1 base-paired flanking sequences to the native IRE in the Aco3C3S construct. Our studies further define the mechanisms underlying the IRP-dependent translational regulatory hierarchy and reveal that Aco2 mRNA species lacking the IRE contribute to the expression of this TCA cycle enzyme.

Surface electrostatics dictate RNA-binding protein CAPRIN1 condensate concentration and hydrodynamic properties.

Biomolecular condensates concentrate proteins, nucleic acids, and small molecules and play an essential role in many biological processes. Their formation is tuned by a balance between energetically favorable and unfavorable contacts, with charge-charge interactions playing a central role in some systems. The positively charged intrinsically disordered carboxy-terminal region of the RNA-binding protein CAPRIN1 is one such example, phase separating upon addition of negatively charged ATP or high concentrations of sodium chloride (NaCl). Using solution NMR spectroscopy, we measured residue-specific near-surface electrostatic potentials (ϕENS) of CAPRIN1 along its NaCl-induced phase separation trajectory to compare with those obtained using ATP. In both cases, electrostatic shielding decreases ϕENS values, yet surface potentials of CAPRIN1 in the two condensates can be different, depending on the amount of NaCl or ATP added. Our results establish that even small differences in ϕENS can significantly affect the level of protein enrichment and the mechanical properties of the condensed phase, leading, potentially, to the regulation of biological processes.

Cold-Induced RNA-Binding Protein and RNA-Binding Motif Protein 3: Two RNA Molecular Chaperones Closely Related to Reproductive Development and Reproductive System Diseases.

Cold-induced RNA-binding protein (CIRP) and RNA-binding motif protein 3 (RBM3) have recently been reported to be involved in cold stress in mammals. These proteins are expressed at low levels in various normal cells, tissues, and organs but can be upregulated upon stimulation by multiple stressors. Studies have shown that CIRP and RBM3 are multifunctional RNA molecular chaperones with different biological functions in various physiological and pathophysiological processes, such as reproductive development, the inflammatory response, the immune response, nerve injury regulation, and tumorigenesis. This paper reviews recent studies on the structure, localization and correlation of CIRP and RBM3 with reproductive development and reproductive system diseases.

Mapping the per-residue surface electrostatic potential of CAPRIN1 along its phase-separation trajectory.

Electrostatic interactions and charge balance are important for the formation of biomolecular condensates involving proteins and nucleic acids. However, a detailed, atomistic picture of the charge distribution around proteins during the phase-separation process is lacking. Here, we use solution NMR spectroscopy to measure residue-specific near-surface electrostatic potentials (ϕENS) of the positively charged carboxyl-terminal intrinsically disordered 103 residues of CAPRIN1, an RNA-binding protein localized to membraneless organelles playing an important role in messenger RNA (mRNA) storage and translation. Measured ϕENS values have been mapped along the adenosine triphosphate (ATP)-induced phase-separation trajectory. In the absence of ATP, ϕENS values for the mixed state of CAPRIN1 are positive and large and progressively decrease as ATP is added. This is coupled to increasing interchain interactions, particularly between aromatic-rich and arginine-rich regions of the protein. Upon phase separation, CAPRIN1 molecules in the condensed phase are neutral (ϕENS [Formula: see text] 0 mV), with ∼five molecules of ATP associated with each CAPRIN1 chain. Increasing the ATP concentration further inverts the CAPRIN1 electrostatic potential, so that molecules become negatively charged, especially in aromatic-rich regions, leading to re-entrance into a mixed phase. Our results collectively show that a subtle balance between electrostatic repulsion and interchain attractive interactions regulates CAPRIN1 phase separation and provides insight into how nucleotides, such as ATP, can induce formation of and subsequently dissolve protein condensates.

ADAR1 prevents autoinflammation by suppressing spontaneous ZBP1 activation.

The RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) limits the accumulation of endogenous immunostimulatory double-stranded RNA (dsRNA)1. In humans, reduced ADAR1 activity causes the severe inflammatory disease Aicardi-Goutières syndrome (AGS)2. In mice, complete loss of ADAR1 activity is embryonically lethal3-6, and mutations similar to those found in patients with AGS cause autoinflammation7-12. Mechanistically, adenosine-to-inosine (A-to-I) base modification of endogenous dsRNA by ADAR1 prevents chronic overactivation of the dsRNA sensors MDA5 and PKR3,7-10,13,14. Here we show that ADAR1 also inhibits the spontaneous activation of the left-handed Z-nucleic acid sensor ZBP1. Activation of ZBP1 elicits caspase-8-dependent apoptosis and MLKL-mediated necroptosis of ADAR1-deficient cells. ZBP1 contributes to the embryonic lethality of Adar-knockout mice, and it drives early mortality and intestinal cell death in mice deficient in the expression of both ADAR and MAVS. The Z-nucleic-acid-binding Zα domain of ADAR1 is necessary to prevent ZBP1-mediated intestinal cell death and skin inflammation. The Zα domain of ADAR1 promotes A-to-I editing of endogenous Alu elements to prevent dsRNA formation through the pairing of inverted Alu repeats, which can otherwise induce ZBP1 activation. This shows that recognition of Alu duplex RNA by ZBP1 may contribute to the pathological features of AGS that result from the loss of ADAR1 function.

ADAR1 averts fatal type I interferon induction by ZBP1.

Mutations of the ADAR1 gene encoding an RNA deaminase cause severe diseases associated with chronic activation of type I interferon (IFN) responses, including Aicardi-Goutières syndrome and bilateral striatal necrosis1-3. The IFN-inducible p150 isoform of ADAR1 contains a Zα domain that recognizes RNA with an alternative left-handed double-helix structure, termed Z-RNA4,5. Hemizygous ADAR1 mutations in the Zα domain cause type I IFN-mediated pathologies in humans2,3 and mice6-8; however, it remains unclear how the interaction of ADAR1 with Z-RNA prevents IFN activation. Here we show that Z-DNA-binding protein 1 (ZBP1), the only other protein in mammals known to harbour Zα domains9, promotes type I IFN activation and fatal pathology in mice with impaired ADAR1 function. ZBP1 deficiency or mutation of its Zα domains reduced the expression of IFN-stimulated genes and largely prevented early postnatal lethality in mice with hemizygous expression of ADAR1 with mutated Zα domain (Adar1mZα/- mice). Adar1mZα/- mice showed upregulation and impaired editing of endogenous retroelement-derived complementary RNA reads, which represent a likely source of Z-RNAs activating ZBP1. Notably, ZBP1 promoted IFN activation and severe pathology in Adar1mZα/- mice in a manner independent of RIPK1, RIPK3, MLKL-mediated necroptosis and caspase-8-dependent apoptosis, suggesting a novel mechanism of action. Thus, ADAR1 prevents endogenous Z-RNA-dependent activation of pathogenic type I IFN responses by ZBP1, suggesting that ZBP1 could contribute to type I interferonopathies caused by ADAR1 mutations.

Theoretical studies on RNA recognition by Musashi 1 RNA-binding protein.

The Musashi (MSI) family of RNA-binding proteins, comprising the two homologs Musashi-1 (MSI1) and Musashi-2 (MSI2), typically regulates translation and is involved in cell proliferation and tumorigenesis. MSI proteins contain two ribonucleoprotein-like RNA-binding domains, RBD1 and RBD2, that bind single-stranded RNA motifs with a central UAG trinucleotide with high affinity and specificity. The finding that MSI also promotes the replication of Zika virus, a neurotropic Flavivirus, has triggered further investigations of the biochemical principles behind MSI-RNA interactions. However, a detailed molecular understanding of the specificity of MSI RBD1/2 interaction with RNA is still missing. Here, we performed computational studies of MSI1-RNA association complexes, investigating different RNA pentamer motifs using molecular dynamics simulations with binding free energy calculations based on the solvated interaction energy method. Simulations with Alphafold2 suggest that predicted MSI protein structures are highly similar to experimentally determined structures. The binding free energies show that two out of four RNA pentamers exhibit a considerably higher binding affinity to MSI1 RBD1 and RBD2, respectively. The obtained structural information on MSI1 RBD1 and RBD2 will be useful for a detailed functional and mechanistic understanding of this type of RNA-protein interactions.