Cryo-EM Structure of the Full-length hnRNPA1 Amyloid Fibril.

Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is a multifunctional RNA-binding protein that is associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis and multisystem proteinopathy. In this study, we have used cryo-electron microscopy to investigate the three-dimensional structure of amyloid fibrils from full-length hnRNPA1 protein. We find that the fibril core is formed by a 45-residue segment of the prion-like low-complexity domain of the protein, whereas the remaining parts of the protein (275 residues) form a fuzzy coat around the fibril core. The fibril consists of two fibril protein stacks that are arranged into a pseudo-21 screw symmetry. The ordered core harbors several of the positions that are known to be affected by disease-associated mutations, but does not encompass the most aggregation-prone segments of the protein. These data indicate that the structures of amyloid fibrils from full-length proteins may be more complex than anticipated by current theories on protein misfolding.

HNRNP A1 Promotes Lung Cancer Cell Proliferation by Modulating VRK1 Translation.

THeterogeneous nuclear ribonucleoprotein (HNRNP) A1 is the most abundant and ubiquitously expressed member of the HNRNP protein family. In recent years, it has become more evident that HNRNP A1 contributes to the development of neurodegenerative diseases. However, little is known about the underlying role of HNRNP A1 in cancer development. Here, we report that HNRNP A1 expression is significantly increased in lung cancer tissues and is negatively correlated with the overall survival of patients with lung cancer. Additionally, HNRNP A1 positively regulates vaccinia-related kinase 1 (VRK1) translation via binding directly to the 3' untranslated region (UTR) of VRK1 mRNA, thus increasing cyclin D1 (CCND1) expression by VRK1-mediated phosphorylation of the cAMP response element-binding protein (CREB). Furthermore, HNRNP A1 binding to the cis-acting region of the 3'UTR of VRK1 mRNA contributes to increased lung cancer cell proliferation. Thus, our study unveils a novel role of HNRNP A1 in lung carcinogenesis via post-transcriptional regulation of VRK1 expression and suggests its potential as a therapeutic target for patients with lung cancer.

Electrostatic modulation of hnRNPA1 low-complexity domain liquid-liquid phase separation and aggregation.

Membrane-less organelles and RNP granules are enriched in RNA and RNA-binding proteins containing disordered regions. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a key regulating protein in RNA metabolism, localizes to cytoplasmic RNP granules including stress granules. Dysfunctional nuclear-cytoplasmic transport and dynamic phase separation of hnRNPA1 leads to abnormal amyloid aggregation and neurodegeneration. The intrinsically disordered C-terminal domain (CTD) of hnRNPA1 mediates both dynamic liquid-liquid phase separation (LLPS) and aggregation. While cellular phase separation drives the formation of membrane-less organelles, aggregation within phase-separated compartments has been linked to neurodegenerative diseases. To understand some of the underlying mechanisms behind protein phase separation and LLPS-mediated aggregation, we studied LLPS of hnRNPA1 CTD in conditions that probe protein electrostatics, modulated specifically by varying pH conditions, and protein, salt and RNA concentrations. In the conditions investigated, we observed LLPS to be favored in acidic conditions, and by high protein, salt and RNA concentrations. We also observed that conditions that favor LLPS also enhance protein aggregation and fibrillation, which suggests an aggregation pathway that is LLPS-mediated. The results reported here also suggest that LLPS can play a direct role in facilitating protein aggregation, and that changes in cellular environment that affect protein electrostatics can contribute to the pathological aggregation exhibited in neurodegeneration.

Thermodynamic stability of hnRNP A1 low complexity domain revealed by high-pressure NMR.

We have investigated the pressure- and temperature-induced conformational changes associated with the low complexity domain of hnRNP A1, an RNA-binding protein able to phase separate in response to cellular stress. Solution NMR spectra of the hnRNP A1 low-complexity domain fused with protein-G B1 domain were collected from 1 to 2500 bar and from 268 to 290 K. While the GB1 domain shows the typical pressure-induced and cold temperature-induced unfolding expected for small globular domains, the low-complexity domain of hnRNP A1 exhibits unusual pressure and temperature dependences. We observed that the low-complexity domain is pressure sensitive, undergoing a major conformational transition within the prescribed pressure range. Remarkably, this transition has the inverse temperature dependence of a typical folding-unfolding transition. Our results suggest the presence of a low-lying extended and fully solvated state(s) of the low-complexity domain that may play a role in phase separation. This study highlights the exquisite sensitivity of solution NMR spectroscopy to observe subtle conformational changes and illustrates how pressure perturbation can be used to determine the properties of metastable conformational ensembles.

Interplay of folded domains and the disordered low-complexity domain in mediating hnRNPA1 phase separation.

Liquid-liquid phase separation underlies the membrane-less compartmentalization of cells. Intrinsically disordered low-complexity domains (LCDs) often mediate phase separation, but how their phase behavior is modulated by folded domains is incompletely understood. Here, we interrogate the interplay between folded and disordered domains of the RNA-binding protein hnRNPA1. The LCD of hnRNPA1 is sufficient for mediating phase separation in vitro. However, we show that the folded RRM domains and a folded solubility-tag modify the phase behavior, even in the absence of RNA. Notably, the presence of the folded domains reverses the salt dependence of the driving force for phase separation relative to the LCD alone. Small-angle X-ray scattering experiments and coarse-grained MD simulations show that the LCD interacts transiently with the RRMs and/or the solubility-tag in a salt-sensitive manner, providing a mechanistic explanation for the observed salt-dependent phase separation. These data point to two effects from the folded domains: (i) electrostatically-mediated interactions that compact hnRNPA1 and contribute to phase separation and (ii) increased solubility at higher ionic strengths mediated by the folded domains. The interplay between disordered and folded domains can modify the dependence of phase behavior on solution conditions and can obscure signatures of physicochemical interactions underlying phase separation.

The nuclear localization sequence mediates hnRNPA1 amyloid fibril formation revealed by cryoEM structure.

Human heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) serves as a key regulating protein in RNA metabolism. Malfunction of hnRNPA1 in nucleo-cytoplasmic transport or dynamic phase separation leads to abnormal amyloid aggregation and neurodegeneration. The low complexity (LC) domain of hnRNPA1 drives both dynamic phase separation and amyloid aggregation. Here, we use cryo-electron microscopy to determine the amyloid fibril structure formed by hnRNPA1 LC domain. Remarkably, the structure reveals that the nuclear localization sequence of hnRNPA1 (termed PY-NLS), which is initially known to mediate the nucleo-cytoplamic transport of hnRNPA1 through binding with karyopherin-ß2 (Kapß2), represents the major component of the fibril core. The residues that contribute to the binding of PY-NLS with Kapß2 also exert key molecular interactions to stabilize the fibril structure. Notably, hnRNPA1 mutations found in familial amyotrophic lateral sclerosis (ALS) and multisystem proteinopathoy (MSP) are all involved in the fibril core and contribute to fibril stability. Our work illuminates structural understandings of the pathological amyloid aggregation of hnRNPA1 and the amyloid disaggregase activity of Kapß2, and highlights the multiple roles of PY-NLS in hnRNPA1 homeostasis.

G-quadruplex-proximity protein labeling based on peroxidase activity.

Peroxidase-proximity protein labeling was performed using a hemin-parallel G-quadruplex (G4) complex. A tyrosine labeling reaction using an N-methyl luminol derivative was accelerated in close proximity to the hemin with enhanced peroxidase activity by binding to parallel G4. The TERRA-hemin complex activated the labeling of many RNA-binding proteins, including heterogeneous nuclear ribonucleoproteins, in a HeLa cell lysate.

Monoclonal antibody against H1N1 influenza virus hemagglutinin cross reacts with hnRNPA1 and hnRNPA2/B1.

Following influenza A vaccination, certain individuals exhibit adverse reactions in the nervous system, which causes a problem with the safety of the influenza A vaccine. However, to the best of our knowledge, the underlying mechanism of this is unknown. The present study revealed that a monoclonal antibody (H1­84mAb) against the H1N1 influenza virus hemagglutinin (HA) protein cross­reacted with an antigen from brain tissue. Total brain tissue protein was immunoprecipitated with this cross­reactive antibody, and mass spectrometry revealed that the bound antigens were heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and hnRNPA2/B1. Subsequently, the two proteins were expressed in bacteria and it was demonstrated that H1­84mAb bound to hnRNPA1 and hnRNPA2/B1. These two proteins were expressed in three segments and the cross­reactivity of H1­84mAb with the glycine (Gly)­rich domains of hnRNPA1 (195aa­320aa) and hnRNPA2/B1 (202aa­349aa) was determined using ELISA blocking experiments. It was concluded that the Gly­rich domains of these two proteins are heterophilic antigens that cross­react with influenza virus HA. The association between the heterophilic antigen Gly­rich domains and the safety of influenza A vaccines remains to be investigated.

Determination of Protein Phase Diagrams by Centrifugation.

Liquid-liquid phase separation (LLPS) underlies the formation of biomolecular condensates, i.e., membrane-less compartments in cells that carry out functions related to RNA metabolism, stress adaptation, transport, or signaling. Examples of such biomolecular condensates are the nucleolus, nuclear speckles, promyelocytic leukemia protein (PML) bodies and paraspeckles in the nucleus, and stress granules and P bodies in the cytoplasm. Other structures in cells that are not typically viewed as bona fide compartments also seem to be formed via LLPS as recently elucidated, including heterochromatin, super-enhancers, and membrane receptor clusters. Key protein and/or RNA components of these biomolecular condensates form a scaffold via LLPS. Other constituents incorporate into this scaffold as clients. To understand the sequence features and interactions that mediate biomolecular condensate formation in cells, it is useful to quantify phase separation of pure components in vitro. Microscopy and turbidity measurements can be used to determine the concentration of a protein above which it phase separates, the so-called saturation concentration. Here, we describe experiments for the determination of full coexistence lines of phase-separating proteins by centrifugation. Coexistence lines are reconstructed from coexisting light and dense phase concentrations of the protein, and we present them as so-called phase diagrams. Phase diagrams allow the quantitative comparison of phase separation for proteins and their mutants under different conditions. They are thus important for our nuanced understanding of the driving forces underlying liquid-liquid phase separation in vitro. Such results have direct applicability for understanding phase separation-driven compartmentalization of cells.

Walking Along a Protein Phase Diagram to Determine Coexistence Points by Static Light Scattering.

The physical process of liquid-liquid phase separation (LLPS), where the drive to minimize global free energy causes a solution to demix into dense and light phases, plays many important roles in biology. It is implicated in the formation of so-called "membraneless organelles" such as nucleoli, nuclear speckles, promyelocytic leukemia protein bodies, P bodies, and stress granules along with the formation of biomolecular condensates involved in transcription, signaling, and transport. Quantitative studies of LLPS in vivo are complicated by the out-of-equilibrium, multicomponent cellular environment. While in vitro experiments with purified biomolecules are inherently an oversimplification of the cellular milieu, they allow probing of the rich physical chemistry underlying phase separation. Critically, with the application of suitable models, the thermodynamics of equilibrium LLPS can inform on the nature of the intermolecular interactions that mediate it. These same interactions are likely to exist in out-of-equilibrium condensates within living cells. Phase diagrams map the coexistence points between dense and light phases and quantitatively describe LLPS by mapping the local minima of free energy versus biomolecule concentration. Here, we describe a light scattering method that allows one to measure coexistence points around a high-temperature critical region using sample volumes as low as 10 µl.

Structure specific recognition of telomeric repeats containing RNA by the RGG-box of hnRNPA1.

The telomere repeats containing RNA (TERRA) is transcribed from the C-rich strand of telomere DNA and comprises of UUAGGG nucleotides repeats in humans. The TERRA RNA repeats can exist in single stranded, RNA-DNA hybrid and G-quadruplex forms in the cell. Interaction of TERRA RNA with hnRNPA1 has been proposed to play critical roles in maintenance of telomere DNA. hnRNPA1 contains an N-terminal UP1 domain followed by an RGG-box containing C-terminal region. RGG-motifs are emerging as key protein motifs that recognize the higher order nucleic acid structures as well as are known to promote liquid-liquid phase separation of proteins. In this study, we have shown that the RGG-box of hnRNPA1 specifically recognizes the TERRA RNA G-quadruplexes that have loops in their topology, whereas it does not interact with the single-stranded RNA. Our results show that the N-terminal UP1 domain in the presence of the RGG-box destabilizes the loop containing TERRA RNA G-quadruplex efficiently compared to the RNA G-quadruplex that lacks loops, suggesting that unfolding of G-quadruplex structures by UP1 is structure dependent. Furthermore, we have compared the telomere DNA and TERRA RNA G-quadruplex binding by the RGG-box of hnRNPA1 and discussed its implications in telomere DNA maintenance.

Valence and patterning of aromatic residues determine the phase behavior of prion-like domains.

Prion-like domains (PLDs) can drive liquid-liquid phase separation (LLPS) in cells. Using an integrative biophysical approach that includes nuclear magnetic resonance spectroscopy, small-angle x-ray scattering, and multiscale simulations, we have uncovered sequence features that determine the overall phase behavior of PLDs. We show that the numbers (valence) of aromatic residues in PLDs determine the extent of temperature-dependent compaction of individual molecules in dilute solutions. The valence of aromatic residues also determines full binodals that quantify concentrations of PLDs within coexisting dilute and dense phases as a function of temperature. We also show that uniform patterning of aromatic residues is a sequence feature that promotes LLPS while inhibiting aggregation. Our findings lead to the development of a numerical stickers-and-spacers model that enables predictions of full binodals of PLDs from their sequences.

Computer-Aided Discovery of Small Molecules Targeting the RNA Splicing Activity of hnRNP A1 in Castration-Resistant Prostate Cancer.

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a versatile RNA-binding protein playing a critical role in alternative pre-mRNA splicing regulation in cancer. Emerging data have implicated hnRNP A1 as a central player in a splicing regulatory circuit involving its direct transcriptional control by c-Myc oncoprotein and the production of the constitutively active ligand-independent alternative splice variant of androgen receptor, AR-V7, which promotes castration-resistant prostate cancer (CRPC). As there is an urgent need for effective CRPC drugs, targeting hnRNP A1 could, therefore, serve a dual purpose of preventing AR-V7 generation as well as reducing c-Myc transcriptional output. Herein, we report compound VPC-80051 as the first small molecule inhibitor of hnRNP A1 splicing activity discovered to date by using a computer-aided drug discovery approach. The inhibitor was developed to target the RNA-binding domain (RBD) of hnRNP A1. Further experimental evaluation demonstrated that VPC-80051 interacts directly with hnRNP A1 RBD and reduces AR-V7 messenger levels in 22Rv1 CRPC cell line. This study lays the groundwork for future structure-based development of more potent and selective small molecule inhibitors of hnRNP A1⁻RNA interactions aimed at altering the production of cancer-specific alternative splice isoforms.

RGG-box in hnRNPA1 specifically recognizes the telomere G-quadruplex DNA and enhances the G-quadruplex unfolding ability of UP1 domain.

hnRNPA1 is a member of heteronuclear ribonucleoproteins that has been shown to promote telomere elongation apart from its roles in RNA transport and alternative splicing. It is a modular protein with an N-terminal domain called UP1 that consists of two RNA Recognition Motifs (RRM1 and RRM2 domains) and a C-terminal region that harbors functional motifs such as RGG-box, a prion-like domain, and a nuclear shuttling sequence. UP1 has been reported to bind and destabilize telomeric DNA G-quadruplexes and thereby participate in DNA telomere remodeling. An RGG-box motif that consists of four RGG repeats (containing arginine and glycine residues) is located C-terminal to the UP1 domain and constitutes an additional nucleic acid and protein-binding domain. However, the precise role of the RGG-box of hnRNPA1 in telomere DNA recognition and G-quadruplex DNA unfolding remains unexplored. Here, we show that the isolated RGG-box interacts specifically with the structured telomere G-quadruplex DNA but not with the single-stranded DNA. Further the interaction of the RGG-box with the G-quadruplex DNA is dependent on the loop nucleotides of the G-quadruplex. Finally, we show that the RGG-box enhances the G-quadruplex unfolding activity of the adjacent UP1 domain. We propose that UP1 and RGG-box act synergistically to achieve complete telomere G-quadruplex DNA unfolding.

Dynamic association of human mRNP proteins with mitochondrial tRNAs in the cytosol.

Cytoplasmic localization, stability, and translation of mRNAs are controlled by their dynamic association of numerous mRNA-binding (mRNP) proteins, including cold shock domain (CSD)-containing proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), and serine/arginine-rich (SR) proteins. Here, we demonstrate that the most abundant human mRNP protein, the CSD-containing Y-box-binding protein 1 (YBX1), the closely related YBX3 protein, and other mRNP proteins, such as SRSF1, SRSF2, SRSF3, hnRNP A1, and H, specifically and efficiently interact with overlapping sets of mitochondrial tRNAs (mt tRNAs). In vitro reconstitution and in vivo binding experiments show that YBX1 recognizes the D- and/or T-stem-loop regions of mt tRNAs through relying on the RNA-binding capacity of its CSD. Cell fractionation and in vivo RNA-protein cross-linking experiments demonstrate that YBX1 and YBX3 interact with mt tRNAs in the cytosol outside of mitochondria. Cell fractionation and fluorescence in situ hybridization experiments provide evidence that mitochondrial autophagy promotes the release of mt tRNAs from the mitochondria into the cytoplasm. Association of mRNP proteins with mt tRNAs is highly dynamic; it is rapidly increased upon transcription inhibition and decreased during apoptosis. Although the cytoplasmic function of mt tRNAs remains elusive, their dynamic interactions with key mRNA-binding proteins may influence cytoplasmic mRNA stability and/or translation.

Structural basis for terminal loop recognition and stimulation of pri-miRNA-18a processing by hnRNP A1.

Post-transcriptional mechanisms play a predominant role in the control of microRNA (miRNA) production. Recognition of the terminal loop of precursor miRNAs by RNA-binding proteins (RBPs) influences their processing; however, the mechanistic basis for how levels of individual or subsets of miRNAs are regulated is mostly unexplored. We previously showed that hnRNP A1, an RBP implicated in many aspects of RNA processing, acts as an auxiliary factor that promotes the Microprocessor-mediated processing of pri-mir-18a. Here, by using an integrative structural biology approach, we show that hnRNP A1 forms a 1:1 complex with pri-mir-18a where both RNA recognition motifs (RRMs) bind to cognate RNA sequence motifs in the terminal loop of pri-mir-18a. Terminal loop binding induces an allosteric destabilization of base-pairing in the pri-mir-18a stem that promotes its downstream processing. Our results highlight terminal loop RNA recognition by RBPs as a potential general principle of miRNA biogenesis and regulation.

HnRNPA1 Specifically Recognizes the Base of Nucleotide at the Loop of RNA G-Quadruplex.

Human telomere RNA performs various cellular functions, such as telomere length regulation, heterochromatin formation, and end protection. We recently demonstrated that the loops in the RNA G-quadruplex are important in the interaction of telomere RNA with heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1). Here, we report on a detailed analysis of hnRNPA1 binding to telomere RNA G-quadruplexes with a group of loop variants using an electrophoretic mobility shift assay (EMSA) and circular dichroism (CD) spectroscopy. We found that the hnRNPA1 binds to RNA G-quadruplexes with the 2'-O-methyl and DNA loops, but fails to bind with the abasic RNA and DNA loops. These results suggested that hnRNPA1 binds to the loop of the RNA G-quadruplex by recognizing the base of the loop's nucleotides. The observation provides the first evidence that the base of the loop's nucleotides is a key factor for hnRNPA1 specifically recognizing the RNA G-quadruplex.

Tandem hnRNP A1 RNA recognition motifs act in concert to repress the splicing of survival motor neuron exon 7.

HnRNP A1 regulates many alternative splicing events by the recognition of splicing silencer elements. Here, we provide the solution structures of its two RNA recognition motifs (RRMs) in complex with short RNA. In addition, we show by NMR that both RRMs of hnRNP A1 can bind simultaneously to a single bipartite motif of the human intronic splicing silencer ISS-N1, which controls survival of motor neuron exon 7 splicing. RRM2 binds to the upstream motif and RRM1 to the downstream motif. Combining the insights from the structure with in cell splicing assays we show that the architecture and organization of the two RRMs is essential to hnRNP A1 function. The disruption of the inter-RRM interaction or the loss of RNA binding capacity of either RRM impairs splicing repression by hnRNP A1. Furthermore, both binding sites within the ISS-N1 are important for splicing repression and their contributions are cumulative rather than synergistic.

Crystal structure of the human heterogeneous ribonucleoprotein A18 RNA-recognition motif.

The heterogeneous ribonucleoprotein A18 (hnRNP A18) is upregulated in hypoxic regions of various solid tumors and promotes tumor growth via the coordination of mRNA transcripts associated with pro-survival genes. Thus, hnRNP A18 represents an important therapeutic target in tumor cells. Presented here is the first X-ray crystal structure to be reported for the RNA-recognition motif of hnRNP A18. By comparing this structure with those of homologous RNA-binding proteins (i.e. hnRNP A1), three residues on one face of an antiparallel ß-sheet (Arg48, Phe50 and Phe52) and one residue in an unstructured loop (Arg41) were identified as likely to be involved in protein-nucleic acid interactions. This structure helps to serve as a foundation for biophysical studies of this RNA-binding protein and structure-based drug-design efforts for targeting hnRNP A18 in cancer, such as malignant melanoma, where hnRNP A18 levels are elevated and contribute to disease progression.

Rules of RNA specificity of hnRNP A1 revealed by global and quantitative analysis of its affinity distribution.

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a multipurpose RNA-binding protein (RBP) involved in normal and pathological RNA metabolism. Transcriptome-wide mapping and in vitro evolution identify consensus hnRNP A1 binding motifs; however, such data do not reveal how surrounding RNA sequence and structural context modulate affinity. We determined the affinity of hnRNP A1 for all possible sequence variants (n = 16,384) of the HIV exon splicing silencer 3 (ESS3) 7-nt apical loop. Analysis of the affinity distribution identifies the optimal motif 5'-YAG-3' and shows how its copy number, position in the loop, and loop structure modulate affinity. For a subset of ESS3 variants, we show that specificity is determined by association rate constants and that variants lacking the minimal sequence motif bind competitively with consensus RNA. Thus, the results reveal general rules of specificity of hnRNP A1 and provide a quantitative framework for understanding how it discriminates between alternative competing RNA ligands in vivo.