A comprehensive understanding of hnRNP A1 role in cancer: new perspectives on binding with noncoding RNA.

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is the most abundant and ubiquitously expressed member of the heterogeneous nuclear ribonucleoproteins family (hnRNPs). hnRNP A1 is an RNA-binding protein associated with complexes active in diverse biological processes such as RNA splicing, transactivation of gene expression, and modulation of protein translation. It is overexpressed in several cancers, where it actively promotes the expression and translation of several key proteins and regulators associated with tumorigenesis and cancer progression. Interesting recent studies have focused on the RNA-binding property of hnRNP A1 and revealed previously under-explored functions of hnRNP A1 in the processing of miRNAs, and loading non-coding RNAs into exosomes. Here, we will report the recent advancements in our knowledge of the role of hnRNP A1 in the biological processes underlying cancer proliferation and growth, with a particular focus on metabolic reprogramming.

Emerging roles of hnRNP A2B1 in cancer and inflammation.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a group of RNA-binding proteins with important roles in multiple aspects of nucleic acid metabolism, including the packaging of nascent transcripts, alternative splicing, transactivation of gene expression, and regulation of protein translation. As a core component of the hnRNP complex in mammalian cells, heterogeneous nuclear ribonucleoprotein A2B1 (hnRNP A2B1) participates in and coordinates various molecular events. Given its regulatory role in inflammation and cancer progression, hnRNP A2B1 has become a novel player in immune response, inflammation, and cancer development. Concomitant with these new roles, a surprising number of mechanisms deemed to regulate hnRNP A2B1 functions have been identified, including post-translational modifications, changes in subcellular localization, direct interactions with multiple DNAs, RNAs, and proteins or the formation of complexes with them, which have gradually made hnRNP A2B1 a molecular target for multiple drugs. In light of the rising interest in the intersection between cancer and inflammation, this review will focus on recent knowledge of the biological roles of hnRNP A2B1 in cancer, immune response, and inflammation.

Transthyretin affects the proliferation and migration of human retinal microvascular endothelial cells in hyperglycemia via hnRNPA2B1.

Transthyretin (TTR) has been proved to repress neovascularization in diabetic retinopathy environment by regulating the molecules in and downstream of the STAT-4/miR-223-3p/FBXW7 signal pathway; however, the details of its direct targets are still not well understood. The interaction between TTR and a target in nucleus of human retinal microvascular endothelial cells (hRECs), heterogeneous nuclear ribonucleoprotein (hnRNP) A2B1, was screened by immunoprecipitation (IP) and mass spectrum (MS), and it was further confirmed by co-immunoprecipitation (co-IP). Regarding ZDOCK analysis using Discovery Studio, the interface and potential binding sites between TTR and hnRNPA2B1 were simulated; mutants were designed in these regions and five soluble ones were recombinantly expressed and prepared; the interaction between TTR and hnRNPA2B1 were disrupted by several mutated residues. In addition, for several mutated TTRs, the inhibition activities against the proliferation, migration and tube formation of hRECs were absent in vitro. Following the disruption of TTR-hnRNPA2B1, the molecules in and downstream of STAT-4/miR-223-3p/FBXW7 signal pathway, including STAT-4, miR-223-3p, FBXW7 p-Akt and Notch1 could not be regulated by TTR mutants; therefore, a TTR-hnRNPA2B1/STAT-4/miR-223-3p/FBXW7 was proposed. In conclusion, this work suggested that TTR should play a physiological role in diabetic environment by the direct binding with hnRNPA2B1, and it provided a theoretical basis for clinical diagnosis, therapy and further application.

SARS-CoV-2 nucleocapsid protein phase-separates with RNA and with human hnRNPs.

Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and assemble within viral factories, dynamic compartments formed within the host cells associated with human stress granules. Here, we test the possibility that the multivalent RNA-binding nucleocapsid protein (N) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) condenses with RNA via liquid-liquid phase separation (LLPS) and that N protein can be recruited in phase-separated forms of human RNA-binding proteins associated with SG formation. Robust LLPS with RNA requires two intrinsically disordered regions (IDRs), the N-terminal IDR and central-linker IDR, as well as the folded C-terminal oligomerization domain, while the folded N-terminal domain and the C-terminal IDR are not required. N protein phase separation is induced by addition of non-specific RNA. In addition, N partitions in vitro into phase-separated forms of full-length human hnRNPs (TDP-43, FUS, hnRNPA2) and their low-complexity domains (LCs). These results provide a potential mechanism for the role of N in SARS-CoV-2 viral genome packing and in host-protein co-opting necessary for viral replication and infectivity.

The SH3 domain of Fyn kinase interacts with and induces liquid-liquid phase separation of the low-complexity domain of hnRNPA2.

Liquid-liquid phase separation of proteins and nucleic acids into membraneless organelles (MLOs) spatially organizes cellular components and reactions. The RNA-binding protein heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2) carries mRNA targets in MLOs called transport granules in neurons and oligodendrocytes. At sites of local translation, hnRNPA2 is phosphorylated by the tyrosine protein kinase Fyn, releasing the mRNA for translation. Fyn recognizes targets through its SH3 domain (Fyn-SH3). However, hnRNPA2 lacks canonical SH3-binding sequences, raising the question of how Fyn-SH3 binds hnRNPA2 in phase-separated transport granules. Here, we characterize the structural details of the interaction of the hnRNPA2 low-complexity domain (LC) with Fyn-SH3 and the effect of Fyn-SH3 on hnRNPA2 phase separation. We combined in vitro microscopy and solution NMR spectroscopy to evaluate assembly of hnRNPA2 and Fyn-SH3 into in vitro phase-separated granules and probe the structural details of their interaction. We observed that Fyn-SH3 induces hnRNPA2 LC phase separation and that Fyn-SH3 is incorporated into in vitro hnRNPA2 LC granules. Moreover, we identified hnRNPA2 LC interaction sites on the surface of Fyn-SH3. Our data offer a structural view of how hnRNPA2 LC may interact with Fyn. To our knowledge, our study provides the first example of a single globular domain inducing phase separation of a disordered MLO scaffold protein.

Lysines in the RNA Polymerase II C-Terminal Domain Contribute to TAF15 Fibril Recruitment.

Many cancer-causing chromosomal translocations result in transactivating protein products encoding FET family (FUS, EWSR1, TAF15) low-complexity (LC) domains fused to a DNA binding domain from one of several transcription factors. Recent work demonstrates that higher-order assemblies of FET LC domains bind the carboxy-terminal domain of the large subunit of RNA polymerase II (RNA pol II CTD), suggesting FET oncoproteins may mediate aberrant transcriptional activation by recruiting RNA polymerase II to promoters of target genes. Here we use nuclear magnetic resonance (NMR) spectroscopy and hydrogel fluorescence microscopy localization and fluorescence recovery after photobleaching to visualize atomic details of a model of this process, interactions of RNA pol II CTD with high-molecular weight TAF15 LC assemblies. We report NMR resonance assignments of the intact degenerate repeat half of human RNA pol II CTD alone and verify its predominant intrinsic disorder by molecular simulation. By measuring NMR spin relaxation and dark-state exchange saturation transfer, we characterize the interaction of RNA pol II CTD with amyloid-like hydrogel fibrils of TAF15 and hnRNP A2 LC domains and observe that heptads far from the acidic C-terminal tail of RNA pol II CTD bind TAF15 fibrils most avidly. Mutation of CTD lysines in heptad position 7 to consensus serines reduced the overall level of TAF15 fibril binding, suggesting that electrostatic interactions contribute to complex formation. Conversely, mutations of position 7 asparagine residues and truncation of the acidic tail had little effect. Thus, weak, multivalent interactions between TAF15 fibrils and heptads throughout RNA pol II CTD collectively mediate complex formation.

Nm23-H1-stabilized hnRNPA2/B1 promotes internal ribosomal entry site (IRES)-mediated translation of Sp1 in the lung cancer progression.

Our recent studies have indicated that specificity protein-1 (Sp1) accumulates substantially in the early stage of lung cancer but is partially decreased in the late stages, which is an important factor in the progression of the cancer. In this study, we found that Nm23-H1 and hnRNPA2/B1 could be recruited to the 5'UTR of Sp1 mRNA. In investigating the clinical relevance of Nm23-H1/Sp1 levels, we found a positive correlation between lung cancer patients with poor prognosis and low levels of Sp1 and Nm23-H1, suggesting an association between Nm23-H1/Sp1 levels and survival rate. Knockdown of Nm23-H1 inhibits lung cancer growth but increases lung cancer cell malignancy, which could be rescued by overexpression of Sp1, indicating that Nm23-H1-induced Sp1 expression is critical for lung cancer progression. We also found that Nm23-H1 increases the protein stability of hnRNPA2/B1and is thereby co-recruited to the 5'UTR of Sp1 mRNA to regulate cap-independent translational activity. Since the Sp1 level is tightly regulated during lung cancer progression, understanding the molecular mechanisms underlying the regulation by Nm23-H1/hnRNPA2B1 of Sp1 expression in the various stages of lung cancer will be beneficial for lung cancer therapy in the future.

RNA-binding proteins with prion-like domains in health and disease.

Approximately 70 human RNA-binding proteins (RBPs) contain a prion-like domain (PrLD). PrLDs are low-complexity domains that possess a similar amino acid composition to prion domains in yeast, which enable several proteins, including Sup35 and Rnq1, to form infectious conformers, termed prions. In humans, PrLDs contribute to RBP function and enable RBPs to undergo liquid-liquid phase transitions that underlie the biogenesis of various membraneless organelles. However, this activity appears to render RBPs prone to misfolding and aggregation connected to neurodegenerative disease. Indeed, numerous RBPs with PrLDs, including TDP-43 (transactivation response element DNA-binding protein 43), FUS (fused in sarcoma), TAF15 (TATA-binding protein-associated factor 15), EWSR1 (Ewing sarcoma breakpoint region 1), and heterogeneous nuclear ribonucleoproteins A1 and A2 (hnRNPA1 and hnRNPA2), have now been connected via pathology and genetics to the etiology of several neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Here, we review the physiological and pathological roles of the most prominent RBPs with PrLDs. We also highlight the potential of protein disaggregases, including Hsp104, as a therapeutic strategy to combat the aberrant phase transitions of RBPs with PrLDs that likely underpin neurodegeneration.

Critical role of hnRNP A1 in activating KRAS transcription in pancreatic cancer cells: A molecular mechanism involving G4 DNA.

KRAS is one of the most mutated genes in human cancer. Its crucial role in the tumourigenesis of pancreatic ductal adenocarcinoma (PDAC) has been widely demonstrated. As this deadly cancer does not sufficiently respond to conventional chemotherapies, it is important to increase our knowledge of pancreatic cancer biology, in particular how oncogenic KRAS is regulated. The promoter of KRAS contains a GA-element composed of runs of guanines that fold into a G4 structure. This unusual DNA conformation is recognized by several nuclear proteins, including MAZ and hnRNP A1. Recent data have revealed that KRAS is interconnected to ILK and hnRNP A1 in a circuitry that enables pancreatic cancer cells to maintain an aggressive phenotype. The present review illustrates recent advances on how KRAS is regulated in pancreatic cancer cells, focusing on the formation of G4 structures in the KRAS promoter and their interaction with hnRNP A1. The newly discovered KRAS-ILK-hnRNP A1 regulatory loop is discussed, emphasizing its potential as a therapeutic target for PDAC-specific molecules. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Amyotrophic Lateral Sclerosis Type 20 - In Silico Analysis and Molecular Dynamics Simulation of hnRNPA1.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that affects the upper and lower motor neurons. 5-10% of cases are genetically inherited, including ALS type 20, which is caused by mutations in the hnRNPA1 gene. The goals of this work are to analyze the effects of non-synonymous single nucleotide polymorphisms (nsSNPs) on hnRNPA1 protein function, to model the complete tridimensional structure of the protein using computational methods and to assess structural and functional differences between the wild type and its variants through Molecular Dynamics simulations. nsSNP, PhD-SNP, Polyphen2, SIFT, SNAP, SNPs&GO, SNPeffect and PROVEAN were used to predict the functional effects of nsSNPs. Ab initio modeling of hnRNPA1 was made using Rosetta and refined using KoBaMIN. The structure was validated by PROCHECK, Rampage, ERRAT, Verify3D, ProSA and Qmean. TM-align was used for the structural alignment. FoldIndex, DICHOT, ELM, D2P2, Disopred and DisEMBL were used to predict disordered regions within the protein. Amino acid conservation analysis was assessed by Consurf, and the molecular dynamics simulations were performed using GROMACS. Mutations D314V and D314N were predicted to increase amyloid propensity, and predicted as deleterious by at least three algorithms, while mutation N73S was predicted as neutral by all the algorithms. D314N and D314V occur in a highly conserved amino acid. The Molecular Dynamics results indicate that all mutations increase protein stability when compared to the wild type. Mutants D314N and N319S showed higher overall dimensions and accessible surface when compared to the wild type. The flexibility level of the C-terminal residues of hnRNPA1 is affected by all mutations, which may affect protein function, especially regarding the protein ability to interact with other proteins.

The LC Domain of hnRNPA2 Adopts Similar Conformations in Hydrogel Polymers, Liquid-like Droplets, and Nuclei.

Many DNA and RNA regulatory proteins contain polypeptide domains that are unstructured when analyzed in cell lysates. These domains are typified by an over-representation of a limited number of amino acids and have been termed prion-like, intrinsically disordered or low-complexity (LC) domains. When incubated at high concentration, certain of these LC domains polymerize into labile, amyloid-like fibers. Here, we report methods allowing the generation of a molecular footprint of the polymeric state of the LC domain of hnRNPA2. By deploying this footprinting technique to probe the structure of the native hnRNPA2 protein present in isolated nuclei, we offer evidence that its LC domain exists in a similar conformation as that described for recombinant polymers of the protein. These observations favor biologic utility to the polymerization of LC domains in the pathway of information transfer from gene to message to protein.

Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization.

Stress granules are membrane-less organelles composed of RNA-binding proteins (RBPs) and RNA. Functional impairment of stress granules has been implicated in amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy-diseases that are characterized by fibrillar inclusions of RBPs. Genetic evidence suggests a link between persistent stress granules and the accumulation of pathological inclusions. Here, we demonstrate that the disease-related RBP hnRNPA1 undergoes liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by a low complexity sequence domain (LCD). While the LCD of hnRNPA1 is sufficient to mediate LLPS, the RNA recognition motifs contribute to LLPS in the presence of RNA, giving rise to several mechanisms for regulating assembly. Importantly, while not required for LLPS, fibrillization is enhanced in protein-rich droplets. We suggest that LCD-mediated LLPS contributes to the assembly of stress granules and their liquid properties and provides a mechanistic link between persistent stress granules and fibrillar protein pathology in disease.

Aggregation of ALS-linked FUS mutant sequesters RNA binding proteins and impairs RNA granules formation.

Protein aggregate/inclusion is one of hallmarks for neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). FUS/TLS, one of causative genes for familial ALS, encodes a multifunctional DNA/RNA binding protein predominantly localized in the nucleus. C-terminal mutations in FUS/TLS cause the retention and the inclusion of FUS/TLS mutants in the cytoplasm. In the present study, we examined the effects of ALS-linked FUS mutants on ALS-associated RNA binding proteins and RNA granules. FUS C-terminal mutants were diffusely mislocalized in the cytoplasm as small granules in transiently transfected SH-SY5Y cells, whereas large aggregates were spontaneously formed in ∼10% of those cells. hnRNP A1, hnRNP A2, and SMN1 as well as FUS wild type were assembled into stress granules under stress conditions, and these were also recruited to FUS mutant-derived spontaneous aggregates in the cytoplasm. These aggregates stalled poly(A) mRNAs and sequestered SMN1 in the detergent insoluble fraction, which also reduced the number of nuclear oligo(dT)-positive foci (speckles) in FISH (fluorescence in situ hybridization) assay. In addition, the number of P-bodies was decreased in cells harboring cytoplasmic granules of FUS P525L. These findings raise the possibility that ALS-linked C-terminal FUS mutants could sequester a variety of RNA binding proteins and mRNAs in the cytoplasmic aggregates, which could disrupt various aspects of RNA equilibrium and biogenesis.

A novel role for the proteasomal chaperone PSMD9 and hnRNPA1 in enhancing IκBα degradation and NF-κB activation - functional relevance of predicted PDZ domain-motif interaction.

PSMD9 is a PDZ domain containing chaperone of proteasome assembly. Based on the ability of PDZ-like domains to recognize C-terminal residues in their interactors, we recently predicted and identified heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) as one of the novel interacting partners of PSMD9. Contingent on the reported role of hnRNPA1 in nuclear factor κB (NF-κB) activation, we tested the role of human PSMD9 and hnRNPA1 in NF-κB signaling. We demonstrated in human embryonic kidney 293 cells that PSMD9 influences both basal and tumor necrosis factor α (TNF-α) mediated NF-κB activation through inhibitor of nuclear factor κB α (IκBα) proteasomal degradation. PSMD9 mediates IκBα degradation through a specific domain-motif interaction involving its PDZ domain and a short linear sequence motif in the C-terminus of hnRNPA1. Point mutations in the PDZ domain or deletion of C-terminal residues in hnRNPA1 disrupt interaction between the two proteins which has a direct influence on NF-κB activity. hnRNPA1 interacts with IκBα directly, whereas PSMD9 interacts only through hnRNPA1. Furthermore, hnRNPA1 shows increased association with the proteasome upon TNF-α treatment which has no such effect in the absence of PSMD9. On the other hand endogenous and trans-expressed PSMD9 are found associated with the proteasome complex. This association is unaffected by PDZ mutations or TNF-α treatment. Collectively, these interactions between IκBα, hnRNPA1 and proteasome bound PSMD9 illustrate a potential mechanism by which ubiquitinated IκBα is recruited on the proteasome for degradation. In this process, hnRNPA1 may act as a shuttle receptor and PSMD9 as a subunit acceptor. The interaction sites of PSMD9 and hnRNPA1 may emerge as a vulnerable drug target in cancer cells which require consistent NF-κB activity for survival.

Molecular basis for the action of a dietary flavonoid revealed by the comprehensive identification of apigenin human targets.

Flavonoids constitute the largest class of dietary phytochemicals, adding essential health value to our diet, and are emerging as key nutraceuticals. Cellular targets for dietary phytochemicals remain largely unknown, posing significant challenges for the regulation of dietary supplements and the understanding of how nutraceuticals provide health value. Here, we describe the identification of human cellular targets of apigenin, a flavonoid abundantly present in fruits and vegetables, using an innovative high-throughput approach that combines phage display with second generation sequencing. The 160 identified high-confidence candidate apigenin targets are significantly enriched in three main functional categories: GTPase activation, membrane transport, and mRNA metabolism/alternative splicing. This last category includes the heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2), a factor involved in splicing regulation, mRNA stability, and mRNA transport. Apigenin binds to the C-terminal glycine-rich domain of hnRNPA2, preventing hnRNPA2 from forming homodimers, and therefore, it perturbs the alternative splicing of several human hnRNPA2 targets. Our results provide a framework to understand how dietary phytochemicals exert their actions by binding to many functionally diverse cellular targets. In turn, some of them may modulate the activity of a large number of downstream genes, which is exemplified here by the effects of apigenin on the alternative splicing activity of hnRNPA2. Hence, in contrast to small-molecule pharmaceuticals designed for defined target specificity, dietary phytochemicals affect a large number of cellular targets with varied affinities that, combined, result in their recognized health benefits.

A guanosine-centric mechanism for RNA chaperone function.

RNA chaperones are ubiquitous, heterogeneous proteins essential for RNA structural biogenesis and function. We investigated the mechanism of chaperone-mediated RNA folding by following the time-resolved dimerization of the packaging domain of a retroviral RNA at nucleotide resolution. In the absence of the nucleocapsid (NC) chaperone, dimerization proceeded through multiple, slow-folding intermediates. In the presence of NC, dimerization occurred rapidly through a single structural intermediate. The RNA binding domain of heterogeneous nuclear ribonucleoprotein A1 protein, a structurally unrelated chaperone, also accelerated dimerization. Both chaperones interacted primarily with guanosine residues. Replacing guanosine with more weakly pairing inosine yielded an RNA that folded rapidly without a facilitating chaperone. These results show that RNA chaperones can simplify RNA folding landscapes by weakening intramolecular interactions involving guanosine and explain many RNA chaperone activities.

hnRNP A1 proofreads 3' splice site recognition by U2AF.

One of the earliest steps in metazoan pre-mRNA splicing involves binding of U2 snRNP auxiliary factor (U2AF) 65 KDa subunit to the polypyrimidine (Py) tract and of the 35 KDa subunit to the invariant AG dinucleotide at the intron 3' end. Here we use in vitro and in vivo depletion, as well as reconstitution assays using purified components, to identify hnRNP A1 as an RNA binding protein that allows U2AF to discriminate between pyrimidine-rich RNA sequences followed or not by a 3' splice site AG. Biochemical and NMR data indicate that hnRNP A1 forms a ternary complex with the U2AF heterodimer on AG-containing/uridine-rich RNAs, while it displaces U2AF from non-AG-containing/uridine-rich RNAs, an activity that requires the glycine-rich domain of hnRNP A1. Consistent with the functional relevance of this activity for splicing, proofreading assays reveal a role for hnRNP A1 in U2AF-mediated recruitment of U2 snRNP to the pre-mRNA.

Camptothecin (CPT) directly binds to human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and inhibits the hnRNP A1/topoisomerase I interaction.

Camptothecin (CPT) is an anti-tumor natural product that forms a ternary complex with topoisomerase I (top I) and DNA (CPT-top I-DNA). In this study, we identified the direct interaction between CPT and human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) using the T7 phage display technology. On an avidin-agarose bead pull down assay, hnRNP A1 protein was selectively pulled down in the presence of C20-biotinylated CPT derivative (CPT-20-B) both in vitro and in vivo. The interaction was also confirmed by an analysis on a quartz-crystal microbalance (QCM) device, yielding a K(D) value of 82.7 nM. A surface plasmon resonance (SPR) analysis revealed that CPT inhibits the binding of hnRNP A1 to top I (K(D): 260 nM) in a non-competitive manner. Moreover, an in vivo drug evaluation assay using Drosophila melanogaster showed that the knockout of the hnRNP A1 homolog Hrb87F gene showed high susceptibility against 5-50 µM of CPT as compared to a wild-type strain. Such susceptibility was specific for CPT and not observed after treatment with other cytotoxic drugs. Collectively, our data suggests that CPT directly binds to hnRNP A1 and non-competitively inhibits the hnRNP A1/top I interaction in vivo. The knockout strain loses the hnRNP A1 homolog as a both CPT-binding partner and naïve brakes of top I, which enhances the formation of the CPT-top I-DNA ternary complexes and subsequently sensitizes the growth inhibitory effect of CPT in D. melanogaster.

IL-6-induced enhancement of c-Myc translation in multiple myeloma cells: critical role of cytoplasmic localization of the rna-binding protein hnRNP A1.

Prior work indicates that IL-6 can stimulate c-Myc expression in multiple myeloma (MM) cells, which is independent of effects on transcription and due to enhanced translation mediated by an internal ribosome entry site in the 5'-UTR of the c-Myc RNA. The RNA-binding protein hnRNP A1 (A1) was also critical to IL-6-stimulated translation. Because A1 shuttles between nucleus and cytoplasm, we investigated whether the ability of IL-6 to enhance Myc translation was mediated by stimulation of A1 shuttling. In MM cell lines and primary specimens, IL-6 increased A1 cytoplasmic localization. In contrast, there was no effect on the total cellular levels of A1. Use of a dominant negative A1 construct, which prevents endogenous A1 from nucleus-to-cytoplasm transit, prevented the ability of IL-6 to enhance Myc internal ribosome entry site activity, Myc protein expression, and MM cell growth. IL-6-stimulated cytoplasmic localization was mediated by alterations in the C-terminal M9 peptide of A1, and this correlated with the ability of IL-6 to induce serine phosphorylation of this domain. A p38 kinase inhibitor prevented IL-6-induced A1 phosphorylation. Thus, IL-6 activates c-Myc translation in MM cells by inducing A1 phosphorylation and cytoplasmic localization in a p38-dependent fashion. These data suggest A1 as a potential therapeutic target in MM.

Extensive association of HuR with hnRNP proteins within immunoselected hnRNP and mRNP complexes.

Regulated gene expression at the post-transcriptional level in higher eukaryotes is based on a network of interactions among RNA-binding proteins (RBPs) operating within multifactorial ribonucleoprotein (RNP) complexes, notably heterogeneous nuclear ribonucleoprotein (hnRNP) and mRNP complexes. We are interested in interactions involving hnRNP proteins participating in several steps of mRNA processing (mainly pre-mRNA splicing) and HuR with an established role in stability/translation of associated mRNAs. hnRNP and HuR proteins have a major nucleoplasmic localization and ability to shuttle between nucleus and cytoplasm. We report here on interactions between hnRNP and HuR proteins that were identified in the context of isolated hnRNP and mRNP complexes. This was done by the application of immunoprecipitation and pull-down assays on different sub-cellular fractions prepared from cells of human and mouse origin, as well as in vivo localization studies. A range of specific associations of HuR with the shuttling hnRNP A1 and A3 and the non-shuttling hnRNP C1/C2 was identified and ascribed discrete properties with respect to stability to RNase A and increasing salt, as well as to cellular distribution. The likelihood of a biological relevance of these associations was tested under heat shock conditions in growing cells, which appeared to affect both the sub-nuclear distribution and interaction of HuR with hnRNPs. The establishment of an extensive association of HuR with hnRNP components of nuclear hnRNP/mRNP and cytoplasmic mRNP complexes supports its broader participation in mRNA processing events than initially anticipated.