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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.

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.

Algorithms designed to identify canonical yeast prions predict that around 250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbour a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here we define pathogenic mutations in PrLDs of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and in one case of familial amyotrophic lateral sclerosis. Wild-type hnRNPA2 (the most abundant isoform of hnRNPA2B1) and hnRNPA1 show an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a ‘steric zipper’ motif in the PrLD, which accelerates the formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Notably, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant steric zipper motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs should therefore be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone. The identification of pathogenic mutations within prion-like domains (PrLDs) of the RNA-binding proteins hnRNPA2B1 and hnRNPA1 add to our understanding of how mutations in these proteins lead to degenerative disease, and highlight the potential importance of PrLDs in degenerative diseases of the nervous system, muscle and bone. Disease link to prion-like RNA-binding protein How do mutations in RNA-binding proteins cause human disease, and neurodegeneration in particular? Hong Joo Kim et al . have identified mutations in two RNA-binding proteins, hnRNPA2B1 and hnRNPA1, in two families with inclusion body myopathy with frontotemporal dementia. Both of the mutations lie within a highly conserved part of a protein domain that has similarities to prion proteins, and a tendency to aggregate. This aggregation is enhanced by the mutations. The mutated prion-like domain of hnRNPA2 can functionally replace that of a yeast prion protein and reproduce its prion-like behaviour. These findings have relevance to the pathogenesis of degenerative diseases and proteinopathies such as amyotrophic lateral sclerosis.

hnRNPA2 is a human ribonucleoprotein (RNP) involved in RNA metabolism. It forms fibrils both under cellular stress and in mutated form in neurodegenerative conditions. Previous work established that the C-terminal low-complexity domain (LCD) of hnRNPA2 fibrillizes under stress, and missense mutations in this domain are found in the disease multisystem proteinopathy (MSP). However, little is known at the atomic level about the hnRNPA2 LCD structure that is involved in those processes and how disease mutations cause structural change. Here we present the cryo-electron microscopy (cryoEM) structure of the hnRNPA2 LCD fibril core and demonstrate its capability to form a reversible hydrogel in vitro containing amyloid-like fibrils. Whereas these fibrils, like pathogenic amyloid, are formed from protein chains stacked into β-sheets by backbone hydrogen bonds, they display distinct structural differences: the chains are kinked, enabling non-covalent cross-linking of fibrils and disfavoring formation of pathogenic steric zippers. Both reversibility and energetic calculations suggest these fibrils are less stable than pathogenic amyloid. Moreover, the crystal structure of the disease-mutation-containing segment (D290V) of hnRNPA2 suggests that the replacement fundamentally alters the fibril structure to a more stable energetic state. These findings illuminate how molecular interactions promote protein fibril networks and how mutation can transform fibril structure from functional to a pathogenic form. hnRNPA2 is involved in RNA metabolism and can form both functional amyloid-like fibrils in membraneless organelles, and pathogenic fibrils in neurodegenerative conditions. Here, the authors present the cryo-EM fibril structure of the wild-type hnRNPA2 low-complexity domain (LCD) and the crystal structure of a LCD segment with the disease causing D290V variant and discuss how mutations can transform fibril structure from a functional to a pathogenic form.

Repeat expansion in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Expanded sense and antisense repeat RNA transcripts in C9orf72 are translated into five dipeptide-repeat proteins (DPRs) in an AUG-independent manner. We previously identified the heterogeneous ribonucleoprotein (hnRNP) A3 as an interactor of the sense repeat RNA that reduces its translation into DPRs. Furthermore, we found that hnRNPA3 is depleted from the nucleus and partially mislocalized to cytoplasmic poly-GA inclusions in C9orf72 patients, suggesting that poly-GA sequesters hnRNPA3 within the cytoplasm. We now demonstrate that hnRNPA3 also binds to the antisense repeat RNA. Both DPR production and deposition from sense and antisense RNA repeats are increased upon hnRNPA3 reduction. All DPRs induced DNA double strand breaks (DSB), which was further enhanced upon reduction of hnRNPA3. Poly-glycine–arginine and poly-proline-arginine increased foci formed by phosphorylated Ataxia Telangiectasia Mutated (pATM), a major sensor of DSBs, whereas poly-glycine–alanine (poly-GA) evoked a reduction of pATM foci. In dentate gyri of C9orf72 patients, lower nuclear hnRNPA3 levels were associated with increased DNA damage. Moreover, enhanced poly-GA deposition correlated with reduced pATM foci. Since cytoplasmic pATM deposits partially colocalized with poly-GA deposits, these results suggest that poly-GA, the most frequent DPR observed in C9orf72 patients, differentially causes DNA damage and that poly-GA selectively sequesters pATM in the cytoplasm inhibiting its recruitment to sites of DNA damage. Thus, mislocalization of nuclear hnRNPA3 caused by poly-GA leads to increased poly-GA production, which partially depletes pATM, and consequently enhances DSB.

Background Heterogeneous nuclear ribonucleoproteins (hnRNPs) of the A/B type (hnRNP A1, A2/B1, A3) are highly related multifunctional proteins participating in alternative splicing by antagonising other splicing factors, notably ASF/SF2. The altered expression pattern of hnRNP A2/B1 and/or splicing variant B1 alone in human lung cancer and their potential to serve as molecular markers for early diagnosis remain issues of intense investigation. The main objective of the present study was to use paired tumour/non-tumour biopsies from patients with non-small cell lung cancer (NSCLC) to investigate the expression profiles of hnRNP A1, A2/B1 and A3 in conjunction with ASF/SF2. Methods We combined western blotting of tissue homogenates with immunohistochemical examination of fixed tissue sections and quantification of mRNA expression levels in tumour versus adjacent normal-looking areas of the lung in the same patient. Results Our study, in addition to clear evidence of mostly uncoupled deregulation of hnRNPs A/B, has revealed hnRNP A1 to be the most deregulated protein with a high frequency of over-expression (76%), followed by A3 (52%) and A2/B1 (43%). Moreover, direct comparison of protein/mRNA levels showed a lack of correlation in the case of hnRNP A1 (as well as of ASF/SF2), but not of A2/B1, suggesting that different mechanisms underlie their deregulation. Conclusion Our results provide strong evidence for the up-regulation of hnRNP A/B in NSCLC, and they support the existence of distinct mechanisms responsible for their deregulated expression.

hnRNP A1 is an RNA-binding protein involved in various aspects of RNA processing. Use of an in vivo cross-linking and immunoprecipitation protocol to find hnRNP A1 RNA targets resulted in the identification of a microRNA (miRNA) precursor, pre-miR-18a. This microRNA is expressed as part of a cluster of intronic RNAs, including miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92, and potentially acts as an oncogene. Here we show that hnRNP A1 binds specifically to the primary RNA sequence pri-miR-18a before Drosha processing. HeLa cells depleted of hnRNP A1 have reduced in vitro processing activity with pri-miR-18a and also show reduced abundances of endogenous pre-miR-18a. Furthermore, we show that hnRNP A1 is required for miR-18a–mediated repression of a target reporter in vivo . These results underscore a previously uncharacterized role for general RNA-binding proteins as auxiliary factors that facilitate the processing of specific miRNAs.

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) is a multifunctional nucleic acid metabolism regulator with established roles in viral infection and tumorigenesis. However, a critical gap exists between the oligomeric state of hnRNP A2/B1 and its function as a nuclear DNA sensor. To address this gap, we generated full-length hnRNP A2/B1 and three domain-truncated variants (△NLS, RRM-PrLD, RRM-RGG) using SUMO/MBP fusion expression systems. To define the nucleic acid binding and oligomeric properties of these variants, we combined SEC with EMSA and ITC. These analyses revealed that full-length hnRNP A2/B1 forms soluble amorphous aggregates, whereas the truncated variants exist as stable homogeneous monomers under in vitro solution conditions. Additionally, results demonstrated that the RRM-RGG (15–250) variant binds to ssDNA but not dsDNA. Notably, SEC combined with CD and AUC confirmed that RRM-RGG (15–250) truncations undergo homodimerization induced by 12nt and 22nt Guanine quadruplex (G4) structure enriched ssDNA, which is abundant in the genomes of diverse viruses. Structure predictions revealed that the C-terminal PrLD, and NLS domain are intrinsically disordered, a feature potentially underlying the protein’s aggregation propensity and crystallization recalcitrance. NPDock simulations demonstrated that G4-structured ssDNA binds and stabilizes the RRM-RGG (15–250) truncation via non-conserved key residues that are distinct from those of other hnRNP family members. This work provides a biophysical basis for hypothesizing that G4-structured ssDNA-dependent dimerization may contribute to the protein’s antiviral function, and establishes a biophysical framework to guide future investigations into the protein’s antiviral mechanism and the design of rational targeted inhibitors.

It is well known that microRNAs (miRs) are abnormally expressed in various cancers and target the messenger RNAs (mRNAs) of cancer-associated genes. While (miRs) are abnormally expressed in various cancers, whether miRs directly target oncogenic proteins is unknown. The present study investigated the inhibitory effects of miR-18a on colon cancer progression, which was considered to be mediated through its direct binding and degradation of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). An MTT assay and xenograft model demonstrated that the transfection of miR-18a induced apoptosis in SW620 cells. A binding assay revealed direct binding between miR-18a and hnRNP A1 in the cytoplasm of SW620 cells, which inhibited the oncogenic functions of hnRNP A1. A competitor RNA, which included the complementary sequence of the region of the miR-18a-hnRNP A1 binding site, repressed the effects of miR-18a on the induction of cancer cell apoptosis. In vitro single and in vivo double isotope assays demonstrated that miR-18a induced the degradation of hnRNP A1. An immunocytochemical study of hnRNP A1 and LC3-II and the inhibition of autophagy by 3-methyladenine and ATG7, p62 and BAG3 siRNA showed that miR-18a and hnRNP A1 formed a complex that was degraded through the autophagolysosomal pathway. This is the first report showing a novel function of a miR in the autophagolysosomal degradation of an oncogenic protein resulting from the creation of a complex consisting of the miR and a RNA-binding protein, which suppressed cancer progression.

RNA molecules are synthesized in the cell nucleus, yet many have to be moved to the cytoplasm to be processed and/or to effect their function. Different classes of RNA are transported from the nucleus by different transport systems. Messenger RNAs (mRNAs) and uridine-rich small nuclear RNAs (U snRNAs) are transcribed by RNA polymerase II and are capped and bound by the cap-binding machinery in the nucleus but are exported by different protein complexes. The feature that distinguishes the two classes of RNA is their length: U snRNAs are short and mRNAs are long. Using an in vitro system and human tissue culture cells, McCloskey et al. (p. 1643 ) show that the length of the RNAs is measured by the heterogeneous nuclear ribonicleoprotein (hnRNP) C tetrameric protein complex. The hnRNP C cannot bind to the short U snRNAs, allowing the U snRNA-specific export adaptor protein, PHAX, to bind and mediate export. Longer mRNAs are bound by hnRNP C, which prevents the binding of PHAX, thus identifying these RNAs for export from the nucleus via the mRNA pathway. A nuclear protein measures the length of newly made RNAs and sorts them into distinct pathways for export. Specific RNA recognition is usually achieved by specific RNA sequences and/or structures. However, we show here a mechanism by which RNA polymerase II (Pol II) transcripts are classified according to their length. The heterotetramer of the heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2 measures the length of the transcripts like a molecular ruler, by selectively binding to the unstructured RNA regions longer than 200 to 300 nucleotides. Thus, the tetramer sorts the transcripts into two RNA categories, to be exported as either messenger RNA or uridine-rich small nuclear RNA (U snRNA), depending on whether or not they are longer than the threshold, respectively. Our findings reveal a new function of the C tetramer and highlight the biological importance of RNA recognition by the length.