Differentiation of memory cells involves DNA-sequence changes in B lymphocytes but is less clearly defined in T cells. RNA rearrangement is identified here as a key event in memory T cell differentiation by analysis of a mouse mutation that altered the proportions of naive and memory T cells and crippled the process of Ptprc exon silencing needed to generate CD45RO in memory T cells. A single substitution in a memory-induced RNA-binding protein, hnRNPLL, destabilized an RNA-recognition domain that bound with micromolar affinity to RNA containing the Ptprc exon-silencing sequence. Hnrpll mutation selectively diminished T cell accumulation in peripheral lymphoid tissues but not proliferation. Exon-array analysis of Hnrpll mutant naive and memory T cells revealed an extensive program of alternative mRNA splicing in memory T cells, coordinated by hnRNPLL. A remarkable overlap with alternative splicing in neural tissues may reflect a co-opted strategy for diversifying memory T cells.

We have cloned and characterized Xlrbpa, a double-stranded RNA-binding protein from Xenopus laevis. Xlrbpa is a protein of 33 kD and contains three tandemly arranged, double-stranded RNA-binding domains (dsRBDs) that bind exclusively to double-stranded RNA in vitro, but fail to bind either single-stranded RNA or DNA. Sequence data and the overall organization of the protein suggest that Xlrbpa is the Xenopus homologue of human TAR-RNA binding protein (TRBP), a protein isolated by its ability to bind to human immunodeficiency virus (HIV) TAR-RNA. In transfection assays, TRBP has also been shown to inhibit the interferon-induced protein kinase PKR possibly by direct physical interaction. To determine the function of Xlrbpa and its human homologue we studied the expression and intracellular distribution of the two proteins. Xlrbpa is ubiquitously expressed with marked quantitative differences amongst all tissues. Xlrbpa and human TRBP can be detected in the cytoplasm and nucleus by immunofluorescence staining and Western blotting. Sedimentation gradient analyses and immunoprecipitation experiments suggest an association of cytoplasmic Xlrbpa with ribosomes. In contrast, a control construct containing two dsRBDs fails to associate with ribosomes in microinjected Xenopus oocytes. Nuclear staining of Xenopus lampbrush chromosome preparations showed the association of the protein with nucleoli, again indicating an association of the protein with ribosomal RNAs. Additionally, Xlrbpa could be located on lampbrush chromosomes and in snurposomes. Immunoprecipitations of nuclear extracts demonstrated the presence of the protein in heterogeneous nuclear (hn) RNP particles, but not in small nuclear RNPs, explaining the chromosomal localization of the protein. It thus appears that Xlrbpa is a general double-stranded RNA-binding protein which is associated with the majority of cellular RNAs, ribosomal RNAs, and hnRNAs either alone or as part of an hnRNP complex.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an abundant nuclear protein that plays an important role in pre-mRNA processing and mRNA export from the nucleus. A1 shuttles rapidly between the nucleus and the cytoplasm, and a 38-amino acid domain, M9, serves as the bidirectional transport signal of A1. Recently, a 90-kD protein, transportin, was identified as the mediator of A1 nuclear import. In this study, we show that transportin mediates the nuclear import of additional hnRNP proteins, including hnRNP F. We have also isolated and sequenced a novel transportin homolog, transportin2, which may differ from transportin1 in its substrate specificity. Immunostaining shows that transportin1 is localized both in the cytoplasm and the nucleoplasm, and nuclear rim staining is also observed. The nuclear localization of A1 is dependent on ongoing RNA polymerase II transcription. Interestingly, a pyruvate kinase-M9 fusion, which normally localizes in the nucleus, also accumulates in the cytoplasm when RNA polymerase II is inhibited. Thus, M9 itself is a specific sensor for transcription-dependent nuclear transport. Transportin1-A1 complexes can be isolated from the cytoplasm and the nucleoplasm, but transportin1 is not detectable in hnRNP complexes. RanGTP causes dissociation of A1-transportin1 complexes in vitro. Thus, it is likely that after nuclear import, A1 dissociates from transportin1 by RanGTP and becomes incorporated into hnRNP complexes, where A1 functions in pre-mRNA processing.

In the ASE/AIE-mediated genetic mechanism for age-related gene regulation, a recently identified age-related homeostasis mechanism, two genetic elements, ASE (age-related stability element) and AIE (age-related increase element as a stem-loop forming RNA), play critical roles in producing specific age-related expression patterns of genes.

Recognition of viral RNA by the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), including RIG-I and MDA5, initiates innate antiviral responses. Although regulation of RLR-mediated signal transduction has been extensively investigated, how the recognition of viral RNA by RLRs is regulated remains enigmatic. In this study, we identified heterogeneous nuclear ribonucleoprotein M (hnRNPM) as a negative regulator of RLR-mediated signaling. Overexpression of hnRNPM markedly inhibited RNA virus-triggered innate immune responses. Conversely, hnRNPM-deficiency increased viral RNA-triggered innate immune responses and inhibited replication of RNA viruses. Viral infection caused translocation of hnRNPM from the nucleus to the cytoplasm. hnRNPM interacted with RIG-I and MDA5, and impaired the binding of the RLRs to viral RNA, leading to inhibition of innate antiviral response. Our findings suggest that hnRNPM acts as an important decoy for excessive innate antiviral immune response.

Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) F/H family are multipurpose RNA binding proteins that participate in most stages of RNA metabolism. Despite having similar RNA sequence preferences, hnRNP F/H proteins function in overlapping and, in some cases, distinct cellular processes. The domain organization of hnRNP F/H proteins is modular, consisting of N-terminal tandem quasi-RNA recognition motifs (F/HqRRM1,2) and a third C-terminal qRRM3 embedded between glycine-rich repeats. The tandem qRRMs are connected through a 10-residue linker, with several amino acids strictly conserved between hnRNP H and F. A significant difference occurs at position 105 of the linker, where hnRNP H contains a proline and hnRNP F an alanine. To investigate the influence of P105 on the conformational properties of hnRNP H, we probed the structural dynamics of its HqRRM1,2 domain with X-ray crystallography, NMR spectroscopy, and small-angle X-ray scattering. The collective results best describe that HqRRM1,2 exists in a conformational equilibrium between compact and extended structures. The compact structure displays an electropositive surface formed at the qRRM1-qRRM2 interface. Comparison of NMR relaxation parameters, including Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion, between HqRRM1,2 and FqRRM1,2 indicates that FqRRM1,2 primarily adopts a more extended and flexible conformation. Introducing the P105A mutation into HqRRM1,2 alters its conformational dynamics to favor an extended structure. Thus, our work demonstrates that the linker compositions confer different structural properties between hnRNP F/H family members that might contribute to their functional diversity.

Telomere integrity in human cells is maintained by the dynamic interplay between telomerase, telomere associated proteins, and DNA repair proteins. These interactions are vital to suppress DNA damage responses and unfavorable changes in chromosome dynamics. The DNA-dependent protein kinase (DNA-PK) is critical for this process. Cells deficient for functional DNA-PKcs show increased rates of telomere loss, accompanied by chromosomal fusions and translocations. Treatment of cells with specific DNA-PK kinase inhibitors leads to similar phenotypes. These observations indicate that the kinase activity of DNA-PK is required for its function at telomeres possibly through phosphorylation of essential proteins needed for telomere length maintenance. Here we show that the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a direct substrate for DNA-PK in vitro. Phosphorylation of hnRNP A1 is stimulated not only by the presence of DNA but also by the telomerase RNA component, hTR. Furthermore, we show that hnRNP A1 is phosphorylated in vivo in a DNA-PK-dependent manner and that this phosphorylation is greatly reduced in cell lines which lack hTR. These data are the first to report that hTR stimulates the kinase activity of DNA-PK toward a known telomere-associated protein, and may provide further insights into the function of DNA-PK at telomeres.

An embryo experiences increasingly complex spatial and temporal patterns of gene expression as it matures, guiding the morphogenesis of its body. Using super-resolution fluorescence microscopy in Drosophila melanogaster embryos, we observed that the nuclear distributions of transcription factors and histone modifications undergo a similar transformation of increasing heterogeneity. This spatial partitioning of the nucleus could lead to distinct local regulatory environments in space and time that are tuned for specific genes. Accordingly, transcription sites driven by different cis-regulatory regions each had their own temporally and spatially varying local histone environments, which could facilitate the finer spatial and temporal regulation of genes to consistently differentiate cells into organs and tissues. Thus, 'nuclear morphogenesis' may be a microscopic counterpart of the macroscopic process that shapes the animal body.

Coordinated assembly of viral and host factors is essential for the successful propagation of viruses as well as the generation of host antiviral response. Previous studies from our group, as well as from other groups, have identified host proteins interacting with various components of the hepatitis E virus (HEV). However, the functional relevance of host protein interactions in HEV replication context has been notably overlooked. The present study reports that heterogeneous nuclear ribonucleoproteins (hnRNPs), namely hnRNPK, hnRNPA2B1, hnRNPH, PCBP1 and PCBP2, interact with HEV RNA promoter and RNA-dependent RNA polymerase to regulate HEV replication. We found that hnRNPK and hnRNPA2B1 are the virus-supportive factors interacting with HEV RNA at promoter regions along with HEV polymerase protein, which are essential for HEV replication in the cells. Contrarily, hnRNPH, PCBP1 and PCBP2 are the antiviral factors that interact exclusively with HEV genomic promoter and inhibit HEV replication in Huh7 S10-3 cells. In vitro RNA-binding assays revealed that the antiviral hnRNP proteins hamper the binding of virus-supportive hnRNP proteins at HEV genomic promoter. In the binding reaction, the binding of HEV polymerase protein to the genomic promoter is slightly affected by the presence of antiviral hnRNPH. In an effort of visualizing the subcellular localization of hnRNP proteins in the HEV replication scenario in the Huh7 cells, we showed that hnRNPK, hnRNPA2B1, hnRNPH, PCBP1 and PCBP2 redistribute from nucleus to cytoplasm. In conclusion, our study highlights the importance of hnRNP proteins in HEV replication regulation.

Heterogeneous ribonucleoprotein A1 (hnRNP A1) is crucial for regulating alternative splicing. Its integrated function within an organism has not, however, been identified. We generated hnRNP A1 knockout mice to study the role of hnRNP A1 in vivo The knockout mice, hnRNP A1-/-, showed embryonic lethality because of muscle developmental defects. The blood pressure and heart rate of the heterozygous mice were higher than those of the wild-type mice, indicating heart function defects. We performed mouse exon arrays to study the muscle development mechanism. The processes regulated by hnRNP A1 included cell adhesion and muscle contraction. The expression levels of muscle development-related genes in hnRNP A1+/- mice were significantly different from those in wild-type mice, as detected using qRT-PCR. We further confirmed the alternative splicing patterns of muscle development-related genes including mef2c, lrrfip1, usp28 and abcc9 Alternative mRNA isoforms of these genes were increased in hnRNP A1+/- mice compared with wild-type mice. Furthermore, we revealed that the functionally similar hnRNP A2/B1 did not compensate for the expression of hnRNP A1 in organisms. In summary, our study demonstrated that hnRNP A1 plays a critical and irreplaceable role in embryonic muscle development by regulating the expression and alternative splicing of muscle-related genes.

HNRNPU encodes the heterogeneous nuclear ribonucleoprotein U, which participates in RNA splicing and chromatin organization. Microdeletions in the 1q44 locus encompassing HNRNPU and other genes and point mutations in HNRNPU cause brain disorders, including early-onset seizures and severe intellectual disability. We aimed to understand HNRNPU's roles in the developing brain. Our work revealed that HNRNPU loss of function leads to rapid cell death of both postmitotic neurons and neural progenitors, with an apparent higher sensitivity of the latter. Further, expression and alternative splicing of multiple genes involved in cell survival, cell motility, and synapse formation are affected following Hnrnpu's conditional truncation. Finally, we identified pharmaceutical and genetic agents that can partially reverse the loss of cortical structures in Hnrnpu mutated embryonic brains, ameliorate radial neuronal migration defects and rescue cultured neural progenitors' cell death.

The biological functions of poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins (hnRNPs) are not well understood. However, it is known that hnRNPs are involved in the regulation of alternative splicing for many genes, including the Ddc gene in Drosophila. Therefore, we first confirmed that poly(ADP-ribose) (pADPr) interacts with two Drosophila hnRNPs, Squid/hrp40 and Hrb98DE/hrp38, and that this function is regulated by Poly(ADP-ribose) Polymerase 1 (PARP1) and Poly(ADP-ribose) Glycohydrolase (PARG) in vivo. These findings then provided a basis for analyzing the role of pADPr binding to these two hnRNPs in terms of alternative splicing regulation. Our results showed that Parg null mutation does cause poly(ADP-ribosyl)ation of Squid and hrp38 protein, as well as their dissociation from active chromatin. Our data also indicated that pADPr binding to hnRNPs inhibits the RNA-binding ability of hnRNPs. Following that, we demonstrated that poly(ADP-ribosyl)ation of Squid and hrp38 proteins inhibits splicing of the intron in the Hsr omega-RC transcript, but enhances splicing of the intron in the Ddc pre-mRNA. Taken together, these findings suggest that poly(ADP-ribosyl)ation regulates the interaction between hnRNPs and RNA and thus modulates the splicing pathways.

Active pre-mRNA splicing occurs co-transcriptionally, and takes place throughout the nucleoplasm of eukaryotic cells. Splicing decisions are controlled by networks of nuclear RNA-binding proteins and their target sequences, sometimes in response to signalling pathways. Sam68 (Src-associated in mitosis 68 kDa) is the prototypic member of the STAR (Signal Transduction and Activation of RNA) family of RNA-binding proteins, which regulate splicing in response to signalling cascades. Nuclear Sam68 protein is concentrated within subnuclear organelles called SLM/Sam68 Nuclear Bodies (SNBs), which also contain some other splicing regulators, signalling components and nucleic acids.

Neurodegeneration, including loss of neurons and axons, is a feature of progressive forms of multiple sclerosis (MS). The mechanisms underlying neurodegeneration are mostly unknown. Research implicates autoimmunity to nonmyelin self-antigens as important contributors to disease pathogenesis. Data from our lab implicate autoimmunity to the RNA binding protein (RBP) heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) as a possible mechanism of neurodegeneration in MS. MS patients make antibodies to hnRNP A1, which have been shown to lead to neuronal dysfunction in vitro. Using an animal model of MS, experimental autoimmune encephalomyelitis (EAE), we show here that injection of anti-hnRNP A1 antibodies, in contrast to control antibodies, resulted in worsened disease and increased neurodegeneration. We found a reduction of NeuN+ neuronal cell bodies in areas of the ventral gray matter of the spinal cord where anti-hnRNP A1 antibodies localized. Neurons displayed increased levels of hnRNP A1 nucleocytoplasmic mislocalization and stress granule formation, both markers of neuronal injury. Anti-hnRNP A1 antibodies were found to surround neuronal cell bodies and interact with CD68+ immune cells via Fc receptors. Additionally, anti-hnRNP A1 antibodies were found within neuronal cell bodies including those of the ventral spinocerebellar tract (VSCT), a tract previously shown to undergo neurodegeneration in anti-hnRNP A1 antibody injected EAE mice. Finally, both immune cells and neurons showed increased levels of inducible nitric oxide synthase, another indicator of cell damage. These findings suggest that autoimmunity to RBPs, such as hnRNP A1, play a role in neurodegeneration in EAE with important implications for the pathogenesis of MS.

Traditionally recognized as an RNA splicing regulator, heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC1/C2) can also bind to double-stranded DNA and function in trans as a vitamin D response element (VDRE)-binding protein. As such, hnRNPC1/C2 may couple transcription induced by the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D) with subsequent RNA splicing. In MG63 osteoblastic cells, increased expression of the 1,25(OH)2D target gene CYP24A1 involved immunoprecipitation of hnRNPC1/C2 with CYP24A1 chromatin and RNA. Knockdown of hnRNPC1/C2 suppressed expression of CYP24A1, but also increased expression of an exon 10-skipped CYP24A1 splice variant; in a minigene model the latter was attenuated by a functional VDRE in the CYP24A1 promoter. In genome-wide analyses, knockdown of hnRNPC1/C2 resulted in 3500 differentially expressed genes and 2232 differentially spliced genes, with significant commonality between groups. 1,25(OH)2D induced 324 differentially expressed genes, with 187 also observed following hnRNPC1/C2 knockdown, and a further 168 unique to hnRNPC1/C2 knockdown. However, 1,25(OH)2D induced only 10 differentially spliced genes, with no overlap with differentially expressed genes. These data indicate that hnRNPC1/C2 binds to both DNA and RNA and influences both gene expression and RNA splicing, but these actions do not appear to be linked through 1,25(OH)2D-mediated induction of transcription.

Heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) is a tumor suppressor protein that binds site- and structure-specifically to RNA sequences to regulate mRNA stability, facilitate alternative splicing, and suppress protein translation on several metastasis-associated mRNAs. Here, we show that hnRNP E1 binds polycytosine-rich DNA tracts present throughout the genome, including those at promoters of several oncogenes and telomeres and monitors genome integrity. It binds DNA in a site- and structure-specific manner. hnRNP E1-knockdown cells displayed increased DNA damage signals including γ-H2AX at its binding sites and also showed increased mutations. UV and hydroxyurea treatment of hnRNP E1-knockdown cells exacerbated the basal DNA damage signals with increased cell cycle arrest, activation of checkpoint proteins, and monoubiquitination of proliferating cell nuclear antigen despite no changes in deubiquitinating enzymes. DNA damage caused by genotoxin treatment localized to hnRNP E1 binding sites. Our work suggests that hnRNP E1 facilitates functions of DNA integrity proteins at polycytosine tracts and monitors DNA integrity at these sites.

Host and viral proteins are involved in dengue virus (DENV) replication. Heterogeneous ribonucleoprotein (hnRNP) C1/C2 are abundant host cellular proteins that exhibit RNA binding activity and play important roles in the replication of positive-strand RNA viruses such as poliovirus and hepatitis C virus. hnRNP C1/C2 have previously been shown to interact with vimentin and viral NS1 in DENV-infected cells; however, their functional role in DENV replication is not clearly understood. In the present study, we investigated the role of hnRNP C1/C2 in DENV replication by using an in vitro model of DENV infection in a hepatocyte cell line (Huh7) and siRNA-mediated knockdown of hnRNP C1/C2.

Multiple sclerosis (MS) is the most common demyelinating disorder of the central nervous system (CNS). Data suggest that antibodies to CNS targets contribute to the pathogenesis of MS. MS patients produce autoantibodies to heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). hnRNP A1 is an RNA binding protein (RBP) overexpressed in neurons that functions in pre-mRNA splicing, mRNA trafficking, and translation. Previously, we showed that anti-hnRNP A1 antibodies entered neuronal cells (in vitro) via clathrin-mediated endocytosis, caused mislocalization of endogenous hnRNP A1 protein and increased markers of neurodegeneration including decreased ATP concentration and apoptosis. In this study, we hypothesized that anti-hnRNP A1 antibodies might cause stress granule formation and altered levels of RNAs and proteins that bind hnRNP A1.

Mitochondrial composition varies by organ and their constituent cell types. This mitochondrial diversity likely determines variations in mitochondrial function. However, the heterogeneity of mitochondria in the brain remains underexplored despite the large diversity of cell types in neuronal tissue. Here, we used molecular systems biology tools to address whether mitochondrial composition varies by brain region and neuronal cell type in mice. We reasoned that proteomics and transcriptomics of microdissected brain regions combined with analysis of single-cell mRNA sequencing (scRNAseq) could reveal the extent of mitochondrial compositional diversity. We selected nuclear encoded gene products forming complexes of fixed stoichiometry, such as the respiratory chain complexes and the mitochondrial ribosome, as well as molecules likely to perform their function as monomers, such as the family of SLC25 transporters. We found that the proteome encompassing these nuclear-encoded mitochondrial genes and obtained from microdissected brain tissue segregated the hippocampus, striatum, and cortex from each other. Nuclear-encoded mitochondrial transcripts could only segregate cell types and brain regions when the analysis was performed at the single-cell level. In fact, single-cell mitochondrial transcriptomes were able to distinguish glutamatergic and distinct types of GABAergic neurons from one another. Within these cell categories, unique SLC25A transporters were able to identify distinct cell subpopulations. Our results demonstrate heterogeneous mitochondrial composition across brain regions and cell types. We postulate that mitochondrial heterogeneity influences regional and cell type-specific mechanisms in health and disease.

Corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) are pivotal mediators of the hormonal response to stressors and are found within neurons of the paraventricular nucleus of the hypothalamus (PVN) and several extrahypothalamic sites where expression is activity-dependent. Previous work has shown increased CRH immunoreactivity in extrahypothalamic sites after kainic-acid (KA)-induced seizures in male rats. This study examined the induction of CRH heterogeneous nuclear RNA (hnRNA), AVP hnRNA and c-fos as a measure of gene transcription and cell activation following kainic-acid (KA)-induced seizures. KA or saline was administered to intact male rats, ovariectomized (OVX) females and OVX females treated with 17beta-estradiol (E2). Animals were sacrificed 0, 15, 60 or 120 min following KA treatment. In the PVN, CRH hnRNA levels were increased by KA treatment at 15, 60, and 120 min. AVP hnRNA and c-fos mRNA in the PVN were also significantly elevated above controls at all time points. Elevations in CRH hnRNA were also identified in hippocampus, the lateral bed nucleus of the stria terminalis (BNST) and globus pallidus at 60 and 120 min following KA and in the piriform cortex, and central nucleus of the amygdala at 120 min after KA. CRH hnRNA levels at 120 min in the PVN, amygdala, cingulate cortex, hippocampus (CA1), piriform cortex, and BNST were lower in OVX+E2 females compared to females without E2. To determine if the increases in CRH hnRNA translated to increased CRH peptide, immunocytochemistry was performed. CRH immunoreactivity was increased in the amygdala, BNST, cingulate cortex, PVN and globus pallidus within 3 h after KA treatment and in the piriform cortex and hippocampus by 6 h after KA. These results suggest a time-dependent activation of the CRH system following activation of kainate receptors, which may result in long-term changes in the expression of extrahypothalamic CRH.