hnRNP C1 Research Articles

A splicing silencer in SMN2 intron 6 is critical in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a fatal neuromuscular disease caused by homozygous deletions or mutations of the SMN1 gene. SMN2 is a paralogous gene of SMN1 and a modifying gene of SMA. A better understanding of how SMN2 exon 7 splicing is regulated helps discover new therapeutic targets for SMA therapy. Based on an antisense walk method to map exonic and intronic splicing silencers (ESSs and ISSs) in SMN2 exon 7 and the proximal regions of its flanking introns, we identified one ISS (ISS6-KH) at upstream of the branch point site in intron 6. By using mutagenesis-coupled RT-PCR with SMN1/2 minigenes, immunochromatography, overexpression and siRNA-knockdown, we found this ISS consists of a bipartite hnRNP A1 binding cis-element and a poly-U sequence located between the proximal hnRNP A1 binding site (UAGCUA) and the branch site. Both HuR and hnRNP C1 proteins promote exon 7 skipping through the poly-U stretch. Mutations or deletions of these motifs lead to efficient SMN2 exon 7 inclusion comparable to SMN1 gene. Furthermore, we identified an optimal antisense oligonucleotide that binds the intron six ISS and causes striking exon 7 inclusion in the SMN2 gene in patient fibroblasts and SMA mouse model. Our findings demonstrate that this novel ISS plays an important role in SMN2 exon 7 skipping and highlight a new therapeutic target for SMA therapy.

Variant in NHLRC2 leads to increased hnRNP C2 in developing neurons and the hippocampus of a mouse model of FINCA\xa0disease

BackgroundFINCA disease is a pediatric cerebropulmonary disease caused by variants in the NHL repeat-containing 2 (NHLRC2) gene. Neurological symptoms are among the first manifestations of FINCA disease, but the consequences of NHLRC2 deficiency in the central nervous system are currently unexplored.MethodsThe orthologous mouse gene is essential for development, and its complete loss leads to early embryonic lethality. In the current study, we used CRISPR/Cas9 to generate an Nhlrc2 knockin (KI) mouse line, harboring the FINCA patient missense mutation (c.442G > T, p.Asp148Tyr). A FINCA mouse model, resembling the compound heterozygote genotype of FINCA patients, was obtained by crossing the KI and Nhlrc2 knockout mouse lines. To reveal NHLRC2-interacting proteins in developing neurons, we compared cortical neuronal precursor cells of E13.5 FINCA and wild-type mouse embryos by two-dimensional difference gel electrophoresis.ResultsDespite the significant decrease in NHLRC2, the mice did not develop severe early onset multiorgan disease in either sex. We discovered 19 altered proteins in FINCA neuronal precursor cells; several of which are involved in vesicular transport pathways and actin dynamics which have been previously reported in other cell types including human to have an association with dysfunctional NHLRC2. Interestingly, isoform C2 of hnRNP C1/C2 was significantly increased in both developing neurons and the hippocampus of adult female FINCA mice, connecting NHLRC2 dysfunction with accumulation of RNA binding protein.ConclusionsWe describe here the first NHLRC2-deficient mouse model to overcome embryonic lethality, enabling further studies on predisposing and causative mechanisms behind FINCA disease. Our novel findings suggest that disrupted RNA metabolism may contribute to the neurodegeneration observed in FINCA patients.

RRM but not the Asp/Glu domain of hnRNP C1/C2is required for splicing regulation of Ron exon 11 pre-mRNA

The Ron proto-oncogene is a human receptor for macrophage-stimulating protein (MSP). The exclusion of exon 11 in alternative splicing generates ΔRON protein that is constitutively activated. Heterogenous ribonucleaoprotein (hnRNP) C1/C2 is one of the most abundant proteins in cells. In this manuscript, we showed that both hnRNP C1 and C2 promoted exon 11 inclusion of Ron pre-mRNA and that hnRNP C1 and hnRNP C2 functioned independently but not cooperatively. Moreover, hnRNP C1 stimulated exon 11 splicing through intron 10 activation but not through intron 11 splicing. Furthermore, we showed that, whereas the RRM domain was required for hnRNP C1 function, the Asp/Glu domain was not. In conclusion, hnRNP C1/C2 promoted exon 11 splicing independently by stimulating intron 10 splicing through RRM but not through the Asp/Glu domain.

Heterogeneous Nuclear Ribonucleoprotein C Proteins Interact with the Human Papillomavirus Type 16 (HPV16) Early 3′-Untranslated Region and Alleviate Suppression of HPV16 Late L1 mRNA Splicing

In order to identify cellular factors that regulate human papillomavirus type 16 (HPV16) gene expression, cervical cancer cells permissive for HPV16 late gene expression were identified and characterized. These cells either contained a novel spliced variant of the L1 mRNAs that bypassed the suppressed HPV16 late, 5'-splice site SD3632; produced elevated levels of RNA-binding proteins SRSF1 (ASF/SF2), SRSF9 (SRp30c), and HuR that are known to regulate HPV16 late gene expression; or were shown by a gene expression array analysis to overexpress the RALYL RNA-binding protein of the heterogeneous nuclear ribonucleoprotein C (hnRNP C) family. Overexpression of RALYL or hnRNP C1 induced HPV16 late gene expression from HPV16 subgenomic plasmids and from episomal forms of the full-length HPV16 genome. This induction was dependent on the HPV16 early untranslated region. Binding of hnRNP C1 to the HPV16 early, untranslated region activated HPV16 late 5'-splice site SD3632 and resulted in production of HPV16 L1 mRNAs. Our results suggested that hnRNP C1 controls HPV16 late gene expression.

Poster #S173 METABOTROPIC GLUTAMATE RECEPTOR 5 DYSREGULATION IN SCHIZOPHRENIA

(MBP), another myelinationand schizophrenia-related gene, is the bestcharacterized QKI mRNA ligand in mice (Larocque et al., 2002). Therefore, we tested this hypothesis using the human immature oligodendrocytic cell line, that levels of hnRNP C1 and C2 would influence the expression of these myelination-related genes. We also attempted to elucidate the functional differences between hnRNP C1 and C2. Methods: We constructed expression vectors for the two hnRNP C variants (hnRNP C1 and C2), and investigated, using the quantitative real-time RT-PCR, whether the overexpression of these proteins would change the expression of myelination-related genes, such as the QKI isoforms (QKI-5, -6, -7, and -7b) and MBP, in the human immature oligodendrocytic cell lime MO3.13 and during differentiation of this cell line by PMA treatment. Results: Endogenous expressions of both hnRNP C1 and C2 were found decreased during MO3.13 differentiation. In the same condition, the levels of expression of QKIs (QKI-5, -6, -7, and -7b) were significantly elevated. The up-regulation of QKIs were not influenced by overexpression of either the hnRNP C or C2. MBP was increased during differentiation of MO3.13, consistent with previous reports (Buntinx et al., 2003; Boscia et al., 2012). The up-regulations were not influenced by overexpression of either the hnRNP C1 or C2. However, intriguingly, MBP was up-regulated under the overexpression of hnRNP C2, but not hnRNP C1, in PMA-untreated cells. Discussion: We observed that the overexpression of hnRNP C1 and C2 do not affect the differential expression of QKIs and MBP during MO3.13 differentiation. But MBP seems to be regulated by hnRNP C2 in PMA-untreated cells. Similarly, we have reported previously that hnRNP C2 regulates MBP expression in a direct fashion, and that such regulation is not mediated by QKI in the human neuroblastoma cell line SK-N-SH (Iwata et al., 2011). Thus, we propose that the mechanism underlying the differential expression of hnRNP C1/2 in schizophrenia patients brains may involve changes of MBP expression. MBP has been found differentially expressed at gene and protein level (Martins-de-Souza et al. 2012; Matthews et al. 2012). Although no functional differences have been reported thus far for the two variants of hnRNP C protein, our findings indicate the existence of distinct functions between hnRNP C1 and C2. Further exploration of separate roles of hnRNP C1 and C2 associated to schizophrenia will lead to a better understanding the pathobiology of this disorder in a molecular level.

Binding Of Immune Serine Proteases To Nucleic Acids Enhances Their Nuclear Localization and Promotes Their Cleavage Of Nucleic Acid-Binding Protein Substrates

When released from cytotoxic T lymphocytes and natural killer cells, Granzyme (Gzm) serine proteases induce programmed cell death of pathogen-infected cells and tumor cells. The Gzms rapidly accumulate in the target cell nucleus by an unknown mechanism. Many of the known substrates of GzmA and GzmB, the most abundant killer cell proteases, bind to DNA or RNA. Gzm substrates predicted by unbiased proteomics studies are also highly enriched for nucleic acid binding proteins. Here we show by fluorescence polarization assays that Gzms bind DNA and RNA with nanomolar affinity. We hypothesized that Gzm binding to nucleic acids enhances nuclear accumulation in target cells and facilitates their cleavage of nucleic acid-binding substrates. In fact, RNase treatment of cell lysates reduced cleavage of RNA binding protein (RBP) targets by GzmA and GzmB. Moreover, adding RNA to recombinant RBP substrates greatly enhanced in vitro cleavage by GzmB, but adding RNA to non-nucleic acid binding proteins did not. For example, exogenous RNA enhanced GzmB cleavage of recombinant hnRNP C1 (an RBP) but not LMNB1 (a non-RBP). In addition, GzmB cleaved the RNA-binding HuR protein efficiently only when it was bound to an HuR-binding RNA oligonucleotide, but not in the presence of an equal amount of non-binding RNA. Thus, nucleic acids facilitate Gzm cleavage of nucleic acid binding substrates.To evaluate whether nucleic acid binding influences Gzm trafficking in target cells, we incubated fixed target cells with RNase and then added Gzms. RNA degradation in target cells reduced Gzm cytosolic localization and increased nuclear accumulation. Similarly, pre-incubating Gzms with exogenous competitor DNA reduced Gzm nuclear localization. The Gzms form a monophyletic clade with other immune serine proteases including neutrophil elastase (NE) and cathepsin G (CATG). Upon neutrophil activation, NE translocates to the nucleus to drive the formation of neutrophil extracellular traps (NETs). NE and CATG, but not non-immune serine proteases such as trypsin and pancreatic elastase, also bind DNA with high affinity and localize to the nucleus of permeabilized cells. Consistent with this finding, competitor DNA also blocks the nuclear localization of NE. Moreover NE and CATG localization to NETs depends on DNA binding. Thus the antimicrobial activity of NETs may depend in part upon the affinity of these proteases for DNA. Our findings indicate that high affinity nucleic acid binding is a conserved and functionally important property of serine proteases involved in cell-mediated immunity. Disclosures:Lieberman:Alnylam Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

A Proteomics Strategy for the Identification of FAT10-Modified Sites by Mass Spectrometry

The ubiquitin-like protein FAT10 (HLA-F adjacent transcript 10) is uniquely expressed in mammals. The fat10 gene is encoded in the MHC class I locus in the human genome and is related to some specific processes, such as apoptosis, immune response, and cancer. However, biological knowledge of FAT10 is limited, owing to the lack of identification of its conjugates. FAT10 covalently modifies proteins in eukaryotes, but only a few substrates of FAT10 have been reported until now, and no FATylated sites have been identified. Here, we report the proteome-scale identification of FATylated proteins by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We identified 175 proteins with high confidence as FATylated candidates. A total of 13 modified sites were identified for the first time by a modified search of the raw MS data. The modified sites were highly enriched with hydrophilic amino acids. Furthermore, the FATylation processes of hnRNP C2, PCNA, and PDIA3 were verified by a coimmunoprecipitation assay. We confirmed that most of the substrates were covalently attached to a FAT10 monomer. The functional distribution of the FAT10 targets suggests that FAT10 participates in various biological processes, such as translation, protein folding, RNA processing, and macromolecular complex assembly. These results should be very useful for investigating the biological functions of FAT10.

Altering the expression balance of hnRNP C1 and C2 changes the expression of myelination-related genes

The expression level of hnRNP C1/C2 protein has been reported to be significantly decreased in the post-mortem brain of schizophrenic patients. In this study, we investigated whether overexpression of the hnRNP C variants hnRNP C1 and C2 changed the expression of myelination-related genes in the human neuroblastoma cell line SK-N-SH. In both hnRNP C1- and C2-overexpressing cells, the expression of quaking (QKI)-6 and QKI-7 significantly increased or decreased compared to the control, respectively. Intriguingly, QKI-5 and myelin basic protein were markedly up- or down-regulated by overexpressing hnRNP C2, respectively. Our findings are the first to demonstrate distinct functions of hnRNP C1 and C2, and may be helpful in understanding the functions of these molecules. These findings indicate that altered expression levels of hnRNP C in the brain of patients with schizophrenia could be involved in the pathophysiology of this disease through alteration of the QKI isoform and myelin basic protein expression.

Heterogeneous ribonucleoprotein C displays a repressor activity mediated by T-cell intracellular antigen-1-related/like protein to modulate Fas exon 6 splicing through a mechanism involving Hu antigen R

T-cell intracellular antigen (TIA)-proteins are known regulators of alternative pre-mRNA splicing. In this study, pull-down experiments and mass spectrometry indicate that TIAR/TIAL1 and hnRNP C1/C2 are associated in HeLa nuclear extracts. Co-immunoprecipitation and GST-pull-down assays confirmed this interaction. Interestingly, binding requires the glutamine-rich (Q-rich) C-terminal domain of TIAR and the leucine-rich plus acidic residues-rich C-terminal domains of hnRNP C1/C2. This interaction also occurs in an RNA-dependent manner. Recombinant GFP-TIAR and RFP-hnRNP C1 proteins display partial nuclear co-localization when overexpressed in HeLa cells, and this requires the Q-rich domain of TIAR. hnRNP C1 overexpression in the presence of rate-limiting amounts of TIAR in HeLa and HEK293 cells affects alternative splicing of Fas and FGFR2 minigenes, promoting Fas exon 6 and FGFR2 exon K-SAM skipping, respectively. The repressor activity of hnRNP C1 on Fas exon 6 splicing is mediated by Hu antigen R (HuR). Experiments involving tethering approaches showed that the repressor capacity of hnRNP C1 is associated with an exonic splicing silencer in Fas exon 6. This effect was reversed by splice-site strengthening and is linked to its basic leucine zipper-like motif. These results suggest that hnRNP C1/C2 acts as a bridge between HuR and TIAR to modulate alternative Fas splicing.

Application of RNase in the purification of RNA-binding proteins

Application of RNase in the purification of RNA-binding proteins

Increased expression of genes in a radioresistant cell strain: modulation of hnRNP E2, Hsp90 and SSBP2 genes in γ-irradiated Chinese hamster V79 cells

The methotrexate-resistant cell strain M5, derived from Chinese hamster lung fibroblast cell line V79, provides a good model to study differential gene expression in response to stress. Genes coding for human homologues of myosin phosphatase (AF458589), filamin-like protein (AAA92644), phosphate transporter 1 (AAH199441) and hnRNP E2 (NP_035172.1) were upregulated in M5 as determined by RAP-PCR. The Gene Expression Array (GEArray) analysis showed increased expression of caspase-6 and Hsp90 in M5 cells compared to that observed in V79 cells. Genomic amplification of SSBP2 as well as its transcriptional upregulation was also detected in M5 cells. Following γ-irradiation, expressions of SSBP2, Hsp90 and hnRNP E2 increased steadily in V79 cells. However, expression of hnRNP K and hnRNP A2 genes decreased at first and then increased, while hnRNP C1 gene expression remained unaffected up to the time studied. Thus, SSBP2, Hsp90 and hnRNP E2 are involved in the radiation response of V79 cells.

A novel nucleocytoplasmic shuttling sequence of DAZAP1, a testis-abundant RNA-binding protein

Deleted in Azoospermia Associated Protein 1 (DAZAP1) is a ubiquitous RNA-binding protein highly expressed in the human and the mouse testes. It shows a dynamic subcellular localization during spermatogenesis, present predominantly in the nuclei of late-stage spermatocytes and round spermatids and translocated to the cytoplasm during spermatid elongation. To test the hypothesis that DAZAP1 shuttles between the nucleus and the cytoplasm, we studied the nuclear transport of DAZAP1 in somatic cells using immunostaining, heterokaryon formation, and mutagenesis. DAZAP1 is detected exclusively in the nucleus and has the ability to shuttle between the nucleus and the cytoplasm using a highly conserved 25 amino acid segment, designated ZNS, at its C terminus. ZNS shares no sequence homology with other known nuclear localization or export signals. Attachment of ZNS to a red fluorescent protein DsRed2 confers the nucleocytoplasmic shuttling ability to that protein. The nuclear localization of DAZAP1 depends on active transcription. In the presence of an RNA polymerase II inhibitor, DAZAP1 is retained in the cytoplasm. DAZAP1 colocalizes with hnRNP A1 and hnRNP C1 in the nucleus and is a component of the heterogeneous nuclear ribonucleoprotein particles. Our results suggest that DAZAP1 plays a key role in mRNA transport during spermatogenesis.

Spotting new DNA damage-responsive chromatin-binding proteins

In response to DNA damage, cells initiate multiple repair mechanisms that all contribute to the survival of both the cell and the organism. These responses are numerous and variable, and can include cell cycle arrest, transcriptional activation of DNA repair genes and relocalization of repair proteins to sites of DNA damage. If all else fails, in multicellular organisms the initiation of apoptosis is also a potential cellular response to DNA damage. Despite a wealth of information about these events, it is clear that we do not yet have a comprehensive picture of the cellular responses to DNA damage. In this issue of the Biochemical Journal, a proteomics approach was used by Lee et al. to identify proteins that bind to chromatin in a DNA damage-inducible manner. The proteins identified, nucleophosmin, hnRNP C1 (heterogeneous nuclear ribonucleoprotein C1) and hnRNP C2, were proteins that would not necessarily have been predicted to behave this way. These studies have the potential to be extended and contribute to our knowledge of the cellular response to DNA damage.

A proteomics approach for the identification of nucleophosmin and heterogeneous nuclear ribonucleoprotein C1/C2 as chromatin-binding proteins in response to DNA double-strand breaks

Double-strand breaks (DSBs) of chromosomal DNA trigger the cellular response that activates the pathways for DNA repair and cell-cycle checkpoints, and sometimes the pathways leading to cell death if the damage is too severe to be tolerated. Evidence indicates that, upon generation of DNA DSBs, many nuclear proteins that are involved in DNA repair and checkpoints are recruited to chromatin around the DNA lesions. In the present study we used a proteomics approach to identify DNA-damage-induced chromatin-binding proteins in a systematic way. Two-dimensional gel analysis for protein extracts of chromatin from DNA-damage-induced and control HeLa cells identified four proteins as the candidates for DNA-damage-induced chromatin-binding proteins. MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analysis identified these proteins to be NPM (nucleophosmin), hnRNP (heterogeneous nuclear ribonucleoprotein) C1, hnRNP C2 and 37-kDa laminin-receptor precursor, and the identity of these proteins was further confirmed by immunoblot analysis with specific antibodies. We then demonstrated with chromatin-binding assays that NPM and hnRNP C1/C2, the abundant nuclear proteins with pleiotropic functions, indeed bind to chromatin in a DNA-damage-dependent manner, implicating these proteins in DNA repair and/or damage response. Immunofluorescence experiments showed that NPM, normally present in the nucleoli, is mobilized into the nucleoplasm after DNA damage, and that neither NPM nor hnRNP C1/C2 is actively recruited to the sites of DNA breaks. These results suggest that NPM and hnRNP C1/C2 may function at the levels of the global context of chromatin, rather than by specifically targeting the broken DNA.

Functional Interaction of Heterogeneous Nuclear Ribonucleoprotein C with Poliovirus RNA Synthesis Initiation Complexes

We had previously demonstrated that a cellular protein specifically interacts with the 3' end of poliovirus negative-strand RNA. We now report the identity of this protein as heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Formation of an RNP complex with poliovirus RNA was severely impaired by substitution of a lysine, highly conserved among vertebrates, with glutamine in the RNA recognition motif (RRM) of recombinant hnRNP C1, suggesting that the binding is mediated by the RRM in the protein. We have also shown that in a glutathione S-transferase (GST) pull-down assay, GST/hnRNP C1 binds to poliovirus polypeptide 3CD, a precursor to the viral RNA-dependent RNA polymerase, 3D(pol), as well as to P2 and P3, precursors to the nonstructural proteins. Truncation of the auxiliary domain in hnRNP C1 (C1DeltaC) diminished these protein-protein interactions. When GST/hnRNP C1DeltaC was added to in vitro replication reactions, a significant reduction in RNA synthesis was observed in contrast to reactions supplemented with wild-type fusion protein. Indirect functional depletion of hnRNP C from in vitro replication reactions, using poliovirus negative-strand cloverleaf RNA, led to a decrease in RNA synthesis. The addition of GST/hnRNP C1 to the reactions rescued RNA synthesis to near mock-depleted levels. Furthermore, we demonstrated that poliovirus positive-strand and negative-strand RNA present in cytoplasmic extracts prepared from infected HeLa cells coimmunoprecipitated with hnRNP C1/C2. Our findings suggest that hnRNP C1 has a role in positive-strand RNA synthesis in poliovirus-infected cells, possibly at the level of initiation.

Heterogeneous nuclear ribonucleoprotein particle A/B proteins and the control of alternative splicing of the mammalian heterogeneous nuclear ribonucleoprotein particle A1 pre-mRNA.

Mammalian pre-messenger RNAs (pre-mRNAs) interact with a distinct set of proteins to form heterogeneous nuclear ribonucleoprotein particles (hnRNPs). More than 25 different hnRNP proteins have been found associated with these complexes (Pinol-Roma et al. 1988; Dreyfuss et al. 1993). The core hnRNP proteins A1, A2, B1, B2, C1, and C2 are the most abundant species and are expressed in actively growing cells to levels that are comparable to core histones (Beyer et al. 1977; Kiledjian et al. 1994). Although several observations support the notion that core hnRNP proteins participate in pre-mRNA processing, the question of whether these proteins act in a sequence-independent or a sequence-specific manner is still a matter of debate (Abdul-Manan and Williams 1996; Abdul-Manan et al. 1996; McAfee et al. 1997). Early investigations have suggested that hnRNP proteins associate randomly with many different types of pre-mRNAs and ribopolymers to form particles sharing common structures and sedimentation properties (McKnight and Miller 1979; Lamb and Daneholt 1979; Pullman and Martin 1983; Thomas et al. 1983; Wilk et al. 1983). While the great abundance of core hnRNP proteins in the nucleus of growing cells (approximately 0.2 mM in a HeLa cell nucleus) can be taken as an argument in favor of generic RNA binding activity, it is unclear what fraction of this population of hnRNP proteins is free to interact with a nascent RNA sequence that exits the RNA polymerase II transcription complex. More recently, the use of antibodies recognizing individual hnRNP proteins has revealed that some hnRNP proteins can be deposited nonrandomly on nascent pre-mRNAs (Pinol-Roma et al. 1989; Matunis et al. 1993; Wurtz et al. 1996), suggesting that binding can be sequence-specific. Given the high abundance of hnRNP proteins and their ability to bind to many types of sequences with a range of affinities (Cobianchi et al. 1988; Conway et al. 1988; Pinol-Roma et al. 1988; LeStourgeon et al. 1990; Casas-Finet et al. 1993; Abdul-Manan and Williams 1996; Abdul-Manan et al. 1996), it has remained unclear to what extent the functions of core hnRNP proteins in nuclear RNA processing always depend on their ability to bind RNA in a sequence-specific manner.

Heterogeneous nuclear ribonucleoprotein C modulates translation of c-myc mRNA in a cell cycle phase-dependent manner.

The c-myc proto-oncogene plays a key role in the proliferation, differentiation, apoptosis, and regulation of the cell cycle. Recently, it was demonstrated that the 5' nontranslated region (5' NTR) of human c-myc mRNA contains an internal ribosomal entry site (IRES). In this study, we investigated cellular proteins interacting with the IRES element of c-myc mRNA. Heterogeneous nuclear ribonucleoprotein C (hnRNP C) was identified as a cellular protein that interacts specifically with a heptameric U sequence in the c-myc IRES located between two alternative translation initiation codons CUG and AUG. Moreover, the addition of hnRNP C1 in an in vitro translation system enhanced translation of c-myc mRNA. Interestingly, hnRNP C was partially relocalized from the nucleus, where most of the hnRNP C resides at interphase, to the cytoplasm at the G(2)/M phase of the cell cycle. Coincidently, translation mediated through the c-myc IRES was increased at the G(2)/M phase when cap-dependent translation was partially inhibited. On the other hand, a mutant c-myc mRNA lacking the hnRNP C-binding site, showed a decreased level of translation at the G(2)/M phase compared to that of the wild-type message. Taken together, these findings suggest that hnRNP C, via IRES binding, modulates translation of c-myc mRNA in a cell cycle phase-dependent manner.

Nuclear DNA Helicase II/RNA Helicase A Binds to Filamentous Actin

Nuclear DNA helicase II (NDH II), also designated RNA helicase A, is a multifunctional protein involved in transcription, RNA processing, and transport. Here we report that NDH II binds to F-actin. NDH II was partially purified from HeLa nuclear extracts by ion-exchange chromatography on Bio-Rex 70 and DEAE-Sepharose. Upon gel-filtration chromatography on Sepharose 4B, partially purified NDH II resolved into two distinct peaks. The first NDH II peak, corresponding to the void volume of Sepharose 4B, displayed coelution with an abundant 42-kDa protein that was subsequently identified as actin. Several nuclear proteins such as RNA polymerase II, the U5 small nuclear ribonucleoprotein (RNP)-associated WD40 protein, and heterogeneous nuclear RNPs (hnRNPs) copurified with NDH II. However, only hnRNPs A1 and C were found together with NDH II and actin polymers during gel filtration. NDH II and hnRNP C from the HeLa nuclear extract coeluted with F-actin on Sepharose 4B in an RNase-resistant manner, whereas hnRNP A1 was nearly completely removed from F-actin-associated hnRNP complexes following RNA digestion. The association of NDH II and hnRNP C with F-actin was abolished by gelsolin, an F-actin-depolymerizing protein that fragments actin polymers into oligomers or monomers. Furthermore, NDH II co-immunoprecipitated with F-actin and hnRNP C, respectively. In vitro translated NDH II coeluted with F-actin on Sepharose 4B, whereas no coelution with F-actin was observed for in vitro translated hnRNP A1 or C1. Binding to F-actin requires an intact C terminus of NDH II and most likely a native protein conformation. Electron microscopy indicated a close spatial proximity among NDH II, hnRNP C, and F-actin within the HeLa nucleus. These results suggest an important function of NDH II in mediating the attachment of hnRNP-mRPP RNP complexes to the actin nucleoskeleton for RNA processing, transport, or other actin-related processes.

Mutational Definition of RNA-binding and Protein-Protein Interaction Domains of Heterogeneous Nuclear RNP C1

The heterogeneous nuclear ribonucleoprotein (hn- RNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of approximately 80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.

Heterogeneous nuclear ribonucleoprotein C binds exclusively to the functionally important UUUUU-motifs in the human papillomavirus type-1 AU-rich inhibitory element

We have previously identified an inhibitory, 57 nt AU-rich sequence in the HPV-1 late 3′ UTR, termed as the HPV-1 AU-rich element ( h1ARE). It contains two types of functionally important motifs: two AUUUA sequences and three UUUUU sequences. We have shown that the h1ARE interacts with heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2 and the ELAV-like HuR protein. While we have shown that HuR binds to both the AUUUA- and the UUUUU-motifs, the interaction between hnRNP C and the h1ARE has not been investigated in detail. Here, we have used recombinant (r)hnRNP C1 to study the interaction between hnRNP C1 and the h1ARE by using the UV cross-linking assay. We demonstrate that (r)hnRNP C1 cross-links specifically to the three functionally important UUUUU-motifs in the h1ARE. In contrast, (r)hnRNP does not UV cross-link to the functionally important AUUUA-motifs in the h1ARE. Conclusively, the binding ability of hnRNP C to the h1ARE correlates with its partially inhibitory function. Additionally, the recombinant AU-rich RNA binding factor 1 (AUF1) was analyzed for binding to the h1ARE by using the UV cross-linking assay, but the results revealed no specificity for the functionally important AUUUA- and UUUUU-motifs.