Case report: Neonatal-onset inflammatory bowel disease due to novel compound heterozygous mutations in DUOX2.

Very Early Onset Inflammatory Bowel Disease (VEO-IBD) is potentially associated with genetic disorders of the intestinal epithelial barrier or inborn errors of immunity (IEI). Dual oxidase 2 (DUOX2), an H2O2-producing NADPH oxidase expressed at apical enterocyte membranes, plays a crucial role in innate defense response. Biallelic DUOX2 mutations have been described only in two patients with VEO-IBD to date. We report the case of a 1-month-old female infant who presented persistent high C-reactive protein (CRP) levels from birth and anemia. Positive occult blood and very high calprotectin in the stool were detected and abdominal ultrasound showed thickened last ileal loop. Full endoscopy evaluation revealed important colon stenosis with multiple pseudo-polyploidy formations that resulted refractory to steroid therapy, requiring a partial colic resection. Histological examination of biopsy samples showed morphological features of IBD. Whole Exome Sequencing (WES) disclosed compound heterozygous variants in the DUOX2 gene: the pathogenic c.2524C>T; p.Arg842Ter and the variant of uncertain significance (VUS) c.3175C>T; p.Arg1059Cys. Molecular and functional studies showed the presence of mutant DUOX2 in the intestinal epithelium of the patient, albeit with at least 50% decreased catalytic activity. In conclusion, we describe the third patient to date with compound heterozygous variants of DUOX2, responsible for monogenic neonatal-IBD. This case expands the knowledge about Mendelian causes of VEO-IBD and DUOX2 deficiency. We suggest that DUOX2 should be part of the diagnostic evaluation of patients with suspected monogenic VEO-IBD.

VEO-IBD NOX1 variant highlights a structural region essential for NOX/DUOX catalytic activity.

Inflammatory bowel diseases (IBD) are chronic intestinal disorders that result from an inappropriate inflammatory response to the microbiota in genetically susceptible individuals, often triggered by environmental stressors. Part of this response is the persistent inflammation and tissue injury associated with deficiency or excess of reactive oxygen species (ROS). The NADPH oxidase NOX1 is highly expressed in the intestinal epithelium, and inactivating NOX1 missense mutations are considered a risk factor for developing very early onset IBD. Albeit NOX1 has been linked to wound healing and host defence, many questions remain about its role in intestinal homeostasis and acute inflammatory conditions. Here, we used in vivo imaging in combination with inhibitor studies and germ-free conditions to conclusively identify NOX1 as essential superoxide generator for microbiota-dependent peroxynitrite production in homeostasis and during early endotoxemia. NOX1 loss-of-function variants cannot support peroxynitrite production, suggesting that the gut barrier is persistently weakened in these patients. One of the loss-of-function NOX1 variants, NOX1 p. Asn122His, features replacement of an asparagine residue located in a highly conserved HxxxHxxN motif. Modelling the NOX1-p22phox complex revealed near the distal heme an internal pocket restricted by His119 and Asn122 that is part of the oxygen reduction site. Functional studies in several human NADPH oxidases show that substitution of asparagine with amino acids with larger side chains is not tolerated, while smaller side chains can support catalytic activity. Thus, we identified a previously unrecognized structural feature required for the electron transfer mechanism in human NADPH oxidases.

[Mechanism of Action of Activated Sludge Properties in Nitrogen Removal by Endogenous Denitrification Through an Intelligent Aeration-controlled A/O Process].

In order to evaluate the mechanism of action of activated sludge properties in nitrogen removal by endogenous denitrification (henceforth EDNR), a new kind of automatic oxygen supply device(AOSD), was applied to the A/O process. The domestication effect of the aeration mode on the activated sludge properties and microbial communities was investigated under the intelligent aeration-controlled A/O process (I-A/O)and the continuous aeration A/O process (C-A/O). The results demonstrated that the effluent NH4+-N and NO2--N components showed obvious accumulation efficiencies and activated sludge generated conspicuous limited bulking in the I-A/O process. Domesticated sludge in the I-A/O process was able to enrich more SCOD to transfer into the polymeric substances as Gly, under a rich exogenous carbon supply state, and stimulated nitrogen removal by endogenous denitrifying under a scarce exogenous carbon supply state. The EDNR rate went up to 0.83 mg·(L·h)-1 in the I-A/O process, which was more than that achieved by the C-A/O process. The microbial communities in the two processes were evaluated by the Illumina HiSeq high-throughput sequencing technology. The results showed that there was no obvious difference in the sludge microbial community diversity between the two processes, but the Candidate division TM7 proliferated in the I-A/O process, and become the abundant taxa to prompt limited filamentous sludge bulking and Gly storage capability enhancement. The oxygen supply mode of AOSD made the activated sludge properties and microbial communities to be screened selectively in the new environment, aerobic heterotrophic bacterial activity to decline, and endogenous denitrifying action to strengthen, which made the I-A/O process implement a kind of dynamic balanced state that limited the DO demand.

Quantitative interaction analysis permits molecular insights into functional NOX4 NADPH oxidase heterodimer assembly.

Protein-protein interactions critically regulate many biological systems, but quantifying functional assembly of multipass membrane complexes in their native context is still challenging. Here, we combined modeling-assisted protein modification and information from human disease variants with a minimal-size fusion tag, split-luciferase-based approach to probe assembly of the NADPH oxidase 4 (NOX4)-p22phox enzyme, an integral membrane complex with unresolved structure, which is required for electron transfer and generation of reactive oxygen species (ROS). Integrated analyses of heterodimerization, trafficking, and catalytic activity identified determinants for the NOX4-p22phox interaction, such as heme incorporation into NOX4 and hot spot residues in transmembrane domains 1 and 4 in p22phox Moreover, their effect on NOX4 maturation and ROS generation was analyzed. We propose that this reversible and quantitative protein-protein interaction technique with its small split-fragment approach will provide a protein engineering and discovery tool not only for NOX research, but also for other intricate membrane protein complexes, and may thereby facilitate new drug discovery strategies for managing NOX-associated diseases.

Modification of histones by sugar ß-N-acetylglucosamine (GlcNAc) occurs on multiple residues, including histone H3 serine 10, and is cell cycle-regulated.

The monosaccharide, ß-N-acetylglucosamine (GlcNAc), can be added to the hydroxyl group of either serines or threonines to generate an O-linked ß-N-acetylglucosamine (O-GlcNAc) residue (Love, D. C., and Hanover, J. A. (2005) Sci. STKE 2005 312, 1-14; Hart, G. W., Housley, M. P., and Slawson, C. (2007) Nature 446, 1017-1022). This post-translational protein modification, termed O-GlcNAcylation, is reversible, analogous to phosphorylation, and has been implicated in many cellular processes. Here, we present evidence that in human cells all four core histones of the nucleosome are substrates for this glycosylation in the relative abundance H3, H4/H2B, and H2A. Increasing the intracellular level of UDP-GlcNAc, the nucleotide sugar donor substrate for O-GlcNAcylation enhanced histone O-GlcNAcylation and partially suppressed phosphorylation of histone H3 at serine 10 (H3S10ph). Expression of recombinant H3.3 harboring an S10A mutation abrogated histone H3 O-GlcNAcylation relative to its wild-type version, consistent with H3S10 being a site of histone O-GlcNAcylation (H3S10glc). Moreover, O-GlcNAcylated histones were lost from H3S10ph immunoprecipitates, whereas immunoprecipitation of either H3K4me3 or H3K9me3 (active or inactive histone marks, respectively) resulted in co-immunoprecipitation of O-GlcNAcylated histones. We also examined histone O-GlcNAcylation during cell cycle progression. Histone O-GlcNAcylation is high in G(1) cells, declines throughout the S phase, increases again during late S/early G(2), and persists through late G(2) and mitosis. Thus, O-GlcNAcylation is a novel histone post-translational modification regulating chromatin conformation during transcription and cell cycle progression.

Molecular characterization of nuclear DNA helicase II (RNA helicase A).

Nuclear DNA helicase II (NDH II) was first isolated from calf thymus using a DNA-unwinding assay. Subsequently it has been shown to be a homologue of human RNA helicase A (RHA) and the maleless protein (MLE) from Drosophila. Accordingly, the protein possesses both DNA and RNA unwinding activities. Also, it can use all four NTPs or dNTPs to fuel the reaction. At its N-terminus it possesses two double-strand RNA binding domains (dsRBD I and II), while the C-terminus comprises an imperfect glycine (G)- and arginine (R)-rich repeat, a so-called RGG-box that preferably binds to ssDNA or ssRNA. Many proteins interact with NDH II both at its N- and C-terminus and thereby mediate transcriptional regulation, RNA processing, and transport, the DNA damage response and genome surveillance. The latter includes the histone variant gamma-H2AX and the Werner syndrome helicase (WRN). Here we describe experimental approaches to obtain mechanistic information about this important nuclear helicase.

A putative colR(XC1049)-colS(XC1050) two-component signal transduction system in Xanthomonas campestris positively regulates hrpC and hrpE operons and is involved in virulence, the hypersensitive response and tolerance to various stresses.

The ColR-ColS two-component signal transduction system was originally characterized as a regulatory system involved in the capacity of root-colonizing biocontrol bacterium Pseudomonas fluorescens to colonize plant roots. There are three pairs of putative colR-colS two-component regulatory systems annotated in the phytopathogen Xanthomonas campestris pathovar campestris. Mutational studies revealed that one of them, named colR(XC1049) and colS(XC1050), is a global regulatory system involved in various cellular processes, including virulence, hypersensitive response and stress tolerance. Growth rate determination showed that, although the colR(XC1049) and colS(XC1050) mutants are not auxotrophic, colR(XC1049) and colS(XC1050) are required for the pathogen to proliferate well in standard media and host plants. Assays of beta-glucuronidase activities of plasmid-driven promoter-gusA reporters and/or semi-quantitative RT-PCR demonstrated that colR(XC1049) and colS(XC1050) positively regulate expression of hrpC and hrpE operons, and that expression of colR(XC1049) and colS(XC1050) is not controlled by key hrp regulators HrpG and HrpX.

Comparative and functional genomics reveals genetic diversity and determinants of host specificity among reference strains and a large collection of Chinese isolates of the phytopathogen Xanthomonas campestris pv. campestris.

BACKGROUND: Xanthomonas campestris pathovar campestris (Xcc) is the causal agent of black rot disease of crucifers worldwide. The molecular genetic diversity and host specificity of Xcc are poorly understood. RESULTS: We constructed a microarray based on the complete genome sequence of Xcc strain 8004 and investigated the genetic diversity and host specificity of Xcc by array-based comparative genome hybridization analyses of 18 virulent strains. The results demonstrate that a genetic core comprising 3,405 of the 4,186 coding sequences (CDSs) spotted on the array are conserved and a flexible gene pool with 730 CDSs is absent/highly divergent (AHD). The results also revealed that 258 of the 304 proved/presumed pathogenicity genes are conserved and 46 are AHD. The conserved pathogenicity genes include mainly the genes involved in type I, II and III secretion systems, the quorum sensing system, extracellular enzymes and polysaccharide production, as well as many other proved pathogenicity genes, while the AHD CDSs contain the genes encoding type IV secretion system (T4SS) and type III-effectors. A Xcc T4SS-deletion mutant displayed the same virulence as wild type. Furthermore, three avirulence genes (avrXccC, avrXccE1 and avrBs1) were identified. avrXccC and avrXccE1 conferred avirulence on the hosts mustard cultivar Guangtou and Chinese cabbage cultivar Zhongbai-83, respectively, and avrBs1 conferred hypersensitive response on the nonhost pepper ECW10R. CONCLUSION: About 80% of the Xcc CDSs, including 258 proved/presumed pathogenicity genes, is conserved in different strains. Xcc T4SS is not involved in pathogenicity. An efficient strategy to identify avr genes determining host specificity from the AHD genes was developed.

Werner syndrome helicase (WRN), nuclear DNA helicase II (NDH II) and histone gammaH2AX are localized to the centrosome.

Werner syndrome helicase (WRN) was found in the centrosome of human cells, both in interphase and in mitosis. Nuclear DNA helicase II (NDH II), also called RNA helicase A (RHA), an interaction partner of WRN, was also present in the centrosome. NDH II localized to the centrosome in interphase but left the centrosome with the ongoing progression of mitosis. The localization of NDH II to the centrosome was hardly affected by cytochalasin D that depolymerizes actin filaments. In contrast, treatment by the microtubules disrupting agent nocodazole strikingly detached NDH II from the centrosome, which was in contrast to WRN that remained there under this condition. Treatment of cells with the DNA damaging agent 4-nitroquinoline-1-oxide (4NQO) released NDH II, but not WRN from the centrosome. Surprisingly, the double-stranded DNA break repair-induced histone variant gammaH2AX was also found in centrosomes of interphase and mitotic cells. Following DNA damage by 4NQO, gammaH2AX left the centrosome with similar kinetics as NDH II. In vitro pull-down assays confirmed a direct physical interaction between these two proteins. Since NDH II associated with gammaH2AX after DNA damage, we suggest that complex formation between NDH II and gammaH2AX may occur in pre-assembled complexes at the centrosome, which are subsequently recruited to sites of damaged DNA for inducing the repair process.

Centrosomal localization of DNA damage checkpoint proteins.

During mitosis, the phosphatidylinositol-3 (PI-3) family-related DNA damage checkpoint kinases ATM and ATR were found on the centrosomes of human cells. ATRIP, an interaction partner of ATR, as well as Chk1 and Chk2, the downstream targets of ATR or ATM, were also localized to the centrosomes. Surprisingly, the DNA-PK inhibitor vanillin enhanced the level of ATM on centrosomes. Accordingly, DNA-PKcs, the catalytic subunit of DNA-PK, was also found on the centrosomes. Vanillin altered the phosphorylation of Chk2 in the centrosomes and in whole cell extracts. Nucleoplasmic ATM co-immunoprecipitated with Ku70/86, the DNA binding subunits of DNA-PK, while vanillin diminished this association. Vanillin did not affect microtubule polymerization at the centrosomes but, surprisingly, caused a transient enhancement of alpha-tubulin foci in the nucleus. Interestingly, gamma-tubulin was also present in the nucleus and co-immunoprecipitated with ATR or BRCA1. DNA damage led to a reduction of the mentioned checkpoint proteins on the centrosomes but increased the level of gamma-tubulin at this organelle. Taken together, these results indicate that DNA damage checkpoint proteins may control the formation of gamma-tubulin and/or the kinetics of microtubule formation at the centrosomes, and thereby couple them to the DNA damage response.

Nuclear DNA helicase II (RNA helicase A) interacts with Werner syndrome helicase and stimulates its exonuclease activity.

Nuclear DNA helicase II (NDH II), alternatively named RNA helicase A, is involved in transcription and RNA processing. Here, we report that NDH II interacts with the Werner syndrome helicase WRN, an enzyme associated with premature aging and predisposition to tumorigenesis. NDH II was co-purified with WRN, DNA polymerase delta, and replication protein A (70 kDa) during several steps of conventional column chromatography. Co-immunoprecipitations revealed an association between NDH II, WRN, and polymerase delta. We demonstrate a direct protein-protein interaction between WRN and NDH II that is mediated by the N-terminal double-strand RNA-binding domain II and C-terminal RGG box of NDH II and the N-terminal exonuclease domain of WRN. WRN inhibited the DNA-dependent NTPase and DNA helicase activities of NDH II. On the other hand, the 3' --> 5' exonuclease activity of WRN was increased by the presence of NDH II. NDH II directly stimulated the exonuclease domain of WRN, whereas the exonuclease domain of WRN suppressed the DNA-dependent (but not RNA-dependent) ATPase activity of NDH II. These results suggest that the double-strand RNA-binding domain II and RGG box of NDH II together form a protein-protein interaction surface that contacts the exonuclease domain of WRN. Furthermore, NDH II enhanced the degradation of D-loop DNA by the WRN exonuclease. Taken together, these results suggest that NDH II plays a role in promoting the DNA processing function of WRN, which in turn might be necessary for maintaining genomic stability.

Actinomycin D induces histone gamma-H2AX foci and complex formation of gamma-H2AX with Ku70 and nuclear DNA helicase II.

Formation of gamma-H2AX foci is a P. O.cellular response to genotoxic stress, such as DNA double strand breaks or stalled replication forks. Here we show that gamma-H2AX foci were also formed when cells were incubated with 0.5 microg/ml DNA intercalating agent actinomycin D. In untreated cells, gamma-H2AX co-immunoprecipitated with Ku70, a subunit of DNA-dependent protein kinase, as well as with nuclear DNA helicase II (NDH II), a DEXH family helicase also known as RNA helicase A or DHX9. This association was increased manifold after actinomycin D treatment. DNA degradation diminished the amount of Ku70 associated with gamma-H2AX but not that of NDH II. In vitro binding studies with recombinant NDH II and H2AX phosphorylated by DNA-dependent protein kinase confirmed a direct physical interaction between NDH II and gamma-H2AX. Thereby, the NDH II DEXH domain alone, i.e. its catalytic core, was able to support binding to gamma-H2AX. Congruently, after actinomycin D treatment, NDH II accumulated in RNA-containing nuclear bodies that predominantly co-localized with gamma-H2AX foci. Taken together, these results suggest that histone gamma-H2AX promotes binding of NDH II to transcriptionally stalled sites on chromosomal DNA.

Nucleolar localization of the human telomeric repeat binding factor 2 (TRF2).

The telomeric repeat binding factor 2 (TRF2) specifically recognizes TTAGGG tandem repeats at chromosomal ends. Unexpectedly immunofluorescence studies revealed a prominent nucleolar localization of TRF2 in human cells, which appeared as discrete dots with sizes similar to those present in the nucleoplasm. The TRF2 dots did not overlap with dots stemming from the upstream binding factor (UBF) or the B23 protein. After treatment with a low concentration of actinomycin D (0.05 microg/ml), TRF2 remained in the nucleolus, although this condition selectively inhibited RNA polymerase I and led to a relocalization of UBF and B23. TRF2 was prominent in the nucleolus at G0 and S but seemed to diffuse out of the nucleolus in G2 phase. During mitosis TRF2 dispersed from the condensed chromosomes and returned to the nucleolus at cytokinesis. Treatment with low doses of actinomycin D delayed the release of TRF2 from the nucleolus as cells progressed from G2 phase into mitosis. With actinomycin D present TRF2 was detected in discrete foci adjacent to UBF in prophase, while in metaphase a complete overlap between TRF2 and UBF was observed. TRF2 was present in DNase-insensitive complexes of nucleolar extracts, whereas DNA degradation disrupted the protein-DNA complexes consisting of Ku antigen and B23. Following treatment with actinomycin D some of the mitotic cells displayed chromosome end-to-end fusions. This could be correlated to the actinomycin D-suppressed relocalization of TRF2 from the nucleolus to the telomeres during mitosis. These results support the view that the nucleolus may sequester TRF2 and thereby influences its telomeric functions.

Multiple functions of nuclear DNA helicase II (RNA helicase A) in nucleic acid metabolism.

Nuclear DNA helicase II (NDH II), or RNA helicase A (RHA), was initially discovered in mammals by conventional protein purification methods. Molecular cloning identified apparent sequence homologies between NDH II and a Drosophila protein named maleless (MLE), the latter being essential for the Drosophila X-chromosome dosage compensation. Increasing amounts of evidence suggest that NDH II is involved in multiple aspects of cellular and viral DNA and RNA metabolism. Moreover the functions of NDH II may have potential clinical implications related to viral infection, autoimmune diseases, or even tumorigenesis.

Nuclear DNA helicase II (RNA helicase A) binds to an F-actin containing shell that surrounds the nucleolus.

Nuclear DNA helicase II (NDH II), alternatively named RNA helicase A (RHA), is an F-actin binding protein that is particularly enriched in the nucleolus of mouse cells. Here, we show that the nucleolar localization of NDH II of murine 3T3 cells depended on an ongoing rRNA synthesis. NDH II migrated out of the nucleolus after administration of 0.05 microg/ml actinomycin D, while nucleolin and the upstream binding factor (UBF) remained there. In S phase-arrested mouse cells, NDH II was frequently found at the nucleolar periphery, where it was accompanied by newly synthesized nucleolar RNA. Human NDH II was mainly distributed through the whole nucleoplasm and not enriched in the nucleoli. However, in the human breast carcinoma cell line MCF-7, NDH II was also found at the nucleolar periphery, together with the tumor suppressor protein p53. Both NDH II and p53 were apparently attached to the F-actin-based filamentous network that surrounded the nucleoli. Accordingly, this subnuclear structure was sensitive to F-actin depolymerizing agents. Depolymerization with gelsolin led to a striking accumulation of NDH II in the nucleoli of MCF-7 cells. This effect was abolished by RNase, which extensively released nucleolus-bound NDH II when added together with gelsolin. Taken together, these results support the idea that an actin-based filamentous network may anchor NDH II at the nucleolar periphery for pre-ribosomal RNA processing, ribosome assembly, and/or transport.

DNA-dependent protein kinase (DNA-PK) phosphorylates nuclear DNA helicase II/RNA helicase A and hnRNP proteins in an RNA-dependent manner.

An RNA-dependent association of Ku antigen with nuclear DNA helicase II (NDH II), alternatively named RNA helicase A (RHA), was found in nuclear extracts of HeLa cells by immunoprecipitation and by gel filtration chromatography. Both Ku antigen and NDH II were associated with hnRNP complexes. Two-dimensional gel electrophoresis showed that Ku antigen was most abundantly associated with hnRNP C, K, J, H and F, but apparently not with others, such as hnRNP A1. Unexpectedly, DNA-dependent protein kinase (DNA-PK), which comprises Ku antigen as the DNA binding subunit, phosphorylated hnRNP proteins in an RNA-dependent manner. DNA-PK also phosphorylated recombinant NDH II in the presence of RNA. RNA binding assays displayed a preference of DNA-PK for poly(rG), but not for poly(rA), poly(rC) or poly(rU). This RNA binding affinity of DNA-PK can be ascribed to its Ku86 subunit. Consistently, poly(rG) most strongly stimulated the DNA-PK-catalyzed phosphorylation of NDH II. RNA interference studies revealed that a suppressed expression of NDH II altered the nuclear distribution of hnRNP C, while silencing DNA-PK changed the subnuclear distribution of NDH II and hnRNP C. These results support the view that DNA-PK can also function as an RNA-dependent protein kinase to regulate some aspects of RNA metabolism, such as RNA processing and transport.

Members of the NF90/NFAR protein group are involved in the life cycle of a positive-strand RNA virus.

A major issue of current virology concerns the characterization of cellular proteins that operate as functional components of the viral multiplication process. Here we describe a group of host factors designated as 'NFAR proteins' that are recruited by the replication machinery of bovine viral diarrhea virus, a close relative of the human pathogen hepatitis C virus. The NFAR proteins associate specifically with both the termini of the viral RNA genome involving regulatory elements in the 5' and 3' non-translated regions. Modification of the protein interaction sites in the 3' non-translated region yielded viral RNAs that were replication deficient. Viral replication was also inhibited by RNAi approaches that reduced the concentration of RNA helicase A, a member of the NFAR group, in the host cell's cytoplasm. Further experimental data suggest that NFAR proteins mediate a circular conformation of the viral genome that may be important for the coordination of translation and replication. Because NFAR proteins are presumed components of the antiviral response, we suspect that viral recruitment may also serve to weaken cellular defense mechanisms.

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.