HNRNPA2B1 | GeneID:3181 | Homo sapiens
[ ] NCBI Entrez Gene
|Gene ID||3181||Official Symbol||HNRNPA2B1|
|Synonyms||DKFZp779B0244; FLJ22720; HNRNPA2; HNRNPB1; HNRPA2; HNRPA2B1; HNRPB1; RNPA2; SNRPB1|
|Full Name||heterogeneous nuclear ribonucleoprotein A2/B1|
|Description||heterogeneous nuclear ribonucleoprotein A2/B1|
|Also Known As||heterogeneous nuclear ribonucleoprotein B1; nuclear ribonucleoprotein particle A2 protein|
|Summary||This gene belongs to the A/B subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs). The hnRNPs are RNA binding proteins and they complex with heterogeneous nuclear RNA (hnRNA). These proteins are associated with pre-mRNAs in the nucleus and appear to influence pre-mRNA processing and other aspects of mRNA metabolism and transport. While all of the hnRNPs are present in the nucleus, some seem to shuttle between the nucleus and the cytoplasm. The hnRNP proteins have distinct nucleic acid binding properties. The protein encoded by this gene has two repeats of quasi-RRM domains that bind to RNAs. This gene has been described to generate two alternatively spliced transcript variants which encode different isoforms. [provided by RefSeq]|
Orthologs and Paralogs
|GeneID:475260||HNRNPA2B1||XP_864274.1||Canis lupus familiaris|
[ ] Monoclonal and Polyclonal Antibodies
|1||abcam||ab31645||hnRNP A2B1 antibody (ab31645); Rabbit polyclonal to hnRNP A2B1|
|2||abcam||ab64800||hnRNP A2B1 antibody (ab64800); Rabbit polyclonal to hnRNP A2B1|
|3||abcam||ab6102||hnRNP A2B1 antibody [DP3B3] (ab6102); Mouse monoclonal [DP3B3] to hnRNP A2B1|
|4||acris||BM4520||HNRNPA2B1; antibody Ab|
|5||acris||BM4520S||HNRNPA2B1; antibody Ab|
|6||scbt||HNRNPA2B1||HNRNPA2B1 Antibody / HNRNPA2B1 Antibodies;|
|7||sigma||R4653||Monoclonal Anti-hnRNP-A2/B1 antibody produced in mouse ;|
|GO:0030530||Component||heterogeneous nuclear ribonucleoprotein complex|
|GO:0043047||Function||single-stranded telomeric DNA binding|
|GO:0000398||Process||nuclear mRNA splicing, via spliceosome|
MicroRNA and Targets
[ ] MicroRNA Sequences and Transcript Targets from miRBase at Sanger
|RNA Target||miRNA #||mat miRNA||Mature miRNA Sequence|
Chemicals and Drugs
[ ] Comparative Toxicogenomics Database from MDI Biological Lab
Curated [chemical–gene interactions|chemical–disease|gene–disease] data were retrieved from the Comparative Toxicogenomics Database (CTD), Mount Desert Island Biological Laboratory, Salisbury Cove, Maine. World Wide Web (URL: http://ctd.mdibl.org/). [Jan. 2009].
|Chemical and Interaction|
Gene and Diseases
Curated [chemical–gene interactions|chemical–disease|gene–disease] data were retrieved from the Comparative Toxicogenomics Database (CTD), Mount Desert Island Biological Laboratory, Salisbury Cove, Maine. World Wide Web (URL: http://ctd.mdibl.org/). [Jan. 2009].
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- [ ] Miyasaka T, et al. (2008) "Interaction of antiproliferative protein Tob with the CCR4-NOT deadenylase complex." Cancer Sci. 99(4):755-761. PMID:18377426
- [ ] Lindahl Allen M, et al. (2007) "Correlation of DNA methylation with histone modifications across the HNRPA2B1-CBX3 ubiquitously-acting chromatin open element (UCOE)." Epigenetics. 2(4):227-236. PMID:18032920
- [ ] Levesque K, et al. (2006) "Trafficking of HIV-1 RNA is mediated by heterogeneous nuclear ribonucleoprotein A2 expression and impacts on viral assembly." Traffic. 7(9):1177-1193. PMID:17004321
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- [ ] Kosturko LD, et al. (2005) "The microtubule-associated protein tumor overexpressed gene binds to the RNA trafficking protein heterogeneous nuclear ribonucleoprotein A2." Mol Biol Cell. 16(4):1938-1947. PMID:15703215
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- [ ] Satoh H, et al. (2004) "Expression of hnRNP A2/B1 proteins in small airway epithelial cells." Int J Mol Med. 14(4):605-608. PMID:15375589
- [ ] Beausoleil SA, et al. (2004) "Large-scale characterization of HeLa cell nuclear phosphoproteins." Proc Natl Acad Sci U S A. 101(33):12130-12135. PMID:15302935
- [ ] Ishikawa M, et al. (2004) "Alterations of heterogeneous nuclear RNP A2 and B1 in the hippocampus of the rat after perforant pathway lesion." Acta Neuropathol. 107(2):144-148. PMID:14608468
- [ ] Sun KH, et al. (2003) "Autoantibodies to dsDNA cross-react with the arginine-glycine-rich domain of heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) and promote methylation of hnRNP A2." Rheumatology (Oxford). 42(1):154-161. PMID:12509629
- [ ] Leonard D, et al. (2003) "hLodestar/HuF2 interacts with CDC5L and is involved in pre-mRNA splicing." Biochem Biophys Res Commun. 308(4):793-801. PMID:12927788
- [ ] Fan X, et al. (2003) "HnRNP A1 and A/B interaction with PABPN1 in oculopharyngeal muscular dystrophy." Can J Neurol Sci. 30(3):244-251. PMID:12945950
- [ ] Li J, et al. (2003) "Regulation of alternative splicing by SRrp86 and its interacting proteins." Mol Cell Biol. 23(21):7437-7447. PMID:14559993
- [ ] Satoh H, et al. (2003) "HnRNP A2/B1 proteins in nontumorous alveolar cells." Lung. 181(4):219-225. PMID:14692562
- [ ] Andersen JS, et al. (2002) "Directed proteomic analysis of the human nucleolus." Curr Biol. 12(1):1-11. PMID:11790298
- [ ] Strausberg RL, et al. (2002) "Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences." Proc Natl Acad Sci U S A. 99(26):16899-16903. PMID:12477932
- [ ] Brumwell C, et al. (2002) "Intracellular trafficking of hnRNP A2 in oligodendrocytes." Exp Cell Res. 279(2):310-320. PMID:12243756
- [ ] Fritsch R, et al. (2002) "Characterization of autoreactive T cells to the autoantigens heterogeneous nuclear ribonucleoprotein A2 (RA33) and filaggrin in patients with rheumatoid arthritis." J Immunol. 169(2):1068-1076. PMID:12097415
- [ ] Zhou Z, et al. (2002) "Comprehensive proteomic analysis of the human spliceosome." Nature. 419(6903):182-185. PMID:12226669
- [ ] Yan-Sanders Y, et al. (2002) "Increased expression of heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP) in pancreatic tissue from smokers and pancreatic tumor cells." Cancer Lett. 183(2):215-220. PMID:12065097
- [ ] Hutchison S, et al. (2002) "Distinct sets of adjacent heterogeneous nuclear ribonucleoprotein (hnRNP) A1/A2 binding sites control 5' splice site selection in the hnRNP A1 mRNA precursor." J Biol Chem. 277(33):29745-29752. PMID:12060656
- [ ] Jurica MS, et al. (2002) "Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis." RNA. 8(4):426-439. PMID:11991638
- [ ] Pioli PA, et al. (2001) "The von Hippel-Lindau protein interacts with heteronuclear ribonucleoprotein a2 and regulates its expression." J Biol Chem. 276(43):40346-40352. PMID:11517223
- [ ] Kim SH, et al. (2001) "Increased protein levels of heterogeneous nuclear ribonucleoprotein A2/B1 in fetal Down syndrome brains." J Neural Transm Suppl. (61):273-280. PMID:11771750
- [ ] Zhou J, et al. (2001) "Differential expression of the early lung cancer detection marker, heterogeneous nuclear ribonucleoprotein-A2/B1 (hnRNP-A2/B1) in normal breast and neoplastic breast cancer." Breast Cancer Res Treat. 66(3):217-224. PMID:11510693
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- [ ] Nichols RC, et al. (2000) "The RGG domain in hnRNP A2 affects subcellular localization." Exp Cell Res. 256(2):522-532. PMID:10772824
- [ ] Hamilton BJ, et al. (1999) "hnRNP A2 and hnRNP L bind the 3'UTR of glucose transporter 1 mRNA and exist as a complex in vivo." Biochem Biophys Res Commun. 261(3):646-651. PMID:10441480
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- [ ] Van Laer L, et al. (1997) "Physical mapping of the HOXA1 gene and the hnRPA2B1 gene in a YAC contig from human chromosome 7p14-p15." Hum Genet. 99(6):831-833. PMID:9187682
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The chemokine receptor CXCR4-mediated signaling cascades play an important role in cell proliferation and migration, but the underlying mechanisms by which the receptor signaling is regulated remain incompletely understood. Here, we demonstrate that CXCR4 was co-immunoprecipitated with cyclophilin A (CyPA) from the lysate of HEK293 cells stably expressing CXCR4. Although both the glutathione S-transferase-CXCR4 N- and C-terminal fusion proteins were associated with the purified CyPA, truncation of the C-terminal domain of CXCR4 robustly inhibited the receptor co-immunoprecipitation with CyPA in intact cells, thereby suggesting a critical role of the receptor C terminus in this interaction. Ligand stimulation of CXCR4 induced CyPA phosphorylation and nuclear translocation, both of which were inhibited by truncation of the C-terminal domain of CXCR4. CyPA was associated with transportin 1, and knockdown of transportin 1 by RNA interference (RNAi) blocked CXCL12-induced nuclear translocation of CyPA, thereby suggesting a transportin 1-mediated nuclear import of CyPA. CyPA formed a complex with heterogeneous nuclear ribonucleoprotein (hnRNP) A2, which underwent nuclear export in response to activation of CXCR4. Interestingly, the CXCR4-mediated nuclear export of hnRNP A2 was blocked by RNAi of CyPA. Moreover, CXCR4-evoked activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was attenuated by CyPA RNAi, by overexpression of a PPIase-deficient mutant of CyPA (CyPA-R55A), and by pretreatment of the immunosuppressive drugs, cyclosporine A and sanglifehrin A. Finally, CXCL12-induced chemotaxis of HEK293 cells stably expressing CXCR4 or Jurkat T cells was inhibited by CyPA RNAi or CsA treatment.
PURPOSE: Development of an early detection marker is one of the most important strategies for improving overall prognosis in lung cancer patients. We previously reported that hnRNP B1--an RNA binding protein--is overexpressed in lung cancer tissue from the early stage of cancer, and found that hnRNP B1 mRNA is detectable in the plasma of lung cancer patients using real-time RT-PCR. The purpose of this study was to establish a quick and simple method for detecting plasma hnRNP B1mRNA for use in screening for lung cancer. METHODS: TRC, a homogenous method for fluorescence real-time monitoring of isothermal RNA amplification using intercalation activating fluorescence DNA probe, was used to detect plasma hnRNP B1 mRNA. RESULTS: The detection limit of hnRNP B1 mRNA by TRC using synthetic control RNA or total RNA derived from a lung cancer cell line was 25 or 8.65 x 10(2) copies, respectively. Using total RNA extracted from 600 mul of plasma, we detected hnRNP B1 mRNA in 39.1% (9/23) of lung cancer patients, with levels ranging from 1.9 to 19,045.5 copies/100 ng RNA, and in 5.2% (5/97) of healthy volunteers. Copy numbers were not associated with age, gender, smoking status, or histological type of cancer. TRC could detect 10(3) copies of hnRNP B1 mRNA in 10 min. CONCLUSION: Detection of plasma hnRNP B1 mRNA by TRC is a quick, easy, and non-invasive method suitable for lung cancer screening.
A disease-causing G-to-T transversion at position +6 of BRCA1 exon 18 induces exclusion of the exon from the mRNA and, as has been suggested by in silico analysis, disrupts an ASF/SF2-dependent splicing enhancer. We show here using a pulldown assay with an internal standard that wild-type (WT) and mutant T6 sequences displayed similar ASF/SF2 binding efficiencies, which were significantly lower than that of a typical exonic splicing enhancer derived from the extra domain A exon of fibronectin. Overexpression or small interfering RNA (siRNA)-mediated depletion of ASF/SF2 did not affect the splicing of a WT BRCA1 minigene but resulted in an increase and decrease of T6 exon 18 inclusion, respectively. Furthermore, extensive mutation analysis using hybrid minigenes indicated that the T6 mutant creates a sequence with a prevalently inhibitory function. Indeed, RNA-protein interaction and siRNA experiments showed that the skipping of T6 BRCA1 exon 18 is due to the creation of a splicing factor-dependent silencer. This sequence specifically binds to the known repressor protein hnRNPA1/A2 and to DAZAP1, the involvement of which in splicing inhibition we have demonstrated. Our results indicate that the binding of the splicing factors hnRNPA1/A2 and DAZAP1 is the primary determinant of T6 BRCA1 exon 18 exclusion.
Tob protein, when overexpressed, suppresses growth of NIH3T3 cells, presumably by regulating expression of various growth-related genes. However, the molecular mechanisms underlying Tob-mediated regulation of gene expression have been obscure. To address this issue we established stable Tob-expressing cell lines and used a proteomics approach to identify Tob-interacting proteins. We found that Tob associates with the CCR4-NOT complex. The carboxyl-terminal half of Tob interacted with Cnot1, a core protein of the CCR4-NOT complex. We further showed that the deadenylase activity associated with the complex was suppressed in vitro by Tob. These results suggest that the antiproliferative activity of Tob is shown post-transcriptionally by controlling the stability of the target mRNAs in addition to its involvement in transcriptional regulation, reported previously.
The methylation-free CpG island encompassing the divergently transcribed promoters of the HNRPA2B1 and CBX3 housekeeping genes possesses a dominant ubiquitously-acting chromatin opening element (UCOE) capability. This element allows reproducible and stable transgene expression including from within centromeric heterochromatin. We present an investigation of DNA methylation and histone modification marks across the HNRPA2B1-CBX3 locus in primary peripheral blood mononuclear cells (PBMCs) to characterise the chromatin structure that underlies UCOE activity. The CpG methylation-free region associated with the UCOE extends into the central areas of HNRPA2B1 and CBX3, with a total length of approximately 5 kb. However, the DNA in the 3' half of both genes is methylated. Histone H4 lysine (K) acetylation shows a broad distribution across both genes, whilst histone H3 lysine acetylation peaks around the transcriptional start sites and drops to background levels at the 3' ends. Higher levels of H3K4 di-methylation are present at the 3' end of the genes in contrast to H3K4 tri-methylation which peaks around the transcriptional start sites. Therefore, methylated DNA in transcribed regions of these genes has been shown here to co-exist with active histone modification marks, indicating that these functionally opposing epigenetic signatures can overlap. This suggests that an extended large region of unmethylated DNA in combination with distinct histone modification patterns are at the basis of UCOE function.
Few details are known about how the human immunodeficiency virus type 1 (HIV-1) genomic RNA is trafficked in the cytoplasm. Part of this process is controlled by the activity of heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2). The role of hnRNP A2 during the expression of a bona fide provirus in HeLa cells is investigated in this study. Using immunofluorescence and fluorescence in situ hybridization techniques, we show that knockdown of hnRNP A2 expression in HIV-1-expressing cells results in the rapid accumulation of HIV-1 genomic RNA in a distinct, cytoplasmic space that corresponds to the microtubule-organizing center (MTOC). The RNA exits in the nucleus and accumulates at the MTOC region as a result of hnRNP A2 knockdown even during the expression of a provirus harboring mutations in the hnRNP A2-response element (A2RE), the expression of which results in nuclear retention of genomic RNA. We also demonstrate that hnRNP A2 expression is required for downstream trafficking of genomic RNA from the MTOC in the cytoplasm. Genomic RNA localization at the MTOC that was both the result of hnRNP A2 knockdown and the overexpression of Rab7-interacting lysosomal protein had little effect on pr55Gag synthesis but negatively influenced virus production and infectivity. These data indicate that altered HIV-1 genomic RNA localization modulates viral assembly and that the MTOC serves as a central site to which HIV-1 genomic RNA converges following its exit from the nucleus, with the host protein, hnRNP A2, playing a central role in taking it to and from this site in the cell.
BACKGROUND: S100 A2 and hnRNP A2/B1 (heterogeneous ribonucleoprotein A2/B1) with its splicing variant hnRNP B1 are proteins which are involved in cellular proliferation, differentiation and protein synthesis and are up-regulated in non-small cell lung cancer (NSCLC). Since previous studies using paraffin-embedded tissues indicated a high potential of these markers for diagnosis and screening the present analysis intended to validate these data applying cryostat sections. METHODS: 78 tumor-infiltrated lung cancer specimens and 66 adjacent histologically tumor-free tissues were analyzed; 11 autopsy specimens from patients who did not suffer from a malignant disease served as a control group. Cryostat sections were stained with monoclonal antibodies against hnRNP A2/B1, hnRNP B1 and S100 A2 and were compared with the previously established immunohistochemical profile of the same patients including EGFR, EGFRvIII, pEGFR, c-erbB-2, c-erbB-3, p53, Ki-67, bcl-2, p120 and microvessel density. Furthermore, these results were correlated with clinical parameters. RESULTS: Expression of hnRNP A2/B1, hnRNP B1 and S100 A2 was increased in the tumor group when compared with the microscopic tumor-free specimens in 10% versus 5% (n.s.), 91% versus 5% and 65% versus 6%, respectively. Increased S100 and A2 hnRNP A2/B1 expressions were negative prognostic factors. With the exception of an increased EGFR expression in hnRNP A2/B1 negative cases the three analyzed markers did not correlate with the immunohistochemical parameters tested previously. CONCLUSION: Comparison between tumor probes and tumor-free specimens of NSCLC patients failed to approve the diagnostic relevance of hnRNP A2/B1 shown in previous studies, whereas hnRNP B1 revealed a high tumor specificity that could be helpful for tumor cell screening. Moreover, S100 A2 and hnRNP A2/B1 confirmed to be valuable prognostic parameters.
By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.
The cellular prion protein (PrP(C)), a highly conserved glycoprotein predominantly expressed by neuronal cells, can convert into an abnormal isoform (PrP(Sc)) and provoke a transmissible spongiform encephalopathy. In spite of many studies, the physiological function of PrP(C) remains unknown. Recent findings suggest that PrP(C) is a multifunctional protein participating in several cellular processes. Using recombinant human PrP as a probe, we performed far-Western immunoblotting (protein overlay assay) to detect cellular PrP(C) interactors. Brain extracts of wild-type and PrP knockout mice were screened by far-Western immunoblotting for PrP-specific interactions. Subsequently, putative ligands were isolated by 2-DE and identified by MALDI-TOF MS, enabling identification of heterogeneous nuclear ribonucleoprotein A2/B1 and aldolase C as novel interaction partners of PrP(C). These data provide the first evidence of a molecule indicating a mechanism for the predicted involvement of PrP(C) in nucleic acid metabolisms. In summary, we have shown the successful combination of 2-DE with far-Western immunoblotting and MALDI-TOF MS for identification of new cellular binding partners of a known protein. Especially the application of this technique to investigate other neurodegenerative diseases is promising.
Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set with a measured error rate as determined by the target-decoy approach, we demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and we present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. We applied our methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where we identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-positive rate of 1.3%.
A hallmark of systemic lupus erythematosus (SLE) is the appearance of autoantibodies to nuclear antigens, including autoantibodies directed to the heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2), which occur in 20% to 30% of SLE patients as well as in animal models of this disease. To investigate the underlying cellular reactivity and to gain further insight into the nature and potential pathogenic role of this autoimmune response we characterized the T cell reactivity against hnRNP-A2 in patients with SLE in comparison to healthy controls. Cellular proliferation of peripheral blood T cells to hnRNP-A2 was determined by [3H]thymidine incorporation and T cell clones (TCCs) specific for hnRNP-A2 were grown by limiting dilution cloning; IFNgamma, IL-4 and IL-10 in culture supernatants were measured by ELISA. Bioactivity of culture supernatants was determined by incubation of anti-CD3/anti-CD28 stimulated peripheral blood CD4+ T cells with supernatants of TCCs. Stimulation assays performed with peripheral blood mononuclear cells of 35 SLE patients and 21 healthy controls revealed pronounced proliferative responses in 66% of SLE patients and in 24% of the controls, which were significantly higher in SLE patients (p < 0.00002). Furthermore, hnRNP-A2 specific TCCs generated from SLE patients (n = 22) contained a relatively high proportion of CD8+ clones and mostly lacked CD28 expression, in contrast to TCCs derived from healthy controls (n = 12). All CD4+ TCCs of patients and all control TCCs secreted IFNgamma and no IL-4. In contrast, CD8+ TCCs of patients secreted very little IFNgamma, while production of IL-10 did not significantly differ from other T cell subsets. Interestingly, all CD8+ clones producing IL-10 in large excess over IFNgamma lacked expression of CD28. Functional assays showed a stimulatory effect of the supernatants derived from these CD8+ CD28- hnRNP-A2 specific TCCs that was similar to that of CD4+ CD28+ clones. Taken together, the pronounced peripheral T cell reactivity to hnRNP-A2 observed in the majority of SLE patients and the distinct phenotype of patient-derived CD8+ TCCs suggest a role for these T cells in the pathogenesis of SLE.
Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a trans-acting RNA-binding protein that mediates trafficking of RNAs containing the cis-acting A2 response element (A2RE). Previous work has shown that A2RE RNAs are transported to myelin in oligodendrocytes and to dendrites in neurons. hnRNP E1 is an RNA-binding protein that regulates translation of specific mRNAs. Here, we show by yeast two-hybrid analysis, in vivo and in vitro coimmunoprecipitation, in vitro cross-linking, and fluorescence correlation spectroscopy that hnRNP E1 binds to hnRNP A2 and is recruited to A2RE RNA in an hnRNP A2-dependent manner. hnRNP E1 is colocalized with hnRNP A2 and A2RE mRNA in granules in dendrites of oligodendrocytes. Overexpression of hnRNP E1 or microinjection of exogenous hnRNP E1 in neural cells inhibits translation of A2RE mRNA, but not of non-A2RE RNA. Excess hnRNP E1 added to an in vitro translation system reduces translation efficiency of A2RE mRNA, but not of nonA2RE RNA, in an hnRNP A2-dependent manner. These results are consistent with a model where hnRNP E1 recruited to A2RE RNA granules by binding to hnRNP A2 inhibits translation of A2RE RNA during granule transport.
Collagen prolyl 4-hydroxylase (C-P4H) alpha-subunit is of regulatory importance in the assembling of C-P4H tetramers, which are necessary for the hydroxylation of procollagen chains. Change in collagen expression by hypoxia or iron diminishment is a significant issue in extracellular matrix remodeling. It was proposed that C-P4H-alpha (I) is regulated at the posttrancriptional level under these conditions. Here we report that the induction of C-P4H-alpha (I) in human fibrosarcoma cells HT1080 by the iron chelator 2,2-dipyridyl is predominantly caused by an enhancement of mRNA stability. This effect is mediated by an increased synthesis and binding of heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1, which interacts with a (U)(16) element located in the 3'-untranslated region of C-P4H-alpha (I) mRNA. Luciferase reporter gene assays depending on C-P4H-alpha (I) 3'-untranslated region and co-transfection with hnRNP-A2/B1 provide evidence that the (U)(16) element is necessary and sufficient for posttranscriptional control of C-P4H-alpha (I) synthesis under the analyzed conditions. Further indication for the significance of hnRNP-A2/B1 in C-P4H-alpha (I) induction was obtained by micro array experiments. In a data set representing 686 independent physiological conditions, we found a significant positive correlation between hnRNP-A2/B1 and C-P4H-alpha (I) mRNAs.
In neural cells, such as oligodendrocytes and neurons, transport of certain RNAs along microtubules is mediated by the cis-acting heterogeneous nuclear ribonucleoprotein A2 response element (A2RE) trafficking element and the cognate trans-acting heterogeneous nuclear ribonucleoprotein (hnRNP) A2 trafficking factor. Using a yeast two-hybrid screen, we have identified a microtubule-associated protein, tumor overexpressed gene (TOG)2, as an hnRNP A2 binding partner. The C-terminal third of TOG2 is sufficient for hnRNP A2 binding. TOG2, the large protein isoform of TOG, is the only isoform detected in oligodendrocytes in culture. TOG coimmunoprecipitates with hnRNP A2 present in the cytoskeleton (CSK) fraction of neural cells, and both coprecipitate with microtubule stabilized pellets. Staining with anti-TOG reveals puncta that are localized in proximity to microtubules, often at the plus ends. TOG is colocalized with hnRNP A2 and A2RE-mRNA in trafficking granules that remain associated with CSK-insoluble tissue. These data suggest that TOG mediates the association of hnRNP A2-positive granules with microtubules during transport and/or localization.
The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a multi-tasking protein that acts in the cytoplasm and nucleus. We have explored the possibility that this protein is associated with telomeres and participates in their maintenance. Rat brain hnRNP A2 was shown to have two nucleic acid binding sites. In the presence of heparin one site binds single-stranded oligodeoxyribonucleotides irrespective of sequence but not the corresponding oligoribonucleotides. Both the hnRNP A2-binding cis-acting element for the cytoplasmic RNA trafficking element, A2RE, and the ssDNA telomere repeat match a consensus sequence for binding to a second sequence-specific site identified by mutational analysis. hnRNP A2 protected the telomeric repeat sequence, but not the complementary sequence, against DNase digestion: the glycine-rich domain was found to be necessary, but not sufficient, for protection. The N-terminal RRM (RNA recognition motif) and tandem RRMs of hnRNP A2 also bind the single-stranded, template-containing segment of telomerase RNA. hnRNP A2 colocalizes with telomeric chromatin in the subset of PML bodies that are a hallmark of ALT cells, reinforcing the evidence for hnRNPs having a role in telomere maintenance. Our results support a model in which hnRNP A2 acts as a molecular adapter between single-stranded telomeric repeats, or telomerase RNA, and another segment of ssDNA.
Glucose transporter-1 (GLUT1) mediates uptake of glucose and is up-regulated in some cancers. The amount of this membrane protein is regulated by a post-transcriptional mechanism in which mRNA binding proteins recognize cis-acting elements in the 3'-untranslated (3'UTR) of the mRNA. To identify cis elements in GLUT1 mRNA we introduced 3'UTR sequences into the 3'UTR of the luciferase gene in a reporter construct. A 30 nt adenosine-uridine-rich element ("GLUT1 AURE") inhibited luciferase activity in HEK-293 cells. This inhibitory effect was confirmed by deleting the GLUT1 AURE from a reporter containing the full-length 3'UTR. Deletion of the GLUT1 AURE caused reporter activity to increase. Deletion of a larger fragment ("Bsu" region) containing the GLUT1 AURE increased reporter activity still further, suggesting that there are additional cis elements in the GLUT1 mRNA. The GLUT1 AURE was also active in GBM-T98G glioblastoma cells. Next, we tested the action of a trans-acting factor, hnRNP A2, on GLUT1 gene expression. We show that a cytoplasmic-localizing isoform of hnRNP A2 binds human GLUT1 RNA by gel-shift assay and by UV-crosslinking. Finally, over-expression of the hnRNP A2 isoform inhibited GLUT1 reporter expression in GBM-T98G cells. These results identify the AURE cis element in human GLUT1 mRNA and show that hnRNP A2 acts on GLUT1 mRNA to inhibit expression of GLUT1 in a brain cancer cell line.
Protein methylation is a stable post-translational modification (PTM) with important biological functions. It occurs predominantly on arginine and lysine residues with varying numbers of methyl groups, such as mono-, di- or trimethyl lysine. Existing methods for identifying methylation sites are laborious, require large amounts of sample and cannot be applied to complex mixtures. We have previously described stable isotope labeling by amino acids in cell culture (SILAC) for quantitative comparison of proteomes. In heavy methyl SILAC, cells metabolically convert [(13)CD(3)]methionine to the sole biological methyl donor, [(13)CD(3)]S-adenosyl methionine. Heavy methyl groups are fully incorporated into in vivo methylation sites, directly labeling the PTM. This provides markedly increased confidence in identification and relative quantitation of protein methylation by mass spectrometry. Using antibodies targeted to methylated residues and analysis by liquid chromatography-tandem mass spectrometry, we identified 59 methylation sites, including previously unknown sites, considerably extending the number of in vivo methylation sites described in the literature.
In order to examine the post-transcriptional regulations in Alzheimer's disease, we employed immunohistochemical techniques and examined the expression of heterogeneous nuclear ribonucleoprotein (hnRNP) B1 in the inferior temporal cortex of subjects with Alzheimer's disease. In the mild cases, intense B1 immunoreactivity was observed in neurons of layer V, and less intense immunoreactivity was observed in layers II and III. The overall distributions and intensities of B1 immunoreactivity were undistinguishable among mild, moderate, and severe cases. Double-immunolabeling with MC1 and B1 demonstrated that B1 immunoreactivity was preserved in the majority of neurofibrillary tangle (NFT)-bearing neurons. Our study suggests that hnRNP B1-associated post-transcriptional regulations are preserved in the inferior temporal cortex of Alzheimer's disease.
The viral proteins A1L, A2L, G8R, and H5R positively modulate vaccinia virus late gene expression. Host-encoded proteins hnRNP A2 and RBM3 may also interact with these viral factors to influence late gene expression. In these studies, a yeast two-hybrid screen and in vitro pulldown and crosslinking experiments were used to investigate protein--protein interactions among these factors. These studies confirmed a previous observation that G8R interacts with itself and A1L. However, self-interactions of A1L and H5R, and interactions between A2L and G8R, A2L and H5R, and H5R and G8R were also observed. In addition, the proteins hnRNP A2 and RBM3 both showed some interaction with A2L. Illustration of these interactions is a step toward understanding the architecture of the late gene transcription complex as it occurs in poxviruses.
The overexpression of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, especially of B1 has been reported informative to detect pre-clinical lung cancer for early detection. However, relatively little work has been performed on the expression of hnRNP A2/B1 in non-malignant airway cells; such information is clearly required to investigate whether overexpression of this protein could be a specific marker for early lung cancer detection. In order to evaluate whether the expression of hnRNP A2/B1 is specific for cancer cells, we investigated the expression of the protein in human small airway epithelial (HSAE) cells. Using immunostaining and Western blotting with monoclonal antibodies, 4G8 (specifically reacts A2) and 2B2 (specific to B1), expression of hnRNP A2/B1 in HSAE cells was evaluated. Northern blotting and quantitative RT-PCR were also performed. In HSAE cells, hnRNP A2 as well as B1 were expressed primarily in the nucleus excluding the nucleolus, although the expression of A2 protein was weaker than that of B1 protein. Staining pattern of hnRNP A2/B1 in HSAE cells was similar to that observed in control cancer cells. Western blotting with 4G8 and 2B2 apparently demonstrated expression of A2 and B1 in HSAE cells. In Northern blotting, both hnRNP A2 mRNA and B1 mRNA were observed in HSAE cells as shown in control cancer cells. hnRNP A2/B1 was apparently expressed in HSAE cells, although the expression in HSAE cells was weaker than that of cancer cells. More quantitative determination of A2/B1 is required to elucidate their significance in early lung cancer detection.
Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.
We examined alterations in post-transcriptional regulation following deafferentation of the perforant pathway by focusing on heterogeneous nuclear ribonucleoprotein (hnRNP) A2 and B1 in rat hippocampi subjected to perforant pathway lesions. In control brains, immunoreactivity to both was observed in the nuclei of neurons throughout the hippocampus using immunohistochemical techniques. From 1 to 14 days post-lesion, a slight increase in A2 immunoreactivity was observed in neurons within the dentate granular layer as well in the pyramidal cells of the cornus Ammon fields ipsilateral to the lesion. In contrast, we observed a marked decrease in B1 immunoreactivity in the same regions at 1, 3 and 7 days post-lesion. All these alterations, however, were transient. A2 immunoreactivity returned to normal levels by 30 days post-lesion, and B1 immunoreactivity had completely recovered by 14 days post-lesion. The results of immunoblot analysis for A2 and B1 were wholly consistent with immunohistochemical observations. Our study suggests that post-transcriptional regulation in the hippocampal neurons changes after a perforant pathway lesion. Our study further suggests that the functions of hnRNPA2 and B1 are different, as each was differentially involved in the plastic response to deafferentation of the perforant pathway.
OBJECTIVE: This study was designed to clarify the internalization of anti-DNA antibodies (anti-DNA) into living cells in the pathogenesis of systemic lupus erythematosus (SLE) using anti-DNA monoclonal antibodies (mAbs). METHODS: Anti-DNA mAbs 9D7, 9D7D2, 9A4, 5E3F5, 12B3H2 and 6E11E3 were prepared by a standard hybridoma procedure to determine the interaction of anti-DNA with proteins in different types of cells. RESULTS: The anti-DNA mAbs reacted with two protein antigens (35 and 50 kDa) in the cells. The 35-kDa antigen was shown to have 100% homology with hnRNP A2. The arginine-glycine-rich domain in hnRNP A2 was found to be the reaction site, and the methylation of hnRNP A2 by PRMT1 (protein arginine methyltransferase 1) was increased by anti-DNA. Moreover, anti-DNA was demonstrated to bind and internalize into the cytoplasm and nucleus. CONCLUSION: Nuclear localizing anti-DNA may cross-react with hnRNP A2 to modulate the inflammatory responses and polarize immune reactions associated with SLE.
hLodestar/HuF2 belongs to the SNF2 family of proteins. This family of proteins has been shown to play a critical role in altering protein-DNA interactions in a variety of cellular contexts. We have identified an unexpected interaction between hLodestar/HuF2 and CDC5L in both the yeast two-hybrid system and HeLa nuclear extract. CDC5L is a well-characterized pre-mRNA splicing factor in yeast and humans. Our findings demonstrate that hLodestar/HuF2 associates with human splicing complexes. We also found that a truncated hLodestar/HuF2 polypeptide that overlaps with the CDC5L-binding region can inhibit pre-mRNA splicing by disrupting spliceosome assembly. These findings indicate that hLodestar/HuF2 may have a role in pre-mRNA splicing. These data are consistent with a close co-ordination of the transcription and splicing pathways in eukaryotes. Although many members of the DExH/D helicase superfamily have been linked to pre-mRNA splicing, this is the first SNF2 family member to be implicated in this pathway.
BACKGROUND: Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive ptosis, dysphagia and proximal limb weakness. The autosomal dominant form of this disease is caused by short expansions of a (GCG)6 repeat to (GCG) in the PABPN1 gene. The mutations lead to the expansion of a polyalanine stretch from 10 to 12-17 alanines in the N-terminus of PABPN1. The mutated PABPN1 (mPABPN1) induces the formation of intranuclear filamentous inclusions that sequester poly(A) RNA and are associated with cell death. METHODS: Human fetal brain cDNA library was used to look for PABPNI binding proteins using yeast two-hybrid screen. The protein interaction was confirmed by GST pull-down and co-immunoprecipitation assays. Oculopharyngeal muscular dystrophy cellular model and OPMD patient muscle tissue were used to check whether the PABPN1 binding proteins were involved in the formation of OPMD intranuclear inclusions. RESULTS: We identify two PABPNI interacting proteins, hnRNP A1 and hnRNP A/B. When co-expressed with mPABPN1 in COS-7 cells, predominantly nuclear protein hnRNP A1 and A/B co-localize with mPABPN1 in the insoluble intranuclear aggregates. Patient studies showed that hnRNP A1 is sequestered in OPMD nuclear inclusions. CONCLUSIONS: The hnRNP proteins are involved in mRNA processing and mRNA nucleocytoplasmic export, sequestering of hnRNPs in OPMD intranuclear aggregates supports the view that OPMD intranuclear inclusions are "poly(A) RNA traps", which would interfere with RNA export, and cause muscle cell death.
SRrp86 is a unique member of the SR protein superfamily containing one RNA recognition motif and two serine-arginine (SR)-rich domains separated by an unusual glutamic acid-lysine (EK)-rich region. Previously, we showed that SRrp86 could regulate alternative splicing by both positively and negatively modulating the activity of other SR proteins and that the unique EK domain could inhibit both constitutive and alternative splicing. These functions were most consistent with the model in which SRrp86 functions by interacting with and thereby modulating the activity of target proteins. To identify the specific proteins that interact with SRrp86, we used a yeast two-hybrid library screen and immunoprecipitation coupled to mass spectrometry. We show that SRrp86 interacts with all of the core SR proteins, as well as a subset of other splicing regulatory proteins, including SAF-B, hnRNP G, YB-1, and p72. In contrast to previous results that showed activation of SRp20 by SRrp86, we now show that SAF-B, hnRNP G, and 9G8 all antagonize the activity of SRrp86. Overall, we conclude that not only does SRrp86 regulate SR protein activity but that it is, in turn, regulated by other splicing factors to control alternative splice site selection.
The overexpression of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, especially of B1, had been reported to detect preclinical lung cancer for early detection. In order to examine whether the expression of the protein is specific for lung cancer, we investigated the expression of the protein in human nonmalignant alveolar cells. Human airway cells obtained by bronchoalveolar lavage (BAL) were analyzed by immunostaining and Western blotting using monoclonal antibodies 4G8 (specifically reacts to A2) and 2B2 (specific to B1). In alveolar cells obtained by BAL, hnRNP A2/B1 proteins were localized primarily in the nucleus, excluding the nucleolus, although the expression of A2 protein was weaker than that of B1 protein. Staining pattern of these cells was similar to those observed in cancer cells. Western blotting with 4G8 and 2B2 demonstrated expression of A2 and B1 proteins in the airway cells. HnRNP A2/B1 proteins were apparently expressed in nonmalignant alveolar cells as well as cancer cells, although the expression in alveolar cells was weaker than that of cancer cells. More quantitative determination of A2/B1 is required to elucidate their significance in early lung cancer detection.
BACKGROUND: The nucleolus is a subnuclear organelle containing the ribosomal RNA gene clusters and ribosome biogenesis factors. Recent studies suggest it may also have roles in RNA transport, RNA modification, and cell cycle regulation. Despite over 150 years of research into nucleoli, many aspects of their structure and function remain uncharacterized. RESULTS: We report a proteomic analysis of human nucleoli. Using a combination of mass spectrometry (MS) and sequence database searches, including online analysis of the draft human genome sequence, 271 proteins were identified. Over 30% of the nucleolar proteins were encoded by novel or uncharacterized genes, while the known proteins included several unexpected factors with no previously known nucleolar functions. MS analysis of nucleoli isolated from HeLa cells in which transcription had been inhibited showed that a subset of proteins was enriched. These data highlight the dynamic nature of the nucleolar proteome and show that proteins can either associate with nucleoli transiently or accumulate only under specific metabolic conditions. CONCLUSIONS: This extensive proteomic analysis shows that nucleoli have a surprisingly large protein complexity. The many novel factors and separate classes of proteins identified support the view that the nucleolus may perform additional functions beyond its known role in ribosome subunit biogenesis. The data also show that the protein composition of nucleoli is not static and can alter significantly in response to the metabolic state of the cell.
The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http:mgc.nci.nih.gov).
Heterogeneous ribonucleoprotein (hnRNP) A2 is a trans-acting factor that mediates intracellular trafficking of specific RNAs containing the A2 response element. HnRNP A2 is localized in the nucleus and also in granules in the perikaryon and processes in oligodendrocytes. The distribution of the cytoplasmic pool of hnRNP A2 is microtubule-dependent. HnRNP A2 is composed of two sequential RNA binding domains (RBDI and RBDII), a glycine-rich domain, and a nuclear import domain (M9). In order to analyze the roles of individual domains in determining the intracellular distribution of hnRNP A2, chimeric mRNAs encoding various domains fused with green fluorescent protein (GFP) were injected into oligodendrocytes, and the subcellular distribution of the GFP hybrid proteins was analyzed by fluorescence microscopy. Chimeric GFP proteins containing the M9 domain were localized to the nucleus. In the absence of the M9 domain, proteins containing the RBDII domain were preferentially concentrated in the distal processes of the cells. Localization of RBDII-containing proteins in the periphery was dependent on the presence of intact microtubules. These data suggest that the RBDII domain of hnRNP A2 targets hnRNP A2 to the periphery of the cell in a microtubule-dependent manner.
The role of autoimmune reactions in the pathogenesis of rheumatoid arthritis (RA) is poorly understood. To address this issue we have investigated the spontaneous T cell response to two well-characterized humoral autoantigens in RA patients and controls: 1) the heterogeneous nuclear ribonucleoprotein A2, i.e., the RA33 Ag (A2/RA33), and 2) filaggrin in unmodified and citrullinated forms. In stimulation assays A2/RA33 induced proliferative responses in PBMC of almost 60% of the RA patients but in only 20% of the controls (patients with osteoarthritis or psoriatic arthritis and healthy individuals), with substantially stronger responses in RA patients (p < 0.00002). Furthermore, synovial T cells of seven RA patients investigated were also clearly responsive. In contrast, responses to filaggrin were rarely observed and did not differ between RA patients and controls. Analysis of A2/RA33-induced cytokine secretion revealed high IFN-gamma and low IL-4 production in both RA and control PBMC, whereas IL-2 production was mainly observed in RA PBMC (p < 0.03). Moreover, A2/RA33-specific T cell clones from RA patients showed a strong Th1 phenotype and secreted higher amounts of IFN-gamma than Th1 clones from controls (p < 0.04). Inhibition experiments performed with mAbs against MHC class II molecules showed A2/RA33-induced T cell responses to be largely HLA-DR restricted. Finally, immunohistochemical analyses revealed pronounced overexpression of A2/RA33 in synovial tissue of RA patients. Taken together, the presence of autoreactive Th1-like cells in RA patients in conjunction with synovial overexpression of A2/RA33 may indicate potential involvement of this autoantigen in the pathogenesis of RA.
The precise excision of introns from pre-messenger RNA is performed by the spliceosome, a macromolecular machine containing five small nuclear RNAs and numerous proteins. Much has been learned about the protein components of the spliceosome from analysis of individual purified small nuclear ribonucleoproteins and salt-stable spliceosome 'core' particles. However, the complete set of proteins that constitutes intact functional spliceosomes has yet to be identified. Here we use maltose-binding protein affinity chromatography to isolate spliceosomes in highly purified and functional form. Using nanoscale microcapillary liquid chromatography tandem mass spectrometry, we identify approximately 145 distinct spliceosomal proteins, making the spliceosome the most complex cellular machine so far characterized. Our spliceosomes comprise all previously known splicing factors and 58 newly identified components. The spliceosome contains at least 30 proteins with known or putative roles in gene expression steps other than splicing. This complexity may be required not only for splicing multi-intronic metazoan pre-messenger RNAs, but also for mediating the extensive coupling between splicing and other steps in gene expression.
Pancreatic cancer is a major cause of deaths in the United States, and has one of the lowest 5-year survival rates. Early diagnosis has not been possible due to the lack of reliable early tumor markers. The heterogeneous nuclear ribonucleoprotein A1/B2 (hnRNP) was recently shown to be up-regulated in the early stage of lung cancer. This protein plays an important role in biogenesis and transport of mRNA. Up-regulation of hnRNP usually precedes morphological differentiation and is considered a good biomarker in the early stages of cancer development. Because smoking is a high risk factor for pancreatic cancer, this study examined the expression of hnRNP in human pancreatic tissues from smokers and non-smokers. A two-fold increase in expression of hnRNP was found overall in smokers when compared to non-smokers and smokers who quit (P<0.05). The increase in expression of hnRNP was higher in female smokers compared to female non-smokers. High levels of expression was also shown in a limited number of human pancreatic adenocarcinomas and two pancreatic tumor cell lines, HPAF-11 and SU 86.86. HP-8, a normal primary pancreatic cell line, did not express hnRNP. These results strongly suggest that up-regulation of hnRNP may be a good candidate for early screening for pancreatic cancer because of its activation in pancreatic tissue from smokers and activation in pancreatic adenocarcinomas. Over-expression of hnRNP has been suggested as evidence that normal transcriptional regulation is altered.
In the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 pre-mRNA, different regions in the introns flanking alternative exon 7B have been implicated in the production of the A1 and A1B mRNA splice isoforms. Among these, the CE1a and CE4 elements, located downstream of common exon 7 and alternative exon 7B, respectively, are bound by hnRNP A1 to promote skipping of exon 7B in vivo and distal 5' splice site selection in vitro. Here, we report that CE1a is flanked by an additional high affinity A1 binding site (CE1d). In a manner similar to CE1a, CE1d affects 5' splice site selection in vitro. Consistent with a role for hnRNP A1 in the activity of CE1d, a mutation that abrogates A1 binding abolishes distal 5' splice site activation. Moreover, the ability of CE1d to stimulate distal 5' splice site usage is lost in an HeLa extract depleted of hnRNP A/B proteins, and the addition of recombinant A1 restores the activity of CE1d. Notably, distal 5' splice site selection mediated by A1 binding sites is not compromised in an extract prepared from mouse cells that are severely deficient in hnRNP A1 proteins. In this case, we show that hnRNP A2 compensates for the A1 deficiency. Further studies with the CE4 element reveal that it also consists of two distinct portions (CE4m and CE4p), each one capable of promoting distal 5' splice site use in an hnRNP A1-dependent manner. The presence of multiple A1/A2 binding sites downstream of common exon 7 and alternative exon 7B probably plays an important role in maximizing the activity of hnRNP A1/A2 proteins.
We describe characterization of spliceosomes affinity purified under native conditions. These spliceosomes consist largely of C complex containing splicing intermediates. After C complex assembly on an MS2 affinity-tagged pre-mRNA substrate containing a 3' splice site mutation, followed by RNase H digestion of earlier complexes, spliceosomes were purified by size exclusion and affinity selection. This protocol yielded 40S C complexes in sufficient quantities to visualize in negative stain by electron microscopy. Complexes purified in this way contain U2, U5, and U6 snRNAs, but very little U1 or U4 snRNA. Analysis by tandem mass spectrometry confirmed the presence of core snRNP proteins (SM and LSM), U2 and U5 snRNP-specific proteins, and the second step factors Prp16, Prp17, Slu7, and Prp22. In contrast, proteins specific to earlier splicing complexes, such as U2AF and U1 snRNP components, were not detected in C complex, but were present in similarly purified H complex. Images of these spliceosomes revealed single particles with dimensions of approximately 270 x 240 A that assort into well-defined classes. These images represent an important first step toward attaining a comprehensive three-dimensional understanding of pre-mRNA splicing.
The product of the von Hippel-Lindau (VHL) tumor suppressor gene, pVHL, functions as a ubiquitin-protein isopeptide ligase in regulating HIF-1 protein turnover, thus accounting for the increased transcription of hypoxia-inducible genes that accompanies VHL mutations. The increased vascular endothelial growth factor mRNA stability in cells lacking pVHL has been hypothesized to be due to a similar regulation of an RNA-binding protein. We report the expression of the GLUT-1 3'-untranslated region RNA-binding protein, heteronuclear ribonucleoprotein (hnRNP) A2, is specifically increased in pVHL-deficient cell lines. Enhanced hnRNP A2 expression was apparent in all cell fractions, including polysomes, where a similar modest effect on hnRNP L (a GLUT-1 and VEGF 3'-untranslated region-binding protein), was seen. Steady state levels of hnRNP A2 mRNA were unaffected. Regulation of hnRNP A2 levels correlated with the ability of pVHL to bind elongin C. Proteasome inhibition of cells expressing wild type pVHL selectively increased cytoplasmic hnRNP A2 levels to that seen in pVHL-deficient cells. Finally, an in vivo interaction between pVHL and hnRNP A2 was demonstrated in both the nucleus and the cytoplasm. Collectively, these data indicate that hnRNP A2 expression is regulated by pVHL in a manner that is dependent on elongin C interactions as well as functioning proteasomes.
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are predominantly nuclear RNA-binding proteins that form complexes with RNA polymerase II transcripts. These proteins play pivotal roles in transcription, pre-mRNA processing in the nucleus, cytoplasmic mRNA translation and its turnover. In addition, hnRNPs have been shown to be essential for embryonic development of Drosophila. Here we studied the protein levels of hnRNPs (A2/B1, H and H') in fetal brain with Down syndrome (DS; n = 5) compared to controls (n = 7). We used two-dimensional (2-D) gel electrophoresis, matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS) and specific software for quantification. hnRNP A2/B1 was significantly increased in fetal DS brain (13.52+/-4.50) compared to controls (9.16+/-1.35), but both hnRNP H and H' were unchanged. Increased hnRNP A2/B1 in fetal DS brain may represent high activity of RNA processing such as RNA trafficking and telomere protection, and/or it could contribute to abnormal development of DS brains. Furthermore, comparable expression of hnRNP H and H' suggest a specific upregulation of hnRNP A2/B.
Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP-A2/B1) is highly expressed during critical stages of lung development and carcinogenesis. To determine if the expression of hnRNP-A2/B1 is an informative biomarker in breast carcinogenesis, we analyzed hnRNP-A2/B1 overexpression by immunohistochemistry in archived specimens. Expression was detected in 48/85 (56.5%) primary invasive breast cancers and 7/72 (9.7%) specimens of normal breast tissue. Northern analysis of breast cancer cells also demonstrated higher level of hnRNP-A2/B1 expression compared to normal or transformed breast cells. Expression of hnRNP-A2/B1 in breast cancer cells was decreased by exposure to retinoids coordinately with decreased cell growth. These results warrant further evaluation of hnRNP-A2/B1 as a marker of breast carcinogenesis.
Heterogeneous nuclear ribonucleoproteins (hnRNPs) A2 and B1 are abundant nuclear proteins that bind to nascent RNAs synthesized by RNA polymerase II. Previously we had found that the splicing isoforms hnRNP B0a/b, from which the ninth exon of the A2/B1 gene is excluded, are abundantly expressed in testis. We postulated that B0a/b are testis-specific isoforms, and investigated the expression of A2/B1 and B0a/b in rat tissues and in postnatal development of rat testes using RNase protection assay, immunoblotting, and immunohistochemistry. We found that hnRNP B0a/b mRNAs are expressed in several tissues but that the testis alone expresses B0a/b proteins. A sequential study using neonatal rat testes demonstrated that B0a/b mRNAs are produced after 17 days of age, but not translated until 4 weeks of age when round spermatids appear in addition to spermatogonia and spermatocytes. Immunohistochemically, hnRNP A2/B1 isoforms are expressed during spermatogenesis from spermatogonia through round spermatids, whereas the expression of A1 is restricted to spermatogonia. This expression pattern in the rat testis is maintained from birth through adulthood. These results suggest that the expression of the hnRNP A2/B1 gene is partly regulated by a testis-specific post-transcriptional mechanism, and that the products of the A2/B1 gene, especially hnRNP B0a/b, are involved in spermatogenesis.
The heterogeneous nuclear ribonucleoproteins (hnRNP) associate with pre-mRNA in the nucleus and play an important role in RNA processing and splice site selection. In addition, hnRNP A proteins function in the export of mRNA to the cytoplasm. Although the hnRNP A proteins are predominantly nuclear, hnRNP A1 shuttles rapidly between the nucleus and the cytoplasm. HnRNP A2, whose cytoplasmic overexpression has been identified as an early biomarker of lung cancer, has been less well studied. Cytosolic hnRNP A2 overexpression has also been noted in brain tumors, in which it has been correlated with translational repression of Glucose Transporter-1 expression. We now examine the role of arginine methylation on the nucleocytoplasmic localization of hnRNP A2 in the HEK-293 and NIH-3T3 mammalian cell lines. Treatment of either cell line with the methyltransferase inhibitor adenosine dialdehyde dramatically shifts hnRNP A2 localization from the nuclear to the cytoplasmic compartment, as shown both by immunoblotting and by immunocytochemistry. In vitro radiolabeling with [(3)H]AdoMet of GST-tagged hnRNP A2 RGG mutants, using recombinant protein arginine methyltransferase (PRMT1), shows (i) that hnRNP A2 is a substrate for PRMT1 and (ii) that methylated residues are found only in the RGG domain. Deletion of the RGG domain (R191-G253) of hnRNP A2 results in a cytoplasmic localization phenotype, detected both by immunoblotting and by immunocytochemistry. These studies indicate that the RGG domain of hnRNP A2 contains sequences critical for cellular localization of the protein. The data suggest that hnRNP A2 may contain a novel nuclear localization sequence, regulated by arginine methylation, that lies in the R191-G253 region and may function independently of the M9 transportin-1-binding region in hnRNP A2.
Recent work identified an RNA binding protein whose presence in brain tumors correlated with translational repression of Glut1 expression. RNase T1 mapping demonstrated that this protein bound an AU-rich response element (AURE) in the Glut1 3'UTR. Facilitated by its differential expression in brain tumor cytosols, we identified this Glut1 RNA binding protein as hnRNP A2. Studies further demonstrated that hnRNP A2 was the major Glut1 RNA binding activity in other cell lines. Recombinant hnRNP A2 exhibited equivalent Glut1 RNA binding specificity, quite distinct from the related AURE binding protein hnRNP A1. These data indicate that hnRNP A2 is the Glut1 AURE binding protein whose cytoplasmic expression in gliomas is associated with translational repression and mRNA instability. Using this approach, we also identified the other major Glut1 3'UTR RNA binding activity as hnRNP L. Stimuli (hypoxia and hypoglycemia) which increase Glut1 mRNA stability selectively decreased polysomal levels of hnRNP A2 and L. Immunoprecipitation demonstrated that hnRNP A2 and L exist as a complex in vivo. As a result of these and other studies, we conclude that hnRNP A2 and L associate in vivo and independently bind the 3'UTR of Glut1 mRNA.
The catalytic subunit of protein kinase CK2 (CK2alpha) was found associated with heterogeneous nuclear ribonucleoprotein particles (hnRNPs) that contain the core proteins A2 and C1-C2. High levels of CK2 activity were also detected in these complexes. Phosphopeptide patterns of hnRNP A2 phosphorylated in vivo and in vitro by protein kinase CK2 were similar, suggesting that this kinase can phosphorylate hnRNPA2 in vivo. Binding experiments using human recombinant hnRNP A2, free human recombinant CK2alpha or CK2beta subunits, reconstituted CK2 holoenzyme and purified native rat liver CK2 indicated that hnRNP A2 associated with both catalytic and regulatory CK2 subunits, and that the interaction was independent of the presence of RNA. However, the capability of hnRNP A2 to bind to CK2 holoenzyme was lower than its binding to the isolated subunits. These data indicate that the association of CK2alpha with CK2beta interferes with the subsequent binding of hnRNP A2. HnRNP A2 inhibited the autophosphorylation of CK2beta. This effect was stronger with reconstituted human recombinant CK2 than with purified native rat liver CK2.
Many important cell mechanisms are carried out and regulated by multi-protein complexes, for example, transcription and RNA processing machinery, receptor complexes and cytoskeletal structures. Most of these complexes remain only partially characterized due to the difficulty of conventional protein analysis methods. The rapid expansion of DNA sequence databases now provides whole or partial gene sequences of model organisms, and recent advances in protein microcharacterization via mass spectrometry allow the possibility of linking these DNA sequences to the proteins in functional complexes. This approach has been demonstrated in organisms whose genomes have been sequenced, such as budding yeast. Here we report the first characterization of an entire mammalian multi-protein complex using these methods. The machinery that removes introns from mRNA precursors--the spliceosome--is a large multi-protein complex. Approximately half of the components excised from a two-dimensional gel separation of the spliceosome were found in protein sequence databases. Using nanoelectrospray mass spectrometry, the remainder were identified and cloned using public expressed sequence tag (EST) databases. Existing EST databases are thus already sufficiently complete to allow rapid characterization of large mammalian protein complexes via mass spectrometry.
Recent reports have demostrated a link between expression of members of the family of heterogeneous nuclear ribonucleoproteins (hnRNPs) and cancer. Overexpression of hnRNP A2/B1 correlated with the eventual development of lung cancer in three different clinical cohorts. We have studied the expression of hnRNP A2/B1 messenger RNA (mRNA) and protein during mammalian development. The expression of hnRNP A2/B1 mRNA and protein are parallel but change dynamically during critical periods in mouse pulmonary development. hnRNP A2/B1 is first detected in the lung in the early pseudoglandular period, peaks at the beginning of the canalicular period, and remains high during the saccular (alveolar) period. In mouse and rat, hnRNP A2/B1 expression is first evident in the earliest lung buds. As lung development progresses, the cuboidal epithelial cells of the distal primitive alveoli show high levels of the ribonucleoprotein, which is almost undetectable in the proximal conducting airways. The expression of hnRNP A2/ B1 is restricted in mature lung. Similar dynamic pattern of expression through lung development was also found in rat and human lung. Upregulated expression of hnRNP A2/B1 at critical periods of lung development was comparable to the level of expression found in lung cancers and preneoplastic lesions and is consistent with hnRNP A2/B1 overexpression playing an oncodevelopmental role.
A cluster of homeobox-containing genes (HOXA) and a heterogeneous nuclear ribonucleoprotein (hnRPA2B1) have both previously been assigned to chromosome 7p15 by in situ hybridization. In this report, we constructed a YAC contig from chromosome 7p14-p15, between markers D7S2496 and D7S1838, and determined the position of the HOXA1 gene and the hnRPA2B1 gene in this YAC contig.
Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a major nuclear protein and one of the major components of the hnRNP core complex in mammalian cells. We first determined the complete sequence of the human gene for hnRNP protein A2 (HNRPA2B1). The human HNRPA2B1 gene exists in a single copy over 9 kb in length. The gene was split into 12 exons, including a 36-nucleotide mini-exon, which was specific to the hnRNP protein B1, providing genetic evidence that the B1 mRNA was generated from the primary HNRPA2B1 transcript by alternative splicing. The 5' region of HNRPA2B1 was GC-rich and contained several DNA motifs for the binding of several transcription factors, which included 2 CCAAT boxes and no TATA sequences. The 5' ends of the mRNA were mapped to multiple positions. These structural features are characteristic of promoter regions of housekeeping genes. Northern blot and RT-PCR analyses of the HNRPA2B1 transcripts revealed levels of B1 mRNA from 2 to 5% of total A2/B1 transcripts and showed that both A2 and B1 mRNAs were transcribed in all human cell lines and mouse tissues studied. The structural and evolutionary characteristics of the A2 and A1 proteins as they relate to each other are discussed.
Heterogeneous nuclear ribonucleoproteins bind to RNA as long as it is transcribed. Since their binding can be sequence-specific, it has been suggested that their expression in different tissues could vary depending on the specific mRNA processing requirements. In order to better establish this possibility we studied the presence of the heterogeneous nuclear ribonucleoproteins A1, A2/B1, C and D in the cell nuclei of different rat tissues by one- and two-dimensional immunoblotting. We found that these proteins were heterogeneously distributed among tissues and that they were found in different proportions.
Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 belongs, with A1, B1 and B2, to the basic protein subset of the hnRNP complex in mammalian cells. All these proteins share a modular structure consisting of two conserved RNA binding domains linked to less conserved Gly-rich domains (2xRBD-Gly). In the framework of our studies on the genetic basis of hnRNP proteins structure and diversity we have isolated and sequenced the A2 gene and compared it to the previously described A1 gene. The A2 gene, which exists in a single copy on Ch. 7 band p15, is split in 12 exons including an alternatively spliced 36 nt mini exon specific for the human hnRNP protein B1. In this work we show that the intron/exon organisation of the A2 gene is identical to that of the A1 gene over the entire length, indicating a common origin by gene duplication. Moreover the comparison of corresponding exons evidences significant conservation also in the apparently divergent Gly-rich domains that could define previously unenvisaged structural and/or functional motifs. The A2 gene promoter is also analysed in comparison to that of the A1 gene.
A patient with Haemophilus aphrophilus endocarditis was successfully treated with ciprofloxacin. The response to treatment with cefotaxime and netilmicin for 12 days was poor but was satisfactory to a 6 weeks' course of ciprofloxacin.
Precursor mRNA is complexed with proteins in the cell nucleus to form heterogeneous nuclear ribonucleoprotein (hnRNP), and these hnRNPs are found associated in vivo with small nuclear RNPs (snRNPs) for the processing of pre-mRNA. In order to better characterize the ATP-independent initial association of U1 snRNP with hnRNP, an important early event in assembly of the spliceosome complex, we have determined some of the components essential to an in vitro reassociation of U1 snRNP with hnRNP. U1 snRNP reassociated in vitro with 40S hnRNP particles from HeLa cells and, similar to the in vivo hnRNP/U1 snRNP association, the in vitro interaction was sensitive to high salt concentrations. U1 snRNP also associated with in vitro reconstituted hnRNP in which bacteriophage MS2 RNA, which lacks introns, was used as the RNA component. Purified snRNA alone would not associate with the MS2 RNA-reconstituted hnRNP, however, intact U1 snRNP did interact with protein-free MS2 RNA. This indicates that the U1 snRNP proteins are required for the hnRNP/U1 snRNP association, but hnRNP proteins are not. Thus, the initial, ATP-independent association of U1 snRNP with hnRNP seems to be mediated by U1 snRNP protein(s) associating with hnRNA without requiring a splice-site sequence. This complex may then be further stabilized by intron-specific interactions and hnRNP proteins, as well as by other snRNPs.