RNA-binding Protein hnRNP Research Articles

Abstract 1852: Identification of the UV-inducible RNA binding protein hnRNP A18 as a new target for melanoma progression

Abstract Melanoma is the most dangerous form of skin cancer. If recognized and treated early, it is nearly 100 percent curable. However, invasive melanoma is hard to treat and consequently presents an extremely poor prognosis with a 5-year survival of about 6% and a median survival duration of 7.5 months. We have identified the UV-inducible RNA binding protein hnRNP A18 as a new target for melanoma progression and responses to anticancer drugs treatments. hnRNP A18 basal levels are up-regulated in melanoma cells as compared to normal melanocytes and UV radiation increases hnRNP A18 levels in 5 of the 6 melanoma cell lines we studied. More over, down regulation of hnRNP A18 reduced by more than 80% melanoma tumor growth in mice xenograft and sensitized melanoma cells to Temozolimide (TMZ) and the hypoxic mimetic agent CoCl2. In addition, levels of hnRNP A18 expression in response to UV radiation are proportional to melanoma cells capacity to survive UV-induced cellular damage. We have identified an hnRNP A18 RNA binding signature motif in the 3′ UTR of key regulatory proteins such as HDAC 8, 9, 11, HIF-1α and ATR that can confer resistance to UV induced DNA damage and hypoxic conditions. Therefore by targeting of a single molecule, hnRNP A18, it may be possible to shut down an entire network of protective proteins conferring melanoma cells, and potentially other solid tumors, growth advantage and resistance to conventional therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1852. doi:1538-7445.AM2012-1852

The Mediator Couples Transcription and Splicing

Transcription is coupled with pre-mRNA splicing in metazoans. In the current issue of Molecular Cell, Huangetal. (2012) show that Med23 interacts with the RNA binding protein hnRNP L to regulate alternative splicing, thus expanding Mediator's function beyond transcriptional control.

Heterogeneous Nuclear Ribonucleoprotein (hnRNP) F Is a Novel Component of Oligodendroglial RNA Transport Granules Contributing to Regulation of Myelin Basic Protein (MBP) Synthesis

Myelin basic protein (MBP) is a major component of central nervous system (CNS) myelin. The absence of MBP results in the loss of almost all compact myelin in the CNS. MBP mRNA is sorted into RNA granules that are transported to the periphery of oligodendrocytes in a translationally inactive state. A central mediator of this transport process is the trans-acting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 that binds to the cis-acting A2-response element in the 3'UTR of MBP mRNA. Recently, we found that activation of the Src family nonreceptor tyrosine kinase Fyn in oligodendrocytes leads to phosphorylation of hnRNP A2 and to increased translation of MBP mRNA. Here, we identify the RNA-binding protein hnRNP F as a novel component of MBP mRNA transport granules. It is associated with hnRNP A2 and MBP mRNA in cytoplasmic granular structures and is involved in post-transcriptional regulation of MBP expression. Fyn kinase activity results in phosphorylation of hnRNP F in the cytoplasm and its release from MBP mRNA and RNA granules. Our results define hnRNP F as a regulatory element of MBP expression in oligodendrocytes and imply an important function of hnRNP F in the control of myelin synthesis.

IL-6-induced Enhancement of c-Myc Translation in Multiple Myeloma Cells: CRITICAL ROLE OF CYTOPLASMIC LOCALIZATION OF THE RNA-BINDING PROTEIN hnRNP A1

Prior work indicates that IL-6 can stimulate c-Myc expression in multiple myeloma (MM) cells, which is independent of effects on transcription and due to enhanced translation mediated by an internal ribosome entry site in the 5'-UTR of the c-Myc RNA. The RNA-binding protein hnRNP A1 (A1) was also critical to IL-6-stimulated translation. Because A1 shuttles between nucleus and cytoplasm, we investigated whether the ability of IL-6 to enhance Myc translation was mediated by stimulation of A1 shuttling. In MM cell lines and primary specimens, IL-6 increased A1 cytoplasmic localization. In contrast, there was no effect on the total cellular levels of A1. Use of a dominant negative A1 construct, which prevents endogenous A1 from nucleus-to-cytoplasm transit, prevented the ability of IL-6 to enhance Myc internal ribosome entry site activity, Myc protein expression, and MM cell growth. IL-6-stimulated cytoplasmic localization was mediated by alterations in the C-terminal M9 peptide of A1, and this correlated with the ability of IL-6 to induce serine phosphorylation of this domain. A p38 kinase inhibitor prevented IL-6-induced A1 phosphorylation. Thus, IL-6 activates c-Myc translation in MM cells by inducing A1 phosphorylation and cytoplasmic localization in a p38-dependent fashion. These data suggest A1 as a potential therapeutic target in MM.

HnRNP C promotes APP translation by competing with FMRP for APP mRNA recruitment to P bodies

Amyloid precursor protein (APP) regulates neuronal synapse function, and its cleavage product Abeta is linked to Alzheimer's disease. Here, we present evidence that the RNA-binding proteins (RBPs) heterogeneous nuclear ribonucleoprotein (hnRNP) C and fragile X mental retardation protein (FMRP) associate with the same APP mRNA coding region element, and they influence APP translation competitively and in opposite directions. Silencing hnRNP C increased FMRP binding to APP mRNA and repressed APP translation, whereas silencing FMRP enhanced hnRNP C binding and promoted translation. Repression of APP translation was linked to colocalization of FMRP and tagged APP RNA within processing bodies; this colocalization was abrogated by hnRNP C overexpression or FMRP silencing. Our findings indicate that FMRP represses translation by recruiting APP mRNA to processing bodies, whereas hnRNP C promotes APP translation by displacing FMRP, thereby relieving the translational block.

Abstract 1595: Phosphomimetic mutation of hnRNP A1 inhibits AKT-dependent IRES function and results in mTOR inhibitor sensitivity

Abstract The relative activity of the Akt kinase has been demonstrated to be a major determinant of sensitivity of tumor cells to mTOR inhibitors. Our previous studies have shown that the multifunctional RNA-binding protein hnRNP A1 regulates a salvage pathway which facilitates IRES-dependent mRNA translation of critical cellular determinants in an Akt-dependent manner following mTOR inhibitor exposure. This salvage pathway functions by stimulating IRES-dependent translation in cells with relatively quiescent Akt resulting in resistance to rapamycin. However, the pathway is repressed in cells with elevated Akt activity rendering cells sensitive to rapamycin induced G1 arrest as a result of the inhibition of global eIF-4E-mediated translation. Akt phosphorylation of hnRNP A1 at serine 199 has been demonstrated to inhibit IRES-mediated translation initiation. Here we describe a phosphomimetic mutant of hnRNP A1 (S199E) which binds both the cyclin D1 and c-MYC IRES RNAs in vitro and results in inhibition of IRES function in dicistronic mRNA reporter assays. Utilizing cells in which Akt is conditionally active, we demonstrate that overexpression of this mutant renders quiescent Akt-containing cells sensitive to rapamycin in vitro and in xenografts. We also demonstrate that activated Akt is strongly correlated with elevated phospho- ser199-hnRNP A1 levels in a panel of 22 glioblastomas. These data demonstrate that the phosphorylation status of hnRNP A1 ser199 regulates the Akt-dependent sensitivity of cells to rapamycin and may have utility as a surrogate biomarker to stratify those tumors which may be most sensitive to mTOR inhibitor therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1595.

The germ cell nuclear proteins hnRNP G-T and RBMY activate a testis-specific exon.

The human testis has almost as high a frequency of alternative splicing events as brain. While not as extensively studied as brain, a few candidate testis-specific splicing regulator proteins have been identified, including the nuclear RNA binding proteins RBMY and hnRNP G-T, which are germ cell-specific versions of the somatically expressed hnRNP G protein and are highly conserved in mammals. The splicing activator protein Tra2β is also highly expressed in the testis and physically interacts with these hnRNP G family proteins. In this study, we identified a novel testis-specific cassette exon TLE4-T within intron 6 of the human transducing-like enhancer of split 4 (TLE4) gene which makes a more transcriptionally repressive TLE4 protein isoform. TLE4-T splicing is normally repressed in somatic cells because of a weak 5′ splice site and surrounding splicing-repressive intronic regions. TLE4-T RNA pulls down Tra2β and hnRNP G proteins which activate its inclusion. The germ cell-specific RBMY and hnRNP G-T proteins were more efficient in stimulating TLE4-T incorporation than somatically expressed hnRNP G protein. Tra2b bound moderately to TLE4-T RNA, but more strongly to upstream sites to potently activate an alternative 3′ splice site normally weakly selected in the testis. Co-expression of Tra2β with either hnRNP G-T or RBMY re-established the normal testis physiological splicing pattern of this exon. Although they can directly bind pre-mRNA sequences around the TLE4-T exon, RBMY and hnRNP G-T function as efficient germ cell-specific splicing co-activators of TLE4-T. Our study indicates a delicate balance between the activity of positive and negative splicing regulators combinatorially controls physiological splicing inclusion of exon TLE4-T and leads to modulation of signalling pathways in the testis. In addition, we identified a high-affinity binding site for hnRNP G-T protein, showing it is also a sequence-specific RNA binding protein.

Ars2 Links the Nuclear Cap-Binding Complex to RNA Interference and Cell Proliferation

Here we identify a component of the nuclear RNA cap-binding complex (CBC), Ars2, that is important for miRNA biogenesis and critical for cell proliferation. Unlike other components of the CBC, Ars2 expression is linked to the proliferative state of the cell. Deletion of Ars2 is developmentally lethal, and deletion in adult mice led to bone marrow failure whereas parenchymal organs composed of nonproliferating cells were unaffected. Depletion of Ars2 or CBP80 from proliferating cells impaired miRNA-mediated repression and led to alterations in primary miRNA processing in the nucleus. Ars2 depletion also reduced the levels of several miRNAs, including miR-21, let-7, and miR-155, that are implicated in cellular transformation. These findings provide evidence for a role for Ars2 in RNA interference regulation during cell proliferation.

A Novel Protein Domain Induces High Affinity Selenocysteine Insertion Sequence Binding and Elongation Factor Recruitment

Selenocysteine (Sec) is incorporated at UGA codons in mRNAs possessing a Sec insertion sequence (SECIS) element in their 3'-untranslated region. At least three additional factors are necessary for Sec incorporation: SECIS-binding protein 2 (SBP2), Sec-tRNA(Sec), and a Sec-specific translation elongation factor (eEFSec). The C-terminal half of SBP2 is sufficient to promote Sec incorporation in vitro, which is carried out by the concerted action of a novel Sec incorporation domain and an L7Ae RNA-binding domain. Using alanine scanning mutagenesis, we show that two distinct regions of the Sec incorporation domain are required for Sec incorporation. Physical separation of the Sec incorporation and RNA-binding domains revealed that they are able to function in trans and established a novel role of the Sec incorporation domain in promoting SECIS and eEFSec binding to the SBP2 RNA-binding domain. We propose a model in which SECIS binding induces a conformational change in SBP2 that recruits eEFSec, which in concert with the Sec incorporation domain gains access to the ribosomal A site.

Haploinsufficiency of the germ cell-specific nuclear RNA binding protein hnRNP G-T prevents functional spermatogenesis in the mouse

Human HNRNPGT, encoding the protein hnRNP G-T, is one of several autosomal retrogenes derived from RBMX. It has been suggested that HNRNPGT functionally replaces the sex-linked RBMX and RBMY genes during male meiosis. We show here that during normal mouse germ cell development, hnRNP G-T protein is strongly expressed during and after meiosis when proteins expressed from Rbmx or Rbmx-like genes are absent. Amongst these Rbmx-like genes, DNA sequence analyses indicate that two other mouse autosomal Rbmx-derived retrogenes have evolved recently in rodents and one already shows signs of degenerating into a non-expressed pseudogene. In contrast, orthologues of Hnrnpgt are present in all four major groups of placental mammals. The sequence of Hnrnpgt is under considerable positive selection suggesting it performs an important germ cell function in eutherians. To test this, we inactivated Hnrnpgt in ES cells and studied its function during spermatogenesis in chimaeric mice. Although germ cells heterozygous for this targeted allele could produce sperm, they did not contribute to the next generation. Chimaeric mice with a high level of mutant germ cells were infertile with low sperm counts and a high frequency of degenerate seminiferous tubules and abnormal sperm. Chimaeras made from a 1:1 mix of targeted and wild-type ES cell clones transmitted wild-type germ cells only. Our data show that haploinsufficiency of Hnrnpgt results in abnormal sperm production in the mouse. Genetic defects resulting in reduced levels of HNRNPGT could, therefore, be a cause of male infertility in humans.

RNA-Binding Proteins hnRNP A2/B1 and CUGBP1 Suppress Fragile X CGG Premutation Repeat-Induced Neurodegeneration in a Drosophila Model of FXTAS

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a recently described neurodegenerative disorder of older adult carriers of premutation alleles (60-200 CGG repeats) in the fragile X mental retardation gene (FMR1). It has been proposed that FXTAS is an RNA-mediated neurodegenerative disease caused by the titration of RNA-binding proteins by the CGG repeats. To test this hypothesis, we utilize a transgenic Drosophila model of FXTAS that expresses a premutation-length repeat (90 CGG repeats) from the 5' UTR of the human FMR1 gene and displays neuronal degeneration. Here, we show that overexpression of RNA-binding proteins hnRNP A2/B1 and CUGBP1 suppresses the phenotype of the CGG transgenic fly. Furthermore, we show that hnRNP A2/B1 directly interacts with riboCGG repeats and that the CUGBP1 protein interacts with the riboCGG repeats via hnRNP A2/B1.

The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a

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

A Family of MicroRNAs Present in Plants and Animals

Although many miRNAs are deeply conserved within each kingdom, none are known to be conserved between plants and animals. We identified Arabidopsis thaliana miR854 and miR855, two microRNAs (miRNAs) with multiple binding sites in the 3' untranslated region (3'UTR) of OLIGOURIDYLATE binding PROTEIN1b (At UBP1b), forming miRNA:mRNA interactions similar to those that cause translational repression/mRNA cleavage in animals. At UBP1b encodes a member of a heterogeneous nuclear RNA binding protein (hnRNP) family. The 3'UTR of At UBP1b is sufficient to repress reporter protein expression in tissues expressing miR854 or miR855 (rosette leaves and flowers, respectively) but not where both miRNAs are absent (cauline leaves). Intergenic regions containing sequences closely resembling miR854 are predicted to fold into stable miRNA precursors in animals, and members of the miR854 family are expressed in Caenorhabditis elegans, Mus musculus, and Homo sapiens, all with imperfect binding sites in the 3'UTR of genes encoding the T cell Intracellular Antigen-Related protein, an hnRNP of the UBP1 family. Potential binding sites for miR854 are absent from UBP1-like genes in fungi lacking the miRNA biogenetic machinery. Our results indicate that plants and animals share miRNAs of the miR854 family, suggesting a common origin of these miRNAs as regulators of basal transcriptional mechanisms.

Molecular and Pharmacologic Suppression of MAPK Activity Rescues Differentiation of BCR/ABL+ Myeloid Progenitors by Releasing C/EBPa from the Inhibitory Effect of hnRNP E2.

Impaired differentiation is a common feature of many hematological malignancies including blast crisis chronic myelogenous leukemia (CML-BC). We previously reported that the inability of CML-BC myeloid progenitors to undergo terminal neutrophil differentiation depends on suppression of C/EBPα expression through the translation inhibitory activity of the BCR/ABL-regulated RNA binding protein hnRNP E2.Here we show that p210-BCR/ABL oncoprotein post-translationally enhances hnRNP E2 expression and translation-regulatory function. In fact, hnRNP E2 protein but not mRNA expression directly correlates with BCR/ABL levels in myeloid progenitors expressing different BCR/ABL levels, in Ph1 K562 and EM3 and BCR/ABL-inducible TonB.210 cells. Likewise, graded BCR/ABL expression in primary Lin− mouse bone marrow cells results in increasing hnRNP E2 levels. This, in turn, inhibits the expression of C/EBPα and that of the C/EBPα-regulated G-CSF (granulocytic-colony stimulating factor) receptor.In these models of CML-BC, increased hnRNP E2 expression results from the BCR/ABL- and ERK1/2-dependent enhancement of hnRNP E2 protein stability, as hnRNP E2 levels are downregulated by treatment with imatinib or the MEK inhibitors PD098059 and U0126, and after expression of dominant negative ERK1/2 mutants. Accordingly, hnRNP E2 protein levels rapidly decrease and are barely detectable in parental 32Dcl3 cells treated for 30 min. with the protein synthesis inhibitor cycloheximide (CHX). By contrast, they remain unchanged in 32D-BCR/ABL cells CHX-treated for 4 hours. Accordingly, hnRNP E2 protein expression is markedly reduced in IL-3-deprived parental but not 32D-BCR/ABL cells through a proteasome-dependent mechanism. Mechanistically, we have evidence that the BCR/ABL-activated ERK1/2 kinases phosphorylate hnRNP E2 on four different sites (S173, S189, T213, and S272), and that serine to alanine substitution of these phosphorylation sites abolishes hnRNP E2 phosphorylation in living cells and in vitro kinase assay. Moreover, the non-phosphatable hnRNP E2S173A, S189A, T213A, S272A mutant is less stable than wild-type and phosphomimetic hnRNP E2.Functionally, suppression of hnRNP E2 phosphorylation/expression by chemical inhibition of ERK1/2 activity results in decreased hnRNP E2 binding to the C-rich element contained in the uORF-spacer region of C/EBPα mRNA. This, in turn, restores C/EBPα expression and allows G-CSF-driven neutrophilic maturation of differentiation-arrested 32D-BCR/ABL cells and Lin− mouse bone marrow cells expressing high levels of p210-BCR/ABL oncoprotein. Likewise, treatment of G-CSF-cultured (48 hours) CML-BCCD34+ bone marrow patient cells with the MEK1 inhibitor U0126 (25 mM, 32 h) destabilizes hnRNP E2, thus restoring C/EBPα expression.Altogether our data not only indicate that the BCR/ABL-induced ERK1/2 activity is essential for the hnRNP E2 stability and suppression of C/EBPα-dependent granulocytic differentiation of CML-BC myeloid progenitors, but they also suggest the potential use of clinically-relevant MAPK inhibitors in the therapy of CML-BC.

BCR/ABL Post-Translationally Enhances hnRNP E2 Expression and Translation-Inhibitory Function through the Activation of ERK1/2 Mitogen-Activated Protein Kinases.

Impaired differentiation is a common feature of many hematological malignancies including blast crisis chronic myelogenous leukemia (CML-BC). We previously reported that expression of the RNA binding protein hnRNP E2 is enhanced in CML-BC patient samples by the activity of BCR/ABL oncogenic kinase which uses hnRNP E2 to suppress translation of C/EBP alpha, the main regulator or granulocytic differentiation. Here we show that hnRNP E2 protein but not mRNA expression correlates with BCR/ABL levels in mouse 32Dcl3 myeloid progenitor cell clones expressing different levels of p210 BCR/ABL, and in the BCR/ABL-inducible TonB.210 cell line. Furthermore, exposure of 32D-BCR/ABL cells to imatinib or to the HSP90 inhibitor 17-AAG, which induces BCR/ABL proteasome-dependent degradation, results in downregulation of hnRNP E2 protein levels. Mechanistically, it appears that increased hnRNP E2 expression depends on BCR/ABL-induced post-translational modifications that increase hnRNP E2 protein stability and prevent its proteasome-dependent degradation. Indeed, we have evidence that hnRNP E2 is associated with PRMT5 arginine-methyltransferase and undergoes phosphorylation in K562 cells. Accordingly, inhibition of the mitogen activated ERK1/2 kinases by PD098059 and U0126 reduces hnRNP E2 levels and, in turn, hnRNP E2 binding to the C-rich element contained in the uORF of c/ebp alpha mRNA. Thus, activation of MAPK might be required for the hnRNP E2-dependent suppression of myeloid maturation in CML-BC.

Squid is required for efficient posterior localization of oskar mRNA during Drosophila oogenesis

The nuclear-cytoplasmic shuttling heterogeneous nuclear RNA-binding protein (hnRNP) Squid (Sqd) is required during Drosophila melanogaster oogenesis, where it plays a critical role in the regulation of the TGFalpha-like molecule Gurken (Grk). Three Sqd isoforms have been described, SqdA, S and B, and two of these, SqdA and SqdS, differentially function in grk mRNA nuclear export, cytoplasmic transport and translational control during oogenesis. Here, we report that Sqd is also required for the regulation of oskar (osk) mRNA, functioning in the cytoplasmic localization of the osk transcript. In oocytes from sqd females, osk mRNA is not efficiently localized to the posterior pole, but rather accumulates at the anterior cortex. Furthermore, anterior patterning defects observed in embryos from sqd females expressing only the SqdS protein isoform suggest that Sqd may also play a role in the translational regulation of the mislocalized osk mRNA. These findings provide additional support for models of mRNA regulation in which cytoplasmic events, such as localization and translational regulation, are coupled. These results also place Sqd among an emerging class of proteins, including such other members as Bruno (Bru) and Hrb27C/Hrp48, which function in multiple aspects of both grk and osk mRNA regulation during Drosophila oogenesis.

Contrasting expression patterns of G2/M phase cyclins in diffuse and intestinal-type gastric cancer

The rate of cell-cycle progression must be tuned in response to nutrient levels to ensure that sufficient materials are synthesized to generate viable daughters. We report that accumulation of the yeast M phase B-cyclin CLB2 mRNA depends on assembly and activation of the heterogeneous nuclear RNA-binding protein (hnRNP) arginine methyltransferase Hmt1, which is promoted by the kinase Dbf2 and countered by the PP2A phosphatase Pph22. Activated Hmt1 methylates hnRNPs, which in turn stabilize CLB2 transcripts. Dbf2 activation of Hmt1 is highly cooperative, producing a sharp increase in CLB2, whereas Pph22 dephosphorylation is graded such that small changes in PP2A activity can cause large shifts in Dbf2-mediated Hmt1 activity. Starvation and rapamycin inhibition of TOR activate Pph22, causing a depletion of CLB2 and delay of M phase. We propose a general model wherein changes to Pph22 activity modulate cyclin mRNA stability to tune cell-cycle progression to environmental conditions.

Heterogeneous RNA-binding Protein M4 Is a Receptor for Carcinoembryonic Antigen in Kupffer Cells

Here we report the isolation of the recombinant cDNA clone from rat macrophages, Kupffer cells (KC) that encodes a protein interacting with carcinoembryonic antigen (CEA). To isolate and identify the CEA receptor gene we used two approaches: screening of a KC cDNA library with a specific antibody and the yeast two-hybrid system for protein interaction using as a bait the N-terminal part of the CEA encoding the binding site. Both techniques resulted in the identification of the rat heterogeneous RNA-binding protein (hnRNP) M4 gene. The rat ortholog cDNA sequence has not been previously described. The open reading frame for this gene contains a 2351-base pair sequence with the polyadenylation signal AATAAA and a termination poly(A) tail. The mRNA shows ubiquitous tissue expression as a 2.4-kilobase transcript. The deduced amino acid sequence comprised a 78-kDa membrane protein with 3 putative RNA-binding domains, arginine/methionine/glutamine-rich C terminus and 3 potential membrane spanning regions. When hnRNP M4 protein is expressed in pGEX4T-3 vector system in Escherichia coli it binds (125)I-labeled CEA in a Ca(2+)-dependent fashion. Transfection of rat hnRNP M4 cDNA into a non-CEA binding mouse macrophage cell line p388D1 resulted in CEA binding. These data provide evidence for a new function of hnRNP M4 protein as a CEA-binding protein in Kupffer cells.

Inhibition of oestrogen receptor activity by the co-repressor HET/SAF-B is relieved by blockade of histone deacetylase activity

The oestrogen receptor (ER) is a member of a superfamily of nuclear transcription factors. When the ER binds oestrogen it undergoes a conformational change that results in dimerisation, binding to specific elements of DNA, and finally altered gene transcription [ 1 Smith D.F. Toft D.O. Steroid receptors and their associated proteins. Mol. Endocrinol. 1993; 7: 4-11 Crossref PubMed Scopus (49) Google Scholar ]. While this model of ER action has held true for the last 30 years, a more complete understanding has revealed that activation of the ER is extremely complex, with regulation by a diverse set of signals and nuclear factors. A poorly described modulator of hormone action is the nuclear matrix, which is a dynamic structure involved in DNA replication, transcription, repair and RNA processing [ 2 Bird R.C. Stein G.S. Lian J.B. Stein J.L. Nuclear structure and gene expression. in: Buetow D.E. Cameron I.L. Padilla G.M. Zimmerman A.M. Cell Biology A Series of Monographs. Academic Press, New York1997 Google Scholar ]. A role for the nuclear matrix in hormone receptor action was postulated many years ago but only recently have specific nuclear matrix proteins been characterised which directly bind to hormone receptors and modulate their activity [ 3 Barrett T.J. Spelsberg T.C. Nuclear matrix and steroid hormone action. Vitamins and Hormones. 1999; 55: 127-163 Crossref PubMed Scopus (17) Google Scholar , 4 Eggert M. Michel J. Schneider S. et al. The glucocorticoid receptor is associated with the RNA-binding nuclear matrix protein hnRNP U. J. Biol. Chem. 1997; 272: 28471-28478 Crossref PubMed Scopus (73) Google Scholar ].

Characterization of the mRNA ligands bound by the RNA binding protein hnRNP A2 utilizing a novel in vivo technique.

Post-transcriptional regulation is an important mechanism in cellular response to stimuli, allowing for the rapid and discrete expression of relevant proteins. Genes regulated by this mechanism have specific cis -acting elements, frequently in their 3' untranslated regions (UTRs), that have been shown to serve as recognition sites for trans -acting RNA-binding proteins. Unfortunately, the identification of specific mRNA ligands for different RNA binding proteins in vivo has been limited by a lack of adequate methodology. We have developed a novel technique that addresses this shortcoming, using immunoprecipitation of RNA binding proteins from polysomes followed by RT-PCR and library screening to identify the in vivo mRNA ligands of RNA binding proteins. Utilizing this approach, we have identified 32 known and 16 novel mRNAs specifically bound by the heterogeneous nuclear ribonucleoprotein (hnRNP) A2. Of the clones identified, 74% contained AU-rich elements and/or poly-uridine tracts in their 3' UTRs, cis -acting elements that have been established as impacting mRNA stability. The high percentage of clones containing these uridine-rich sequences compares favorably with the high affinity binding of poly-uridine RNA by hnRNP A2 in vitro. These data thus support the representative nature of the technique.