Search PubMed⌕ Search

Biomedical subjects

J D Keene

Publications and source records attributed to J D Keene.

At least 19 recordsLinked to original sources
PubMed2001

Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome.

Fragile X syndrome results from the absence of the RNA binding FMR protein. Here, mRNA was coimmunoprecipitated with the FMRP ribonucleoprotein complex and used to interrogate microarrays. We identified 432 associated mRNAs from mouse brain. Quantitative RT-PCR confirmed some to be >60-fold enriched in the immunoprecipitant. In parallel studies, mRNAs from polyribosomes of fragile X cells were used to probe microarrays. Despite equivalent cytoplasmic abundance, 251 mRNAs had an abnormal polyribosome profile in the absence of FMRP. Although this represents <2% of the total messages, 50% of the coimmunoprecipitated mRNAs with expressed human orthologs were found in this group. Nearly 70% of those transcripts found in both studies contain a G quartet structure, demonstrated as an in vitro FMRP target. We conclude that translational dysregulation of mRNAs normally associated with FMRP may be the proximal cause of fragile X syndrome, and we identify candidate genes relevant to this phenotype.

Amino Acid Sequence↗
PubMed2001

Ribonucleoprotein infrastructure regulating the flow of genetic information between the genome and the proteome.

Following transcription and splicing, each mRNA of a mammalian cell passes into the cytoplasm where its fate is in the hands of a complex network of ribonucleoproteins (mRNPs). The success or failure of a gene to be expressed depends on the performance of this mRNP infrastructure. The entry, gating, processing, and transit of each mRNA through an mRNP network helps determine the composition of a cell's proteome. The machinery that regulates storage, turnover, and translational activation of mRNAs is not well understood, in part, because of the heterogeneous nature of mRNPs. Recently, subsets of cellular mRNAs clustered as members of mRNP complexes have been identified by using antibodies reactive with RNA-binding proteins, including ELAV/Hu, eIF-4E, and poly(A)-binding proteins. Cytoplasmic ELAV/Hu proteins are involved in the stability and translation of early response gene (ERG) transcripts and are expressed predominately in neurons. mRNAs recovered from ELAV/Hu mRNP complexes were found to have similar sequence elements, suggesting a common structural linkage among them. This approach opens the possibility of identifying transcripts physically clustered in vivo that may have similar fates or functions. Moreover, the proteins encoded by physically organized mRNAs may participate in the same biological process or structural outcome, not unlike operons and their polycistronic mRNAs do in prokaryotic organisms. Our goal is to understand the organization and flow of genetic information on an integrative systems level by analyzing the collective properties of proteins and mRNAs associated with mRNPs in vivo.

Animals↗
PubMed2000

Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays.

Genomic array technologies provide a means for profiling global changes in gene expression under a variety of conditions. However, it has been difficult to assess whether transcriptional or posttranscriptional regulation is responsible for these changes. Additionally, fluctuations in gene expression in a single cell type within a complex tissue like a tumor may be masked by overlapping profiles of all cell types in the population. In this paper, we describe the use of cDNA arrays to identify subsets of mRNAs contained in endogenous messenger ribonucleoprotein complexes (mRNPs) that are cell type specific. We identified mRNA subsets from P19 embryonal carcinoma stem cells by using mRNA-binding proteins HuB, eIF-4E, and PABP that are known to play a role in translation. The mRNA profiles associated with each of these mRNPs were unique and represented gene clusters that differed from total cellular RNA. Additionally, the composition of mRNAs detected in HuB-mRNP complexes changed dramatically after induction of neuronal differentiation with retinoic acid. We suggest that the association of structurally related mRNAs into mRNP complexes is dynamic and may help regulate posttranscriptional events such as mRNA turnover and translation. Recovering proteins specifically associated with mRNP complexes to identify and profile endogenously clustered mRNAs should provide insight into structural and functional relationships among gene transcripts and/or their protein products. We have termed this approach to functional genomics ribonomics and suggest that it will provide a useful paradigm for organizing genomic information in a biologically relevant manner.

Animals↗
PubMed2000

Human La antigen is required for the hepatitis C virus internal ribosome entry site-mediated translation.

The 5'-noncoding region (5'-NCR) of the hepatitis C virus (HCV) RNA genome serves as an internal ribosome entry site (IRES) and mediates translation initiation in a cap-independent manner. Previously, we reported the interaction between La antigen and the HCV IRES, which appeared to occur in the context of initiator AUG. It was further shown that HCV IRES-mediated translation was stimulated in the presence of human La antigen. In this study, we have defined the cis- and trans-acting elements responsible for La-5'-NCR interactions and established the dependence of the HCV IRES efficiency on cellular La antigen. During the La-IRES interaction, initiator AUG but not the neighboring codons was found to be the direct target of La binding. The C terminus effector domain-dependent modulation of La binding to the HCV IRES is demonstrated by deletion and substitution mutagenesis of the protein. An RNA systematic evolution of ligands by exponential enrichment (SELEX), generated against La protein that selectively binds La in HeLa lysates and competes for the protein binding to the 5'-NCR, was used to demonstrate the requirement of La for the HCV IRES function in the context of mono- and dicistronic mRNAs. Sequestration of La antigen by the RNA SELEX in HeLa translation lysates blocked the HCV and poliovirus IRES-mediated translation in vitro. The functional requirement of La protein for the HCV IRES activity was further established in a liver-derived cell line and in an add-back experiment in which the inhibited IRES was rescued by recombinant human La. These results strongly argue for the novel role of La protein during selection of the initiator AUG and its participation during internal initiation of translation of the HCV RNA genome.

5' Untranslated Regions↗
PubMed2000

Inhibition of polyglutamine protein aggregation and cell death by novel peptides identified by phage display screening.

Proteins with expanded polyglutamine domains cause eight inherited neurodegenerative diseases, including Huntington's, but the molecular mechanism(s) responsible for neuronal degeneration are not yet established. Expanded polyglutamine domain proteins possess properties that distinguish them from the same proteins with shorter glutamine repeats. Unlike proteins with short polyglutamine domains, proteins with expanded polyglutamine domains display unique protein interactions, form intracellular aggregates, and adopt a novel conformation that can be recognized by monoclonal antibodies. Any of these polyglutamine length-dependent properties could be responsible for the pathogenic effects of expanded polyglutamine proteins. To identify peptides that interfere with pathogenic polyglutamine interactions, we screened a combinatorial peptide library expressed on M13 phage pIII protein to identify peptides that preferentially bind pathologic-length polyglutamine domains. We identified six tryptophan-rich peptides that preferentially bind pathologic-length polyglutamine domain proteins. Polyglutamine-binding peptide 1 (QBP1) potently inhibits polyglutamine protein aggregation in an in vitro assay, while a scrambled sequence has no effect on aggregation. QBP1 and a tandem repeat of QBP1 also inhibit aggregation of polyglutamine-yellow fluorescent fusion protein in transfected COS-7 cells. Expression of QBP1 potently inhibits polyglutamine-induced cell death. Selective inhibition of pathologic interactions of expanded polyglutamine domains with themselves or other proteins may be a useful strategy for preventing disease onset or for slowing progression of the polyglutamine repeat diseases.

Amino Acid Sequence↗
PubMed1999

ELAV tumor antigen, Hel-N1, increases translation of neurofilament M mRNA and induces formation of neurites in human teratocarcinoma cells.

Human ELAV proteins are implicated in cell growth and differentiation via regulation of mRNA expression in the cytoplasm. In human embryonic teratocarcinoma (hNT2) cells transfected with the human neuronal ELAV-like protein, Hel-N1, neurites formed, yet cells were not terminally differentiated. Cells in which neurite formation was associated with Hel-N1 overexpression, also expressed increased levels of endogenous neurofilament M (NF-M) protein, which distributed along the neurites. However, steady-state levels of NF-M mRNA remained similar whether or not hNT2 cells were transfected with Hel-N1. These findings suggest that turnover of NF-M mRNA was not affected by Hel-N1 expression, despite the fact that Hel-N1 can bind to the 3' UTR of NF-M mRNA and was found directly associated with NF-M mRNA in transfected cells. Analysis of the association of NF-M mRNA with the translational apparatus in Hel-N1 transfectants showed nearly complete recruitment to heavy polysomes, indicating that Hel-N1 caused an increase in translational initiation. Our results suggest that the stability and/or translation of ARE-containing mRNAs can be regulated independently by the ELAV protein, Hel-N1, depending upon sequence elements in the 3' UTRs and upon the inherent turnover rates of the mRNAs that are bound to Hel-N1 in vivo.

3' Untranslated Regions↗
PubMed1999

ELAV proteins stabilize deadenylated intermediates in a novel in vitro mRNA deadenylation/degradation system.

We have developed an in vitro mRNA stability system using HeLa cell cytoplasmic S100 extracts and exogenous polyadenylated RNA substrates that reproduces regulated aspects of mRNA decay. The addition of cold poly(A) competitor RNA activated both a sequence-specific deadenylase activity in the extracts as well as a potent, ATP-dependent ribonucleolytic activity. The rates of both deadenylation and degradation were up-regulated by the presence of a variety of AU-rich elements in the body of substrate RNAs. Competition analyses demonstrated that trans-acting factors were required for RNA destabilization by AU-rich elements. The approximately 30-kD ELAV protein HuR specifically bound to RNAs containing an AU-rich element derived from the TNF-alpha mRNA in the in vitro system. Interaction of HuR with AU-rich elements, however, was not associated with RNA destabilization. Interestingly, recombinant ELAV proteins specifically stabilized deadenylated intermediates generated from the turnover of AU-rich element-containing substrate RNAs. These data suggest that mammalian ELAV proteins play a role in regulating mRNA stability by influencing the access of degradative enzymes to RNA substrates.

Adenosine Triphosphate↗
PubMed1998

Messenger ribonucleoprotein complexes containing human ELAV proteins: interactions with cytoskeleton and translational apparatus.

Mammalian ELAV proteins bind to polyadenylated messenger RNAs and have specificity for AU-rich sequences. Preferred binding sites in vitro include the AUUUA pentamer and related sequences present in the 3' untranslated regions of many growth regulatory mRNAs. Human ELAV (hELAV) proteins have been implicated in post-transcriptional regulation of gene expression by their effects on the stability and translatability of growth regulatory mRNAs. We have examined the intracellular localization of ELAV proteins in neurons and in tumor cells of neuronal origin using indirect immunofluorescence, confocal microscopy and biochemical separation. Mammalian neuronal ELAV proteins are found predominantly in the cytoplasm of cells in mRNP complexes termed alpha complexes which, when associated with polysomes, form large and high density ss complexes, as assayed by glycerol and accudenz gradients, respectively. Puromycin, cytochalasin or EDTA treatments disrupt beta complexes causing the release of alpha complexes, which then appear, by confocal microscopy, as large hELAV mRNP granules associated with microtubules. Association of partially purified hELAV mRNP alpha complexes with microtubules was confirmed by in vitro reconstitution assays. Furthermore, colchicine treatment of cells suggested that association of hELAV mRNP alpha complexes with microtubules is also necessary for the formation of ss complexes. Our data suggest a model in which a subset of mRNAs is associated with microtubules as ELAV mRNP particles (alpha complexes) which, in turn, associate with polysomes to form a translational apparatus (beta complex) that is, through polysomes, associated with the microfilament cytoskeletal network. hELAV proteins in these mRNP granules may affect post-transcriptional regulation of gene expression via the intracellular transport, localization and/or translation of growth regulatory mRNAs.

Cytoskeleton↗
PubMed1998

ELAV protein HuA (HuR) can redistribute between nucleus and cytoplasm and is upregulated during serum stimulation and T cell activation.

ELAV proteins are implicated in regulating the stability and translation of cytokine and growth regulatory mRNAs such as GM-CSF, IL-2, c-myc, c-fos and GLUT1 by binding to their AU-rich 3'UTRs. The tissue-specific ELAV protein HuB (aka. Hel-N1) is predominantly cytoplasmic and has been shown to stabilize GLUT1 and c-myc mRNAs and to increase their translation following ectopic expression in 3T3-L1 cells. We report that the most widely expressed mouse ELAV protein, mHuA, is predominately nuclear in cultured NIH-3T3 cells, but is localized in the cytoplasm during early G1 of the cell cycle. Therefore, much like the primarily cytoplasmic HuB, HuA becomes temporally localized in the cytoplasm where it can potentially regulate the stability or translation of bound mRNAs. Moreover, we report that stimulation of mouse spleen cells using either mitogenic or sub-mitogenic levels of anti-CD3/CD28 resulted in a dramatic increase in the level of HuA. Upregulation of HuA corresponds to previously documented increases in cytokine expression which are due to increased mRNA stability following T cell activation. Consistent with these findings, HuA was down regulated in quiescent cells and upregulated in 3T3 cells following serum stimulation. The increase of murine HuA during the cell cycle closely resembles that of cyclin B1 which peaks in G2/M. Together with our earlier studies, these data indicate that mammalian ELAV proteins function during cell growth and differentiation due in part to their effects on posttranscriptional stability and translation of multiple growth regulatory mRNAs. This supports the hypothesis that ELAV proteins can function as transacting factors which affect a default pathway of mRNA degradation involved in the expression of growth regulatory proteins.

3T3 Cells↗
PubMed1997

Ectopic expression of Hel-N1, an RNA-binding protein, increases glucose transporter (GLUT1) expression in 3T3-L1 adipocytes.

3T3-L1 preadipocytes ectopically expressing the mammalian RNA-binding protein Hel-N1 expressed up to 10-fold more glucose transporter (GLUT1) protein and exhibited elevated rates of basal glucose uptake. Hel-N1 is a member of the ELAV-like family of proteins associated with the induction and maintenance of differentiation in various species. ELAV proteins are known to bind in vitro to short stretches of uridylates in the 3' untranslated regions (3'UTRs) of unstable mRNAs encoding growth-regulatory proteins involved in transcription and signal transduction. GLUT1 mRNA also contains a large 3'UTR with a U-rich region that binds specifically to Hel-N1 in vitro. Analysis of the altered GLUT1 expression at the translational and posttranscriptional levels suggested a mechanism involving both mRNA stabilization and accelerated formation of translation initiation complexes. These findings are consistent with the hypothesis that the Hel-N1 family of proteins modulate gene expression at the level of mRNA in the cytoplasm.

3T3 Cells↗
PubMed1996

RNA recognition by autoantigens and autoantibodies.

The La, Ro, Sm and RNP autoantigens have been intensely studied over the past decade since cDNAs encoding autoantigens have been available. Most of these autoantigens are closely associated with RNA in RNP particles and molecular studies have provided insights into their modes of recognition and binding to RNA. For example, a common RNA Recognition Motif (RRM) was found to be a critical component of the RNA-binding domain of these autoantigens and the three dimensional structure of the RRM has been solved. As described in other articles in this series, the presence of La, Ro, Sm and RNP autoantibodies correlates with disease subsets, such as Sjogren's syndrome, systemic lupus erythematous and other connective tissue diseases. Immunological analysis of sera from autoimmune patients using recombinant autoantigens has revealed that multiple epitopes reside along the proteins and these represent both continuous and discontinuous (conformational) autotopes. Findings to date support a model of autoantibody induction which involves the direct presentation of proteinaceous autoantigens to the immune system. Circumstantial evidence has suggested that immunological crossreactivity between systemic autoantigens and structural components of infectious agents may play an initial role in the autoimmune response to certain antigens. However, the etiology of autoimmune diseases is probably multifactoral with genetic and other immune features acting on the organismal level. In addition, RNA molecules themselves can be autoantigens with higher order structural conformations which are recognized by RNP-type autoantibodies. Immune crossreactivity and/or direct presentation may generate autoantibodies reactive with conformational RNA epitopes. If crossreactivity with components of cellular or infectious agents give rise to RNA epitopes, they may represent structural or functional mimetics of the primary epitopes that actually drive the response. These ideas are discussed with respect to the role of mimetic processes in molecular recognition during autoimmunity.

Animals↗
PubMed1996

RNA surfaces as functional mimetics of proteins.

Accumulating evidence suggests that RNA molecules can form surfaces that mimic those of proteins. Reactivity of autoantibodies with RNA surfaces may be due to cross-reactivity between a protein epitope and the RNA. The structural mimicry detected by an autoantibody may reflect functional mimicry.

Animals↗