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Jack D Keene

Publications and source records attributed to Jack D Keene.

16 recordsLinked to original sources
PubMed2005

Stable ribosome binding to the endoplasmic reticulum enables compartment-specific regulation of mRNA translation.

In eukaryotic cells, protein synthesis is compartmentalized; mRNAs encoding secretory/membrane proteins are translated on endoplasmic reticulum (ER)-bound ribosomes, whereas mRNAs encoding cytosolic proteins are translated on free ribosomes. mRNA partitioning between the two compartments occurs via positive selection: free ribosomes engaged in the translation of signal sequence-encoding mRNAs are trafficked from the cytosol to the ER. After translation termination, ER-bound ribosomes are thought to dissociate, thereby completing a cycle of mRNA partitioning. At present, the physiological basis for termination-coupled ribosome release is unknown. To gain insight into this process, we examined ribosome and mRNA partitioning during the unfolded protein response, key elements of which include suppression of the initiation stage of protein synthesis and polyribosome breakdown. We report that unfolded protein response (UPR)-elicited polyribosome breakdown resulted in the continued association, rather than release, of ER-bound ribosomes. Under these conditions, mRNA translation in the cytosol was suppressed, whereas mRNA translation on the ER was sustained. Furthermore, mRNAs encoding key soluble stress proteins (XBP-1 and ATF-4) were translated primarily on ER-bound ribosomes. These studies demonstrate that ribosome release from the ER is termination independent and identify new and unexpected roles for the ER compartment in the translational response to induction of the unfolded protein response.

Animals↗
PubMed2005

Post-transcriptional operons and regulons co-ordinating gene expression.

Experiments reported over the past several years, including genome-wide microarray approaches, have demonstrated that many eukaryotic RNA-binding proteins (RBPs) associate with multiple messenger RNAs (mRNAs) both in vitro and in vivo. This multi-targeted binding property of RBPs has led to a model of regulated gene expression in eukaryotes that we termed the post-transcriptional operon. This concept was established by an analogy between polycistronic mRNAs that are generated from bacterial operons, and the co-ordinated regulation of multiple monocistronic mRNAs by RBPs. Post-transcriptional operons represent a powerful mechanism to organize and express genetic information as functionally related combinations of monocistronic mRNAs. In fact, much of the diversification of individual proteomes may be determined by the combinatorial properties of post-transcriptional operons. This review examines data supporting the role of post-transcriptional operons and regulons in organizing genetic information and co-ordinating expression of functionally related transcripts from their origins at transcription to their subsequent splicing, export and translation.

Bacteria↗
PubMed2004

RNA-binding proteins to assess gene expression states of co-cultivated cells in response to tumor cells.

BACKGROUND: Tumors and complex tissues consist of mixtures of communicating cells that differ significantly in their gene expression status. In order to understand how different cell types influence one another's gene expression, it will be necessary to monitor the mRNA profiles of each cell type independently and to dissect the mechanisms that regulate their gene expression outcomes. RESULTS: In order to approach these questions, we have used RNA-binding proteins such as ELAV/Hu, poly (A) binding protein (PABP) and cap-binding protein (eIF-4E) as reporters of gene expression. Here we demonstrate that the epitope-tagged RNA binding protein, PABP, expressed separately in tumor cells and endothelial cells can be used to discriminate their respective mRNA targets from mixtures of these cells without significant mRNA reassortment or exchange. Moreover, using this approach we identify a set of endothelial genes that respond to the presence of co-cultured breast tumor cells. CONCLUSION: RNA-binding proteins can be used as reporters to elucidate components of operational mRNA networks and operons involved in regulating cell-type specific gene expression in tissues and tumors.

Animals↗
PubMed2004

La gets its wings.

Explore the source record for details and available documents.

Autoantigens↗
PubMed2004

Gene expression analysis of messenger RNP complexes.

RNA-binding proteins can organize messenger RNAs (mRNAs) into structurally and functionally related subsets, thus facilitating the coordinate production of gene classes necessary for complex cellular processes. Historically, in vitro methods primarily have been used to identify individual targets of mRNA-binding proteins. However, more direct methods are required for the identification of endogenously associated RNAs and their cognate proteins. To better understand posttranscriptional mRNA organization within the cell, we developed a systems biology approach to identify multiple-endogenous mRNA transcripts associated with RNA-binding proteins. This approach, termed ribonomics, takes advantage of high-throughput genomic array technologies that have greatly advanced the study of global gene expression changes. This chapter describes techniques for purifying mRNA-protein complexes (mRNPs) and identifying the associated mRNAs

Blotting, Western↗
PubMed2004

Biotinylated tags for recovery and characterization of ribonucleoprotein complexes.

Determining the in vivo targets of RNA-binding proteins and characterizing the posttranscriptional networks in which they participate constitute major challenges in the post-genomic era. An important step in this direction is the development of methods that permit efficient recovery of ribonucleoprotein (RNP) complexes. We present an improved methodology for efficient isolation of mammalian cell RNPs in which a biotin acceptor peptide (BAP) is used to tag RNA-binding proteins. BAP-tagged RNA-binding proteins can be biotinylated in vivo by co-expression of the Escherichia coli BirA enzyme. RNP recovery was obtained using streptavidin sepharose beads, and messenger RNAs (mRNAs) were identified using multiprobe RNase protection assays and cDNA microarrays. Using this approach we efficiently recovered and quantified RNAs bound to cytoplasmic poly(A)-binding protein (PABP) and to nuclear human transformer 2 (hTra-2) with minimal background.

Biotinylation↗
PubMed2003

Genome-wide regulatory analysis using en masse nuclear run-ons and ribonomic profiling with autoimmune sera.

Coordinated gene expression is influenced by transcriptional and posttranscriptional events and is necessary for efficient cell growth and differentiation. Genomic array technologies have afforded great advances in identifying global changes of gene expression in response to a variety of environmental stimuli. However, it has been a challenge to assess whether a concomitant effect on protein expression reflects the coordinated regulation of distinct subsets of mRNAs detected by cDNA arrays [Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 7018]. We have expanded the utility of cDNA arrays by using them to assist in elucidating combinatorial posttranscriptional eukaryotic operons [Mol. Cell 9 (2002) 1161]. In this study, we have used two mRNA partitioning methods in which: (1) subsets of mRNAs are isolated as endogenous mRNP complexes using autoimmune patient sera, and (2) transcriptional contributions to gene expression are assessed using cDNA array analysis of an en masse nuclear run-on assay (emRUN). The combination of these methods can provide an additional 'systems biology' discovery approach to gene expression analysis based upon the physical partitioning of mRNA subsets, as well as a functional partitioning of transcriptional and posttranscriptional processes. We demonstrate how these approaches can reduce transcriptomic complexity by partitioning mRNAs into biologically relevant subsets in order to derive information about the expression of multiple, but functionally linked, genes.

Autoimmune Diseases↗
PubMed2003

RNA-binding protein HuR enhances p53 translation in response to ultraviolet light irradiation.

Exposure to short-wavelength UV light (UVC) strongly induces p53 expression. In human RKO colorectal carcinoma cells, this increase was not due to elevated p53 mRNA abundance, cytoplasmic export of p53 mRNA, or UVC-triggered stabilization of the p53 protein. Instead, p53 translation was potently enhanced after UVC irradiation. The 3' UTR of p53 was found to be a target of the RNA-binding protein HuR in a UVC-dependent manner in vitro and in vivo. HuR-overexpressing RKO cells displayed elevated p53 levels, whereas cells expressing reduced HuR showed markedly diminished p53 abundance and p53 translation. Our results demonstrate a role for HuR in binding to the p53 mRNA and enhancing its translation.

3' Untranslated Regions↗
PubMed2003

Differential phosphorylation and subcellular localization of La RNPs associated with precursor tRNAs and translation-related mRNAs.

The La protein facilitates the production of tRNAs in the nucleus and the translation of specific mRNAs in the cytoplasm. We report that human La that is phosphorylated on serine 366 (pLa) is nucleoplasmic and associated with precursor tRNAs and other nascent RNA polymerase III transcripts while nonphosphorylated (np)La is cytoplasmic and associated with a subset of mRNAs that contain 5'-terminal oligopyrimidine (5'TOP) motifs known to control protein synthesis. Thus, La ribonucleoproteins (RNP) exist in distinct states that differ in subcellular localization, serine 366 phosphorylation, and associated RNAs. These results are consistent with a model in which the relative concentrations of the La S366 isoforms in different subcellular compartments in conjunction with the relative concentrations of specific RNA ligands in these compartments determine the differential association of npLa and pLa with their respective classes of associated RNAs.

Antibodies, Phospho-Specific↗
PubMed2003

Posttranscriptional generation of macromolecular complexes.

Discrete classes of mRNAs that encode functionally related proteins are associated with sequence-specific RNA-binding proteins in yeast and mammalian cells. recently reported that pre-mRNAs encoding components of inhibitory synapses are bound to neuron-specific Nova RNA-binding proteins.

Animals↗
PubMed2003

Partitioning and translation of mRNAs encoding soluble proteins on membrane-bound ribosomes.

In eukaryotic cells, it is generally accepted that protein synthesis is compartmentalized; soluble proteins are synthesized on free ribosomes, whereas secretory and membrane proteins are synthesized on endoplasmic reticulum (ER)-bound ribosomes. The partitioning of mRNAs that accompanies such compartmentalization arises early in protein synthesis, when ribosomes engaged in the translation of mRNAs encoding signal-sequence-bearing proteins are targeted to the ER. In this report, we use multiple cell fractionation protocols, in combination with cDNA microarray, nuclease protection, and Northern blot analyses, to assess the distribution of mRNAs between free and ER-bound ribosomes. We find a broad representation of mRNAs encoding soluble proteins in the ER fraction, with a subset of such mRNAs displaying substantial ER partitioning. In addition, we present evidence that membrane-bound ribosomes engage in the translation of mRNAs encoding soluble proteins. Single-cell in situ hybridization analysis of the subcellular distribution of mRNAs encoding ER-localized and soluble proteins identify two overall patterns of mRNA distribution in the cell-endoplasmic reticular and cytosolic. However, both partitioning patterns include a distinct perinuclear component. These results identify previously unappreciated roles for membrane-bound ribosomes in the subcellular compartmentalization of protein synthesis and indicate possible functions for the perinuclear membrane domain in mRNA sorting in the cell.

Cell Fractionation↗
PubMed2002

Ribonomics: identifying mRNA subsets in mRNP complexes using antibodies to RNA-binding proteins and genomic arrays.

Although in vitro methods have been used to identify putative targets of mRNA-binding proteins, direct in vivo methods are needed to identify endogenously associated mRNAs and their cognate proteins. Therefore, we have developed high-throughput methods to identify structurally and/or functionally related mRNA transcripts through their endogenous association with RNA-binding proteins. We have termed the identification and analysis of mRNA subsets using RNA-associated proteins ribonomics, and have established four primary steps for the method: (1) isolation of endogenous mRNA-protein complexes (mRNPs) under optimized conditions, (2) the en masse characterization of the protein and mRNA components associated with the targeted mRNP complexes, (3) identification of sequences or structural similarities among members of the mRNA subset, and (4) determination of functional relationships among the protein products coded for by members of the mRNA subset. We have hypothesized that mRNAs are organized into structurally and functionally linked groups to better affect information transfer through coordinate gene expression. The functional consequences of such organization would be to facilitate the production of proteins that regulate processes necessary for growth and differentiation. This article describes a series of biochemical techniques that deal with the first two steps of ribonomic profiling: purifying endogenous mRNP complexes and identifying multiple mRNA targets using microarray analysis.

Animals↗
PubMed2002

Eukaryotic mRNPs may represent posttranscriptional operons.

Genomic array analysis of endogenous mammalian ribonucleoproteins has recently revealed three novel findings: (1) mRNA binding proteins are associated with unique subpopulations of messages, (2) the compositions of these mRNA subsets can vary with growth conditions, and (3) the same mRNA species can be found in multiple mRNP complexes. Based on these and other findings, we propose a model of posttranscriptional gene expression in which mRNA binding proteins regulate mRNAs as subpopulations during cell growth and development. This model predicts that functionally related genes are regulated posttranscriptionally as groups by specific mRNA binding proteins that recognize sequence elements in common among the mRNAs.

Animals↗
PubMed2002

Autoimmune epitopes in messenger RNA.

Patients with systemic autoimmune disorders produce autoantibodies against sequence-specific conformational RNA epitopes on U1 snRNA, 28S rRNA, and transfer RNAs. The molecular basis for immunological reactivity with these highly abundant and stable RNAs is not understood. Here, we report the existence of discrete RNA epitopes in messenger RNAs that are generally less abundant and less stable than snRNAs and tRNAs. An iterative selection and amplification procedure using pooled autoimmune patient sera identified immunoreactive mRNA species. Following deconvolution of the pools to identify the reactive sera, several mRNAs recognized by these autoantibodies were cloned and sequenced. Detailed analysis using one particular serum indicated reactivity against the messages encoding alternative splicing factor (ASF/SF2) and calmodulin. Deletion and site-directed mutagenesis determined that an epitope recognized by this serum is located in a 17-base stem-loop structure common to both messages. This serum was then used to immunoprecipitate native mRNAs encoding ASF/SF2 and calmodulin from total HeLa cell RNA. Our results demonstrate that despite its low abundance and instability, messenger RNA is capable of reacting with autoantibodies generated during an autoimmune response. These data are consistent with direct presentation as a model to explain the generation of RNA conformation-specific autoantibodies.

Autoantibodies↗
PubMed2002

A phosphorylated cytoplasmic autoantigen, GW182, associates with a unique population of human mRNAs within novel cytoplasmic speckles.

A novel human cellular structure has been identified that contains a unique autoimmune antigen and multiple messenger RNAs. This complex was discovered using an autoimmune serum from a patient with motor and sensory neuropathy and contains a protein of 182 kDa. The gene and cDNA encoding the protein indicated an open reading frame with glycine-tryptophan (GW) repeats and a single RNA recognition motif. Both the patient's serum and a rabbit serum raised against the recombinant GW protein costained discrete cytoplasmic speckles designated as GW bodies (GWBs) that do not overlap with the Golgi complex, endosomes, lysosomes, or peroxisomes. The mRNAs associated with GW182 represent a clustered set of transcripts that are presumed to reside within the GW complexes. We propose that the GW ribonucleoprotein complex is involved in the posttranscriptional regulation of gene expression by sequestering a specific subset of gene transcripts involved in cell growth and homeostasis.

Amino Acid Motifs↗