Search PubMed⌕ Search

Biomedical subjects

Michael B Mathews

Publications and source records attributed to Michael B Mathews.

18 recordsLinked to original sources
PubMed2005

Polymorphic CUG repeats in human mRNAs and their effects on gene expression.

Expanded CUG repeats in the 3'-untranslated region (UTR) of the gene encoding myotonic dystrophy protein kinase (DMPK) cause myotonic dystrophy type 1 disease (DM1). The presence of such repeats has been found to impede gene expression at several levels in model systems. We took a bioinformatic approach to survey all human mRNA sequences for polymorphic CUG repeats. Our survey revealed that CUG repeats occur widely in various regions of mRNAs, with higher frequency in protein coding regions than 5'-UTRs or 3'-UTRs. About 30 genes were found to contain CUG repeats that are polymorphic in the number of repeats, suggesting the potential to expand or shrink. However, long polymorphic repeats were restricted to the 3'-UTR of the DMPK gene and the coding region of the ribosomal protein L14 gene. Using cell-free translation systems, we showed that extended CUG repeats can inhibit protein synthesis in vitro in the rabbit reticulocyte lysate, but not in wheat germ extracts, consistent with our previous finding of an interaction of CUG repeats with the protein kinase PKR. In transfected cells, CUG repeats can inhibit gene expression both in cis and in trans. However, observations with PKR-minus cells indicate that these effects are not primarily attributable to the interaction of extended CUG repeats with PKR. Northwestern blotting detected the presence in human cells of more CUG-binding proteins than are currently known.

Animals↗
PubMed2005

RNA binding and phosphorylation determine the intracellular distribution of nuclear factors 90 and 110.

Members of the nuclear factor 90 (NF90) family of human double-stranded RNA (dsRNA) binding proteins are phosphorylated and translocate into the cytoplasm with the onset of mitosis. We investigated the mechanism of translocation for NF90 and NF110, its larger splice variant. During interphase, NF90 is predominantly nuclear, NF110 is exclusively nuclear, and both are bound to RNA. About half of the NF90 is tethered in the nucleus by RNA bound to the protein's dsRNA-binding motifs. The nuclear localization of NF110 is also dependent on RNA binding but is independent of these motifs, and is governed by contacts made to the protein's unique C terminus. During mitosis, about half of the cytoplasmic NF90 becomes dissociated from RNA, but phosphorylation does not impair the binding affinity of either NF90 or NF110 for dsRNA. We conclude that NF90 and NF110 engage RNA differentially and translocate from the nucleus to the cytoplasm in mitosis because phosphorylation disturbs their interactions with other nuclear proteins.

Alternative Splicing↗
PubMed2005

Granulin and granulin repeats interact with the Tat.P-TEFb complex and inhibit Tat transactivation.

The cellular positive transcription elongation factor b (P-TEFb), containing cyclin T1 and cyclin-dependent kinase 9 (CDK9), interacts with the human immunodeficiency virus, type 1 (HIV-1) regulatory protein Tat to enable viral transcription and replication. Cyclin T1 is an unusually long cyclin and is engaged by cellular regulatory proteins. Previous studies showed that the granulin/epithelin precursor (GEP) binds the histidine-rich region of cyclin T1 and inhibits P-TEFb function. GEP is composed of repeats that vary in sequence and properties. GEP also binds directly to Tat. Here we show that GEP and some of its constituent granulin repeats can inhibit HIV-1 transcription via Tat without directly binding to cyclin T1. The interactions of granulins with Tat and cyclin T1 differ with respect to their binding sites and divalent cation requirements, and we identified granulin repeats that bind differentially to Tat and cyclin T1. Granulins DE and E bind Tat but do not interact directly with cyclin T1. These granulins are present in complexes with Tat and P-TEFb in which Tat forms a bridge between the cellular proteins. Granulins DE and E repress transcription from the HIV-1 LTR and gene expression from the viral genome, raising the possibility of developing granulin-based inhibitors of viral infection.

Amino Acid Sequence↗
PubMed2004

MLE functions as a transcriptional regulator of the roX2 gene.

Dosage compensation is a process that equalizes transcription activity between the sexes. In Drosophila, two non-coding RNA, roX1 and roX2, and at least six protein regulators, MSL-1, MSL-2, MSL-3, MLE, MOF, and JIL-1, have been identified as essential for dosage compensation. Although there is accumulating evidence of the intricate functional and physical interactions between protein and RNA regulators, little is known about how roX RNA expression and function are modulated in coordination with protein regulators. In this report, we have found that a relatively short (about 350 bp) upstream genomic region of the roX2 gene, Prox2, harbors an activity that drives transcription of the downstream gene. Our study has shown that MLE can stimulate the transcription activity of Prox2 and that MLE associates with Prox2 through direct interaction with a newly identified 54-bp repeat, Prox. Our observations suggest a novel mechanism by which roX2 RNA is regulated at the transcriptional level.

Animals↗
PubMed2004

The double-stranded-RNA-binding motif: interference and much more.

RNA duplexes have been catapulted into the spotlight by the discovery of RNA interference and related phenomena. But double-stranded and highly structured RNAs have long been recognized as key players in cell processes ranging from RNA maturation and localization to the antiviral response in higher organisms. Penetrating insights into the metabolism and functions of such RNAs have come from the identification and study of proteins that contain the double-stranded-RNA-binding motif.

Amino Acid Sequence↗
PubMed2003

Selective regulation of gene expression by nuclear factor 110, a member of the NF90 family of double-stranded RNA-binding proteins.

Members of the nuclear factor 90 (NF90) family of double-stranded RNA (dsRNA)-binding proteins have been implicated in several biological processes including the regulation of gene expression. cDNA sequences predict that the proteins have a functional nuclear localization signal and two dsRNA-binding motifs (dsRBMs), and are identical at their N termini. Isoforms are predicted to diverge at their C termini as well as by the insertion of four amino acid residues (NVKQ) between the two dsRBMs. In this study, we verified the expression of four of the isoforms by cDNA cloning and mass spectrometric analysis of proteins isolated from human cells. Cell fractionation studies showed that NF90 and its heteromeric partner, NF45, are predominantly nuclear and largely chromatin-associated. The C-terminally extended NF90 species, NF110, are almost exclusively chromatin-bound. Both NF110 isoforms are more active than NF90 isoforms in stimulating transcription from the proliferating cell nuclear antigen reporter in a transient expression system. NF110b, which carries the NVKQ insert, was identified as the strongest activator. It stimulated transcription of some, but not all, promoters in a fashion that suggested that it functions in concert with other transcription factors. Finally, we demonstrate that NF110b associates with the dsRBM-containing transcriptional co-activator, RNA helicase A, independently of RNA binding.

Amino Acid Sequence↗
PubMed2003

Differential activation of Tat variants in mitogen-stimulated cells: implications for HIV-1 postintegration latency.

Like other HIV-1 (human immunodeficiency virus type 1) proteins, Tat undergoes rapid mutation and occurs in numerous sequence variants in nature. Virus isolated from patients often has defects in Tat that lower its activity. The levels of P-TEFb, an essential cellular cofactor for Tat, are elevated by T-cell activation. To test the hypothesis that stimulation of P-TEFb levels might compensate for attenuation of Tat activity, we generated Tat constructs with a range of transactivation function. Transactivation by the Tat mutants correlated with their ability to bind to P-TEFb in vitro. Treatment of U937 cells with the phorbol ester PMA (phorbol myristate acetate) induced P-TEFb and stimulated Tat transactivation for alleles with basal transcription activity above a threshold (>5% compared to wild-type). Highly active alleles (>66% of wild-type) were stimulated to a lesser extent than those with activity in the intermediate range. Thus, attenuation of Tat function, in concert with low levels of P-TEFb activity, could serve to keep the virus in a latent state in quiescent cells yet permit viral replication after cell activation.

Amino Acid Sequence↗
PubMed2003

RNA helicase A interacts with dsDNA and topoisomerase IIalpha.

RNA helicase A (RHA) is a multifunctional protein involved in various nuclear processes such as transcription and RNA export. It is believed that the interacting factors play important roles in determining the functional specificity of RHA. Here we show that RHA directly interacts with double-stranded (ds) nucleic acids (NAs) and assembles complexes with topoisomerase IIalpha. First, electrophoresis mobility shift assays demonstrate that RHA interacts with dsDNAs of different lengths ranging from 15 to 104 bp. Secondly, the binding of RHA to closed circular dsDNA stimulates the relaxation reaction catalyzed by either calf thymus topoisomerase I or HeLa topoisomerase IIalpha. Thirdly, immunoprecipitation, coupled with western blot analysis using anti-RHA and anti-topoisomerase IIalpha antibodies, shows that RHA and topoisomerase IIalpha assemble a complex in the presence of as yet unknown RNA molecules and additional protein factors such as Ubc9. Our observation suggests physical and functional interaction between RHA and topoisomerase IIalpha, which, perhaps, play important roles in regulating chromatin structure. The putative role of RHA-topoisomerase IIalpha complex in RNA polymerase II-mediated transcription is discussed.

Adenosine Triphosphatases↗
PubMed2003

Functional cloning of genes encoding dsRNA binding proteins.

Over a dozen human genes code for proteins that specifically bind to double-stranded RNA. These proteins have been implicated in several important cellular processes such as transcriptional activation, inhibition of translational initiation, RNA editing, mRNA localization, signal transduction, and posttranscriptional gene silencing (PTGS or RNAi). The recent discovery that PTGS or RNAi is a dsRNA-mediated pathway has further added to the study of dsRNA binding proteins. A method that enables the cloning of genes encoding dsRNA binding proteins would greatly facilitate the identification and study of these molecules. Here we describe a method for isolating such genes from an expression library using radiolabeled poly(I):poly(C) as a binding substrate.

Cloning, Molecular↗
PubMed2003

A naturally occurring substitution in human immunodeficiency virus Tat increases expression of the viral genome.

A natural amino acid substitution in the human immunodeficiency virus type 1 (HIV-1) transcriptional activator Tat increases its activity and compensates for deleterious mutations elsewhere in the Tat protein. Substitution of asparagine for threonine 23 increases Tat transactivation of the HIV-1 promoter and the binding of Tat to the cellular kinase positive transcription elongation factor b (P-TEFb). Of nine other position 23 mutations tested, only the serine substitution retained wild-type activity. Correspondingly, asparagine is the most frequent amino acid at this position in HIV-1 isolates, followed by threonine and serine. Asparagine is prevalent in Tat proteins of viruses in clades A, C, and D, which are major etiologic agents of AIDS. We suggest that selection for asparagine in position 23 confers an advantage to the virus, since it can compensate for deleterious mutations in Tat. It may also support the replication of otherwise less fit drug-resistant viruses and permit the emergence of virulent strains.

Amino Acid Sequence↗
PubMed2003

The human I-mfa domain-containing protein, HIC, interacts with cyclin T1 and modulates P-TEFb-dependent transcription.

Positive transcription elongation factor b (P-TEFb) hyperphosphorylates the carboxy-terminal domain of RNA polymerase II, permitting productive transcriptional elongation. The cyclin T1 subunit of P-TEFb engages cellular transcription factors as well as the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. To identify potential P-TEFb regulators, we conducted a yeast two-hybrid screen with cyclin T1 as bait. Among the proteins isolated was the human I-mfa domain-containing protein (HIC). HIC has been reported to modulate expression from both cellular and viral promoters via its C-terminal cysteine-rich domain, which is similar to the inhibitor of MyoD family a (I-mfa) protein. We show that HIC binds cyclin T1 in yeast and mammalian cells and that it interacts with intact P-TEFb in mammalian cell extracts. The interaction involves the I-mfa domain of HIC and the regulatory histidine-rich region of cyclin T1. HIC also binds Tat via its I-mfa domain, although the sequence requirements are different. HIC colocalizes with cyclin T1 in nuclear speckle regions and with Tat in the nucleolus. Expression of the HIC cDNA modulates Tat transactivation of the HIV-1 long terminal repeat (LTR) in a cell type-specific fashion. It is mildly inhibitory in CEM cells but stimulates gene expression in HeLa, COS, and NIH 3T3 cells. The isolated I-mfa domain acts as a dominant negative inhibitor. Activation of the HIV-1 LTR by HIC in NIH 3T3 cells occurs at the RNA level and is mediated by direct interactions with P-TEFb.

Animals↗
PubMed2003

The growth factor granulin interacts with cyclin T1 and modulates P-TEFb-dependent transcription.

Cyclin T1, together with the kinase CDK9, is a component of the transcription elongation factor P-TEFb which binds the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. P-TEFb facilitates transcription by phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. Cyclin T1 is an exceptionally large cyclin and is therefore a candidate for interactions with regulatory proteins. We identified granulin as a cyclin T1-interacting protein that represses expression from the HIV-1 promoter in transfected cells. The granulins, mitogenic growth factors containing repeats of a cysteine-rich motif, were reported previously to interact with Tat. We show that granulin formed stable complexes in vivo and in vitro with cyclin T1 and Tat. Granulin bound to the histidine-rich domain of cyclin T1, which was recently found to bind to the CTD, but not to cyclin T2. Binding of granulin to P-TEFb inhibited the phosphorylation of a CTD peptide. Granulin expression inhibited Tat transactivation, and tethering experiments showed that this effect was due, at least in part, to a direct action on cyclin T1 in the absence of Tat. In addition, granulin was a substrate for CDK9 but not for the other transcription-related kinases CDK7 and CDK8. Thus, granulin is a cellular protein that interacts with cyclin T1 to inhibit transcription.

3T3 Cells↗
PubMed2003

RNA binding and intramolecular interactions modulate the regulation of gene expression by nuclear factor 110.

Nuclear factor 110 (NF110) belongs to the nuclear factor 90 (NF90) family of double-stranded RNA (dsRNA) binding proteins that regulate gene expression at the transcriptional level in vertebrates. The proteins are identical at their N terminus, which functions as a negative regulatory region, but have distinct C termini as a result of alternate splicing. Maximal transcriptional activity of NF110 requires its C-terminal domain and a central domain that contains a nuclear localization signal and two dsRNA-binding motifs (dsRBMs). We find that dsRNA binding is reduced by RGG and GQSY motifs present in the C-terminal region. To directly evaluate the role of RNA binding in transactivation, we conducted site-directed mutagenesis to substitute conserved residues in one or both of the dsRBMs. The mutations reduced the ability of NF110 to stimulate gene expression to an extent that paralleled the mutants' reduced ability to bind dsRNA. Full activity was restored when the dsRBM-containing region of NF110 was replaced with the RNA-binding region of the protein kinase PKR. Finally, NF110-mediated transactivation was inhibited by cotransfection of a plasmid encoding an artificial highly structured RNA. These data suggest that NF110 and its homologs are regulated by cis-acting domains present in some of the protein isoforms, and via interactions with RNAs that bind to their dsRBMs. We propose a model in which structured RNAs regulate gene expression by modulating transcription through interactions with members of the NF90 protein family.

Base Sequence↗
PubMed2002

Neoplastic progression in melanoma and colon cancer is associated with increased expression and activity of the interferon-inducible protein kinase, PKR.

The interferon-inducible, double-stranded RNA (dsRNA)-activated protein kinase, PKR, plays key roles in regulation of cell growth and differentiation, and has been postulated as a tumor suppressor. Downstream effectors of PKR include the translation initiation factor, eIF2alpha, and the transcription factor, NF-kappaB. We found elevated levels of PKR protein, dsRNA-dependent PKR autophosphorylation activity, and phosphorylated eIF2alpha in melanoma cells compared to nontransformed melanocytes in culture. Treatment with interferon-alpha2b further induced PKR expression and activity. Immunohistochemical analysis of primary melanomas demonstrated minimal PKR immunoreactivity, but melanoma lymph node metastases expressed a high level of PKR protein. Furthermore, analysis of colon cancer specimens revealed that transformation from normal mucosa to adenomas and carcinomas was coincident with an increase in PKR expression. These data do not support the concept of PKR as a classic tumor suppressor but instead suggest that PKR upregulation occurs at defined steps in cancer progression, probably as a cellular response to neoplasia.

Colonic Neoplasms↗
PubMed2002

The 3'-untranslated regions of cytoskeletal muscle mRNAs inhibit translation by activating the double-stranded RNA-dependent protein kinase PKR.

Cytoskeletal proteins are associated with actin in the microfilaments and have a major role in microfilament assembly and function. The expression of some of these proteins has been implicated in cell growth and transformation. Specifically, the 3'-untranslated regions (3'-UTRs) of tropomyosin, troponin and cardiac actin can induce muscle cell differentiation and appear to function as tumor suppressors. These RNA sequences are predicted to fold to form secondary structures with extended stretches of duplex. We show that the 3'-UTRs of the cytoskeletal mRNAs interact with the RNA-binding domain of the RNA-activated protein kinase PKR. Correspondingly, these RNAs activate PKR in vitro and inhibit globin translation in the rabbit reticulocyte lysate translation system. These data are consistent with a mechanism whereby PKR mediates the differentiation- and tumor-related actions of the cytoskeletal 3'-UTR sequences.

3' Untranslated Regions↗
PubMed2002

Lost in translation.

Explore the source record for details and available documents.

5' Untranslated Regions↗
PubMed2002

The RNA binding protein nuclear factor 90 functions as both a positive and negative regulator of gene expression in mammalian cells.

Nuclear factor 90 (NF90) was originally isolated in a complex that binds to the antigen recognition response element (ARRE-2) present in the interleukin-2 promoter. To characterize the transcriptional properties of NF90 in mammalian cells, we examined its ability to modulate promoter function in cellular transfection assays. NF90-Gal4 fusion proteins inhibited transcription from the adenovirus major late promoter in a fashion that was dependent on Gal4 targeting. Conversely, NF90 activated the cytomegalovirus immediate-early promoter, to which it was not targeted. These effects required distinct but overlapping domains in the C terminus of NF90, which contains a functional nuclear localization signal and two double-stranded-RNA binding motifs. NF90 is present in cellular complexes together with the NF45 protein. Transfection assays showed that NF45 binds NF90 strongly and stimulates its ability to activate but not to inhibit gene expression. This report characterizes NF90 as both a positive and negative regulator of gene expression, depending on the promoter context, and suggests a role for NF45 as a regulator of NF90.

Adenoviridae↗