Showing papers by "Michael R. Green published in 1995"
TL;DR: It is shown that PTB can regulate alternative splicing by selectively repressing 3' splice sites that contain a PTB-binding protein, which can help explain the ability of Sxl to regulate splicing of transformer pre-mRNA and autoregulatesplicing of its own pre- mRNA.
Abstract: In higher eukaryotes, the polypyrimidine-tract (Py-tract) adjacent to the 3' splice site is recognized by several proteins, including the essential splicing factor U2AF65, the splicing regulator Sex-lethal (Sxl), and polypyrimidine tract-binding protein (PTB), whose function is unknown. Iterative in vitro genetic selection was used to show that these proteins have distinct sequence preferences. The uridine-rich degenerate sequences selected by U2AF65 are similar to those present in the diverse array of natural metazoan Py-tracts. In contrast, the Sxl-consensus is a highly specific sequence, which can help explain the ability of Sxl to regulate splicing of transformer pre-mRNA and autoregulate splicing of its own pre-mRNA. The PTB-consensus is not a typical Py-tract; it can be found in certain alternatively spliced pre-mRNAs that undergo negative regulation. Here it is shown that PTB can regulate alternative splicing by selectively repressing 3' splice sites that contain a PTB-binding site.
544 citations
TL;DR: A yeast two-hybrid screen is used to clone human Rev interacting protein (hRIP), which specifically interacts with the Rev effector domain, which has homology with nucleoporins, a class of proteins that mediate nucleocytoplasmic transport and are those expected of a Rev cellular cofactor.
Abstract: The Rev protein of human immunodeficiency virus type 1 (HIV-1) facilitates the nuclear export of unspliced and partly spliced viral RNAs. Rev contains an RNA binding domain, required for interaction with HIV-1 RNA, and an effector domain, required for RNA-bound Rev to function. The Rev effector domain is believed to interact with a cellular cofactor required for the Rev response and thus HIV-1 replication. Here we report the use of a yeast two-hybrid screen to clone human Rev interacting protein (hRIP), which specifically interacts with the Rev effector domain. This hRIP protein has homology with nucleoporins, a class of proteins that mediate nucleocytoplasmic transport. These and other properties of hRIP are those expected of a Rev cellular cofactor.
271 citations
TL;DR: It is suggested that activation of HSF1 by stress in humans is controlled by two regulatory mechanisms that separately confer heat shock-induced DNA binding and transcriptional stimulation.
Abstract: Human heat shock factor 1 (HSF1) stimulates transcription from heat shock protein genes following stress. We have used chimeric proteins containing the GAL4 DNA binding domain to identify the transcriptional activation domains of HSF1 and a separate domain that is capable of regulating activation domain function. This regulatory domain conferred heat shock inducibility to chimeric proteins containing the activation domains. The regulatory domain is located between the transcriptional activation domains and the DNA binding domain of HSF1 and is conserved between mammalian and chicken HSF1 but is not found in HSF2 or HSF3. The regulatory domain was found to be functionally homologous between chicken and human HSF1. This domain does not affect DNA binding by the chimeric proteins and does not contain any of the sequences previously postulated to regulate DNA binding of HSF1. Thus, we suggest that activation of HSF1 by stress in humans is controlled by two regulatory mechanisms that separately confer heat shock-induced DNA binding and transcriptional stimulation.
191 citations
TL;DR: It is shown that no specific sequence of the leucine zipper is required for a Tax response, and Tax provides a model for how a single auxiliary factor can regulate multiple sequence-specific DNA-binding proteins.
Abstract: HUMAN T-cell leukaemia virus type I (HTLV-I) Tax protein increases the DNA binding of many cellular transcription factors that contain a basic regiona¤-leucine zipper (bZIP) DNA-binding domain1a¤-3. bZIP domains comprise a leucine-rich dimerization motif and a basic region that mediates DNA contact. How Tax recognizes diverse bZIPs is not understood. Here we show that no specific sequence of the leucine zipper is required for a Tax response. In contrast, the basic region is essential for the Tax-mediated DNA-binding increase, which can be eliminated by single substitutions of several conserved amino acids. Surprisingly, Tax alters the relative affinity of a bZIP for different DNA binding sites. Thus, through recognition of the conserved basic region, Tax increases DNA binding and modifies DNA site selection. Tax provides a model for how a single auxiliary factor can regulate multiple sequence-specific DNA-binding proteins.
134 citations
TL;DR: Evidence is provided that diverse activators recruit TFIIB to the promoter by a related mechanism, and this central step in transcriptional activation is sensitive to promoter architecture, and is required for each new round of transcription.
58 citations
Journal Article•
TL;DR: Using site-specific photochemical cross-linking, this work has identified two proteins that specifically interact with the branch point during the splicing reaction and indicates that the branches point is sequentially recognized by distinct splicing factors in the course of thesplicing reaction.
Abstract: Splicing of pre-mRNAs occurs via a lariat intermediate in which an intronic adenosine, embedded within a branch point sequence, forms a 2',5'-phosphodiester bond (RNA branch) with the 5' end of the intron. How the branch point is recognized and activated remains largely unknown. Using site-specific photochemical cross-linking, we have identified two proteins that specifically interact with the branch point during the splicing reaction. U2AF65, an essential splicing factor that binds to the adjacent polypyrimidine tract, crosslinks to the branch point at the earliest stage of spliceosome formation in an ATP-independent manner. A novel 28-kDa protein, which is a constituent of the mature spliceosome, contacts the branch point after the first catalytic step. Our results indicate that the branch point is sequentially recognized by distinct splicing factors in the course of the splicing reaction.
50 citations
Journal Article•
TL;DR: The differential effects of the aptamer probe on protein:protein interactions suggest a model for how the transcription factor binding sites on the surface of the Tax protein are organized, which is consistent with data from a variety of other studies.
Abstract: Nucleic acid aptamers isolated from random sequence pools have generally proven useful at inhibiting the interactions of nucleic acid binding proteins with their cognate nucleic acids. In order to develop reagents that could also be used to study protein:protein interactions, we have used in vitro selection to search for RNA aptamers that could interact with the transactivating protein Tax from human T-cell leukemia virus. Tax does not normally bind to nucleic acids, but instead stimulates transcription by interacting with a variety of cellular transcription factors, including the cyclic AMP-response element binding protein (CREB), NF-kappa B, and the serum response factor (SRF). Starting from a pool of greater than 10(13) different RNAs with a core of 120 random sequence positions, RNAs were selected for their ability to be co-retained on nitrocellulose filters with Tax. After five cycles of selection and amplification, a single nucleic acid species remained. This aptamer was found to bind Tax with high affinity and specificity, and could disrupt complex formation between Tax and NF-kappa B, but not with SRF. The differential effects of our aptamer probe on protein:protein interactions suggest a model for how the transcription factor binding sites on the surface of the Tax protein are organized. This model is consistent with data from a variety of other studies.
46 citations
01 Jan 1995
TL;DR: Most messenger RNAs in higher eukaryotes are transcribed as precursors containing intervening sequences (introns), which are removed by a sophisticated processing machinery that splices together the sequences which form the mature mRNA.
Abstract: Most messenger RNAs in higher eukaryotes are transcribed as precursors containing intervening sequences (introns). Introns are removed by a sophisticated processing machinery that splices together the sequences (exons) which form the mature mRNA (reviewed in chapters 3–5). Although the origin of introns is unclear, cells have learned to harness the splicing process to regulate gene expression. Retaining an intron or using alternative splice sites can be used to switch genes on or off, or to synthesize proteins with differences in their structural domains. The protein variants may differ in the presence or absence of a nuclear localization signal, a membrane attachment region, a phosphorylation site, a dimerization motif, a transcriptional activation domain, etc. These differences have important consequences for the functional properties of the protein, and even for the physiology and identity of the cell. A single alternative splicing decision can, for example, define the sex of flies, trigger programmed cell death or turn a nonmetastatic tumor into a malignant cancer. Table 6.1 summarizes some of these effects.
21 citations
TL;DR: It is shown that Tat activation can be reconstructed on a completely synthetic promoter lacking all cis-acting elements unique to the human immunodeficiency virus long terminal repeat.
Abstract: Lentivirus Tat proteins comprise a novel class of RNA-binding transcriptional activators that are essential for viral replication. In this study, we performed a series of protein fusion experiments to delineate the minimal protein domains and promoter elements required for Tat action. We show that a 15-amino-acid region of equine infectious anemia virus (EIAV) Tat protein, when fused to the GAL4 or LexA DNA binding domain, can activate transcription in appropriate promoter contexts. In the natural human immunodeficiency virus type 1 long terminal repeat, activation by Tat is dependent on multiple binding sites for the cellular transcription factor SP1. We delineate a 114-amino-acid region of the SP1 glutamine-rich activation domain that when fused to the GAL4 DNA binding domain can support transcription activation by Tat. Using these Tat and SP1 derivatives, we show that Tat activation can be reconstructed on a completely synthetic promoter lacking all cis-acting elements unique to the human immunodeficiency virus long terminal repeat. Our results indicate that lentivirus Tat proteins have essential properties of typical cellular transcriptional activators and define useful reagents for studying the detailed mechanism of Tat action.
15 citations
Patent•
10 Jul 1995TL;DR: In this article, the authors proposed a method of detecting leukemic cells in a biological sample, the method includes measuring the activity of a RING3-related kinase in the sample, an increase in the activity relative to a control being indicative of the presence of leukemia cells.
Abstract: New nuclear kinases that are regulated by signal transduction and that participate in phosphorylation cascades which regulate transcription and related methods for regulating transcription. The novel nuclear kinases play a vital role in gene expression, particularly with regard to leukemia in humans. The kinase is (i) substantially exclusively intranuclearly localized; (ii) capable of autophosphorylation; (iii) selectively bindable with antibodies raised against the RING3 portion of GST-RING3; (iv) of a molecular weight of from about 82.5 to about 92.7 kilodaltons; and (v) includes peptide sequences Asp-Ser-Asn Pro-Asp-Glu-Ile-Glu-Ile-Asp-Phe-Glu-Thr-Leu-Lys-Pro-Thr-Thr-Leu (SEQ ID NO: 1) and Ala-Val-His-Glu-Gln-Leu-Ala-Ala-Leu-Ser-Gln-Ala-Pro (SEQ ID NO: 2). The invention features a method of detecting leukemic cells in a biological sample, the method includes measuring the activity of a RING3-related kinase in the sample, an increase in the activity relative to a control being indicative of the presence of leukemic cells.
2 citations
Patent•
06 Dec 1995
TL;DR: In this paper, the authors proposed a high-throughput screening method to identify inhibitors of hepatitis B Virus (HBV) replication which act by inhibiting the ability of the pX protein of HBV to promote DNA binding of bZIP-containing transcription factors.
Abstract: The present invention provides high-throughput screening methods to identify inhibitors of Hepatitis B Virus (HBV) replication which act by inhibiting the ability of the pX protein of HBV to promote DNA binding of bZIP-containing transcription factors. The invention also provides methods for inhibiting replication of HBV by introducing into injected calls an inhibitor molecule that interferes with the binding of the pX protein to a bZIP domain in a bZIP-containing protein.