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J L Manley

Publications and source records attributed to J L Manley.

At least 37 records · Page 2Linked to original sources
PubMed2000

The Drosophila TATA binding protein contains a strong but masked activation domain.

TATA binding protein (TBP) is a critical transcription factor involved in transcription by all three RNA polymerases (RNAPs). Studies using in vitro systems and yeast have shown that the C-terminal core domain (CTD) of TBP is necessary and sufficient for many TBP functions, but the significance of the N-terminal domain (NTD) of TBP is still obscure. Here, using transient expression assays in Drosophila Schneider cells, we show that the NTD of Drosophila TBP (dTBP) strongly activates transcription when fused to the GAL4 DNA binding domain (DBD). Strikingly, the activity of the NTD is completely repressed in the context of full-length dTBP. In contrast to the much weaker activation obtained by either full-length dTBP or the dTBP CTD fused to the GAL4 DBD, activation by the NTD is dependent on the presence of GAL4 binding sites and is susceptible to the effects of a dominant negative TFIIB mutant, TFIIB deltaC202, a property observed previously with certain authentic activation domains. Activation by the NTD, but not full-length dTBP or the CTD, seems to be mediated by the action of a strong activation domain, likely a glutamine-rich region. In conclusion, the dTBP NTD can behave as a very strong activator that is masked in the full-length protein, suggesting possible roles for the dTBP NTD in RNAP II-mediated transcription.

Animals↗
PubMed1999

Functional interaction of BRCA1-associated BARD1 with polyadenylation factor CstF-50.

Polyadenylation of messenger RNA precursors requires a complex protein machinery that is closely integrated with the even more complex transcriptional apparatus. Here a polyadenylation factor, CstF-50 (cleavage stimulation factor), is shown to interact in vitro and in intact cells with a nuclear protein of previously unknown function, BRCA1-associated RING domain protein (BARD1). The BARD1-CstF-50 interaction inhibits polyadenylation in vitro. BARD1, like CstF-50, also interacts with RNA polymerase II. These results indicate that BARD1-mediated inhibition of polyadenylation may prevent inappropriate RNA processing during transcription, perhaps at sites of DNA repair, and they reveal an unanticipated integration of diverse nuclear events.

Antibodies, Monoclonal↗
PubMed1999

Phosphorylated RNA polymerase II stimulates pre-mRNA splicing.

RNA polymerase II (RNAP II) is responsible for transcription of mRNA precursors in eukaryotic cells. Recent studies, however, have suggested that RNAP II also participates in subsequent RNA processing reactions through interactions between the carboxy-terminal domain (CTD) of the RNAP II largest subunit and processing factors. Using reconstituted in vitro splicing assays, we show that RNAP II functions directly in pre-mRNA splicing by influencing very early steps in assembly of the spliceosome. We demonstrate that the phosphorylation status of the CTD dramatically affects activity: Hyperphosphorylated RNAP IIO strongly activates splicing, whereas hypophosphorylated RNAP IIA can inhibit the reaction.

Cell Nucleus↗
PubMed1999

Determinants of SR protein specificity.

The SR (Ser-Arg) proteins are a family of nuclear factors that play multiple important roles in splicing of mRNA precursors in metazoan organisms, functioning in both constitutive and regulated splicing. Certain of these functions are redundant, such that any single SR proteins will suffice, but other functions are unique and are specific to a given family member. A number of studies during the past year have investigated the basis for SR protein specificity.

Animals↗
PubMed1999

Allosteric regulation of even-skipped repression activity by phosphorylation.

The Drosophila homeodomain protein Even-skipped (Eve) is a well characterized transcriptional repressor. Here, we show that Eve's ability to function in vitro is negatively regulated by phosphorylation. DNA-binding activity was unaffected by phosphorylation, but phosphorylated Eve was unable to interact with the TATA-binding protein (TBP), a known target for repression. Unexpectedly, phosphorylation of the Eve N terminus, which is dispensable for repression and TBP binding, was necessary and sufficient to inactivate Eve. LiCl, which specifically inhibits glycogen synthase kinase-3 (GSK-3), reduced Eve phosphorylation in nuclear extract and blocked inhibition of repression. In addition, Eve was phosphorylated and inactivated by purified GSK-3 beta plus casein kinase II. Our results suggest a novel mechanism of transcriptional control involving phosphorylation-induced allosteric interference with a repressive protein-protein interaction.

Allosteric Regulation↗
PubMed1999

The protein kinase Clk/Sty directly modulates SR protein activity: both hyper- and hypophosphorylation inhibit splicing.

The splicing of mammalian mRNA precursors requires both protein phosphorylation and dephosphorylation, likely involving modification of members of the SR protein family of splicing factors. Several kinases have been identified that can phosphorylate SR proteins in vitro, and transfection assays have provided evidence that at least one of these, Clk/Sty, can modulate splicing in vivo. But evidence that a specific kinase can directly affect the splicing activity of SR proteins has been lacking. Here, by using purified recombinant Clk/Sty, a catalytically inactive mutant, and individual SR proteins, we show that Clk/Sty directly affects the activity of SR proteins, but not other essential splicing factors, in reconstituted splicing assays. We also provide evidence that both hyper- and hypophosphorylation inhibit SR protein splicing activity, repressing constitutive splicing and switching alternative splice site selection. These findings indicate that Clk/Sty directly and specifically influences the activity of SR protein splicing factors and, importantly, show that both under- and overphosphorylation of SR proteins can modulate splicing.

Alternative Splicing↗
PubMed1998

Phosphorylation-dephosphorylation differentially affects activities of splicing factor ASF/SF2.

SR proteins are a conserved family of splicing factors that function in both constitutive and activated splicing. We reported previously that phosphorylation of the SR protein ASF/SF2 enhances its interaction with the U1 snRNP-specific 70K protein and is required for the protein to function in splicing, while other studies have provided evidence that subsequent dephosphorylation can also be required for SR protein function, at least in constitutive splicing. We now show that the phosphorylation status of ASF/SF2 can differentially affect several properties of the protein. In keeping with a dynamic cycle of phosphorylation-dephosphorylation during splicing, ASF/SF2 phosphorylation was found to affect interaction with several putative protein targets in different ways: positively, negatively or not at all. Extending these results, we also show that, in contrast to constitutive splicing, dephosphorylation is not required for ASF/SF2 to function as a splicing activator. We discuss these results with respect to the differential protein-protein interactions that must occur during constitutive and activated splicing.

Adenosine Triphosphate↗
PubMed1998

RNA polymerase II is an essential mRNA polyadenylation factor.

Production of messenger RNA in eukaryotic cells is a complex, multistep process. mRNA polyadenylation, or 3' processing, requires several protein factors, including cleavage/polyadenylation-specificity factor (CPSF), cleavage-stimulation factor, two cleavage factors and poly(A) polymerase. These proteins seem to be unnecessary for other steps in mRNA synthesis such as transcription and splicing, and factors required for these processes were not considered to be essential for polyadenylation. Nonetheless, these reactions may be linked so that they are effectively coordinated in vivo. For example, the CTD carboxy-terminal domain of the largest subunit of RNA polymerase II (RNAP II) is required for efficient splicing and polyadenylation in vivo, and CPSF is brought to a promoter by the transcription factor TFIID and transferred to RNAP II at the time of transcription initiation. These findings suggest that polyadenylation factors can be recruited to an RNA 3'-processing signal by RNAP II, where they dissociate from the polymerase and initiate polyadenylation. Here we present results that extend this model by showing that RNAP II is actually required, in the absence of transcription, for 3' processing in vitro.

Animals↗
PubMed1998

The upstream sequence element of the C2 complement poly(A) signal activates mRNA 3' end formation by two distinct mechanisms.

The poly(A) signal of the C2 complement gene is unusual in that it possesses an upstream sequence element (USE) required for full activity in vivo. We describe here in vitro experiments demonstrating that this USE enhances both the cleavage and poly(A) addition reactions. We also show that the C2 USE can be cross-linked efficiently to a 55-kD protein that we identify as the polypyrimidine tract-binding protein (PTB), implicated previously in modulation of pre-mRNA splicing. Mutation of the PTB-binding site significantly reduces the efficiency of the C2 poly(A) site both in vivo and in vitro. Furthermore, addition of PTB to reconstituted processing reactions enhances cleavage at the C2 poly(A) site, indicating that PTB has a direct role in recognition of this signal. The C2 USE, however, also increases the affinity of general polyadenylation factors independently for the C2 poly(A) signal as detected by enhanced binding of cleavage-stimulaton factor (CstF). Strikingly, this leads to a novel CstF-dependant enhancement of the poly(A) synthesis phase of the reaction. These studies both emphasize the interconnection between splicing and polyadenylation and indicate an unexpected flexibility in the organization of mammalian poly(A) sites.

Binding Sites↗
PubMed1998

Genetic analysis of the SR protein ASF/SF2: interchangeability of RS domains and negative control of splicing.

The SR proteins constitute a family of splicing factors, highly conserved in metazoans, that contain one or two amino-terminal RNA-binding domains (RBDs) and a region enriched in arginine/serine repeats (RS domain) at the carboxyl terminus. Previous studies have shown that SR proteins possess distinct RNA-binding specificities that likely contribute to their unique functions, but it is unclear whether RS domains have specific roles in vivo. Here, we used a genetic system developed in the chicken B cell line DT40 to address this question. Expression of chimeric proteins generated by fusion of the RS domains of heterologous SR proteins, or a human TRA-2 protein, with the RBDs of ASF/SF2 allowed cell growth following genetic inactivation of endogenous ASF/SF2, indicating that RS domains are interchangeable for all functions required to maintain cell viability. However, a chimera containing the RS domain from a related splicing factor, U2AF65, could not rescue viability and was inactive in in vitro splicing assays, suggesting that this domain performs a distinct function. We also used the DT40 system to show that depletion of ASF/SF2 affects splicing of specific transcripts in vivo. Although splicing of several simple constitutive introns was not significantly affected, the alternative splicing patterns of two model pre-mRNAs switched in a manner consistent with predictions from previous studies. Unexpectedly, ASF/SF2 depletion resulted in a substantial increase in splicing of an HIV-1 tat pre-mRNA substrate, indicating that ASF/SF2 can repress tat splicing in vivo. These results provide the first demonstration that an SR protein can influence splicing of specific pre-mRNAs in vivo.

Alternative Splicing↗
PubMed1998

Human Tra2 proteins are sequence-specific activators of pre-mRNA splicing.

The RNA-binding protein Tra2 is an important regulator of sex determination in Drosophila. Recently, two mammalian Tra2 homologs of unknown function have been described. Here, we show that human Tra2 proteins are present in HeLa cell nuclear extracts and that they bind efficiently and specifically to a previously characterized pre-mRNA splicing enhancer element. Indeed, both purified proteins bound preferentially to RNA sequences containing GAA repeats, characteristic of many enhancer elements. Neither Tra2 protein functioned in constitutive splicing in vitro, but both activated enhancer-dependent splicing in a sequence-specific manner and restored it after inhibition with competitor RNA. Our findings indicate that mammalian Tra2 proteins are sequence-specific splicing activators that likely participate in the control of cell-specific splicing patterns.

Animals↗
PubMed1998

Inhibition of poly(A) polymerase requires p34cdc2/cyclin B phosphorylation of multiple consensus and non-consensus sites.

We showed previously that p34(cdc2)/cyclin B (MPF) hyperphosphorylates poly(A) polymerase (PAP) during M-phase of the cell cycle, causing repression of its enzymatic activity. Mutation of three cyclin-dependent kinase (cdk) consensus sites in the PAP C-terminal regulatory domain prevented complete phosphorylation and MPF-mediated repression. Here we show that PAP also contains four nearby non-consensus cdk sites that are phosphorylated by MPF. Remarkably, full phosphorylation of all these cdk sites was required for repression of PAP activity, and partial phosphorylation had no detectable effect. The consensus sites were phosphorylated in vitro at a 10-fold lower concentration of MPF than the non-consensus sites. Consistent with this, during meiotic maturation of Xenopus oocytes, consensus sites were phosphorylated prior to the non-consensus sites at metaphase of meiosis I, and remained so throughout maturation, while the non-consensus sites did not become fully phosphorylated until after 12 h of metaphase II arrest. We propose that PAP's multiple cdk sites, and their differential sensitivity to MPF, provide a mechanism to link repression specifically to late M-phase. We discuss the possibility that this reflects a general means to control the timing of cdk-dependent regulatory events during the cell cycle.

Amino Acid Sequence↗
PubMed1998

High mobility group protein-1 (HMG-1) is a unique activator of p53.

The binding of p53 protein to DNA is stimulated by its interaction with covalent as well as noncovalent modifiers. We report the identification of a factor from HeLa nuclear extracts that activates p53 DNA binding. This factor was purified to homogeneity and identified as the high mobility group protein, HMG-1. HMG-1 belongs to a family of highly conserved chromatin-associated nucleoproteins that bend DNA and facilitate the binding of various transcription factors to their cognate DNA sequences. We demonstrate that recombinant His-tagged HMG-1 enhances p53 DNA binding in vitro and also that HMG-1 and p53 can interact directly in vitro. Unexpectedly, HMG-1 also stimulates DNA binding by p53Delta30, a carboxy-terminally deleted form of the protein that is considered to be constitutively active, suggesting that HMG-1 stimulates p53 by a mechanism that is distinct from other known activators of p53. Finally, using transient transfection assays we show that HMG-1 can increase p53 and p53Delta30-mediated transactivation in vivo. HMG-1 promotes the assembly of higher order p53 nucleoprotein structures, and these data, along with the fact that HMG-1 is capable of bending DNA, suggest that HMG-1 may activate p53 DNA binding by a novel mechanism involving a structural change in the target DNA.

DNA↗
PubMed1998

Levels of polyadenylation factor CstF-64 control IgM heavy chain mRNA accumulation and other events associated with B cell differentiation.

Cleavage stimulation factor (CstF) is one of the multiple factors required for mRNA polyadenylation. The concentration of one CstF subunit (CstF-64) increases during activation of B cells, and this is sufficient to switch IgM heavy chain mRNA expression from membrane-bound form to secreted form. To extend this observation, we disrupted the endogenous CstF-64 gene in the B cell line DT40 and replaced it with a regulatable transgene. Strikingly, a 10-fold decrease in CstF-64 concentration did not markedly affect cell growth but specifically and dramatically reduced accumulation of IgM heavy chain mRNA. Further reduction caused reversible cell cycle arrest in G0/G1 phase, while depletion resulted in apoptotic cell death. Our results indicate that CstF-64 plays unexpected roles in regulating gene expression and cell growth in B cells.

Animals↗
PubMed1998

A UV-crosslinkable interaction in human U6 snRNA.

U6 snRNA is the most conserved of all the snRNAs involved in pre-mRNA splicing, and likely plays an important role in splicing catalysis. Using a U6 snRNA fragment encompassing residues 25-99, we have identified a strong, UV-sensitive tertiary intramolecular interaction. A 5' deletion that removed sequences up to nt 37 only slightly reduced crosslinking, but further deletion of 11 bases, eliminating the nearly invariant ACAGAGA sequence, essentially abolished crosslinking, as did deletion of sequences 3' of 82A. The crosslinked residues were mapped to 44G in the ACAGAGA sequence and to 81C, the nucleotide at the base of the U6 intramolecular helix, opposite the G of the invariant AGC trinucleotide. This interaction is striking in that it has the potential to juxtapose invariant regions of U6 believed to play critical roles in splicing catalysis.

Base Sequence↗
PubMed1998

Even-skipped represses transcription by binding TATA binding protein and blocking the TFIID-TATA box interaction.

The Drosophila homeodomain protein Even-skipped (Eve) is a transcriptional repressor, and previous studies have suggested that it functions by interfering with the basal transcription machinery. Here we describe experiments indicating that the mechanism of Eve repression involves a direct interaction with the TATA binding protein (TBP) that blocks binding of TBP-TFIID to the promoter. We first compared Eve activities in in vitro transcription systems reconstituted with either all the general transcription factors or only TBP, TFIIB, TFIIF30, and RNA polymerase II. In each case, equivalent and very efficient levels of repression were observed, indicating that no factors other than those in the minimal system are required for repression. We then show that Eve can function efficiently when its recognition sites are far from the promoter and that the same regions of Eve required for repression in vivo are necessary and sufficient for in vitro repression. This includes, in addition to an Ala-Pro-rich region, residues within the homeodomain. Using GAL4-Eve fusion proteins, we demonstrate that the homeodomain plays a role in repression in addition to DNA binding, which is to facilitate interaction with TBP. Single-round transcription experiments indicate that Eve must function prior to TBP binding to the promoter, suggesting a mechanism whereby Eve represses by competing with the TATA box for TBP binding. Consistent with this, excess TATA box-containing oligonucleotide is shown to specifically and efficiently disrupt the TBP-Eve interaction. Importantly, we show that Eve binds directly to TFIID and that this interaction can also be disrupted by the TATA oligonucleotide. We conclude that Eve represses transcription via a direct interaction with TBP that blocks TFIID binding to the promoter.

Animals↗
PubMed1998

Deregulation of poly(A) polymerase interferes with cell growth.

Vertebrate poly(A) polymerase (PAP) contains a catalytic domain and a C-terminal Ser-Thr-rich regulatory region. Consensus and nonconsensus cyclin-dependent kinase (cdk) sites are conserved in the Ser-Thr-rich region in vertebrate PAPs. PAP is phosphorylated by cdc2-cyclin B on these sites in vitro and in vivo and is inactivated by hyperphosphorylation in M-phase cells, when cdc2-cyclin B is active. In the experiments described here, we undertook a genetic approach in chicken DT40 cells to study the function of PAP phosphorylation. We found that PAP is highly conserved in chicken and is essential in DT40 cells. While cells could tolerate reduced levels of PAP, even modest overexpression of either wild-type PAP or a mutant PAP with two consensus cdk sites mutated (cdk- PAP) was highly deleterious and at a minimum resulted in reduced growth rates. Importantly, cells that expressed cdk- PAP had a significantly lower growth rate than did cells that expressed similar levels of wild-type PAP, which was reflected in increased accumulation of cells in the G0-G1 phase of the cell cycle. We propose that the lower growth rate is due to the failure of hyperphosphorylation and thus M-phase inactivation of cdk- PAP.

Amino Acid Sequence↗