PubMed Health⌕ Search

Biomedical subjects

J L Manley

Publications and source records attributed to J L Manley.

At least 55 records · Page 3Linked to original sources
PubMed1998

Phosphorylation modulates direct interactions between the Toll receptor, Pelle kinase and Tube.

Determination of dorsal/ventral polarity in Drosophila requires 12 genetically defined, maternally encoded proteins. These include Toll, a transmembrane receptor, Pelle, a ser/thr protein kinase and Tube, all of which function intracytoplasmically to initiate the cascade that ultimately activates Dorsal, an NF-kappaB family transcription factor. Here we describe biochemical interactions between recombinant Toll, Pelle and Tube that provide insights into early events in activation of the signaling cascade. We first show that Pelle binds directly to a region within the Toll intracytoplasmic domain, providing the first evidence that these two evolutionarily conserved molecules physically interact. We then demonstrate that Pelle can be autophosphorylated, and that this prevents binding to Toll as well as Tube. Autophosphorylation occurs in the N-terminal, death-domain-containing region of Pelle, which is dispensable for binding to Toll but required for enzymatic activity. We also show that Pelle phosphorylates Toll, within the region required for Pelle interaction, but this phosphorylation can be blocked by a previously characterized inhibitory domain at the Toll C terminus. These and other results allow us to propose a model by which multiple phosphorylation-regulated interactions between these three proteins lead to activation of the Dorsal signaling pathway.

Animals↗
PubMed1997

Creatine phosphate, not ATP, is required for 3' end cleavage of mammalian pre-mRNA in vitro.

The poly(A) tail of a mammalian mRNA is generated by endonucleolytic cleavage and poly(A) addition. Previous studies conducted with nuclear extracts suggested an ATP requirement for the cleavage step. We have reexamined the cofactor requirement, initially with the SV40 late pre-mRNA, which requires for cleavage four protein factors, cleavage and polyadenylation specificity factor, cleavage stimulation factor, cleavage factor I, and cleavage factor II. Using highly purified preparations of these factors, which lacked detectable creatine phosphokinase and ATPase activities, creatine phosphate (CP) was, surprisingly, found to be sufficient to promote efficient cleavage. Although other phosphate compounds substituted poorly or not at all for CP, another phosphoguanidine, arginine phosphate, was fully functional. Notably, ATP was neither necessary nor sufficient, and could in fact inhibit the reaction. Treatment of the purified factors with hexokinase plus glucose (to deplete any contaminating ATP) was without effect, as was addition of EDTA. Using 32P-labeled CP, we found that neither hydrolysis of CP nor phosphate transfer from CP occurred during the cleavage reaction. CP also allowed cleavage of the adenovirus 2 L3 pre-mRNA. However, in this case, ATP both enhanced the reaction and influenced the precise site of cleavage, perhaps reflecting the requirement of poly(A) polymerase for cleavage of this RNA. These results indicate that ATP is not essential for 3' pre-mRNA cleavage and that CP or a related compound can function as a necessary cofactor.

Adenosine Triphosphate↗
PubMed1997

Transcription factor TFIID recruits factor CPSF for formation of 3' end of mRNA.

Initiation of transcription by RNA polymerase II from a promoter region on DNA requires the assembly of several initiation factors to form a preinitiation complex. Assembly of this complex is initiated by the binding of the transcription factor TFIID, composed of the TATA-box binding protein (TBP) and TBP-associated factors (TAF[II]s), to the promoter. We have now characterized an immunopurified TFIID complex which we unexpectedly find contains the cleavage-polyadenylation specificity factor (CPSF), one of the factors required for formation of the 3' end of messenger RNA. CPSF is brought to the preinitiation complex by TFIID, but after transcription starts, CPSF dissociates from TFIID and becomes associated with the elongating polymerase. We also show that overexpression of recombinant TBP in HeLa cells decreases polyadenylation without affecting the correct initiation of transcription of the reporter gene. This indicates that, owing to incomplete assembly of TFIID on recombinant TBP, CPSF is not brought to the promoter and therefore polyadenylation becomes less efficient. Our observations have thus revealed a link between transcription initiation and elongation by RNA polymerase II and processing of the 3' end of mRNA.

Amino Acid Sequence↗
PubMed1997

Sequence-specific RNA binding by an SR protein requires RS domain phosphorylation: creation of an SRp40-specific splicing enhancer.

We showed previously that ASF/SF2, a member of the SR protein family of splicing factors, can activate a splicing enhancer element composed of high-affinity ASF/SF2 binding sites. To determine whether other SR proteins can behave similarly, we selected a high-affinity RNA-binding site (B1) for the SR protein SRp40. Strikingly, the success of this selection was completely dependent on phosphorylation of the RS domain, as unphosphorylated SRp40 failed to select specific sequences. We show that three copies of B1 function as a strong splicing enhancer, activating an intron with suboptimal splicing signals in nuclear extracts. Enhancer activity in S100 extracts (which lack SR proteins) required SRp40 and a nuclear fraction previously found to be required for ASF/SF2-dependent splicing. Importantly, enhancer activity was lost when SRp40 was replaced by ASF/SF2 or SC35, and SRp40 was the only classical SR protein found to be associated with the enhancer. Together, our results indicate that phosphorylation-dependent, sequence-specific RNA binding can impart unique activities to individual SR proteins.

Binding Sites↗
PubMed1997

Phosphorylation of the ASF/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing.

ASF/SF2 is a member of a conserved family of splicing factors known as SR proteins. These proteins, which are necessary for splicing in vitro, contain one or two amino-terminal RNP-type RNA-binding domains and an extensively phosphorylated carboxy-terminal region enriched in repeating Arg-Ser dipeptides (RS domains). Previous studies have suggested that RS domains participate in protein-protein interactions with other RS domain-containing proteins. Here we provide evidence that the RS domain of unphosphorylated recombinant ASF/SF2 is necessary, but not sufficient, for binding to the U1 snRNP-specific 70-kD protein (70K) in vitro. An apparent interaction of the isolated RS domain with 70K was observed if contaminating RNA was not removed, suggesting a nonspecific bridging between the basic RS domain, RNA, and 70K. In vitro phosphorylation of recombinant ASF/SF2 both significantly enhanced binding to 70K and also eliminated the RS domain-RNA interaction. Providing evidence that these interactions are relevant to splicing, ASF/SF2 can bind selectively to U1 snRNP in an RS domain-dependent, phosphorylation-enhanced manner. We also describe conditions that reveal for the first time a phosphorylation requirement for ASF/SF2 splicing activity in vitro.

Arginine↗
PubMed1997

Regulation of pre-mRNA splicing in metazoa.

Recent studies have identified additional cis-acting sequence elements that can either positively or negatively regulate pre-mRNA splicing. Trans-acting protein factors involved in tissue-specific splicing regulation have been characterized. The functions of splicing factors have been explored in vivo and certain splicing factors have been demonstrated to be essential for proper development and cell viability. Kinases that can specifically phosphorylate splicing factors have been identified and phosphorylation has been shown to influence the activity of splicing factors, consistent with a role for phosphorylation in splicing regulation.

Animals↗
PubMed1997

RNA recognition by the human polyadenylation factor CstF.

Polyadenylation of mammalian mRNA precursors requires at least two signal sequences in the RNA: the nearly invariant AAUAAA, situated 5' to the site of polyadenylation, and a much more variable GU- or U-rich downstream element. At least some downstream sequences are recognized by the heterotrimeric polyadenylation factor CstF, although how, and indeed if, all variations of this diffuse element are bound by a single factor is unknown. Here we show that the RNP-type RNA binding domain of the 64-kDa subunit of CstF (CstF-64) (64K RBD) is sufficient to define a functional downstream element. Selection-amplification (SELEX) experiments employing a glutathione S-transferase (GST)-64K RBD fusion protein selected GU-rich sequences that defined consensus recognition motifs closely matching those present in natural poly(A) sites. Selected sequences were bound specifically, and with surprisingly high affinities, by intact CstF and were functional in reconstituted, CstF-dependent cleavage assays. Our results also indicate that GU- and U-rich sequences are variants of a single CstF recognition motif. For comparison, SELEX was performed with a GST fusion containing the RBD from the apparent yeast homolog of CstF-64, RNA15. Strikingly, although the two RBDs are almost 50% identical and yeast poly(A) signals are at least as degenerate as the mammalian downstream element, a nearly invariant 12-base U-rich sequence distinct from the CstF-64 consensus was identified. We discuss these results in terms of the function and evolution of mRNA 3'-end signals.

Binding Sites↗
PubMed1997

The human U6 snRNA intramolecular helix: structural constraints and lack of sequence specificity.

Splicing of mRNA precursors occurs in a massive structure known as the spliceosome and requires the function of several small nuclear RNAs (snRNAs). A number of studies have suggested potentially important roles for two snRNAs, U2 and U6, in splicing catalysis. These two RNAs interact extensively with each other, as well as with the pre-mRNA, and possible similarities with catalytic RNAs have been noted. An important feature of the U2-U6 complex is an intramolecular helix in U6, which forms in conjunction with activation of the spliceosome. Here we describe a detailed genetic analysis of residues that make up this helix in human U6 snRNA, using an in vivo assay in which splicing of a test pre-mRNA is dependent on exogenous U6 snRNA. Our results show that many, but not all, positions tested are sensitive to mutation. Unexpectedly, base pairing is fully compatible with function at all positions, and at many is both necessary and sufficient. For example, conversion of two noncanonical A-C pairs to G-C pairs did not affect splicing, nor did conversion of an A-G to C-G. Extension of the helix by a base pair was also tolerated, provided that base pairing was maintained. Most notable was the behavior of a bulged U (U74), which has been suggested previously to be of particular importance. Although U74 was sensitive to substitution or deletion, incorporation into the helix by insertion of an A across from it was without effect, even in the context of a second helix-stabilizing mutation. We discuss these results in terms of possible mechanisms by which U6 snRNA might function in splicing catalysis.

Base Composition↗
PubMed1996

The polyadenylation factor CstF-64 regulates alternative processing of IgM heavy chain pre-mRNA during B cell differentiation.

The switch from membrane-bound to secreted-form IgM that occurs during differentiation of B lymphocytes has long been known to involve regulated processing of the heavy chain pre-mRNA. Here, we show that accumulation of one subunit of an essential polyadenylation factor (CstF-64) is specifically repressed in mouse primary B cells and that overexpression of CstF-64 is sufficient to switch heavy chain expression from membrane-bound (microm) to secreted form (micros). We further show that CstF-64 is limiting for formation of intact CstF, that CstF has a higher affinity for the microm poly(A) site than for the micros site, and that the microm site is stronger in a reconstituted in vitro processing reaction. Our results indicate that CstF-64 plays a key role in regulating IgM heavy chain expression during B cell differentiation.

Alternative Splicing↗
PubMed1996

Cell-cycle related regulation of poly(A) polymerase by phosphorylation.

The poly(A) tail found on almost all eukaryotic messenger RNAs is important in enhancing translation initiation and determining mRNA stability. Control of poly(A)-tail synthesis thus has the potential to be a key regulatory step in gene expression and is indeed known to be important during early development in many organisms. To study a possible basis for such regulation, we examined phosphorylation of poly(A) polymerase (PAP) by p34(cdc2)/cyclin B (maturation/mitosis-promoting factor, MPF). We show here that PAP can be phosphorylated in vivo and in vitro by MPF. Consistent with this, PAP becomes hyperphosphorylated both during meiotic maturation of Xenopus laevis oocytes and in HeLa cells arrested at M phase, times in the cell-cycle when MPF is known to be active. We show further that hyperphosphorylation by MPF dramatically reduces the activity of purified PAP, and that PAP isolated from mitotic HeLa cells is similarly inhibited by hyperphosphorylation. This repression probably contributes to the well established reductions in poly(A)+ RNA and/or protein synthesis known to occur in M-phase cells.

Animals↗
PubMed1996

Transcriptional repression by p53 involves molecular interactions distinct from those with the TATA box binding protein.

In addition to serving a role as a DNA binding-dependent transcriptional activator, p53 has been reported to repress a variety of promoters that lack p53 binding sites. Data from recent studies have suggested that this activity is mediated via an interaction between p53 and the TATA box binding protein (TBP). To investigate the functional relevance of this interaction in vivo, we have performed transient transfection assays in Drosophila Schneider cells. Wild-type p53 was found to repress expression from TATA box- but not initiator (Inr)-containing promoters activated by GAL4-VP16, GAL4-ftzQ or Sp1. A mutant p53(His175), defective in DNA binding and transcriptional activation, also inhibited TATA-dependent transcription activated by Sp1. However, p53 was unable to repress a basal TATA promoter stimulated by overexpression of TBP. Furthermore, overexpression of TBP failed to rescue the p53-mediated repression of activated transcription and a p53 mutant with its N-terminal TBP interaction domain intact, but defective in transcriptional activation and binding to TBP-associated factors (TAFs), was similarly defective in transcriptional repression. These data suggest that a p53-TBP interaction is not sufficient for transcriptional repression by p53 and that repression involves an interaction between p53 and other factors, such as TAFs, that are required for activated but not basal transcription. We suggest that p53-mediated repression results from squelching of a factor limiting for activated transcription from TATA- but not Inr-containing promoters.

Animals↗
PubMed1996

Targeted disruption of an essential vertebrate gene: ASF/SF2 is required for cell viability.

Alternative splicing factor/splicing factor 2 (ASF/SF2) is the prototype of a family of nuclear proteins highly conserved throughout metazoa, the SR (serine/arginine) proteins. Based largely on in vitro studies, SR proteins have been suggested to play important roles in constitutive and alternative splicing of pre-mRNAs. Here we describe the development of a genetic system employing the chicken B-cell line DT40 to study the function of ASF/SF2 in vivo. The high level of homologous recombination and rapid growth rate of these cells allowed us to show first that ASF/SF2 is an essential gene, and then to perform targeted disruption of both ASF/SF2 alleles, by creating a cell line in which the only source of ASF/SF2 is a human cDNA controlled by a tetracycline (tet)-repressible promoter. We show that addition of tet to these cells results in rapid depletion of ASF/SF2, concomitant accumulation of incompletely processed pre-mRNA, and subsequent cell death. The tet-induced lethality could be rescued by plasmids expressing wild-type ASF/SF2, but not several mutant derivatives, or other SR proteins. Heterozygous cell lines overexpressing human ASF/SF2 displayed significant reductions of endogenous ASF/SF2 mRNA, suggesting that ASF/SF2 mRNA levels are controlled by an autoregulatory loop. This system provides a novel method for genetic analysis of factors that function in basic processes in vertebrate cells.

Alleles↗
PubMed1996

Mechanisms of transcriptional activation and repression can both involve TFIID.

Regulation of transcription involves the activities of activators and repressors. Recent experiments have provided evidence that the function of both types of regulators can involve interactions with one or more component of the basal transcription machinery. A principal target appears to be TFIID, which consists of the TATA binding protein (TBP) and associated factors (TAFs). Here we describe experiments that provide added support for the idea that interactions affecting TFIID can play important roles in both activation and repression. We show, using transfection assays in Drosophila Schneider cells, that recruitment of TBP to a promoter as a GAL4-TBP fusion protein can provide a substantial activation of transcription. The conserved core of TBP is necessary and sufficient for this effect, which was observed with both TATA-containing and TATA-lacking basal promoters. These findings extend experiments performed in yeast, and strengthen the idea that recruitment of TBP (TFIID) can be an important mechanism of activation. We also provide further support for the idea that TBP can be a target for a transcriptional repressor, the Drosophila Even-skipped protein (Eve). We present evidence that the homeodomain, which is necessary for binding TBP in vitro, can also be required for repression in vivo, independent of its role in DNA binding. On the other hand, deletion of the alanine/proline-rich region that is essential for repression in vivo and TBP binding in vitro does not significantly affect DNA binding by the purified protein. These results strengthen the view that TBP, either directly or indirectly as a component of TFIID, can be a target of both activators and repressors.

Animals↗
PubMed1996

Functional interactions between the pelle kinase, Toll receptor, and tube suggest a mechanism for activation of dorsal.

A complex signal transduction pathway functions in the early Drosophila embryo to establish dorsal-ventral polarity. Activation of this pathway results in the nuclear transport of the protein dorsal (dl), a member of the rel/NF-kappaB family of transcription factors. Genetic studies have identified three intracellular components whose activity is required for activation of dl: Toll, a transmembrane receptor; pelle (pll), a serine/threonine protein kinase; and tube, a protein of unknown function. Here we examine the activities of these proteins when coexpressed in Drosophila Schneider cells. Coexpression of pll with dl enhanced dl nuclear localization and resulted in a modest increase in transcriptional activity. However, when pll was coexpressed with a specific mutant derivative of Toll (TlNaeI), although not with wild-type Toll, a striking synergistic activation of dl was detected. Unexpectedly, coexpression of pll plus TlNaeI, in the absence of dl, resulted in a similar synergistic activation of a GAL4-tube fusion protein. Based on these and other results, we propose a model in which pll receives a signal from activated Toll and phosphorylates tube, which then participates directly in dl activation.

Animals↗
PubMed1996

Interaction between the U1 snRNP-A protein and the 160-kD subunit of cleavage-polyadenylation specificity factor increases polyadenylation efficiency in vitro.

We have previously shown that the U1 snRNP-A protein (U1A) interacts with elements in SV40 late polyadenylation signal and that this association increases polyadenylation efficiency. It was postulated that this interaction occurs to facilitate protein-protein association between components of the U1 snRNP and proteins of the polyadenylation complex. We have now used GST fusion protein experiments, coimmunoprecipitations and Far Western blot analyses to demonstrate direct binding between U1A and the 160-kD subunit of cleavage-polyadenylation specificity factor (CPSF). In addition, Western blot analyses of fractions from various stages of CPSF purification indicated that U1A copurified with CPSF to a point but could be separated in the highly purified fractions. These data suggest that U1A protein is not an integral component of CPSF but may be able to interact and affect its activity. In this regard, the addition of purified, recombinant U1A to polyadenylation reactions containing CPSF, poly(A) polymerase, and a precleaved RNA substrate resulted in concentration-dependent increases in both the level of polyadenylation and poly(A) tail length. In agreement with the increase in polyadenylation efficiency caused by U1A, recombinant U1A stabilized the interaction of CPSF with the AAUAAA-containing substrate RNA in electrophoretic mobility shift experiments. These findings suggest that, in addition to its function in splicing, U1A plays a more global role in RNA processing through effects on polyadenylation.

Glutathione Transferase↗