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J L Manley

Publications and source records attributed to J L Manley.

At least 73 records · Page 4Linked to original sources
PubMed1996

The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution.

Mammalian Clk/Sty is the prototype for a family of dual specificity kinases (termed LAMMER kinases) that have been conserved in evolution, but whose physiological substrates are unknown. In a yeast two-hybrid screen, the Clk/Sty kinase specifically interacted with RNA binding proteins, particularly members of the serine/arginine-rich (SR) family of splicing factors. Clk/Sty itself has an serine/arginine-rich non-catalytic N-terminal region which is important for its association with SR splicing factors. In vitro, Clk/Sty efficiently phosphorylated the SR family member ASF/SF2 on serine residues located within its serine/arginine-rich region (the RS domain). Tryptic phosphopeptide mapping demonstrated that the sites on ASF/SF2 phosphorylated in vitro overlap with those phosphorylated in vivo. Immunofluorescence studies showed that a catalytically inactive form of Clk/Sty co-localized with SR proteins in nuclear speckles. Overexpression of the active Clk/Sty kinase caused a redistribution of SR proteins within the nucleus. These results suggest that Clk/Sty kinase directly regulates the activity and compartmentalization of SR splicing factors.

Amino Acid Sequence↗
PubMed1996

Complex alternative RNA processing generates an unexpected diversity of poly(A) polymerase isoforms.

Multiple forms of poly(A) polymerase (PAPs I, II, and III) cDNA have previously been isolated from bovine, human, and/or frog cDNA libraries. PAPs I and II are long forms of the enzyme that contain four functional domains: an apparent ribonucleoprotein-type RNA-binding domain, a catalytic region that may be related to the polymerase module, two nuclear localization signals (NLSs I and 2), and a C-terminal Ser/Thr-rich region. PAP III would encode a truncated protein that lacks the NLSs and the S/T-rich region. To investigate further the structure and expression of these forms, we isolated the mouse PAP gene and an intronless pseudogene from a mouse liver genomic library. The structure of the gene indicates that different forms of PAP are produced by alternative splicing (PAPs I and II) or by competition between polyadenylation and splicing (PAP III). The pseudogene appears to reflect yet another form of long PAP, which we call PAP IV. Mouse PAP III and two additional truncated forms, PAPs V and VI, which would be produced by use of poly(A) sites in adjacent introns, were also isolated from a mouse brain cDNA library. RNase protection and reverse transcription-PCR analyses showed that PAP II, V, and VI are expressed in all tissues tested but that PAP I and/or IV and III are tissue specific. However, immunoblot analysis detected only the long forms, raising the possibility that the short-form RNAs are not translated. Purified recombinant baculovirus-expressed PAPs were tested in several in vitro assays, and the short forms were found to be inactive. We discuss the possible significance of this complex expression pattern.

Alternative Splicing↗
PubMed1996

Functional interaction between p53, the TATA-binding protein (TBP), andTBP-associated factors in vivo.

The transcriptional activator p53 is known to interact with components of the general transcription factor TFIID in vitro. To examine the relevance of these associations to transcriptional activation in vivo, plasmids expressing a p53-GAL4 chimera and Drosophila TATA-binding protein (dTBP) were transfected into Drosophila Schneider cells. p53-GAL4 and dTBP displayed a markedly synergistic effect on activated transcription from a GAL4 site-containing reporter that was at least 10-fold greater than observed with other activators tested. A mutant p53 previously shown to be defective in both transcriptional activation in vivo and in binding to TBP-associated factors (TAFs) in vitro, although still capable of binding dTBP, did not cooperate with dTBP, suggesting that TAFs may contribute to this synergy. Providing further support for this possibility, transfected dTBP assembled into rapidly sedimenting complexes and could be immunoprecipitated with anti-TAF antibodies. While overexpression of any of several TAFs did not affect basal transcription, in either the presence or the absence of cotransfected dTBP, overexpression of TAFII230 inhibited transcriptional activation mediated by p53-GAL4 as well as by GAL4-VP16 and Sp1. Overexpression of TAFII40 and TAFII60 also inhibited activation by p53-GAL4 but had negligible effects on activation by GAL4-VP16 and Sp1, while TAFII110 did not affect any of the activators. TAF-mediated inhibition of activated transcription could be rescued by high levels of exogenous dTBP, which also restored full synergy. These data demonstrate for the first time that functional interactions can occur in vivo between TBP, TAFs, and p53.

Animals↗
PubMed1995

The 160-kD subunit of human cleavage-polyadenylation specificity factor coordinates pre-mRNA 3'-end formation.

Cleavage-polyadenylation specificity factor (CPSF) is a multisubunit protein that plays a central role in 3' processing of mammalian pre-mRNAs. CPSF recognizes the AAUAAA signal in the pre-mRNA and interacts with other proteins to facilitate both RNA cleavage and poly(A) synthesis. Here we describe the isolation of cDNAs encoding the largest subunit of CPSF (160K) as well as characterization of the protein product. Antibodies raised against the recombinant protein inhibit polyadenylation in vitro, which can be restored by purified CPSF. Extending previous studies, which suggested that 160K contacts the pre-mRNA, we show that purified recombinant 160K can, by itself, bind preferentially to AAUAAA-containing RNAs. While the sequence of 160K reveals similarities to the RNP1 and RNP2 motifs found in many RNA-binding proteins, no clear match to a known RNA-binding domain was found, and RNA recognition is therefore likely mediated by a highly diverged or novel structure. We also show that 160K binds specifically to both the 77K (suppressor of forked) subunit of the cleavage factor CstF and to poly(A) polymerase (PAP). These results provide explanations for previously observed cooperative interactions between CPSF and CstF, which are responsible for poly(A) site specification, and between CPSF and PAP, which are necessary for synthesis of the poly(A) tail. Also supporting a direct role for 160K in these interactions is the fact that 160K by itself retains partial ability to cooperate with CstF in binding pre-mRNA and, unexpectedly, inhibits PAP activity in in vitro assays. We discuss the significance of these multiple functions and also a possible evolutionary link between yeast and mammalian polyadenylation suggested by the properties and sequence of 160K.

Amino Acid Sequence↗
PubMed1995

Chromosomal localization of mouse and human genes encoding the splicing factors ASF/SF2 (SFRS1) and SC-35 (SFRS2).

The mammalian SR-type splicing factors ASF/SF2 and SC-35 play crucial roles in pre-mRNA splicing and have been shown to shift splice site choice in vitro. We have mapped the ASF/SF2 gene in mice and humans and the SC-35 gene in mice. Somatic cell hybrid mapping of the human ASF/SF2 gene (SFRS1 locus) reveals that it resides on chromosome 17, and fluorescence in situ hybridization refines this localization to 17q21.3-q22. Recombinant inbred mapping of the mouse ASF/SF2 gene (Sfrs1 locus) and the mouse SC-35 gene (Sfrs2 locus) demonstrates that both genes are located in a part of mouse chromosome 11 that is homologous to human chromosome 17. Mapping of Sfrs1 using F1 hybrid backcross mice between the strains C57BL/6 and DDK places Sfrs1 very near the marker D11Mit38 and indicates that the ASF/SF2 gene is closely linked to the Ovum mutant locus.

Alternative Splicing↗
PubMed1995

U1 snRNP-ASF/SF2 interaction and 5' splice site recognition: characterization of required elements.

Members of the SR family of proteins, can collaborate with U1 snRNP in the recognition of 5' splice sites in pre-messenger RNAs. We have previously shown that purified U1 snRNP and ASF/SF2 form a ternary complex with pre-mRNA, which is dependent on a functional 5' splice site. In this manuscript we dissect the requirements for the formation of this complex. Sequences in the pre-mRNA, domains in ASF/SF2 and components of the U1 snRNP particle are shown to be required for complex formation. We had shown that sequences at the 5' splice site of PIP7. A are necessary and now we show these are sufficient for complex formation. Furthermore, we show that one functional RNA binding domain and the RS domain are both required for ASF/SF2 to participate in complex formation. The RNA binding domains were redundant in this assay, suggesting that either domain can interact with the pre-messenger RNA. Finally, our experiments show no function for the U1-specific A protein in complex formation, whereas a function for U1-specific C protein was strongly suggested. The study of the earliest interactions between pre-mRNA and splicing factors suggests a model for 5' splice site recognition.

Animals↗
PubMed1995

The human splicing factors ASF/SF2 and SC35 possess distinct, functionally significant RNA binding specificities.

ASF/SF2 and SC35 belong to a highly conserved family of nuclear proteins that are both essential for splicing of pre-mRNA in vitro and are able to influence selection of alternative splice sites. An important question is whether these proteins display distinct RNA binding specificities and, if so, whether this influences their functional interactions with pre-mRNA. To address these issues, we first performed selection/amplification from pools of random RNA sequences (SELEX) with portions of the two proteins comprising the RNA binding domains (RBDs). Although both molecules selected mainly purine-rich sequences, comparison of individual sequences indicated that the motifs recognized are different. Binding assays performed with the full-length proteins confirmed that ASF/SF2 and SC35 indeed have distinct specificities, and at the same time provided evidence that the highly charged arginine-serine region of each protein is not a major determinant of specificity. In the case of ASF/SF2, evidence is presented that binding specificity involves cooperation between the protein's two RBDs. Finally, we demonstrate that an element containing three copies of a high-affinity ASF/SF2 binding site constitutes a powerful splicing enhancer. In contrast, a similar element consisting of three SC35 sites was inactive. The ASF/SF2 enhancer can be activated specifically in splicing-deficient S100 extracts by recombinant ASF/SF2 in conjunction with one or more additional protein factors. These and other results suggest a central role for ASF/SF2 in the function of purine-rich splicing enhancers.

Animals↗
PubMed1995

A novel U2-U6 snRNA structure is necessary for mammalian mRNA splicing.

Splicing of mRNA precursors requires a complex and dynamic set of RNA-RNA base-pairing interactions in which the U2 and U6 snRNAs play central roles. Using a genetic suppression assay, we refine and extend a U2-U6 snRNA structure that may comprise the catalytic center of the spliceosome. We first show that a critical U2-U6 helix proven in yeast, helix Ia, is also essential for mammalian splicing. Mutations in the adjacent helix Ib, however, cannot be similarly suppressed, and relevant residues in both U2 and U6 are shown to participate in intramolecular, rather than intermolecular, base-pairing. We next demonstrate the requirement for a novel U2-U6 helix, helix III, which involves bases extending 3' from the branch site recognition sequence in U2 and 5' from an evolutionarily invariant sequence in U6 implicated previously in 5' splice site recognition. This configuration suggests that helix III may help juxtapose the pre-mRNA 5' splice site and branch site. We provide evidence for this by demonstrating that a branch site mutation can be suppressed by a mutation in the 5' splice site, provided that compensatory changes are made in the appropriate bases in U2 and U6. Our results provide new insights into how U2 and U6 snRNAs interact with each other and with the pre-mRNA to initiate the first catalytic step in splicing.

Base Sequence↗
PubMed1995

Cooperation between core promoter elements influences transcriptional activity in vivo.

Core promoters for RNA polymerase II frequently contain either (or both) of two consensus sequence elements, a TATA box and/or an initiator (Inr). Using test promoters consisting of prototypical TATA and/or Inr elements, together with binding sites for sequence-specific activators, we have analyzed the function of TATA and Inr elements in vivo. In the absence of activators, the TATA element was significantly more active than the Inr, and the combination of elements was only slightly more effective than the TATA-only promoter. In the presence of any of several coexpressed activator proteins, the TATA elements was again most active, but here addition of the Inr allowed significant increases in activity, indicating a cooperative interaction between the two elements. An interesting exception was observed with the activator Sp1, which was more effective with the Inr-only promoter, and addition of a TATA box did not enhance activity. Finally, in all cases the TATA plus Inr promoters were found to be partially or completely resistant to the dominant negative effects of a transcription factor TFIIB mutant previously shown to interfere with expression from TATA-only promoters. This result strengthens the conclusion that TATA and Inr elements can cooperate in vivo.

Animals↗
PubMed1995

Regulation of dorsal in cultured cells by Toll and tube: tube function involves a novel mechanism.

We described previously a transient cotransfection assay that allows us to study regulation of the Drosophila Dorsal protein (dl) in cultured cells. For example, we showed that over-expression of the Toll transmembrane receptor was sufficient to cause relocalization of dl from the cytoplasm to the nucleus. Here we present data that the tube protein, shown previously by genetic studies to act downstream of Toll, can function in a novel way to enhance dl activity. In the absence of dl, or when dl is cytoplasmic, tube is also found in the cytoplasm of transfected cells. But when dl is localized to the nucleus, so is tube. tube can then function to enhance reporter gene expression, either by cooperation with dl or as a GAL4-tube fusion protein. tube thus appears capable of acting both as a chaperon or escort for dl as it moves to the nucleus, and then as a transcriptional coactivator. We also show that the intracytoplasmic domain of Toll, and specifically the region sharing homology with the interleukin-1 receptor, is sufficient to induce dl-tube nuclear translocation.

Animals↗
PubMed1995

A complex protein assembly catalyzes polyadenylation of mRNA precursors.

Nearly every eukaryotic mRNA contains a poly(A) tail at its 3' end. Unlike the factors that catalyze other RNA processing reactions, the basal polyadenylation machinery consists entirely of proteins. As with other steps in gene expression, however, this machinery is complex, consisting of multiple separable factors. Two multisubunit factors are required for specification of the poly(A) site and formation of a stable protein-RNA complex. Two additional proteins, as well as poly(A) polymerase, join the complex and are required for cleavage of the pre-mRNA and synthesis of the poly(A) tail, respectively. These and other properties suggest at least a superficial similarity between transcription initiation and polyadenylation.

Animals↗
PubMed1995

A direct interaction between a glutamine-rich activator and the N terminus of TFIIB can mediate transcriptional activation in vivo.

Studies examining the mechanism by which transcriptional activators function have suggested that the general transcription factor IIB (TFIIB) can be a target for certain regulatory proteins. For example, we showed previously that expression of a mutant form of TFIIB can specifically inhibit activation in vivo mediated by the strong, glutamine-rich activator protein GAL4-ftzQ. Using transient cotransfection assays, we have defined the regions in both GAL4-ftzQ and TFIIB that are required for activity in vivo and provide evidence that a potential zinc finger structure at the N terminus of TFIIB is necessary for the observed functional interaction between the two proteins. Using a protein binding assay, we have demonstrated that GAL4-ftzQ can specifically interact with TFIIB in vitro. This interaction requires the same regions in both molecules necessary for function in vivo and is reduced or eliminated by mutations predicted to disrupt the zinc finger in TFIIB. These results support the idea that a direct interaction between a regulatory protein and TFIIB can be important for transcriptional activation in vivo and, combined with previous data of others, suggest that different activators can function by contacting distinct regions of TFIIB.

Amino Acid Sequence↗
PubMed1995

The transcriptional repressor even-skipped interacts directly with TATA-binding protein.

The Drosophila homeodomain protein Even-skipped (Eve) has previously been shown to function as a sequence-specific transcriptional repressor, and in vitro and in vivo experiments have shown that the protein can actively block basal transcription. However, the mechanism of repression is not known. Here, we present evidence establishing a direct interaction between Eve and the TATA-binding protein (TBP). Using cotransfection assays with minimal basal promoters whose activity can be enhanced by coexpression of TBP, we found that Eve could efficiently block, or squelch, this enhancement. Squelching did not require Eve DNA-binding sites on the reporter plasmids but was dependent on the presence of the Eve repression domain. Further support for an in vivo interaction between the Eve repression domain and TBP was derived from a two-hybrid-type assay with transfected cells. Evidence that Eve and TBP interact directly was provided by in vitro binding assays, which revealed a specific protein-protein interaction that required an intact Eve repression domain and the conserved C terminus of TBP. The Eve homeodomain was also required for these associations, suggesting that it may function in protein-protein interactions. We also show that a previously characterized artificial repression region behaves in a manner similar to that of the Eve repression domain, including its ability to squelch TBP-enhanced expression in vivo and to bind TBP specifically in vitro. Our results suggest a model for transcriptional repression that involves an interaction between Eve and TBP.

Amino Acid Sequence↗
PubMed1995

Overexpression of the SR proteins ASF/SF2 and SC35 influences alternative splicing in vivo in diverse ways.

The SR proteins are a family of essential splicing factors highly conserved throughout metazoa. Here we examine the effects of two prototypical SR proteins, ASF/SF2 and SC35, when overexpressed by transfection in cultured cells together with plasmids encoding alternatively spliced model transcripts. As expected from past work, both proteins were found to affect alternative splicing, but differences as well as similarities in their behavior were observed. With adenovirus E1a pre-mRNA, ASF/SF2 caused shifts in alternative splicing similar to those observed previously, and the effects of mutations, in the protein and the pre-mRNA, were largely consistent with in vitro results. For example, the C-terminal RS domain was not required to alter splice site selection. SC35 overexpression also altered E1a splicing, but the pattern was distinct from that detected with ASF/SF2, indicating that the two proteins can function differently in vivo. Unexpectedly, with SV40 early pre-mRNA, overexpression of either protein resulted in a marked inhibition of splicing, with the downstream small t 5' splice site more sensitive than the upstream large T 5' splice site. This is essentially the opposite of what has been observed when the concentration of these proteins is increased in vitro. The RS domain was necessary but not sufficient for this effect. Finally, overexpression of SC35, but not ASF/SF2, resulted in substantial accumulation of the unspliced SV40 pre-mRNA, which was efficiently transported to the cytoplasm. This finding suggests that SC35 may play an unanticipated role in mRNA stability and/or transport.

Adenovirus E1A Proteins↗
PubMed1994

A polyadenylation factor subunit is the human homologue of the Drosophila suppressor of forked protein.

Polyadenylation of messenger RNA precursors is a complex process that requires multiple protein factors (for reviews, see refs 1, 2). Cleavage stimulation factor (CstF) is one of these, functioning together with cleavage-polyadenylation specificity factor, two cleavage factors, and poly(A)+ polymerase. CstF is composed of three subunits of M(r) 77, 64 and 50K. The 64K and 50K subunits contain, respectively, an RNP-type RNA-binding domain that contacts the pre-mRNA and transducin repeats characteristic of G-protein beta-subunits. Here we report the cloning and characterization of the 77K subunit of human CstF (referred to as 77K). We show that the 77K subunit is required for formation of active CstF and bridges the 64K and 50K subunits. Sequence analyses indicate that the 77K subunit is the homologue of the protein encoded by the Drosophila melanogaster suppressor of forked (su(f)) gene. Mutations in su(f) can enhance or suppress the effects of transposable element insertions, and our data indicate that this is due to changes in polyadenylation. Both the 77K subunit and the su(f) protein share homology with Saccharomyces cerevisiae RNA14, previously shown to be involved in mRNA metabolism. Our results thus also indicate that components of the complex polyadenylation machinery are conserved from yeast to man.

Amino Acid Sequence↗
PubMed1994

The human splicing factor ASF/SF2 can specifically recognize pre-mRNA 5' splice sites.

ASF/SF2 is a human protein previously shown to function in in vitro pre-mRNA splicing as an essential factor necessary for all splices and also as an alternative splicing factor, capable of switching selection of 5' splice sites. To begin to study the protein's mechanism of action, we have investigated the RNA binding properties of purified recombinant ASF/SF2. Using UV crosslinking and gel shift assays, we demonstrate that the RNA binding region of ASF/SF2 can interact with RNA in a sequence-specific manner, recognizing the 5' splice site in each of two different pre-mRNAs. Point mutations in the 5' splice site consensus can reduce binding by as much as a factor of 100, with the largest effects observed in competition assays. These findings support a model in which ASF/SF2 aids in the recognition of pre-mRNA 5' splice sites.

Base Sequence↗
PubMed1994

Protein-protein interactions and 5'-splice-site recognition in mammalian mRNA precursors.

Exactly how specific splice sites are recognized during the processing of complex precursor messenger RNAs is not clear. Small nuclear ribonucleoprotein particles (snRNPs) are involved, but are not sufficient by themselves to define splice sites. Now a human protein essential for splicing in vitro, called alternative splicing factor/splicing factor 2, is shown to cooperate with the U1 snRNP particle in binding pre-mRNA. This cooperation is probably achieved by specific interactions between the arginine/serine-rich domain of the splicing factor and a similar region in a U1 snRNP-specific protein.

Adenoviridae↗