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J L Manley

Publications and source records attributed to J L Manley.

At least 109 records · Page 6Linked to original sources
PubMed1991

SV40 T-antigen-binding sites within the 5'-flanking regions of human U1 and U2 genes.

The 5' flanking regions of the genes (U1 and U2) encoding the human U1 and U2 small nuclear RNAs (snRNAs) each contain sequences that bind specifically to the simian virus (SV40) large tumor antigen (T.Ag). Substitution of these sites with sequences that lack T.Ag-binding sites did not block accumulation of U1 or U2 snRNA in a variety of cell types, but deletion of these regions resulted in the total loss of expression. Thus, these sequences may serve only a spacing function, and the T.Ag-binding sites appear not to be necessary for expression. However, coexpression of T.Ag markedly reduced expression of a U1 gene containing a high-affinity T.Ag-binding site (from the SV40 genome) in place of the U1 T.Ag-binding site. In contrast, coexpression of T.Ag enhanced synthesis of U2, but not U1, snRNA, independent of the presence of the T.Ag-binding sites. Thus, while the consensus T.Ag-binding sites within the U1 and U2 promoter regions do not appear to influence expression, the binding of SV40 T.Ag to a high-affinity site can lead to significant repression of a strong snRNA promoter, and T.Ag can enhance expression of another in the absence of a known binding site.

Antigens, Polyomavirus Transforming↗
PubMed1991

Primary structure and expression of bovine poly(A) polymerase.

Poly(A) polymerase has a critical role in the synthesis of messenger RNA in eukaryotic cells. The isolation and characterization of complementary DNAs encoding bovine poly(A) polymerase is described here. The predicted sequences of the mRNA and protein reveal features that provide insights into how the enzyme functions and how it might be regulated. Poly(A) polymerase expressed from a cloned cDNA is fully functional in in vitro assays, and mutational analyses have identified a putative regulatory domain that enhances, but is not essential for, activity.

Amino Acid Sequence↗
PubMed1991

Base pairing between U2 and U6 snRNAs is necessary for splicing of a mammalian pre-mRNA.

Splicing of pre-messenger RNA in eukaryotic cells occurs in a multicomponent complex termed the spliceosome, which contains small nuclear ribonucleoprotein particles (snRNPs), protein factors and substrate pre-mRNA. Assembly of the spliceosome involves the stepwise binding of snRNPs and protein factors to the pre-mRNA through a poorly understood mechanism which probably involves specific RNA-RNA, RNA-protein and protein-protein interactions. Of particular interest are the interactions between snRNPs, which are likely to be important not only for assembly of the spliceosome but also for catalysis. U1 snRNP interacts with the 5' splice site and U2 snRNP with the branch site of the pre-mRNA; both of these interactions involve Watson-Crick base pairing. But very little is known about how other factors such as the U4/U6 and U5 snRNPs reach the spliceosome and function in splicing. Here we report evidence that U6 snRNA interacts directly with U2 snRNA by a mechanism involving base-pairing, and that this interaction can be necessary for splicing of a mammalian pre-mRNA in vivo.

Base Composition↗
PubMed1991

Primary structure of the human splicing factor ASF reveals similarities with Drosophila regulators.

We described previously the purification of a human protein, called alternative splicing factor (ASF), that can switch utilization of alternative 5' splice sites in an SV40 early pre-mRNA. We now report the isolation of a cDNA, designated ASF-1, that encodes this protein. ASF-1 consists of 248 amino acid residues, including an 80 residue RNA-binding domain at its N-terminus and a 50 residue C-terminal region that is 80% serine plus arginine. ASF-1 produced in E. coli can activate splicing in vitro and switch 5' splice-site utilization, establishing that the recombinant protein is sufficient to supply these activities. Analysis of additional cDNAs revealed that ASF pre-mRNA can itself be alternatively spliced, surprisingly, by utilization of a shared 5' splice site and two closely spaced 3' splice sites. Use of the upstream site results in a second mRNA (ASF-2) in which translation of the downstream exon occurs extensively in an alternative reading frame distinct from ASF-1.

Amino Acid Sequence↗
PubMed1991

A nuclear micrococcal-sensitive, ATP-dependent exoribonuclease degrades uncapped but not capped RNA substrates.

We have developed an assay for an exoribonuclease present in HeLa cell nuclear extracts that degrades capped but not uncapped RNA substrates, and used it to partially purify and characterize such an activity. Capped and uncapped transcripts of different sizes (37-317 nt) were incubated with fractionated nuclear extracts, and in all cases the capped RNAs were stable while their uncapped counterparts were completely degraded. No changes in activity were detected when cap analogs were included in reaction mixtures, suggesting that the stability of capped RNAs was not due to a cap binding protein. The exoribonuclease was shown to be specific for RNA, and to function processively with either substrates containing 5'-hydroxyl or 5'-phosphorylated ends. The products were predominantly 5'-mononucleotides, and no detectable intermediates were observed at any reaction time points. Sedimentation analysis suggests that the native size of the nuclease is 7.4S or approximately 150 kDa. Interestingly, a nucleoside triphosphate was found to be necessary for specific and complete degradation of the uncapped RNAs. Finally, micrococcal nuclease (MN) pretreatment of the partially purified enzyme inhibited its activity. As several controls indicated that this was not due to non-specific effects of MN, this finding suggests that the exoribonuclease contains an essential RNA component.

Adenosine Triphosphate↗
PubMed1991

Activation and repression of transcription by the gap proteins hunchback and Krüppel in cultured Drosophila cells.

We have studied the ability of the Drosophila gap proteins Krüppel and hunchback to function as transcriptional regulators in cultured cells. Both proteins bind to specific sites in a 100-bp DNA fragment located upstream of the segment polarity gene engrailed, which also contains functional binding sites for a number of homeo box proteins. The hunchback protein is a strikingly concentration-dependent activator of transcription, capable of functioning both by itself and also synergistically with the pair-rule proteins fushi tarazu and paired. In contrast, Krüppel is a transcriptional repressor that can block transcription induced either by hunchback or by several different homeo box proteins. While repression of the homeo box protein activators requires a Krüppel-binding site on the DNA, repression of hunchback can occur efficiently in the absence of a Krüppel-binding site. We discuss the possible molecular mechanisms underlying these activities, as well as the potential significance of these results with respect to segmentation in Drosophila.

Animals↗
PubMed1991

A novel protein factor is required for use of distal alternative 5' splice sites in vitro.

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites.

Adenovirus Early Proteins↗
PubMed1990

Sequence specificity of the human mRNA N6-adenosine methylase in vitro.

N6-adenosine methylation is a frequent modification of mRNAs and their precursors, but little is known about the mechanism of the reaction or the function of the modification. To explore these questions, we developed conditions to examine N6-adenosine methylase activity in HeLa cell nuclear extracts. Transfer of the methyl group from S-[3H methyl]-adenosylmethionine to unlabeled random copolymer RNA substrates of varying ribonucleotide composition revealed a substrate specificity consistent with a previously deduced consensus sequence, Pu[G greater than A]AC[A/C/U]. 32-P labeled RNA substrates of defined sequence were used to examine the minimum sequence requirements for methylation. Each RNA was 20 nucleotides long, and contained either the core consensus sequence GGACU, or some variation of this sequence. RNAs containing GGACU, either in single or multiple copies, were good substrates for methylation, whereas RNAs containing single base substitutions within the GGACU sequence gave dramatically reduced methylation. These results demonstrate that the N6-adenosine methylase has a strict sequence specificity, and that there is no requirement for extended sequences or secondary structures for methylation. Recognition of this sequence does not require an RNA component, as micrococcal nuclease pretreatment of nuclear extracts actually increased methylation efficiency.

Base Sequence↗
PubMed1990

A protein factor, ASF, controls cell-specific alternative splicing of SV40 early pre-mRNA in vitro.

SV40 early pre-mRNA is alternatively spliced by utilization of two different 5' splice sites and a shared 3' splice site to produce large T and small t mRNAs. The ratio of small t to large T mRNAs produced in human embryonic kidney 293 cells is 10- to 20-fold greater than in other mammalian cells, suggesting the existence of a 293 cell-specific factor that modulates alternative splicing. Here we show that nuclear extracts from 293 cells give rise to significantly more small t splicing than do extracts from HeLa cells. Using an in vitro complementation assay, we have characterized and extensively purified a factor from 293 extracts that brings about striking increases in small t splicing with concomitant decreases in large T splicing. The factor is heat sensitive and micrococcal nuclease resistant, suggesting that it is a protein lacking an accessible RNA component. Purification of the alternative splicing factor indicates that the activity is contained in one of several possibly related polypeptides of 30-35 kd.

Biological Factors↗
PubMed1990

Polyoma virus small tumor antigen pre-mRNA splicing requires cooperation between two 3' splice sites.

We have studied splicing of the polyoma virus early region pre-mRNA in vitro. This RNA is alternatively spliced in vivo to produce mRNA encoding the large, middle-sized (MTAg), and small (StAg) tumor antigens. Our primary interest was to learn how the 48-nucleotide StAg intron is excised, because the length of this intron is significantly less than the apparent minimum established for mammalian introns. Although the products of all three splices are detected in vitro, characterization of the pathway and sequence requirements of StAg splicing suggests that splicing factors interact with the precursor RNA in an unexpected way to catalyze removal of this intron. Specifically, StAg splicing uses either of two lariat branch points, one of which is located only 4 nucleotides from the 3' splice site. Furthermore, the StAg splice absolutely requires that the alternative MTAg 3' splice site, located 14 nucleotides downstream of the StAg 3' splice site, be intact. Insertion mutations that increase or decrease the quality of the MTAg pyrimidine stretch enhance or repress StAg as well as MTAg splicing, and a single-base change in the MTAg AG splice acceptor totally blocks both splices. These results demonstrate the ability of two 3' splice sites to cooperate with each other to bring about removal of a single intron.

Antigens, Polyomavirus Transforming↗
PubMed1990

A multisubunit factor, CstF, is required for polyadenylation of mammalian pre-mRNAs.

We have purified and characterized a factor required for accurate polyadenylation of mammalian pre-mRNAs in vitro. This factor, called cleavage-stimulation factor (CstF), is composed of three distinct polypeptide subunits of 77, 64, and 50 kD. Using monoclonal antibodies directed against the 64- and 50-kD subunits, we show that CstF is required for efficient cleavage of polyadenylation substrates. Furthermore, CstF present in unfractionated nuclear extracts interacts with pre-mRNAs containing the signal sequence AAUAAA, but not AAGAAA, in such a manner that the 64-kD subunit can be cross-linked to the RNA by UV light. This polypeptide is thus identical to the previously described 64-kD nuclear protein that binds to AAUAAA-containing RNAs. Finally, indirect immunofluorescence of fixed cells indicates that CstF is distributed diffusely throughout the nucleus in a granular pattern distinct from the "speckled" pattern displayed by factors involved in pre-mRNA splicing, but similar to that of heterogeneous nuclear ribonucleoproteins. A model is presented in which CstF binds specifically to nascent RNA polymerase II transcripts and, by interacting with other factors, results in a rapid initiation of 3'-end processing of pre-mRNAs.

Antibodies, Monoclonal↗
PubMed1990

A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates.

A 64-kilodalton (kDa) polypeptide that is cross-linked by UV light specifically to polyadenylation substrate RNAs containing a functional AAUAAA element has been identified previously. Fractionated HeLa nuclear components that can be combined to regenerate efficient and accurate polyadenylation in vitro have now been screened for the presence of the 64-kDa protein. None of the individual components contained an activity which could generate the 64-kDa species upon UV cross-linking in the presence of substrate RNA. It was necessary to mix two components, cleavage stimulation factor and specificity factor, to reconstitute 64-kDa protein-RNA cross-linking. The addition of cleavage factors to this mixture very efficiently reconstituted the AAUAAA-specific 64-kDa protein-RNA interaction. The 64-kDa protein, therefore, is present in highly purified, reconstituted polyadenylation reactions. However, it is necessary to form a multicomponent complex to efficiently cross-link the protein to a substrate RNA.

Base Sequence↗
PubMed1990

The Drosophila even-skipped promoter is transcribed in a stage-specific manner in vitro and contains multiple, overlapping factor-binding sites.

To investigate the factors contributing to regulation of expression of the Drosophila segmentation gene even-skipped (eve), we have analyzed both the in vitro transcription and eve-promoter-binding proteins in embryo extracts. We show that the eve promoter is accurately and efficiently expressed in nuclear extracts derived from Drosophila embryos and that transcription is more efficient in extracts prepared from embryos at early stages of development than in those from older embryos, broadly reproducing the temporal pattern of expression observed in vivo. This stage-specific expression is dependent on sequences upstream of the eve transcription start site which contain multiple binding sites for at least two distinct proteins present in embryo nuclei. One of these proteins, the binding sites for which correspond to the sequences required for stage-specific expression, appears to be the previously described GAGA factor. Although the binding activity of the GAGA factor remains constant, the level of the binding activity of the other protein, which we have called the TCCT factor, changes during the course of embryogenesis. Activity is first detected 3 to 5 h after fertilization and decreases during later stages of embryogenesis. We discuss the possibility that the TCCT factor plays a role in the maintenance or refinement of the eve expression pattern.

Animals↗
PubMed1989

The graded distribution of the dorsal morphogen is initiated by selective nuclear transport in Drosophila.

The maternal morphogen dorsal (dl) plays a key role in the establishment of dorsal-ventral polarity in Drosophila. We present evidence that the graded distribution of dl protein is initiated by selective nuclear transport. The dl protein is uniformly distributed throughout the cytoplasm of early embryos, but approximately 90 min after fertilization, dl protein present in ventral but not dorsal regions is selectively transported to the nucleus. Mutations in maternally active genes that regulate dl disrupt this transport process, resulting in an inactive, cytoplasmically localized form of the dl protein. Selective nuclear transport of dl protein was reproduced in tissue culture cells. The wild-type dl protein is largely restricted to the cytoplasm, while truncated proteins are predominantly localized within the nucleus. Transient cotransfection assays suggest that dl activates expression from several promoters in an apparently sequence-independent manner. We discuss the role of nuclear transport as a regulated process in gene expression and development.

Amino Acid Sequence↗
PubMed1989

Oligonucleotide-targeted degradation of U1 and U2 snRNAs reveals differential interactions of simian virus 40 pre-mRNAs with snRNPs.

We have investigated the roles of U1 and U2 snRNP particles in SV40 pre-mRNA splicing by oligonucleotide-targeted degradation of U1 or U2 snRNAs in Xenopus laevis oocytes. Microinjection of oligonucleotides complementary to regions of U1 or U2 RNAs either in the presence or absence of SV40 DNA resulted in specific cleavage of the corresponding snRNA. Unexpectedly, degradation of U1 or U2 snRNA was far more extensive when the oligonucleotide was injected without, or prior to, introduction of viral DNA. In either co-injected or pre-injected oocytes, these oligonucleotides caused a dramatic reduction in the accumulation of spliced SV40 mRNA expressed from the viral late region, and a commensurate increase in unspliced late RNA. When pre-injected, two different U2 specific oligonucleotides also inhibited the formation of both large and small tumor antigen spliced early mRNAs. However, even when, by pre-injection of a U1 5' end-specific oligonucleotide, greater than 95% degradation of the U1 snRNA 5' ends occurred in oocytes, no reduction in early pre-mRNA splicing was observed. In contrast, the same U1 5' end oligonucleotide, when added to HeLa splicing extracts, substantially inhibited the splicing of SV40 early pre-mRNA, indicating that U1 mRNP is not totally dispensable for early splicing. These findings confirm and extend our earlier observations which suggested that different pre-mRNAs vary in their requirements for snRNPs.

Animals↗
PubMed1989

A CCAAT box sequence in the adenovirus major late promoter functions as part of an RNA polymerase II termination signal.

Termination of transcription by RNA polymerase II has been shown in several cases to require a functional poly(A) addition site, although the actual termination event occurs further downstream. To define in more detail the sequences required for termination, we mapped the site at which transcription terminates on a chimeric plasmid template that contains the adenovirus MLP directing transcription of the SV40 early region. Termination in cells transfected with this plasmid occurs within a discrete promoter-proximal region that contains an inverted CCAAT-box sequence. This region of the MLP was also capable of directing termination, in an orientation-dependent manner, when inserted downstream of the poly(A) site in the plasmid template. In addition, in adenovirus-infected cells, transcription initiated from upstream promoters on the adenovirus chromosome terminates within the same MLP promoter-proximal region, both establishing the physiological relevance of the observed CCAAT-box dependent termination, and also suggesting a possible function for transcription termination in adenovirus infection.

Adenoviridae↗