PubMed Health⌕ Search

Biomedical subjects

J L Manley

Publications and source records attributed to J L Manley.

At least 19 recordsLinked to original sources
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↗
PubMed1992

A human polyadenylation factor is a G protein beta-subunit homologue.

Cleavage stimulation factor (CstF) is one of the multiple factors required for polyadenylation of mammalian pre-mRNAs in vitro. We have shown previously that this factor is composed of three distinct subunits of 77, 64, and 50 kDa, and that the 64-kDa subunit can be UV-cross-linked to RNA in a polyadenylation signal (AAUAAA)-dependent manner. By molecular cloning, the 64-kDa subunit was shown to contain a ribonucleoprotein-type RNA binding domain and a novel repeat structure. To study the functions of the other subunits, we have now isolated cDNAs encoding the 50-kDa subunit of human CstF. This subunit shares extensive homology with mammalian G protein beta-subunits and has a characteristic repeat structure (transducin repeat), in which an approximately 44-amino acid-long sequence is repeated seven times. To our knowledge, the 50-kDa subunit is the first example of a functional beta-subunit-like protein in vertebrates. Possible roles of the transducin repeat, both in CstF function specifically and in other beta-subunit homologues more generally, are discussed.

Amino Acid Sequence↗
PubMed1992

Characterization of the multisubunit cleavage-polyadenylation specificity factor from calf thymus.

Cleavage-polyadenylation specificity factor (CPSF) is one of five separable factors known to be required for 3' cleavage and polyadenylation of mRNA precursors in vitro. Previous studies have shown that the cleavage and poly(A) addition reactions can be uncoupled in vitro and have suggested that CPSF may be the only factor essential for both of these subreactions. Here we report the purification of CPSF to near homogeneity from calf thymus and show that the purified factor contains three polypeptides of 165, 105, and 70 kDa. These polypeptides cosediment precisely with CPSF activity, which has a sedimentation coefficient of 11.5 S. Consistent with previous reports from our laboratory, purified CPSF does not contain a detectable RNA component, indicating that it is a multisubunit protein and not a small nuclear ribonucleoprotein. Extensively purified bovine CPSF can function with human poly(A) polymerase to bring about AAUAAA-dependent poly(A) addition or with human cleavage factors to catalyze accurate 3' cleavage of a pre-mRNA substrate. UV cross-linking and gel retention analyses demonstrate that highly purified CPSF interacts with one of these cleavage factors, the multisubunit cleavage-stimulation factor, to facilitate stable binding of both to an AAUAAA-containing pre-mRNA. Likewise, evidence is presented indicating that poly(A) polymerase and CPSF can interact directly.

Animals↗
PubMed1992

The human 64-kDa polyadenylylation factor contains a ribonucleoprotein-type RNA binding domain and unusual auxiliary motifs.

Cleavage stimulation factor is one of the multiple factors required for 3'-end cleavage of mammalian pre-mRNAs. We have shown previously that this factor is composed of three subunits with estimated molecular masses of 77, 64, and 50 kDa and that the 64-kDa subunit can be UV-crosslinked to RNA in a polyadenylylation signal (AAUAAA)-dependent manner. We have now isolated cDNAs encoding the 64-kDa subunit of human cleavage stimulation factor. The 64-kDa subunit contains a ribonucleoprotein-type RNA binding domain in the N-terminal region and a repeat structure in the C-terminal region in which a pentapeptide sequence (consensus MEARA/G) is repeated 12 times and the formation of a long alpha-helix stabilized by salt bridges is predicted. An approximately 270-amino acid segment surrounding this repeat structure is highly enriched in proline and glycine residues (approximately 20% for each). When cloned 64-kDa subunit was expressed in Escherichia coli, an N-terminal fragment containing the RNA binding domain bound to RNAs in a polyadenylylation-signal-independent manner, suggesting that the RNA binding domain is directly involved in the binding of the 64-kDa subunit to pre-mRNAs.

Amino Acid Sequence↗
PubMed1992

Alternatively spliced transcripts of the Drosophila tramtrack gene encode zinc finger proteins with distinct DNA binding specificities.

A protein present in nuclear extracts of Drosophila embryos binds multiple sites in the promoter and genetically defined autoregulatory element of the pair-rule gene even-skipped (eve). We reported here the isolation of a cDNA encoding this binding activity, the sequence of which identifies it as the 69 kDa zinc finger tramtrack (ttk) protein. As ttk was previously implicated in controlling the expression of another pair-rule gene, fushi tarazu (ftz), our findings suggest that ttk plays a role in the regulation of at least two developmentally important genes. An additional ttk-related cDNA clone was isolated which gives rise to an 88 kDa protein with an alternative set of zinc fingers having a DNA binding specificity distinct from that of the 69 kDa protein. Both proteins were shown to be encoded by the ttk gene through alternative splicing, providing the first example of the use of this mechanism to generate related proteins with distinct DNA binding specificities. Whole mount in situ hybridization analysis revealed different patterns of embryonic expression of the two ttk mRNA isoforms.

Amino Acid Sequence↗
PubMed1992

Ectopic expression of the Drosophila tramtrack gene results in multiple embryonic defects, including repression of even-skipped and fushi tarazu.

The tramtrack (ttk) gene of Drosophila encodes 69-kDa and 88-kDa proteins through alternative splicing of the primary ttk transcript. The two proteins share a common amino-terminal sequence, but contain different carboxy-terminal portions, each of which has a distinct zinc finger domain with a unique DNA binding specificity. The 69-kDa ttk protein has been shown to bind multiple sites within important regulatory elements of the pair-rule genes even-skipped (eve) and fushi tarazu (ftz), and it has been suggested that this protein may function as a repressor of ftz transcription. Here we present evidence that the 69-kDa ttk protein can indeed repress expression not only of ftz, but also of eve. Ectopic expression of the 69-kDa protein, but not of the 88-kDa form, was found to nearly abolish the striped patterns of expression of both eve and ftz in transgenic embryos. These findings, coupled with our detection of significant levels of ttk protein in ovaries and 0-2-h embryos, support the idea that maternally supplied ttk protein serves to prevent premature activation of eve and ftz, thereby helping to establish the timing of the onset of zygotic expression of these two genes. Furthermore, gross defects in the larval cuticle resulting from misexpression of the 69-kDa protein suggest that this protein performs additional functions in the early embryo.

Animals↗
PubMed1992

The Drosophila RNA-binding protein RBP1 is localized to transcriptionally active sites of chromosomes and shows a functional similarity to human splicing factor ASF/SF2.

An RNA-binding protein gene (rbp1) from Drosophila melanogaster, encoding an RNA recognition motif and an Arg-Ser rich (RS) domain, has been characterized. The predicted amino acid sequence of rbp1 is similar to those of the human splicing factor ASF/SF2, the Drosophila nuclear phosphoprotein SRp55, and the Drosophila puff-associated protein B52. Northern and immunohistochemical analyses showed that rbp1 is expressed at all stages in all tissues and that the RBP1 protein is localized to the nucleus. Consistent with a role in mRNA metabolism, indirect immunofluorescence reveals that the RBP1 protein colocalizes with RNA polymerase II on larval salivary gland polytene chromosomes. RBP1 protein made in Escherichia coli was tested for splicing activity using human cell extracts in which ASF has been shown previously both to activate splicing and to affect the choice of splice sites in alternatively spliced pre-mRNAs. In these assays, RBP1 protein, like ASF, is capable of both activating splicing and switching splice site selection. However, in each case, clear differences in the behavior of the two proteins were detected, suggesting that they have related but not identical functions. The general nuclear expression pattern, colocalization on chromosomes with RNA polymerase II, the similarity to ASF/SF2, SRp55, and B52, along with the effect on alternative splicing shown in vitro, suggest that rbp1 is involved in the processing of precursor mRNAs.

Amino Acid Sequence↗
PubMed1992

TFIID can be rate limiting in vivo for TATA-containing, but not TATA-lacking, RNA polymerase II promoters.

We have studied the effect of exogenous expression of the basal transcription factor TFIID on the activities of several different TATA-containing and TATA-lacking promoters. Overexpression of TFIID from a transfected plasmid in Drosophila Schneider cells resulted in substantial concentration-dependent increases in expression from a cotransfected minimal TATA-containing promoter. Overexpression of TFIID activated expression from all TATA-containing promoters tested, with the maximum level of activation being inversely proportional to the strength of the promoter. In contrast, expression from TATA-less promoters was not enhanced, and could in fact be reduced, by increased expression of TFIID. Consistent with these findings overexpression of TFIID had opposite effects on Sp1-mediated activation observed from minimal synthetic promoters consisting of Sp1-binding sites and either a TATA box or initiator element. We discuss the significance of these results in terms of the role of TFIID in the initiation of transcription and as a possible regulatory target for expression from TATA-containing promoters, as well as the role TFIID may play in expression from TATA-less promoters.

Animals↗
PubMed1992

Selective nuclear transport of the Drosophila morphogen dorsal can be established by a signaling pathway involving the transmembrane protein Toll and protein kinase A.

Establishment of dorsal-ventral polarity in the early Drosophila embryo requires a concentration gradient of the maternal morphogen dorsal (dl). This concentration gradient is established by selective nuclear transport of dl so that dl protein is present only in ventral nuclei. The activity of 11 genes is required for dl nuclear localization. One of these genes, Toll, encodes a transmembrane protein that appears to play the most direct role in regulating dl localization. We have examined the effects of Toll on dl in cotransfected Schneider cells to gain insight into the nature of the interaction between these proteins. We have found that Toll can enhance the nuclear localization of dl and, independently, the ability of dl to activate transcription once in the nucleus. We present evidence that the signaling pathway from Toll to dl involves protein kinase A (PKA) and that nuclear transport and activation of dl results from phosphorylation of dl by PKA. We discuss the significance of these results with respect both to Drosophila embryogenesis and to the regulation of the mammalian transcription factor NF-kappa B.

Animals↗
PubMed1992

Multiple functional domains of human U2 small nuclear RNA: strengthening conserved stem I can block splicing.

We showed previously that a branch site mutation in simian virus 40 early pre-mRNA that prevented small t antigen mRNA splicing could be efficiently suppressed by a compensatory mutation in a coexpressed U2 small nuclear (sn) RNA gene. We have now generated second-site mutations in this suppressor gene to investigate regions of U2 RNA required for function. A number of mutations in a putative stem at the 5' end of the molecule inhibited splicing, indicating that bases in this region are important for activity. However, several lines of evidence suggested that formation of the entire stem is not essential for splicing. Indeed, mutations that strengthen the stem actually inhibited splicing, and evidence that this prevents a required base-pairing interaction with U6 snRNA is presented. These results suggest that the relative stabilities of competing intra- and intermolecular base-pairing interactions play an important role in the splicing reaction. Mutations in a conserved single-stranded region immediately 3' to the branch site recognition sequence all inhibited splicing, indicating that this region is required for U2 function, although its exact role remains unknown. Finally, two mutations in the loop of stem IV at the 3' end of the molecule, which destroy the binding site of U2 sn ribonucleoprotein B", prevented small t splicing; this finding contrasts with previous studies which utilized different assay systems. Analysis of the accumulation and subcellular localization of all of the mutant RNAs showed that they were similar to those of the parental suppressor U2 RNA, indicating that the effects observed indeed reflect defects in splicing.

Base Sequence↗
PubMed1992

Multiple activities of the human splicing factor ASF.

The effects of human alternative splicing factor, ASF, on in vitro splicing of adenovirus E1A pre-mRNA were examined. E1A pre-mRNA is a complex substrate, and splicing in HeLa cell nuclear extracts produces six different RNAs using three alternative 5' splice sites and two 3' splice sites. Addition of excess ASF to splicing reactions produced a simplified splicing pattern, in which only one spliced product, 13S RNA, was detected. Inhibition of 12S and 9S splicing, which use 5' splice sites upstream of the 13S 5' splice site, extends previous observations that when multiple 5' splice sites compete for the same 3' splice site, ASF causes preferential selection of the proximal 5' splice site. However, inhibition of the other splices, which use a different upstream 3' splice site, represents a novel activity of ASF, as competition between 5' splice sites is not involved. The effect of ASF on 12S splicing was found to depend on its position relative to competing 5' splice sites, indicating that the ability of ASF to activate proximal 5' splice sites is position- but not sequence-dependent. Finally, addition of small amounts of ASF to ASF-lacking S100 extract was able to activate distal as well as proximal 5' splice sites in two of three pre-mRNAs tested, indicating that in these cases changes in the concentration of ASF alone can be sufficient to modulate alternative 5' splice site selection.

Adenovirus Early Proteins↗
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↗