Biomedical subjects
P A Sharp
Publications and source records attributed to P A Sharp.
Transcriptional regulation of the murine 3' IgH enhancer by OCT-2.
Targeted disruption of either of the B cell-specific transcription factors Oct-2 or OCA-B/BOB-1/OBF-1 dramatically affects B cell terminal differentiation. The 3' enhancer of immunoglobulin heavy chain (IgH) locus is important for transcription of the locus in terminal plasma cells. Allele-specific suppression of mutant Oct-2 binding sites in this enhancer by a variant Oct-2 protein revealed that in a mature B cell line this enhancer was specifically dependent upon Oct-2, as contrasted to the closely related Oct-1 transcription factor. Phosphorylation of the Oct-2 protein was important for this activation and was synergistic for coactivation by the OCA-B factor. These results indicate that Oct-2 and OCA-B interact with the 3' enhancer in regulation of the IgH locus during B cell activation.
PUF60: a novel U2AF65-related splicing activity.
We have identified a new pyrimidine-tract binding factor, PUF, that is required, together with U2AF, for efficient reconstitution of RNA splicing in vitro. The activity has been purified and consists of two proteins, PUF60 and the previously described splicing factor p54. p54 and PUF60 form a stable complex in vitro when cotranslated in a reaction mixture. PUF activity, in conjunction with U2AF, facilitates the association of U2 snRNP with the pre-mRNA. This reaction is dependent upon the presence of the large subunit of U2AF, U2AF65, but not the small subunit U2AF35. PUF60 is homologous to both U2AF65 and the yeast splicing factor Mud2p. The C-terminal domain of PUF60, the PUMP domain, is distantly related to the RNA-recognition motif domain, and is probably important in protein-protein interactions.
Tat-SF1 protein associates with RAP30 and human SPT5 proteins.
The potent transactivator Tat recognizes the transactivation response RNA element (TAR) of human immunodeficiency virus type 1 and stimulates the processivity of elongation of RNA polymerase (Pol) II complexes. The cellular proteins Tat-SF1 and human SPT5 (hSPT5) are required for Tat activation as shown by immunodepletion with specific sera and complementation with recombinant proteins. In nuclear extracts, small fractions of both hSPT5 and Pol II are associated with Tat-SF1 protein. Surprisingly, the RAP30 protein of the heterodimeric transcription TFIIF factor is associated with Tat-SF1, while the RAP74 subunit of TFIIF is not coimmunoprecipitated with Tat-SF1. Overexpression of Tat-SF1 and hSPT5 specifically stimulates the transcriptional activity of Tat in vivo. These results suggest that Tat-SF1 and hSPT5 are indispensable cellular factors supporting Tat-specific transcription activation and that they may interact with RAP30 in controlling elongation.
Selection in vitro of novel ribozymes from a partially randomized U2 and U6 snRNA library.
Combinatorial libraries related to spliceosomal U2 and U6 snRNAs were tested for catalytic reactions typical of the splicing of nuclear pre-mRNAs. Ribozymes with four different activities were selected based on covalent bond formation to a substrate RNA. The first activity was reversible self-cleavage; ribozymes self-cleaved then ligated the 5'-hydroxyl group of the substrate oligonucleotide to their 2',3'-cyclic phosphate intermediate. The second activity was 2',5'-branch formation by the attack of a substrate 2'-hydroxyl group on the 5'-terminal triphosphate of the ribozyme transcript, releasing pyrophosphate. The third ribozyme activity was similar to reversible self-cleavage but was a three-step reaction. This ribozyme self-cleaved, then cleaved the substrate in trans, and then ligated the substrate 3' cleavage product to its cyclic phosphate intermediate. This three-step pathway shares similarities with the pathway of tRNA splicing. The fourth activity was 2',3'-branch formation; to form this unusual branch, a 2'-hydroxyl of the substrate attacked an internal phosphate of the ribozyme, releasing an oligonucleotide leaving group. The isolation of branching activities by the in vitro selection protocol was unanticipated and was due to surprising properties of reverse transcriptase, which can read through 2',5'- or 2',3'-branches and efficiently perform non-templated intramolecular jumps.
A coactivator of pre-mRNA splicing.
The nuclear matrix antigen recognized by the monoclonal antibody (mAb) B1C8 is a novel serine (S) and arginine (R)-rich protein associated with splicing complexes and is named here SRm160 (SR-related matrix protein of 160 kD). SRm160 contains multiple SR repeats, but unlike proteins of the SR family of splicing factors, lacks an RNA recognition motif. SRm160 and a related protein SRm300 (the 300-kD nuclear matrix antigen recognized by mAb B4A11) form a complex that is required for the splicing of specific pre-mRNAs. The SRm160/300 complex associates with splicing complexes and promotes splicing through interactions with SR family proteins. Binding of SRm160/300 to pre-mRNA is normally also dependent on U1 snRNP and is stabilized by U2 snRNP. Thus, SRm160/300 forms multiple interactions with components bound directly to important sites within pre-mRNA. The results suggest that a complex of the nuclear matrix proteins SRm160 and SRm300 functions as a coactivator of pre-mRNA splicing.
Evolutionary fates and origins of U12-type introns.
U2-type and U12-type introns are spliced by distinct spliceosomes in eukaryotic nuclei. A classification method was devised to distinguish these two types of introns based on splice site sequence properties and was used to identify 56 different genes containing U12-type introns in available genomic sequences. U12-type introns occur with consistently low frequency in diverse eukaryotic taxa but have almost certainly been lost from C. elegans. Comparisons with available homologous sequences demonstrate subtype switching of U12 introns between termini of AT-AC and GT-AG as well as conversion of introns from U12-type to U2-type and provide evidence for a fission/fusion model in which the two splicing systems evolved in separate lineages that were fused in a eukaryotic progenitor.
Alternative splicing of the fibronectin EIIIB exon depends on specific TGCATG repeats.
The fibronectin EIIIB exon is alternatively spliced in a cell-type-specific manner, and TGCATG repeats in the intron downstream of EIIIB have been implicated in this regulation. Analysis of the intron sequence from several vertebrates shows that the pattern of repeats in the 3' half of the intron is evolutionarily conserved. Point mutations in certain highly conserved repeats greatly reduce EIIIB inclusion, suggesting that a multicomponent complex may recognize the repeats. Expression of the SR protein SRp40, SRp20, or ASF/SF2 stimulates EIIIB inclusion. Studies of the interplay between mutations in the repeats and SRp40-stimulated inclusion suggest that the repeats are recognized in many, if not all, cell types, and that EIIIB inclusion may be regulated by quantitative changes in multiple factors.
Classification of introns: U2-type or U12-type.
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Ultraspiracle: an invertebrate nuclear receptor for juvenile hormones.
Juvenile hormones (JH), a sesquiterpenoid group of ligands that regulate developmental transitions in insects, bind to the nuclear receptor ultraspiracle (USP). In fluorescence-based binding assays, USP protein binds JH III and JH III acid with specificity, adopting for each ligand a different final conformational state. JH III treatment of Saccharomyces cerevisiae expressing a LexA-USP fusion protein stabilizes an oligomeric association containing this protein, as detected by formation of a protein-DNA complex, and induces USP-dependent transcription in a reporter assay. We propose that regulation of morphogenetic transitions in invertebrates involves binding of JH or JH-like structures to USP.
Design of TATA box-binding protein/zinc finger fusions for targeted regulation of gene expression.
Fusing the TATA box-binding protein (TBP) to other DNA-binding domains may provide a powerful way of targeting TBP to particular promoters. To explore this possibility, a structure-based design strategy was used to construct a fusion protein, TBP/ZF, in which the three zinc fingers of Zif268 were linked to the COOH terminus of yeast TBP. Gel shift experiments revealed that this fusion protein formed an extraordinarily stable complex when bound to the appropriate composite DNA site (half-life up to 630 h). In vitro transcription experiments and transient cotransfection assays revealed that TBP/ZF could act as a site-specific repressor. Because the DNA-binding specificities of zinc finger domains can be systematically altered by phage display, it may be possible to target such TBP/zinc finger fusions to desired promoters and thus specifically regulate expression of endogenous genes.
Mechanisms involved in increased iron uptake across rat duodenal brush-border membrane during hypoxia.
1. Chronic hypoxia enhances intestinal iron transport but the cellular processes involved are poorly understood. In order to assess the effects of 3 days of hypoxia on iron uptake across the duodenal brush-border membrane, we have measured the membrane potential difference (Vm) of villus-attached enterocytes by direct microelectrode impalement and have used semi-quantitative autoradiography to study changes in expression of iron uptake during enterocyte maturation. 2. Hypoxia increased duodenal Vm (-57.7 vs. -49.3 mV, P < 0.001). Ion substitution experiments revealed that hyperpolarization was due, at least in part, to a reduction in brush-border Na+ permeability. 3. Autoradiography revealed that hypoxia increased by 6-fold the rate of iron accumulation during enterocyte transit along the lower villus and enhanced by 3-fold the maximal accumulation of iron. Depolarization of the brush border, using a high-K(+)-containing buffer, caused a proportionally greater reduction in iron uptake in control compared with hypoxic tissue suggesting that the raised iron uptake is only partly driven by brush-border hyperpolarization. 4. We conclude that hypoxia increases the expression of iron transport in duodenal brush-border membrane and an enhanced electrical driving force may be involved in this response.
Topoisomerase I enhances TFIID-TFIIA complex assembly during activation of transcription.
The mechanism of coactivation by DNA topoisomerase I (topo I) was examined in a highly defined in vitro transcription system containing Pol II and purified factors. Both stimulation of the basal reaction and coactivation occurred dependent on TAF(II)s. Activation was first observed at the TFIID-TFIIA stage of initiation and maximal activation required the concomitant presence of TFIID, TFIIA, topo I, and activator. Electrophoretic mobility shift assay demonstrated a dramatic enhancement in the formation of the TFIID-TFIIA complex by topo I and activator, dependent on the TAF(II)s. DNase I footprinting confirmed this recruitment. A catalytically inactive topo I, which coactivated transcription, similarly stimulated the rapid formation of the TFIID-TFIIA complex in the presence of activator. A camptothecin-mediated DNA cleavage assay demonstrated the recruitment of topo I to the template by TFIID. Topo I likely functions during activation by enhancing the formation of an active TFIID-TFIIA complex on the promoter.
SC35-mediated reconstitution of splicing in U2AF-depleted nuclear extract.
Assembly of the mammalian spliceosome is known to proceed in an ordered fashion through several discrete complexes, but the mechanism of this assembly process may not be universal. In an early step, pre-mRNAs are committed to the splicing pathway through association with U1 small nuclear ribonucleoprotein (snRNP) and non-snRNP splicing factors, including U2AF and members of the SR protein family. As a means of studying the steps of spliceosome assembly, we have prepared HeLa nuclear extracts specifically depleted of the splicing factor U2AF. Surprisingly, the SR protein SC35 can functionally substitute for U2AF65 in the reconstitution of pre-mRNA splicing in U2AF-depleted extracts. This reconstitution is substrate-specific and is reminiscent of the SC35-mediated reconstitution of splicing in extracts depleted of U1 snRNP. However, SC35 reconstitution of splicing in U2AF-depleted extracts is dependent on the presence of functional U1 snRNP. These observations suggest that there are at least three distinguishable mechanisms for the binding of U2 snRNP to the pre-mRNA, including U2AF-dependent and -independent pathways.
A minimal spliceosomal complex A recognizes the branch site and polypyrimidine tract.
The association of U2 snRNP with the pre-mRNA branch region is a critical step in the assembly of spliceosomal complexes. We describe an assembly process that reveals both minimal requirements for formation of a U2 snRNP-substrate RNA complex, here designated the Amin complex, and specific interactions with the branch site adenosine. The substrate is a minimal RNA oligonucleotide, containing only a branch sequence and polypyrimidine tract. Interactions at the branch site adenosine and requirements for polypyrimidine tract-binding proteins for the Amin complex are the same as those of authentic prespliceosome complex A. Surprisingly, Amin complex formation does not require U1 snRNP or ATP, suggesting that these factors are not necessary for stable binding of U2 snRNP per se, but rather are necessary for accessibility of components on longer RNA substrates. Furthermore, there is an ATP-dependent activity that releases or destabilizes U2 snRNP from branch sequences. The simplicity of the Amin complex will facilitate a detailed understanding of the assembly of prespliceosomes.
Early diabetes-induced changes in rat jejunal glucose transport and the response to insulin.
The effects of 1 day of streptozotocin-induced diabetes in rats on glucose transport across the brush border membrane (BBM) and basolateral membrane (BLM) prepared from jejunal enterocytes has been studied. The effects on glucose transport of treatment of diabetic animals with insulin to reduce to normal the elevated blood glucose levels has also been assessed. The maximum capacity (Vmax) for SGLT1-mediated glucose uptake by BBM vesicles was unaffected by diabetes or insulin treatment of diabetic rats. In contrast, Vmax for BLM glucose uptake was increased by 206% in diabetes, a response that could not be reversed by treatment with insulin. Western blotting of BBM for SGLT1 protein revealed a single band with a molecular weight of 73 kDa and the intensity of this band was unaffected by diabetes. However, an increased level of GLUT2 was noted in diabetic BLM and this was not a consequence of changes in glycaemic or insulin status. Diabetes hyperpolarised the BBM, implying an increased driving force for Na(+)-sugar co-transport but insulin treatment only partially reversed this enhanced potential difference. Benzamil (2 microns), an epithelial Na+ channel blocker, hyperpolarised the BBM of control but not diabetic enterocytes, implying that a reduced Na+ permeability was responsible for the diabetic hyperpolarisation. It was concluded that in early diabetes, before the onset of hyperphagia, a greater driving force for Na(+)-dependent BBM sugar transport together with increased GLUT2 activity at the BLM promotes sugar movement across the enterocyte. Possible triggers for the transport responses are discussed.
Streptozotocin diabetes and the expression of GLUT1 at the brush border and basolateral membranes of intestinal enterocytes.
Changes in membrane expression of sodium-dependent glucose transporter (SGLT1) and glucose transporter isoform (GLUT2) protein have been implicated in the increased intestinal glucose transport in streptozotocin-diabetes. The possible involvement of GLUT1 in the transport response, however, has not previously been studied. Using confocal microscopy on tissue sections and Western blotting of purified brush border membrane (BBM) and basolateral membrane (BLM), we have examined enterocyte expression of GLUT1 in untreated and in 1 and 21 day streptozotocin diabetic rats. In control enterocytes, GLUT1 was absent at the BBM and detected at low levels at the BLM. Diabetes resulted in a 4- to 5-fold increased expression of GLUT1 at the BLM and the protein could also be readily detected at the BBM. Insulin treatment of diabetic rats increased GLUT1 level at the BBM but was without effect on expression of the protein at the BLM.
Tat-SF1: cofactor for stimulation of transcriptional elongation by HIV-1 Tat.
Tat may stimulate transcriptional elongation by recruitment of a complex containing Tat-SF1 and a kinase to the human immunodeficiency virus-type 1 (HIV-1) promoter through a Tat-TAR interaction. A complementary DNA for the cellular activity, Tat-SF1, has been isolated. This factor is required for Tat trans-activation and is a substrate of an associated cellular kinase. Cotransfection with the complementary DNA for Tat-SF1 specifically modulates Tat activation. Tat-SF1 contains two RNA recognition motifs and a highly acidic carboxyl-terminal half. It is distantly related to EWS and FUS/TLS, members of a family of putative transcription factors with RNA recognition motifs that are associated with sarcomas.