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Biomedical subjects

W Keller

Publications and source records attributed to W Keller.

At least 55 records · Page 3Linked to original sources
PubMed1997

The major yeast poly(A)-binding protein is associated with cleavage factor IA and functions in premessenger RNA 3'-end formation.

Polyadenylation of premessenger RNAs occurs posttranscriptionally in the nucleus of eukaryotic cells by cleavage of the precursor and polymerization of adenosine residues. In the yeast Saccharomyces cerevisiae, the mature poly(A) tail ranges from 60 to 70 nucleotides. 3'-end processing can be reproduced in vitro with purified factors. The cleavage reaction requires cleavage factors I and II (CF I and CF II), whereas polyadenylation involves CF I, polyadenylation factor I (PFI), and poly(A) polymerase (Pap1p). CF I has recently been separated into two factors, CF IA and CF IB. We have independently purified CF IA and found that five polypeptides cofractionate with the activity. They include Rna14p, Rna15p, Pcf11p, a new protein called Clp1p, and remarkably, the major poly(A)-binding protein Pab1p. Extracts from strains where the PAB1 gene is mutated or deleted are active for cleavage but generate transcripts bearing abnormally long poly(A) tracts. Complementation with recombinant Pab1p not only restores the length of the poly(A) tails to normal, but also triggers a poly(A) shortening activity. In addition, a monoclonal Pab1p antibody prevents the formation of poly(A) tails in extracts or in a reconstituted system. Our data support the notion that Pab1p is involved in the length control of the poly(A) tails of yeast mRNAs and define a new essential function for Pab1p in the formation of mature mRNAs.

Poly(A)-Binding Proteins↗
PubMed1997

The 30-kD subunit of mammalian cleavage and polyadenylation specificity factor and its yeast homolog are RNA-binding zinc finger proteins.

Cleavage and polyadenylation specificity factor (CPSF), a key component of the mammalian RNA 3'-end processing machinery, consists of four subunits of 160, 100, 73, and 30 kD. Here we report the isolation and characterization of a cDNA encoding the 30-kD polypeptide. Antibodies raised against this protein inhibit cleavage and polyadenylation and coimmunoprecipitate the other CPSF subunits. The protein sequence contains five C3H-zinc-finger repeats and a putative RNA-binding zinc knuckle motif at the carboxyl terminus. Consistent with this observation, the in vitro translated 30-kD protein binds RNA polymers with a distinct preference for poly(U). In addition, an essential S. cerevisiae gene, YTH1, was cloned which is 40% identical to CPSF 30K at the protein level. Extracts prepared from a conditional yth1 mutant have normal cleavage activity, but fail to polyadenylate the upstream cleavage product. Efficient polyadenylation activity can be restored by the addition of purified polyadenylation factor I (PF I). We demonstrate that Yth1p is a component of PF I that interacts in vivo and in vitro with Fip1p, a known PF I subunit.

Amino Acid Sequence↗
PubMed1997

Randomised trial of transjugular-intrahepatic-portosystemic shunt versus endoscopy plus propranolol for prevention of variceal rebleeding.

BACKGROUND: The transjugular-intrahepatic-portosystemic shunt is a new interventional treatment for portal hypertension. The aim of our study was to compare the transjugular shunt with endoscopic treatment for the prophylaxis of recurrent variceal bleeding. METHODS: Between March, 1993, and March, 1996, 126 patients with variceal bleeding were randomly assigned either transjugular shunt (n = 61) or endoscopic treatment (n = 65). Patients were followed up for a median of 14 (IQR 8-25) months and 13 (8-25) months, respectively. In 31 (51%) of the shunted patients, simultaneous transjugular-variceal embolisation was done at the time of shunt placement. Endoscopic treatment consisted of sclerotherapy and/or banding ligation and was combined with propranolol medication. FINDINGS: Technical success was achieved in all patients assigned to the shunt group. During follow-up, the cumulative 1-year variceal rebleeding rates in the shunted and endoscopically treated patients were 15% and 41% and the 2-year rates were 21% and 52% (p = 0.001), respectively. In nine (12%) patients from the endoscopic group treatment failed and the patients received the transjugular-shunt treatment. A total of 19 bleeding episodes from any source occurred in 15 patients in the shunt group compared with 100 episodes in 33 patients in the endoscopic group. There was no difference in survival with estimated 1-year survival rates for shunted and endoscopically treated patients of 90% and 89%, and 2-year survival rates of 79% and 82%, respectively. The incidence of clinically significant hepatic encephalopathy after 1 year was higher in the shunt group (36% vs 18%, p = 0.011). INTERPRETATION: These results suggest, that the transjugular shunt is more effective than endoscopic treatment in prevention of variceal rebleeding but has a considerable risk of hepatic encephalopathy. Survival is similar in the two groups.

Adult↗
PubMed1997

Purification of human double-stranded RNA-specific editase 1 (hRED1) involved in editing of brain glutamate receptor B pre-mRNA.

RNAs encoding subunits of glutamate-gated ion channel receptors are posttranscriptionally modified by RNA editing and alternative splicing. The change in amino acid sequence caused by RNA editing can affect both the kinetics and the permeability of the ion channel receptors to cations. Here, we report the purification of a 90-kDa double-stranded RNA-specific adenosine deaminase from HeLa cell nuclear extract that specifically edits the glutamine codon at position 586 in the pre-mRNA of the glutamate receptor B subunit. Site-specific deamination of an adenosine to an inosine converts the glutamine codon to that of arginine. Recently, a gene encoding a double-stranded-specific editase (RED1) was cloned from a rat brain cDNA library. Antibodies generated against the deaminase domain of its human homolog specifically recognized and inhibited the activity of the 90-kDa enzyme, indicating that we have purified hRED1 the human homolog of rat RED1. This enzyme is distinct from double-stranded RNA-specific adenosine deaminase which we and others have previously purified and cloned.

Adenosine Deaminase↗
PubMed1997

A comparison of mammalian and yeast pre-mRNA 3'-end processing.

Many components of the mammalian and yeast pre-mRNA 3'-end-processing machinery have recently been purified and cDNAs or genes coding for these factors have been cloned. Most of the factors consist of multiple subunits, some of which serve to bind the RNA substrate, others of which are involved in forming a complex network of protein-protein interactions. Most of the mammalian 3'-end-processing factors are similar in their amino acid sequence to the yeast factors, indicating that they have a common evolutionary history.

Animals↗
PubMed1997

Promoter sequences from two different Brassica napus tapetal oleosin-like genes direct tapetal expression of beta-glucuronidase in transgenic Brassica plants.

To investigate the sequences responsible for the regulated expression of tapetal-specific oleosin-like genes, ca. 2 kb of the 5'-upstream regions from two divergent genes, OlnB;4 and OlnB;13, were isolated, sequenced and fused to the reporter gene beta-glucuronidase for study in transgenic Brassica napus plants. Although the proteins encoded by these two genes are highly divergent, except for the conserved oleosin-like domain, the first 250 bp of their 5'-upstream regions was 86% identical, including a region of 150 bp upstream from the TATA box. Analysis of 42 independent transformants by histochemical and fluorometric methods showed that both promoters directed tapetal-specific expression that peaked at the 4 mm flower bud stage.

Base Sequence↗
PubMed1997

Two forms of human double-stranded RNA-specific editase 1 (hRED1) generated by the insertion of an Alu cassette.

The double-stranded RNA-specific editase 1 (RED1/ADAR2) is implicated in the editing of precursor-mRNAs (pre-mRNA) encoding subunits of glutamate receptors (GluRs) in brain. Site-specific deamination of adenosine to inosine alters the codon at the Q/R site in GluR-B rendering the heteromeric receptor impermeable to Ca2+ ions. We cloned human RED1 (hRED1/hADAR2) cDNAs from a brain cDNA library. The human enzyme is 95% identical to the rat homologue. We characterized two alternatively spliced forms that differed by the presence of an Alu-J cassette in the deaminase domain. For the long form containing the Alu cassette, we isolated cDNA clones with an alternative C-terminus and 3'-UTR. An 8.8-kb transcript of hRED1 is most abundant in brain and heart, and lower levels are detected in other tissues. In vitro editing assays with purified recombinant hRED1 containing or lacking the Alu-J cassette revealed that both forms of the protein have the same substrate specificity, but differ in their catalytic activity.

Adenosine Deaminase↗
PubMed1996

The Effect of Impeller Type on Floc Size and Structure during Shear-Induced Flocculation

The effect of impeller type and shear rate on the evolution of floc size and structure during shear-induced flocculation of polystyrene particles with aluminum sulfate is investigated by image analysis. One radial flow (six-blade Rushton turbine) and two axial flow (three-blade fluid foil, four-blade 45° pitch) impeller configurations are examined. The steady state average floc size is shown to depend on the frequency of recirculation to the impeller zone and its characteristic velocity gradient. The concepts of fractal geometry are used to characterize the floc structure. For all impellers, the two-dimensional floc fractal dimension, Dpf, increases during floc growth, indicating formation of more open structures. Later on, Dpf levels off at a steady state value as breakage becomes significant and the floc size distribution approaches steady state. The shear rate does not affect the steady state Dpf of the flocs within experimental uncertainty.

Journal Article↗
PubMed1996

Sequence similarity between the 73-kilodalton protein of mammalian CPSF and a subunit of yeast polyadenylation factor I.

The 3' ends of most eukaryotic messenger RNAs are generated by endonucleolytic cleavage and polyadenylation. In mammals, the cleavage and polyadenylation specificity factor (CPSF) plays a central role in both steps of the processing reaction. Here, the cloning of the 73-kilodalton subunit of CPSF is reported. Sequence analyses revealed that a yeast protein (Ysh1) was highly similar to the 73-kD polypeptide. Ysh1 constitutes a new subunit of polyadenylation factor I (PFI), which has a role in yeast pre-mRNA 3'-end formation. This finding was unexpected because in contrast to CPSF, PFI is only required for the polyadenylation reaction. These results contribute to the understanding of how 3'-end processing factors may have evolved.

Amino Acid Sequence↗
PubMed1996

The Schizosaccharomyces pombe pla1 gene encodes a poly(A) polymerase and can functionally replace its Saccharomyces cerevisiae homologue.

We have isolated the poly(A) polymerase (PAP) encoding gene pla1 [for poly(A) polymerase] from the fission yeast Schizosaccharomyces pombe. Protein sequence alignments with other poly(A) polymerases reveal that pla1 is more closely related to Saccharomyces cerevisiae PAP than to bovine PAP. The two yeast poly(A) polymerases share significant sequence homology not only in the generally conserved N-terminal part but also in the C-terminus. Furthermore, pla1 rescues a S. cerevisiae PAP1 disruption mutant. An extract from the complemented strain is active in the specific in vitro polyadenylation assay. In contrast, recombinant PLA1 protein can not replace bovine PAP in the mammalian in vitro polyadenylation assay. These results indicate a high degree of conservation of the polyadenylation machinery among the evolutionary diverged budding and fission yeasts.

Amino Acid Sequence↗
PubMed1996

Structural requirements for RNA editing in glutamate receptor pre-mRNAs by recombinant double-stranded RNA adenosine deaminase.

Pre-mRNAs for brain-expressed ionotropic glutamate receptor subunits undergo RNA editing by site-specific adenosine deamination, which alters codons for molecular determinants of channel function. This nuclear process requires double-stranded RNA structures formed by exonic and intronic sequences in the pre-mRNA and is likely to be catalyzed by an adenosine deaminase that recognizes these structures as a substrate. DRADA, a double-stranded RNA adenosine deaminase, is a candidate enzyme for L-glutamate-activated receptor channel (GluR) pre-mRNA editing. We show here that DRADA indeed edits GluR pre-mRNAs, but that it displays selectivity for certain editing sites. Recombinantly expressed DRADA, both in its full-length form and in an N-terminally truncated version, edited the Q/R site in GluR6 pre-mRNA and the R/G site but not the Q/R site of GluR-B pre-mRNA. This substrate selectivity correlated with the base pairing status and sequence environment of the editing-targeted adenosines. The Q/R site of GluR-B pre-mRNA was edited by an activity partially purified from HeLa cells and thus differently structured editing sites in GluR pre-mRNAs appear to be substrates for different enzymatic activities.

Adenosine↗
PubMed1996

Mutational analysis of mammalian poly(A) polymerase identifies a region for primer binding and catalytic domain, homologous to the family X polymerases, and to other nucleotidyltransferases.

We have tested deletion and substitution mutants of bovine poly(A) polymerase, and have identified a small region that overlaps with a nuclear localization signal and binds to the RNA primer. Systematic mutagenesis of carboxylic amino acids led to the identification of three aspartates that are essential for catalysis. Sequence and secondary structure comparisons of regions surrounding these aspartates with sequences of other polymerases revealed a significant homology to the palm structure of DNA polymerase beta, terminal deoxynucleotidyltransferase and DNA polymerase IV of Saccharomyces cerevisiae, all members of the family X of polymerases. This homology extends as far as cca: tRNA nucleotidyltransferase and streptomycin adenylyltransferase, an antibiotic resistance factor.

Amino Acid Sequence↗
PubMed1996

Purification and characterization of human cleavage factor Im involved in the 3' end processing of messenger RNA precursors.

Six different protein factors are required for the specific cleavage and polyadenylation of pre-mRNA in mammals. Whereas four of them have been purified and most of their components cloned, cleavage factor Im (CF Im) and cleavage factor IIm (CF IIm) remained poorly characterized. We report here the separation of CF Im from CF 11m and the purification of CF Im to near homogeneity. Three polypeptides of 68, 59, and 25 kDa copurify with CF Im activity. All three polypeptides can be UV cross-linked to a cleavage and polyadenylation substrate in the presence of a large excess of unspecific competitor RNA, but not to a splicing-only substrate. No additional protein factor is required for the binding of CF Im to pre-mRNA. Gel retardation experiments confirmed the results obtained by UV cross-linking. In addition, we could show that CF Im stabilizes the binding of the cleavage and polyadenylation specificity factor (CPSF) to pre-mRNA and that CPSF and CF Im together form a slower migrating complex with pre-mRNA than the single protein factors. Cleavage stimulation factor (CstF) and poly(A) polymerase (PAP) had no detectable effect on the binding of CF Im to pre-mRNA. Furthermore, the CstF-CPSF-RNA as well as the CstF-CPSF-PAP-RNA complex are supershifted and stabilized upon the addition of CF Im.

Base Sequence↗
PubMed1996

Crystal structure of dimeric HIV-1 capsid protein.

X-ray diffraction analysis of a human immunodeficiency virus (HIV-1) capsid (CA) protein shows that each monomer within the dimer consists of seven alpha-helices, five of which are arranged in a coiled coil-like structure. Sequence assignments were made for two of the helices, and tentative connectivity of the remainder of the protein was confirmed by the recent solution structure of a monomeric N-terminal fragment. The C-terminal third of the protein is mostly disordered in the crystal. The longest helices in the coiled coil-like structure are separated by a long, highly antigenic peptide that includes the binding site of an antibody fragment complexed with CA in the crystal. The site of binding of the Fab, the position of the antigenic loop and the site of cleavage between the matrix protein and CA establish the side of the dimer that would be on the exterior of the retroviral core.

Amino Acid Sequence↗
PubMed1996

The biochemistry of polyadenylation.

During the synthesis of mRNA in the nucleus, 3'-ends are generated by endonucleolytic cleavage followed by polyadenylation. The machinery responsible for this simple reaction is surprisingly complex. In vitro reconstitution of 3'-end processing has demonstrated the importance of cooperative interactions in RNA recognition and catalysis. However, the inventory of processing factors is still incomplete and important mechanistic questions have not yet been answered.

Animals↗
PubMed1995

Crystal structure of a bZIP/DNA complex at 2.2 A: determinants of DNA specific recognition.

The X-ray structure of the GCN4-bZIP protein bound to DNA containing the ATF/CREB recognition sequence has been refined at 2.2 A. The water-mediated interactions between the basic domain and DNA are revealed, and combined with a more accurate description of the direct contacts, further clarify how binding specificity is achieved. Water molecules extend the interactions of both invariant basic domain residues, asparagine 235 and arginine 243, beyond their direct base contacts. The slight bending of the basic domain alpha-helix around the DNA facilitates the linking of arginine 241, 243 and 245 to main-chain carbonyl oxygen atoms via water molecules, apparently stabilizing interactions with the DNA.

Amino Acid Sequence↗
PubMed1995

Cloning of cDNAs encoding the 160 kDa subunit of the bovine cleavage and polyadenylation specificity factor.

3'-processing of mRNA precursors depends on several protein factors. One of them, cleavage and polyadenylation specificity factor (CPSF) is required for the cleavage of the mRNA precursor or as well as for the tail elongation reaction. We have obtained complementary DNA encoding the 160 kDa subunit, which had previously been shown to interact with the AAUAAA polyadenylation signal. The cDNAs code for an open reading frame of 1444 amino acids. The translated protein has a calculated molecular weight of 161 kDa and a predicted pl of 6.2. Polyclonal antibodies raised against a bacterially expressed fragment of the cDNA recognise 160 kDa subunit of purified calf thymus CPSF. The sequence contains a possible nuclear localisation signal but none of the known RNA binding motifs. It does, however, show sequence similarities to a UV-damaged DNA binding protein (UVdDb).

Amino Acid Sequence↗