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T Pieler

Publications and source records attributed to T Pieler.

At least 55 records · Page 3Linked to original sources
PubMed1993

Gene structure and alternative splicing of XFG 5-1, a X. laevis Zn finger protein with RNA homopolymer binding activity.

We describe the fine-structure of the Xenopus laevis XFG 5-1 gene which codes for an RNA homopolymer binding Zn finger protein of the FAR (Finger Associated Repeat) subfamily. The gene contains six exons, i.e., a leader exon (I), four exons (II-V), each of them encoding one individual copy of the FAR repeat, and one exon (VI) encoding the linker as well as the complete multifinger-region of the corresponding protein. Isolation and characterization of distinct cDNAs revealed that primary transcripts are alternatively spliced, thereby leading either to mRNAs containing different copy numbers of the FAR repeat or, by utilization of an alternative splice acceptor site in front of exon VI, to an extension of the linker region between the FAR repeats and the multifinger domain. We also describe the fine-structure of a closely related gene, termed XFG 5-2, which is located downstream to the XFG 5-1 gene. The general structural organization in both genes is identical, but point mutations should give rise to a XFG 5-2 protein with a different number of Zn finger units.

Alternative Splicing↗
PubMed1993

TFIIIA: nine fingers--three hands?

Over ten years ago transcription factor IIIA (TFIIIA) was identified as the only protein that binds specifically to both RNA and DNA. Although many details of its biochemical and biological activities have since been discovered, the complicated, multifunctional nature of this protein is still not fully understood. This article reviews interesting and perhaps important developments in the TFIIIA story.

Amino Acid Sequence↗
PubMed1992

RNA and DNA binding zinc fingers in Xenopus TFIIIA.

The nine tandem zinc finger repeats in the 5S gene-specific transcription factor IIIA (TFIIIA) from Xenopus mediate specific binding to 5S DNA as well as to 5S ribosomal RNA. A comparative functional analysis of a systematic set of TFIIIA zinc finger combinations reveals that most, if not all, participate in both DNA and RNA binding. Minimal sets of fingers sufficient for DNA and RNA recognition are different. In RNA binding, most finger elements are found to be functionally equivalent. However, the nonessential finger 6 exhibits RNA binding characteristics distinct from the other eight modules. The secondary/tertiary structure of the central domain in 5S RNA, not its primary sequence, is found to carry the essential structural information for TFIIIA binding in Xenopus oocytes. Taken together, our findings suggest that RNA and DNA binding are overlapping, though separable functions of the nine zinc finger elements in TFIIIA, occurring via fundamentally different molecular mechanisms.

Animals↗
PubMed1992

Assembly and nuclear transport of the U4 and U4/U6 snRNPs.

We have analyzed the assembly of the spliceosomal U4/U6 snRNP by injecting synthetic wild-type and mutant U4 RNAs into the cytoplasm of Xenopus oocytes and determining the cytoplasmic-nuclear distribution of U4 and U4/U6 snRNPs by CsCl density gradient centrifugation. Whereas the U4 snRNP was localized in both the cytoplasmic and nuclear fractions, the U4/U6 snRNP was detected exclusively in the nuclear fraction. Cytoplasmic-nuclear migration of the U4 snRNP did not depend on the stem II nor on the 5' stem-loop region of U4 RNA. Our data provide strong evidence that, following the cytoplasmic assembly of the U4 snRNP, the interaction of the U4 snRNP with U6 RNA/RNP occurs in the nucleus; furthermore, cytoplasmic-nuclear transport of the U4 snRNP is independent of U4/U6 snRNP assembly.

Animals↗
PubMed1992

Molecular cloning, expression and in vitro functional characterization of Myb-related proteins in Xenopus.

Two cDNAs encoding Myb-related proteins have been cloned from Xenopus laevis and they have been termed Xmyb1 and Xmyb2. The Xmyb1 cDNA clone codes for an open reading frame of 733 amino acids and exhibits a high degree of similarity over the entire predicted protein sequence with the human B-Myb protein. Xmyb2 is a partial cDNA clone encoding three copies of amino-terminal tandem repeat elements typical for the Myb DNA-binding domain. The predicted protein sequence is most closely related to the human A-Myb gene product. In vitro translation of two deletion mutants of Xmyb1, truncated in the 3'-portion of the open reading frame, results in protein products which cross-react with polyvalent as well as monoclonal antibodies directed against the human c-Myb protein. The same two XMyb1 proteins, which both contain the complete set of aminoterminal repeats, specifically bind to the c-Myb-specific DNA binding sequence as evidenced by electrophoretic mobility shift analysis in vitro. RNA expression profiles of Xmyb1 and -2 are very different from each other; Xmyb1 is present throughout oogenesis and early Xenopus embryogenesis; in adult tissue it is primarily detected in blood. In contrast, Xmyb2 is expressed at only very low levels during oogenesis, not detectable in embryonic RNA preparations, and in adult tissue it is predominantly expressed in testis, with only a very low level seen in blood.

Amino Acid Sequence↗
PubMed1992

Nuclear transport and phosphorylation of the RNA binding Xenopus zinc finger protein XFG 5-1.

XFG 5-1 is a Krüppel-type Xenopus zinc finger protein with specific RNA homopolymer binding activity in vitro. In the oocyte, the protein is distributed between nucleus and cytoplasm; the nuclear fraction, not the cytoplasm, contains phosphorylated isoform(s) of XFG 5-1. In vitro phosphorylation by use of oocyte/egg extracts or purified casein kinase II is specific to the amino-terminal portion of the protein. The carboxy-terminal zinc finger domain contains a signal sufficient for nuclear transport. Overexpression of either full length XFG 5-1 or of the carboxy-terminal portion alone, which maintains RNA binding and nuclear import activities, was achieved in Xenopus embryos by mRNA injection. This treatment did not result in impaired regulation of development, suggesting that XFG 5-1 functions in a way distinct from the mode of action exemplified in the Drosophila zinc finger protein Krüppel.

Amino Acid Sequence↗
PubMed1991

The maternal store of zinc finger protein encoding mRNAs in fully grown Xenopus oocytes is not required for early embryogenesis.

A large family of C2H2 (Krüppel-like) zinc finger protein genes is maternally transcribed in Xenopus oocytes; many of the corresponding mRNAs are actively translated post-fertilization, before the onset of zygotic activation of transcription. With the aim of asking if any of these stored mRNAs have a function in Xenopus development, we made use of antisense oligonucleotide mediated, targeted RNA destruction. Injected oocytes lose the entire pool of C2H2 zinc finger protein encoding mRNAs. They are indistinguishable from control oocytes in their abilities to mature in vitro and to be fertilized in vitro. Embryos generated from such oocytes develop normally until tadpole stage. These findings do not rule out the possibility that C2H2 zinc finger protein genes are involved in developmental control in Xenopus. However, they do suggest that the biological function for at least some of the early expressed zinc finger proteins in Xenopus differs in important aspects from the way Krüppel or other DNA binding factors act as developmental regulators in Drosophila.

Amino Acid Sequence↗
PubMed1991

Structure, expression and in vitro functional characterization of a novel RNA binding zinc finger protein from Xenopus.

Large multigene families of zinc finger proteins are expressed in vertebrates. One way of approaching their function is to characterize their structure, expression and biochemical properties. XFG 5-1 is a Xenopus zinc finger protein which is widely transcribed in oocytes, embryos and adult tissues. It carries a novel, non-finger repeat structure, which is common to a subfamily of Xenopus zinc finger proteins. The bacterially expressed protein exhibits specific RNA homopolymer binding activities with the zinc finger domain being sufficient for this ability. These findings suggest that XFG 5-1 serves a general biological function involving its RNA binding capacity.

Amino Acid Sequence↗
PubMed1990

A transcription stimulatory factor binds to the upstream region of Xenopus 5 S RNA and tRNA genes.

Upstream sequence elements modulate transcription efficiency and competition strength of eucaryotic 5 S rRNA and tRNA genes transcribed by RNA polymerase III. We report on the chromatographic separation and functional characterization of at least two components involved in this process: one has a DNA binding activity and interacts with the immediate upstream region of Xenopus tRNA and 5 S rRNA genes, and a second component, which by itself does not interact with the upstream region of these genes, is required together with the DNA binding activity for transcription stimulation in vitro.

Animals↗
PubMed1990

Protein-mediated nuclear export of RNA: 5S rRNA containing small RNPs in xenopus oocytes.

We have analyzed RNP formation and nucleocytoplasmic migration of 5S RNA and 5S RNA variants transcribed from microinjected genes in Xenopus oocytes. Using antisera against three different proteins we find that newly transcribed nuclear 5S rRNA transiently interacts with La antigen. The La protein is then replaced by either ribosomal protein L5 or the 5S gene-specific transcription factor IIIA (TFIIIA), and each of these two RNPs migrates out of the nucleus and accumulates in the cytoplasm. RNA molecules that are impaired in their ability to interact with L5 and TFIIIA are retained in the nucleus. Thus, L5 and TFIIIA define a new functional class of proteins involved in the nuclear export of RNA. In addition, we show that RNP migration depletes the nucleus of TFIIIA, resulting in a loss of transcription competence for newly injected 5S rRNA genes.

Animals↗
PubMed1990

The Xenopus laevis poly(A) binding protein is composed of multiple functionally independent RNA binding domains.

A family of eukaryotic RNA binding proteins is defined by the conserved RNP motif. The poly(A) binding protein has four such motifs. We report on the isolation and structural characterization of several variant cDNA clones, as well as of a gene encoding this protein in Xenopus laevis embryos. Wild-type protein as well as truncated versions carrying isolated single motifs or artificial combinations of two and more such elements were characterized for their ability to bind specifically to RNA homopolymers. Three of the isolated repeats were functional in specific RNA binding, whereas the N-terminal RNP motif was non-functional. Combinatorial effects in RNA binding were measured with constructs carrying multiple repeats, which were not predictable from the activity of isolated domains.

Amino Acid Sequence↗
PubMed1989

Second-order repeats in Xenopus laevis finger proteins.

The primary structure of 342 finger repeats encoded in 42 different cDNA clones isolated from Xenopus laevis oocyte and gastrula cDNA libraries has been determined. Comparative sequence analysis of the predicted protein sequences results in a consensus repeat sequence that has an extended conserved segment of 16 amino acid residues, including the evolutionary conserved H/C link element, connected to a highly variable segment that is located in the finger loop region. Groups of tandem finger repeats are found to be organized in distinct higher-order structural units, with a pair of mutually distinct fingers being the most frequently observed second-order repeat unit. Structural features observed are discussed in respect to existing models for Zn finger structure and function.

Amino Acids↗
PubMed1989

Common mechanisms of promoter recognition by RNA polymerases II and III.

Recent results indicate that RNA polymerase III can use upstream promoters that are structurally and functionally very similar to those recognized by RNA polymerase II. The demonstration that RNA polymerases II and III can use the same transcription factors emphasizes the fundamental similarities between these distinct activities. It is also clear now that transcription factors can be functionally interchanged between distantly related species, indicating that the basic structures involved in promoter recognition are highly conserved throughout evolution.

Base Sequence↗
PubMed1989

Evolutionary conserved modules associated with zinc fingers in Xenopus laevis.

Many DNA-binding proteins that are involved in the differential regulation of gene expression are composed of multiple discrete modules. Association of the homeobox-encoded helix-turn-helix DNA-binding motif with conserved modules, such as the paired box or the POU domain, has led to the definition of structurally and functionally related subfamilies of regulatory proteins. The zinc finger, which is the second major nucleic acid-binding motif characterized to date, defines large multigene families in higher eukaryotes; we have isolated more than 100 Xenopus finger protein-encoding cDNAs and in this study we show that at least 10 of these clones share extensive sequence homologies in a region of more than 200 amino acids in the N-terminal nonfinger portion of the predicted proteins, which is connected to variable finger clusters. We refer to this element as a finger-associated boxes (FAX) domain. Cross-hybridization with human genomic DNA indicates that the finger-associated boxes domain is evolutionary conserved. Northern blot analysis shows that the corresponding genes are differentially expressed in the course of early Xenopus embryogenesis.

Amino Acid Sequence↗
PubMed1988

Oocyte and somatic 5S ribosomal RNA and 5S RNA encoding genes in Xenopus tropicalis.

We have investigated the structure of oocyte and somatic 5S ribosomal RNA and of 5S RNA encoding genes in Xenopus tropicalis. The sequences of the two 5S RNA families differ in four positions, but only one of these substitutions, a C to U transition in position 79 within the internal control region of the corresponding 5S RNA encoding genes, is a distinguishing characteristic of all Xenopus somatic and oocyte 5S RNAs characterized to date, including those from Xenopus laevis and Xenopus borealis. 5S RNA genes in Xenopus tropicalis are organized in clusters of multiple repeats of a 264 base pair unit; the structural and functional organization of the Xenopus tropicalis oocyte 5S gene is similar to the somatic but distinct from the oocyte 5S DNA in Xenopus laevis and Xenopus borealis. A comparative sequence analysis reveals the presence of a strictly conserved pentamer motif AAAGT in the 5'-flanking region of Xenopus 5S genes which we demonstrate in a separate communication to serve as a binding signal for an upstream stimulatory factor.

Animals↗
PubMed1988

The finger motif defines a multigene family represented in the maternal mRNA of Xenopus laevis oocytes.

We have screened Xenopus laevis cDNA and genomic libraries for finger motif encoding sequences by use of a synthetic oligonucleotide probe coding for a stretch of conserved amino acids, the H/C-link, which joins individual finger loops in several multi-fingered proteins. Our studies reveal that a large number of different cDNA clones encode amino acid sequences predicting multiple units of the metal-coordinating finger structure. Derived proteins are different from each other as well as from the two examples of Xenopus finger proteins reported to date, TFIIIA and X.fin. The 109 finger repeats characterized are derived from 14 different cDNA clones and have been analysed for the presence of conserved and highly variable amino acids, revealing a close structural relatedness among each other as well as with a few selected finger domains from Drosophila and mouse proteins. The results from this comparative sequence analysis are also discussed in terms of the existing models for DNA binding. All sequences are identified in an ovary cDNA library but the patterns of mRNA level for individual finger clones vary greatly during early development. The prevalence of these structures in the oocyte suggests that part of the maternal information for the realization of the developmental program utilized in Xenopus embryogenesis might be transmitted in the form of regulatory, nucleic-acid-binding proteins.

Amino Acid Sequence↗
PubMed1987

The 5S gene internal control region is composed of three distinct sequence elements, organized as two functional domains with variable spacing.

Systematic oligonucleotide-directed mutagenesis within the internal control region of the Xenopus laevis somatic 5S RNA gene identifies three distinct sequence elements that regulate transcription activity: box A, containing the common, conserved class III promoter domain, and two 5S-gene-specific segments, termed intermediate element and box C. Analysis of the individual steps in the formation of the stable initiation complex reveals that the two 5S-gene-specific elements are the main determinants for the stable binding of TFIIIA. In contrast, TFIIIC binding appears to be dependent on interactions with TFIIIA and on direct DNA interactions in box A as well as probably in box C. Alterations of the spacing between the two major promoter domains of from -3 to +10 nucleotides are tolerated, although they reduce transcription activity and were found to prevent the formation of a stable initiation complex.

Animals↗