Tomas Pieler

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.

Regionalized metabolic activity establishes boundaries of retinoic acid signalling

The competence of a cell to respond to the signalling molecule retinoic acid (RA) is thought to depend largely on its repertoire of cognate zinc finger nuclear receptors. XCYP26 is an RA hydroxylase that is expressed differentially during early Xenopus development. In Xenopus embryos, XCYP26 can rescue developmental defects induced by application of exogenous RA, suggesting that the enzymatic modifications introduced inhibit RA signalling activities in vivo. Alterations in the expression pattern of a number of different molecular markers for neural development induced upon ectopic expression of XCYP26 reflect a primary function of RA signalling in hindbrain development. Progressive inactivation of RA signalling results in a stepwise anteriorization of the molecular identity of individual rhombomeres. The expression pattern of XCYP26 during gastrulation appears to define areas within the prospective neural plate that develop in response to different concentrations of RA. Taken together, these observations appear to reflect an important regulatory function of XCYP26 for RA signalling; XCYP26‐mediated modification of RA modulates its signalling activity and helps to establish boundaries of differentially responsive and non‐responsive territories.

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.

Zinc finger proteins: what we know and what we would like to know.

The molecular description of diverse nucleic acid binding proteins has led to the definition of structurally related families of such polypeptides. They often carry multiple modular functional domains, and one conserved motif responsible for nucleic acid binding is the zinc finger repeat. It has originally been discovered in TFIIIA, a 5S gene specific transcription factor from Xenopus. This protein contains nine tandem repeats of a sequence element referred to as the C2H 2 zinc finger since it contains highly conserved pairs of cysteine and histidine residues (Miller et al., 1985; Brown et al., 1985), which fold each of these sequences about a zinc atom (Diakun et al., 1986). Following its discovery in TFIIIA, variants of the zinc finger motif have been detected in many other, often regulatory proteins. It should be pointed out that the term zinc finger has been applied to several other structures such as the cysteine rich DNA binding domain of ligand dependent nuclear receptors (Evans, 1988; Green and Chambon, 1988; Beato, 1989) or a group of retroviral RNA binding proteins (Katz and Jentoft, 1989). Here, we will focus our discussion on the C2H 2 class of zinc finger proteins (ZFPs), of which TFIIIA is a prototype.

Cytoplasmic retention and nuclear import of 5S ribosomal RNA containing RNPs.

Nuclear export of newly transcribed 5S ribosomal RNA in Xenopus oocytes occurs in the context of either a complex with the ribosomal protein L5 (5S RNP) or with the transcription factor IIIA (7S RNP). Here we examine nuclear import of 5S RNA, L5 and TFIIIA. The 5S RNP shuttles between nucleus and cytoplasm and only 5S RNA variants which can bind to L5 gain access to the nucleus. The 7S RNP is retained in the cytoplasm. Only TFIIIA which is not bound to 5S RNA is imported into the nucleus. As a novel mechanism for cytoplasmic retention, we propose that RNA binding masks a nuclear localization sequence in TFIIIA. In contrast to the nuclear import of L5, import of TFIIIA is sensitive towards the nuclear localization sequence (NLS) competitor p(lys)‐BSA, suggesting that these two proteins make use of different import pathways.

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.

Elr-type proteins protect Xenopus Dead end mRNA from miR-18-mediated clearance in the soma

Segregation of the future germ line defines a crucial cell fate decision during animal development. In Xenopus, germ cells are specified by inheritance of vegetally localized maternal determinants, including a group of specific mRNAs. Here, we show that the vegetal localization elements (LE) of Xenopus Dead end (XDE) and of several other germ-line-specific, vegetally localized transcripts mediate germ cell-specific stabilization and somatic clearance of microinjected reporter mRNA in Xenopus embryos. The part of XDE-LE critical for somatic RNA clearance exhibits homology to zebrafish nanos1 and appears to be targeted by Xenopus miR-18 for somatic mRNA clearance. Xenopus Elr-type proteins of the vegetal localization complex can alleviate somatic RNA clearance of microinjected XDE-LE and endogenous XDE mRNA. ElrB1 synergizes with Xenopus Dead end protein in the stabilization of XDE-LE mRNA. Taken together, our findings unveil a functional link of vegetal mRNA localization and the protection of germ-line mRNAs from somatic clearance.

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.

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.

Assembly and nuclear transport of the U4 and U4U6 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.