The primary structure of rat ribosomal protein L9

The Drosophila homeobox gene fushi tarazu (ftz) is expressed in a highly dynamic striped pattern in early embryos. A key regulatory element that controls the ftz pattern is the ftz proximal enhancer, which mediates positive autoregulation via multiple binding sites for the Ftz protein. In addition, the enhancer is necessary for stripe establishment prior to the onset of autoregulation. We previously identified nine binding sites for multiple Drosophila nuclear proteins in a core 323-bp region of the enhancer. Three of these nine sites interact with the same cohort of nuclear proteins in vitro. We showed previously that the nuclear receptor Ftz-F1 interacts with this repeated module. Here we purified additional proteins interacting with this module from Drosophila nuclear extracts. Peptide sequences of the zinc finger protein Ttk and the transcription factor Adf-1 were obtained. While Ttk is thought to be a repressor of ftz stripes, we have shown that both Adf-1 and Ftz-F1 activate transcription in a binding site-dependent fashion. These two proteins are expressed ubiquitously at the time ftz is expressed in stripes, suggesting that either may activate striped expression alone or in combination with the Ftz protein. The roles of the nine nuclear factor binding sites were tested in vivo, by site-directed mutagenesis of individual and multiple sites. The three Ftz-F1-Adf-1-Ttk binding sites were found to be functionally redundant and essential for stripe expression in transgenic embryos. Thus, a biochemical analysis identified cis-acting regulatory modules that are required for gene expression in vivo. The finding of repeated binding sites for multiple nuclear proteins underscores the high degree of redundancy built into embryonic gene regulatory networks.Embryonic development is controlled by the differential expression of regulatory genes. This process can be readily analyzed in the fruit fly, Drosophila melanogaster, since the basic body plan of the fly is established within the first few hours of embryogenesis by interacting sets of regulatory genes that are expressed in tightly regulated spatial and temporal patterns (1,42,44). One example of this is the homeobox gene fushi tarazu (ftz), which is expressed in a seven-stripe pattern in cellularizing embryos. The expression of ftz in these seven stripes directs the development of alternate body segments. In ftz mutant embryos, these segments are missing; the remaining portions of the embryo are fused, resulting in approximately half-sized embryos that die before hatching (20,36,38,61). Similarly, ectopic expression of ftz throughout the embryo is lethal, demonstrating that ftz expression must be restricted to its sevenstripe domain for development to proceed normally (57).Multiple regulatory elements dispersed over a distance of at least 10 kb interact to generate the ftz seven-stripe expression pattern (26,27). One key regulatory element is the ftz upstream element (Ϫ3.6 to Ϫ6 kb), which contains two independently acting enhancers, the distal and proximal enhan...

Section: Resultsmentioning

confidence: 98%

A Binding Site for Multiple Transcriptional Activators in the fushi tarazu Proximal Enhancer Is Essential for Gene Expression In Vivo

Han

et al. 1998

“…2C). Similar to rpL12, rpL23a is a 60S subunit component that binds to the 28S rRNA (16,37). The influence of importin 11 on the nuclear localization of rpL12 was further confirmed in cells overexpressing importin 11(78-975).…”

Section: Vol 22 2002 Importin 11 Mediates Rpl12 Import 1267mentioning

confidence: 84%

Ribosomal Protein L12 Uses a Distinct Nuclear Import Pathway Mediated by Importin 11

Plafker

Macara

2002

Ribosome biogenesis requires the nuclear translocation of ribosomal proteins from their site of synthesis in the cytoplasm to the nucleus. Analyses of the import mechanisms have revealed that most ribosomal proteins can be delivered to the nucleus by multiple transport receptors (karyopherins or importins). We now provide evidence that ribosomal protein L12 (rpL12) is distinguished from the bulk of ribosomal proteins because it accesses the importin 11 pathway as a major route into the nucleus. rpL12 specifically and directly interacted with importin 11 in vitro and in vivo. Both rpL12 binding to and import by importin 11 were inhibited by another importin 11 substrate, UbcM2, indicating that these two cargoes may bind overlapping sites on the transport receptor. In contrast, the import of rpL23a, a ribosomal protein that uses the general ribosomal protein import system, was not competed by UbcM2, and in an in vitro binding assay, importin 11 did not bind to the nuclear localization signal of rpL23a. Furthermore, in a transient transfection assay, the nuclear accumulation of rpL12 was increased by coexpressed importin 11, but not by other importins. These data are consistent with importin 11 being a mediator of rpL12 nuclear import. Taken together, these results indicate that rpL12 uses a distinct nuclear import pathway that may contribute to a mechanism for regulating ribosome synthesis and/or maturation.Assembly of the 40S and 60S subunits of the eukaryotic ribosome takes place within the nucleolar compartment of the nucleus. As a result, the ϳ75 ribosomal proteins necessary for ribosomal assembly and maturation must be imported into the nucleus and targeted to the nucleolus following their synthesis in the cytoplasm. Although the sizes of these proteins are well below the diffusion limit of the nuclear pore, ribosomal protein import requires energy and soluble nuclear transport factors (15, 32) that include a class of import receptors, often called karyopherins or importins. It has been reasoned (32) that such rapid, active transport is necessary because of the short cytoplasmic half-lives (2 to 3 min) of many ribosomal proteins (38,39).Importins recognize specific nuclear localization signals (NLSs) encoded in the sequences of their cargo proteins. The importins also interact with nucleoporins, which are components of the nuclear pore complex. This interaction permits a facilitated diffusive translocation of the importin-cargo complex through the pores. Within the nucleus, the importins bind to Ran:GTP, which triggers cargo release. The importin-Ran: GTP complex then recycles back to the cytoplasm, where the GTP on Ran is hydrolyzed, and Ran:GDP dissociates from the importins (all reviewed in references 5, 10,

“…The human clone encodes many Arg and Gly residues at the N terminus, but the Arg residues in particular are not conserved in the rat clone. It is possible that they are sites of posttranslational modification for the dimethylation of Arg residues, as a Gly residue is usually situated directly C terminal to the modified Arg residue and may be required for recognition by the methyltransferase (13,40,51). There are six Arg/Gly repeats within 40 amino acids in the human dsRNA adenosine deaminase.…”

mentioning

confidence: 99%

Cloning of cDNAs encoding mammalian double-stranded RNA-specific adenosine deaminase

O’Connell

Krause

Higuchi

et al. 1995

248

231

Double-stranded RNA (dsRNA)-specific adenosine deaminase converts adenosine to inosine in dsRNA. The protein has been purified from calf thymus, and here we describe the cloning of cDNAs encoding both the human and rat proteins as well as a partial bovine clone. The human and rat clones are very similar at the amino acid level except at their N termini and contain three dsRNA binding motifs, a putative nuclear targeting signal, and a possible deaminase motif. Antibodies raised against the protein encoded by the partial bovine clone specifically recognize the calf thymus dsRNA adenosine deaminase. Furthermore, the antibodies can immunodeplete a calf thymus extract of dsRNA adenosine deaminase activity, and the activity can be restored by addition of pure bovine deaminase. Staining of HeLa cells confirms the nuclear localization of the dsRNAspecific adenosine deaminase. In situ hybridization in rat brain slices indicates a widespread distribution of the enzyme in the brain. Double-stranded RNA (dsRNA)-specific adenosine deaminase is a mammalian RNA-modifying enzyme that was discovered upon performing antisense experiments with Xenopus laevis (2, 36). The enzyme destabilizes duplex RNA by converting adenosine to inosine by hydrolytic deamination (34), which results in unstable U ⅐ I base pairs so that the dsRNA becomes unwound. While in vitro there is no sequence specificity for substrate RNAs, the activity is specific for dsRNAs, and a minimum of 100 bp of either intermolecular or intramolecular duplex seems to be required for efficient modification (30).The biological function of the enzyme as well as the full range of its physiological substrate RNAs are not known. The deaminase has been implicated in editing the RNAs of certain subunits of glutamate-gated ion channel receptors in the brain, because of the dependence of this editing event on intramolecular RNA ⅐ RNA base pairing (19,24,37). The dsRNA adenosine deaminase appears also to be responsible for the generation of defective measles virus with biased hypermutations that result in a lethal central nervous system disease, measles inclusion body encephalitis (4).This enzyme is of interest not only because of its assumed biological functions but also because its mechanism of action is intriguing. dsRNA differs from duplex DNA in forming an A-type helix. In A-form RNA, the minor groove is shallow and wide (10 to 11 Å [1 Å ϭ 0.1 nm]) and the major groove is deep and narrow (3.5 Å) (38). RNA-binding proteins usually bind to the major groove at the end of a helix or at a bulge in RNA structures where the major groove is more accessible (38). The binding of human immunodeficiency virus Tat protein to its target RNA TAR is an example, as the binding occurs on the major groove in the region of a bulge (35,47,48). The dsRNA adenosine deaminase binds to continuous duplex RNA; therefore, it must be able to recognize and access the adenosines that it deaminates even though this amino group is at position 6 of the base and lies in the major groove. This position is inacc...