Crystallization and characterization of Smaug: a novel RNA-binding motif

During Drosophila embryogenesis, Smaug protein represses translation of Nanos through an interaction with a specific element in its 3(')UTR. The repression occurs in the bulk cytoplasm of the embryo; Nanos is, however, successfully translated in the specialized cytoplasm of the posterior pole. This generates a gradient of Nanos emanating from the posterior pole that is essential for organizing proper abdominal segmentation. To understand the structural basis of RNA binding and translational control, we have crystallized a domain of Drosophila Smaug that binds RNA. The crystals belong to the space group R3 with unit cell dimensions of a=b=129.3A, c=33.1A, alpha=beta=90 degrees, gamma=120 degrees and diffract to 1.80A with synchrotron radiation. Initial characterization of this domain suggests that it encodes a novel RNA-binding motif.     

A testis cytoplasmic RNA-binding protein that has the properties of a translational repressor.

Translation of the mouse protamine 1 (Prm-1) mRNA is repressed for several days during male germ cell differentiation. With the hope of cloning genes that regulate the translational repression of Prm-1, we screened male germ cell cDNA expression libraries with the 3' untranslated region of the Prm-1 RNA. From this screen we obtained two independent clones that encode Prbp, a Prm-1 RNA-binding protein. Prbp contains two copies of a double-stranded-RNA-binding domain. In vitro, the protein binds to a portion of the Prm-1 3' untranslated region previously shown to be sufficient for translational repression in transgenic mice, as well as to poly(I). poly(C). Prbp protein is present in multiple forms in cytoplasmic extracts prepared from wild-type mouse testes and is absent from testes of germ cell-deficient mouse mutants, suggesting that Prbp is restricted to the germ cells of the testis. Immunocytochemical localization confirmed that Prbp is present in the cytoplasmic compartment of late-stage meiotic cells and haploid round spermatids. Recombinant Prbp protein inhibits the translation of multiple mRNAs in a wheat germ lysate, suggesting that Prbp acts to repress translation in round spermatids. While this protein lacks complete specificity for Prm-1-containing RNAs in vitro, the properties of Prbp are consistent with it acting as a general repressor of translation.     

RNA-binding properties of a translational activator, the adenovirus L4 100-kilodalton protein.

The adenovirus L4 100-kDa nonstructural protein (100K protein) is required for efficient initiation of translation of viral late mRNA species during the late mRNA species during the late phase of infection (B. W. Hayes, G. C. Telling, M. M. Myat, J. F. Williams, and S. J. Flint, J. Virol. 64:2732-2742, 1990). The RNA-binding properties of this protein were analyzed in an immunoprecipitation assay with the 100K-specific monoclonal antibody 2100K-1 (C. L. Cepko and P. A. Sharp, Virology 129:137-154, 1983). Coprecipitation of the 100K protein and 3H-infected cell RNA was demonstrated. The RNA-binding activity of the 100K protein was inhibited by single-stranded DNA but not by double-stranded DNA, double-stranded RNA, or tRNA. Competition assays were used to investigate the specificity with which the 100K protein binds to RNA in vitro. Although the protein exhibited a strong preference for the ribohomopolymer poly(U) or poly(G), no specific binding to viral mRNA species could be detected; uninfected or adenovirus type 5-infected HeLa cell poly(A)-containing and poly(A)-lacking RNAs were all effective inhibitors of binding of the protein to viral late mRNA. Similar results were obtained when the binding of the 100K protein to a single, in vitro-synthesized L2 mRNA was assessed. The poly(U)-binding activity of the 100K protein was used to compare the RNA-binding properties of the 100K protein prepared from cells infected by adenovirus type 5 and the H5ts1 mutant (B. W. Hayes, G. C. Telling, M. M. Myat, J. F. Williams, and S. J. Flint, J. Virol. 64:2732-2742, 1990). A temperature-dependent decrease in H5ts1 100K protein binding was observed, correlating with the impaired translational function of this protein in vivo. By contrast, wild-type 100K protein RNA binding was unaffected by temperature. These data suggest that the 100K protein acts to increase the translational efficiency of viral late mRNA species by a mechanism that involves binding to RNA.     

MAGOH interacts with a novel RNA-binding protein

MAGOH is the human homologue of Drosophila mago nashi, a protein that is required for normal germ plasm development in the Drosophila embryo. Using human MAGOH as a bait protein in a yeast two-hybrid screen, we recovered four independent cDNA clones that encode different lengths of a novel protein containing a conserved RNA-binding region. This gene, designated RBM8, encodes a 173-aa protein that was shown to have an apparent molecular mass of 26 kDa, as demonstrated by in vitro translation assay. The interaction between MAGOH and RBM8 was demonstrated by both yeast two-hybrid and GST fusion protein pull-down assays. Like MAGOH, RBM8 gene is expressed ubiquitously in human tissues; three species of RBM8 mRNA were detected. Also similar to MAGOH, RBM8 expression is serum inducible in quiescent NIH3T3 fibroblast cells.     

The hinge region operates as a stability switch in cGMP-dependent protein kinase I alpha

The molecular mechanism of cGMP-dependent protein kinase activation by its allosteric regulator cyclic-3',5'-guanosine monophosphate (cGMP) has been intensely studied. However, the structural as well as thermodynamic changes upon binding of cGMP to type I cGMP-dependent protein kinase are not fully understood. Here we report a cGMP-induced shift of Gibbs free enthalpy (DeltaDeltaGD) of 2.5 kJ.mol-1 as determined from changes in tryptophan fluorescence using urea-induced unfolding for bovine PKG Ialpha. However, this apparent increase in overall stability specifically excluded the N-terminal region of the kinase. Analyses of tryptic cleavage patterns using liquid chromatography-coupled ESI-TOF mass spectrometry and SDS/PAGE revealed that cGMP binding destabilizes the N-terminus at the hinge region, centered around residue 77, while the C-terminus was protected from degradation. Furthermore, two recombinantly expressed mutants: the deletion fragment Delta1-77 and the trypsin resistant mutant Arg77Leu (R77L) revealed that the labile nature of the N-terminus is primarily associated with the hinge region. The R77L mutation not only stabilized the N-terminus but extended a stabilizing effect on the remaining domains of the enzyme as well. These findings support the concept that the hinge region of PKG acts as a stability switch.     

Smaug, a novel and conserved protein, contributes to repression of nanos mRNA translation in vitro

Proper deployment of Nanos protein at the posterior of the Drosophila embryo, where it directs posterior development, requires a combination of RNA localization and translational controls. These controls ensure that only the posteriorly-localized nanos mRNA is translated, whereas unlocalized nanos mRNA is translationally repressed. Here we describe cloning of the gene encoding Smaug, an RNA-binding protein that interacts with the sequences, SREs, in the nanos mRNA that mediate translational repression. Using an in vitro translation assay, we demonstrate that SRE-dependent repression occurs in extracts from early stage embryos. Immunodepletion of Smaug from the extracts eliminates repression, consistent with the notion that Smaug is involved. Smaug is a novel gene and the existence of potential mammalian Smaug homologs raises the possibility that Smaug represents a new class of conserved translational repressor.     

RNA-binding protein hoip accelerates polyQ-induced neurodegeneration in Drosophila

Abnormal polyglutamine (polyQ) expansion in the N-terminal domain of the human androgen receptor (hAR) is known to cause spinobulbar muscular atrophy (SBMA), a hereditary human neurodegenerative disorder. To explore the molecular mechanisms of neurodegeneration in SBMA, we genetically screened modulators of neurodegeneration in a Drosophila SBMA experimental model system. We identified hoip as an accelerator of polyQ-induced neurodegeneration. We found that hoip forms a complex with 18s rRNA together nop56 and nop5 proteins, whose human homologs are known to form a snoRNP complex involved in ribosomal RNA processing. Significantly, the levels of mutant polyQ-hAR were up-regulated in a mutant line overexpressing hoip. Consistently, severe neurodegeneration phenotype (rough eye) was also observed in both nop56 and nop5 overexpression mutant lines. These findings suggest that the process of neurodegeneration induced by abnormal polyQ expansion in the hAR may be regulated by the activity of snoRNP complex.