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.     

Short communication. A high level of atmospheric oxygen, as occurred toward the end of the Cretaceous period, increases leaf diffusion conductance

Leaf diffusion conductance much increased when Xanthium strumarium (C3), Atriplex prostata (C3) and Flaveria pringlei (C3), F. sonorensis (C3/C4), F. floridana (C3/C4), and F. trinervia (C4) plants were grown under relatively high (28 kPa) ambient oxygen. This phenomenon, which increases sensitivity to water stress, could have adversely affected vegetation during the late Cretaceous, high O2 episode.Keywords: Palaeo-atmosphere, stomata, oxygen, Cretaceous.      

A heteromeric RNA-binding protein is involved in maintaining acrophase and period of the circadian clock

The RNA-binding protein CHLAMY1 from the green alga Chlamydomonas reinhardtii consists of two subunits. One (named C1) contains three lysine homology motifs and the other (named C3) has three RNA recognition motifs. CHLAMY1 binds specifically to uridine-guanine-repeat sequences and its circadian-binding activity is controlled at the posttranslational level, presumably by time-dependent formation of protein complexes consisting of C1 and C3 or C1 alone. Here we have characterized the role of the two subunits within the circadian system by measurements of a circadian rhythm of phototaxis in strains where C1 or C3 are either up- or down-regulated. Further, we have measured the rhythm of nitrite reductase activity in strains with reduced levels of C1 or C3. In case of changes in the C3 level (both increases and decreases), the acrophase of the phototaxis rhythm and of the nitrite reductase rhythm (C3 decrease) was shifted by several hours from subjective day (maximum in wild-type cells) back towards the night. In contrast, both silencing and overexpression of C1 resulted in disturbed circadian rhythms and arrhythmicity. Interestingly, the expression of C1 is interconnected with that of C3. Our data suggest that CHLAMY1 is involved in the control of the phase angle and period of the circadian clock in C. reinhardtii.     

Free left arms as precursor molecules in the evolution of Alu sequences

Summary The dimeric Alu molecule of human and other primates is composed of a left and a right arm that are very similar but show characteristic differences. If the Alu sequence has arisen through the fusion of monomeric precursor molecules, the traces of such precursor genes are expected still to be present in contemporary primate DNA. We report finding seven independent human DNA sequences that qualify s descendants of a left-arm precursor gene. Some characteristics in primary and secondary structures of these sequences are described.     

Defining the orientation of the tandem fusions that occurred during the evolution of Indian muntjac chromosomes by BAC mapping

The Indian muntjac (Muntiacus muntjak vaginalis) has a karyotype of 2n=6 in the female and 7 in the male, the karyotypic evolution of which through extensive tandem fusions and several centric fusions has been well-documented by recent molecular cytogenetic studies. In an attempt to define the fusion orientations of conserved chromosomal segments and the molecular mechanisms underlying the tandem fusions, we have constructed a highly redundant (more than six times of whole genome coverage) bacterial artificial chromosome (BAC) library of Indian muntjac. The BAC library contains 124,800 clones with no chromosome bias and has an average insert DNA size of 120 kb. A total of 223 clones have been mapped by fluorescent in situ hybridization onto the chromosomes of both Indian muntjac and Chinese muntjac and a high-resolution comparative map has been established. Our mapping results demonstrate that all tandem fusions that occurred during the evolution of Indian muntjac karyotype from the acrocentric 2n=70 hypothetical ancestral karyotype are centromere–telomere (head–tail) fusions.     

Evidence that Con A induces cytotoxicity in the same subclass of T cells as does alloimmunization.

We have studied the induction of cytoxic activity in murine T cells by the T cell mitogen Con A. Here we report the results of experiments that indicate that this cytotoxicity develops in the same class of T cells potentially activatable to cytotoxicity by immunization with allogeneic cells. Cytotoxic activity does not result from activation of cells of the T-helper class by PHA, and extensive reduction of the proportion of cells of the T-helper class by in vivo treatment with ATS does not comparably reduce the cytotoxicity developed in response to Con A. The Con A-activated cytotoxic cell sediments as a large cell. Furthermore, spleen cell populations previously immunized to alloantigens in vivo develop greatly increased cytotoxicity specific for the alloantigen of the immunizing haplotype after culture with Con A in vitro.     

The chaperonin of the archaeon Sulfolobus solfataricus is an RNA-binding protein that participates in ribosomal RNA processing.

The 60 kDa molecular chaperones (chaperonins) are high molecular weight protein complexes having a characteristic double-ring toroidal shape; they are thought to aid the folding of denatured or newly synthesized polypeptides. These proteins exist as two functionally similar, but distantly related families, one comprising the bacterial and organellar chaperonins and another (the so-called CCT-TRiC family) including the chaperonins of the archaea and the eukaryotes. Although some evidence exists that the archaeal chaperonins are implicated in protein folding, much remains to be learned about their precise cellular function. In this work, we report that the chaperonin of the thermophilic archaeon Sulfolobus solfataricus is an RNA-binding protein that interacts specifically in vivo with the 16S rRNA and participates in the maturation of its 5' extremity in vitro. We further show that the chaperonin binds RNA as the native heterooligomeric complex and that RNA binding and processing are inhibited by ATP. These results agree with previous reports indicating a role for the bacterial/organellar chaperonins in RNA protection or processing and suggest that all known chaperonin families share specific and evolutionarily ancient functions in RNA metabolism.     

The multiple RNA-binding domains of the mRNA poly(A)-binding protein have different RNA-binding activities.

The poly(A)-binding protein (PABP) is the major mRNA-binding protein in eukaryotes, and it is essential for viability of the yeast Saccharomyces cerevisiae. The amino acid sequence of the protein indicates that it consists of four ribonucleoprotein consensus sequence-containing RNA-binding domains (RBDs I, II, III, and IV) and a proline-rich auxiliary domain at the carboxyl terminus. We produced different parts of the S. cerevisiae PABP and studied their binding to poly(A) and other ribohomopolymers in vitro. We found that none of the individual RBDs of the protein bind poly(A) specifically or efficiently. Contiguous two-domain combinations were required for efficient RNA binding, and each pairwise combination (I/II, II/III, and III/IV) had a distinct RNA-binding activity. Specific poly(A)-binding activity was found only in the two amino-terminal RBDs (I/II) which, interestingly, are dispensable for viability of yeast cells, whereas the activity that is sufficient to rescue lethality of a PABP-deleted strain is in the carboxyl-terminal RBDs (III/IV). We conclude that the PABP is a multifunctional RNA-binding protein that has at least two distinct and separable activities: RBDs I/II, which most likely function in binding the PABP to mRNA through the poly(A) tail, and RBDs III/IV, which may function through binding either to a different part of the same mRNA molecule or to other RNA(s).     

Identification of pp68 as the Tyrosine-phosphorylated Form of SYNCRIP/NSAP1. A cytoplasmic RNA-binding protein

Recently we reported that osmotic shock increased the insulin-stimulated tyrosine phosphorylation of a 68-kDa RNA-binding protein in 3T3-L1 adipocytes (Hresko, R. C., and Mueckler, M. (2000) J. Biol. Chem. 275, 18114-18120). In this present study we have identified, by MALDI mass spectrometry, pp68 as the tyrosine-phosphorylated form of synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP)/NSAP1, a newly discovered cytoplasmic RNA-binding protein. Both SYNCRIP and pp68 were enriched in free polysomes found in low density microsomes isolated from 3T3-L1 adipocytes. In vitro phosphorylation studies revealed that SYNCRIP, once extracted from low density microsomes, can be tyrosine phosphorylated using purified insulin receptor. Binding of RNA to SYNCRIP specifically inhibited its in vitro phosphorylation but had no effect on receptor autophosphorylation or on the ability of the receptor to phosphorylate a model substrate, RCM-lysozyme. These results raise the possibility that regulation of mRNA translation or stability by insulin may involve the phosphorylation of SYNCRIP.     

A protease that increases during a period of enzymic and metabolic adjustment in Tetrahymena.

The specific activity of a neutral protease (assayed at pH 8, using azocasein as substrate) in Tetrahymena doubled or tripled within a few hours after the onset of shaking of statically grown, stationary phase cultures. The increase occurred during a period when several peroxisomal enzymes were decreasing. The increase was prevented by actinomycin D or cycloheximide, both of which also prevented the decrease in peroxisomal enzymes. Protease activity towards hemoglobin at pH 3.6 increases during this period, but to a lesser extent, while activity towards BANA (α-N-benzoyl-d,l-arginine 2-naphthylamide) was almost unchanged. The three protease activities have been partially purified by gel filtration and affinity chromatography, and are indistinguishable on this basis. Chromatography on DEAE-Sephadex yields three peaks having activity towards BANA but not towards hemoglobin and azocasein, and two peaks having activity towards all three substrates. The activities towards azocasein and hemoglobin are also indistinguishable on the basis of sensitivity to a variety of inhibitors, to temperature, and chromatography on CM Sephadex. The partially purified protease has an absolute sulfhydryl requirement when azocasein is used as substrate and is inhibited by leupeptin, chymostatin, TLCK, TPCK, and iodacetamide but not by pepstatin or PMSF. Activity towards BANA is much more susceptible to these inhibitors than is that towards azocasein. About half of the activity towards azocasein sediments with the large particle (40,000g-min) fraction. The distribution between two components of this fraction resembles that of a lysosomal marker. However, the activity did not follow the distribution of marker enzymes of any of the typical cell organelles when either subfraction was centrifuged through a sucrose density gradient, nor did it follow; the distribution pattern of the other two protease activities. Much of the activity, in fact, remained at the top of the gradient, even after repeated washings of the particulate fraction or fractionation in the presence of a membrane-stabilizing agent or a protease inhibitor. The protease or proteases appears to be in part responsible for the rapid loss of enzyme activity that is characteristic of Tetrahymena homogenates. The existence of a protease that can attack cellular enzymes at physiological pH suggests that extralysosomal breakdown of proteins can occur in a eukaryotic cell and may be of importance in the regulation of cellular enzyme levels.     

A genetic element that increases the frequency of gene amplification.

A repetitive mammalian genetic element, HSAG-1, has been shown to promote the amplification of the vector, pSV2-DHFR, containing the functional cDNA for dihydrofolate reductase (DHFR). LR-73 cells, a line of Chinese hamster ovary cells, were transfected with this recombinant construct or with the parent vector, then subjected to culture in selective medium containing steadily increasing concentrations of methotrexate, a drug which specifically inhibits DHFR. Cultures transfected with the HSAG-1-containing construct acquired drug resistance faster than those transfected with the parent vector. This acceleration of acquisition of drug resistance was due to an increased probability of the generation and subsequent selection of cellular variants with increased copy numbers of the vector. The effect has also been observed in CHO(DHFR-) and HeLa cell lines. Possible mechanisms for the effect of the HSAG-1 element on gene amplification are discussed.     

Changes in ornithine transcarbamylase activity and protein level during perinatal period in rat liver. Effects of actinomycin D

Ornithine transcarbamylase activity and immunoreactive enzyme level are compared during perinatal period and in adult rat. Ornithine transcarbamylase activity regularly rises during late fetal period and presents a marked increase 24 hours after birth. Immunoreactive enzyme level does not correlate with this developmental pattern. Ornithine transcarbamylase level increases from 0.06 mg on day 19.5 of pregnancy to 0.417 mg/g liver on day 21.5 and remains constant after birth (0.418 mg/g liver). These results suggest that inactive mitochondrial ornithine transcarbamylase accumulates before birth and that the postnatal increase in enzyme activity is mainly associated with an activation. Furthermore, the paradoxical effect of actinomycin D on ornithine transcarbamylase activity is associated with an increase in enzyme level (about 25%).     

Structural basis of the RNA-binding specificity of human U1A protein.

The RNP domain is a very common eukaryotic protein domain involved in recognition of a wide range of RNA structures and sequences. Two structures of human U1A in complex with distinct RNA substrates have revealed important aspects of RNP-RNA recognition, but have also raised intriguing questions concerning the origin of binding specificity. The beta-sheet of the domain provides an extensive RNA-binding platform for packing aromatic RNA bases and hydrophobic protein side chains. However, many interactions between functional groups on the single-stranded nucleotides and residues on the beta-sheet surface are potentially common to RNP proteins with diverse specificity and therefore make only limited contribution to molecular discrimination. The refined structure of the U1A complex with the RNA polyadenylation inhibition element reported here clarifies the role of the RNP domain principal specificity determinants (the variable loops) in molecular recognition. The most variable region of RNP proteins, loop 3, plays a crucial role in defining the global geometry of the intermolecular interface. Electrostatic interactions with the RNA phosphodiester backbone involve protein side chains that are unique to U1A and are likely to be important for discrimination. This analysis provides a novel picture of RNA-protein recognition, much closer to our current understanding of protein-protein recognition than that of DNA-protein recognition.     

Molecular evolution of the major capsid protein VP1 of enterovirus 70.

Nucleotide sequences of the genome RNA encoding capsid protein VP1 (918 nucleotides) of 18 enterovirus 70 (EV70) isolates collected from various parts of the world in 1971 to 1981 were determined, and nucleotide substitutions among them were studied. The genetic distances between isolates were calculated by the pairwise comparison of nucleotide difference. Regression analysis of the genetic distances against time of isolation of the strains showed that the synonymous substitution rate was very high at 21.53 x 10(-3) substitution per nucleotide per year, while the nonsynonymous rate was extremely low at 0.32 x 10(-3) substitution per nucleotide per year. The rate estimated by the average value of synonymous and nonsynonymous substitutions (W.-H. Li, C.-C. Wu, and C.-C. Luo, Mol. Biol. Evol. 2:150-174, 1985) was 5.00 x 10(-3) substitution per nucleotide per year. Taking the average value of synonymous and nonsynonymous substitutions as genetic distances between isolates, the phylogenetic tree was inferred by the unweighted pairwise grouping method of arithmetic average and by the neighbor-joining method. The tree indicated that the virus had evolved from one focal place, and the time of emergence was estimated to be August 1967 +/- 15 months, 2 years before first recognition of the pandemic of acute hemorrhagic conjunctivitis. By superimposing every nucleotide substitution on the branches of the phylogenetic tree, we analyzed nucleotide substitution patterns of EV70 genome RNA. In synonymous substitutions, the proportion of transitions, i.e., C<==>U and G<==>A, was found to be extremely frequent in comparison with that reported on other viruses or pseudogenes. In addition, parallel substitutions (independent substitutions at the same nucleotide position on different branches, i.e., different isolates, of the tree) were frequently found in both synonymous and nonsynonymous substitutions. These frequent parallel substitutions and the low nonsynonymous substitution rate despite the very high synonymous substitution rate described above imply a strong restriction on nonsynonymous substitution sites of VP1, probably due to the requirement for maintaining the rigid icosahedral conformation of the virus.