Calcium/calmodulin-dependent kinases are involved in growth,thermotolerance, oxidative stress survival,and fertility in Neurospora crassa

Calcium/calmodulin-dependent kinases (Ca²⁺/CaMKs) are Ser/Thr protein kinases that respond to change in cytosolic free Ca²⁺ ([Ca²⁺]_c) and play multiple cellular roles in organisms ranging from fungi to humans. In the filamentous fungus Neurospora crassa, four Ca²⁺/CaM-dependent kinases, Ca²⁺/CaMK-1 to 4, are encoded by the genes NCU09123, NCU02283, NCU06177, and NCU09212, respectively. We found that camk-1 and camk-2 are essential for full fertility in N. crassa. The survival of ?camk-2 mutant was increased in induced thermotolerance and oxidative stress conditions. In addition, the ?camk-1 ?camk-2, ?camk-4 ?camk-2, and ?camk-3 ?camk-2 double mutants display slow growth phenotype, reduced aerial hyphae, decreased thermotolerance, and increased sensitivity to oxidative stress, revealing the genetic interactions among these kinases. Therefore, Ca²⁺/CaMKs are involved in growth, thermotolerance, oxidative stress tolerance, and fertility in N. crassa.     

Structural and functional properties of the evolutionarily ancient Y-box family of nucleic acid binding proteins

The Y-box proteins are the most evolutionarily conserved nucleic acid binding proteins yet defined in bacteria, plants and animals. The central nucleic acid binding domain of the vertebrate proteins is 43% identical to a 70-amino-acid-long protein (CS7.4) from E. coli. The structure of this domain consists of an antiparallel fivestranded β-barrel that recognizes both DNA and RNA. The diverse biological roles of these Y-box proteins range from the control of the E. coli cold-shock stress response to the translational masking of messenger RNA in vertebrate gametes. This review discusses the organization of the prokaryotic and eukaryotic Y-box proteins, how they interact with nucleic acids, and their biological roles, both proven and potential.     

Concentration and Localization of Coexpressed ELAV/Hu Proteins Control Specificity of mRNA Processing

Neuronally coexpressed ELAV/Hu proteins comprise a family of highly related RNA binding proteins which bind to very similar cognate sequences. How this redundancy is linked to in vivo function and how gene-specific regulation is achieved have not been clear. Analysis of mutants in Drosophila ELAV/Hu family proteins ELAV, FNE, and RBP9 and of genetic interactions among them indicates that they have mostly independent roles in neuronal development and function but have converging roles in the regulation of synaptic plasticity. Conversely, ELAV, FNE, RBP9, and human HuR bind ELAV target RNA in vitro with similar affinities. Likewise, all can regulate alternative splicing of ELAV target genes in nonneuronal wing disc cells and substitute for ELAV in eye development upon artificially increased expression; they can also substantially restore ELAV''s biological functions when expressed under the control of the elav gene. Furthermore, ELAV-related Sex-lethal can regulate ELAV targets, and ELAV/Hu proteins can interfere with sexual differentiation. An ancient relationship to Sex-lethal is revealed by gonadal expression of RBP9, providing a maternal fail-safe for dosage compensation. Our results indicate that highly related ELAV/Hu RNA binding proteins select targets for mRNA processing through alteration of their expression levels and subcellular localization but only minimally by altered RNA binding specificity.     

Cycloheximide Can Induce Bax/Bak Dependent Myeloid Cell Death Independently of Multiple BH3-Only Proteins

Apoptosis mediated by Bax or Bak is usually thought to be triggered by BH3-only members of the Bcl-2 protein family. BH3-only proteins can directly bind to and activate Bax or Bak, or indirectly activate them by binding to anti-apoptotic Bcl-2 family members, thereby relieving their inhibition of Bax and Bak. Here we describe a third way of activation of Bax/Bak dependent apoptosis that does not require triggering by multiple BH3-only proteins. In factor dependent myeloid (FDM) cell lines, cycloheximide induced apoptosis by a Bax/Bak dependent mechanism, because Bax^-/-Bak^-/- lines were profoundly resistant, whereas FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Addition of cycloheximide led to the rapid loss of Mcl-1 but did not affect the expression of other Bcl-2 family proteins. In support of these findings, similar results were observed by treating FDM cells with the CDK inhibitor, roscovitine. Roscovitine reduced Mcl-1 abundance and caused Bax/Bak dependent cell death, yet FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Therefore Bax/Bak dependent apoptosis can be regulated by the abundance of anti-apoptotic Bcl-2 family members such as Mcl-1, independently of several known BH3-only proteins.     

The Nop5-L7A-fibrillarin RNP complex and a novel box C/D containing sRNA of Halobacterium salinarum NRC-1

RNA 2′O-methylation is a frequent modification of rRNA and tRNA and supposed to influence RNA folding and stability. Ribonucleoprotein (RNP) complexes, containing the proteins Nop5, L7A, fibrillarin, and a box C/D sRNA, are guided for 2′O-methylation by interactions of their RNA component with their target RNA. In vitro complex assembly was analyzed for several thermophilic Archaea but in vivo studies are rare, even unavailable for halophilic Archaea. To analyze the putative box C/D RNP complex in the extremely halophilic Halobacterium salinarum NRC-1 we performed pull-down analysis and identified the proteins Nop5, L7A, and fibrillarin and the tRNA^Trp intron, as a typical box C/D sRNA of this RNP complex in vivo. We show for the first time a ribonucleolytic activity of the purified RNP complex proteins, as well as for the RNP complex containing pull-down fractions. Furthermore, we identified a novel RNA (OE4630R-3′sRNA) as part of the complex, containing the typical boxes C/D and C′/D′ sequence motifs and being twice as abundant as the tRNA^Trp intron.     

Tyrosylprotein Sulfotransferase-2 Expression Is Required for Sulfation of RNase 9 and Mfge8 in Vivo

Protein-tyrosine sulfation is mediated by two Golgi tyrosyl-protein sulfotransferases (TPST-1 and TPST-2) that are widely expressed in vivo. However, the full substrate repertoire of this enzyme system is unknown and thus, our understanding of the biological role(s) of tyrosine sulfation is limited. We reported that whereas Tpst1^-/- male mice have normal fertility, Tpst2^-/- males are infertile despite normal spermatogenesis. However, Tpst2^-/- sperm are severely defective in their motility in viscous media and in their ability to fertilize eggs. These findings suggest that sulfation of unidentified substrate(s) is crucial for normal sperm function. We therefore sought to identify tyrosine-sulfated proteins in the male genital tract using affinity chromatography on PSG2, an anti-sulfotyrosine monoclonal antibody, followed by mass spectrometry. Among the several candidate tyrosine-sulfated proteins identified, RNase 9 and Mfge8 were examined in detail. RNase 9, a catalytically inactive RNase A family member of unknown function, is expressed only in the epididymis after onset of sexual maturity. Mfge8 is expressed on mouse sperm and Mfge8^-/- male mice are subfertile. Metabolic labeling coupled with sulfoamino acid analysis confirmed that both proteins are tyrosine-sulfated and both proteins are expressed at comparable levels in wild type, Tpst1^-/-, and Tpst2^-/- epididymides. However, we demonstrate that RNase 9 and Mfge8 are tyrosine-sulfated in wild type and Tpst1^-/-, but not in Tpst2^-/- mice. These findings suggest that lack of sulfation of one or both of these proteins may contribute mechanistically to the infertility of Tpst2^-/- males.Protein-tyrosine sulfation is a post-translational modification described over 50 years ago (1). Tyrosine-sulfated proteins and/or tyrosylprotein sulfotransferase activity have been described in many species in the plant and animal kingdoms (2, 3). In humans, dozens of tyrosine-sulfated proteins have been identified. These include certain adhesion molecules, G-protein-coupled receptors, coagulation factors, serpins, extracellular matrix proteins, hormones, and others. It has been demonstrated that some of these proteins require tyrosine sulfation for optimal function (3).In mice and humans, protein-tyrosine sulfation is mediated by one of two tyrosylprotein sulfotransferases called TPST-1² and TPST-2 (4–6). Mouse TPST-1 and TPST-2 are 370- and 376-residue type II transmembrane proteins, respectively. Each has a short N-terminal cytoplasmic domain followed by a single ≈17-residue transmembrane domain, a membrane proximal ≈40-residue stem region, and a luminal catalytic domain containing four conserved Cys residues and two N-glycosylation sites. The amino acid sequence of human and mouse TPST-1 are ≈96% identical and human and mouse TPST-2 have a similar degree of identity. TPST-1 is ≈65–67% identical to TPST-2 in both mice and humans. TPST-1 and TPST-2 are broadly expressed in human and murine tissues and cell lines and are co-expressed in most, if not all, cell types (3).A variety of biochemical studies have shown that protein-tyrosine sulfation occurs exclusively in the trans-Golgi network (7, 8). This conclusion has been strengthened by more recent immunofluorescence studies showing that a TPST-1/enhanced green fluorescent protein fusion protein co-localizes with golgin-97, a marker for the trans-Golgi network (9). Thus, protein-tyrosine sulfation occurs only on proteins that transit the secretory pathway and occurs well after protein folding and disulfide formation are complete and after N- and O-linked glycosylation are initiated.To gain an understanding of the biological importance of TPSTs, we have generated TPST-deficient mice by targeted disruption of either the Tpst1 or Tpst2 gene. Our studies of Tpst1^-/- mice revealed unexpected but modest effects on body weight and fecundity (10). Tpst1^-/- mice appear healthy but have ≈5% lower average body weight than wild type mice. Fertility of Tpst1^-/- males and females per se was normal. However, Tpst1^-/- females have smaller litters than wild type females due to embryonic lethality between 8.5 and 15.5 days post coitum.In our studies of Tpst2^-/- mice we found that Tpst2^-/- males were infertile, in contrast to Tpst1^-/- males that have normal fertility (11). We found that Tpst2^-/- males were eugonadal and have normal spermatogenesis. Epididymal sperm from Tpst2^-/- males were normal in number, morphology, and motility and appeared to capacitate in vitro and undergo acrosome exocytosis in response to agonist. However, Tpst2^-/- sperm are severely defective in motility in viscous media and in their ability to fertilize zona pellucida (ZP)-intact eggs. In addition, in vitro fertilization experiments revealed that Tpst2^-/- sperm had reduced ability to adhere to the egg plasma membrane, but were able to undergo membrane fusion with the egg.These findings suggest that tyrosine sulfation of one or more substrates is crucial for normal sperm function. However, there are no proteins directly involved in sperm function that are known to be tyrosine-sulfated. The luteinizing hormone receptor and follicle-stimulating hormone receptor are the only proteins important in reproductive biology that are known to be tyrosine-sulfated. Both receptors have been shown to be sulfated at a membrane proximal site in their respective N-terminal extracellular domains that are conserved in many species including the mouse (12). Sulfation of these receptors has been shown to be required for optimal affinity of their cognate ligands in vitro. However, our observations that serum LH, FSH, and testosterone levels are normal in Tpst2^-/- males coupled with the observation that spermatogenesis is normal excludes defective sulfation of these receptors as an explanation for infertility of Tpst2^-/- males (11).In this study, we sought to identify tyrosine-sulfated proteins expressed in the male genital tract that may provide clues as to the mechanism for the infertility of Tpst2^-/- male mice. Among the several candidate tyrosine-sulfated proteins that were identified, RNase 9 and Mfge8 were of particular interest. RNase 9 is a catalytically inactive RNase A family member of unknown function and is expressed only in the epididymis after onset of sexual maturity (13). Mfge8 is expressed on mouse sperm and Mfge8^-/- male mice have been reported to be subfertile (14). Metabolic labeling coupled with sulfoamino acid analysis confirmed that both proteins are tyrosine-sulfated. We also showed that both proteins are expressed at comparable levels in wild type, Tpst1^-/-, and Tpst2^-/- epididymides, and that RNase 9 and Mfge8 are sulfated in wild type and Tpst1^-/- mice, but not in Tpst2^-/- mice. Therefore, lack of sulfation of one or both of these proteins may contribute mechanistically to the infertility of Tpst2^-/- male mice.     

Isolation of novel pre-mRNA splicing mutants of Schizosaccharomyces pombe

New prp (pre-mRNA processing) mutants of the fission yeast Schizosaccharomyces pombe were isolated from a bank of 700 mutants that were either temperature sensitive (ts^-) or cold sensitive (cs^-) for growth. The bank was screened by Northern blot analysis with probes complementary to S. pombe U6 small nuclear RNA (sn RNA), the gene for which has a splicesomal (mRNA-type) intron. We identified 12 prp mutants that accumulated the U6 snRNA precursor at the nonpermissive temperature. All such mutants were also found to have defects in an early step of TFIID pre-mRNA splicing at the nonpermissive temperature. Complementation analyses showed that seven of the mutants belong to six new complementation groups designated as prp8 and prp10-prp14, whereas the five other mutants were classified into the known complementation groups prp1, prp2 and prp3. Interestingly, some of the isolated prp mutants produced elongated cells at the nonpermissive temperature, which is a phenotype typical of cell division cycle (cdc) mutants. Based on these findings, we propose that some of the wild-type products from these prp ⁺ genes play important roles in the cellular processes of pre-mRNA splicing and cell cycle progression.     

Enrichment for temperature-sensitive and auxotrophic mutants in Saccharomyces cerevisiae by tritium suicide

Tritium suicide is shown to be an efficient technique for mutant enrichment in Saccharomyces cerevisiae. Decays from incorporated [5-³H]uridine and tritiated amino acids proved equally effective in inducing suicide; in cultures labeled to a specific activity of 50 dpm/cell, the viability fell to 2% after 12 days' storage at 4°. Mutagenized cultures were labeled with either [5-³H]uridine or a mixture of tritiated amino acids under conditions where auxotrophic mutants and temperature-sensitive mutants in RNA or protein synthesis would not incorporate a significant amount of the tritiated percursor. When survival fell to 2%, the percentages of both auxotrophic and temperature-sensitive mutants were 10-fold higher among these survivors than in the original mutagenized culture, regardless of the radioactive precursor used.     

Knockout of Multiple Arabidopsis Cation/H+ Exchangers Suggests Isoform-Specific Roles in Metal Stress Response,Germination and Seed Mineral Nutrition

Cation/H⁺ exchangers encoded by CAX genes play an important role in the vacuolar accumulation of metals including Ca²⁺ and Mn²⁺. Arabidopsis thaliana CAX1 and CAX3 have been previously shown to differ phylogenetically from CAX2 but the physiological roles of these different transporters are still unclear. To examine the functions and the potential of redundancy between these three cation transporters, cax1/cax2 and cax2/cax3 double knockout mutants were generated and compared with wild type and cax single knockouts. These double mutants had equivalent metal stress responses to single cax mutants. Both cax1 and cax1/cax2 had increased tolerance to Mg stress, while cax2 and cax2/cax3 both had increased sensitivity to Mn stress. The cax1/cax2 and cax2/cax3 mutants did not exhibit the deleterious developmental phenotypes previously seen with the cax1/cax3 mutant. However, these new double mutants did show alterations in seed germination, specifically a delay in germination time. These alterations correlated with changes in nutrient content within the seeds of the mutants, particularly the cax1/cax2 mutant which had significantly higher seed content of Ca and Mn. This study indicates that the presence of these Arabidopsis CAX transporters is important for normal germination and infers a role for CAX proteins in metal homeostasis within the seed.     

Visualization of the reconstituted FRGY2-mRNA complexes by electron microscopy

Xenopus oocytes store large quantities of translationally dormant mRNA in the cytoplasm as storage messenger ribonucleoprotein particles (mRNPs). The Y-box proteins, mRNP3 and FRGY2/mRNP4, are major RNA binding components of maternal storage mRNPs in oocytes. In this study, we show that the FRGY2 proteins form complexes with mRNA, which leads to mRNA stabilization and translational repression. Visualization of the FRGY2-mRNA complexes by electron microscopy reveals that FRGY2 packages mRNA into a compact RNP. Our results are consistent with a model that the Y-box proteins function in packaging of mRNAs to store them stably for a long time in the oocyte cytoplasm.