A role for the exosome in the in vivo degradation of unstable mRNAs

In mammals, the mRNAs encoding many proteins involved in inflammation bear destabilizing AU-rich elements (AREs) in the 3'-untranslated region. The exosome, a complex of 3' --> 5' exonucleases, is rate limiting in the destruction of such mRNAs in a mammalian in vitro system, but a role in vivo has not been demonstrated. The phenomenon of ARE-mediated degradation also occurs in the protist parasite Trypanosoma brucei. Messenger RNAs with 3'-untranslated region U-rich elements, which strongly resemble AREs, are extremely unstable in the trypanosome form that parasitizes mammals. The first step in degradation of these mRNAs in vivo is rapid destruction of the 3'-untranslated region; subsequently the mRNA is destroyed by exonucleases acting in both 5' --> 3' and 3' --> 5' directions. We here investigated the roles of three subunits of the trypanosome exosome complex, RRP45, RRP4, and CSL4, in this process, depleting the individual subunits in vivo by inducible RNA interference. RRP45 depletion, which probably disrupts exosome integrity, caused a delay in the onset of degradation of the very unstable RNAs, but did not affect degradation of more stable species. Depletion of RRP4 or CSL4 does not affect the stability of the residual exosome and did not change mRNA degradation kinetics. We conclude that the exosome is required for the initiation of rapid degradation of unstable mRNAs in trypanosomes.     

When do parasites fail to speciate in response to host speciation?

Cospeciation generally increases the similarity between host and parasite phylogenies. Incongruence between host and parasite phylogenies has previously been explained in terms of host switching, sorting, and duplication events. Here, we describe an additional process, failure of the parasite to speciate in response to host speciation, that may be important in some host-parasite systems. Failure to speciate is likely to occur when gene flow among parasite populations is much higher than that of their hosts. We reconstructed trees from mitochondrial and nuclear DNA sequences for pigeons and doves (Aves: Columbiformes) and their feather lice in the genus Columbicola (Insecta: Phthiraptera). Although comparisons of the trees from each group revealed a significant amount of cospeciation, there was also a significant degree of incongruence. Cophylogenetic analyses generally indicated that host switching may be an important process in the history of this host-parasite association. Using terminal sister taxon comparisons, we also identified three apparent cases where the host has speciated but the associated parasite has not. In two of these cases of failure to speciate, these comparisons involve allopatric sister taxa of hosts whose lice also occur on hosts sympatric with both of the allopatric sisters. These additional hosts for generalist lice may promote gene flow with lice on the allopatric sister species. Relative rate comparisons for the mitochondrial cytochrome oxidase I gene indicate that molecular substitution occurs about 11 times faster in lice than in their avian hosts.     

Can animals recall the past and plan for the future?

According to the 'mental time travel hypothesis' animals, unlike humans, cannot mentally travel backwards in time to recollect specific past events (episodic memory) or forwards to anticipate future needs (future planning). Until recently, there was little evidence in animals for either ability. Experiments on memory in food-caching birds, however, question this assumption by showing that western scrub-jays form integrated, flexible, trial-unique memories of what they hid, where and when. Moreover, these birds can adjust their caching behaviour in anticipation of future needs. We suggest that some animals have elements of both episodic-like memory and future planning.     

Heart and liver defects and reduced transforming growth factor beta2 sensitivity in transforming growth factor beta type III receptor-deficient embryos

The type III transforming growth factor beta (TGFbeta) receptor (TbetaRIII) binds both TGFbeta and inhibin with high affinity and modulates the association of these ligands with their signaling receptors. However, the significance of TbetaRIII signaling in vivo is not known. In this study, we have sought to determine the role of TbetaRIII during development. We identified the predominant expression sites of TbetaRIII mRNA as liver and heart during midgestation and have disrupted the murine TbetaRIII gene by homologous recombination. Beginning at embryonic day 13.5, mice with mutations in TbetaRIII developed lethal proliferative defects in heart and apoptosis in liver, indicating that TbetaRIII is required during murine somatic development. To assess the effects of the absence of TbetaRIII on the function of its ligands, primary fibroblasts were generated from TbetaRIII-null and wild-type embryos. Our results indicate that TbetaRIII deficiency differentially affects the activities of TGFbeta ligands. Notably, TbetaRIII-null cells exhibited significantly reduced sensitivity to TGFbeta2 in terms of growth inhibition, reporter gene activation, and Smad2 nuclear localization, effects not observed with other ligands. These data indicate that TbetaRIII is an important modulator of TGFbeta2 function in embryonic fibroblasts and that reduced sensitivity to TGFbeta2 may underlie aspects of the TbetaRIII mutant phenotype.     

Novel application of a fibrin cell block method to evaluate melanocytic cell populations

Confirming melanocytic lineage and purity is important for experiments using cultured human melanocytes. The objective of this study was to develop a simple, reliable method to evaluate and archive cultured melanocytic cells. Melanocytes were isolated from adult skin biopsies or from neonatal foreskins using standard culturing methods. Fibrin cell blocks (FCBs) were prepared from cultured cells at passages two and six. Fibrin blocks were paraffin-embedded and sectioned for immunohistochemical (CD68, Melan-A, and HMB-45) and H & E staining. Flow cytometry was performed (Melan-A) at passage six. A mixing experiment with cultured melanocytes and fibroblasts was performed and cell population purity was determined by manual counts of positively staining cells in the FCBs and by flow cytometry. The FCB method of evaluating population purity was validated experimentally and by correlation with flow cytometry results. Preparation of a FCB followed by immunohistochemical staining is an easy and inexpensive way to confirm melanocytic lineage, estimate population purity, and provide a permanent archive of cultured cells.     

The expression level determines the surface distribution of the transferrin receptor in Trypanosoma brucei

The transferrin receptor (TfR) of Trypanosoma brucei is a heterodimer attached to the surface membrane by a glycosylphosphatidylinositol (GPI) anchor. The TfR is restricted to the flagellar pocket, a deep invagination of the plasma membrane. The membrane of the flagellar pocket and the rest of the cell surface are continuous, and the mechanism that selectively retains the TfR in the pocket is unknown. Here, we report that the TfR is retained in the flagellar pocket by a specific and saturable mechanism. In bloodstream-form trypanosomes transfected with the TfR genes, TfR molecules escaped flagellar pocket retention and accumulated on the entire surface, even at modest (threefold) overproduction levels. Similar surface accumulation was observed when the TfR levels were physiologically upregulated threefold when trypanosomes were starved for transferrin. These results suggest that the TfR flagellar pocket retention mechanism is easily saturated and that control of the expression level is critical to maintain the restricted surface distribution of the receptor.     

Multiple roles for the C-terminal domain of eIF5 in translation initiation complex assembly and GTPase activation

eIF5 stimulates the GTPase activity of eIF2 bound to Met-tRNA(i)(Met), and its C-terminal domain (eIF5-CTD) bridges interaction between eIF2 and eIF3/eIF1 in a multifactor complex containing Met-tRNA(i)(Met). The tif5-7A mutation in eIF5-CTD, which destabilizes the multifactor complex in vivo, reduced the binding of Met-tRNA(i)(Met) and mRNA to 40S subunits in vitro. Interestingly, eIF5-CTD bound simultaneously to the eIF4G subunit of the cap-binding complex and the NIP1 subunit of eIF3. These interactions may enhance association of eIF4G with eIF3 to promote mRNA binding to the ribosome. In vivo, tif5-7A eliminated eIF5 as a stable component of the pre-initiation complex and led to accumulation of 48S complexes containing eIF2; thus, conversion of 48S to 80S complexes is the rate-limiting defect in this mutant. We propose that eIF5-CTD stimulates binding of Met-tRNA(i)(Met) and mRNA to 40S subunits through interactions with eIF2, eIF3 and eIF4G; however, its most important function is to anchor eIF5 to other components of the 48S complex in a manner required to couple GTP hydrolysis to AUG recognition during the scanning phase of initiation.     

Mutational analysis of the RNA component of Saccharomyces cerevisiae RNase MRP reveals distinct nuclear phenotypes

The 340-nucleotide RNA component of Saccharomyces cerevisiae RNase MRP is encoded by the single-copy essential gene, NME1. To gain additional insight into the proposed structure and functions of this endoribonuclease, we have extensively mutagenized the NME1 gene and characterized yeast strains expressing mutated forms of the RNA using a gene shuffle technique. Strains expressing each of 26 independent mutations in the RNase MRP RNA gene were characterized for their ability to grow at various temperatures and on various carbon sources, stability of the RNase MRP RNA and processing of the 5.8S rRNA (a nuclear function of RNase MRP). 11 of the mutations resulted in a lethal phenotype, six displayed temperature-conditional lethality, and several preferred a non-fermentable carbon source for growth. In those mutants that exhibited altered growth phenotypes, the severity of the growth defect was directly proportional to the severity of the 5.8S rRNA processing defect in the nucleus. Together this analysis has defined essential regions of the RNase MRP RNA and provides evidence that is consistent with the proposed function of the RNase MRP enzyme.     

Characterization of promoter function and cell-type-specific expression from viral vectors in the nervous system

Viral vectors have become important tools to effectively transfer genes into terminally differentiated cells, including neurons. However, the rational for selection of the promoter for use in viral vectors remains poorly understood. Comparison of promoters has been complicated by the use of different viral backgrounds, transgenes, and target tissues. Adenoviral vectors were constructed in the same vector background to directly compare three viral promoters, the human cytomegalovirus (CMV) immediate-early promoter, the Rous sarcoma virus (RSV) long terminal repeat, and the adenoviral E1A promoter, driving expression of the Escherichia coli lacZ gene or the gene for the enhanced green fluorescent protein. The temporal patterns, levels of expression, and cytotoxicity from the vectors were analyzed. In sensory neuronal cultures, the CMV promoter produced the highest levels of expression, the RSV promoter produced lower levels, and the E1A promoter produced limited expression. There was no evidence of cytotoxicity produced by the viral vectors. In vivo analyses following stereotaxic injection of the vector into the rat hippocampus demonstrated differences in the cell-type-specific expression from the CMV promoter versus the RSV promoter. In acutely prepared hippocampal brain slices, marked differences in the cell type specificity of expression from the promoters were confirmed. The CMV promoter produced expression in hilar regions and pyramidal neurons, with minimal expression in the dentate gyrus. The RSV promoter produced expression in dentate gyrus neurons. These results demonstrate that the selection of the promoter is critical for the success of the viral vector to express a transgene in specific cell types.