Nucleolar localization elements of Xenopus laevis U3 small nucleolar RNA

The Nucleolar Localization Elements (NoLEs) of Xenopus laevis U3 small nucleolar RNA (snoRNA) have been defined. Fluorescein-labeled wild-type U3 snoRNA injected into Xenopus oocyte nuclei localized specifically to nucleoli as shown by fluorescence microscopy. Injection of mutated U3 snoRNA revealed that the 5' region containing Boxes A and A', known to be important for rRNA processing, is not essential for nucleolar localization. Nucleolar localization of U3 snoRNA was independent of the presence and nature of the 5' cap and the terminal stem. In contrast, Boxes C and D, common to the Box C/D snoRNA family, are critical elements for U3 localization. Mutation of the hinge region, Box B, or Box C' led to reduced U3 nucleolar localization. Results of competition experiments suggested that Boxes C and D act in a cooperative manner. It is proposed that Box B facilitates U3 snoRNA nucleolar localization by the primary NoLEs (Boxes C and D), with the hinge region of U3 subsequently base pairing to the external transcribed spacer of pre-rRNA, thus positioning U3 snoRNA for its roles in rRNA processing.     

Multiple G1 regulatory elements control the androgen-dependent proliferation of prostatic carcinoma cells

Prostatic epithelial cells and most primary prostate tumors are dependent on androgen for growth, but how androgen regulates cellular proliferation remains unsolved. Using poorly understood mechanisms, recurrent tumor cells evade the androgen requirement. We utilized androgen-dependent prostatic tumor cells to demonstrate that androgen exerts its effect on the cell cycle by influencing specific aspects of G1-S progression. Androgen depletion of these cells results in early G1 arrest, characterized by reduced cyclin-dependent kinase activity, and underphosphorylated retinoblastoma tumor suppressor protein (RB). The reduction in kinase activity was partially attributed to reduction of specific G1 cyclins and alternate regulation of cyclin-dependent kinase inhibitors. Using this information, we developed a reliable assay to assess the ability of specific G1 regulatory proteins to circumvent these controls and promote androgen-independent growth. As expected, inactivation of RB was required for progression through the cell cycle. Surprisingly, overexpression of G1 cyclins, which drives RB phosphorylation, was insufficient to promote androgen-independent cell cycle progression. Introduction of viral oncoproteins did promote G1-S progression in the absence of androgen, dependent on their ability to sequester RB and related proteins. These results provide the first evidence that multiple elements governing the G1-S transition dictate androgen-dependent growth, and the formation of androgen-independent prostatic tumors may be because of misregulation of these processes.     

Assembly of specific SR protein complexes on distinct regulatory elements of the Drosophila doublesex splicing enhancer

The Drosophila doublesex female-specific splicing enhancer consists of two classes of regulatory elements, six 13-nucleotide repeat sequences, and a single purine-rich element (PRE). Here, we show that the Drosophila regulatory proteins Transformer (Tra) and Transformer 2 (Tra2) recruit different members of the SR family of splicing factors to the repeats and the PRE. The complexes formed on the repeats in HeLa cell extract consist of Tra, Tra2, and the SR protein 9G8. in Drosophila Kc cell extract, Tra and Tra2 recruit the SR protein RBP1 to the repeats. These proteins are arranged in a specific order on the repeats, with the SR protein at the 5' end of each repeat, and Tra2 at each 3' end. Although Tra did not cross-link strongly to the repeats, its presence was essential for the binding of Tra2 to the 3' end of the repeat. Individual SR proteins were also recruited to the PRE by Tra and Tra2, but in this case they were SF2/ASF and dSRp30 in HeLa and Drosophila cell extracts, respectively. The binding of Tra2, Tra, and the specific SR proteins to the repeats or the PRE was highly cooperative within each complex. Thus, Tra2, which contains a single RNA binding domain, can recognize distinct sequences in the repeats and the PRE in conjunction with specific SR proteins. These observations show that the protein composition of each complex is determined by the RNA recognition sequence and specific interactions between SR proteins and Tra and Tra2.     

Identification of an mRNA-binding protein and the specific elements that may mediate the pH-responsive induction of renal glutaminase mRNA

Various segments of the 3'-nontranslated region of the renal glutaminase (GA) mRNA were tested for their ability to enhance turnover and pH responsiveness. The combined effects were retained in the 340-base R-2 segment. However, the combined R-1 and R-3 fragments also imparted a partial destabilization and pH responsiveness to a chimeric beta-globin mRNA. RNA electrophoretic mobility shift assays indicated that cytosolic extracts of rat renal cortex contain a protein that binds to the R-2 and R-3 RNAs. The binding observed with the R-2 RNA was mapped to a direct repeat of an 8-base AU sequence. This binding was effectively competed with an excess of the same RNA, but not by adjacent or unrelated RNAs. UV cross-linking experiments identified a 48-kDa protein that binds to the AU repeats of the R-2 RNA. The apparent binding of this protein was greatly reduced in renal cytosolic extracts prepared from acutely acidotic rats. Two related RNA sequences in the R-3 segment also exhibited specific binding. However, the latter binding was more effectively competed by R-2 RNA than by itself, indicating that the homologous sites may be weaker binding sites for the same 48-kDa protein. Thus, a single protein may bind specifically to multiple instability elements within the 3'-nontranslated region of the GA mRNA and mediate its pH-responsive stabilization.     

Regulation of distinct steps of angiogenesis by different angiogenic molecules

This study was designed to determine the relative activity of basic fibroblast growth factor (bFGF), vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), platelet-derived growth factor (PDGF), platelet-derived endothelial cell growth factor (PD-ECGF), hepatocyte growth factor (HGF), and interleukin-8 (IL-8) in regulating endothelial cell division, migration, degradation of the extracellular matrix (ECM), morphogenesis, and survival. Human umbilical vein endothelial cells (HUVEC) were treated with different concentrations of the six cytokines. bFGF was the most potent mitogen followed by VEGF/VPF and PD-ECGF. VEGF/VPF and bFGF also enhanced the survival of the endothelial cells in serum-free medium. Interstitial collagenase (MMP-1) and urokinase plasminogen activator (uPA) were significantly upregulated only by bFGF. HGF, bFGF, and VEGF/VPF induced chemotactic migration of the endothelial cells, but only HGF (scatter factor) enhanced nondirectional motility. The organization of endothelial cells to form tubes on Matrigel was induced by bFGF and, to a lesser extent, by VEGF/VPF and IL-8. Permeability across endothelial cell monolayers was induced only by VEGF/VPF. These data demonstrate that different angiogenic molecules differentially regulate distinct steps in the process of angiogenesis, suggesting that any given molecule may be necessary but in itself insufficient for establishment of a viable vasculature.     

Demonstration of mRNA editing and localization of guide RNA genes in kinetoplast-mitochondria of the plant trypanosomatid Phytomonas serpens

The adoptive transfer of naive CD4(+) T cell receptor (TCR) transgenic T cells was used to investigate the mechanisms by which the adjuvant lipopolysaccharide (LPS) enhance T cell clonal expansion in vivo. Subcutaneous administration of soluble antigen (Ag) resulted in rapid and transient accumulation of the Ag-specific T cells in the draining lymph nodes (LNs), which was preceded by the production of interleukin (IL)-2. CD28-deficient, Ag-specific T cells produced only small amounts of IL-2 in response to soluble Ag and did not accumulate in the LN to the same extent as wild-type T cells. Injection of Ag and LPS, a natural immunological adjuvant, enhanced IL-2 production and LN accumulation of wild-type, Ag-specific T cells but had no significant effect on CD28-deficient, Ag-specific T cells. Therefore, CD28 is critical for Ag-driven IL-2 production and T cell proliferation in vivo, and is essential for the LPS-mediated enhancement of these events. However, enhancement of IL-2 production could not explain the LPS-dependent increase of T cell accumulation because IL-2-deficient, Ag-specific T cells accumulated to a greater extent in the LN than wild-type T cells in response to Ag plus LPS. These results indicate that adjuvants improve T cell proliferation in vivo via a CD28-dependent signal that can operate in the absence of IL-2.     

Searching for regulatory elements in human noncoding sequences

Injury type and severity among front outboard occupants of passenger vehicles struck in the side by another passenger vehicle and recorded in the United States National Accident Sampling System Crashworthiness Data System were examined in relation to the location of impact, the angle of impact, occupant gender and age, seat belt use, the weight and body style of the side-impacted vehicle, and the weight and body style of the striking vehicle. Elderly occupants were three times as likely as younger occupants in similar crashes to be seriously injured. Serious injuries were also more likely for occupants seated on the struck side and occupants of lightweight passenger vehicles. After accounting for vehicle weight differences, struck-side occupants of cars were still much more likely to be seriously injured than struck-side occupants of light trucks. However, among occupants seated on the side of the vehicle opposite the impact, the likelihood of serious injury was higher for those seated in light trucks.     

Abnormal localization of iron regulatory protein in Alzheimer's disease

A role for altered iron metabolism in the pathogenesis of Alzheimer's disease has been suggested by several reports associating the cardinal neuropathologic lesions with markers of free radical-induced damage and redox-active iron. We hypothesized that the abnormal distribution of iron in Alzheimer brain might result from alterations in iron regulatory proteins (IRP) such as IRP-1 and IRP-2, the main control elements of cellular iron homeostasis. Here, we report that while IRP-1 is present at similar levels in both Alzheimer and control brain tissue, IRP-2 shows striking differences and is associated with intraneuronal lesions, including neurofibrillary tangles, senile plaque neurites and neuropil threads. Since IRP-2 colocalizes with redox-active iron, our results suggest that alterations in IRP-2 might be directly linked to impaired iron homeostasis in Alzheimer's disease.     

Automatic detection of conserved RNA structure elements in complete RNA virus genomes

We propose a new method for detecting conserved RNA secondary structures in a family of related RNA sequences. Our method is based on a combination of thermodynamic structure prediction and phylogenetic comparison. In contrast to purely phylogenetic methods, our algorithm can be used for small data sets of approximately 10 sequences, efficiently exploiting the information contained in the sequence variability. The procedure constructs a prediction only for those parts of sequences that are consistent with a single conserved structure. Our implementation produces reasonable consensus structures without user interference. As an example we have analysed the complete HIV-1 and hepatitis C virus (HCV) genomes as well as the small segment of hantavirus. Our method confirms the known structures in HIV-1 and predicts previously unknown conserved RNA secondary structures in HCV.     

Heparin inhibits acetylcholine receptor aggregation at two distinct steps in the agrin-induced pathway

Muscle cells depend on motoneurons for the initiation of postsynaptic differentiation during early development of the neuromuscular junction. Motoneurons secrete specific isoforms of the extracellular matrix protein agrin which trigger the aggregation of acetylcholine receptors (AChRs) on the muscle surface. Both motoneuron- and agrin-induced AChR aggregation are inhibited by heparin. Here we show that this inhibition is due to two separate and distinguishable mechanisms. At high concentrations, heparin directly binds to agrin isoforms which contain the peptide KSRK, resulting in a virtually complete inhibition of AChR clustering. Heparin and other polyanions do not bind to agrin splicing variants without KSRK insert. Isoforms containing or lacking the KSRK insert have a high potency to induce AChR aggregation in the presence of an activating eight-amino-acid insert. This activity is inhibited by low concentrations of heparin even in the absence of any binding of heparin to agrin. Therefore, this second type of inhibition is due to the interaction of heparin with a downstream component of the agrin-induced clustering pathway. Binding of heparin to this yet unidentified component substantially decreases, but does not completely abolish AChR aggregation. The inhibition is particularly strong on myotubes which have not completely matured in culture.     

Lack of Drosophila cytoskeletal tropomyosin affects head morphogenesis and the accumulation of oskar mRNA required for germ cell formation

Drosophila encodes five muscle and one cytoskeletal isoform of the actin-binding protein tropomyosin. We have identified a lack-of-function mutation in the cytoskeletal isoform (cTmII). Zygotic mutant embryos show a defect in head morphogenesis, while embryos lacking maternal cTmII are defective in germ cell formation but otherwise give rise to viable adults. oskar mRNA, which is required for both germ cell formation and abdominal segmentation, fails to accumulate at the posterior pole in these embryos. nanos mRNA, however, which is required exclusively for abdominal segmentation, is localized at wild-type levels. These results indicate that head morphogenesis and the accumulation of high levels of oskar mRNA necessary for germ cell formation require tropomyosin-dependent cytoskeleton.     

Different core promoters possess distinct regulatory activities in the Drosophila embryo

There are numerous examples of shared enhancers interacting with just a subset of target promoters. In some cases, specific enhancer-promoter interactions depend on promoter competition, whereby the activation of a preferred target promoter precludes expression of linked genes. Here, we employ a transgenic embryo assay to obtain evidence that promoter selection is influenced by the TATA element. Both the AE1 enhancer from the Drosophila Antennapedia gene complex (ANT-C) and the IAB5 enhancer from the Bithorax complex (BX-C) preferentially activate TATA-containing promoters when challenged with linked TATA-less promoters. In contrast, the rho neuroectoderm enhancer (NEE) does not discriminate between these two classes of promoters. Thus, certain upstream activators, such as Ftz, prefer TATA-containing promoters, whereas other activators, including Dorsal, work equally well on both classes of promoters. These results provide in vivo evidence that different core promoters possess distinct regulatory activities. We discuss the possibility that an invariant TFIID complex can adopt different conformations on the core promoter.