Quantitative profiling of in vivo-assembled RNA-protein complexes using a novel integrated proteomic approach

Identification of proteins in RNA-protein complexes is an important step toward understanding regulation of RNA-based processes. Because of the lack of appropriate methodologies, many studies have relied on the creation of in vitro assembled RNA-protein complexes using synthetic RNA and cell extracts. Such complexes may not represent authentic RNPs as they exist in living cells as synthetic RNA may not fold properly and nonspecific RNA-protein interactions can form during cell lysis and purification processes. To circumvent limitations in current approaches, we have developed a novel integrated strategy namely MS2 in vivo biotin tagged RNA affinity purification (MS2-BioTRAP) to capture bona fide in vivo-assembled RNA-protein complexes. In this method, HB-tagged bacteriophage protein MS2 and stem-loop tagged target or control RNAs are co-expressed in cells. The tight association between MS2 and the RNA stem-loop tags allows efficient HB-tag based affinity purification of authentic RNA-protein complexes. Proteins associated with target RNAs are subsequently identified and quantified using SILAC-based quantitative mass spectrometry. Here the 1.2 kb internal ribosome entry site (IRES) from lymphoid enhancer factor-1 mRNA has been used as a proof-of-principle target RNA. An IRES target was chosen because of its importance in protein translation and our limited knowledge of proteins associated with IRES function. With a conventionally translated target RNA as control, 36 IRES binding proteins have been quantitatively identified including known IRES binding factors, novel interacting proteins, translation initiation factors (eIF4A-1, eIF-2A, and eIF3g), and ribosomal subunits with known noncanonical actions (RPS19, RPS7, and RPL26). Validation studies with the small molecule eIF4A-1 inhibitor Hippuristanol shows that translation of endogenous lymphoid enhancer factor-1 mRNA is especially sensitive to eIF4A-1 activity. Our work demonstrates that MS2 in vivo biotin tagged RNA affinity purification is an effective and versatile approach that is generally applicable for other RNA-protein complexes.     

Prolyl Endopeptidase: Inhibition In Vivo by N-Benzyloxycarbonyl-Prolyl-Prolinal

The activity of prolyl endopeptidase in homogenates of mouse tissues was determined 30 min after intraperitoneal injection of N-benzyloxycarbonyl-prolyl-prolinal (1.25 mg/kg), a potent transition state analog inhibitor (K1 = 14 nM) of prolyl endopeptidase (EC 3.4.21.26). A more than 85% decrease of enzyme activity was obtained in all tissues. The in vivo degradation of potential prolyl endopeptidase substrates was studied by following the release of sulfamethoxazole from N-benzyloxycarbonylglycyl-prolyl-sulfamethoxazole, a model synthetic substrate of the enzyme. When this substrate was given intraperitoneally, its enzymatic degradation was blocked after administration of the inhibitor in a dose- and time-dependent manner, indicating inhibition of the enzyme in vivo. Of interest is the long duration of the inhibition. After a relatively low inhibitor dose (5 mg/kg) significant inhibition was seen in most tissues even after 6 h. The brain was particularly sensitive to the effect of the inhibitor. Since prolyl endopeptidase readily degrades many proline-containing neuropeptides, the inhibitor should be of value in studies on the role of the enzyme in neuropeptide metabolism.     

Transcriptional regulation restricting bone sialoprotein gene expression to both hypertrophic chondrocytes and osteoblasts

This study identifies a cis-acting element that confers tissue-restricted expression to the bone sialoprotein (BSP) gene. Using both gain of function and loss-of function studies, we demonstrate that this element acts as a tissue specific enhancer of BSP expression in osteoblasts and hypertrophic chondrocytes but does not function in non-hypertrophic chondrocytes or fibroblasts. Furthermore, our data demonstrate that binding of this element occurs in correlation with active BSP expression. While Dlx5 has been implicated as the tissue-specific regulator of BSP expression through direct DNA binding at an element with homology to the one under study here, our results demonstrate that Dlx5 does not act as a positive regulator of BSP expression. Finally, mutational analyses of this element demonstrate that while there is homology to putative homeodomain binding elements, this site is unlikely to bind homeodomain factors including Dlx5. Thus, these studies identify an important cis-acting element in the BSP promoter that acts as a tissue-specific enhancer of BSP expression in both osteoblasts and hypertrophic chondrocytes. As such this is the first demonstration of a common regulatory mechanism utilized by both chondrocytes and osteoblasts for the tissue-restricted expression of the BSP gene.     

A high-resolution gene expression atlas of epistasis between gene-specific transcription factors exposes potential mechanisms for genetic interactions

Karrikins are a family of compounds produced by wildfires that can stimulate the germination of dormant seeds of plants from numerous families. Seed plants could have ‘discovered’ karrikins during fire-prone times in the Cretaceous period when flowering plants were evolving rapidly. Recent research suggests that karrikins mimic an unidentified endogenous compound that has roles in seed germination and early plant development. The endogenous signalling compound is presumably not only similar to karrikins, but also to the related strigolactone hormones.     

Multiple deficiencies in antioxidant enzymes in mice result in a compound increase in sensitivity to oxidative stress

To examine the effect of compound deficiencies in antioxidant defense, we have generated mice (Sod2^+/−/Gpx1^−/−) that are deficient in Mn superoxide dismutase (MnSOD) and glutathione peroxidase 1 (Gpx1) by breeding Sod2^+/− and Gpx1^−/− mice together. Although Sod2^+/−/Gpx1^−/− mice showed a 50% reduction in MnSOD and no detectable Gpx1 activity in either mitochondria or cytosol in all tissues, they were viable and appeared normal. Fibroblasts isolated from Sod2^+/−/Gpx1^−/− mice were more sensitive (4- to 6-fold) to oxidative stress (t-butyl hydroperoxide or γ irradiation) than fibroblasts from wild-type mice, and were twice as sensitive as cells from Sod2^+/− or Gpx1^−/− mice. Whole-animal studies demonstrated that survival of the Sod2^+/−/Gpx1^−/− mice in response to whole body γ irradiation or paraquat administration was also reduced compared with that of wild-type, Sod2^+/−, or Gpx1^−/− mice. Similarly, endogenous oxidative stress induced by cardiac ischemia/reperfusion injury led to greater apoptosis in heart tissue from the Sod2^+/−/Gpx1^−/− mice than in that from mice deficient in either MnSOD or Gpx1 alone. These data show that Sod2^+/−/Gpx1^−/− mice, deficient in two mitochondrial antioxidant enzymes, have significantly enhanced sensitivity to oxidative stress induced by exogenous insults and to endogenous oxidative stress compared with either wild-type mice or mice deficient in either MnSOD or Gpx1 alone.     

Targeted Deletion of Kynurenine 3-Monooxygenase in Mice: A NEW TOOL FOR STUDYING KYNURENINE PATHWAY METABOLISM IN PERIPHERY AND BRAIN*

Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway (KP) of tryptophan degradation, has been suggested to play a major role in physiological and pathological events involving bioactive KP metabolites. To explore this role in greater detail, we generated mice with a targeted genetic disruption of Kmo and present here the first biochemical and neurochemical characterization of these mutant animals. Kmo^−/− mice lacked KMO activity but showed no obvious abnormalities in the activity of four additional KP enzymes tested. As expected, Kmo^−/− mice showed substantial reductions in the levels of its enzymatic product, 3-hydroxykynurenine, in liver, brain, and plasma. Compared with wild-type animals, the levels of the downstream metabolite quinolinic acid were also greatly decreased in liver and plasma of the mutant mice but surprisingly were only slightly reduced (by ∼20%) in the brain. The levels of three other KP metabolites: kynurenine, kynurenic acid, and anthranilic acid, were substantially, but differentially, elevated in the liver, brain, and plasma of Kmo^−/− mice, whereas the liver and brain content of the major end product of the enzymatic cascade, NAD⁺, did not differ between Kmo^−/− and wild-type animals. When assessed by in vivo microdialysis, extracellular kynurenic acid levels were found to be significantly elevated in the brains of Kmo^−/− mice. Taken together, these results provide further evidence that KMO plays a key regulatory role in the KP and indicate that Kmo^−/− mice will be useful for studying tissue-specific functions of individual KP metabolites in health and disease.     

Pseudomonas marginalis: its degradative capability on organic nitriles and amides

Pseudomonas marginalis, capable of utilizing acetonitrile as the sole source of carbon and nitrogen, was isolated from an industrial waste site. P. marginalis metabolized acetonitrile into ammonia and acetate. The minimal inhibitory concentration values of different nitriles and amides for P. marginalis were in the range 5–300 mM. The bacterium was able to transform high-molecular-mass nitrile compounds and their respective amides into ammonia. The data from substrate-dependent kinetics showed that the K _m and V _max values of P. marginalis for acetonitrile were 33 mM and 67 nmol oxygen consumed min^–1 (ml cell suspension)^–1 respectively. The study with [¹⁴C]acetonitrile indicated that nearly 66% of the carbon was released as ¹⁴CO₂ and 12% was associated with the biomass. The enzyme system involved in the hydrolysis of acetonitrile was shown to be intracellular and inducible. The specific activities of the enzymes nitrile aminohydrolase and amidase were determined in the cell-free extracts of P. marginalis. Both the enzymes could hydrolyze a wide range of nitriles and amides. The present study suggests that the biodegradation of organic nitriles and the bioproduction of organic acids may be achieved with the cells of P. marginalis.     

Spermatophoric reaction reappraised: Novel insights into the functioning of the loliginid spermatophore based on Doryteuthis plei (Mollusca: Cephalopoda)

During copulation, spermatophores produced by male coleoid cephalopods undergo the spermatophoric reaction, a complex process of evagination that culminates in the attachment of the spermatangium (everted spermatophore containing the sperm mass) on the female's body. To better understand this complicated phenomenon, the present study investigated the functional morphology of the spermatophore of the squid Doryteuthis plei applying in vitro analysis of the reaction, as well as light and electron microscopy investigation of spermatangia obtained either in vitro, or naturally attached on females. Hitherto unnoticed functional features of the loliginid spermatophore require a reappraisal of some important processes involved in the spermatophoric reaction. The most striking findings concern the attachment mechanism, which is not carried out solely by cement adhesive material, as previously believed, but rather by an autonomous, complex process performed by multiple structures during the spermatophoric reaction. During evagination, the ejaculatory apparatus provides anchorage on the targeted tissue, presumably due to the minute stellate particles present in the exposed spiral filament. Consequently, the ejaculatory apparatus maintains the attachment of the tip of the evaginating spermatophore until the cement body is extruded. Subsequently, the cement body passes through a complex structural rearrangement, which leads to the injection of both its viscid contents and pointed oral region onto the targeted tissue. The inner membrane at the oral region of the cement body contains numerous stellate particles attached at its inner side; eversion of this membrane exposes these sharp structures, which presumably adhere to the tissue and augment attachment. Several naturally attached spermatangia were found with their bases implanted at the deposition sites, and the possible mechanisms of perforation are discussed based on present evidence. The function of the complex squid spermatophore and its spermatophoric reaction is revisited in light of these findings. J. Morphol. 2012. © 2011 Wiley Periodicals, Inc.     

Form and function of tentacles in pteriomorphian bivalves

Tentacles are remarkable anatomical structures in invertebrates for their diversity of form and function. In bivalves, tentacular organs are commonly associated with protective, secretory, and sensory roles. However, anatomical details are available for only a few species, rendering the diversity and evolution of bivalve tentacles still obscure. In Pteriomorphia, a clade including oysters, scallops, pearl oysters, and relatives, tentacles are abundant and diverse. We investigated tentacle anatomy in the group to understand variation, infer functions, and investigate patterns in tentacle diversity. Six species from four pteriomorphian families (Ostreidae, Pinnidae, Pteriidae, and Spondylidae) were collected and thoroughly investigated with integrative microscopy techniques, including histology, scanning electron microscopy, and confocal microscopy. Tentacles can be classified as middle fold tentacles (MFT) and inner fold tentacles (IFT) according to their position with respect to the folds of the mantle margin. While MFT morphology indicates intense secretion of mucosubstances, no evidence for secretory activity was found for IFT. However, both tentacle types have appropriate ciliary distribution and length to promote mucus transportation for cleaning and lubrication. Protective and sensory functions are discussed based on different lines of evidence, including secretion, cilia distribution, musculature, and innervation. Our results support the homology of MFT and IFT only for Pterioidea and Ostreoidea, considering their morphology, the presence of ciliated receptors at the tips, and branched innervation pattern. This is in accordance with recent phylogenetic hypotheses that support the close relationship between these superfamilies. In contrast, major structural differences indicate that MFT and IFT are probably not homologous across all pteriomorphians. By applying integrative microscopy, we were able to reveal anatomical elements that are essential for the understanding of homology and function when dealing with such superficially similar structures.