Regulation of Alternative Polyadenylation by U1 snRNPs and SRp20

Although considerable information is currently available about the factors involved in constitutive vertebrate polyadenylation, the factors and mechanisms involved in facilitating communication between polyadenylation and splicing are largely unknown. Even less is known about the regulation of polyadenylation in genes in which 3′-terminal exons are alternatively recognized. Here we demonstrate that an SR protein, SRp20, affects recognition of an alternative 3′-terminal exon via an effect on the efficiency of binding of a polyadenylation factor to an alternative polyadenylation site. The gene under study codes for the peptides calcitonin and calcitonin gene-related peptide. Its pre-mRNA is alternatively processed by the tissue-specific inclusion or exclusion of an embedded 3′-terminal exon, exon 4, via factors binding to an intronic enhancer element that contains both 3′ and 5′ splice site consensus sequence elements. In cell types that preferentially exclude exon 4, addition of wild-type SRp20 enhances exon 4 inclusion via recognition of the intronic enhancer. In contrast, in cell types that preferentially include exon 4, addition of a mutant form of SRp20 containing the RNA-binding domain but missing the SR domain inhibits exon 4 inclusion. Inhibition is likely at the level of polyadenylation, because the mutant SRp20 inhibits binding of CstF to the exon 4 poly(A) site. This is the first demonstration that an SR protein can influence alternative polyadenylation and suggests that this family of proteins may play a role in recognition of 3′-terminal exons and perhaps in the communication between polyadenylation and splicing.     

Bacterial Contamination of Drinking Water Supplies in a Modern Rural Neighborhood

On six occasions during a 15-month period, the private well and spring water supplies in a modern rural neighborhood of 78 households were examined for total coliforms, fecal coliforms, Staphylococcus aureus, and standard plate count bacteria. More than one-third of the water supplies were unsatisfactory on at least one occasion as judged by standard plate counts over 10³/ml and the presence of coliforms, fecal coliforms, and/or S. aureus. Citrobacter freundii, Klebsiella pneumoniae, and Escherichia coli were the most frequently isolated total coliforms. At least 12 other genera of bacteria were identified from standard plate count agar. Coliform contamination was found to be higher after periods of rainfall, and high standard plate counts were more prevalent during warmer weather. These observations probably reflect leakage of surface water into improperly sealed wells or aquifer contamination during winter and the lack of chlorination to control microbial regrowth during the warm season. An inverse correlation was found between the presence of high standard plate counts and incidence of coliforms. Consumer education and at least a twice yearly monitoring of private water supplies (winter and summer) are suggested methods to signal that treatment may be necessary to reduce the risk of waterborne disease.     

Crag Is a GEF for Rab11 Required for Rhodopsin Trafficking and Maintenance of Adult Photoreceptor Cells

Rhodopsins (Rhs) are light sensors, and Rh1 is the major Rh in the Drosophila photoreceptor rhabdomere membrane. Upon photoactivation, a fraction of Rh1 is internalized and degraded, but it remains unclear how the rhabdomeric Rh1 pool is replenished and what molecular players are involved. Here, we show that Crag, a DENN protein, is a guanine nucleotide exchange factor for Rab11 that is required for the homeostasis of Rh1 upon light exposure. The absence of Crag causes a light-induced accumulation of cytoplasmic Rh1, and loss of Crag or Rab11 leads to a similar photoreceptor degeneration in adult flies. Furthermore, the defects associated with loss of Crag can be partially rescued with a constitutive active form of Rab11. We propose that upon light stimulation, Crag is required for trafficking of Rh from the trans-Golgi network to rhabdomere membranes via a Rab11-dependent vesicular transport.     

Remodeling of an identified motoneuron during metamorphosis: central and peripheral actions of ecdysteroids during regression of dendrites and motor terminals

During metamorphosis of the moth Manduca sexta, an identified leg motoneuron, the femoral depressor motoneuron (FeDe MN), undergoes reorganization of its central and peripheral processes. This remodeling is under the control of two insect hormones: the ecdysteroids and juvenile hormone (JH). Here, we asked whether peripheral or central actions of the ecdysteroids influenced specific regressive aspects of MN remodeling. We used stable hormonal mimics to manipulate the hormonal environment of either the FeDe muscle or the FeDe MN soma. Our results demonstrate that motor-terminal retraction and dendritic regression can be experimentally uncoupled, indicating that central actions of ecdysteroids trigger dendritic regression whereas peripheral actions trigger terminal retraction. Our results further demonstrate that discrete aspects of motor-terminal retraction can also be experimentally uncoupled, suggesting that they also are regulated differently.     

Distal recognition site for classical pathway convertase located in the C345C/netrin module of complement component C5

Previous studies focused on indels in the complement C345 protein family identified a number of potential protein-protein interaction sites in components C3 and C5. Here, one of these sites in C5, near the alpha-chain C terminus, was examined by alanine-scanning mutagenesis at 16 of the 18 non-alanine residues in the sequence KEALQIKYNFSF RYIYPLD. Alanine substitutions affected activities in the highly variable manner characteristic of binding sites. Substitutions at the lysine or either phenylalanine residue in the central KYNFSF sequence had the greatest effects, yielding mutants with <20% of the normal activity. These three mutants were also resistant to the classical pathway (CP) C5 convertase, with sensitivities roughly proportional to their hemolytic activities, but had normal susceptibilities to the cobra venom factor (CVF)-dependent convertase. Synthetic peptide MGKEALQIKYNFS-NH2 was found similarly to inhibit CP but not CVF convertase activation, and the effects of alanine substitutions in this peptide largely reflected those of the equivalent mutations in C5. These results indicate that residues KYNFSF form a novel, distal binding site for the CP, but not CVF convertase. This site lies approximately 880 residues downstream of the convertase cleavage site within a module that has been independently named C345C and NTR; this module is found in diverse proteins including netrins and tissue inhibitors of metalloproteinases.     

swinger: a user‐friendly computer program to establish captive breeding groups that minimize relatedness without pedigree information

Captive breeding programmes are often a necessity for the continued persistence of a population or species. They typically have the goal of maintaining genetic diversity and minimizing inbreeding. However, most captive breeding programmes have been based on the assumption that the founding breeders are unrelated and outbred, even though in situ anthropogenic impacts often mean these founders may have high relatedness and substantial inbreeding. In addition, polygamous group‐breeding species in captivity often have uncertain pedigrees, making it difficult to select the group composition for subsequent breeding. Molecular‐based estimates of relatedness and inbreeding may instead be used to select breeding groups (≥two individuals) that minimize relatedness and filter out inbred individuals. swinger constructs breeding groups based on molecular estimates of relatedness and inbreeding. The number of possible combinations of breeding groups quickly becomes intractable by hand. swinger was designed to overcome this major issue in ex situ conservation biology. The user can specify parameters within swinger to reach breeding solutions that suit the mating system of the target species and available resources. We provide evidence of the efficiency of the software with an empirical example and using simulations. The only data required are a typical molecular marker data set, such as a microsatellite or SNP data set, from which estimates of inbreeding and pairwise relatedness may be obtained. Such molecular data sets are becoming easier to gather from non‐model organisms with next‐generation sequencing technology. swinger is an open‐source software with a user‐friendly interface and is available at http://www.molecularecology.flinders.edu.au/molecular-ecology-lab/software/swinger/swinger/ and https://github.com/Yuma248/Swinger .     

Functional and crystal structure analysis of active site adaptations of a potent anti-angiogenic human tRNA synthetase

Higher eukaryote tRNA synthetases have expanded functions that come from enlarged, more differentiated structures that were adapted to fit aminoacylation function. How those adaptations affect catalytic mechanisms is not known. Presented here is the structure of a catalytically active natural splice variant of human tryptophanyl-tRNA synthetase (TrpRS) that is a potent angiostatic factor. This and related structures suggest that a eukaryote-specific N-terminal extension of the core enzyme changed substrate recognition by forming an active site cap. At the junction of the extension and core catalytic unit, an arginine is recruited to replace a missing landmark lysine almost 200 residues away. Mutagenesis, rapid kinetic, and substrate binding studies support the functional significance of the cap and arginine recruitment. Thus, the enzyme function of human TrpRS has switched more to the N terminus of the sequence. This switch has the effect of creating selective pressure to retain the N-terminal extension for functional expansion.     

<Emphasis Type="Italic">Marcetia candolleana</Emphasis> (<Emphasis Type="Italic">Melastomeae</Emphasis> – <Emphasis Type="Italic">Melastomataceae</Emphasis>), a new species from Bahia (Brazil)

Summary   Marcetia candolleana A. K. A. Santos & A. B. Martins, is apparently restricted to Mucugê, Bahia (Brazil), where it occurs in areas of campo rupestre vegetation. This new species is closely related to the sympatric M. mucugensis Wurdack, but can be easily recognised by its semi-prostate to procumbent habit, reddish glandular-hirsute indument, loose and flexuous branches, leaves with inconspicuous reticulation on the abaxial surface, connectives very shortly prolonged below the thecae, style curved towards the apex, not exceeding the anthers, and pendulous fruit.

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Resumo   Marcetia candolleana A. K. A. Santos & A. B. Martins, é aparentemente restrita a Mucugê, Bahia (Brasil), onde ocorre em áreas de campo rupestre. Esta nova espécie é proximamente relacionada à M. mucugensis Wurdack, mas pode ser facilmente reconhecida por seu hábito semi-prostado a procumbente, indumento glandular-hirsuto, vináceo, ramos flexuosos, folhas inconspicuamente reticuladas na face abaxial, conectivos muito curtamente prolongados abaixo das tecas, estilete curvo no ápice, n?o ultrapassando o comprimento das anteras, e fruto pêndulo.

     

Jelly belly trans‐synaptic signaling to anaplastic lymphoma kinase regulates neurotransmission strength and synapse architecture

In Drosophila, the secreted signaling molecule Jelly Belly (Jeb) activates anaplastic lymphoma kinase (Alk), a receptor tyrosine kinase, in multiple developmental and adult contexts. We have shown previously that Jeb and Alk are highly enriched at Drosophila synapses within the CNS neuropil and neuromuscular junction (NMJ) and postulated a conserved intercellular signaling function. At the embryonic and larval NMJ, Jeb is localized in the motor neuron presynaptic terminal whereas Alk is concentrated in the muscle postsynaptic domain surrounding boutons, consistent with anterograde trans‐synaptic signaling. Here, we show that neurotransmission is regulated by Jeb secretion by functional inhibition of Jeb–Alk signaling. Jeb is a novel negative regulator of neuromuscular transmission. Reduction or inhibition of Alk function results in enhanced synaptic transmission. Activation of Alk conversely inhibits synaptic transmission. Restoration of wild‐type postsynaptic Alk expression in Alk partial loss‐of‐function mutants rescues NMJ transmission phenotypes and confirms that postsynaptic Alk regulates NMJ transmission. The effects of impaired Alk signaling on neurotransmission are observed in the absence of associated changes in NMJ structure. Complete removal of Jeb in motor neurons, however, disrupts both presynaptic bouton architecture and postsynaptic differentiation. Nonphysiologic activation of Alk signaling also negatively regulates NMJ growth. Activation of Jeb–Alk signaling triggers the Ras‐MAP kinase cascade in both pre‐ and postsynaptic compartments. These novel roles for Jeb–Alk signaling in the modulation of synaptic function and structure have potential implications for recently reported Alk functions in human addiction, retention of spatial memory, cognitive dysfunction in neurofibromatosis, and pathogenesis of amyotrophic lateral sclerosis. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013