Specific protein domains mediate cooperative assembly of HuR oligomers on AU-rich mRNA-destabilizing sequences

The RNA-binding factor HuR is a ubiquitously expressed member of the Hu protein family that binds and stabilizes mRNAs containing AU-rich elements (AREs). Hu proteins share a common domain organization of two tandemly arrayed RNA recognition motifs (RRMs) near the N terminus, followed by a basic hinge domain and a third RRM near the C terminus. In this study, we engineered recombinant wild-type and mutant HuR proteins lacking affinity tags to characterize their ARE-binding properties. Using combinations of electrophoretic mobility shift and fluorescence anisotropy-based binding assays, we show that HuR can bind ARE substrates as small as 13 nucleotides with low nanomolar affinity, but forms cooperative oligomeric protein complexes on ARE substrates of at least 18 nucleotides in length. Analyses of deletion mutant proteins indicated that RRM3 does not contribute to high affinity recognition of ARE substrates, but is required for cooperative assembly of HuR oligomers on RNA. Finally, the hinge domain between RRM2 and RRM3 contributes significant binding energy to HuR.ARE complex formation in an ARE length-dependent manner. The hinge does not enhance RNA-binding activity by increased ion pair formation despite extensive positive charge within this region, and it does not thermodynamically stabilize protein folding. Together, the results define distinct roles for the HuR hinge and RRM3 domains in formation of cooperative HuR.ARE complexes in solution.     

Fasting and glucose effects on pituitary leptin expression: is leptin a local signal for nutrient status?

Leptin, a potent anorexigenic hormone, is found in the anterior pituitary (AP). The aim of this study was to determine whether and how pituitary leptin-bearing cells are regulated by nutritional status. Male rats showed 64% reductions in pituitary leptin mRNA 24 hr after fasting, accompanied by significant (30-50%) reductions in growth hormone (GH), prolactin, and luteinizing hormone (LH), and 70-80% reductions in target cells for gonadotropin-releasing hormone or growth hormone-releasing hormone. There was a 2-fold increase in corticotropes. Subsets (22%) of pituitary cells coexpressed leptin and GH, and <5% coexpressed leptin and LH, prolactin, thyroid-stimulating hormone, or adrenocorticotropic hormone. Fasting resulted in significant (55-75%) losses in cells with leptin proteins or mRNA, and GH or LH. To determine whether restoration of serum glucose could rescue leptin, LH, and GH, additional fasted rats were given 10% glucose water for 24 hr. Restoring serum glucose in fasted rats resulted in pituitary cell populations with normal levels of leptin and GH and LH cells. Similarly, LH and GH cells were restored in vitro after populations from fasted rats were treated for as little as 1 hr in 10-100 pg/ml leptin. These correlative changes in pituitary leptin, LH, and GH, coupled with leptin's rapid restoration of GH and LH in vitro, suggest that pituitary leptin may signal nutritional changes. Collectively, the findings suggest that pituitary leptin expression could be coupled to glucose sensors like glucokinase to facilitate rapid responses by the neuroendocrine system to nutritional cues.     

Mitochondrial protein-import machinery: correlating structure with function

Most mitochondrial proteins are synthesized in the cytosol, translocated into the organelle and directed along specific sorting pathways. Over the past 20 years, >30 proteins have been identified as having key roles in mitochondrial protein import. Recently, the elucidation of the structures of several import components has provided fresh insight into the import process. Here, we review the different pathways involved in sorting proteins into mitochondrial subcompartments. Along the way, we highlight the available structural information about the protein-import machinery and discuss how these structures correlate with previously ascribed functions. Future challenges for the cell biologists will be to use this structural information to test specific hypotheses addressing the molecular mechanisms of mitochondrial protein import.     

Genome-wide analysis of signatures of selection in populations of African honey bees (Apis mellifera) using new web-based tools

Background

With the development of inexpensive, high-throughput sequencing technologies, it has become feasible to examine questions related to population genetics and molecular evolution of non-model species in their ecological contexts on a genome-wide scale. Here, we employed a newly developed suite of integrated, web-based programs to examine population dynamics and signatures of selection across the genome using several well-established tests, including F_ST, pN/pS, and McDonald-Kreitman. We applied these techniques to study populations of honey bees (Apis mellifera) in East Africa. In Kenya, there are several described A. mellifera subspecies, which are thought to be localized to distinct ecological regions.

Results

We performed whole genome sequencing of 11 worker honey bees from apiaries distributed throughout Kenya and identified 3.6 million putative single-nucleotide polymorphisms. The dense coverage allowed us to apply several computational procedures to study population structure and the evolutionary relationships among the populations, and to detect signs of adaptive evolution across the genome. While there is considerable gene flow among the sampled populations, there are clear distinctions between populations from the northern desert region and those from the temperate, savannah region. We identified several genes showing population genetic patterns consistent with positive selection within African bee populations, and between these populations and European A. mellifera or Asian Apis florea.

Conclusions

These results lay the groundwork for future studies of adaptive ecological evolution in honey bees, and demonstrate the use of new, freely available web-based tools and workflows (http://usegalaxy.org/r/kenyanbee) that can be applied to any model system with genomic information.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1712-0) contains supplementary material, which is available to authorized users.     

The peripheral nervous system supports blood cell homing and survival in the Drosophila larva

Interactions of hematopoietic cells with their microenvironment control blood cell colonization, homing and hematopoiesis. Here, we introduce larval hematopoiesis as the first Drosophila model for hematopoietic colonization and the role of the peripheral nervous system (PNS) as a microenvironment in hematopoiesis. The Drosophila larval hematopoietic system is founded by differentiated hemocytes of the embryo, which colonize segmentally repeated epidermal-muscular pockets and proliferate in these locations. Importantly, we show that these resident hemocytes tightly colocalize with peripheral neurons and we demonstrate that larval hemocytes depend on the PNS as an attractive and trophic microenvironment. atonal (ato) mutant or genetically ablated larvae, which are deficient for subsets of peripheral neurons, show a progressive apoptotic decline in hemocytes and an incomplete resident hemocyte pattern, whereas supernumerary peripheral neurons induced by ectopic expression of the proneural gene scute (sc) misdirect hemocytes to these ectopic locations. This PNS-hematopoietic connection in Drosophila parallels the emerging role of the PNS in hematopoiesis and immune functions in vertebrates, and provides the basis for the systematic genetic dissection of the PNS-hematopoietic axis in the future.     

Spatially resolved characterization of biogenic manganese oxide production within a bacterial biofilm

Pseudomonas putida strain MnB1, a biofilm-forming bacterial culture, was used as a model for the study of bacterial Mn oxidation in freshwater and soil environments. The oxidation of aqueous Mn+2 [Mn+2(aq)] by P. putida was characterized by spatially and temporally resolving the oxidation state of Mn in the presence of a bacterial biofilm, using scanning transmission X-ray microscopy (STXM) combined with near-edge X-ray absorption fine structure (NEXAFS) spectroscopy at the Mn L2,3 absorption edges. Subsamples were collected from growth flasks containing 0.1 and 1 mM total Mn at 16, 24, 36, and 48 h after inoculation. Immediately after collection, the unprocessed hydrated subsamples were imaged at a 40-nm resolution. Manganese NEXAFS spectra were extracted from X-ray energy sequences of STXM images (stacks) and fit with linear combinations of well-characterized reference spectra to obtain quantitative relative abundances of Mn(II), Mn(III), and Mn(IV). Careful consideration was given to uncertainty in the normalization of the reference spectra, choice of reference compounds, and chemical changes due to radiation damage. The STXM results confirm that Mn+2(aq) was removed from solution by P. putida and was concentrated as Mn(III) and Mn(IV) immediately adjacent to the bacterial cells. The Mn precipitates were completely enveloped by bacterial biofilm material. The distribution of Mn oxidation states was spatially heterogeneous within and between the clusters of bacterial cells. Scanning transmission X-ray microscopy is a promising tool for advancing the study of hydrated interfaces between minerals and bacteria, particularly in cases where the structure of bacterial biofilms needs to be maintained.     

Synthesis and cellular studies of porphyrin-cobaltacarborane conjugates

The total syntheses of five new porphyrin-cobaltacarborane conjugates (1-5) have been achieved in 88-98% yields in a single-step reaction between a nucleophilic meso-pyridyl-containing porphyrin and zwitterionic cobaltacarborane [3,3'-Co(8-C(4)H(8)O(2)-1,2-C(2)B(9)H(10))(1',2'-C(2)B(9)H(11))]. These unique zwitterionic compounds have one to four cobaltabisdicarbollide anions conjugated to the porphyrin macrocycle via (CH(2)CH(2)O)(2) chains. The X-ray structure of one of these conjugates (1) is presented and discussed. The cellular uptake, cytotoxicity, and subcellular localization of cobaltacarboraneporphyrins 1-5 were investigated in human HEp2 cells. The number and distribution of cobaltacarborane residues linked to the porphyrin macrocycle has a significant effect on the cellular uptake of the conjugates.     

A novel interaction of Cap-binding protein complexes eukaryotic initiation factor (eIF) 4F and eIF(iso)4F with a region in the 3'-untranslated region of satellite tobacco necrosis virus

Satellite tobacco necrosis virus (STNV) RNA is naturally uncapped at its 5' end and lacks polyadenylation at its 3' end. Despite lacking these two hallmarks of eukaryotic mRNAs, STNV-1 RNA is translated very efficiently. A approximately 130-nucleotide translational enhancer (TED), located 3' to the termination codon, is necessary for efficient cap-independent translation of STNV-1 RNA. The STNV-1 TED RNA fragment binds to the eukaryotic cap-binding complexes, initiation factor (eIF) 4F and eIF(iso)4F, as measured by nitrocellulose binding and fluorescence titration. STNV-1 TED is a potent inhibitor of in vitro translation when added in trans. This inhibition is reversed by the addition of eIF4F or eIF(iso)4F, and the subunits of eIF4F and eIF(iso)4F cross-link to STNV-1 TED, providing additional evidence that these factors interact directly with STNV-1 TED. Deletion mutagenesis of the STNV-1 TED indicates that a minimal region of approximately 100 nucleotides is necessary to promote cap-independent translation primarily through interaction with the cap binding subunits (eIF4E or eIF(iso)4E) of eIF4F or eIF(iso)4F.