Gemin5 Binds to the Survival Motor Neuron mRNA to Regulate SMN Expression

Reduced expression of SMN causes spinal muscular atrophy, a severe neurodegenerative disease. Despite the importance of maintaining SMN levels, relatively little is known about the mechanisms by which SMN levels are regulated. We show here that Gemin5, the snRNA-binding protein of the SMN complex, binds directly to the SMN mRNA and regulates SMN expression. Gemin5 binds with high specificity, both in vitro and in vivo, to sequence and structural elements in the SMN mRNA 3′-untranslated region that are reminiscent of the snRNP code to which Gemin5 binds on snRNAs. Reduction of Gemin5 redistributes the SMN mRNA from heavy polysomes to lighter polysomes and monosomes, suggesting that Gemin5 functions as an activator of SMN translation. SMN protein is not stoichiometrically present on the SMN mRNA with Gemin5, but the mRNA-binding activity of Gemin5 is dependent on SMN levels, providing a feedback mechanism for SMN to regulate its own expression via Gemin5. This work both reveals a new autoregulatory pathway governing SMN expression, and identifies a new mechanism through which SMN can modulate specific mRNA expression via Gemin5.     

Genomic correlates of recombination rate and its variability across eight recombination maps in the western honey bee (Apis mellifera L.)

Background

Meiotic recombination has traditionally been explained based on the structural requirement to stabilize homologous chromosome pairs to ensure their proper meiotic segregation. Competing hypotheses seek to explain the emerging findings of significant heterogeneity in recombination rates within and between genomes, but intraspecific comparisons of genome-wide recombination patterns are rare. The honey bee (Apis mellifera) exhibits the highest rate of genomic recombination among multicellular animals with about five cross-over events per chromatid.

Results

Here, we present a comparative analysis of recombination rates across eight genetic linkage maps of the honey bee genome to investigate which genomic sequence features are correlated with recombination rate and with its variation across the eight data sets, ranging in average marker spacing ranging from 1 Mbp to 120 kbp. Overall, we found that GC content explained best the variation in local recombination rate along chromosomes at the analyzed 100 kbp scale. In contrast, variation among the different maps was correlated to the abundance of microsatellites and several specific tri- and tetra-nucleotides.

Conclusions

The combined evidence from eight medium-scale recombination maps of the honey bee genome suggests that recombination rate variation in this highly recombining genome might be due to the DNA configuration instead of distinct sequence motifs. However, more fine-scale analyses are needed. The empirical basis of eight differing genetic maps allowed for robust conclusions about the correlates of the local recombination rates and enabled the study of the relation between DNA features and variability in local recombination rates, which is particularly relevant in the honey bee genome with its exceptionally high recombination rate.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1281-2) contains supplementary material, which is available to authorized users.     

The arcuate nucleus and neuropeptide Y contribute to the antitumorigenic effect of calorie restriction

Calorie restriction (CR) is known to have profound effects on tumor incidence. A typical consequence of CR is hunger, and we hypothesized that the neuroendocrine response to CR might in part mediate CR's antitumor effects. We tested CR under appetite suppression using two models: neuropeptide Y (NPY) knockout mice and monosodium glutamate-injected mice. While CR was protective in control mice challenged with a two-stage skin carcinogenesis model, papilloma development was neither delayed nor reduced by CR in the monosodium glutamate-treated and NPY knockout mice. Adiponectin levels were also not increased by CR in the appetite-suppressed mice. We propose that some of CR's beneficial effects cannot be separated from those imposed on appetite, and that NPY neurons in the arcuate nucleus of the hypothalamus are involved in the translation of reduced intake to downstream physiological and functional benefits.     

Identification of adult mineralized tissue zebrafish mutants

Zebrafish craniofacial, skeletal, and tooth development closely resembles that of higher vertebrates. Our goal is to identify viable adult zebrafish mutants that can be used as models for human mineralized craniofacial, dental, and skeletal system disorders. We used a large-scale forward-genetic chemical N-ethyl-nitroso-urea mutagenesis screen to identify 17 early lethal homozygous recessive mutants with defects in craniofacial cartilage elements, and 7 adult homozygous recessive mutants with mineralized tissue phenotypes including craniofacial shape defects, fused sutures, dysmorphic or missing skeletal elements, scoliosis, and neural arch defects. One mutant displayed both an early lethal homozygous phenotype and an adult heterozygous phenotype. These results extend the utility of the zebrafish model beyond the embryo to study human bone and cartilage disorders.     

Branch point identification and sequence requirements for intron splicing in Plasmodium falciparum

Splicing of mRNA is an ancient and evolutionarily conserved process in eukaryotic organisms, but intron-exon structures vary. Plasmodium falciparum has an extreme AT nucleotide bias (>80%), providing a unique opportunity to investigate how evolutionary forces have acted on intron structures. In this study, we developed an in vivo luciferase reporter splicing assay and employed it in combination with lariat isolation and sequencing to characterize 5' and 3' splicing requirements and experimentally determine the intron branch point in P. falciparum. This analysis indicates that P. falciparum mRNAs have canonical 5' and 3' splice sites. However, the 5' consensus motif is weakly conserved and tolerates nucleotide substitution, including the fifth nucleotide in the intron, which is more typically a G nucleotide in most eukaryotes. In comparison, the 3' splice site has a strong eukaryotic consensus sequence and adjacent polypyrimidine tract. In four different P. falciparum pre-mRNAs, multiple branch points per intron were detected, with some at U instead of the typical A residue. A weak branch point consensus was detected among 18 identified branch points. This analysis indicates that P. falciparum retains many consensus eukaryotic splice site features, despite having an extreme codon bias, and possesses flexibility in branch point nucleophilic attack.     

Activation of human macrophages by bacterial components relieves the restriction on replication of an interferon-inducing parainfluenza virus 5 (PIV5) P/V mutant

Macrophages regulate immune responses during many viral infections, and can be a major determinant of pathogenesis, virus replication and immune response to infection. Here, we have addressed the question of the outcome of infection of primary human macrophages with parainfluenza virus 5 (PIV5) and a PIV5 mutant (P/V-CPI-) that is unable to counteract interferon (IFN) responses. In cultures of na?ve monocyte-derived macrophages (MDMs), WT PIV5 established a highly productive infection, whereas the P/V-CPI- mutant was restricted for replication in MDMs by IFN-beta. Restricted replication in vitro was relieved in MDM that had been activated by prior exposure to heat killed Gram positive bacteria, including Listeria monocytogenes, Streptococcus pyogenes, and Bacillus anthracis. Enhanced replication of the P/V mutant in MDM previously activated by bacterial components correlated with a reduced ability to produce IFN-beta in response to virus infection, whereas IFN signaling was intact. Activated MDM were found to upregulate the synthesis of IRAK-M, which has been previously shown to negatively regulate factors involved in TLR signaling and IFN-beta production. We discuss these results in terms of the implications for mixed bacteria-virus infections and for the use of live RNA virus vectors that have been engineered to be attenuated for IFN sensitivity.     

Natural variation in decision-making behavior in Drosophila melanogaster

There has been considerable recent interest in using Drosophila melanogaster to investigate the molecular basis of decision-making behavior. Deciding where to place eggs is likely one of the most important decisions for a female fly, as eggs are vulnerable and larvae have limited motility. Here, we show that many natural genotypes of D. melanogaster prefer to lay eggs near nutritious substrate, rather than in nutritious substrate. These preferences are highly polymorphic in both degree and direction, with considerable heritability (0.488) and evolvability.Relative preferences are modulated by the distance between options and the overall concentration of ethanol, suggesting Drosophila integrate many environmental factors when making oviposition decisions. As oviposition-related decisions can be efficiently assessed by simply counting eggs, oviposition behavior is an excellent model for understanding information processing in insects. Associating natural genetic polymorphisms with decision-making variation will shed light on the molecular basis of host choice behavior, the evolutionary maintenance of genetic variation, and the mechanistic nature of preference variation in general.     

Formulating a fluorogenic assay to evaluate S-adenosyl-L-methionine analogues as protein methyltransferase cofactors

Protein methyltransferases (PMTs) catalyze arginine and lysine methylation of diverse histone and nonhistone targets. These posttranslational modifications play essential roles in regulating multiple cellular events in an epigenetic manner. In the recent process of defining PMT targets, S-adenosyl-L-methionine (SAM) analogues have emerged as powerful small molecule probes to label and profile PMT targets. To examine efficiently the reactivity of PMTs and their variants on SAM analogues, we transformed a fluorogenic PMT assay into a ready high throughput screening (HTS) format. The reformulated fluorogenic assay is featured by its uncoupled but more robust character with the first step of accumulation of the commonly-shared reaction byproduct S-adenosyl-L-homocysteine (SAH), followed by SAH-hydrolase-mediated fluorogenic quantification. The HTS readiness and robustness of the assay were demonstrated by its excellent Z' values of 0.83-0.95 for the so-far-examined 8 human PMTs with SAM as a cofactor (PRMT1, PRMT3, CARM1, SUV39H2, SET7/9, SET8, G9a and GLP1). The fluorogenic assay was further implemented to screen the PMTs against five SAM analogues (allyl-SAM, propargyl-SAM, (E)-pent-2-en-4-ynyl-SAM (EnYn-SAM), (E)-hex-2-en-5-ynyl-SAM (Hey-SAM) and 4-propargyloxy-but-2-enyl-SAM (Pob-SAM)). Among the examined 8 × 5 pairs of PMTs and SAM analogues, native SUV39H2, G9a and GLP1 showed promiscuous activity on allyl-SAM. In contrast, the bulky SAM analogues, such as EnYn-SAM, Hey-SAM and Pob-SAM, are inert toward the panel of human PMTs. These findings therefore provide the useful structure-activity guidance to further evolve PMTs and SAM analogues for substrate labeling. The current assay format is ready to screen methyltransferase variants on structurally-diverse SAM analogues.