RNA degradation is required for the germ-cell to maternal transition in Drosophila.

In sexually reproducing animals, the oocyte contributes a large supply of RNAs that are essential to launch development upon fertilization. The mechanisms that regulate the composition of the maternal RNA contribution during oogenesis are unclear. Here, we show that a subset of RNAs expressed during the early stages of oogenesis is subjected to regulated degradation during oocyte specification. Failure to remove these RNAs results in oocyte dysfunction and death. We identify the RNA-degrading Super Killer complex and No-Go Decay factor Pelota as key regulators of oogenesis via targeted degradation of specific RNAs expressed in undifferentiated germ cells. These regulators target RNAs enriched for cytidine sequences that are bound by the polypyrimidine tract binding protein Half pint. Thus, RNA degradation helps orchestrate a germ cell-to-maternal transition that gives rise to the maternal contribution to the zygote.

Drug Discovery Insights from Medicinal Beetles in Traditional Chinese Medicine.

Traditional Chinese medicine (TCM) was the primary source of medical treatment for the people inhabiting East Asia for thousands of years. These ancient practices have incorporated a wide variety of materia medica including plants, animals and minerals. As modern sciences, including natural products chemistry, emerged, there became increasing efforts to explore the chemistry of this materia medica to find molecules responsible for their traditional use. Insects, including beetles have played an important role in TCM. In our survey of texts and review articles on TCM materia medica, we found 48 species of beetles from 34 genera in 14 different families that are used in TCM. This review covers the chemistry known from the beetles used in TCM, or in cases where a species used in these practices has not been chemically studied, we discuss the chemistry of closely related beetles. We also found several documented uses of beetles in Traditional Korean Medicine (TKM), and included them where appropriate. There are 129 chemical constituents of beetles discussed.

Plant-derived isoquinoline alkaloids that target ergosterol biosynthesis discovered by using a novel antifungal screening tool.

The ergosterol pathway is a prime antifungal target as it is required for fungal survival, yet is not involved in human homeostasis. Methods to study the ergosterol pathway, however, are often time-consuming. The minimum inhibitory concentration (MIC) assay is a simple research tool that determines the lowest concentration at which a novel antimicrobial is active in vitro with limited scope to determine the mechanism of action for a drug. In this study, we show that by adding hydrogen peroxide, an oxidative stressor, or glutathione (GSH), an antioxidant, to modify a commonly performed MIC assay allowed us to screen selectively for new antifungal drugs that target ergosterol biosynthesis in fungi. A human pathogen and dermatophyte, Microsporum gypseum, was used as a test organism. When exposed to ergosterol targeting drugs, the hydrogen peroxide treatment significantly decreased fungal survival by reducing ergosterol in the cell wall, whereas GSH increased survival of M. gypseum. Further, by performing a series of experiments with M. gypseum and Trichophyton rubrum, it was determined that the oxidative stress from hydrogen peroxide causes cell death at different developmental stages based on fungal species. These findings allow us to describe a simple, high-throughput method for simultaneously screening new antifungal drugs for activity and effects on the ergosterol pathway. By using this tool, two isoquinoline alkaloids were discovered to be potent inhibitors of ergosterol biosynthesis in vitro by reducing the amount of ergosterol without affecting the expression of 1,3-ß-glucan. Both compounds also significantly reduced the severity of acanthosis, hyperkeratosis, spongiosis and dermal edema in vivo.

Tunable Transcriptional Interference at the Endogenous Alcohol Dehydrogenase Gene Locus in Drosophila melanogaster.

Neighboring sequences of a gene can influence its expression. In the phenomenon known as transcriptional interference, transcription at one region in the genome can repress transcription at a nearby region in cis Transcriptional interference occurs at a number of eukaryotic loci, including the alcohol dehydrogenase (Adh) gene in Drosophila melanogasterAdh is regulated by two promoters, which are distinct in their developmental timing of activation. It has been shown using transgene insertion that when the promoter distal from the Adh start codon is deleted, transcription from the proximal promoter becomes de-regulated. As a result, the Adh proximal promoter, which is normally active only during the early larval stages, becomes abnormally activated in adults. Whether this type of regulation occurs in the endogenous Adh context, however, remains unclear. Here, we employed the CRISPR/Cas9 system to edit the endogenous Adh locus and found that removal of the distal promoter also resulted in the untimely expression of the proximal promoter-driven mRNA isoform in adults, albeit at lower levels than previously reported. Importantly, transcription from the distal promoter was sufficient to repress proximal transcription in larvae, and the degree of this repression was dependent on the degree of distal promoter activity. Finally, upregulation of the distal Adh transcript led to the enrichment of histone 3 lysine 36 trimethylation over the Adh proximal promoter. We conclude that the endogenous Adh locus is developmentally regulated by transcriptional interference in a tunable manner.

Exceptional Nuclease Resistance of Paranemic Crossover (PX) DNA and Crossover-Dependent Biostability of DNA Motifs.

Nanometer-sized features and molecular recognition properties make DNA a useful material for nanoscale construction, but degradation in biological fluids poses a considerable roadblock to biomedical applications of DNA nanotechnology. Here, we report the remarkable biostability of a multistranded motif called paranemic crossover (PX) DNA. Compared to double stranded DNA, PX DNA has dramatically enhanced (sometimes >1000 fold) resistance to degradation by four different nucleases, bovine and human serum, and human urine. We trace the cause of PX's biostability to DNA crossovers, showing a continuum of protection that scales with the number of crossovers. These results suggest that enhanced biostability can be engineered into DNA nanostructures by adopting PX-based architectures or by strategic crossover placement.

Sequential Regulation of Maternal mRNAs through a Conserved cis-Acting Element in Their 3' UTRs.

Maternal mRNAs synthesized during oogenesis initiate the development of future generations. Some maternal mRNAs are either somatic or germline determinants and must be translationally repressed until embryogenesis. However, the translational repressors themselves are temporally regulated. We used polar granule component (pgc), a Drosophila maternal mRNA, to ask how maternal transcripts are repressed while the regulatory landscape is shifting. pgc, a germline determinant, is translationally regulated throughout oogenesis. We find that different conserved RNA-binding proteins bind a 10-nt sequence in the 3' UTR of pgc mRNA to continuously repress translation at different stages of oogenesis. Pumilio binds to this sequence in undifferentiated and early-differentiating oocytes to block Pgc translation. After differentiation, Bruno levels increase, allowing Bruno to bind the same sequence and take over translational repression of pgc mRNA. We have identified a class of maternal mRNAs that are regulated similarly, including zelda, the activator of the zygotic genome.

DNA targeting and cleavage by an engineered metalloprotein dimer.

Nature has illustrated through numerous examples that protein dimerization has structural and functional advantages. We previously reported the design and characterization of an engineered "metallohomeodomain" protein (C2) based on a chimera of the EF-hand Ca-binding motif and the helix-turn-helix motif of homeodomains (Lim and Franklin in Protein Sci. 15:2159-2165, 2004). This small metalloprotein binds the hard metal ions Ca(II) and Ln(III) and interacts with DNA with modest sequence preference and affinity, yet exhibits only residual DNA cleavage activity. Here we have achieved substantial improvement in function by constructing a covalent dimer of this C2 module (F2) to create a larger multidomain protein. As assayed via fluorescence spectroscopy, this F2 protein binds Ca(II) more avidly (25-fold) than C2 on a per-domain basis; in gel shift selection experiments, metallated F2 exhibits a specificity toward 5'-TAATTA-3' sequences. Finally, Ca(2)F2 cleaves plasmid DNA and generates a linear product in a Ca(II)-dependent way, unlike the CaC2 monomer. To the best of our knowledge this activation of Ca(II) in the context of an EF-hand binding motif is unique and represents a significant step forward in the design of artificial metallonucleases by utilizing biologically significant metal ions.

Sequence preference in DNA binding: de novo designed helix-turn-helix metallopeptides recognize a family of DNA target sites.

The DNA-binding behavior and target sequences of two designed metallopeptides have been investigated with an iterative electrophoresis mobility shift assay followed by PCR amplification, and by circular dichroism spectroscopy. Peptides P3W and P5b were designed based on the structural similarity of the helix-turn-helix motif of homeodomains and the EF-hand motifs of calmodulin, as previously described for P3W. Like P3W, P5b binds both Eu(III) (K(d) = 12.6 +/- 1.9 microM) and Ca(II) (K(d) = 70 +/- 8 microM) with reasonable affinity. Binding selection from a library of randomized 8-mer DNA oligonucleotide sequences identified one target family for CaP5b [5'-pur-T-pur-G-(G/C)-3'], and two target sites for CaP3W [5'-(A/T)-G-G-G-(T/C)-3' and 5'-A-T-(G/T)-T-G-3']. Circular dichroism studies indicate that unlike EuP3W, EuP5b is poorly folded in the absence of DNA. In the presence of DNA containing target-binding sites for both peptides, both EuP3W and EuP5b increase in helical content, in the latter case significantly. These results suggest that EuP5b binding to target DNA involves an induced-fit mechanism. These small chimeric metallopeptides have been found to bind selectively to DNA targets, analogous to natural protein-DNA interactions. This corroborates our earlier conclusions (J. Am. Chem. Soc. 125:6656, 2003) that sequence-preferential DNA cleavage by Ce(IV)P3W was due to sequence recognition.