Sex-steroid hormones are essential for normal reproductive activity in both sexes in all vertebrates. Estrogens are required for ovarian differentiation during a critical developmental stage and promote the growth and differentiation of the female reproductive system following puberty. Recent studies have shown that environmental estrogens influence the developing reproductive system as well as gametogenesis, especially in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor-ligand interactions in Elasmobranchii, we cloned a single estrogen receptor (ESR) from two shark species, the cloudy catshark (Scyliorhinus torazame) and whale shark (Rhincodon typus) and used an ERE-luciferase reporter assay system to characterize the interaction of these receptors with steroidal and other environmental estrogens. In the transient transfection ERE-luciferase reporter assay system, both shark ESR proteins displayed estrogen-dependent activation of transcription, and shark ESRs were more sensitive to 17beta-estradiol compared with other natural and synthetic estrogens. Further, the environmental chemicals, bisphenol A, nonylphenol, octylphenol and DDT could activate both shark ESRs. The assay system provides a tool for future studies examining the receptor-ligand interactions and estrogen disrupting mechanisms in Elasmobranchii.

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SHORT ABSTRACTThis paper summarizes how to visualize the flexible inter-domain movements of CRISPR-associated protein Cas9 using single molecule FRETLONG ABSTRACTThe CRISPR-associated protein Cas9 is widely used as a genome editing tool because of its ability to be programmed to cleave any DNA sequence that is followed by a protospacer adjacent motif. The continuing expansion of Cas9 technologies has stimulated studies regarding the molecular basis of the Cas9 catalytic process. Here we summarize methods for single molecule FRET (smFRET) to visualize the inter-domain movements of Cas9 protein. Our measurements and analysis demonstrate flexible and reversible movements of the Cas9 domains. Such flexible movements allow Cas9 to adopt transient conformations beyond those solved by crystal structures and play important roles in the Cas9 catalytic process. In addition to the smFRET measurement itself, to obtain precise results, it is necessary to validate Cas9 catalytic activity. Also, fluorescence anisotropy data are required to interpret smFRET data properly. Thus, in this paper, we describe the details of these important additional experiments for smFRET measurements.

Background Argonaute proteins play a central role in RNA silencing by forming protein-small RNA complexes responsible for the silencing process. While most Argonaute proteins have a short N-terminal region, Argonaute2 in Drosophila melanogaster (DmAgo2) harbors a long and unique N-terminal region. Previous in vitro biochemical studies have shown that the loss of this region does not impair the RNA silencing activity of the complex. However, an N-terminal mutant of Drosophila melanogaster has demonstrated abnormal RNA silencing activity. To explore the causes of this discrepancy between in vitro and in vivo studies, we investigated the biophysical properties of the region. The N-terminal region is highly rich in glutamine and glycine residues, which is a well-known property for prion-like domains, a subclass of amyloid-forming peptides. Therefore, the possibility of the N-terminal region functioning as an amyloid was tested. Results Our in silico and biochemical assays demonstrated that the N-terminal region exhibits amyloid-specific properties. The region indeed formed aggregates that were not dissociated even in the presence of sodium dodecyl sulfate. Also, the aggregates enhanced the fluorescence intensity of thioflavin-T, an amyloid detection reagent. The kinetics of the aggregation followed that of typical amyloid formation exhibiting self-propagating activity. Furthermore, we directly visualized the aggregation process of the N-terminal region under fluorescence microscopy and found that the aggregations took fractal or fibril shapes. Together, the results indicate that the N-terminal region can form amyloid-like aggregates. Conclusions Many other amyloid-forming peptides have been reported to modulate the function of proteins through their aggregation. Therefore, our findings raise the possibility that aggregation of the N-terminal region regulates the RNA silencing activity of DmAgo2.

Argonaute proteins play a central role in RNA silencing by forming protein-small RNA complexes responsible for the silencing process. While most Argonaute proteins have a short N-terminal region, Argonaute2 in Drosophila melanogaster (DmAgo2) harbors a long and unique N-terminal region. Previous in vitro biochemical studies have shown that the loss of this region does not impair the RNA silencing activity of the complex. However, an N-terminal mutant of Drosophila melanogaster has demonstrated abnormal RNA silencing activity. To explore the causes of this discrepancy between in vitro and in vivo studies, we investigated the biophysical properties of the region. Because the N-terminal region is highly rich in glutamine and glycine residues, which is a well-known property for prion-like domains (PrLD), the possibility of the N-terminal region functioning as a PrLD was tested. Our biochemical assays demonstrated that the N-terminal region can form aggregates that are not dissociated even in the presence of SDS. Also, the aggregates enhanced the fluorescence intensity of thioflavin-T, an amyloid detection reagent. The kinetics of the aggregation followed that of typical amyloid formation exhibiting the self-propagating activity. Further, we directly visualized the aggregation process of the N-terminal region under fluorescence microscopy and found that the aggregations took fractal or fibril shapes. Together, the results indicate that the N-terminal region is a PrLD. Many other PrLDs have been reported to modulate the function of proteins through their aggregation. Therefore, our results raise the possibility that aggregation of the N-terminal region regulates the RNA silencing activity of DmAgo2.