Paramutation in Drosophila Requires Both Nuclear and Cytoplasmic Actors of the piRNA Pathway and Induces Cis-spreading of piRNA Production

Transposable element activity is repressed in the germline in animals by PIWI-interacting RNAs (piRNAs), a class of small RNAs produced by genomic loci mostly composed of TE sequences. The mechanism of induction of piRNA production by these loci is still enigmatic. We have shown that, in Drosophila melanogaster, a cluster of tandemly repeated P-lacZ-white transgenes can be activated for piRNA production by maternal inheritance of a cytoplasm containing homologous piRNAs. This activated state is stably transmitted over generations and allows trans-silencing of a homologous transgenic target in the female germline. Such an epigenetic conversion displays the functional characteristics of a paramutation, i.e., a heritable epigenetic modification of one allele by the other. We report here that piRNA production and trans-silencing capacities of the paramutated cluster depend on the function of the rhino, cutoff, and zucchini genes involved in primary piRNA biogenesis in the germline, as well as on that of the aubergine gene implicated in the ping-pong piRNA amplification step. The 21-nt RNAs, which are produced by the paramutated cluster, in addition to 23- to 28-nt piRNAs are not necessary for paramutation to occur. Production of these 21-nt RNAs requires Dicer-2 but also all the piRNA genes tested. Moreover, cytoplasmic transmission of piRNAs homologous to only a subregion of the transgenic locus can generate a strong paramutated locus that produces piRNAs along the whole length of the transgenes. Finally, we observed that maternally inherited transgenic small RNAs can also impact transgene expression in the soma. In conclusion, paramutation involves both nuclear (Rhino, Cutoff) and cytoplasmic (Aubergine, Zucchini) actors of the piRNA pathway. In addition, since it is observed between nonfully homologous loci located on different chromosomes, paramutation may play a crucial role in epigenome shaping in Drosophila natural populations.     

Fast adaptive responses in the oral jaw of Lake Victoria cichlids

Rapid morphological changes in response to fluctuating natural environments are a common phenomenon in species that undergo adaptive radiation. The dramatic ecological changes in Lake Victoria provide a unique opportunity to study environmental effects on cichlid morphology. This study shows how four haplochromine cichlids adapted their premaxilla to a changed diet over the past 30 years. Directly after the diet change toward larger and faster prey in the late 1980s, the premaxilla (upper jaw) changed in a way that is in agreement with a more food manipulating feeding style. During the 2000s, two zooplanktivorous species showed a reversal of morphological changes after returning to their original diet, whereas two other species showed no reversal of diet and morphology. These rapid changes indicate a potential for extremely fast adaptive responses to environmental fluctuations, which are likely inflicted by competition release and increase, and might have a bearing on the ability of haplochromines to cope with environmental changes. These responses could be due to rapid genetic change or phenotypic plasticity, for which there is ample evidence in cichlid fish structures associated with food capture and processing. These versatile adaptive responses are likely to have contributed to the fast adaptive radiation of haplochromines.     

Structure,Dynamics, and Substrate Specificity of the OprO Porin from Pseudomonas aeruginosa

The outer membrane (OM) of Gram-negative bacteria functions as a selective permeability barrier between cell and environment. For nutrient acquisition, the OM contains a number of channels that mediate uptake of small molecules by diffusion. Many of these channels are specific, i.e., they prefer certain substrates over others. In electrophysiological experiments, the OM channels OprP and OprO from Pseudomonas aeruginosa show a specificity for phosphate and diphosphate, respectively. In this study we use x-ray crystallography, free-energy molecular dynamics (MD) simulations, and electrophysiology to uncover the atomic basis for the different substrate specificity of these highly similar channels. A structural analysis of OprP and OprO revealed two crucial differences in the central constriction region. In OprP there are two tyrosine residues, Y62 and Y114, whereas the corresponding residues in OprO are phenylalanine F62 and aspartate D114. To probe the importance of these two residues in generating the different substrate specificities, the double mutants were generated in silico and in vitro. Applied-field MD simulations and electrophysiological experiments demonstrated that the double mutations interchange the phosphate and diphosphate specificities of OprP and OprO. Our findings outline a possible strategy to rationally design channel specificity by modification of a small number of residues that may be applicable to other pores as well.     

Molecular Insights into the Coding Region Determinant-binding Protein-RNA Interaction through Site-directed Mutagenesis in the Heterogeneous Nuclear Ribonucleoprotein-K-homology Domains

The ability of its four heterogeneous nuclear RNP-K-homology (KH) domains to physically associate with oncogenic mRNAs is a major criterion for the function of the coding region determinant-binding protein (CRD-BP). However, the particular RNA-binding role of each of the KH domains remains largely unresolved. Here, we mutated the first glycine to an aspartate in the universally conserved GXXG motif of the KH domain as an approach to investigate their role. Our results show that mutation of a single GXXG motif generally had no effect on binding, but the mutation in any two KH domains, with the exception of the combination of KH3 and KH4 domains, completely abrogated RNA binding in vitro and significantly retarded granule formation in zebrafish embryos, suggesting that any combination of at least two KH domains cooperate in tandem to bind RNA efficiently. Interestingly, we found that any single point mutation in one of the four KH domains significantly impacted CRD-BP binding to mRNAs in HeLa cells, suggesting that the dynamics of the CRD-BP-mRNA interaction vary over time in vivo. Furthermore, our results suggest that different mRNAs bind preferentially to distinct CRD-BP KH domains. The novel insights revealed in this study have important implications on the understanding of the oncogenic mechanism of CRD-BP as well as in the future design of inhibitors against CRD-BP function.     

Reduced genetic diversity in Bearded Vultures Gypaetus barbatus in Southern Africa

The Bearded Vulture Gypaetus barbatus occurs throughout its range in small and dwindling population fragments with limited genetic differentiation between populations, suggesting that the species might be managed as a single entity. The numbers of East and Southern African Bearded Vultures included in previous studies were small, so we determine the genetic variation within, evolutionary placement of and connectivity among sub‐Saharan African populations. Mitochondrial DNA fragment analyses detected little or no differentiation between populations in Ethiopia and Southern Africa, with reduced haplotype diversity in Southern Africa compared with populations in the Northern Hemisphere. The results inform conservation management of this species globally and locally, and offer guidelines for translocations should populations continue to decline.     

The BRCA1 Tumor Suppressor Binds to Inositol 1,4,5-Trisphosphate Receptors to Stimulate Apoptotic Calcium Release

The inositol 1,4,5-trisphosphate receptor (IP₃R) is a ubiquitously expressed endoplasmic reticulum (ER)-resident calcium channel. Calcium release mediated by IP₃Rs influences many signaling pathways, including those regulating apoptosis. IP₃R activity is regulated by protein-protein interactions, including binding to proto-oncogenes and tumor suppressors to regulate cell death. Here we show that the IP₃R binds to the tumor suppressor BRCA1. BRCA1 binding directly sensitizes the IP₃R to its ligand, IP₃. BRCA1 is recruited to the ER during apoptosis in an IP₃R-dependent manner, and, in addition, a pool of BRCA1 protein is constitutively associated with the ER under non-apoptotic conditions. This is likely mediated by a novel lipid binding activity of the first BRCA1 C terminus domain of BRCA1. These findings provide a mechanistic explanation by which BRCA1 can act as a proapoptotic protein.