Valeria Specchia

Hsp90 prevents phenotypic variation by suppressing the mutagenic activity of transposons

The canalization concept describes the resistance of a developmental process to phenotypic variation, regardless of genetic and environmental perturbations, owing to the existence of buffering mechanisms. Severe perturbations, which overcome such buffering mechanisms, produce altered phenotypes that can be heritable and can themselves be canalized by a genetic assimilation process. An important implication of this concept is that the buffering mechanism could be genetically controlled. Recent studies on Hsp90, a protein involved in several cellular processes and development pathways, indicate that it is a possible molecular mechanism for canalization and genetic assimilation. In both flies and plants, mutations in the Hsp90-encoding gene induce a wide range of phenotypic abnormalities, which have been interpreted as an increased sensitivity of different developmental pathways to hidden genetic variability. Thus, Hsp90 chaperone machinery may be an evolutionarily conserved buffering mechanism of phenotypic variance, which provides the genetic material for natural selection. Here we offer an additional, perhaps alternative, explanation for proposals of a concrete mechanism underlying canalization. We show that, in Drosophila, functional alterations of Hsp90 affect the Piwi-interacting RNA (piRNA; a class of germ-line-specific small RNAs) silencing mechanism leading to transposon activation and the induction of morphological mutants. This indicates that Hsp90 mutations can generate new variation by transposon-mediated ‘canonical’ mutagenesis.

Transposons, environmental changes, and heritable induced phenotypic variability

The mechanisms of biological evolution have always been, and still are, the subject of intense debate and modeling. One of the main problems is how the genetic variability is produced and maintained in order to make the organisms adaptable to environmental changes and therefore capable of evolving. In recent years, it has been reported that, in flies and plants, mutations in Hsp90 gene are capable to induce, with a low frequency, many different developmental abnormalities depending on the genetic backgrounds. This has suggested that the reduction of Hsp90 amount makes different development pathways more sensitive to hidden genetic variability. This suggestion revitalized a classical debate around the original Waddington hypothesis of canalization and genetic assimilation making Hsp90 the prototype of morphological capacitor. Other data have also suggested a different mechanism that revitalizes another classic debate about the response of genome to physiological and environmental stress put forward by Barbara McClintock. That data demonstrated that Hsp90 is involved in repression of transposon activity by playing a significant role in piwi-interacting RNA (piRNAs)-dependent RNA interference (RNAi) silencing. The important implication is that the fixed phenotypic abnormalities observed in Hsp90 mutants are probably related to de novo induced mutations by transposon activation. In this case, Hsp90 could be considered as a mutator. In the present theoretical paper, we discuss several possible implications about environmental stress, transposon, and evolution offering also a support to the concept of evolvability.

aubergine Gene Overexpression in Somatic Tissues of auberginesting Mutants Interferes With the RNAi Pathway of a yellow Hairpin dsRNA in Drosophila melanogaster

AUBERGINE (AUB) is a member of the PPD family of proteins. These proteins are implicated in RNA interference. In this article we demonstrate that the expression of the aub gene and protein increase in aubsting mutants. We used a genetic method to test whether aubsting overexpression could interfere with proper functioning of the process of RNA interference in somatic tissues of Drosophila melanogaster. This method is based on a transgenic line bearing a construct in which a fragment of the yellow (y) gene is cloned to form an inverted repeat (y-IR) under the control of the upstream activation sequence (UAS) of the yeast transcriptional activator GAL4. The UAS-y-IR transgene and the Act5C-GAL4 driver were brought together on chromosome 3 via recombination. In the resulting strain (Act5C-y-IR), transcriptional activation by GAL4 constitutively produces a dsRNA hairpin bearing cognate sequences to the yellow gene causing continuing degradation of y mRNA resulting in yellow1 (y1) phenocopies. In this genetic background, the mutation of any factor involved in RNAi should repress degradation of y mRNA, restoring the wild-type phenotype. We employed this genetic approach to show that an increased amount of AUBERGINE interferes with the regular functioning of the somatic RNAi pathway.

The Drosophila fragile X mental retardation protein participates in the piRNA pathway

ABSTRACT RNA metabolism controls multiple biological processes, and a specific class of small RNAs, called piRNAs, act as genome guardians by silencing the expression of transposons and repetitive sequences in the gonads. Defects in the piRNA pathway affect genome integrity and fertility. The possible implications in physiopathological mechanisms of human diseases have made the piRNA pathway the object of intense investigation, and recent work suggests that there is a role for this pathway in somatic processes including synaptic plasticity. The RNA-binding fragile X mental retardation protein (FMRP, also known as FMR1) controls translation and its loss triggers the most frequent syndromic form of mental retardation as well as gonadal defects in humans. Here, we demonstrate for the first time that germline, as well as somatic expression, of Drosophila Fmr1 (denoted dFmr1), the Drosophila ortholog of FMRP, are necessary in a pathway mediated by piRNAs. Moreover, dFmr1 interacts genetically and biochemically with Aubergine, an Argonaute protein and a key player in this pathway. Our data provide novel perspectives for understanding the phenotypes observed in Fragile X patients and support the view that piRNAs might be at work in the nervous system.

Functional Characterization of the Bari1 Transposition System

The transposons of the Bari family are mobile genetic elements widespread in the Drosophila genus. However, despite a broad diffusion, virtually no information is available on the mechanisms underlying their mobility. In this paper we report the functional characterization of the Bari elements transposition system. Using the Bari1 element as a model, we investigated the subcellular localization of the transposase, its physical interaction with the transposon, and its catalytic activity. The Bari1 transposase localized in the nucleus and interacted with the terminal sequences of the transposon both in vitro and in vivo, however, no transposition activity was detected in transposition assays. Profiling of mRNAs expressed by the transposase gene revealed the expression of abnormal, internally processed transposase transcripts encoding truncated, catalytically inactive transposase polypeptides. We hypothesize that a post-transcriptional control mechanism produces transposase-derived polypeptides that effectively repress transposition. Our findings suggest further clues towards understanding the mechanisms that control transposition of an important class of mobile elements, which are both an endogenous source of genomic variability and widely used as transformation vectors/biotechnological tools.

The “Special” crystal-Stellate System in Drosophila melanogaster Reveals Mechanisms Underlying piRNA Pathway-Mediated Canalization

The Stellate-made crystals formation in spermatocytes is the phenotypic manifestation of a disrupted crystal-Stellate interaction in testes of Drosophila melanogaster. Stellate silencing is achieved by the piRNA pathway, but many features still remain unknown. Here we outline the important role of the crystal-Stellate modifiers. These have shed light on the piRNA pathways that defend genome integrity against transposons and other repetitive elements in the gonads. In particular, we illustrate the finding that HSP90 participates in the molecular pathways of piRNA production. This observation has relevance for the mechanisms underlying the evolutionary canalization process.

Porin isoform 2 has a different localization in Drosophila melanogaster ovaries than porin 1

Eukaryotic porins or VDACs are a class of transmembrane proteins mainly localized in the outer mitochondrial membrane, whose function is to allow the diffusion of metabolites between the cytosol and the mitochondrion. In Drosophila melanogaster, as in other organisms, a small family of genes encoding porins has been discovered from the sequence of the genome. It is of general interest to understand whether these genes represent functional entities or not, and whether their product is associated to any particular tissue. In previous work we reported about the transcriptional and translational analysis of porin 1 and porin 2, and we proposed the specific presence of the latter in spermatozoa of the fly. In this paper we performed real time RT-PCR quantification of porin 1 and porin 2 transcripts in germ cells of the fly. It indicates that Porin 1 is abundantly expressed in both male and female tissues; Porin 2 instead, is very abundant in testis and it is present in ovaries as well, but in a small amount. The immuno-histological stain of ovaries shows that Porin isoform 1 is selectively targeted to follicular cells while Porin isoform 2 is present in mitochondria of the epithelial sheath cells of the ovariole. The implications of the subcellular distribution of these porin isoforms and the specific localization in germ tissues are discussed.

The future of biotic indices in the ecogenomic era: Integrating (e)DNA metabarcoding in biological assessment of aquatic ecosystems

The bioassessment of aquatic ecosystems is currently based on various biotic indices that use the occurrence and/or abundance of selected taxonomic groups to define ecological status. These conventional indices have some limitations, often related to difficulties in morphological identification of bioindicator taxa. Recent development of DNA barcoding and metabarcoding could potentially alleviate some of these limitations, by using DNA sequences instead of morphology to identify organisms and to characterize a given ecosystem. In this paper, we review the structure of conventional biotic indices, and we present the results of pilot metabarcoding studies using environmental DNA to infer biotic indices. We discuss the main advantages and pitfalls of metabarcoding approaches to assess parameters such as richness, abundance, taxonomic composition and species ecological values, to be used for calculation of biotic indices. We present some future developments to fully exploit the potential of metabarcoding data and improve the accuracy and precision of their analysis. We also propose some recommendations for the future integration of DNA metabarcoding to routine biomonitoring programs.

dFmr1 Plays Roles in Small RNA Pathways of Drosophila melanogaster

Fragile-X syndrome is the most common form of inherited mental retardation accompanied by other phenotypes, including macroorchidism. The disorder originates with mutations in the Fmr1 gene coding for the FMRP protein, which, with its paralogs FXR1 and FXR2, constitute a well-conserved family of RNA-binding proteins. Drosophila melanogaster is a good model for the syndrome because it has a unique fragile X-related gene: dFmr1. Recently, in addition to its confirmed role in the miRNA pathway, a function for dFmr1 in the piRNA pathway, operating in Drosophila gonads, has been established. In this review we report a summary of the piRNA pathways occurring in gonads with a special emphasis on the relationship between the piRNA genes and the crystal-Stellate system; we also analyze the roles of dFmr1 in the Drosophila gonads, exploring their genetic and biochemical interactions to reveal some unexpected connections.

Novel mutants of the aubergine gene

ABSTRACT Aubergine is an RNA-binding protein of the Piwi clade, functioning in germline in the piRNA pathway that silences transposons and repetitive sequences. Several mutations of this gene exist, but they mostly result in truncated proteins or correspond to mutations that also affect neighboring genes. We have generated complete aubergine knock-out mutants that do not disrupt the neighboring genes. These novel mutants are characterized by PCR and sequencing. Their nature is confirmed by female sterility and by the presence of crystals in testes, common to the aubergine loss of function mutations. These mutants provide novel and more appropriate tools for the study of the piRNA pathway that controls genome stability.