The characterization of amphibian nucleoplasmins yields new insight into their role in sperm chromatin remodeling

BACKGROUND: Nucleoplasmin is a nuclear chaperone protein that has been shown to participate in the remodeling of sperm chromatin immediately after fertilization by displacing highly specialized sperm nuclear basic proteins (SNBPs), such as protamine (P type) and protamine-like (PL type) proteins, from the sperm chromatin and by the transfer of histone H2A-H2B. The presence of SNBPs of the histone type (H type) in some organisms (very similar to the histones found in somatic tissues) raises uncertainty about the need for a nucleoplasmin-mediated removal process in such cases and poses a very interesting question regarding the appearance and further differentiation of the sperm chromatin remodeling function of nucleoplasmin and the implicit relationship with SNBP diversity The amphibians represent an unique opportunity to address this issue as they contain genera with SNBPs representative of each of the three main types: Rana (H type); Xenopus (PL type) and Bufo (P type). RESULTS: In this work, the presence of nucleoplasmin in oocyte extracts from these three organisms has been assessed using Western Blotting. We have used mass spectrometry and cloning techniques to characterize the full-length cDNA sequences of Rana catesbeiana and Bufo marinus nucleoplasmin. Northern dot blot analysis shows that nucleoplasmin is mainly transcribed in the egg of the former species. Phylogenetic analysis of nucleoplasmin family members from various metazoans suggests that amphibian nucleoplasmins group closely with mammalian NPM2 proteins. CONCLUSION: We have shown that these organisms, in striking contrast to their SNBPs, all contain nucleoplasmins with very similar primary structures. This result has important implications as it suggests that nucleoplasmin's role in chromatin assembly during early zygote development could have been complemented by the acquisition of a new function of non-specifically removing SNBPs in sperm chromatin remodeling. This acquired function would have been strongly determined by the constraints imposed by the appearance and differentiation of SNBPs in the sperm

The gene transformer-2 of Anastrepha fruit flies (Diptera, Tephritidae) and its evolution in insects

<p>Abstract</p> <p>Background</p> <p>In the tephritids <it>Ceratitis</it>, <it>Bactrocera </it>and <it>Anastrepha</it>, the gene <it>transformer </it>provides the memory device for sex determination via its auto-regulation; only in females is functional Tra protein produced. To date, the isolation and characterisation of the gene <it>transformer-2 </it>in the tephritids has only been undertaken in <it>Ceratitis</it>, and it has been shown that its function is required for the female-specific splicing of <it>doublesex </it>and <it>transformer </it>pre-mRNA. It therefore participates in <it>transformer </it>auto-regulatory function. In this work, the characterisation of this gene in eleven tephritid species belonging to the less extensively analysed genus <it>Anastrepha </it>was undertaken in order to throw light on the evolution of <it>transformer-2</it>.</p> <p>Results</p> <p>The gene <it>transformer-2 </it>produces a protein of 249 amino acids in both sexes, which shows the features of the SR protein family. No significant partially spliced mRNA isoform specific to the male germ line was detected, unlike in <it>Drosophila</it>. It is transcribed in both sexes during development and in adult life, in both the soma and germ line. The injection of <it>Anastrepha transformer-2 </it>dsRNA into <it>Anastrepha </it>embryos caused a change in the splicing pattern of the endogenous <it>transformer </it>and <it>doublesex </it>pre-mRNA of XX females from the female to the male mode. Consequently, these XX females were transformed into pseudomales. The comparison of the eleven <it>Anastrepha </it>Transformer-2 proteins among themselves, and with the Transformer-2 proteins of other insects, suggests the existence of negative selection acting at the protein level to maintain Transformer-2 structural features.</p> <p>Conclusions</p> <p>These results indicate that <it>transformer-2 </it>is required for sex determination in <it>Anastrepha </it>through its participation in the female-specific splicing of <it>transformer </it>and <it>doublesex </it>pre-mRNAs. It is therefore needed for the auto-regulation of the gene <it>transformer</it>. Thus, the <it>transformer/transfomer-2 > doublesex </it>elements at the bottom of the cascade, and their relationships, probably represent the ancestral state (which still exists in the Tephritidae, Calliphoridae and Muscidae lineages) of the extant cascade found in the Drosophilidae lineage (in which <it>tra </it>is just another component of the sex determination gene cascade regulated by <it>Sex-lethal</it>). In the phylogenetic lineage that gave rise to the drosophilids, evolution co-opted for <it>Sex-lethal</it>, modified it, and converted it into the key gene controlling sex determination.</p

Extracellular Vesicles Enriched in Connexin 43 Promote a Senescent Phenotype in Bone and Synovial Cells Contributing to Osteoarthritis Progression

[Abstract] The accumulation of senescent cells is a key characteristic of aging, leading to the progression of age-related diseases such as osteoarthritis (OA). Previous data from our laboratory has demonstrated that high levels of the transmembrane protein connexin 43 (Cx43) are associated with a senescent phenotype in chondrocytes from osteoarthritic cartilage. OA has been reclassified as a musculoskeletal disease characterized by the breakdown of the articular cartilage affecting the whole joint, subchondral bone, synovium, ligaments, tendons and muscles. However, the mechanisms that contribute to the spread of pathogenic factors throughout the joint tissues are still unknown. Here, we show for the first time that small extracellular vesicles (sEVs) released by human OA-derived chondrocytes contain high levels of Cx43 and induce a senescent phenotype in targeted chondrocytes, synovial and bone cells contributing to the formation of an inflammatory and degenerative joint environment by the secretion of senescence-associated secretory associated phenotype (SASP) molecules, including IL-1ß and IL-6 and MMPs. The enrichment of Cx43 changes the protein profile and activity of the secreted sEVs. Our results indicate a dual role for sEVs containing Cx43 inducing senescence and activating cellular plasticity in target cells mediated by NF-kß and the extracellular signal-regulated kinase 1/2 (ERK1/2), inducing epithelial-to-mesenchymal transition (EMT) signalling programme and contributing to the loss of the fully differentiated phenotype. Our results demonstrated that Cx43-sEVs released by OA-derived chondrocytes spread senescence, inflammation and reprogramming factors involved in wound healing failure to neighbouring tissues, contributing to the progression of the disease among cartilage, synovium, and bone and probably from one joint to another. These results highlight the importance for future studies to consider sEVs positive for Cx43 as a new biomarker of disease progression and new target to treat OA.This work was supported in part through funding from Health Institute ‘Carlos III’ (ISCIII, Spain), the European Regional Development Fund, ‘A way of making Europe’ from the European Union (to MDM): grant PI19/00145; a grant from the Joint Transnational Call for Proposals for “European Innovative Research & Technological Development Projects in Nanomedicine” EURONANOMED III (AC21_2/00026) (to MDM); a grant from Xunta de Galicia (IN607B2020/12) (to MDM) and from H2020, Future and Emerging Technologies (grant 858014 “PANACHE”) to MDM. MV-E was funded with a predoctoral (ED481A-2015/188) and post-doctoral fellowship (IN606B-2019/004) from Xunta de Galicia. AG-C was funded with a predoctoral fellowship (FIS20/00310) from ISCIII. PC-F was funded with a post-doctoral fellowship and a grant from Xunta de Galicia (INB606B 2017/014 and IN606C 2021/006). We thank members of the CellCOM group for helpful technical suggestion, María Dolores Álvarez Alvariño (CHUS) for generously collecting tissue samples in the operating room after surgery and Arantxa Tabernero (INCYL, University of Salamanca) for kindly providing the human Cx43 plasmid used in this studyXunta de Galicia; IN607B2020/12Xunta de Galicia; ED481A-2015/188Xunta de Galicia; IN606B-2019/004Xunta de Galicia; INB606B 2017/014Xunta de Galicia; IN606C 2021/00

The comparative study of five sex-determining proteins across insects unveils high rates of evolution at basal components of the sex determination cascade

29 p.-4 fig.-1 tab. supl.In insects, the sex determination cascade is composed of genes that interact with each other in a strict hierarchical manner, constituting a co-adapted gene complex built in reverse order from bottom to top. Accordingly, ancient elements at the bottom are expected to remain conserved ensuring the correct functionality of the cascade. In the present work we have studied the levels of variation displayed by five key components of the sex determination cascade across 59 insect species, including Sex-lethal, transformer, transformer-2, fruitless, doublesex and sister-of-Sex-lethal (a paralog of Sxl encompassing sex-independent functions). Surprisingly, our results reveal that basal components of the cascade (doublesex, fruitless) seem to evolve more rapidly than previously suspected. Indeed, in the case of Drosophila, these proteins evolve more rapidly than the master regulator Sex-lethal. These results agree with the notion suggesting that genes involved in early aspects of development will be more constrained due to the large deleterious pleiotropic effects of mutations, resulting in increased levels of purifying selection at top positions of the cascade. The analyses of the selective episodes involved in the recruitment of Sxl into sex determining functions in Drosophila further support this idea, suggesting the presence of bursts of adaptive selection in thecommon ancestor of drosophilids, followed by purifying selection preserving the master regulatory role of this protein. Altogether, this work underscores the importance of the position of sex determining genes in the cascade, constituting a major constraint shaping the molecular evolution of the insect sex determination pathway.The present work was supported by start-up funds from the College of Arts and Sciences at Florida International University (CAS-FIU) to J.M.E.-L.Peer reviewe

An unusual role for doublesex in sex determination in the dipteran Sciara

59 p.-8 fig. Título del preprint:The gene doublesex of dipteran Sciara does not follow the expression pattern observed in insects.The gene doublesex, which is placed at the bottom of the sex determination gene cascade, plays the ultimate discriminatory role for sex determination in insects. In all insects where this gene has been characterized, the dsx pre-mRNA follows a sex-specific splicing pattern producing male- and female-specific mRNAs, encoding the male-DSXM and female-DSXF proteins,which determine male and female development, respectively. The present paper reports the isolation and characterization of the gene doublesex of dipteran Sciara insects. The Sciara doublesex gene is constitutively transcribed during development and adult life of males and females. Sciara had no sex-specific doublesex mRNAs but the same transcripts, produced by alternative splicing of its primary transcript, were present in both sexes, although their relative abundance is sex-specific. However, only the female DSXF protein, but not the male DSXM protein, was produced at similar amounts in both sexes. An analysis of the expression of female and male Sciara DSX proteins in Drosophila showed that these proteins conserved female and male function, respectively, on the control of Drosophila yolk-protein genes. The molecular evolution of gene doublesex of all insects where this gene has been characterized revealed that Sciara doublesex displays a considerable degree of divergence with respect to the rest of dipterans, as suggested by its basal position within the doublesex phylogeny, its gene organization and its splicing pattern. It is proposed that if the gene doublesex were needed for Sciara sex determination, this gene would not play the ultimate discriminatory role for sex determination performed in insects.L Sánchez had financial support (BFU2005-03000 and BFU2008-00474) from the Spanish government during the time the group was doing the work presented in this manuscript. However, he had to close his laboratory —with the consequent disappearance of the group— because his grant was not renewed it due to the economical crisis in Spain. L Sánchez thanks very much to JL Barbero for permitting us to finish this work in his laboratory and sponsored by his grant.N

The gene transformer of anastrepha fruit flies (Diptera, tephritidae) and its evolution in insects.

In the tephritids Ceratitis capitata and Bactrocera oleae, the gene transformer acts as the memory device for sex determination, via an auto-regulatory function; and functional Tra protein is produced only in females. This paper investigates the evolution of the gene tra, which was characterised in twelve tephritid species belonging to the less extensively analysed genus Anastrepha. Our study provided the following major conclusions. Firstly, the memory device mechanism used by this gene in sex determination in tephritids likely existed in the common ancestor of the Ceratitis, Bactrocera and Anastrepha phylogenetic lineages. This mechanism would represent the ancestral state with respect to the extant cascade seen in the more evolved Drosophila lineage. Secondly, Transformer2-specific binding intronic splicing silencer sites were found in the splicing regulatory region of transformer but not in doublesex pre-mRNAs in these tephritids. Thus, these sites probably provide the discriminating feature for the putative dual splicing activity of the Tra-Tra2 complex in tephritids. It acts as a splicing activator in dsx pre-mRNA splicing (its binding to the female-specific exon promotes the inclusion of this exon into the mature mRNA), and as a splicing inhibitor in tra pre-mRNA splicing (its binding to the male-specific exons prevents the inclusion of these exons into the mature mRNA). Further, a highly conserved region was found in the specific amino-terminal region of the tephritid Tra protein that might be involved in Tra auto-regulatory function and hence in its repressive splicing behaviour. Finally, the Tra proteins conserved the SR dipeptides, which are essential for Tra functionality