Anastasios Vourekas

In Drosophila melanogaster and many other metazoans, the specification of germ cells requires cytoplasmic inheritance of maternally synthesized RNA and protein determinants, which are assembled in electron-dense cytoplasmic structures known as germ or polar granules, found at the posterior end of the oocytes. Recent studies have shown that the formation of germ granules is dependent on the interaction of proteins containing tudor domains with the piwi-interacting RNA (piRNA)-binding Piwi proteins, and such interactions are dependent on symmetrically dimethylated arginines (sDMAs) of Piwi proteins. Tudor-Piwi interactions are crucial and are conserved in the germ cells of sexually reproducing animals, including mammals. In the September 1, 2010, issue of Genes & Development, Liu and colleagues (pp. 1876-1881) use a combination of genetics, biochemistry, and crystallography to uncover the molecular and structural details of how Tudor recognizes and binds the sDMAs of the Piwi protein Aubergine.

Piwi proteins such as Drosophila Aubergine (Aub) and mouse Miwi are essential for germline development and for primordial germ cell (PGC) specification. They bind piRNAs and contain symmetrically dimethylated arginines (sDMAs), catalyzed by dPRMT5. PGC specification in Drosophila requires maternal inheritance of cytoplasmic factors, including Aub, dPRMT5, and Tudor (Tud), that are concentrated in the germ plasm at the posterior end of the oocyte. Here we show that Miwi binds to Tdrd6 and Aub binds to Tudor, in an sDMA-dependent manner, demonstrating that binding of sDMA-modified Piwi proteins with Tudor-domain proteins is an evolutionarily conserved interaction in germ cells. We report that in Drosophila tud(1) mutants, the piRNA pathway is intact and most transposons are not de-repressed. However, the localization of Aub in the germ plasm is severely reduced. These findings indicate that germ plasm assembly requires sDMA modification of Aub by dPRMT5, which, in turn, is required for binding to Tudor. Our study also suggests that the function of the piRNA pathway in PGC specification may be independent of its role in transposon control.

Recent studies have uncovered an unexpected relationship between factors that are essential for germline development in Drosophila melanogaster: the arginine protein methyltransferase 5 (dPRMT5/Csul/Dart5) and its cofactor Valois, methylate the Piwi family protein Aub, enabling it to bind Tudor. The RNA helicase Vasa is another essential protein in germline development. Here, we report that mouse (mouse Vasa homolog), Xenopus laevis, and D. melanogaster Vasa proteins contain both symmetrical and asymmetrical dimethylarginines. We find that dPRMT5 is required for the production of sDMAs of Vasa in vivo. Furthermore, we find that the mouse Vasa homolog associates with Tudor domain-containing proteins, Tdrd1 and Tdrd6, as well as the Piwi proteins, Mili and Miwi. Arginine methylation is thus emerging as a conserved and pivotal post-translational modification of proteins that is essential for germline development.

More than half of human cancers have aberrantly upregulated phosphoinositide signals; yet how phospholipid signals are controlled during tumorigenesis is not fully understood. We report here that TIPE3 (TNFAIP8L3) is the transfer protein of phosphoinositide second messengers that promote cancer. High-resolution crystal structure of TIPE3 shows a large hydrophobic cavity that is occupied by a phospholipid-like molecule. TIPE3 preferentially captures and shuttles two lipid second messengers, i.e., phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, and increases their levels in the plasma membrane. Notably, human cancers have markedly upregulated TIPE3 expression. Knocking out TIPE3 diminishes tumorigenesis, whereas enforced TIPE3 expression enhances it in vivo. Thus, the function and metabolism of phosphoinositide second messengers are controlled by a specific transfer protein during tumorigenesis.

Type 2 diabetes mellitus (T2DM) is a complex disease characterized by the inability of the insulin-producing β cells in the endocrine pancreas to overcome insulin resistance in peripheral tissues. To determine if microRNAs are involved in the pathogenesis of human T2DM, we sequenced the small RNAs of human islets from diabetic and nondiabetic organ donors. We identified a cluster of microRNAs in an imprinted locus on human chromosome 14q32 that is highly and specifically expressed in human β cells and dramatically downregulated in islets from T2DM organ donors. The downregulation of this locus strongly correlates with hypermethylation of its promoter. Using HITS-CLIP for the essential RISC-component Argonaute, we identified disease-relevant targets of the chromosome 14q32 microRNAs, such as IAPP and TP53INP1, that cause increased β cell apoptosis upon overexpression in human islets. Our results support a role for microRNAs and their epigenetic control by DNA methylation in the pathogenesis of T2DM.

Piwi interacting RNAs (piRNAs) are small (∼25 to ∼30 nucleotide) and are expressed in the germline. piRNAs bind to the Piwi subclade of Argonaute proteins and form the core ribonucleoproteins (RNPs) of piRNPs. We describe a method for the massive identification of piRNAs from immunopurified piRNPs. This strategy may also be used for immunopurification and directional sequencing of RNAs from other RNPs that contain small RNAs.

The effect of macrolide antibiotic spiramycin on RNase P holoenzyme and M1 RNA from Escherichia coli was investigated. Ribonuclease P (RNase P) is a ribozyme that is responsible for the maturation of 5' termini of tRNA molecules. Spiramycin revealed a dose-dependent activation on pre-tRNA cleavage by E. coli RNase P holoenzyme and M1 RNA. The K s and V max, as well as the K s(app) and V max(app) values of RNase P holoenzyme and M1 RNA in the presence or absence of spiramycin, were calculated from primary and secondary kinetic plots. It was found that the activity status of RNase P holoenzyme and M1 RNA is improved by the presence of spiramycin 18- and 12-fold, respectively. Primer extension analysis revealed that spiramycin induces a conformational change of the P10/11 structural element of M1 RNA, which is involved in substrate recognition.

Whereas the importance of microRNA (miRNA) for the development of several tissues is well established, its role in the intestine is unknown. We aimed to quantify the complete miRNA expression profile of the mammalian intestinal mucosa and to determine the contribution of miRNAs to intestinal homeostasis using genetic means.

Comparative in silico analyses of bacterial RNase P enzymes clustered their RNA subunits in type A RNA, found in Escherichia coli, and in type B, found in Bacillus subtilis. Zymomonas mobilis RNase P consists of one protein (Zmo-RnpA) and one type A RNA (RPR) subunit containing the P19 element, present in many RNase P RNAs of any structure class but lacking in the E. coli RNase P RNA. To investigate the putative role of the P19 stem, we constructed a P19 deletion RNA mutant (ΔP19RPR) and performed detailed kinetic analysis of reconstituted enzymes in the presence of the homologous Zmo-RnpA protein or Eco-RnpA protein from E. coli. The deletion of P19 perturbs the monovalent ion requirements. The Mg(2+) requirement for the ΔP19RPR holoenzyme was almost identical to that for the wtRPR holoenzyme at Mg(2+) concentrations of ≤25 mM. Interestingly, enzymes reconstituted with Eco-RnpA protein, relative to those assembled with Zmo-RnpA, exhibited enhanced activity in the presence of ΔP19RPR, suggesting that Eco-RnpA protein can effectively replace its Z. mobilis counterpart. Homologous and heterologous reconstituted enzymes in the presence of ΔP19RPR exhibited differences in their Km values and catalytic efficacies. Overall, the presence of the P19 stem points toward an adaption during the co-evolution of Zmo-RnpA and RPR that is essential for stabilizing the overall structure of the Z. mobilis RNase P. Finally, our results are in line with existing structural data on RNase P enzymes and provide biochemical support for the possible role of appended domains in RNase P RNA subunits.

Dictyostelium discoideum nuclear RNase P is a ribonucleoprotein complex that displays similarities with its counterparts from higher eukaryotes such as the human enzyme, but at the same time it retains distinctive characteristics. In the present study, we report the molecular cloning and interaction details of DRpp29 and RNase P RNA, two subunits of the RNase P holoenzyme from D. discoideum. Electrophoretic mobility shift assays exhibited that DRpp29 binds specifically to the RNase P RNA subunit, a feature that was further confirmed by the molecular modeling of the DRpp29 structure. Moreover, deletion mutants of DRpp29 were constructed in order to investigate the domains of DRpp29 that contribute to and/or are responsible for the direct interaction with the D. discoideum RNase P RNA. A eukaryotic specific, lysine- and arginine-rich region was revealed, which seems to facilitate the interaction between these two subunits. Furthermore, we tested the ability of wild-type and mutant DRpp29 to form active RNase P enzymatic particles with the Escherichia coli RNase P RNA.

A cell-free system derived from Dictyostelium discoideum has been used to study the kinetics of inhibition of RNase P by puromycin, amicetin and blasticidin S. Detailed kinetic analysis showed that the type of inhibition of RNase P activity by puromycin is simple competitive, whereas the type of inhibition by amicetin and blasticidin S is simple non-competitive. On the basis of Ki values amicetin is stronger inhibitor than puromycin and blasticidin S.

Ribonuclease P (RNase P) is ubiquitous and essential Mg(2+)-dependent endoribonuclease that catalyzes the 5'-maturation of transfer RNAs. RNase P and the ribosome are so far the only ribozymes known to be conserved in all kingdoms of life. Eukaryotic RNase P activity has been detected in nuclei, mitochondria and chloroplasts and demonstrates great variability in sequence and subunit composition. In the last few years we have developed methodologies and pursued projects addressing the occurrence, distribution and the potential physiological role of RNase P in human epidermal keratinocytes. In view of the vital importance of lymphocytes for an effective immune system and their successful application after transfection with RNase P-associated external guide sequences in gene therapy, we concerned ourselves with the isolation and characterization of RNase P of peripheral human lymphocytes. We developed a method described herein, that will enable the study of the possible involvement of this ribozyme in the pathogenetic mechanisms of diverse autoimmune, inflammatory and neoplastic cutaneous disorders and may facilitate the further development of RNase P-based technology for gene therapy of infectious and neoplastic dermatoses.

RNA molecules play critical roles in cell biology, and novel findings continuously broaden their functional repertoires. Apart from their well-documented participation in protein synthesis, it is now apparent that several noncoding RNAs (i.e., micro-RNAs and riboswitches) also participate in the regulation of gene expression. The discovery of catalytic RNAs had profound implications on our views concerning the evolution of life on our planet at a molecular level. A characteristic attribute of RNA, probably traced back to its ancestral origin, is the ability to interact with and be modulated by several ions and molecules of different sizes. The inhibition of ribosome activity by antibiotics has been extensively used as a therapeutical approach, while activation and substrate-specificity alteration have the potential to enhance the versatility of ribozyme-based tools in translational research. In this review, we will describe some representative examples of such modulators to illustrate the potential of catalytic RNAs as tools and targets in research and clinical approaches.

BACKGROUND: Validation of physiologic miRNA targets has been met with significant challenges. We employed HITS-CLIP to identify which miRNAs participate in liver regeneration, and to identify their target mRNAs.

RESULTS: miRNA recruitment to the RISC is highly dynamic, changing more than five-fold for several miRNAs. miRNA recruitment to the RISC did not correlate with changes in overall miRNA expression for these dynamically recruited miRNAs, emphasizing the necessity to determine miRNA recruitment to the RISC in order to fully assess the impact of miRNA regulation. We incorporated RNA-seq quantification of total mRNA to identify expression-weighted Ago footprints, and developed a microRNA regulatory element (MRE) prediction algorithm that represents a greater than 20-fold refinement over computational methods alone. These high confidence MREs were used to generate candidate 'competing endogenous RNA' (ceRNA) networks.

CONCLUSION: HITS-CLIP analysis provide novel insights into global miRNA:mRNA relationships in the regenerating liver.

RNase P is an essential and ubiquitous endonuclease that mediates the maturation of the 5' ends of all precursor tRNA molecules. The holoenzyme from Dictyostelium discoideum possesses RNA and protein subunits essential for activity, but the exact composition of the ribonucleoprotein complex is still under investigation. Bioinformatic analysis of D. discoideum genome identified seven open reading frames encoding candidate RNase P protein subunits. The gene named drpp30 encodes a protein with a predicted molecular mass of 40.7 kDa that clusters with Rpp1 and Rpp30 RNase P protein subunits from Saccharomyces cerevisiae and human respectively, which have significantly lower molecular masses. Cloning and heterologous expression of DRpp30 followed by immunochemical analysis of RNase P active fractions demonstrates its association with RNase P holoenzyme. Furthermore, we show that DRpp30 can bind D. discoideum RNase P RNA and tRNA transcripts in vitro, giving a first insight of its possible role in D. discoideum RNase P function. Homology modeling using as a template the archaeal Ph1887p, and molecular dynamics simulations of the modeled structure suggest that DRpp30 adopts a TIM-barrel fold.

Ribonuclease P is an essential enzyme that matures the 5' ends of all primary tRNA transcripts. RNase P enzymes contain a similar in size RNA subunit which is absolutely required for catalysis. The holoenzyme from Dictyostelium discoideum possesses an essential for activity RNA subunit but the exact protein composition is still under investigation. Bioinformatic analysis of D. discoideum sequencing data returned seven ORFs homologous to previously characterized RNase P protein subunits from human. In the present study, DRpp20 and DRpp40 were cloned and characterized. These proteins apart from the noted similarity possess idiosyncratic regions. Immunobiochemical analysis presented herein indicates their direct involvement in the formation of the ribonucleoprotein complex of D. discoideum RNase P holoenzyme.

Ribonuclease P (RNase P) is a key enzyme in tRNA biogenesis that catalyses the endonucleolytic cleavage of tRNA precursors and generates their mature 5' ends. The activity of this ribozyme has never been isolated from living human tissues and data about epidermal tRNA biogenesis are not available. The purpose of the present study was to isolate and purify RNase P from human epidermis and to investigate the in vitro effects of retinoids on its activity. Enzyme isolation and purification from homogenates of keratinocytes derived after trypsinization from dispase-separated human epidermis were carried out using phosphocellulose chromatography. The optimal activity of the enzyme was found at 100 mM NH4Cl and 5 mM MgCl2 at pH 7.5 and 37 degrees C. All-trans retinoic acid and acitretin revealed a dose-dependent inhibitory effect on RNase P activity. The isolation of RNase P activity from human epidermis, reported here for the first time, will enable the investigation of the possible involvement of this ribozyme in the regulation of epidermal differentiation and proliferation and the evaluation of its significance for the pathogenesis and gene therapy of various cutaneous disorders.

Catalytic RNAs, known as ribozymes, act as true enzymes and are implicated in important biological processes, such as protein synthesis, mRNA splicing, transcriptional regulation and retroviral replication. Ribozymes are capable of serving as a new molecular target for a variety of drugs and as a reliable screening system for their biological activity.

Heat shock protein 27 (Hsp27), apart from its protective function in response to stress, is implicated in the regulation of cell growth, differentiation and apoptosis. Data on the expression of Hsp27 in the developing human epidermis are sparse and partially conflicting. Thus, the purpose of the present study was to investigate Hsp27 expression during the morphogenesis of human epidermis. Skin biopsies and dispase-separated epidermal sheets obtained from 7 human embryos (7 and 8 weeks estimated gestational age, EGA), from 79 human fetuses (9-23 weeks EGA) and from 10 healthy adult volunteers were investigated by immunohistochemistry and Western blotting, respectively. The earliest detection of Hsp27 expression was found by immunohistochemistry at the 12th week EGA (basal and intermediate layer) and by Western blotting at the 9th week EGA. From the 16th to the 23rd week EGA immunoreactivity was not detectable in the basal layer, whereas in the overlying layers it revealed a differentiation-related pattern. The simultaneous onset of epidermal stratification and Hsp27 expression (9th week EGA) and the alterations of the latter in the subsequent stages of development, suggest that this stress protein may be involved in the molecular events underlying human epidermal morphogenesis.

The specific targeting and inactivation of gene expression represents nowadays the goal of the mainstream basic and applied biomedical research. Both researchers and pharmaceutical companies, taking advantage of the vast amount of genomic data, have been focusing on effective endogenous mechanisms of the cell that can be used against abnormal gene expression. In this context, RNA represents a key molecule that serves both as tool and target for deploying molecular strategies based on the suppression of genes of interest. The main RNA-mediated therapeutic methodologies, deriving from studies on catalytic activity of ribozymes, blockage of mRNA translation and the recently identified RNA interference, will be discussed in an effort to understand the utilities of RNA as a central molecule during gene expression.