The Role of ARID1A in Endometrial Cancer and the Molecular Pathways Associated With Pathogenesis and Cancer Progression.

AT-rich interaction domain 1A gene (ARID1A) encodes for a subunit of the switch/sucrose non-fermentable (SWI/SNF) complex, a chromatin remodeling complex, and it has been implicated in the pathogenesis of various cancer types. In this review, we discuss how ARID1A is linked to endometrial cancer and what molecular pathways are affected by mutation or inhibition of ARID1A. We also discuss the potential use of ARID1A not only as a prognostic biomarker, but also as a target for therapeutic interventions.

Construction of an M1GS Ribozyme for Targeted and Rapid mRNA Cleavage; Application on the Ets-2 Oncogene.

BACKGROUND: RNase P-mediated cleavage of target RNAs has been proposed as a promising tool for gene silencing. Ets-2 proto-oncogene controls the expression of a wide variety of genes involved in cancer and immunity. OBJECTIVE: Construction of a functional RNase P-based ribozyme (M1GS303) that targets Ets-2 mRNA. METHODS: The accessible sites for targeting of Ets-2 mRNA were identified by footprinting analysis. M1GS303 ribozyme was constructed by cloning. The activity of the ribozyme in the presence or absence of spiramysin in E. coli cells and human cell lines was quantified by RT-PCR. The efficiency of the ribozyme in silencing the endogenous expression of Ets-2 in human cell lines was examined by RT-PCR, western blot and immunofluorescence analysis. RESULTS: In E. coli cells co-transformed with plasmids bearing M1GS303 and the ets-2 target gene, Ets-2 mRNA was decreased by 93% 12h after IPTG induction in the absence, and after 4h in the presence of spiramycin. Ets-2 was rapidly downregulated in the human embryonic kidney cell line HEK293 and the T-cell line Jurkat transfected with an M1GS303 plasmid; the silencing effect of M1GS303 was considerably faster when the cells were cultured with spiramycin. In Jurkat cells, Ets-2-downregulation resulted in upregulation of the expression of IL-2, IL-4 and IFN-α cytokine genes that have Ets-2 binding sites on their promoters, whereas it had no effect on the expression of the IL-10 gene that lacks Ets-2 binding sites on its promoter. CONCLUSIONS: M1GS303 ribozyme cleaves effectively Ets-2 mRNA in bacteria and mammalian cells, and its activity is enhanced by spiramycin. Downregulation of ets-2 gene in the T-cell line Jurkat upregulates IL-2, IL-4 and IFN-α cytokine genes. M1GS technology may be a better alternative to conventional gene-interference therapies and the delineation of the effects of gene silencing in various pathologies.

The dual role of LSD1 and HDAC3 in STAT5-dependent transcription is determined by protein interactions, binding affinities, motifs and genomic positions.

STAT5 interacts with other factors to control transcription, and the mechanism of regulation is of interest as constitutive active STAT5 has been reported in malignancies. Here, LSD1 and HDAC3 were identified as novel STAT5a interacting partners in pro-B cells. Characterization of STAT5a, LSD1 and HDAC3 target genes by ChIP-seq and RNA-seq revealed gene subsets regulated by independent or combined action of the factors and LSD1/HDAC3 to play dual role in their activation or repression. Genes bound by STAT5a alone or in combination with weakly associated LSD1 or HDAC3 were enriched for the canonical STAT5a GAS motif, and such binding induced activation or repression. Strong STAT5 binding was seen more frequently in intergenic regions, which might function as distal enhancer elements. Groups of genes bound weaker by STAT5a and stronger by LSD1/HDAC3 showed an absence of the GAS motif, and were differentially regulated based on their genomic binding localization and binding affinities. These genes exhibited increased binding frequency in promoters, and in conjunction with the absence of GAS sites, the data indicate a requirement for stabilization by additional factors, which might recruit LSD1/HDAC3. Our study describes an interaction network of STAT5a/LSD1/HDAC3 and a dual function of LSD1/HDAC3 on STAT5-dependent transcription, defined by protein-protein interactions, genomic binding localization/affinity and motifs.

On the role of the appended P19 element in type A RNAs of bacterial RNase P.

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.

Domain architecture of the DRpp29 protein and its interaction with the RNA subunit of Dictyostelium discoideum RNase P.

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.

Altered occludin expression in brain capillaries induced by obstructive jaundice in rats.

The development of increased oxidative stress in the context of obstructive cholestasis has been proven in various rats' organs including the brain. The present study aimed to detect alterations of tight junction-associated occludin in rat brain capillaries after bile duct ligation (BDL). In experiment 1, occludin expression was evaluated by Western blot analysis in 5 animals 10 days after BDL and compared with 5 sham-operated ones. In experiment 2, groups of 9 animals each were used to assess occludin levels on the 1st, 5th, and 10th days after BDL and to associate these measurements with the in vivo superoxide radical production measured by means of an ultrasensitive fluorescent assay. The results indicated that occludin expression in BDL animals, as opposed to sham-operated, was significantly reduced at every time point studied, being lowest in the rats remaining on BDL condition for 10 days. Moreover, it was demonstrated that the time-dependent downregulation of occludin expression in the brain endothelial was significantly correlated with the time-dependent increase of brain superoxide radical level, implying a relationship between these two abnormalities. In conclusion, the evidence presented herein suggests the implication of occludin and, therefore, of blood-brain barrier in the pathophysiology of extrahepatic cholestasis.

Partial purification and characterization of RNase P from human peripheral lymphocytes.

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.

Insights into functional modulation of catalytic RNA activity.

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.

Activation of bacterial ribonuclease P by macrolides.

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.

RNA-mediated therapeutics: from gene inactivation to clinical application.

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.