Exosome mediated delivery of functional nucleic acid nanoparticles (NANPs).

RNAi-based technologies have shown biomedical potential; however, safe and efficient delivery of RNA remains a barrier for their broader clinical applications. Nucleic acid nanoparticles (NANPs) programmed to self-assemble and organize multiple therapeutic nucleic acids (TNAs) also became attractive candidates for diverse therapeutic options. Various synthetic nanocarriers are used to deliver TNAs and NANPs, but their clinical translation is limited due to immunotoxicity. Exosomes are cell-derived nanovesicles involved in cellular communication. Due to their ability to deliver biomolecules, exosomes are a novel delivery choice. In this study, we explored the exosome-mediated delivery of NANPs designed to target GFP. We assessed the intracellular uptake, gene silencing efficiency, and immunostimulation of exosomes loaded with NANPs. We also confirmed that interdependent RNA/DNA fibers upon recognition of each other after delivery, can conditionally activate NF-kB decoys and prevent pro-inflammatory cytokines. Our study overcomes challenges in TNA delivery and demonstrates future studies in drug delivery systems.

saRNA-guided Ago2 targets the RITA complex to promoters to stimulate transcription.

Small activating RNAs (saRNAs) targeting specific promoter regions are able to stimulate gene expression at the transcriptional level, a phenomenon known as RNA activation (RNAa). It is known that RNAa depends on Ago2 and is associated with epigenetic changes at the target promoters. However, the precise molecular mechanism of RNAa remains elusive. Using human CDKN1A (p21) as a model gene, we characterized the molecular nature of RNAa. We show that saRNAs guide Ago2 to and associate with target promoters. saRNA-loaded Ago2 facilitates the assembly of an RNA-induced transcriptional activation (RITA) complex, which, in addition to saRNA-Ago2 complex, includes RHA and CTR9, the latter being a component of the PAF1 complex. RITA interacts with RNA polymerase II to stimulate transcription initiation and productive elongation, accompanied by monoubiquitination of histone 2B. Our results establish the existence of a cellular RNA-guided genome-targeting and transcriptional activation mechanism and provide important new mechanistic insights into the RNAa process.

Inducing gene expression by targeting promoter sequences using small activating RNAs.

Vector-based systems comprised of exogenous nucleic acid sequences remain the standard for ectopic expression of a particular gene. Such systems offer robust overexpression, but have inherent drawbacks such as the tedious process of construction, excluding sequences (e.g. introns and untranslated regions) important for gene function and potential insertional mutagenesis of host genome associated with the use of viral vectors. We and others have recently reported that short double-stranded RNAs (dsRNAs) can induce endogenous gene expression by targeting promoter sequences in a phenomenon referred to as RNA activation (RNAa) and such dsRNAs are termed small activating RNAs (saRNAs). To date, RNAa has been successfully utilized to induce the expression of different genes such as tumor suppressor genes. Here, we describe a detailed protocol for target selection and dsRNA design with associated experiments to facilitate RNAa in cultured cells. This technique may be applied to selectively activate endogenous gene expression for studying gene function, interrogating molecular pathways and reprogramming cell fate.

Upregulation of Cyclin B1 by miRNA and its implications in cancer.

It is largely recognized that microRNAs (miRNAs) function to silence gene expression by targeting 3'UTR regions. However, miRNAs have also been implicated to positively-regulate gene expression by targeting promoter elements, a phenomenon known as RNA activation (RNAa). In the present study, we show that expression of mouse Cyclin B1 (Ccnb1) is dependent on key factors involved in miRNA biogenesis and function (i.e. Dicer, Drosha, Ago1 and Ago2). In silico analysis identifies highly-complementary sites for 21 miRNAs in the Ccnb1 promoter. Experimental validation identified three miRNAs (miR-744, miR-1186 and miR-466d-3p) that induce Ccnb1 expression in mouse cell lines. Conversely, knockdown of endogenous miR-744 led to decreased Ccnb1 levels. Chromatin immunoprecipitation (ChIP) analysis revealed that Ago1 was selectively associated with the Ccnb1 promoter and miR-744 increased enrichment of RNA polymerase II (RNAP II) and trimethylation of histone 3 at lysine 4 (H3K4me3) at the Ccnb1 transcription start site. Functionally, short-term overexpression of miR-744 and miR-1186 resulted in enhanced cell proliferation, while prolonged expression caused chromosomal instability and in vivo tumor suppression. Such phenotypes were recapitulated by overexpression of Ccnb1. Our findings reveal an endogenous system by which miRNA functions to activate Ccnb1 expression in mouse cells and manipulate in vivo tumor development/growth.

Distinct activities of several RNase J proteins in methanogenic archaea.

RNA degradation plays an important role in the control of gene expression in all domains of life, including Archaea. While analyzing RNA degradation in different archaea, we faced an interesting situation. The members of a group of methanogenic archaea, including Methanocaldococcus jannaschii, contain neither the archaeal exosome nor RNase II/R homologs. However, looking for potential ribonucleases revealed proteins related to the recently discovered ribonuclease RNase J. RNase J is unique among known ribonucleases because it may combine endo- and 5'→3' exo-ribonucleolytic activities in a single polypeptide. Here, we report the characterization of the ribonuclease activities of three RNase J homologs encoded in the genome of the methanogenic archaeon Methanocaldococcus jannaschii. The analysis of the recombinant archaeal proteins purified from E. coli revealed an optimal activity at 60°C. Whereas mjRNase J1 and -J3 displayed exclusively 5'→3' exonucleolytic activity, mjRNase J2 is an endonuclease with no apparent exonuclease activity. The exonucleolytic activity of both mjRNase J1 and -J3 is enhanced in molecules harboring monophosphate at the 5' end. mjRNase J3, and to some extent mjRNase J2, degrade ssDNA. Together, these results reveal that in archaea lacking the exosome and RNase II/R, RNA and perhaps also DNA are possibly degraded by the coordinated activities of several RNase J proteins. Unlike bacteria, in archaea RNase J proteins provide separately the exo- and endonucleolytic activities that are probably essential for RNA degradation.

Small RNA and transcriptional upregulation.

Small RNA molecules, such as microRNA and small interfering RNA, have emerged as master regulators of gene expression through their ability to suppress target genes in a phenomenon collectively called RNA interference (RNAi). There is growing evidence that small RNAs can also serve as activators of gene expression by targeting gene regulatory sequences. This novel mechanism, known as RNA activation (RNAa), appears to be conserved in at least mammalian cells and triggered by both endogenous and artificially designed small RNAs. RNAa depends on Argonaute proteins, but possesses kinetics distinct from that of RNAi. Epigenetic changes are associated with RNAa and may contribute to transcriptional activation of target genes, but the underlying mechanism remains elusive. Given the potential of RNAa as a molecular tool for studying gene function and as a therapeutic for disease, further research is needed to completely elucidate its molecular mechanism in order to refine the rules for target selection and improve strategies for exploiting it therapeutically. WIREs RNA 2011 2 748-760 DOI: 10.1002/wrna.90 For further resources related to this article, please visit the WIREs website.

Mycoplasma gallisepticum as the first analyzed bacterium in which RNA is not polyadenylated.

The addition of poly(A)-tails to RNA is a phenomenon common to almost all organisms. In addition to most eukaryotic mRNAs possessing a stable poly(A)-tail, RNA is polyadenylated as part of a degradation mechanism in prokaryotes, organelles, and the eukaryotic nucleus. To date, only very few systems have been described wherein RNA is metabolized without polyadenylation, including several archaea and yeast mitochondria. The minimal genome of the parasitic bacteria, Mycoplasma, does not encode homologs of any known polyadenylating enzyme. Here, we analyze polyadenylation in Mycoplasma gallisepticum. Our results suggest this organism as being the first described bacterium in which RNA is not polyadenylated.

Polynucleotide phosphorylase and the archaeal exosome as poly(A)-polymerases.

The addition of poly(A)-tails to RNA is a phenomenon common to almost all organisms. Not only homopolymeric poly(A)-tails, comprised exclusively of adenosines, but also heteropolymeric poly(A)-rich extensions, which include the other three nucleotides as well, have been observed in bacteria, archaea, chloroplasts, and human cells. Polynucleotide phosphorylase (PNPase) and the archaeal exosome, which bear strong similarities to one another, both functionally and structurally, were found to polymerize the heteropolymeric tails in bacteria, spinach chloroplasts, and archaea. As phosphorylases, these enzymes use diphosphate nucleotides as substrates and can reversibly polymerize or degrade RNA, depending on the relative concentrations of nucleotides and inorganic phosphate. A possible scenario, illustrating the evolution of RNA polyadenylation and its related functions, is presented, in which PNPase (or the archaeal exosome) was the first polyadenylating enzyme to evolve and the heteropolymeric tails that it produced, functioned in a polyadenylation-stimulated RNA degradation pathway. Only at a later stage in evolution, did the poly(A)-polymerases that use only ATP as a substrate, hence producing homopolymeric adenosine extensions, arise. Following the appearance of homopolymeric tails, a new role for polyadenylation evolved; RNA stability. This was accomplished by utilizing stable poly(A)-tails associated with the mature 3' ends of transcripts. Today, stable polyadenylation coexists with unstable heteropolymeric and homopolymeric tails. Therefore, the heteropolymeric poly(A)-rich tails, observed in bacteria, organelles, archaea, and human cells, represent an ancestral stage in the evolution of polyadenylation.

Detection and characterization of polyadenylated RNA in Eukarya, Bacteria, Archaea, and organelles.

The posttranscriptional addition of poly(A) extensions to RNA is a phenomenon common to almost all organisms. In eukaryotes, a stable poly(A) tail is added to the 3'-end of most nucleus-encoded mRNAs, as well as to mitochondrion-encoded transcripts in animal cells. In prokaryotes and organelles, RNA molecules are polyadenylated as part of a polyadenylation-stimulated RNA degradation pathway. In addition, polyadenylation of nucleus-encoded transcripts in yeast and human cells was recently reported to promote RNA degradation. Not only homopolymeric poly(A) tails, composed exclusively of adenosines, but also heteropolymeric poly(A)-rich extensions, which include the other three nucleotides as well, have been observed in bacteria, archaea, chloroplasts, and human cells. In most instances, the detection of nonabundant truncated transcripts with posttranscriptionally added poly(A) or poly(A)-rich extensions serves as a telltale sign of the presence of a polyadenylation-stimulated RNA degradation pathway. In this chapter, we describe several methods found to be efficient in detecting and characterizing polyadenylated transcripts in bacteria, archaea, organelles, and nucleus-encoded RNAs. Detailed protocols for the oligo(dT)- and circularized reverse transcription (cRT) PCR methods, as well as the ribonuclease digestion method, are outlined, along with examples of results obtained with these techniques.

Analysis of the human polynucleotide phosphorylase (PNPase) reveals differences in RNA binding and response to phosphate compared to its bacterial and chloroplast counterparts.

PNPase is a major exoribonuclease that plays an important role in the degradation, processing, and polyadenylation of RNA in prokaryotes and organelles. This phosphorolytic processive enzyme uses inorganic phosphate and nucleotide diphosphate for degradation and polymerization activities, respectively. Its structure and activities are similar to the archaeal exosome complex. The human PNPase was recently localized to the intermembrane space (IMS) of the mitochondria, and is, therefore, most likely not directly involved in RNA metabolism, unlike in bacteria and other organelles. In this work, the degradation, polymerization, and RNA-binding properties of the human PNPase were analyzed and compared to its bacterial and organellar counterparts. Phosphorolytic activity was displayed at lower optimum concentrations of inorganic phosphate. Also, the RNA-binding properties to ribohomopolymers varied significantly from those of its bacterial and organellar enzymes. The purified enzyme did not preferentially bind RNA harboring a poly(A) tail at the 3' end, compared to a molecule lacking this tail. Several site-directed mutations at conserved amino acid positions either eliminated or modified degradation/polymerization activity in different manners than observed for the Escherichia coli PNPase and the archaeal and human exosomes. In light of these results, a possible function of the human PNPase in the mitochondrial IMS is discussed.

RNA polyadenylation and degradation in different Archaea; roles of the exosome and RNase R.

Polyadenylation is a process common to almost all organisms. In eukaryotes, stable poly(A)-tails, important for mRNA stability and translation initiation, are added to the 3' ends of most mRNAs. Contrarily, polyadenylation can stimulate RNA degradation, a phenomenon witnessed in prokaryotes, organelles and recently, for nucleus-encoded RNA as well. Polyadenylation takes place in hyperthermophilic archaea and is mediated by the archaeal exosome, but no RNA polyadenylation was detected in halophiles. Here, we analyzed polyadenylation in the third archaea group, the methanogens, in which some members contain genes encoding the exosome but others lack these genes. Polyadenylation was found in the methanogen, Methanopyrus kandleri, containing the exosome genes, but not in members which lack these genes. To explore how RNA is degraded in the absence of the exosome and without polyadenylation, we searched for the exoribonuclease that is involved in this process. No homologous proteins for any other known exoribonuclease were detected in this group. However, the halophilic archaea contain a gene homologous to the exoribonuclease RNase R. This ribonuclease is not able to degrade structured RNA better than PNPase. RNase R, which appears to be the only exoribonucleases in Haloferax volcanii, was found to be essential for viability.

RNA polyadenylation in Archaea: not observed in Haloferax while the exosome polynucleotidylates RNA in Sulfolobus.

The addition of poly(A) tails to RNA is a phenomenon common to all organisms examined so far. No homologues of the known polyadenylating enzymes are found in Archaea and little is known concerning the mechanisms of messenger RNA degradation in these organisms. Hyperthermophiles of the genus Sulfolobus contain a protein complex with high similarity to the exosome, which is known to degrade RNA in eukaryotes. Halophilic Archaea, however, do not encode homologues of these eukaryotic exosome components. In this work, we analysed RNA polyadenylation and degradation in the archaea Sulfolobus solfataricus and Haloferax volcanii. No RNA polyadenylation was detected in the halophilic archaeon H. volcanii. However, RNA polynucleotidylation occurred in hyperthermophiles of the genus Sulfolobus and was mediated by the archaea exosome complex. Together, our results identify the first organism without RNA polyadenylation and show a polyadenylation activity of the archaea exosome.

Domain analysis of the chloroplast polynucleotide phosphorylase reveals discrete functions in RNA degradation, polyadenylation, and sequence homology with exosome proteins.

The molecular mechanism of mRNA degradation in the chloroplast consists of sequential events, including endonucleolytic cleavage, the addition of poly(A)-rich sequences to the endonucleolytic cleavage products, and exonucleolytic degradation. In spinach chloroplasts, the latter two steps of polyadenylation and exonucleolytic degradation are performed by the same phosphorolytic and processive enzyme, polynucleotide phosphorylase (PNPase). An analysis of its amino acid sequence shows that the protein is composed of two core domains related to RNase PH, two RNA binding domains (KH and S1), and an alpha-helical domain. The amino acid sequence and domain structure is largely conserved between bacteria and organelles. To define the molecular mechanism that controls the two opposite activities of this protein in the chloroplast, the ribonuclease, polymerase, and RNA binding properties of each domain were analyzed. The first core domain, which was predicted to be inactive in the bacterial enzymes, was active in RNA degradation but not in polymerization. Surprisingly, the second core domain was found to be active in degrading polyadenylated RNA only, suggesting that nonpolyadenylated molecules can be degraded only if tails are added, apparently by the same protein. The poly(A) high-binding-affinity site was localized to the S1 domain. The complete spinach chloroplast PNPase, as well as versions containing the core domains, complemented the cold sensitivity of an Escherichia coli PNPase-less mutant. Phylogenetic analyses of the two core domains showed that the two domains separated very early, resulting in the evolution of the bacterial and organelle PNPases and the exosome proteins found in eukaryotes and some archaea.

RNA polyadenylation and degradation in cyanobacteria are similar to the chloroplast but different from Escherichia coli.

The mechanism of RNA degradation in Escherichia coli involves endonucleolytic cleavage, polyadenylation of the cleavage product by poly(A) polymerase, and exonucleolytic degradation by the exoribonucleases, polynucleotide phosphorylase (PNPase) and RNase II. The poly(A) tails are homogenous, containing only adenosines in most of the growth conditions. In the chloroplast, however, the same enzyme, PNPase, polyadenylates and degrades the RNA molecule; there is no equivalent for the E. coli poly(A) polymerase enzyme. Because cyanobacteria is a prokaryote believed to be related to the evolutionary ancestor of the chloroplast, we asked whether the molecular mechanism of RNA polyadenylation in the Synechocystis PCC6803 cyanobacteria is similar to that in E. coli or the chloroplast. We found that RNA polyadenylation in Synechocystis is similar to that in the chloroplast but different from E. coli. No poly(A) polymerase enzyme exists, and polyadenylation is performed by PNPase, resulting in heterogeneous poly(A)-rich tails. These heterogeneous tails were found in the amino acid coding region, the 5' and 3' untranslated regions of mRNAs, as well as in rRNA and the single intron located at the tRNA(fmet). Furthermore, unlike E. coli, the inactivation of PNPase or RNase II genes caused lethality. Together, our results show that the RNA polyadenylation and degradation mechanisms in cyanobacteria and chloroplast are very similar to each other but different from E. coli.