Análise da expressão gênica de pequenos RNAs derivados de tRNAs (tRFs) em plantas

Small non coding RNAs emerged as important characters in several biology aspects. Among then, the most studied are microRNAs (miRNAs) and short interfering RNAs (siRNAs), that regulate their target gene post-transcriptionally in plants, animals and RNAi pathway intermediates, respectively. Both of classes have similar biogenesis being processed by Dicer enzymes and subsequent association with Argonaute enzymes. In plants, miRNAs and siRNAs have important functions in development, genome integrity and biotic and abiotic stress responses. The advances in high-throughtput sequencing and in silico analisys provide the uncover of new small non coding RNAs classes, many of them with unknown functions and biogenesis. tRNA derived small RNAs (tRFs) are a small non coding RNA class, that have as precursor a tRNA molecule. These were uncovers in the last decade in many organisms and, recently, in plants. Recent works detected tRFs from different sizes, with different source portions of the mature tRNA molecule (5’ end; 3’ end, anti-codon loop) and some from the tRNA precursor (pre-tRNA), suggesting that may be a novel class of small RNA and not random degradation products. Works in humans showed that some tRFs are processed by the Dicer enzymes, have association with the Argonaute enzymes and cell differentiation, tumor appearance and gene silencing related functions. Works in Arabidopsis and pumpkin (Cucurbita maxima) showed, respectively, that the tRFs have nutritional stress response possible functions and long distance signaling function between source and drain tissues, and may affect the translation. The tRFs biogenesis in plants are, until now an unknown, absence information about it in the literature and its possible biological functions are few studied yet, making then interesting target for studies among the small non coding RNAs in plants

Efficient RNAi-induced Protein Knockdown in Somatic Cells Using Diced or Chemically Produced Small Interfering RNAs (siRNA)

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specific corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specific mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most efficient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specific and unique siRNA sequences (Stealth RNai (TM)). Materials, Methods & Results: For in vitro-produced siRNAs, two segments of the human Ku70 (167 bp in exon 5; and 249 bp in exon 13; NM001469) and Xrcc4 (172 bp in exon 2; and 108 bp in exon 6; NM003401) genes were chosen to generate dsRNA for subsequent "Dicing" to create mixtures of siRNAs. The Diced fragments of siRNA for each gene sequence were pooled and stored at -80 degrees C. Alternatively, chemically synthesized Stealth siRNAs were designed and generated to match two very specific gene sequence regions for each target gene of interest (Ku70 and Xrcc4). HCT116 cells were plated at 30% confluence in 24- or 6-well culture plates. The next day, cells were transfected by lipofection with either Diced or Stealth siRNAs for Ku70 or Xrcc4, in duplicate, at various doses, with blank and sham transfections used as controls. Cells were harvested at 0, 24, 48, 72 and 96 h post-transfection for protein determination. The knockdown of specific targeted gene products was quantified by Western blot using GAPDH as control. Transfection of gene-specific siRNA to either Ku70 or Xrcc4 with both Diced and Stealth siRNAs resulted in a down regulation of the targeted proteins to approximately 10 to 20% of control levels 48 h after transfection, with recovery to pre-treatment levels by 96 h. Discussion: By transfecting cells with Diced or chemically synthesized Stealth siRNAs, Ku70 and Xrcc4, two highly expressed proteins in cells, were effectively attenuated, demonstrating the great potential for the use of both siRNA production strategies as tools to perform loss of function experiments in mammalian cells. In fact, down-regulation of Ku70 and Xrcc4 has been shown to reduce the activity of the non-homologous end joining DNA pathway, a very desirable approach for the use of homologous recombination technology for gene targeting or knockout studies. Stealth RNAi (TM) was developed to achieve high specificity and greater stability when compared with mixtures of enzymatically-produced (Diced) siRNA fragments. In this study, both siRNA approaches inhibited the expression of Ku70 and Xrcc4 gene products, with no detectable toxic effects to the cells in culture. However, similar knockdown effects using Diced siRNAs were only attained at concentrations 10-fold higher than with Stealth siRNAs. The application of RNAi technology will expand and continue to provide new insights into gene regulation and as potential applications for new therapies, transgenic animal production and basic research.

Metodologie per l'analisi statistica di dati di sequencing relativi a un esperimento di rna interference

La RNA interference è un processo attraverso il quale alcuni piccoli frammenti di RNA (19-25 nucleotidi) sono in grado di silenziare l'espressione genica. La sua scoperta, nel 1998, ha rivoluzionato le concezioni della biologia molecolare, minando le basi del cosiddetto Dogma Centrale. Si è visto che la RNAi riveste ruoli fondamentali in meccanismi di regolazione genica, nello spegnimento dell'espressione e funziona come meccanismo di difesa innata contro varie tipologie di virus. Proprio a causa di queste implicazioni richiama interesse non solo dal punto di vista scientifico, ma anche da quello medico, in quanto potrebbe essere impiegata per lo sviluppo di nuove cure. Nonostante la scoperta di tale azione desti la curiosità e l'interesse di molti, i vari processi coinvolti, soprattutto a livello molecolare, non sono ancora chiari. In questo lavoro si propongono i metodi di analisi di dati di un esperimento prodotto dall'Istituto di Biologia molecolare e cellulare di Strasburgo. Nell'esperimento in questione vengono studiate le funzioni che l'enzima Dicer-2 ha nel pathway - cioè la catena di reazioni biomolecolari - della RNA interference durante un'infezione virale nel moscerino della frutta Drosophila Melanogaster. Per comprendere in che modo Dicer-2 intervenga nel silenziamento bisogna capire in quali casi e quali parti di RNA vengono silenziate, a seconda del diverso tipo di mutazione dell'enzima stesso. Dunque è necessario sequenziare l'RNA nelle diverse condizioni sperimentali, ottenendo così i dati da analizzare. Parte dei metodi statistici che verranno proposti risultano poco convenzionali, come conseguenza della peculiarità e della difficoltà dei quesiti che l'esperimento mette in luce. Siccome le tematiche affrontate richiedono un approccio sempre più interdisciplinare, è aumentata considerevolmente la richiesta di esperti di altri settori scientifici come matematici, informatici, fisici, statistici e ingegneri. Questa collaborazione, grazie a una diversità di approccio ai problemi, può fornire nuovi strumenti di comprensione in ambiti che, fino a poco tempo fa, rientravano unicamente nella sfera di competenza dei biologi.

Pdsg1 and Pdsg2, novel proteins involved in developmental genome remodelling in Paramecium

The epigenetic influence of maternal cells on the development of their progeny has long been studied in various eukaryotes. Multicellular organisms usually provide their zygotes not only with nutrients but also with functional elements required for proper development, such as coding and non-coding RNAs. These maternally deposited RNAs exhibit a variety of functions, from regulating gene expression to assuring genome integrity. In ciliates, such as Paramecium these RNAs participate in the programming of large-scale genome reorganization during development, distinguishing germline-limited DNA, which is excised, from somatic-destined DNA. Only a handful of proteins playing roles in this process have been identified so far, including typical RNAi-derived factors such as Dicer-like and Piwi proteins. Here we report and characterize two novel proteins, Pdsg1 and Pdsg2 (Paramecium protein involved in Development of the Somatic Genome 1 and 2), involved in Paramecium genome reorganization. We show that these proteins are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development. Our results suggest an association between RNA-mediated trans-generational epigenetic signal and chromatin modifications in the process of Paramecium genome reorganization.

INVESTIGATING THE ROLES OF THE p63 ISOFORMS IN THE MICRORNA BIOGENESIS PATHWAY

MicroRNAs play roles in various biological processes like development, tumorigenesis, metastasis and pluripotency. My thesis work has demonstrated roles for p63, a p53 family member, in the upstream regulation of microRNA biogenesis. The p63 gene has a complex gene structure and has multiple isoforms. The TAp63 isoforms contain an acidic transcription activation domain. The ΔNp63 isoforms, lack the TA domain, but have a proline rich region critical for gene transactivation. To understand the functions of these isoforms, the Flores lab generated TAp63 and ΔNp63 conditional knock out mice. Using these mice and tissues and cells from these mice we have found that TAp63 transcriptionally regulates Dicer while ΔNp63 transcriptionally regulates DGCR8. TAp63 -/- mice are highly tumor prone. These mice develop metastatic mammary adenocarcinomas, squamous cell carcinomas, and lung adenocarcinomas to distant sites including the liver, lungs, and brain. I found that TAp63 suppresses metastasis by transcriptionally activating Dicer. TAp63 and Dicer levels were very low or lost in high grade human tumors like mammary adenocarcinomas, squamous cell carcinomas, and lung adenocarcinomas. Expression of Dicer in these tumor cell lines reduced their invasiveness. Using ΔNp63 -/- mice, I found that ΔNp63 transcriptionally activates DGCR8, resulting in a miRNA profile that is critical to reprogram cells to pluripotency. Analysis of epidermal cells derived from ΔNp63 -/- mice revealed that these cells expressed markers of pluripotency, including Sox2, Oct 4 and Nanog; however, genome-wide analysis revealed a novel profile of genes that are common between ΔNp63 -/- epidermal cells and embryonic stem cells. I also found that mouse cells depleted of ΔNp63 form chimeric mice and teratomas in SCID mice, demonstrating that ΔNp63 deficient cells are pluripotent. Further, I found that restoration of DGCR8 in ΔNp63 -/- epidermal cells reduces their pluripotency and induces terminal differentiation. I also demonstrated that iMS (induced multipotent stem) cells could be generated using human keratinocytes by knockdown of ∆Np63 or DGCR8. Taken together, my work has placed p63 and its isoforms at a critical node in controlling miRNA biogenesis.

RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway

RNA interference (RNAi) is widely used to silence genes in plants and animals. it operates through the degradation of target mRNA by endonuclease complexes guided by approximately 21 nucleotide (nt) short interfering RNAs (siRNAs). A similar process regulates the expression of some developmental genes through approximately 21 nt microRNAs. Plants have four types of Dicer-like (DCL) enzyme, each producing small RNAs with different functions. Here, we show that DCL2, DCL3 and DCL4 in Arabidopsis process both replicating viral RNAs and RNAi-inducing hairpin RNAs (hpRNAs) into 22-, 24- and 21 nt siRNAs, respectively, and that loss of both DCL2 and DCL4 activities is required to negate RNAi and to release the plant's repression of viral replication. We also show that hpRNAs, similar to viral infection, can engender long-distance silencing signals and that hpRNA-induced silencing is suppressed by the expression of a virus-derived suppressor protein. These findings indicate that hpRNA-mediated RNAi in plants operates through the viral defence pathway.

PAMP-triggered immunity against Pseudomonas syringae involves microRNA-mediated regulation of several uncharacterized R genes

Two main types of noncoding small RNA molecules have been found in plants: microRNAs (miRNAs) and small interfering RNAs (siRNAs). They differ in their biogenesis and mode of action, but share similar sizes (20-24 nt). Their precursors are processed by Dicer-Like RNase III (dcl) proteins present in Arabidopsis thaliana, and in their mature form can act as negative regulators of gene expression, being involved in a vast array of plant processes, including plant development, genomic integrity or response to stress. Small-RNA mediated regulation can occurs at transcriptional level (TGS) or at post-transcriptional level (PTGS). In recent years, the role of gene silencing in the regulation of expression of genes related to plant defence responses against bacterial pathogens is becoming clearer. Comparisons carried out in our lab between the expression profiles of different mutants affected in gene silencing, and plants challenged with Pseudomonas syringae pathovar tomato DC3000, led us to identify a set of uncharacterized R genes, belonging to the TIR-NBS-LRR gene family, differentially expressed in these conditions. Through the use of bioinformatics tools, we found a miRNA* of 22 nt putatively responsible for down-regulating expression of these R genes through the generation of siRNAs. We have also found that the corresponding pri-miRNA is down-regulated after PAMP-perception in a SA-dependent manner. We also demonstrate that plants with altered levels of miRNA* (knockdown lines or overexpression lines) exhibit altered PTI-associated phenotypes, suggesting a role for this miRNA* in this defence response against bacteria. In addition we identify one of the target genes as a negative regulator of defence response against Pseudomonas syringae.

Diseño de un vector no viral basado en nanopartículas lipídicas para el tratamiento de la hepatitis C mediante ARN de interferencia

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Targeting cancer cells using LNA-modified aptamer-siRNA chimeras

Aptamers are chimerized with drug or antisense oligos or nanoparticles to generate targeted therapeutics for cancer. Aptamer chimerized siRNA rescues nonspecific delivery and, thereby, enhances the availability of siRNA to target cells. EpCAM RNA aptamer (EpApt or Ep) has potential for siRNA chimerization due to its secondary structure. Stathmin and survivin proteins are reported to aid oncogenicity in retinoblastoma (RB), breast cancer and other cancers. Thus, chimerization of EpCAM Apt with siRNA against survivin and stathmin, respectively, was performed by incorporating Locked Nucleic Acid (LNA) modification. The LNA-modified chimeric aptamers were stable until 96 h and got internalized into RB, WERI-Rb1 and breast cancer, MDAMB453 cell lines. The constructs were studied using the recombinant dicer enzyme for the siRNA generation. Quantitative polymerase chain reaction and immunofluorescence by microscopic analysis of chimeras in vitro exhibited silencing of stathmin and survivin in the RB and breast cancer model. The chimeric constructs showed significant inhibition of cell proliferation of breast cancer cells. Thus, LNA-modified aptamer-based siRNA delivery aids in cell targeting and necessitates further studies in animal models.