RNA interference (RNAi) is a diverse, conserved regulatory mechanism in eukaryotic cells, which silences the target gene expression in a homology-dependent manner. Although it has been well documented that RNAi is an antiviral mechanism in plants and insects, it is still unclear whether RNAi naturally limits viral infections in vertebrates. Viruses are masters of adopting strategies to subvert cellular defense mechanisms. Not only can viruses use elaborate strategies to suppress the effects of defensive RNAi, but they can also redirect or interfere with cellular functions orchestrated by endogenous small RNAs. In our work we have focused on studying the relationship of human adenovirus type 5 (Ad5) infection and the RNAi/miRNA pathways. We show that Ad5 infection inhibits RNAi by blocking the activity of Dicer and the RNA-induced silencing complex (RISC). For Dicer inhibition, the virus-associated RNAs, VA RNAI and VA RNAII bind Dicer through their terminal stems and are cleaved by Dicer into functional small RNAs that are incorporated into active RISC. Furthermore, by cloning small RNAs, we found that approximately 80% of Ago2-containing RISC immunopurified from late infected cells was associated with VA RNA-derived small RNAs (mivaRNAs). Interestingly, the small RNAs derived from VA RNAII, the minor VA RNA species, appear to be the major mivaRNAs occupying RISC and associate with polyribosomes, which indicates their potential roles as miRNAs regulating translation of cellular mRNAs. During our previous work, we observed that the strand bias of VA RNAI derived small RNA (mivaRI) incorporating into active RISC varied in the different viable Ad5 mutant viruses infected cells. It has been reported that Ad5 VA RNAI had two transcription initiation sites, which produced two clusters of VA RNAI with 3 nt difference at their 5’ end. Our data show that this heterogeneity resulted in a dramatic difference in mivaRI guide strand selection. Collectively, our data contributes to understanding the interplay between virus and host. This study would be beneficial in designing optimal adenovirus vectors for therapeutic RNAi application.

MicroRNAs (miRNAs) play a vital role in differentiation, proliferation and tumorigenesis by binding to messenger RNAs (mRNA) and inhibiting translation. To initiate an investigation into the identification of miRNAs in the domestic dog, an emerging model for human disease, a comparison of the human and canine genetic databases was conducted. The bioinformatics work revealed significant conservation of miRNA genes between the two species. Proof of principle experiments, including serial dilutions and sequencing, were performed to verify that primers made to amplify human mature miRNAs can be used to amplify canine miRNAs, providing that the mature sequences are conserved. TaqMan® Real-time RT-PCR, a sensitive and specific method, was used to isolate the first miRNA mature products from canine tissues. The expression levels of miR-17-3p, miR-17-5p, miR-18, miR-19a, miR-19b, miR-20, and miR-92 were evaluated in five canine tissues (heart, lung, brain, kidney, and liver). Because miRNAs have been found to act as both tumor suppressors and oncogenes in several different cancers, expression patterns of ten miRNAs (miR-15a, miR-16, miR-17-5p, miR-21, miR-29b, miR-125b, miR-145, miR-155, miR-181b, let-7f) known to be associated with human breast cancer were compared between malignant canine mammary tumors (n=6) and normal canine mammary tissue (n=10). Resulting data revealed miR-29b and miR-21 to have a statistically significant (p<0.05) up-regulation in cancerous samples. Overall expression patterns showed nine of the ten miRNAs follow the same pattern of expression in the domestic dog as the human, while the miR-145 expression does not show a difference between the normal and cancerous samples.

RNA interference, or RNAi, is a cellular mechanism that describes the sequence-specific post transcriptional gene silencing observed in plants, fungi, and metazoans, facilitated by short double-stranded RNAs and microRNAs (miRNAs) with sequence complementarity to target mRNAs. Many of the regulatory mechanisms of the RNAi pathway by which these small miRNAs are first processed, from primary transcripts to precursor miRNA stemloops and then to mature miRNAs, by the multiprotein complexes Drosha and Dicer, respectively, still remain unknown. Within the miRNA biogenesis pathway, there is strong evidence pointing to the terminal loop region as an important regulatory determinant of miRNA maturation. To further elucidate the terminal loop's exerted control over miRNA processing, we propose a combined in vitro / in vivo selection experiment of a randomized pri-miRNA terminal loop library in search of an optimally processed pre-miRNA substrate. Here, we report the isolation of a premiRNA terminal loop sequence that is favorably processed by Drosha in vivo but also functions as an effective cis-inhibitor of further pre-miRNA processing by downstream Dicer. This terminal loop also demonstrated modular properties of Dicer inhibition in two different miRNAs, and should prove useful in further elucidating the mechanisms of miRNA processing in context of a newly proposed Dicer cleavage model (Gu et al. 2012). In combination, these findings may have important implications in both Drosha and Dicer's direct role in gene expression and miRNA biogenesis, the regulatory proteins that modulate their respective functions, as well as the potential development of new design rules for the more efficient processing and targeting of miRNA-based technology and RNAi therapeutics. / text

RNA molecules isolated from FFPE samples are highly fragmented and modified, and generally deemed unsuitable for downstream gene expression profiling. With the development of molecular biology, there has been growing interest in profiling archival FFPE samples. Successful profiling of transcripts from FFPE samples would greatly expand tissue sources for large scale gene expression studies; also it would pave the way for future applications on the type of tissue readily available in the clinical setting. So far, there is a lack of systemic studies evaluating the quality of RNA isolated from routinely processed FFPE samples, and it has remained difficult to assess how well FFPE-derived RNA mirrors the status of RNA isolated before fixation. In this project, the similarity of miRNA and mRNA profiles between matched frozen and FFPE lymphoid hyperplasia tissues (N=7 for miRNA comparison, N=4 for mRNA comparison) were evaluated. We found consistently good correlation (mean of Pearson coefficient=0.939, mean of Spearman coefficient=0.905, mean of Kendall tau=0.744) between matched frozen and FFPE-derived miRNA profiles, suggesting FFPE samples may retain miRNA expression information quite well. This has major positive implications for research using FFPE samples, as miRNA profiling becomes more prominent in bioprofiling studies. On the contrary, mRNA isolated from FFPE samples showed less correlation (Spearman coefficient less than 0.75) with its frozen counterpart on the Agilent microarray platform. With a post extraction heat treatment aimed at reversing base modifications and cross linking structures, obvious global mRNA quality improvement was observed in cases where samples appeared to be heavily cross linked, but was less effective and even detrimental in cases where cross linking was less prominent. This research suggests that the extent of cross linking may be critical in terms of determining whether a particular FFPE tissue will become a useful source of mRNA for global profiling studies / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-09-26 10:49:50.044

Prion diseases are curious neurodegenerative diseases characterized by the conversion of a cellular protein, PrPC, into an infectious isoform, PrPSc. One of the earliest hallmarks of disease and concurrent with prion deposition, is the activation of the brain’s principal immune effector cells, microglia. In prion disease, activated microglia synthesize fairly low levels of pro-inflammatory cytokines, presumably to ameliorate the severe pathology that can arise in host tissue as a result of an acute inflammatory response. The specific stimuli and signaling pathways that lead to this modulation of function are as yet unknown. However, the involvement of miRNAs, a recently identified class of regulatory molecules, is likely. Recently, miR-146a was found to be upregulated in the brains of prion infected mice. In addition, its expression was found to be enriched in cells of microglial origin. It was hypothesized that, given the immunomodulatory role ascribed to miR-146a in macrophages, upregulation of miR-146a may function to attenuate the microglial immune response to prion infection. The first objective was to identify inflammatory related miRNAs associated with prion disease in microglia. Using Taqman Low Density Arrays, allowing for the detection of hundreds of miRNAs at once, the miRNAs of microglia treated with inflammatory agonists were profiled. The miRNA profile of activated microglia was found to be similar to that of macrophages. Furthermore, among the miRNAs profiled, miR-146a and miR-155 were the most highly induced and persistently expressed over 24 hours. The second objective was to investigate miR-146a induction. Therefore, microglia were treated with various agonists and miR-146a expression was determined using Taqman miR-146a assays. Although treatment with a PrP-mimic did not induce miR-146a expression, stimulation of TLRs 1, 2, 4, and 5, resulted in significant over-expression similar to what has been described previously. Moreover, in contrast to the rapid and transient induction of inflammatory mediators, miR-146a follows alternate kinetics functioning to prolong the dampening of the innate immune response following activation via TLR4 and TLR2. By employing a functional proteomic strategy, the third objective was to identify miR-146a regulated proteins. First, miR-146a expression was manipulated using miR-146a mimics and miR-146a inhibitors. Secondly, the functional model was validated by confirming decreased expression of IL6 by ELISA in miR-146a over-expressing microglia cells. Lastly, using Tandem Mass Tag labels to discriminate between treatment group (miR-146a mimic and TLR2 agonist) and control group (scrambled-miR and TLR2 agonist), the effect of miR-146a on the proteome was determined. In total, 172 proteins were identified as being miR-146a regulated and gene ontology assignment resulted in an over-representation of proteins involved in cellular dynamics capable of altering the activation state of microglia. After filtering for bioinformatically predicted targets and those implicated in a similar genomic study, it was decided to further investigate proteins ARF6, RhoA and NOS2 based on their role in modulating the phagocytic potential of microglia. The final objective was to validate miR-146a putative direct targets identified from the proteomics analysis. Luciferase expression of the 3’UTR of targets upon transfection with miR-146a were determined. Based on luciferase analysis, NOS2 appears to be directly targeted by miR-146a and this was also confirmed by western blot. While production of NOS2 by microglia under an acute activation state serves to support and protect CNS homeostasis, chronic expression of this factor can lead to neurotoxicity. Therefore, miR-146a appears to have an overarching role in altering microglial activation during prion disease thus protecting neurons from bystander damage. Taken together, these results suggest that miR-146a could play an important role in the prion disease process by specifically attenuating the microglial induced immune response. Therefore, manipulation of miR-146a may represent a novel therapeutic strategy. Furthermore, given that miR-146a de-regulation has been observed in other neurodegenerative diseases, these results may well extend beyond the realm of prion disease. Lastly, although practical limitations relating to the sensitivity of the comparative proteomics methodology meant that it alone were not sufficient to identify miRNA targets, an integrated approach that takes into consideration genomic and bioinformatic strategies is most promising.

miRNAs are a species of small-regulatory RNA that post-transcriptionally regulate gene expression via the RNA induced silencing complex (RISC). They are encoded ubiquitously among animals and plants, and have recently been shown to be encoded by the majority of herpesviruses. It seems likely that herpesvirus encoded miRNAs have evolved as a tool for the manipulation of host-cellular and viral-gene expression during infection. Human cytomegalovirus (HCMV) is a clinically important herpesvirus that represents a significant cause of morbidity and mortality in the immune-compromised. HCMV encodes as many as 25 miRNAs during infection, but the function of the majority of these is not known. Identifying the targets of HCMV miRNAs will not only establish a basis for understanding the role of miRNAs within the context of HCMV infection, but also provide a means for discovering novel host-virus interactions. Using RISC immunoprecipitation and siRNA screening, host-cellular targets of viral miRNAs that play important roles in the biology of HCMV were identified. ATP6VOC, a key component of the vacuolar-ATPase, was shown to be a target of miR-US25-1 and subsequent siRNA knockdown of ATP6VOC resulted in the almost complete inhibition of infectious virion production. Despite this, ATP6VOC knock-down did not inhibit viral entry, DNA synthesis, or gene expression, highlighting a possible role for ATP6VOC in the assembly and egress of HCMV. A critical step in HCMV assembly and egress is the formation of the juxta-nuclear virion assembly compartment (VAC). The HCMV VAC is derived from host-cellular endocytic and secretory vacuoles, and is crucial for the efficient nuclear egress of nucleocapsids, cyotplasmic tegumentation, final envelopment, and the egress of mature virions. Using siRNA knock-down, immunofluorescence-microscopy, and western-blot analysis, a crucial role for ATP6VOC and v-ATPase function in the formation of the VAC was demonstrated. siRNA knock-down of ATP6VOC resulted in a failure in the reorganisation of trans-golgi and early-endosomal compartments during infection, resulting in a failure in VAC formation. These findings demonstrate a crucial role for ATP6VOC during infection, and in so doing identify a novel host factor that is required for HCMV assembly.

Two types mechanisms that control gene expression involve cis-regulatory factors and trans-regulatory factors. Cis-acting regulatory RNAs include targeted messenger RNA (mRNA) specificity and AU-rich elements (AREs). AU-rich mRNAs are a subcategory of mRNAs that have AREs in their 3'-Untranslated Regions (UTRs). These ARE-genes have been observed to correlate with rapid mRNA decay patterns. They comprise approximately 12% of all transcripts and are known to encode for a group of proteins that have involvement in the inflammatory response. Trans-acting regulatory mechanisms are micro RNAs (miRNAs) in eukaryotes, and small RNAs (sRNA) in prokaryotes. Misregulation of these mechanisms can lead to many disease states if rapid mRNA decay does not occur, leading to tumorigenesis, and eventually, different types of cancer. In this project, the TNF-α ARE was studied in both serum-positive and quiescent G0 conditions in order to analyze whether the translation of the gene differed in any respect due to the binding of a known miRNA called miR-130a. Additionally, both serum-positive and one-day serum-starved quiescent G0 conditions were analyzed for eIF5B and FXR1 levels to analyze whether there was a correlation between the two proteins.

MicroRNAs (miRNAs) have been identified as a key regulator in various biological processes and different diseases including cancer, heart disease, and viral infections. In the context of virus-host interactions, previous genome wide functional screen involving overexpression and inhibition of murine miRNAs in vitro identified several miRNAs that suppressed viral replication in diverse herpesviruses including herpes simplex virus 1 (HSV-1), murine cytomegalovirus (MCMV) and murine gamma herpesvirus 68 (MHV-68) (Santhakumar, D. et al, 2010). One of the top broad-spectrum anti-viral miRNAs, miR-542-5p, also suppressed human cytomegalovirus (HCMV) as well as a Semliki Forest virus (SFV) and two subtypes of influenza A virus (H1N1 & H3N2) in vitro. Following the previous study, this thesis focuses on generation of in vivo anti-viral efficacy data using miR-542-5p against two diverse viruses: MCMV and influenza (H1N1) in a pre-clinical model (mouse). One of the key challenges for generating in vivo efficacy data with miRNAs is the choice of delivery vehicle. To address this issue the first part of the project focused on optimising delivery conditions (dose, route of administration) for miR-542-5p mimic to target the lungs of mice (as both MCMV and H1N1 replicate in lungs). Initially, delivery was optimised using two cationic polymers: linear polyethylenimine (in vivo-JetPEI) and branched polyethylenimine (25KDa bPEI) that have been widely used previously to deliver nucleic acids in mouse. In parallel, two novel delivery systems were tested as an alternative delivery vehicle for miRNA mimics: biodegradable cationic lipids (Lifectin) and exosomes, natural vesicles produced by cells that can transport RNA. Results from in vivo delivery studies indicate that nebulisation of miR-542-5p mimic complexed with bPEI (25KDa) showed a more significant increase in the level of miRNA in the lung compared to other delivery systems and did not result in an immune response. Using bPEI as the delivery vehicle, the miR-542-5p mimic was administered to mouse lungs to test its anti-viral function against H1N1 and MCMV. Delivery of the miR-542-5p mimic resulted in 4.6 fold reduction of H1N1 virus titre in lungs (averaged across multiple experiments). The miR-542-5p mimic also had a 2 fold reduction in MCMV titre in the lungs. These data confirm the broad-spectrum anti-viral effect of miR-542-5p in mouse as observed in previous in vitro studies. Preliminary microarray analysis of genes regulated by miR-542-5p in vitro suggest this miRNA may target multiple genes required by diverse viruses during their life cycles and may modulate the PI3K-Akt signalling pathway.

Many cellular functions depend on the tightly regulated expression of various proteins. Canonical control of the protein expression is associated with transcriptional regulation. However, the small non-coding RNAs called microRNAs (miRNAs) were identified as post-transcriptional regulators of gene expression. In a typical manner, miRNAs originate similarly to the coding RNAs and are processed in a multi-step maturation process. It has been shown that miRNAs are very important for the proper functioning of tissues. Interestingly, the human nervous system contains over 70% of all miRNAs; thus, the maturation process has to be tightly regulated. However, despite the important role of miRNAs, little is known about the mechanisms regulating their biogenesis. In my PhD project, I showed that during early stages of neuronal differentiation, Lin28a controls levels of neuro-specific miRNA-9. I demonstrated that Lin28a binds to the conserved terminal loop (CTL) of pre-miRNA-9 and decreases the cellular levels of miRNA-9 during retinoic acid-mediated neuronal differentiation of mouse teratocarcinoma P19 cells. I revealed that the Lin28a-mediated inhibition of miRNA-9 production was uridylation-independent. Furthermore, constitutive expression of GFP-tagged Lin28a reduced the levels of let-7a but not miRNA-9, whereas untagged Lin28a inhibited both miR-9 and let-7a during the course of neuronal differentiation. Using small RNAseq analysis of P19 cells with constitutive expression of Lin28a I showed that it controls many more miRNAs than previously recognised. Intriguingly, many miRNAs were upregulated by Lin28a overexpression. I demonstrated with high-throughput, the limited function of GFP-tagged Lin28a results, and I also showed that untagged Lin28a inhibits the production of a number of brain-specific miRNAs including miRNA-9. Finally, I revealed that 3’-5’exoribonuclease Dis3l2 was responsible for uridylation-independent degradation of pre-miRNA-9. Altogether, my results provided evidence that Lin28a has both positive and negative roles in the regulation of miRNA production and has a dual role in triggering pre-miRNA degradation.

L’objectif de mon projet principal de thèse est de déterminer la fonction biologique d’un lncARN conservés chez le zebrafish que nous avons appelé libra. La séquence de libra étant hautement homologue à la région 3’UTR de la protéine Nrep. Ces deux transcrits, libra et Nrep, contiennent en effet un site de liaison au miARN profondément conservé et inhabituellement complémentaire au miR-29. En utilisant à le modèle souris et les cellules murines, nous avons décrypté la relation régulatrice entre ce transcrit conservé dans l’évolution des vertébrés et la voie métabolique des miARN. Nous avons montré que Nrep limite le domaine d’expression de miR-29 au cervelet, et qu’il le déstabilise en rognant sa séquence. Notre travail révèle donc le premier exemple de dégradation endogène ciblée des miARN (ou TDMD). De plus, un ensemble d’expériences in vivo sur les modèles zebrafish et souris, nous a permis de démontrer que libra et Nrep contrôlent tout les deux le comportement animal. Via la perturbation génétique du site de liaison au miARN de Nrep murin, nous avons observé que ce gène régule le dosage du miR29 de part son site de liaison aux miARN, et que cette régulation est nécessaire à un comportement animal normal. Dans la seconde partie de ma thèse, je décris une stratégie exploré afin de déréguler les lncARN de la manière la moins invasive possible. Les lncARN sont actuellement neutralisés par des approches qui introduisent de vastes changements de séquence au niveau génomique. Nous avons donc développer une stratégie in vivo, appliquée au zebrafish, qui inactive les lncARN via l’insertion génomique d’une séquence ribozyme autoclivante ou d’un signal polyA prématuré. / The goal of my main thesis project was to determine the biological function of a deeply conserved zebrafish long noncoding RNAs (lncRNA) which we called libra. libra shows sequence similarity with the 3'UTR of the NREP a protein coding transcript. Both libra and Nrep contain a deeply conserved and unusually complementary microRNA (miRNA) binding site for miR-29. Using both the mouse model and mouse cell lines, we deciphered the regulatory relationship between this conserved transcript and the miRNA pathway. We showed that Nrep restricts the spatial expression domain of miR-29 in the cerebellum and that it destabilizes miR-29 through 3' trimming. Until now, only viral transcripts and artificial reporters engineered to contain highly complementary miRNA binding sites have been shown to regulate miRNAs in this fashion. Thus, our work uncovers the first example of endogenous target-directed miRNA degradation (TDMD). In addition, through a set of in vivo experiments in zebrafish and mouse, we showed that both libra and Nrep control normal animal behavior. By genetically disrupting the miR-29 binding site in Nrep in mouse, we showed that Nrep regulates miR-29 dosage through its miR-29 site and controls animal behavioral. In a second part of my thesis I describe a strategy to genetically downregulate lncRNAs in a minimally invasive manner. Approaches to knock-out lncRNAs that do not introduce vast sequence changes at the genomic level have not been adequately developed yet. I present our in vivo strategy applied to the zebrafish model using a genomic knock-in of a self-cleaving ribozyme sequence and a premature poly(A) signal to knock-out lncRNAs.