Utilizing Selected Di- and Trinucleotides of siRNA to Predict RNAi Activity

Small interfering RNAs (siRNAs) induce posttranscriptional gene silencing in various organisms. siRNAs targeted to different positions of the same gene show different effectiveness; hence, predicting siRNA activity is a crucial step. In this paper, we developed and evaluated a powerful tool named “siRNApred” with a new mixed feature set to predict siRNA activity. To improve the prediction accuracy, we proposed 2-3NTs as our new features. A Random Forest siRNA activity prediction model was constructed using the feature set selected by our proposed Binary Search Feature Selection (BSFS) algorithm. Experimental data demonstrated that the binding site of the Argonaute protein correlates with siRNA activity. “siRNApred” is effective for selecting active siRNAs, and the prediction results demonstrate that our method can outperform other current siRNA activity prediction methods in terms of prediction accuracy

Endogenous Small RNAs in the \u3cem\u3eDrosophila\u3c/em\u3e Soma: A Dissertation

Since the discovery in 1993 of the first small silencing RNA, a dizzying number of small RNAs have been identified, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). These classes differ in their biogenesis, modes of target regulation and in the biological pathways they regulate. Historically, siRNAs were believed to arise only from exogenous double-stranded RNA triggers in organisms lacking RNA-dependent RNA polymerases. However, the discovery of endogenous siRNAs in flies expanded the biological significance of siRNAs beyond viral defense. By high throughput sequencing we identified Drosophila endosiRNAs as 21 nt small RNAs, bearing a 2´-O-methyl group at their 3´ ends, and depleted in dicer-2 mutants. Methylation of small RNAs at the 3´ end in the soma, is a consequence of assembly into a mature Argonaute2-RNA induced silencing complex. In addition to endo-siRNAs, we observed certain miRNAs or their miRNA* partners loading into Argonaute2. We discovered, that irrespective of its biogenesis, a miRNA duplex can load into either Argonaute (Ago1 or Ago2), contingent on its structural and sequence features, followed by assignment of one of the strands in the duplex as the functional or guide strand. Usually the miRNA strand is selected as the guide in complex with Ago1 and miRNA* strand with Ago2. In our efforts towards finding 3´ modified small RNAs in the fly soma, we also discovered 24-28nt small RNAs in certain fly genotypes, particularly ago2 and dcr-2mutants. 24-28nt small RNAs share many features with piRNAs present in the germline, and a significant fraction of the 24-28nt small RNAs originate from similar transposon clusters as somatic endo-siRNAs. Therefore the same RNA can potentially act as a precursor for both endo-siRNA and piRNA-like small RNA biogenesis. We are analyzing the genomic regions that spawn somatic small RNAs in order to understand the triggers for their production. Ultimately, we want to attain insight into the underlying complexity that interconnects these small RNA pathways. Dysregulation of small RNAs leads to defects in germline development, organogenesis, cell growth and differentiation. This thesis research provides vital insight into the network of interactions that fine-tune the small RNA pathways. Understanding the flow of information between the small RNA pathways, a great deal of which has been revealed only in the recent years, will help us comprehend how the pathways compete and collaborate with each other, enabling each other’s optimum function

Antisense Therapy

Antisense Therapy offers a comprehensive, state-of-the art perspective on the role of antisense therapy in the treatment of human disease, with a special focus on cancer. Use of antisense oligonucleotides is a growing field of pharmaceutical and biotech companies and research programs for treatment of several diseases. This book summarizes and presents the best updates, therapeutic principles, methods, and applications in the field and offers meaningful information to move treatment discovery forward

Applications of genome editing tools in drug discovery and basic research

Since the discovery of the DNA double helix, major advances in biology have been; the development of recombinant DNA technology in the 1970s, methods to amplify DNA and gene targeting technology in the late 1980s. In organisms such as yeast and mice, the ability to accurately add or delete genetic information transformed biology, allowing an unmatched level of precision in studies of gene function. But, the ability to easily and specifically edit the genetic material of other cells and organisms remained impossible until recently for molecular biologists. The recent advent of programmable nucleases has dramatically changed the efficiency and speed of genome manipulation in several model organisms including cultured cells, as well as whole animals and plants. These tools opened up a powerful technique for biology research now called “genome editing” or “genome engineering” (Carroll, 2011; Hsu et al., 2014; Kim and Kim, 2014). In the first half of my doctoral studies, I developed genome-editing strategies to discover drug targets for a rare genetic disease called Friedreich’s Ataxia. Friedreich’s Ataxia (FRDA) is a neurodegenerative disease caused by deficiency of the mitochondrial protein frataxin (FXN) (Campuzano et al., 1997). This deficiency results from an expansion of a trinucleotide GAA repeat in the first intron of the FXN gene (Campuzano et al., 1996; Durr et al., 1996). Therapeutics that reactivate FXN gene expression are expected to be beneficial to FRDA patients (Gottesfeld, 2007). However, high-throughput screening (HTS) for FXN activators has so far met with limited success because current cellular models do not accurately assess endogenous FXN gene regulation. Here I used genome-editing technologies to generate a cellular model in which a luciferase reporter is introduced into the endogenous FXN locus. Using this system in a high-throughput genomic screen, we discovered novel inhibitors of FXN-luciferase expression. I confirmed that reducing expression of one of these inhibitors, PRKD1, led to an increase in FXN expression in FRDA patient fibroblasts (Villasenor et al., 2015). We then used reprogramming technologies to create a disease-relevant situation and test small molecules that specifically modulate PRKD1. We found that WA-21-JO19, a chemical inhibitor of PRKD1, increases FXN expression levels in iPSC-derived FRDA patient neurons. This approach, developed at the interface between academic and pharmaceutical research, demonstrates how a combination of genome editing, cellular reprogramming, and high-throughput biology can generate an effective novel drug discovery platform. In the second part of my doctoral work, we developed an interface between genome editing and proteomics to isolate native protein complexes produced from their natural genomic contexts. In many biological processes, proteins act as members of protein complexes. Understanding the molecular composition of protein complexes is a key task towards explaining their function in the cell. Conventional affinity purification followed by mass spectrometry analysis is a broadly applicable method to decipher molecular interaction networks and infer protein function. However, traditional affinity purification methods are limited by a number of factors such as antibody specificity and are sensitive to perturbations induced by overexpressed target proteins. Here, we combined genome editing with tandem affinity purification to circumvent current limitations. I uncovered subunits and interactions among well-characterized complexes and report the isolation of novel Mettl3-binding partners. The multi-protein complex composed of two active methyltransferases Mettl3 and Mettl14 mediates methylation of adenosines at position N6 on RNA molecules (Bokar et al., 1994; Bokar et al., 1997; Liu et al., 2014). N6-methyladenosine is the most abundant internal modification in eukaryotic mRNA and is often found on introns, which implies that methylation occurs co-transcriptionally (Fu et al., 2014). My work identified a set of nuclear RNA binding proteins, which specifically interact with the Mettl3-Mettl14 complex. We are currently testing the ability of these factors to function as “recruiters” of the Mettl3-Mettl14 complex to nascent mRNAs in the cell nucleus. In summary, our approach solidly establishes how a combination of genome editing and proteomics can simplify explorations of protein complexes as well as the study of post-translational modifications. In addition, this approach opens up new opportunities to study native protein complexes in a wide variety of cells and model organisms and will likely enable the systematic investigation of mammalian proteome function

Targeting and function of mammalian microRNAs

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2009.Cataloged from PDF version of thesis.Includes bibliographical references.In the span of a few short years, animal microRNAs have become recognized as broad regulators of gene expression, largely in part due to our improved understanding of how animal microRNAs recognize their targets. Crucial to microRNA targeting are the ~7-nt seed sites complementary to nucleotides 2-8 at the 5' end of the microRNA. We show that protein-coding genes preferentially expressed at the same time and place as a highly expressed microRNA have evolved their 3' UTR sequence to specifically avoid seed sites matching that microRNA. In contrast, conserved sites appear to be preferentially expressed in developmental states prior to microRNA expression, and are downregulated upon induction of that microRNA. Combined with the result that both conserved and nonconserved seed sites are generally functional, our findings extend the direct and indirect influence of mammalian microRNAs to the majority of protein-coding genes. Although seed sites account for much of the specificity of microRNA regulation, they are not always sufficient for repression, suggesting the contribution of additional specificity determinants. Combining independent computational and experimental approaches, we found five general features associated with site efficacy: AU-rich nucleotide composition near the site, proximity to sites for coexpressed microRNAs, pairing outside of the seed region at microRNA nucleotides 13-16, and positioning within the 3' UTR at least 15nt from the stop codon and away from the center of long UTRs. By incorporating these five features, we are able to explain much of the differences in site efficacy for both exogenously added microRNAs and for endogenous microRNA-message interactions. We further refined the seed site motif involved in microRNA repression, by demonstrating experimentally an Adenosine preference across from the unpaired first nucleotide of the microRNA and ranking the relative effectiveness of different classes of seed sites. Although sites lacking perfect seed pairing were generally ineffective, a fraction of these sites were supplemented by detectable compensatory 3' pairing. In addition, by extending our conservation analysis to 11 genomes, we show that the confidence with which conserved target sites can be predicted is a function of the conservation of the seed site itself relative to the conservation of surrounding sequence. This allows individual conserved sites to be assigned a confidence score reflecting the likelihood that the site is being conserved due to selection rather than by chance.by Kyle Kai-How Farh.Ph. D

Transcriptional Regulation by the Oncogenic ZNF217/CoREST Complex

The ZNF217 transcription factor is an oncogene found within the 20q13 amplicon and is amplified and overexpressed in many cancers including breast and ovarian. Overexpression of ZNF217 leads to increased cell proliferation, survival, and causes resistance to TGFβ\u27s anti-proliferative effects. ZNF217 is a core constituent of a transcriptional complex that includes CoREST, HDAC1/2, LSD1, and the CtBP1/2. In this study, I have combined genome-wide biochemical approaches to identify genes directly regulated by ZNF217. I have identified the tumor suppressor and cell cycle inhibitor, p15ink4b, as a direct target of the ZNF217 complex and demonstrated that ZNF217 represses the p15ink4b gene by promoting a repressive chromatin environment and facilitating promoter DNA hypermethylation that involves a novel interaction with DNMT3A. Furthermore, treatment of cells with TGFβ triggers DNA demethylation of the p15ink4b promoter and the release of ZNF217/CoREST/DNMT3A complex. Subsequently, a novel activation complex is recruited that consists of SMAD2/3, CBP, and the DNA glycosylase TDG which precedes increases in p15ink4b protein expression. Knockdown of TDG, or its functional homolog MBD4, prevents TGF-β-dependent demethylation of the p15ink4b promoter suggesting that the demethylation occurs through an active mechanism and is required for TGFβ dependant activation of gene expression. DNA immunoprecipitation experiments indicate that 5mC undergoes conversion to 5hmC in response to TGFβ treatment. AID/APOBEC2 deaminases are also required for the DNA demethylation by TGFβ supporting a mechanism whereby 5mC is hydroxylated to 5hmC and then deaminated to 5hmU which is reverted to the unmethylated cytosine by the BER enzymes. Overexpression of ZNF217 inhibits promoter demethylation and expression of the p15ink4b gene in response to TGFβ by preventing recruitment of SMAD2/3/TDG complex. These findings suggest that the coregulator balance at promoters of genes is an important determinant of gene regulation and oncogenic amplifications such as ZNF217 can upset this balance causing deregulation of many genes. Taken together, these results establish the ZNF217 complex as a negative regulator of the p15ink4b gene and may constitute an important link between amplification of ZNF217, increased cell proliferation and loss of TGFβ responsiveness in cancer

Oncogene and Cancer

This book describes a course of cancer growth starting from normal cells to cancerous form and the genomic instability, the cancer treatment as well as its prevention in form of the invention of a vaccine. Some diseases are also discussed in detail, such as breast cancer, leucaemia, cervical cancer, and glioma. Understanding cancer through its molecular mechanism is needed to reduce the cancer incidence. How to treat cancer more effectively and the problems like drug resistance and metastasis are very clearly illustrated in this publication as well as some research result that could be used to treat the cancer patients in the very near future. The book was divided into six main sections: 1. HER2 Carcinogenesis: Etiology, Treatment and Prevention; 2. DNA Repair Mechanism and Cancer; 3. New Approach to Cancer Mechanism; 4. New Role of Oncogenes and Tumor Suppressor Genes; 5. Non Coding RNA and Micro RNA in Tumorigenesis; 6. Oncogenes for Transcription Factor

Terrae Incognitae: Integrative Genomic Analysis of Hnrnp L Splicing Regulation

Alternative splicing is a critical component of human gene control that generates functional diversity from a limited genome. Defects in alternative splicing are associated with disease in humans. Alternative splicing is regulated developmentally and physiologically by the combinatorial actions of cis- and trans-acting factors, including RNA binding proteins that regulate splicing through sequence-specific interactions with pre-mRNAs. In T cells, the splicing regulator hnRNP L is an essential factor that regulates alternative splicing of physiologically important mRNAs, however the broader physical and functional impact of hnRNP L remains unknown. In this dissertation, I present analysis of hnRNP L-RNA interactions with CLIP-seq, which identifies transcriptome-wide binding sites and uncovers novel functional targets. I then use functional genomics studies to define pre-mRNA processing alterations induced by hnRNP L depletion, chief among which is cassette-type alternative splicing. Finally, I use integrative genomic analysis to identify a direct role for hnRNP L in repression of exon inclusion and an indirect role for enhancing exon inclusion that supports a novel regulatory interplay between hnRNP L and chromatin. In two appendices, I present CLIP-seq studies of two additional RNA binding proteins: the splicing factor CELF2 and the RNA helicase DDX17. In conclusion, I provide comparisons of these three CLIP-seq studies, providing high-level insights into the capabilities and limitations of CLIP-seq. In sum, this dissertation expands our knowledge of hnRNP L splicing control in the context of broader studies of RNA binding proteins in multiple cell types and conditions