Replication of genetic material is the most important and central process during the viral life cycle. Most RNA viruses assign one or more proteins translated from their own genome for replicating genomic RNAs. Understanding the various biochemical activities of these replication proteins is the aim of this dissertation research. The replicase proteins of Turnip crinkle virus (TCV) and Tomato bushy stunt virus (TBSV) were selected for this study. Both viruses have small, messenger-sense, single-stranded RNA genomes. Replicase proteins p28/p88 of TCV and p33/p92 of TBSV- were expressed and purified from E. coli as N-terminal fusions to maltose binding protein. In vitro assays revealed that the recombinant p88 has RNA-dependent RNA polymerase (RdRp) and RNAbinding activities. Deletion of the N-terminal p28 domain in p88 resulted in a highly active RdRp, while further deletions at both N- and C-terminal ends abolished RdRp activity. Comparison of p88, the N-terminal p28-deletion mutant of p88 and a TCV RdRp preparation obtained from infected plants revealed remarkable similarities in RNA template recognition and plus and minus strands synthesis. Contrary to recombinant TCV RdRp activities under similar experimental conditions. p33 preferentially binds to singlestranded (ss) RNA with positive cooperativity in vitro. The RNA binding activity was mapped to arginine/proline-rich motif (RPR-motif) at the C-terminus of p33 and the corresponding sequence in p92. The non-overlapping C-terminal domain of p92 also contained additional RNA-binding regions that flank the conserved RdRp motifs on both sides. Cooperative RNA binding by p33 suggested inter-molecular interactions between p33 monomers. Indeed the yeast two-hybrid and surface plasmon resonance assays revealed interactions between p33 and p33 and also between p33 and p92. The sequence involved in the protein-protein interactions was mapped to the C-terminal region in p33, proximal to RPR-motif. Within this region, mutations introduced at two short stretches of amino acid residues were found to affect p33:p33 and p33:p92 interactions in vivo and also decreased the replication of a TBSV-defective interfering RNA in yeast, a model system, supporting the significance of these protein interactions in tombusvirus replication.

Synthetic biology, which aims to enable the design and assembly of customized biological systems, holds great promise for delivering solutions to numerous modern day challenges in agriculture, sustainable energy production, and medicine. However, at its current stage, synthetic biology is not yet equipped with the necessary tools and understanding to reprogram the immensely complex molecular environment of the cell beyond simple proof of concept demonstrations. One current objective within synthetic biology is to create robust tools that can be used to manipulate biological systems in a predictable and reliable manner. While many transcription-based control devices have been reported, little consideration has been given to the eukaryotic protein translation apparatus as a target for engineering gene-regulatory tools. In this work, we explore the potential for reprogramming the protein synthesis machinery for cell engineering. We begin in Chapter 1 by reviewing canonical protein synthesis and survey the assortment of translation reprogramming mechanisms that exist in nature, focusing on the role of RNA in these processes. We then cover previous efforts to engineer the protein synthesis machinery and discuss their methodological approaches. Lastly, we examine potential opportunities for engineering protein synthesis that have not yet been explored. RNA’s prominent role in protein synthesis and its amenability to high-throughput in vitro selection approaches raises the possibility that the translation apparatus could be engineered through in vitro directed evolution of its RNA components. In Chapter 2, we develop an experimental framework for identifying mRNA sequence elements that reprogram protein synthesis, focusing on stop codon readthrough. By adapting a previously developed in vitro selection technology called mRNA display, we demonstrate that molecules of RNA derived from expansive libraries of random sequences can be enriched as a result of their translation reprogramming activity. We then analyze these stop codon readthrough signals and propose the use of these sequences for enhanced unnatural amino acid incorporation technologies. In Chapter 3, we apply this very same selection principle for the in vitro directed evolution of RNA sequences that stimulate -1 programmed ribosomal frameshifting. Then, using previously reported RNA aptamers, we rationally engineer RNA switches that regulate translation reading frame in response to small molecule inputs. To further optimize switch performance, an in vivo directed evolution platform was established. We explore the utility of these RNA switches, particularly their ability to regulate multi-protein stoichiometry, for performing cellular logic operations and controlling cell fate. A major focus of translation engineering has been the incorporation of unnatural amino acids for fluorescent labeling of proteins in living cells. The successful achievement of this goal will require small molecule fluorophores with desirable biological properties, as well as robust synthetic methods for their production. In Chapter 4, we present a scalable approach to oxazine and xanthene fluorophores that utilizes a general diaryl ether synthetic intermediate. Finally, in Chapter 5, we describe a photoactivatable oxazine fluorophore and demonstrate its utility as a live-cell imaging reagent with applicability to advanced microscopy techniques.

The Pentatricopeptide Repeat (PPR) gene family produces RNA-binding proteins that target organellar transcripts. The PPR family is expanded in land plants, with nearly 450 genes identified in Arabidopsis thaliana. In plants with a Cytoplasmic Male Sterility (CMS) phenotype, members of the PPR family can act as a RESTORER OF FERTILITY (Rf) and are part of a subset of genes called RESTORER OF FERTILITY-LIKE (RFL). Unlike other PPR transcripts, RFL transcripts are targets of both microRNA (miRNA) and trans-acting siRNA (tasiRNA) and produce secondary siRNA after initial miRNA- or tasiRNA-guided cleavage. We utilized the A. lyrata genome assembly and high-throughput sequencing of small RNA to examine the evolutionary dynamics of the PPR gene family and the pattern of small RNA targeting of RFL transcripts. We found an expanded set of 539 PPR genes in A. lyrata, 51 of which were in the RFL group, often in multiple collinear copies when compared to their A. thaliana orthologs. In-species RFL paralogs appear to be more related to one another than to their collinear orthologs, which is possible evidence of gene conversion or ectopic recombination. miRNA targeting of RFL transcripts is largely conserved with nearly two-thirds of all target sites maintained. TasiRNA targeting was less conserved with roughly one-third of comparable validated tasiRNA targets maintained in both species. However, when clusters of potential tasiRNA targets were considered, roughly two-thirds of target sites are conserved. Production of secondary siRNA from A. lyrata PPR transcripts is less well defined than in A. thaliana, with strong signals coming from phases that are not concordant with the miRNA- or tasiRNA-guided cleavage sites. / Graduation date: 2013

No description available.

Fused in sarcoma (FUS) is an RNA binding protein involved in DNA repair and RNA metabolism, including mRNA transcription, splicing, transport and translation. FUS is genetically and pathologically associated with rare and aggressive forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To explore the mechanisms by which pathogenic mutations in FUS cause neurodegeneration in ALS-FUS, we generated a series of FUS knock-in mouse lines that express the equivalent of the ALS-associated mutant proteins FUSP525L and FUSΔEX14 at physiological levels from the FUS locus. We demonstrate that heterozygous mutant FUS mice show progressive, age-dependent loss of vulnerable subpopulations of spinal motor neurons. While ALS-associated mutations in FUS lead to partial loss of function, we provide genetic evidence that the motor neuron phenotype observed is a consequence of a dose-dependent gain of function, associated with the insolubility of FUS and related RNA binding proteins (RBPs). Furthermore, we show that motor neuron degeneration is driven by cell autonomous mechanisms, associated with mutant FUS-independent inflammatory changes. In this faithful mouse model of ALS-FUS, we demonstrate that an antisense oligonucleotide (ASO) targeting the FUS transcript (ION363) results in the efficient silencing of both wild type and mutant FUS alleles, and that postnatal reduction of FUS protein levels in the brain and spinal cord delays disease onset in this mouse model of ALS-FUS. In a first-in-human trial of ION363, we demonstrate that repeated, intrathecal injections of this candidate therapeutic in an ALS patient with a FUSP525L mutation leads to the efficient silencing of both wild type and mutant FUS in the central nervous system, and a reduction in the burden of FUS aggregates that are a pathological hallmark of ALS-FUS. In mouse genetic and human clinical studies, we provide evidence in support of a therapeutic strategy by which silencing of the FUS gene may be used to prevent or delay disease onset in pre-symptomatic carriers of pathogenic FUS mutations, or to slow disease progression in symptomatic ALS- and FTD-FUS patients. In addition, we use this newly generated model to investigate the role of potential modifiers of FUS toxicity, including hnRNP U and UPF1, and study the role of chronic neuroinflammation in the disease progression that could lead to the development of novel therapeutics to provide immediate clinical benefit to patients with ALS-FUS.

By means of reverse-transcription polymerase chain reaction, overexpression of ZNF146 was detected in two human HCC cell lines HepG2 and Hep3B and a clear relationship between HCC and overexpression of ZNF146 has been established. Subcellular localization of ZNF146 protein in liver cells was studied by generation and expression of a green fluorescent protein (GFP) fusion protein. The nuclear localization and the reported DNA binding ability of ZNF146 protein provided a hint that ZNF146 may carry out its function in the cell system by interacting with specific genomic DNA sequences. Recombinant ZNF146 protein was expressed using bacterial and yeast system for the genomic DNA pull down assay in the identification of potential interacting genomic DNA sequences. Several potential genomic DNA sequences that interact with ZNF146 were identified and the gene MDM2 is the one of the candidates that is directly related to human carcinogenesis. MDM2 is a negative regulator of the tumor suppresser protein p53. Deregulation of MDM2 will impair the cell's ability in cell cycle arrest, DNA repair and apoptosis upon induced DNA damage. / Hepatocellular carcinoma (HCC) is a type of primary malignant liver tumor. And is one of the most frequent malignancies worldwide. The focus of this research project is the characterization of a Kruppel zinc finger protein, zinc Finger Protein 146 (ZNF146) using HCC as a disease model. The aim of this project is to understand the functional role ZNF146 and try to explore the mechanism of how ZNF146 might be involved in the carcinogenesis of HCC. / In order to have a better understanding with the protein ZNF146, SUMOylation properties of this protein has been studied. SUMO1 modification on ZNF146 has already been reported. And in our study, experimental result demonstrated that ZNF146 is also modified by SUMO2 and SUMO3 in liver cells. Other than the SUMOylation sites for SUMO1 protein which has been reported, modification sites for SUMO2 at the K247 and K275 positions were mapped, while K191R, K219R, K247R, K256R and K275R, five positions were mapped for SUMO3 modification. A more complete picture of the SUMOylation properties of ZNF146 has been revealed. Since we hypothesized that ZNF146 is related to the p53 tumor suppressor, cell cycle control and DNA repair pathway, a cell cycle study using flow cytometry was performed for the investigation of the effect on cell cycle regulation by ZNF146 overexpression. In our study, ZNF146 overexpression promoted the G1/S transition in the cell division cycle, which indicated that liver cells were more active for the progression of cell cycle. / On the other hand, using cDNA microarray technology expression profiles of ZNF146 overexpressing and non-overexpressing liver cell lines were compared and with real-time polymerase chain reaction, six candidate genes CRLF1, IFI44, ST6GAL1, LOC441601, IL18 and RAD17 were confirmed with their deregulation induced by the overexpression of ZNF146. Four of the candidates, IFI44, LOC441601, IL18 and RAD17 were found to be related to the p53 tumor suppressor activity or DNA damage, repair response and control. This observation, together with the result of genomic DNA pull down assay, gives us a hint that ZNF146 is possibly involved in liver carcinogenesis by affecting DNA repair and cell cycle control upon induced DNA damage. / The gene ZNF146 codes for a member of the Kruppel zinc finger proteins, however ZNF146 protein is different from most members of the Kruppel zinc finger proteins subfamily. It encodes a 33 kDa protein solely composed of 10 zinc finger motifs and is devoid of any non-zinc finger regulatory domain for interactions with other proteins. ZNF146 overexpression has been reported in a number of cancers including colon cancer and pancreatic carcinoma. However, the functional role of ZNF146 overexpression in tumorigenesis is yet to be solved and not much research on how ZNF146 might be invovled in the establishment of HCC was published. / To conclude, the experimental results of this study support the hypothesis that ZNF146 overexpression may deregulating the cell division cycle and some genes differentially regulated upon over-expression of ZNF146 are related to the regulations of DNA damage response. Future research on ZNF146 can be focused on the detail regulatory pathway of ZNF146 overexpression and its interaction between the p53 tumor suppressor, DNA damage response and cell cycle regulation, and a fuller picture of how ZNF146 overexpression might induce hepatocarcinogenesis can be revealed. / Yeung, Tsz Lun. / Adviser: Miu Yee (Mary) Waye. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3329. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 287-304). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Non-protein coding eukaryotic genome sequences often referred to as junk DNA are estimated to encode several non-coding RNAs (ncRNAs) which may account for nearly 98% of all genomic output in humans. The output of such a wide spread transcription in eukaryotes consists of intronic, antisense and small RNAs. In addition to the classical ncRNAs such as rRNA, tRNA and small nucleolar RNAs, the eukaryotic genome encodes two distinct categories of ncRNAs, referred to as small ncRNAs and long mRNA–like ncRNAs (mlncRNAs). The long ncRNAs, which are transcribed by RNA Polymerase II, spliced and polyadenylated, are implicated in a number of regulatory processes such as imprinting, X-chromosome inactivation, DNA demethylation, transcription, RNA interference, chromatin structure dynamics and antisense mediated regulation. Expression of noncoding RNAs is altered during stress conditions and a large number of such transcripts have been identified of late. This study has identified a novel ncRNA whose expression is upregulated during viral infection of mouse brain. While we have named this RNA as VINC or virus inducible ncRNA, others have named it as NEAT1 (Hutchinson et al., 2007) and Men (Sunwoo et al., 2008). VINC/NEAT1/Men is associated with a distinct nuclear domain called paraspeckles Using a cell line as well as an animal model system we have investigated VINC in great detail and based on these studies we report that VINC is a nuclear ncRNA that localizes to paraspeckles and it interacts with the paraspeckle protein, P54nrb in both cell line model system as well as in animal tissues by a combination of in vitro and in vivo methods. We have also mapped the domains within VINC that are involved in P54nrb interactions. Till date, the only other RNA known to localise to paraspeckles is CTN-RNA. While CTN-RNA is a protein coding RNA, VINC does not code for a protein and thus VINC is the first ncRNA to be localized to paraspeckles. Further, the mechanism of nuclear retention of these two paraspeckle RNAs appears to be distinct. In case of CTN-RNA, it has been clearly shown that it is A-I edited and such hyperedited RNAs are retained by the p54/nrb mediated complex in nucleus (Zhang and Carmichael, 2001). However the mechanism by which VINC is retained in nucleus is not clear. There is apparently no A-I editing in VINC and hence VINC retention in the nucleus by binding to nuclear proteins such as p54/nrb might involve a different mechanism. It is well established of late that nuclear matrix retains RNAs and that there is a population of poly (A) RNA that is retained in nucleus (Huang et al.,1994 ; Carter et al.,1991). However the significance of such retention is not clear but it is believed that it might be important for some constitutive functions in nucleus (Nickerson et al., 1989). More investigations are needed to understand the exact functions of nuclear RNAs such as VINC in supporting the nuclear architecture. P54nrb is a multi functional nuclear protein that mediates most of its functions in association with PSF (Shav-Tal and Zipori, 2002). Phosphorylation status of P54nrb is a key determinant for its localisation to various nuclear regions. P54nrb is a multiphosphorylated protein during mitosis and its phosphorylation is mediated by PIN-1 at its C-terminus (Proteau et al., 2005). Tyrosine phosphorylation of P54nrb is essential for it to be retained in nuclear matrix (Otto et al., 2001). The N-terminal phosphorylation is speculated but not much has been investigated. The protein has two distinct RNA recognition motifs (RRMs) in its N-terminus that are responsible for its RNA binding activity. The significance of the p54/nrb-PSF heterodimer cannot be undermined as they have been shown to be important during HIV replication. The dimer is recruited by viral machinery and P54nrb has been shown to be exported to cytosol for binding to replicative complexes (Zolotukhin et al., 2003). During adenoviral replication in nucleus many SR proteins are recruited to viral replication foci and rearrangement of speckle components happen. It has been shown with respect to speckles that nuclear domains are highly dynamic and exchange of proteins depends upon the transcriptional status of cell (Lamond and Spector, 2003). Flaviviral replication complexes are hosted in nucleus and ~20% of this complex docks in nucleus and serves as an alternate site for viral replication. The presence of viral replicative complexes alters the nuclear organisation and hence modulation of gene expression is expected (Uchil et al., 2006). The up regulation of nuclear ncRNA such as VINC is definitively one of those events associated with viral replication and definitively one needs to study the various changes carefully to understand the role of VINC in virus life cycle and/or viral pathogenesis. VINC interaction with the multi-functional nuclear protein P54nrb raises interesting aspects related to function of P54nrb in JEV infection. Knockdown of P54nrb in human myeloid cell line results in abnormal size of paraspeckles and impairs chondrogenesis (Hata et al., 2008). PSF-P54nrb complex can divert many of HIV gag RNA complexes to paraspeckles thus trying to restrict viral replication. However the exact relationship between paraspeckles and its constituent proteins is not clear. The presence of ncRNA adds another new dimension to paraspeckles. It is unclear whether the ncRNA VINC is essential for paraspeckle structure but a recent study indicates that Men (VINC/NEATI) RNA may be essential for paraspeckle formation (Sunwoo et al., 2008). The exact function VINC in neuronal as well as non-neuronal cell nuclei remains elusive and more investigations are need to understand these aspects.

[Truncated abstract] Breast cancer is the cause of significant suffering and death in our community. It is now estimated that the risk of developing breast cancer for an Australian woman before the age of 85 is 1 in 8, with this risk rising for unknown reasons. While mortality rates from breast cancer are falling due to increased awareness and early detection, few new treatments have been developed from an advanced understanding of the molecular basis of the disease. From decades of scientific research it is clear that estrogen (E2) has a large role to play in breast cancer. However, the basic mechanism behind E2 action in breast cancer remains unclear. E2 plays a fundamental role in breast cancer cell proliferation and is highly expressed in breast cancers, thus, it is important to understand both E2 and its receptor, the estrogen receptor (ER). The ER is a member of the nuclear receptor (NR) superfamily. The NR superfamily consists of a large group of proteins which regulate a large number of homeostatic proteins together with regulator proteins termed coregulators and corepressors. SRA (steroid receptor RNA activator) is the only known RNA coactivator and augments transactivation by NRs. SRA has been demonstrated to play an important role in mediating E2 action (Lanz et al., 1999; Lanz et al., 2003) and its expression is aberrant in many human breast tumors, suggesting a potential role in breast tumorigenesis (Murphy et al., 2000). Despite evidence that an alternative splice variant of SRA exists as a protein (Chooniedass-Kothari et al., 2004), it has been conclusively shown that SRA can function as an RNA transcript to coactivate NR transcription (Lanz et al., 1999; Lanz et al., 2002; Lanz et al., 2003). The precise mechanism by which SRA augments ER activity remains unknown. However, it is currently hypothesized that SRA acts as an RNA scaffold for other coregulators at the transcription initiation site. Several SRA stem loops have been identified as important for SRA function, including structure (STR) 1, 5 and 7 (Lanz et al., 2002; Zhao et al., 2007). Previously, I sought to identify SRA-binding proteins using a specific stem-loop structure of SRA (STR7) that was identified as both important for its coactivator function (Lanz et al., 2002) and also as a target for proteins from breast cancer cell extracts (Hatchell, 2002). From a yeast E. Hatchell Abstract iii III hybrid screen using STR7 as bait, I identified a novel protein which was named SLIRP (Patent Number: WO/2007/009194): SRA stem-Loop Interacting RNA-binding Protein (Hatchell, 2002; Hatchell et al., 2006). '...' This thesis demonstrates that SLIRP modulates NR transactivation, provides mechanistic insight into interactions between SRA, SRC-1, HSP-60 and NCoR and suggests that SLIRP may regulate mitochondrial function. These studies contribute significantly to the growing field of NR biology, and contribute more specifically to the elucidation of estrogen action in breast cancer. Furthermore, it lays a strong and exciting foundation for further studies to evaluate SLIRP as a biomarker and potential therapeutic target in hormone dependent cancers.

Thesis (Ph.D.). / Written for the Dept. of Plant Science. Title from title page of PDF (viewed 2008/01/12). Includes bibliographical references.

Nucleic acids and proteins, some of the building blocks of life, are not static structures but highly dynamic entities that need to interact with one another to meet cellular demands. The work presented in this thesis focuses on the application of highly sensitive fluorescence methods, both at ensemble and single-molecule level, to determine the dynamics and structure of specific biomolecular interactions with nanometer resolution and in temporal scales from nanoseconds to minutes, which includes most biologically relevant processes. The main aims of my PhD can be classified in three areas: i) exploring new fluorescent sensors with increased specificity for certain nucleic acid structures; ii) understanding how some of these nucleic acids sense the presence of small molecules in the cellular environment and trigger gene regulation by altering their structure; and iii) understanding how certain molecular machines, such as helicase proteins, are able to unwind the DNA double helix by using chemical energy in the form of ATP hydrolysis.