In eukaryotes, the spliceosome is a macromolecular ribonucleoprotein machine that excises introns from pre-mRNAs through two sequential transesterification reactions. The chemistry and fidelity of pre-mRNA splicing are dependent upon a series of spliceosomal rearrangements, which are mediated by trans-acting splicing factors. One key class of these factors is the DEAH-box ATPase subfamily of proteins, whose members couple ATP hydrolysis to promote RNP structural rearrangements within the spliceosome. This is typified by Prp22, which promotes release of the spliced mRNA from the spliceosome and ensures fidelity of the second step of splicing. This role is well documented through classical biochemical and yeast genetics methods. Yet very little is known regarding the comings and goings of Prp22 relative to the spliceosome. My thesis research investigated the dynamics of Prp22 during splicing by using single-molecule fluorescence methods that allowed direct observation of these events. To do this, I helped construct a toolkit that combined yeast genetics, chemical biology and Colocalization Single Molecule Spectroscopy (CoSMoS) with in vitro splicing assays. Specifically, my thesis research consisted of CoSMoS splicing experiments in which fluorescently labeled pre-mRNA, spliceosome components and Prp22 were directly visualized and analyzed. Using these methods, I found that Prp22’s interactions with the spliceosome are highly dynamic and reversible. By simultaneously monitoring Prp22 and individual spliceosome subcomplexes, I was able to frame these Prp22 binding events in context relative to specific steps in spliceosome assembly and splicing. These experiments provide insight into how Prp22 promotes mRNA release from the spliceosome and maintains splicing fidelity.

<p>There are several techniques and methods on how to capture data during a Live Box response in computer forensics, but the key towards these acquisitions is to keep the collected data admissible in a judicial court process. Different approaches during a Live Box examination will lead to data changes in the computer, due to the volatile nature of data stored in memory. The inevitable changes of volatile data are what cause the controversy when admitting digital evidence to court room proceedings.</p> <p>The main goal of this dissertation was to create a process model, titled Live Box Computer Preservation Response(LBCPR), that would assist in ensuing validity, reliably and accuracy of evidence in a court of law. This approach maximizes the admissibly of digital data derived from a Live Box response. </p> <p>The LBCPR was created to meet legal and technical requirements in acquiring data from a live computer. With captured Live Box computer data, investigators can further add value to their investigation when processing and analyzing the captured data set, that would have otherwise been permanently unrecoverable upon powering down the machine. By collecting the volatile data prior to conducting Dead Box forensics, there is an increased amount of information that that can be a utilized to understand the state of the machine upon collection when combined with the stored data contents. </p> <p>This study created a comparative analysis on data collection with the LBCPR method versus traditional Dead Box forensics techniques, further proving the expected results of Live Box techniques capturing volatile data. However, due to the structure of the LBCPR, there were enhanced capabilities of obtaining value from the randomization of memory dumps, because of the assistance of the collected logs in the process model. In addition, with the legal admissibility focus, there was incorporation of techniques to keep data admissible in a court of law. </p>

Positive-stranded (+)RNA viruses replicate inside cells and depend on many cellular factors to complete their infection cycle. In the meanwhile, (+)RNA viruses face the host innate immunity, such as cell-intrinsic restriction factors that could block virus replication. Firstly, I have established that the plant DDX17-like RH30 DEAD-box helicase conducts strong inhibitory function on tombusvirus replication when expressed in plants and yeast surrogate host. This study demonstrates that RH30 blocks the assembly of viral replicase complex, the activation of RNA-dependent RNA polymerase function of p92pol and viral RNA template recruitment. In addition, the features rendering the abundant plant DEAD-box helicases either antiviral or pro-viral functions in tombusvirus replication are intriguing. I found the reversion of the antiviral function of DDX17-like RH30 DEAD-box helicase and the coopted pro-viral DDX3-like RH20 helicase due to deletion of unique N-terminal domains. The discovery of the sequence plasticity of DEAD-box helicases that can alter recognition of different cis-acting elements in the viral genome illustrates the evolutionary potential of RNA helicases in the arms race between viruses and their hosts. Moreover, I discovered that Xpo1 possesses an anti-viral function and exports previously characterized cell-intrinsic restriction factors (CIRFs) from the nucleus to the replication compartment of tombusviruses. Altogether, in my PhD studies, I found plant RH30 DEAD-box helicase is a potent host restriction factor inhibiting multiple steps of the tombusvirus replication. In addition, I provided the evidence supporting that the Nterminal domain determines the functions of antiviral DDX17-like RH30 DEAD-box helicase and pro-viral DDX3-like RH20 DEAD-box helicase in tombusvirus replication. Moreover, I discovered the emerging significance of the Xpo1-dependent nuclear export pathway in tombusvirus replication.

No description available.

All organisms exist in some sort of symbiosis with their environment. The food we eat, air we breathe, and things we touch all have their own microbiota and we interact with these microbiota on a daily basis. As such, we employ a method of compartmentalization in order to keep foreign entities outside of the protected internal environments of the body. However, as other organisms seek to replicate themselves, they may invade our sterile compartments in order to do so. To protect ourselves from unfettered replication of pathogens or from cellular damage, we have developed a series of receptors and signaling pathways that detect foreign bodies as well as abnormal signals from our own perturbed cells. The downstream effector molecules that these signaling pathways initiate can be toxic and damaging to both pathogen and host, so special care is given to the regulation of these systems. One method of regulation is the production of endogenous small ribonucleic acids that can regulate the expression of various receptors and adaptors in the immune signaling pathways. In this dissertation, I present work that establishes an important protein in small ribonucleic acid regulation, Dicer, as an essential protein for regulating the innate immune response to immuno-stimulatory nucleic acids as well as regulating the productive infection of encephalomyocarditis virus. Depleting Dicer from murine embryonic fibroblasts renders a disparate type I interferon response where nucleic acid stimulation in the Dicer null cells fails to produce an appreciable interferon response while infection with the paramyxovirus, Sendai, induces a more robust interferon response than the wild-type control. Additionally, I show that Dicer plays a vital role in controlling infection by the picornavirus, encephalomyocarditis virus. Encephalomyocarditis virus fails to grow efficiently in Dicer null cells due to the inability for the virus to bind to the outside of the cell, suggesting that Dicer has a role in modulating viral infection by affecting host cellular protein levels. Together, this work identifies Dicer as a key protein in viral innate immunology by regulating both the growth of virus and also the immune response generated by exposure to pathogen associated molecular patterns. Understanding this regulation will be vital for future development of small molecule therapeutics that can either modulate the innate immune response or directly affect viral growth.

No description available.

L’ARN hélicase à boite DEAD de la levure S.cerevisiae Ded1 est une protéine essentielle dont la fonction a été conservée au cours de l’évolution. Ses homologues fonctionnels sont impliqués dans le développement et le cycle cellulaire. Ded1 a longtemps été associée à l’étape de scanning de la région 5’UTR des ARNm au niveau de l’initiation de la traduction. Nous avons utilisé différentes approches comme les co-immunoprécipitations, des analyses de spectrométrie de masse, des tests de complémentation génétique, de séparation des complexes sur gradients de saccharose, des expériences de localisation in situ et d’enzymologie pour montrer que Ded1 interagissait physiquement avec des complexes cytoplasmique et nucléaire de liaison à la coiffe des ARNm. Nous avons également montré que Ded1 peut passer du noyau vers le cytoplasme par différentes voies d’export nucléaire. De façon intéressante, ses partenaires protéines sont capables de stimuler son activité ATPase. De plus, nous avons montré qu’il existait un lien génétique entre Ded1 et ses partenaires. Nous avons également montré que Ded1 colocalise partiellement avec ses partenaires dans des gradients de saccharose, suggérant que Ded1 pourrait être associée à certains mRNPs. Nos résultats encore préliminaires indiquent que Ded1 pourrait s’associer à d’autre ARNs coiffés. Ainsi, Ded1 pourrait remodeler les complexes associés à différentes étapes de la vie des ARN coiffés. / The budding yeast DEAD-box RNA helicase Ded1 is an essential yeast protein that is closely related to a subfamily of DEAD-box proteins that are involved in developmental and cell-cycle regulation. Ded1 is generally considered to be a translation-initiation factor that helps the 40S ribosome scan the mRNA from the 5' 7-methylguanosine cap to the AUG start codon. We have used IgG pulldown experiments, mass spectroscopy analyses, genetic experiments, saccharose gradients, in situ localizations, and enzymatic assays to show that Ded1 is a cap-associated factor that actively shuttles between the cytoplasm and the nucleus. We show that Ded1 physically interacts with various cap-associated factors and that its enzymatic activity is stimulated by these factors. By using various mutated proteins, we show that Ded1 is genetically linked to these factors. Ded1 comigrates with these factors on saccharose gradients, but the peak of Ded1 sediments slightly heavier than for the other factors, which suggests that Ded1 is predominately associated with a subset of the mRNPs. Finally, purification of the protein complexes associated with Ded1 and subsequent analysis by nanoLC-MS/MS indicates that Ded1 is associated with both nuclear and cytoplasmic mRNPs. Preliminary experiements showed that Ded1 can associate with other capped RNA. We conclude that Ded1 may function as a remodeling factor that is needed to form the different complexes associated with the different processing steps of the capped RNA.

Transposon silencing is required to maintain genome stability. The non-coding piRNAs effectively suppress of transposon activity during germline development. In the Drosophila female germline, long precursors of piRNAs are transcribed from discrete heterochromatic clusters and then processed into primary piRNAs in the perinuclear nuage. However, the detailed mechanism of piRNA biogenesis, specifically how the nuclear and cytoplasmic processes are connected, is not well understood. The nuclear DEAD box protein UAP56 has been previously implicated in protein-coding gene transcript splicing and export. I have identified a novel function of UAP56 in piRNA biogenesis. In Drosophila egg chambers, UAP56 co-localizes with the cluster-associated HP1 variant Rhino. Nuage is a germline-specific perinuclear structure rich in piRNA biogenesis proteins, including Vasa, a DEAD box with an established role in piRNA production. Vasa-containing nuage granules localize directly across the nuclear envelope from cluster foci containing UAP56 and Rhino, and cluster transcripts immunoprecipitate with both Vasa and UAP56. Significantly, a charge-substitution mutation that alters a conserved surface residue in UAP56 disrupts co-localization with Rhino, germline piRNA production, transposon silencing, and perinuclear localization of Vasa. I therefore propose that UAP56 and Vasa function in a piRNA-processing compartment that spans the nuclear envelope.

No description available.

A cellular pre-mRNA undergoes various post-transcriptional processing events, including capping, splicing and polyadenylation prior to nuclear export. Splicing is particularly important for mRNA nuclear export as two distinct multi-protein complexes, known as human TREX (hTREX) and the exon-junction complex (EJC), are recruited to the mRNA in a splicing-dependent manner. In contrast, a number of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic mRNAs lack introns and are exported by the virus-encoded ORF57 protein. Herein we show that ORF57 binds to intronless viral mRNAs and functions to recruit the complete hTREX complex, but not the EJC, in order assemble an export component viral ribonucleoprotein particle (vRNP). The formation of this vRNP is mediated by a direct interaction between ORF57 and the hTREX export adapter protein, Aly. Aly in turn interacts directly with the DEAD-box protein UAP56, which functions as a bridge to recruit the remaining hTREX proteins to the complex. Moreover, we show that a point mutation in ORF57 which disrupts the ORF57-Aly interaction leads to a failure in the ORF57-mediated recruitment of the entire hTREX complex to the intronless viral mRNA and inhibits the mRNAs subsequent nuclear export and virus replication. Furthermore, we have utilised a trans-dominant Aly mutant to prevent the assembly of the complete ORF57-hTREX complex; this results in a vRNP consisting of viral mRNA bound to ORF57, Aly and the nuclear export factor, TAP. Strikingly, although both the export adapter Aly and the export factor TAP were present on the viral mRNP, a dramatic decrease in intronless viral mRNA export and virus replication was observed in the absence of the remaining hTREX components (UAP56 and hTHO-complex). Together, these data provide the first direct evidence that the complete hTREX complex is essential for the export of KSHV intronless mRNAs and infectious virus production.