The splicing of precursor messenger RNAs (pre-mRNAs) plays an essential role in dictating the mature mRNA profiles of eukaryotic cells. Mis-regulation of splicing, due to mutations in pre-mRNAs or in components of the splicing machinery, is associated with many diseases. Therefore, knowledge of pre-mRNA splicing mechanisms is required to understand gene expression regulation during states of homeostasis and disease, and for the development of therapeutic interventions.Splicing is catalyzed by the spliceosome, a dynamic and protein-rich ribozyme composed of five small nuclear ribonucleoproteins (snRNPs) and ~170 auxiliary factors. Early interactions that occur in prespliceosomal complexes formed by the 5′- and 3′-splice-site bound U1 and U2 snRNPs are responsible for committing introns for removal. However, the mechanisms underlying these early interactions remain to be fully characterized for understanding the influence of alternative splicing factors and the impact of recurrent disease-associated mutations in prespliceosomal proteins. The goal of my dissertation research was to delineate the role of the U1 small nuclear RNA (snRNA) during prespliceosome assembly. By applying a cellular minigene reporter assay and a variety of in vitro techniques including cell-free protein expression, UV-crosslinking, electrophoretic mobility shift assays, surface plasmon resonance, and RNA affinity purification, my work establishes critical roles for the U1 snRNA stem-loops 3 (SL3) and 4 (SL4) in formation of intron definition interactions during prespliceosome assembly. Previously, the SL4 of the U1 snRNA was shown to form a molecular bridge across introns by contacting the U2-specific splicing factor 3A1 (SF3A1). I identified the Ubiquitin-like domain of SF3A1 as a non-canonical RNA binding domain responsible for U1-SL4 binding. I also determined a role for the SL3 region of the U1 snRNA in splicing and characterized the spliceosomal RNA helicase UAP56 as an SL3 interacting protein. By knocking-down the SL3- and SL4-interacting proteins, I confirmed that U1 splicing activity in vivo relies on UAP56 and SF3A1 and that their functions are interdependent. These findings, in addition to the observations made using in vitro splicing assays, support a model whereby UAP56, through its interaction with U1-SL3, enhances the cross-intron interaction between U1-SL4 and SF3A1 to promote prespliceosome formation.

Necroptosis is a pro-inflammatory mechanism of programmed cell death. It has been implicated in many diseases such as inflammatory diseases, neurodegenerative diseases, cancer and during viral infections. The focus of this research work was to establish the relationship between poxvirus pathogenesis and necroptosis, and the translation implications of necroptosis in oncolytic virotherapy. Vaccinia virus (VACV) is the currently used vaccine for smallpox and it has also been developed as a vaccine vector for several pathogens. E3L is one of the key innate immune evasion genes of VACV and it encodes E3 protein composed of dsRNA binding domain in the C-terminus and Z-NA-binding domain (Z-NA BD) in the N terminus. Both domains are necessary for type 1 interferon resistance and pathogenesis. Recently, it has been shown that in in vitro, the N-terminus of E3 is necessary to inhibit necroptosis occurring through the host-encoded cellular proteins RIP3 and Z-NA-binding protein DAI interaction leading to phosphorylation of MLKL, the key executioner step in the pathway. The research work presented here clearly demonstrates that in a mouse model, the N-terminus of VACV E3 is necessary to inhibit necroptosis during pathogenesis in mice. Another poxvirus belonging to the same family as VACV is monkeypox virus (MPXV) and is an emerging human pathogen. MPXV contains a natural truncation in the N-terminus of its E3 homologue, F3. The results indicate that during MPXV infection in mice, pathogenesis was higher only in DAI knockout mice and not in MLKL knockout mice, suggesting that DAI is possibly activating other proteins not leading to necroptosis. The characterization of VACV as an oncolytic virus was carried out with a focus on future clinical trials. In this study, a pan screening was conducted in various cancer cell lines as many cancers downregulate necroptotic proteins. The results reveal that the N-terminal deletion mutant of VACV selectively replicates in cancer cell lines with a deficient necroptotic pathway and thus, can be used as a potential treatment against specific tumors and evidently, provides abundant scope for future studies.

Stress granules are cytoplasmic foci that form in response to various types of cellular stress, including viral infection. They contain mRNA, translation initiation factors, the small ribosomal subunit, RNA binding proteins, and other unique components depending on the type of stress the cell is under. Stress granules are thought to store these components until the stress as passed at which time the mRNA resumes translation. They also have an active role in the cell's antiviral response and are required for efficient induction of the interferon pathway. There are many viruses that induce or interfere with stress granules, including poliovirus. Poliovirus is a positive sense RNA virus that is part of the Picornaviridae family. Stress granules in poliovirus infected cells differ from stress granules in cells undergoing other types of stress because they contain the RNA binding protein Sam68, their formation is dependent on RNA export by the Crm1 pathway, and they are induced by poliovirus cleavage of eIF4G and PABP. It was found previously that Sam68 is found in the stress granules of poliovirus infected HeLa cells but not in oxidative stress of heat shock induced stress granules. My research shows that this finding is true in other cell lines and thus represents a biologically significant finding. The Crm1 pathway exports snRNAs and some mRNAs, rRNAs, and proteins. To determine which of these classes of RNA is necessary for stress granule formation in poliovirus infected cells but not in cells undergoing other types of stress, plasmids with modified PHAX protein were used to isolate the snRNA export pathway. More work needs to be done to determine the impact of snRNA export on stress granule formation. This research could eventually help us better understand the cell's anti-viral response and have implications for how we treat viral infections.