The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 10, 2010). Vita. Thesis advisor: David Pintel "August 2008" Includes bibliographical references

Caspase-9 has two splice variants, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b, and dysregulation of caspase-9 splice variant ratio or expression of caspase-9b isoform has been linked to augmentation of the anchorage-independent growth and tumorigenic capacity of non-small cell lung cancer (NSCLC) cells. This study focuses on cell signaling pathway(s) regulating the alternative splicing of caspase-9 pre-mRNA and mechanistic roles of caspase-9b in a certain oncogenic/survival pathway. In regards to the former, we have identified hnRNP U as a novel splice-enhancer associated with exon 3 of caspase-9 (C9/E3). Moreover, hnRNP U binds specifically to C9/E3 at an RNA cis-element previously reported as the binding site for the splicing repressor, hnRNP L. Phosphorylated hnRNP L interferes with hnRNP U for binding to C9/E3, and our results demonstrate the importance of the phosphoinositide 3-kinase/AKT pathway in modulating the association of hnRNP U to C9/E3. Overall, a mechanistic model has been revealed where hnRNP U competes with hnRNP L for C9/E3 binding to enhance the inclusion of the four-exon cassette, and this splice-enhancing effect is blocked by the AKT pathway via phosphorylation of hnRNP L. As to the latter aim, it is unknown about the mechanistic roles of caspase-9b besides the inhibitory effect on caspase-9a processing. In this study, caspase-9b has been demonstrated to have a dual function in regulating the survival/oncogenic nuclear factor κB (NF-κB) pathway, which is independent from modulating caspase-9a activation. In particular, caspase-9b has been shown to activate the canonical arm and inhibit the non-canonical arm of the NF-κB pathway by destabilizing NF-κB inhibitor alpha (IκB-α) and NF-κB-inducing kinase (NIK). Importantly, this new role for caspase-9b contributes to the enhanced survival and anchorage-independent growth of NSCLC cells conferred by caspase-9b expression. Further mechanistic studies have demonstrated a direct association of caspase-9b with the cellular inhibitor of apoptosis 1 (cIAP1), a regulatory factor in both arms of the NF-κB network, via its IAP-binding motif. Through this interaction, caspase-9b induces the E3 ligase activity of cIAP1, which regulates NF-κB activation, and promotes the survival, anchorage-independent growth and tumorigenicity of NSCLC cells. Overall, a novel tumorigenic mechanism has been identified, by which alternative mRNA processing regulates the NF-κB signaling independent of external agonist.

It was shown that 95 % of human multi-exon genes are alternatively spliced and the regulation of alternative splicing is extremely complex. Most pre-mRNA splicing events occur co- transcriptionally and there is increasing body of evidence, that chromatin modifications play an important role in the regulation of alternative splicing. Here we showed that inhibition of histone deacetylases (HDACs) modulates alternative splicing of ~700 genes via induction of histone H4 acetylation and increase of Pol II elongation rate along alternative region. We identified HDAC1 the catalytic activity of which is responsible for changes in alternative splicing. Then, we analyzed whether acetylhistone binding protein Brd2 regulates alternative splicing and showed that Brd2 occupies promoter regions of targeted genes and controls alternative splicing of ~300 genes. Later we showed that knockdown of histone acetyltransferase p300 promotes inclusion of the alternative fibronectin (FN1) EDB exon. p300 associates with CRE sites in the promoter via the CREB transcription factor. We created mini-gene reporters driven by an artificial promoter containing CRE sites. Both deletion and mutation of the CRE site affected EDB alternative splicing in the same manner as the p300 knockdown. Next we showed that p300 controls histone...

Alternative splicing (AS) is involved in expansion of transcriptome and proteome during cell growth, cell death, pluripotency, cell differentiation and development. There is increasing evidence to suggest that splicing decisions are made when the nascent RNA is still associated with chromatin. Here, I studied regulation of AS via chromatin modification with main focus on histone acetylation. First, we demonstrate that activity of histone deacetylases (HDACs) influences splice site selection in 700 genes. We provided evidence that HDAC inhibition induces histone H4 acetylation and increases RNA Polymerase II (RNA Pol II) processivity along an alternatively spliced element. In addition, HDAC inhibition reduces co-transcriptional association of the splicing regulator SRp40 with the target fibronectin exon. Further we showed that histone acetylation reader, Brd2 protein, affect transcription of 1450 genes. Besides, almost 290 genes change their AS pattern upon Brd2 depletion. We study distribution of Brd2 along the target and control genes and find that Brd2 is specifically localized at promoters of target genes only. Surprisingly, Brd2 interaction with chromatin cannot be explained solely by histone acetylation, which suggests that other protein-domains (in addition to bromodomains) are important for...

During mRNA synthesis, pre-mRNAs must be cleaved and polyadenylated at their 3'-end to be fully mature, before being exported from the nucleus. In yeast, there is a large protein machinery comprised of dozens of proteins that work together to perform these two reactions. Some of these proteins are capable of recognizing and binding key sequence elements in the pre-mRNA, effectively directing where in the transcript the cleavage and polyadenylation occur. In this thesis, recently reported structural findings related to the pre-mRNA 3'-end processing machinery are summarized. Within this machinery, the Cleavage Factor IA (CF-IA) complex is comprised of the Rna14, Rna15, and Pcf11 and Clp1 proteins. Results reported here include the crystal structure of the Rna14-Rna15 complex, which indicates that the Rna14 protein forms a dimer that has inherent conformational variability. The Rna15 protein binds to the C-terminal domain of Rna14, and is connected to the Rna14 HAT domain by a flexible linker, which may indicate that Rna15 functions somewhat independently of the Rna14 HAT domain. The complete CF-IA complex is explored in detail, including protein-protein interactions within the complex and the stoichiometric ratios of CF-IA components. Unlike previous reports, results indicate that CF-IA may form a dimer with a 2:2:2:2 stoichiometry of Rna14:Rna15:Clp1:Pcf11. Also reported are projects unrelated to CF-IA, including the crystal structure of the biotin-dependent alpha(6)beta(6) geranyl-CoA carboxylase (GCC) holoenzyme. Comparison of GCC to the closely related 3-methylcrotonyl CoA carboxylase (MCC) holoenzyme reveals a conserved domain swap in the carboxyltransferase (CT) domains of both enzymes. This domain swap is not present in the related biotin-dependent carboxylases propionyl-CoA carboxylase (PCC) and acetyl-CoA carboxylase (ACC), which may indicate a distinct lineage for biotin-dependent carboxylases that target the γ-carbon. In addition, comparison of the two structures also reveals a conserved Phe191 in MCC that is absent in GCC. Phe191 blocks a key substrate-binding pocket and explains the differences in substrate-specificities between MCC and GCC. The role of Phe191 is tested by site-directed mutagenesis to a Glycine to open the pocket in MCC and by mutating a structurally equivalent Glycine to Phe to close the pocket in GCC. These mutations can convert MCC to a GCC and vice versa.

Hereditary hearing loss is the most common sensory disorder, affecting 1 in 500 newborns. There are more than 538 million individuals with genetic hearing loss worldwide and this number is expected to grow to 1 billion over the next three decades. Currently, the only option for individuals with hearing loss is mechanical intervention such as hearing aids or cochlear implants. In the past decade, many studies have highlighted the need for personalized gene therapy or molecular intervention to treat genetic deafness. However, in order to fulfill this vision a comprehensive understanding of the intricate mutation-gene-phenotype nuances and relationships is required. Toward this goal, we unraveled novel mutation-gene-phenotype associations and mechanisms in four deafness-causing genes (CIB2, COL11A1, CEACAM16 and DFNA5), by using a combination of in-depth phenotyping, human genetics, cutting edge genomic technologies, murine mutant models, and functional assays. These novel insights revealed mutations in CIB2 do not cause Usher Syndrome, mutations in COL11A1 can cause either non-syndromic or syndromic hearing loss, CEACAM16-related deafness is due to two distinct mechanisms, loss of function and gain of function, and coding variants can influence mRNA assembly and cause DFNA5-related hearing loss. Elucidating these novel mutation-gene-phenotype relationships has improved our knowledge of the pathogenic mechanisms underlying hearing loss and provided much needed answers to individuals seeking a diagnosis for their deafness. Recognizing the complexities associated with genetic hearing loss and the challenges in interpreting the clinical significance of genetic variants, we established the first deafness-specific variant database, the Deafness Variation Database (DVD), which classifies over 876,000 variants across 152 deafness-associated genes. This breadth of data provided us with a unique opportunity to explore the molecular landscape of deafness. We show that over 96% of coding variants are rare and novel and that mutational signatures are unique to each gene and are driven by minor allele frequency thresholds, variant effect, and protein domain. The mutational landscape we define shows complex gene-specific variability, making an understanding of these nuances foundational for improved accuracy in variant interpretation. Overall the work presented in this thesis improves our understanding of deafness biology, identifies novel targets for therapeutics and enhances clinical decision-making.

No description available.

During an adenovirus infection the accumulation of alternatively spliced mRNAs is subjected to a tight temporal regulation. The IIIa protein is a structural protein expressed exclusively late after infection. To study the significance of the restricted IIIa protein expression we used a Tet-ON regulated adenoviral vector to overexpress the IIIa protein during the early phase of infection. The results show that unregulated IIIa protein expression caused a reduction in late viral protein accumulation and a slight block of viral DNA replication. Further, the results indicate that IIIa splicing might be subjected to a regulation via a feed back loop stimulating its own expression. To improve the efficacy of vectors for regulated transgene expression, we constructed binary adenoviral vectors based on the Tet-ON and Tet-OFF systems. These vectors encode both the transcriptional activator and the tetracycline-regulated expression cassette from the same viral unit, ensuring that each infected cell will express both the activator and the reporter gene. In model experiments this system was shown to result in a tight control of gene expression with no detectable background expression of the transgene and induction levels reaching 500-600 fold. Introduction of dsRNA into a cell will induce a sequence specific degradation of the homologous mRNA via a mechanism named RNA interference (RNAi). The adenovirus VA RNAs are short highly structured RNAs that are expressed in large amounts late during an adenovirus infection. Here we showed that both VA RNAI and VA RNAII functions as virus-encoded suppressors of RNAi, by interfering with the activity of Dicer, the enzyme that cleaves the initial dsRNA to short-interfering RNAs (siRNAs) that mediate RNAi. Further, the VA RNAs themselves are substrates for Dicer and are cleaved into siRNAs in vivo that are incorporated into active RNA-induced silencing complexes. There is a great interest in developing novel therapeutic strategies based on RNAi. We constructed adenoviral vectors that express short hairpin RNAs, which in vivo will be cleaved to siRNAs that induce sequence-specific RNAi. We compared the efficiency of RNAi induced by vectors based on the viral VA RNAI and the human U6 promoters. Our results suggest that under conditions where the recombinant virus does not replicate, the VA RNA promoter is more effective in down regulating target gene expression, whereas the U6 promoter was more effective under replicative conditions.

This review article presents the splicing process during messenger RNA maturation and how it is regulated by different Cis-regulatory RNA-sequence elements and splicing factors. A more detailed description of the process alternative splicing and its importance to the function of genes from the model organism Arabidopsis thaliana is also given. A single eukaryotic gene can by the process alternative splicing (AS) give rise to a number of functionally mature mRNA-molecules, which in turn encodes for structurally and/or functionally different proteins. During the course of evolution, the process alternative splicing has thus shown to be effective in increasing transcriptome and proteome diversity of most eukaryotic organisms. This suggests therefore that the dominant theory in molecular biology, a gene encodes for a protein, needs to be corrected. A future challenge is to determine the function of the proteins obtained from a given gene by alternative splicing.

Although many group I (GI) introns can self-splice in vitro, their splicing is promoted by proteins in vivo. Only a few splicing factors that specifically promote GI intron splicing have been identified, however, none are from chloroplasts, which is the subject of this study. In previous work from our lab, a strategy was developed to identify splicing factors for chloroplast GI introns of Chlamydomonas by using suppressor genetics. A mutant with reduced splicing of the chloroplast 23S rRNA intron (Cr.LSU) was generated. Then, 3 nuclear suppressors (7120, 71N1 and 7151) with substantially restored splicing of Cr.LSU were isolated and partially characterized. However, the suppressor gene(s) were not identified. In this study, I have used genetic mapping to make a renewed attempt to isolate these genes. Using polymorphisms between the 137C strain that was used for suppressor isolation, and a new strain of C.reinhardtii (S1D2), the nuclear suppressor mutations in 7120 and 71N1 were mapped to a region on chromosome III that is essentially devoid of recombination. Based on the recombination maps, the suppressor gene in 7120 is located within a ~418-kb region from bp 2,473,064 to 2,891,232, whereas the suppressor in 71N1 is likely located within a ~236-kb subregion from bp 2,473,064 to 2,709,377. It is possible that these mutations are in the same gene; however, the maps could not be refined further due to the lack of recombination in this 418-kb region. I also attempted to compare the genomic sequence of the 7120 suppressor, which was obtained by next-generation sequencing, with the Chlamydomonas reference genome (JGI, v.4). Next-generation sequencing of 7120 revealed the existence of abundant repetitive sequences and transposable elements clustered in a ~40-kb subregion of the recombinationally suppressed 418-kb region on chromosome III. I suggest that the high frequency of repetitive sequences and transposable elements in this region may be the reason for the suppressed recombination. Searching for candidate genes in the mapped region led me to examine a novel protein that was predicted to have a putative chloroplast transit-peptide, and an RNA binding domain. Further bioinformatic analysis revealed a single rhodanese domain with an active-site cysteine. The protein was expressed in E.coli as the full-length and predicted mature forms, plus a small His-tag. The purified mature protein had rhodanese catalytic activity, based on the fact that it was able to transfer sulfur from thiosulfate to cyanide. Also, western blot analysis with a polyclonal antibody produced in rabbits showed that the cellular protein migrated on SDS gels close to the mature protein and faster than the full-length protein, indicative of an organelle-targeted protein. The antibody also showed that the cellular protein co-fractionated with chloroplasts. To gain insight into its in vivo function, the gene was knocked down using the tandem RNAi system (Rohr et al., 2004), which produced strains (5) with reductions of 31% to 76% in the mRNA level, and ~30% to ~60% in the protein level. These strains were sensitive to bright light, and had reduced rates of growth under all conditions, which are characteristics of chloroplast translation mutants. Thus, chloroplast protein synthesis was examined by radioisotope pulse-labeling in the presence of cycloheximide, which showed that the RNAi strains were broadly and negatively affected, and RT-PCR and northern blot revealed only normal chloroplast mRNA levels. These data have identified a new rhodanese-family enzyme that is required for chloroplast translation, which I have designated “CRLT”, for chloroplast rhodanese-like translation. / text