Indiana University-Purdue University Indianapolis (IUPUI) / RNA-binding proteins (RBPs) are vital post-transcriptional regulatory molecules in transcriptome of mammalian species. It necessitates studying their expression dynamics to extract how post-transcriptional networks work in various mammalian tissues. RNA binding proteins (RBPs) play important roles in controlling the post-transcriptional fate of RNA molecules, yet their evolutionary dynamics remains largely unknown. As expression profiles of genes encoding for RBPs can yield insights about their evolutionary trajectories on the post-transcriptional regulatory networks across species, we performed a comparative analyses of RBP expression profiles across 8 tissues (brain, cerebellum, heart, lung, liver, lung, skeletal muscle, testis) in 11 mammals (human, chimpanzee, gorilla, orangutan, macaque, rat, mouse, platypus, opossum, cow) and chicken & frog (evolutionary outgroups). Noticeably, orthologous gene expression profiles suggest a significantly higher expression level for RBPs than their non-RBP gene counterparts, which include other protein-coding and non-coding genes, across all the mammalian tissues studied here. This trend is significant irrespective of the tissue and species being compared, though RBP gene expression distribution patterns were found to be generally diverse in nature. Our analysis also shows that RBPs are expressed at a significantly lower level in human and mouse tissues compared to their expression levels in equivalent tissues in other mammals: chimpanzee, orangutan, rat, etc., which are all likely exposed to diverse natural habitats and ecological settings compared to more stable ecological environment humans and mice might have been exposed, thus reducing the need for complex and extensive post-transcriptional control. Further analysis of the similarity of orthologous RBP expression profiles between all pairs of tissue-mammal combinations clearly showed the grouping of RBP expression profiles across tissues in a given mammal, in contrast to the clustering of expression profiles for non-RBPs, which frequently grouped equivalent tissues across diverse mammalian species together, suggesting a significant evolution of RBPs expression after speciation events. Calculation of species specificity indices (SSIs) for RBPs across various tissues, to identify those that exhibited restricted expression to few mammals, revealed that about 30% of the RBPs are species-specific in at least one tissue studied here, with lung, liver, kidney & testis exhibiting a significantly higher proportion of species specifically expressed RBPs. We conducted a differential expression analysis of RBPs in human, mouse and chicken tissues to study the evolution of expression levels in recently evolved species (i.e., humans and mice) than evolutionarily-distant species (i.e., chickens). We identified more than 50% of the orthologous RBPs to be differentially expressed in at least one tissue, compared between human and mouse, but not so between human and an outgroup chicken, in which RBP expression levels are relatively conserved. Among the studied tissues (brain, liver and kidney) showed a higher fraction of differentially expressed RBPs, which may suggest hyper- regulatory activities by RBPs in these tissues with species evolution. Overall, this study forms a foundation for understanding the evolution of expression levels of RBPs in mammals, facilitating a snapshot of the wiring patterns of post-transcriptional regulatory networks in mammalian genomes. In our second study, we focused on elucidating novel features of post-transcriptional regulatory molecules called as circRNA from LongPolyA RNA-sequence data. The debate over presence of nonlinear exon splicing such as exon-shuffling or formation of circularized forms has finally come to an end as numerous repertoires have shown of their occurrence and presence through transcriptomic analyses. It is evident from previous studies that along with consensus-site splicing non-consensus site splicing is robustly occurring in the cell. Also, in spite of applying different high-throughput approaches (both computational and experimental) to determine their abundance, the signal is consistent and strongly conforming the plausible circularization mechanisms. Earlier studies hypothesized and hence focused on the ribo-minus non-polyA RNA-sequence data to identify circular RNA structures in cell and compared their abundance levels with their linear counterparts. Thus far, the studies show their conserved nature across tissues and species also that they are not translated and preferentially are without poly (A) tail, with one to five exons long. Much of this initial work has been performed using non-polyA sequencing thus probably underestimates the abundance of circular RNAs originating from long poly (A) RNA isoforms. Our hypothesis is if the circular RNA events are not the artifact of random events, but has a structured and defined mechanism for their formation, then there would not be biases on preferential selection / leaving of polyA tails, while forming the circularized isoforms. We have applied an existing computational pipeline from earlier studies by Memczack et. al., on ENCODE cell-lines long poly (A) RNA-sequence data. With the same pipeline, we achieve a significant number of circular RNA isoforms in the data, some of which are overlapping with known circular RNA isoforms from the literature. We identified an approach and worked upon to identify the precise structure of circular RNA, which is not plausible from the existing computational approaches. We aim to study their expression profiles in normal and cancer cell-lines, and see if there exists any pattern and functional significance based on their abundance levels in the cell.

Indiana University-Purdue University Indianapolis (IUPUI) / In response to different environmental stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) rapidly reduces protein synthesis, which lowers energy expenditure and facilitates reprogramming of gene expression to remediate stress damage. Central to the changes in gene expression, eIF2 phosphorylation also enhances translation of ATF4, a transcriptional activator of genes subject to the Integrated Stress Response (ISR). The ISR increases the expression of genes important for alleviating stress, or alternatively triggering apoptosis. One ISR target gene encodes the transcriptional regulator CHOP whose accumulation is critical for stress-induced apoptosis. In this dissertation research, I show that eIF2 phosphorylation induces preferential translation of CHOP by a mechanism involving a single upstream ORF (uORF) located in the 5’-leader of the CHOP mRNA. In the absence of stress and low eIF2 phosphorylation, translation of the uORF serves as a barrier that prevents translation of the downstream CHOP coding region. Enhanced eIF2 phosphorylation during stress facilitates ribosome bypass of the uORF, and instead results in the translation of CHOP. Stable cell lines were also constructed that express CHOP transcript containing the wild type uORF or deleted for the uORF and each were analyzed for expression changes in response to the different stress conditions. Increased CHOP levels due to the absence of inhibitory uORF sensitized the cells to stress-induced apoptosis when compared to the cells that express CHOP mRNA containing the wild type uORF. This new mechanism of translational control explains how expression of CHOP and the fate of cells are tightly linked to the levels of phosphorylated eIF2 and stress damage.

Indiana University-Purdue University Indianapolis (IUPUI) / In response to different environmental stresses, phosphorylation of eIF2 (eIF2P) represses global translation coincident with preferential translation of ATF4. ATF4 is a transcriptional activator of the integrated stress response, a program of gene expression involved in metabolism, nutrient uptake, anti-oxidation, and the activation of additional transcription factors, such as CHOP/GADD153, that can induce apoptosis. Although eIF2P elicits translational control in response to many different stress arrangements, there are selected stresses, such as exposure to UV irradiation, that do not increase ATF4 expression despite robust eIF2P. In this study we addressed the underlying mechanism for variable expression of ATF4 in response to eIF2P during different stress conditions and the biological significance of omission of enhanced ATF4 function. We show that in addition to translational control, ATF4 expression is subject to transcriptional regulation. Stress conditions such as endoplasmic reticulum stress induce both transcription and translation of ATF4, which together enhance expression of ATF4 and its target genes in response to eIF2P. By contrast, UV irradiation represses ATF4 transcription, which diminishes ATF4 mRNA available for translation during eIF2∼P. eIF2P enhances cell survival in response to UV irradiation. However, forced expression of ATF4 and its target gene CHOP leads to increased sensitivity to UV irradiation. In this study, we also show that C/EBPβ is a transcriptional repressor of ATF4 during UV stress. C/EBPβ binds to critical elements in the ATF4 promoter resulting in its transcriptional repression. The LIP isoform of C/EBPβ, but not the LAP version is regulated following UV exposure and directly represses ATF4 transcription. Loss of the LIP isoform results in increased ATF4 mRNA levels in response to UV irradiation, and subsequent recovery of ATF4 translation, leading to enhanced expression of its target genes. Together these results illustrate how eIF2P and translational control, combined with transcription factors regulated by alternative signaling pathways, can direct programs of gene expression that are specifically tailored to each environmental stress.

Text in English / Abstracts in English and Venda / This study has investigated the effectiveness of available tools used for translating from English into Tshivenḓa and vice versa with the aim to investigate and determine the effectiveness of these tools. This study dealt with the problem of lack of effective translation tools used to translate between English and Tshivenḓa. Tshivenḓa is one of South Africa’s minority languages. Its (Tshivenḓa) lack of effective translation tools negatively affects language practitioners’ work. This situation is perilous for translation quality assurance. Translation tools, both computer technology and non-computer technology tools abound for developed languages such as English, French and others. Based on the results of this research project, the researcher did make recommendations that could remedy the situation. South Africa is a democratic country that has a number of language-related policies. This then creates a conducive context for stakeholders with language passion to fully develop Tshivenḓa language in all dimensions. The fact is that all languages have evolved and they were all underdeveloped. This vividly shows that Tshivenḓa language development is also possible just like Afrikaans, which never existed on earth before 1652. It (Afrikaans) has evolved and overtaken all indigenous South African languages. This study did review the literature regarding translation and translation tools. The literature was obtained from both published and unpublished sources. The study has used mixed methods research, i.e. quantitative and qualitative research methods. These methods successfully complemented each other throughout the entire research. Data were gathered through questionnaires and interviews wherein both open and closed-ended questions were employed. Both purposive/judgemental and snowball (chain) sampling have been applied in this study. Data analysis was addressed through a combination of methods owing to the nature of mixed methods research. Guided by analytic comparison approach when grouping together related data during data analysis and presentation, both statistical and textual analyses have been vital in this study. Themes were constructed to lucidly present the gathered data. At the last chapters, the researcher discussed the findings and evaluated the entire research before making recommendations and conclusion. / Iyi ṱhoḓisiso yo ita tsedzuluso nga ha kushumele kwa zwishumiswa zwi re hone zwine zwa shumiswa u pindulela u bva kha luambo lwa English u ya kha Tshivenḓa na u bva kha Tshivenḓa u ya kha English ndivho I ya u sedzulusa na u lavhelesa kushumele kwa izwi zwishumiswa uri zwi a thusa naa. Ino ṱhoḓisiso yo shumana na thaidzo ya ṱhahelelo ya zwishumiswa zwa u pindulela zwine zwa shumiswa musi hu tshi pindulelwa vhukati ha English na Tshivenḓa. Tshivenḓa ndi luṅwe lwa nyambo dza Afrika Tshipembe dzine dza ambiwa nga vhathu vha si vhanzhi. U shaea ha zwishumiswa zwa u pindulela zwine zwa shuma nga nḓila I thusaho zwi kwama mushumo wa vhashumi vha zwa nyambo nga nḓila I si yavhuḓi. Iyi nyimele I na mulingo u kwamaho khwaḽithi ya zwo pindulelwaho. Zwishumiswa zwa u pindulela, zwa thekhnoḽodzhi ya khomphiyutha na zwi sa shumisi thekhnoḽodzhi ya khomphiyutha zwo ḓalesa kha nyambo dzo bvelelaho u tou fana na kha English, French na dziṅwe. Zwo sendeka kha mvelelo dza ino thandela ya ṱhoḓisiso, muṱoḓisisi o ita themendelo dzine dza nga fhelisa thaidzo ya nyimele. Afrika Tshipembe ndi shango ḽa demokirasi ḽine ḽa vha na mbekanyamaitele dzo vhalaho nga ha dzinyambo. Izwi zwi ita uri hu vhe na nyimele ine vhafaramikovhe vhane vha funesa nyambo vha kone u bveledza Tshivenḓa kha masia oṱhe. Zwavhukuma ndi zwa uri nyambo dzoṱhe dzi na mathomo nahone dzoṱhe dzo vha dzi songo bvelela. Izwi zwi ita uri zwi vhe khagala uri luambo lwa Tshivenḓa na lwone lu nga bveledzwa u tou fana na luambo lwa Afrikaans lwe lwa vha lu si ho ḽifhasini phanḓa ha ṅwaha wa 1652. Ulu luambo (Afrikaans) lwo vha hone shangoni lwa mbo bveledzwa lwa fhira nyambo dzoṱhe dza fhano hayani Afrika Tshipembe. Kha ino ṱhoḓisiso ho vhaliwa maṅwalwa ane a amba nga ha u pindulela na nga ha zwishumiswa zwa u pindulela. Maṅwalwa e a vhalwa o wanala kha zwiko zwo kanḓiswaho na zwiko zwi songo kanḓiswaho. Ino ṱhoḓisiso yo shumisa ngona dza ṱhoḓisiso dzo ṱanganyiswaho, idzo ngona ndi khwanthithethivi na khwaḽithethivi. Idzi ngona dzo shumisana zwavhuḓisa kha ṱhoḓisiso yoṱhe. Data yo kuvhanganywa hu tshi khou shumiswa dzimbudziso na u tou vhudzisa hune afho ho shumiswa mbudziso dzo vuleaho na dzo valeaho. Ngona dza u nanga sambula muṱoḓisisi o shumisa khaṱulo yawe uri ndi nnyi ane a nga vha a na data yo teaho na u humbela vhavhudziswa uri vha bule vhaṅwe vhathu vha re na data yo teaho ino ṱhoḓisiso. viii Tsenguluso ya data ho ṱanganyiswa ngona dza u sengulusa zwo itiswa ngauri ṱhoḓisiso ino yo ṱanganyisa ngona dza u ita ṱhoḓisiso. Sumbanḓila ho shumiswa tsenguluso ya mbambedzo kha u sengulusa data. Data ine ya fana yo vhewa fhethu huthihi musi hu tshi khou senguluswa na u vhiga. Tsenguluso I shumisaho mbalo/tshivhalo (khwanthithethivi) na I shumisaho maipfi kha ino ngudo dzo shumiswa. Ho vhumbiwa dziṱhoho u itela u ṱana data ye ya kuvhanganywa. Ngei kha ndima dza u fhedza, muṱodisisi o rera nga ha mawanwa, o ṱhaṱhuvha ṱhoḓisiso yoṱhe phanḓa ha u ita themendelo na u vhina. / African Languages / M.A. (African Languages)

Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer disease (AD) results, in part, from the excess accumulation of the amyloid-β peptide (Aβ) as neuritic plaques in the brain. The short Aβ peptide is derived from a large transmembrane precursor protein, APP. Two different proteolytic enzymes, BACE1 and the gamma-secretase complex, are responsible for cleaving Aβ peptide from APP through an intricate processing pathway. Dysregulation of APP and BACE1 levels leading to excess Aβ deposition has been implicated in various forms of AD. Thus, a major goal in this dissertation was to discover novel regulatory pathways that control APP and BACE1 expression as a means to identify novel drug targets central to the Aβ-generating process. MicroRNAs (miRNA) are short, non-coding RNAs that act as post-transcriptional regulators of gene expression through specific interactions with target mRNAs. Global analyses predict that over sixty percent of human transcripts contain evolutionarily conserved miRNA target sites. Therefore, the specific hypothesis tested was that miRNA are relevant regulators of APP and BACE1 expression. In this work, several specific miRNA were identified that regulate APP protein expression (miR-101, miR-153 and miR-346) or BACE1 expression (miR-339-5p). These miRNAs mediated their post-transcriptional effects via interactions with specific target sites in the APP and BACE1 transcripts. Importantly, these miRNA also altered secretion of Aβ peptides in primary human fetal brain cultures. Surprisingly, miR-346 stimulated APP expression via target sites in the APP 5’-UTR. The mechanism of this effect appears to involve other RNA-binding proteins that bind to the APP 5’-UTR. Expression analyses demonstrated that these miRNAs are expressed to varying degrees in the human brain. Notably, miR-101, miR-153 and miR-339-5p are dysregulated in the AD brain at various stages of the disease. The work in this dissertation supports the hypothesis that miRNAs are important regulators of APP and BACE1 expression and are capable of altering Aβ homeostasis. Therefore, these miRNA may possibly serve as novel therapeutic targets for AD.

Indiana University-Purdue University Indianapolis (IUPUI) / RNA polymerase II (RNAPII) is regulated by multiple modifications to the C-terminal domain (CTD) of the largest subunit, Rpb1. This study has focused on the relationship between hyperphosphorylation of the CTD and RNAPII turnover and proteolytic degradation as well as post-translational modifications of the globular core of RNAPII. Following tandem affinity purification, western blot analysis showed that MG132 treated RTR1 ERG6 deletion yeast cells have accumulation of total RNAPII and in particular, the hyperphosphorylated form of the protein complex. In addition, proteomic studies using MuDPIT have revealed increased interaction between proteins of the ubiquitin-proteasome degradation system in the mutant MG132 treated yeast cells as well as potential ubiquitin and phosphorylation sites in RNAPII subunits, Rpb6 and Rpb1, respectively. A novel Rpb1 phosphorylation site, T1471-P, is located in the linker region between the CTD and globular domain of Rpb1 and will be the focus of future studies to determine biological significance of this post-translational modification.

Indiana University-Purdue University Indianapolis (IUPUI) / Translation Initiation in protein synthesis is a crucial step controlling gene expression that enhanced by eukaryotic translation initiation factors (eIFs). eIF3a, the largest subunit of eIF3 complexes, has been shown to regulate protein synthesis and cellular response to cisplatin treatment. Its expression has also been shown to negatively associate with prognosis. In this study, we tested a hypothesis that eIF3a regulates synthesis of proteins important for repair of double strand DNA breaks induced by ionizing radiation (IR). We found that eIF3a up-regulation sensitizes cellular response to IR while its knockdown causes resistance to IR. We also found that eIF3a over-expression increases IR-induced DNA damage and decreases Non-Homologous End Joining (NHEJ) activity by suppressing expression level of NHEJ repair proteins such as DNA-PKcs and vice versa. Together, we conclude that eIF3a plays an important role in cellular response to DNA-damaging treatments by regulating synthesis of DNA repair proteins and, thus, eIIF3a likely plays an important role in the outcome of cancer patients treated with DNA-damaging strategies including ionizing radiation.