Chicken fetuses exposed to chronic hypoxia suffer from growth retardation and induces an overall sympathetic activity, including elevation of the concentration of circulating catecholamines. Simultaneously, hypoxic fetuses display a lowered β-adrenoreceptor (βAR) density in myocardial tissue. In vertebrates, β1AR and β2AR are the most important signalling pathways for acute elevation of cardiac performance. The aim of this study was to see how chronic hypoxia affects the level of messenger RNA (mRNA) for the β1AR in the fetal chicken heart at different developmental ages. The broiler chicken is a suitable model organism for studying the progression of heart failure because the fast growth rate requires a large increase in blood perfusion at the end of fetal development. The β1AR sequence of the broiler chicken is 1587 bp and located on chromosome 6. When running a PCR for quantification of the sequence, primers for almost the whole sequence failed (1404 bp) and so did primers of 1193 bp; instead primers of 692 bp of the sequence were used and made quantification possible. Similar results were obtained from both the heart and liver of day 15 fetal chickens. The PCR product was cloned into a TOPO vector and sent for sequencing, to enable the making of a probe for a northern blot analysis of the mRNA in the fetal chicken hearts.

RNA-binding proteins (RBP) shuttle between cellular compartments either constitutively or in response to stress and regulate localization, translation and turn over of mRNAs. In our laboratory, cytosolic proteome map of Phanerochaete chrysosporium was established and upon Pb exposure, the changes in cytosolic protein expressions were determined. The identified RBPs were a newly induced polyadenylate-binding protein (RRM superfamily) as well as two up-regulated proteins, namely splicing factor RNPS1 and ATP-dependent RNA helicase, all being very important candidates of post-transcriptional control in response to stress. This finding inspired us to conduct Real Time PCR studies in order to have a better understanding of the changes in the expression of corresponding genes at mRNA level in response to Pb exposure, thus the present study aims at examining the effect of lead exposure on the transcript levels of the genes coding for ATP-dependent RNA helicase, splicing factor RNPS1 and polyadenylate binding protein. As shown via expression analysis based on Real Time PCR, the mRNA level of splicing factor RNPS1 showed 2.68, 2.62 and 4.86 fold increases in a dose-dependent manner when the cells were grown for 40 h in the presence of 25, 50 and 100 &micro / M Pb, repectively. ATP-dependent RNA helicase mRNA level showed no significant increase in response to 25 &micro / M Pb exposure while increased 2 and 1.84 fold in response to 50 and 100 &micro / M Pb, respectively. Polyadenylate binding protein mRNA levels revealed no significant increase when exposed to 25, 50 and 100 &micro / M Pb. As to the mRNA dynamics as a function of duration of lead exposure, the mRNA level of this protein showed 2.54-fold increase upon 1 h exposure to 100 &micro / M Pb. Splicing factor RNPS1 mRNA level showed a significant increase of 19.22 fold at 2nd h of 50 &micro / M Pb exposure. Expression level of ATP-dependent RNA helicase was not affected by the time of exposure to Pb.

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Regulation of gene expression is a complex process in mammalian cells with many levels of control. In recent years non-coding RNAs in the form of microRNAs (miRNA) have surfaced as important regulators of protein coding genes, with biologically important roles in development, differentiation and cell growth. In this dissertation the complex interactions between miRNAs and mRNAs in ovarian cancer are investigated using a combination of computational and experimental techniques. In vitro studies and current models predict that increases in levels of miRNA should result in corresponding decreases in the levels of targeted mRNAs due to miRNA induced degradation. Profiling the global miRNA and mRNA expression patterns in epithelial ovarian cancer cells from patients and surface epithelial cells from normal ovaries reveal only ~11% of predicted targets of miRNAs are inversely correlated in vivo. In an effort to dissect the mechanisms behind these unexpected observations single miRNA transfection experiments are carried out followed by gene expression profiling. Analysis of genes altered following these transfections reveal majority of the altered genes are not direct targets of the miRNAs. Network analysis however suggests that miRNAs may target "hub genes" to cause altered expression in downstream transcripts. Pathway enrichment analysis of altered genes demonstrates miRNAs may regulate specific pathways rather than causing random off-target effects. Finally investigation of miRNA regulation reveals miRNAs may also affect the levels of other miRNAs, which may indirectly affect more genes downstream. Together these results provide a detailed view of the mechanisms employed by miRNAs to regulate the expression of hundreds of genes in ovarian cancer cells.

Androgens and the androgen receptor (AR) play important roles in virilization, spermatogenesis, and sexual behavior in vertebrates. An understanding of the distribution and levels of expression of the ARs on the cellular and tissue level demonstrates the pattern of responsiveness to the androgenic hormones in a given organism. In this study, a fragment of the AR gene was cloned and sequenced from the bonnethead shark, Sphyrna tiburo, an elasmobranch species with a well-defined annual reproductive cycle. Acquiring this gene sequence facilitated the construction of species-specific AR polymerase chain reaction (PCR) primers and species-specific AR mRNA probes that were used to screen reproductive tissues for evidence of AR gene expression using reverse transcription (RT)-PCR and in situ hybridization (ISH), respectively. The RT-PCR screens demonstrated AR gene expression in the testes, epididymides, seminal vesicles, and claspers of male sharks. The use of relative PCR revealed that these organs have variable levels of AR gene expression that significantly differ with the stage of the shark's seasonal reproductive cycle. ISH results localized the AR RNA in the interstitial cells, Sertoli cells, and developing sperm of the testes, and mature spermatozoa within the seminal vesicles and the epididymides. Immunocytochemical methods used to detect the AR protein using a rabbit polyclonal antibody, PG-21, produced comparable results in the shark testes but did not yield positive results in the seminal vesicles or the epididymides. However, the Leydig gland, whose secretions contribute to the seminal fluid, demonstrated consistent AR immunoreactivity. Results of ICC in male and female embryos of S. tiburo revealed AR protein in the developing kidney but not in the embryonic reproductive structures. By characterizing AR distribution in the reproductive tract of male S. tiburo, this study provides the basis for future research on the direct and indirect effects of androgenic hormones in this species.

Prediction of outcome in colorectal cancer (CRC) is currently based on the TNM staging classification; however, histopathological classification alone is insufficient for accurately predicting survival in stage II and III patients. Studies indicate that microarray gene expression profiles can predict survival in CRC. We hypothesize that tumor gene expression in combination with clinical parameters, is a better predictor of outcome in stage II and III colorectal cancers than the TNM stage classification alone. Clinical records and follow-up data were retrospectively reviewed for 58 Stage II and Stage III patients with primary colorectal cancer, who did not receive any neoadjuvant therapy preoperatively and whose samples had been previously analyzed for gene expression profiles using the Affymetrix U 133a Gene chip. For molecular classification of patients as being at high or low risk for poor survival, samples were divided into two clusters by hierarchical cluster analysis of genes selected by SAM. Univariate and multivariate analyses using Cox proportional hazard models were done to identify significant prognostic factors. The 3-year and 5-year survival estimates were 72.41% (SE=5.8%) and 55.17% (SE=6.7%), respectively, for all 58 patients. Univariate analysis showed that advanced stage, older age, high-risk molecular classification, positive lymph nodes were the statistically significant prognostic factors of poor survival (p<0.05), while gender, preoperative CEA level, and family history of CRC in first degree relatives were not statistically significant. In multivariate analysis molecular classification, age and body mass index were independent significant prognostic factors. In Cox proportional hazard model, the estimated hazard ratios for Stage III vs II was 2.45 (95%CI: 0.85-7.04), for high vs low molecular risk was 3.83 (95%CI: 1.22-12.06) and old vs young age was 3.72 (95%CI: 1.2-11.49). Model containing clinical stage in conjunction with molecular risk, body mass index, and age was a stronger indicator of clinical outcome (p= 0.0056) than model with clinical stage alone. Gene expression profiles predict survival independent of clinical parameters, and the addition of gene expression profiles to stage is more predictive of survival than stage alone. Further analysis needs to be done to validate the molecular classification on an independent dataset.

The function and evolution of introns have been topics of great interest since introns were discovered in the 1970s. Introns that interrupt protein-coding regions have the most obvious potential to affect coding sequences; therefore, their evolution have been studied most intensively. Splicing of introns within untranslated regions does not contribute directly to the diversity of proteins, yet ~35% of human transcripts contain introns within the 5' untranslated region (UTR). The evolution and possible functions of 5'UTR introns (5UIs) remain largely unexplored. Here we undertook a genome-wide functional analysis of 5UIs. Our main results are as follows: First, the distribution of these introns in the human genome is nonrandom. While genes with regulatory roles are enriched in having 5UIs, genes encoding proteins that are targeted to the endoplasmic reticulum and mitochondria are surprisingly depleted of these introns. Second, we offered and supported a model whereby gene encoding secretory and nuclear-encoded mitochondrial proteins share a common regulatory mechanism at the level of mRNA export, which is dependent on the absence of 5'UTR introns. Specifically, the upstream element in a given transcript, be it an intron or RNA elements near the 5' end of coding sequences (CDS), dictates the mRNA export pathway used. Finally, we discovered a strong correlation between existence of 5'UTR introns and sequence features near the 5' end of CDS. We developed an integrated machine-learning framework that can predict absence of 5UIs using solely the sequence near the 5' end of CDS. Our model achieved >80% accuracy when validated against nuclear-encoded mitochondrial transcripts. Specific RNA elements predictive of 5UI absence are found in ~40% of human transcripts spanning a wide spectrum of functions. By analyzing hundreds of large-scale datasets, we functionally characterized the transcripts with these RNA elements; revealing their association with translational regulation. These RNA elements were bound by proteins interacting with the Exon Junction Complex in vivo suggesting a molecular mechanism that links these elements to their downstream effects in mRNA export and translational regulation. While some 5'UTR introns might be evolving neutrally, our results, taken together, suggest that complex evolutionary forces are acting on this distinct class of introns.

Innate immune signaling pathways initiate host defenses against viral pathogens. Receptors specific for viral nucleic acids activate these pathways culminating in cell-to-cell communication and/or cell death. In mammals, this cell- to-cell communication is achieved through the production of interferons and pro- inflammatory cytokines, which activate antiviral defenses in uninfected neighboring cells and instruct adaptive immune responses. The production of these signaling molecules is essential for the defense against viral infection, but must also be tightly regulated to prevent unnecessary inflammation. As an antiviral defense, cell death is also an effective mechanism to limit viral replication and spread but comes with the cost of tissue damage and inflammation. Therefore, regulating these antiviral responses is critical for controlling the spread of infection as well as preventing unnecessary pathologies related to excessive signaling. Hundreds of genes are involved in controlling these immune responses and a wide variety of mechanisms are utilized to regulate them. One mechanism to regulate gene function is the generation of protein variants through alternative translation. While polycistronic transcripts are a common feature of bacterial and viral gene expression, the process of alternative translation as a means to regulate gene function is not a feature generally attributed to mammalian mRNA. This dissertation describes a novel regulator of antiviral signaling that is generated through alternative translation. Expression of the transcript encoding the antiviral adaptor protein, MAVS, results in the production of two proteins: the full-length MAVS adaptor and a truncated variant, miniMAVS. Production of these proteins is in part regulated by cis-acting elements that control translation initiation. Production of miniMAVS regulates antiviral signaling by limiting interferon production induced by full-length MAVS, whereas both MAVS variants positively regulate cell death. To identify other examples of alternative translation in mammalian cells a genome-wide ribosomal profiling technique was used to generate a candidate list of antiviral truncation variants. This dissertation therefore demonstrates that protein variants generated through alternative translation of polycistronic mRNAs can be an effective mechanism for immune regulation and may be more common than previously understood.

RNA localization, a regulated step of gene expression, is fundamentally important in development and differentiation. In multidisciplinary experiments, we discovered that RNA (mis)localization underlies the human disease myotonic dystrophy (DM). DM, the most prevalent adult muscular dystrophy, is caused independently by two alleles: DM1 is characterized by a (CTG)n expansion in the DM kinase (DMPK) gene 3' untranslated region while DM2 has a mutation in a small presumptive RNA binding protein. These analyses were guided by disease characteristics and have provided insights to DM's cytopathology, cell biology and molecular genetics. Examining muscle biopsies, it is demonstrated here that DM kinase mRNA is specifically subcellularly localized within normal human muscle and that DM kinase mRNA harboring the 3’UTR mutation (DM1) is mislocalized in DM patient muscle to cytoplasmic areas characteristic of DM disease pathology. Thus, the disease mutation alters the cellular distribution of the effected message. DMPK mRNA mislocalization causes altered DM kinase protein localization, correlates with novel phosphoprotein appearance and can account for DM’s diseased phenotype. While we were fortunate to access DM patient tissue to establish these key findings, the system does not lend itself to experimental manipulation. Hence, I established a disease- relevant tissue culture system, which recapitulates DMPK trafficking, Employing this system; I elucidate a complementary role for the DM2 gene product as a localization factor for DMPK mRNA (DM1 gene product). Comprehensive RNA-protein interaction experiments reveal the DM2 protein specifically and selectively recognizes a small, definitive area within the DMPK RNA 3'UTR. Detailed biochemical, cytological and functional experiments reveal 1) the DM2 protein colocalizes with DMPK mRNA, 2) the small area of the DMPK 3’UTR bound by pDM2 acts to properly localize a reporter construct and 3) disruption of the DM2 protein results in DMPK mRNA mislocalization. These data establish mRNA localization as a vital process underlying human disease etiology. Moreover, they reveal DM1 and DM2 gene products function in the same molecular pathway and that mutation of either causes DMPK mRNA mislocalization, leading to disease. These data have apparent application to several neuromuscular disorders and open a plethora of novel research avenues, both basic and applied. / text

Eukaryotic gene expression requires hundreds of proteins and several RNA factors to facilitate nuclear RNA processing. These RNA processing events include RNA transcription, pre-mRNA splicing, pre-ribosomal RNA (pre-rRNA) processing and trafficking of RNA to its proper location in the cell. As we learn more about the molecular details of the factors governing these highly coordinated processes it is becoming increasingly clear that a subset of factors participate in multiple RNA processing pathways to ensure faithful gene expression. Our work completes the characterization of the Abelson pre-mRNA splicing mutants. We have discovered that the prp27-1 splicing mutant is a severe loss of function allele of RAT1, an essential 5’→3’ exoribonuclease. Several alleles of RAT1 have been previously isolated with each conferring an array of phenotypes thus making the elucidation of its essential in vivo function difficult. We set out to determine how mutations within a specific region determines the RNA processing pathway in which Rat1p has been implicated to function within. In our analysis of Rat1p function we discovered the prp27-1 allele exhibits novel 3’ end processing defects never reported in previous rat1 mutants. We performed mutational analysis to examine the coupling of 5’ and 3’ exonucleolytic activities in nuclear RNA processing events. Through our study we have discovered a means by which the cell coordinately regulates the nuclear RNA degradation complexes to ensure efficient processing of pre-RNAs for the faithful execution of eukaryotic gene expression. Additionally, we offer evidence in support of role for Rat1p in promoting mitotic events in vivo. / text