In budding yeast, the Trf4/5p-Air1/2p-Mtr4p polyadenylation (TRAMP) complex recognizes unwanted RNA transcripts in the nucleus and then targets them to the nuclear exosome for rapid degradation, constituting an important pathway of nuclear RNA quality control. Each pre-mRNA splicing event unavoidably generates a RNA side-product that should be recognized by TRAMP and then removed by the nuclear exosome to prevent the potentially harmful sequestration of splicing factors and/or ribonucleotides. While successful pre-mRNA splicing inevitably produces a spliced-out intron, errors in pre-mRNA splicing lead to the emergence of either an abnormal splicing intermediate, or a splicing-incompetent pre-mRNA that cannot be properly spliced. However, it remains unclear how and when these RNA side-products of pre-mRNA splicing are recognized by TRAMP. In this study, chromatin immunoprecipitation (ChIP) was applied to demonstrate that both TRAMP and the nuclear exosome component Rrp6p are cotranscriptionally recruited to nascent RNA transcripts, particularly to intronic sequences, indicating that splicing side-products are recognized by TRAMP and committed to subsequent nuclear-exosome-mediated degradation in a cotranscriptional manner. Deletion of TRF4, of both AIR1 and AIR2, or of RRP6, resulted in accumulation of unspliced pre-mRNAs. Surprisingly, while such pre-mRNAs accumulated in rrp6 cells owing to defects in pre-mRNA degradation, the same phenotype in trf4 and air1air2 cells involved splicing defects, demonstrating that only TRAMP, but not the nuclear exosome, contributes to optimal pre-mRNA splicing. Consistent with a direct stimulatory role for TRAMP in pre-mRNA splicing, negative genetic interactions and physical interactions between Trf4p and several splicing factors were observed, and that Trf4p was further shown to be required for optimal recruitment of the splicing factor Msl5p. The direct facilitation of pre-mRNA splicing by TRAMP may act as a fail-safe mechanism to ensure the cotranscriptional recruitment of TRAMP to nascent intron-containing transcripts before or during pre-mRNA splicing, such that the subsequently generated spliced-out introns, abnormal splicing intermediates, or splicing-incompetent pre-mRNAs can be recognized immediately by TRAMP, and then targeted to the nuclear exosome for prompt degradation before their potentially harmful accumulation. Since most TRAMP and nuclear exosome components found in budding yeast also contain functional human homologs, this work provides important insights into how splicing side-products are rapidly degraded by the nuclear RNA quality control system in human cells, which have a much higher frequency of introns within their genome, and certainly require a much more efficient pathway for the removal of an increased amount of splicing side-products due to the greater number of splicing events. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

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Thesis (Ph.D.) -- University of Adelaide, Dept. of Biochemistry, 1976? / Typescript (photocopy).

Thesis (Ph.D.) -- University of Adelaide, Dept. of Biochemistry, 1977. / Typescript (photocopy).

Thesis (M.S.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains vi, 56 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 55-56).

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In this work, rainbow trout were used as a model system to examine the regulation of insulin (INS)- and insulin receptor (IR)-encoding mRNA expression profiles. INS- and IR-encoding mRNAs were isolated, cloned, and sequenced; and shown to be differentially expressed within and among multiple tissue types. Regulation was examined through various nutritional and hormonal treatments (in vivo and in vitro). A real-time quantitative-PCR assay was developed to measure the respective levels of mRNA expression. Fasting, growth hormone (GH), and somatostatin (SS) differentially regulated INS and IR mRNAs within selected tissues, in vivo. Glucose, GH, SS, and insulin-like growth factor-1 (IGF-I) differentially regulated INS and IR mRNAs within selected tissues, in vitro. The results of this dissertation research display the identification and differential regulation of multiple INS- and IR-encoding mRNAs and suggest that independent mechanisms may serve to regulate the various isoforms in a tissue-specific manner. Future studies are also suggested.

The glycolytic enzyme enolase is an abundant, immunodominant antigen of the fungal pathogen Candida albicans. A C. albicans enolase cDNA was used to study the genetic organization and expression of enolase genes. Experimental results were consistent with a model predicting four enolase genes per diploid genome. Enolase steady state mRNA levels increased during logarithmic growth, but were unaffected by pH, growth form or carbon source.

Polyribosome aggregation state in growing tissues of barley and wheat leaf or stems of pea and squash was studied in relation to seedling growth and water status of the growing tissue in plants at various levels of osmotic stress. It was found to be highly correlated with water potential and osmotic potential of the growing tissue and with leaf or stem elongation rate. Stress rapidly reduced polyribosome content and water status in growing tissues of barley leaves; changes were slow and slight in the non-growing leaf blade. Membrane-bound and free polyribosomes were equally sensitive to stress-induced disaggregation. Incorporation of ³²PO₄³⁻ into ribosomal RNA was rapidly inhibited by stress, but stability of poly(A) ⁺RNA relative to ribosomal RNA was similar in stressed and unstressed tissues, with a half-life of about 12 hours. Stress also caused progressive loss of poly(A) ⁺RNA from these tissues. Quantitation of poly(A) and in vitro messenger template activity in polysome gradient fractions showed a shift of activity from the polysomal region to the region of 20-60 S in stressed plants. Messenger RNA in the 20-60 S region coded for the same peptides as mRNA found in the polysomal fraction. Nonpolysomal and polysome-derived messenger ribonucleoprotein complexes (mRNP) were isolated, and characteristic proteins were found associated with either fraction. Polysomal mRNP from stressed or unstressed plants were translated with similar efficiency in a wheat germ cell-free system; activity of nonpolysomal mRNP was variable, but usually less than that of polysomal mRNP. Deproteinization of mRNP failed to improve its activity. No inhibition of translation of poly(A) ⁺RNA by nonpolysomal mRNP was observed in mixing experiments with the wheat germ cell-free system. It was concluded that no translational inhibitory activity was associated with nonpolysomal mRNP from barley prepared as described.