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Browsing Theses and Dissertations by Author "Abdel-Ghany, Salah, advisor"

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    Functional analyses of splice variants of the splicing regulator SR45 in abiotic stresses in Arabidopsis
    (Colorado State University. Libraries, 2013) Albaqami, Mohammed M., author; Reddy, A. S. N., advisor; Abdel-Ghany, Salah, advisor; Byrne, Patrick, committee member
    Alternative splicing, a post-transcriptional regulatory mechanism of gene expression, produces multiple mRNAs from a single gene. Alternative splicing increases proteome complexity and regulates gene expression through multiple mechanisms. A number of stresses have been shown to regulate alternative splicing of precursor mRNAs in plants and change transcriptome complexity. Serine/arginine-rich (SR) and SR-like proteins that regulate splicing also undergo extensive alternative splicing in response to various stresses. SR45, an SR-like protein, interacts with several spliceosomal proteins such as U170K, SCL33, U2AF35, and also with an intronic sequence of SR30 and regulates alternative splicing of pre-mRNAs of several other SR genes. It has been previously shown that SR45 pre-mRNA undergoes alternative splicing and produces two alternatively spliced mRNA isoforms (long and short) and the proteins coded by these two isoforms differ in eight amino acids. The two isoforms have distinct biological functions during development where the long isoform is important for flower development while the short isoform is necessary for normal root growth. In this work, I have studied the roles of SR45 and its splice variants in heat and salt tolerance using SR45 mutant (sr45) and transgenic lines complemented with either the long or short isoform. I have found that at different developmental stages sr45 shows high sensitivity to heat stress and salt stress as compared to wild type. The sensitivity of sr45 to heat and salt stresses is rescued by the long isoform but not the short one, suggesting that only the long isoform functions in these stresses. Further molecular analyses have revealed that the relative expression and the splicing pattern of heat shock factors (HSFs), heat shock proteins (HSPs), salt overly sensitive (SOS) genes, ABA signaling pathway genes, and other stress-responsive genes are affected in the sr45 mutant and the long isoform is needed for normal splicing and expression of these genes. Furthermore, an in vitro binding assay showed that SR45 binds to an alternatively spliced intron of HsfA2, suggesting that SR45 directly regulates alternative splicing and expression of HsfA2 under heat stress. In addition to misregulation of expression and splicing of some salt stress responsive genes in the mutant, new splicing isoforms that are affected in the mutant are identified, suggesting the importance of SR45 in fine-tuning gene expression under salt stress. In conclusion, results presented here demonstrate that SR45 functions as a positive regulator of tolerance to two abiotic stresses by modulating the expression and splicing of several stress responsive genes. Further, I show that only the long isoform confers tolerance to these abiotic stresses.