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Date of Award
Doctor of Philosophy (PhD)
Splicing is a crucial step of processing pre-mRNA molecules for precise flow of genetic information from DNA to proteins, where introns are removed and exons of pre-mRNA are joined together to form mature mRNA. The variability in splicing pattern generate a wide array of mature mRNAs from a limited set of genes enabling greater protein diversity with different functions. This process is carried out by a megadalton complex called the spliceosome that consists of more than 200 proteins and snRNA molecules. Previous studies have shown that alterations to a DNA sequence of spliceosome proteins introduce errors in the splicing process that leads to incorrect splicing. Many spliceosome proteins have been implicated in diseases like neurodegenerative disorders, retinitis pigmentosa, cancer and spinal muscular atrophy. Thus, it is important to understand the role of individual spliceosome proteins in the splicing process and their effect on splicing fidelity.
The spliceosome undergoes a series of transitions from a pre-catalytic state (complex A) to catalytically active state (complex C) during the splicing process. A subset of spliceosome proteins forms the core component of the spliceosome that are present throughout the splicing process, while other proteins are transient that bind and leave the complex at different points during the splicing process. Our lab previously characterized loss-of-function mutations in four different spliceosome proteins that act as suppressors of splice site mutations in Chlamydomonas reinhardtii. Interestingly, three out of four proteins are part of the small group of proteins that joins the catalytically active spliceosome complex C late in the splicing process. This dissertation focuses on understanding the role of two of these spliceosome proteins, DGCR14 and FRA10AC1. I analyzed the splicing patterns in dgr14 and fra10 mutants in a wild-type background and in double mutants with a mutation that affects nonsense mediated decay (NMD) to capture the breadth of global splicing changes incurred by the spliceosome mutants. The study demonstrates that NMD is an active pathway in C. reinhardtii and degrades non-functional transcripts as shown by analyzing a nonsense mutation in the SMG1 gene, which disrupts the NMD pathway. Next, I show that the two splicing factors affect the 3’ and 5’ splice site choice and their absence specifically weakens the selectivity of weak 3’ splice site. Also, the newly formed alternate 3’ splice site demonstrate significant decrease in the splicing fidelity at 3’ end. This suggests that the two splicing factors affect the splicing fidelity even though they join the spliceosome complex at late stage, pointing towards a potential proof-reading mechanism in splicing. Further work to investigate the interaction of these factors with other spliceosome proteins and 5’ and 3’ splice site is warranted.
To capture the extent to which alternative splicing is active and functional in C. reinhardtii, I analyzed the existing RNA-seq data from Zones et al., 2015 study, obtained during the diurnal cell cycle of C. reinhardtii. The analysis shows that alternative splicing events are temporally regulated during the cell cycle and identified a subset of events that show periodic changes during the diurnal cell cycle. Many of these events introduce premature termination codon (PTC) in the transcript that potentially makes them NMD targets. This study also demonstrates a potential AS mediated regulation of ODC1 gene which encodes for ornithine decarboxylase enzyme, during light to dark transition during the diurnal cell cycle. Our findings are concordant with previous studies that show light-mediated activation of the ODC1 activity in C. reinhardtii and tobacco plants.
Together my research work in this dissertation reveals new insights into the post-transcriptional regulation of genes by alternative splicing in C. reinhardtii and highlights the role of non-core spliceosome proteins in maintaining the splicing fidelity and selection of weak splice sites.
Chair and Committee
Gary D. Stormo Susan Dutcher
Ting Wang, James G. Umen, Hani Zaher, Tim Schedl,
Pandey, Manishi, "Systematic Investigation of Alternative Splicing and Conserved Spliceosome Factors in Chlamydomonas reinhardtii" (2019). Arts & Sciences Electronic Theses and Dissertations. 1852.
Available for download on Tuesday, August 15, 2119