Browsing by Author "Reddy, A. S. N., advisor"
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Item Open Access Alternative splicing and its regulatory mechanisms in photosynthetic eukaryotes(Colorado State University. Libraries, 2011) Link, Alicia, author; Reddy, A. S. N., advisor; Stack, Stephen, committee member; Lapitan, Nora, committee memberIn recent years, alternative splicing (AS) of pre-mRNAs, which generates multiple transcripts from a single gene, has emerged as an important process in general proteome diversity and in regulatory gene expression in multicellular eukaryotes. In Arabidopsis over 40% of intron-containing genes are alternatively spliced. However, mechanisms by which AS is regulated in plants are not fully understood, primarily due to the lack of an in vitro splicing system derived from plants. Furthermore, the extent of AS in simple unicellular photosynthetic eukaryotes from which plants have evolved is also not known. My research addresses these two attributes of splicing in plants. In Part 1 of my thesis, I have investigated an aspect of AS regulation in plants. We have previously shown that an SR-related splicing regulator called SR45 regulates AS of pre-mRNAs in Arabidopsis by altering splice site selection (Ali et al. 2007). In this work using bimolecular fluorescent complements, I have demonstrated that SR45 interacts with U2AF35, an important spliceosomal protein involved in 3' splice site selection in plant cells. This interaction takes place in the nucleus, specifically in the subnuclear domains called speckles, which are known to contain splicing regulators and other proteins involved in transcription. My work has shown that SR45 interacts with both paralogs of U2AF35 and I mapped the domains in SR45 that are involved in its interaction with U2AF35. In addition, my studies have revealed interaction of the paralogs as hetero- and homodimers. Interestingly, U2AF35 was found to interact with U1-70K, a key protein involved in 5' splice site selection. Based on this work and previous work in our laboratory, a model is proposed that explains the role of SR45 in splice site selection. In the second part of my work I studied the extent of alternative splicing (AS) in the unicellular green alga Chlamydomonas, that shares a common ancestor with land plants. In collaboration with Dr. Asa Ben Hur's lab, we have performed a comprehensive analysis of AS in Chlamydomonas reinhardtii using both computational and experimental methods. Our results show that AS is common in Chlamydomonas, but its extent is less than what is observed in land plants. However, the relative frequency of different splicing events in Chlamydomonas is very similar to higher plants. We have found that a large number of genes undergo alternative splicing, and together with the simplicity of the system and the use of available molecular and genetic tools. This organism is an experimental system to investigate the mechanisms involved in alternative splicing. To further validate predicted splice variants, we performed extensive analysis of AS for two genes, which not only confirmed predictions but also revealed novel splice variants, suggesting that the extent of AS is higher than we predicted. AS can also play a role in the regulation of gene expression through processes such as regulated unproductive splicing and translation (RUST) that involves nonsense-mediated decay (NMD), a mechanism of mRNA surveillance that degrades transcripts containing premature termination codons (PTCs). The basic mechanism of NMD relies upon many factors, but there are three critical proteins, termed the UP-frameshift (UPF) proteins due to their ability to up-regulate suppression of nonsense transcripts. UPF1, UPF2, and UPF3 appear to be conserved across animals and plants. Our analysis of AS has found that in Chlamydomonas, many splice variants have a premature termination codon (PTC). However, to date, the mechanism of NMD has not been investigated in Chlamydomonas. Analysis of the Chlamydomonas genome sequence shows that UPF1, 2, and 3 proteins are present, and we have shown that they share some sequence similarity with both plants and humans, indicating that the process of NMD may be present in this organism. To address the role of UPFs in NMD in Chlamydomonas, we have utilized the artificial miRNA approach. I have generated stably transformed Chlamydomonas cell lines that are expressing amiRNA for UPF1 and UPF3 that will be useful in analyzing NMD of selected genes as well as all PTC-containing transcripts globally.Item Open Access Analysis of genome-wide targets of Arabidopsis signal responsive 1 (AtSR1) transcription factor and its transcript stability in response to stress(Colorado State University. Libraries, 2017) Abdel-Hameed, Amira, author; Reddy, A. S. N., advisor; Bush, Daniel, committee member; Leach, Jan, committee member; Abdel-Ghany, Salah, committee memberAbiotic and biotic stresses cause significant yield losses in all crops. Acquisition of stress tolerance in plants requires rapid reprogramming of gene expression. SR1/CAMTA3, a member of signal responsive transcription factors (TFs), functions both as a positive and a negative regulator of biotic stress responses and as a positive regulator of cold stress-induced gene expression. Using high throughput RNA-seq, we identified ~3000 SR1-regulated genes. Promoters of about 60% of the differentially expressed genes have a known DNA binding site for SR1, suggesting that they are likely direct targets. Gene ontology analysis of SR1-regulated genes confirmed previously known functions of SR1 and uncovered a potential role for this TF in salt stress. Our results showed that SR1 mutant is more tolerant to salt stress than the wild type and complemented line. Improved tolerance of sr1 seedlings to salt is accompanied with the induction of salt-responsive genes. Furthermore, ChIP-PCR results showed that SR1 binds to promoters of several salt-responsive genes. These results suggest that SR1 acts as a negative regulator of salt tolerance by directly repressing the expression of salt-responsive genes. Overall, this study identified SR1-regulated genes globally and uncovered a previously uncharacterized role for SR1 in salt stress response. Soil salinity, one of the most prevalent environmental stresses, causes enormous losses in global crop yields every year. Therefore, it is imperative to generate salt tolerant cultivars. To achieve this goal, it is essential to understand the mechanisms by which plants respond to and cope with salt stress. Stress-induced reprogramming of gene expression at multiple levels contributes to the survival of plants under adverse environmental conditions. The control of mRNA stability is one of the post-transcriptional mechanisms that is highly regulated under stress conditions leading to changes in expression pattern of many genes. In this study, we show that salt stress increases the level of SR1 mRNA, by enhancing its stability. Multiple lines of evidence indicate that ROS generated by NADPH oxidase activity mediate salt-induced SR1 transcript stability. Furthermore, cycloheximide (CHX), a protein synthesis inhibitor, also increased SR1 mRNA stability, albeit to a higher level than in the presence of salt, suggesting a role for one or more labile proteins in SR1 mRNA turnover. Similar to salt, ROS generated by NADPH oxidase is also involved in CHX-induced SR1 mRNA accumulation. To gain further insights into mechanisms involved in saltand CHX-induced SR1 stability, the roles of different mRNA degradation pathways were examined in mutants that are impaired in either nonsense-mediated decay (NMD) or mRNA decapping pathways. These studies have revealed that neither the NMD pathway nor the decapping of SR1 mRNA is required for its decay. However, decapping activity is required for saltand CHXaccumulation of SR1 mRNA. To identify any specific regions within the open reading frame of the SR1 transcript (~3 kb) that are responsible for the salt-induced accumulation of SR1 mRNA, we generated transgenic lines expressing several truncated versions of the SR1 coding region in the sr1 mutant background. Then, we analyzed accumulation of each version in response to salt stress and CHX. Interestingly, we identified a 500 nts region in the 3' end of the SR1 coding sequence to be required for both saltand CHX-induced stability of SR1 mRNA. Potential mechanisms by which this region confers SR1 transcript stability in response to salt and CHX are discussed.Item Open Access 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 memberAlternative 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.Item Open Access Functional analysis of three Arabidopsis SR proteins (SCL33, SC35, SCL30A) in plant development and splicing(Colorado State University. Libraries, 2012) Thomas, Julie, author; Reddy, A. S. N., advisor; Bedinger, Pat, committee member; Pilon, Marinus, committee member; Wilusz, Jeff, committee memberTo view the abstract, please see the full text of the document.Item Open Access Gene expression regulation by a stress-responsive transcription factor in rice seedlings(Colorado State University. Libraries, 2019) Williams, Seré., author; Reddy, A. S. N., advisor; Leach, Jan, committee member; Bush, Daniel, committee memberStress physiology is an inherently complex field. As plants cannot leave their environment when it becomes unfavorable, they have developed multiple mechanisms to cope with stresses. Many of these are unique to plants compared to mobile organisms. Plant stress physiology is of interest not only for this reason, but because the human population relies on agriculture for food. Additionally, our ecosystem relies on plants as primary producers as an integral component of life on earth. Plant stress physiology at the molecular level involves a symphony of signaling cascades that reshape cell physiology and communicate the stress signal to the whole plant and even nearby organisms. Over the last thirty years, enormous progress has been made to identify key genes, hormones, and signaling pathways that are involved in plant stress responses. To this end, we have yet to understand a cohesive picture of how plants respond to a combination of stresses. Given the variety of biotic stresses from bacteria, fungi, viruses, nematodes, and herbivores and their interaction with abiotic stresses including environmental extremes and resource availability, continued efforts are needed to understand the molecular nuances of plant stress responses. Not only are stresses variable and unique, plants have evolved to thrive in specific habitats, thereby developing unique strategies to cope with local environments. For example, rice grows well in flooded soils which would induce a stress-response in typical, non-aquatic organisms. Therefore, stress response will need to be decoded at the level of the organism. The goal of this work is to better elucidate stress response in rice. Specifically, I have looked at the influence of a transcription factor, SIGNAL RESPONSIVE 1 (OsSR1), that is regulated by Ca2+/CaM and known to be a dynamic regulator in a myriad of stresses in Arabidopsis. I have generated complemented lines of Ossr1 mutant and OsSR1 overexpressor transgenic rice lines. When compared with WT and mutant lines, these lines showed a range of OsSR1 expression. These lines will be of great help in deciphering the action of this transcription factor. Homozygous SR1 complemented and overexpressor lines along with WT and Ossr1 mutant will be used in future studies to better understand the action of SR1 in stress response in rice. Additionally, I performed a factorial global gene expression analysis using RNA-seq with WT and Ossr1 lines at the seedling stage in control and drought conditions, which will serve as a breeding ground for hypothesis generation and testing in future studies. Significant differentially expressed (DE) genes show down-regulation of genes encoding serine threonine-protein kinase receptor (SRK)-receptors, kinases, TCP family transcription factor, cytokinin-modifying enzyme and up-regulation of aquaporin, sucrose synthase, G-protein-related, and ferredoxin-nitrate reductase in the mutant when compared to WT. In response to polyethylene glycol (PEG)-induced drought stress, the mutant up-regulated transcription factors (homeobox [HOX]- containing TFs, WRKY, and DIVARICATA), signaling proteins (protein phosphatases), late embryogenesis abundant protein 1 (LEA1), nodulin-related genes, and senescence-associated gene 21 (SAG21), while down-regulating a CaM-dependent protein kinase, efflux transporters, peroxidases, aquaporins, and disease-related genes including Pathogenesis-related protein PRB1-2, disease resistance protein RPS2, and NB-ARC domain-containing protein. Lastly, significant DE genes in the WT illuminate how this important crop plant responds when exposed to PEG-induced drought. Drought induced the expression of MAPKKKs, ethylene-responsive transcription factors (ERFs), HOX TFs, as well as zinc-finger proteins and protein phosphatase 2Cs. In drought, WT down-regulated glycol-lipid transfer proteins, aquaporins, and salt stress-induced proteins. Gene ontology (GO) analysis of significant DE genes showed enrichment of GO terms related to membranes, oxidative stress, response to stimulus, and transcription regulation in both the WT and mutant when exposed to PEG. Future work will analyze the promoters of candidate genes for the OsSR1 DNA-binding motif (CG-1) to identify direct targets of OsSR1. Rice is the model organism for monocots and provides 15% of the calories consumed by humans. This study and other studies based on this work will help in elucidating the functions of this stress-responsive transcription factor, OsSR1, in this important crop plant.Item Open Access Identification of direct targets of serine/arginine-rich 45 protein isoforms by TRIBE (Targets of RNA-binding proteins Identified By Editing) in Arabidopsis thaliana(Colorado State University. Libraries, 2021) Huynh, Nikki, author; Reddy, A. S. N., advisor; Garrity, Deborah, committee member; Wilusz, Jeffrey, committee memberTo view the abstract, please see the full text of the document.Item Open Access In vitro and in vivo studies on pre-mRNA splicing in plants(Colorado State University. Libraries, 2017) Albaqami, Mohammed M., author; Reddy, A. S. N., advisor; Wilusz, Jeffrey, committee member; Ben-Hur, Asa, committee member; Montgomery, Tai, committee memberTo view the abstract, please see the full text of the document.Item Open Access Isolation and characterization of proteins that interact with a pollen-specific calmodulin-binding protein(Colorado State University. Libraries, 2008) Shin, Sung-Bong, author; Reddy, A. S. N., advisorCalcium and calmodulin, a calcium sensor, are implicated in pollen germination and tube growth. However, the mechanisms by which calcium and calmodulin regulate these processes are largely unknown. Calcium bound calmodulin regulates diverse cellular processes by modulating the activity of other proteins called calmodulin-binding proteins. Maize pollen-specific calmodulin-binding protein (MPCBP) and its homolog (NPG1, no pollen germination) from Arabidopsis were isolated previously. Studies with a knockout mutant have shown that AtNPG1 is not necessary for pollen development but is essential for pollen germination. Analysis of the Arabidopsis genome sequence with AtNPG1 revealed the presence of two other proteins (AtNPGR1, NPG-Related1; AtNPGR2, NPG-Related 2) that are closely related to AtNPG1. To gain insights into the function of AtNPG1 and AtNPGRs, I focused my research on characterization of these proteins. Specifically, my research focused on in vivo localization of AtNPG1 in pollen grain and tube, interaction between AtNPGs, isolation and characterization of AtNPG1 interacting proteins, and functional analysis of AtNPGR1 in plant development. Transgenic plants containing GFP fused to AtNPG1 promoter showed GFP expression only in mature and germinating pollen, suggesting that the promoter is active only in pollen. Localization of GFP-AtNPG1, driven by AtNPG1 promoter, during different stages of pollen germination revealed uniform cytosolic distribution of GFP-AtNPG1 in the growing pollen tube that was similar to GFP alone. However, the observed uniform localization of GFP-AtNPG1 is not due to degraded fusion protein. AtNPGRs, like AtNPG1, bind calmodulin in a calcium-dependent manner. The calmodulin-binding domain in AtNPGs was mapped to a short region. AtNPG1 and AtNPGRs have several tetratricopeptide repeats (TPRs) that are known to be involved in protein-protein interaction. I tested the interaction among AtNPGs using the yeast two-hybrid analysis. AtNPG1-BD interacted with itself-AD and AtNPGR1-AD and AtNPGR2-AD. AtNPGR1-BD interacted with itself-AD, AtNPG1-AD and AtNPGR2-AD. However, AtNPGR2-BD did not interact with AtNPG1-AD or AtNPGR1-AD and showed a very weak interaction with itself-AD. To study the role of AtNPG1, AtNPG1 interacting proteins from a petunia pollen library were isolated in a yeast two-hybrid screen and identified as pectate lyase-like proteins. Using in vivo and in vitro protein-protein interaction assays, I show that AtNPGs interacts with four Arabidopsis pectate lyase-like (PLL) proteins with the highest similarity to petunia PLLs. Truncated AtNPG1 lacking the TPR 1 did not interact with most of partners or showed drastically decreased interaction with some proteins, suggesting that the TPR 1 domain is essential for this interaction. To understand the role of Arabidopsis PLL proteins, we characterized these using molecular and biochemical tools. Of the 26 Arabidopsis PLLs, fourteen were expressed in pollen and four AtPLLs were highly expressed. These four AtPLLs showed expression in other tissues also. Analysis of pectate lyase activity in Arabidopsis tissues (flower, root, stem, and leaf) revealed enzyme activity in all four tissues and the activity varied depending on the buffer pH. To see if AtNPG1 interacting AtPLLs have enzyme activity, four AtPLLs were expressed in bacteria or yeast and assayed for their enzyme activity under different conditions with different substrates. None of the AtPLLs expressed by bacterial or yeast showed pectate lyase activity. To discover the role of AtPLL in Arabidopsis development, one AtPLL mutant, atpll8, was isolated. Phenotypic analysis of atpll8 under different growth condition showed no significant differences as compared to wild type. AtNPGR1, unlike AtNPG1, is expressed in tissues other than pollen. To understand the role of AtNPGR1 in plant development, I isolated an atnpgr1 knockout mutant and characterized its phenotype under different growth conditions. The atnpgr1 showed a sugar resistance phenotype, suggesting that it might be involved in sugar sensing and/or signaling pathway. Expression of hexokinase (Hxk), an important component in sugar signaling in plants, and other genes in the Hxk pathway, revealed that NPGR1 might be involved in an Hxk independent pathway.