Author ORCID Identifier
Date Available
1-28-2018
Year of Publication
2017
Degree Name
Doctor of Philosophy (PhD)
Document Type
Doctoral Dissertation
College
Agriculture, Food and Environment
Department/School/Program
Plant and Soil Sciences
First Advisor
Dr. Arthur G. Hunt
Abstract
Drought stress is considered one of the most devastating abiotic stress factors that limit crop productivity for modern agriculture worldwide. There is a large range of physiological and biochemical responses induced by drought stress. The responses range from physiological and biochemical to regulation at transcription and posttranscriptional levels. Post-transcription, the products encoded by eukaryotic genes must undergo a series of modifications to become a mature mRNA. Polyadenylation is an important one in terms of regulation. Polyadenylation impacts gene expression through determining the coding and regulation potential of the mRNA, especially when different mRNAs from the same gene may be polyadenylated at more than one position. This alternative polyadenylation (APA) has numerous potential effects on gene regulation and function. I have studied the impact of drought stress on APA, testing the hypothesis that drought stress may give rise to changes in the usage of poly(A) sites generating different mRNA isoforms. The results showed that usage of poly(A) sites that lie within 5’-UTRs and coding sequence (CDS) changes more than usage of sites in other regions due to drought stress.
Alternative polyadenylation is meditated by the polyadenylation complex of proteins that are conserved in eukaryotic cells. The Arabidopsis CPSF30 protein (AtCPSF30), which is an RNA-binding endonuclease subunit of the polyadenylation complex, plays an important role in controlling APA. Previous study showed that poly(A) site choice changes on a large scale in oxidative stress tolerant 6 (oxt6), a mutant lacking AtCPSF30. Within the mutant/WT genotypes, there are three classes of poly(A) site, wild type specific, oxt6 specific, and common (both in wild type and mutant). The wild type specific and oxt6 specific mRNAs make up around 70% of the total of all mRNA species. I hypothesize that the stability of these various mRNA isoforms should be different, and that this is a possible way that AtCPSF30 regulates gene expression. I tested this by assessing the influence poly(A) sites can have on the mRNA isoform’s stability in the wild type and oxt6 mutant. My results show that most mRNA isoforms show similar stability profiles in the wild-type and mutant plants. However, the mRNA isoforms derived from polyadenylation within CDS are much more stable in the mutant than the wild-type. These results implicate AtCPSF30 in the process of non-stop mRNA decay.
Messenger RNA polyadenylation occurs in the nucleus, and the subunits of the polyadenylation complex that meditate this process are expected to reside within the nucleus. However, AtCPSF30 by itself localizes not only to the nucleus, but also to the cytoplasm. AtCPSF30 protein contains three predicted CCCH-type zinc finger motifs. The first CCCH motif is the primary motif that is responsible for the bulk of its RNA-binding activity. It can bind with calmodulin, but the RNA-binding activity of AtCPSF30 is inhibited by calmodulin in a calcium-dependent manner. The third CCCH motif is associated with endonuclease activity. Previous studies demonstrated that the endonuclease activity of AtCPSF30 can be inhibited by disulfide reducing agents. These published results suggest that there are proteins that interact with AtCPSF30 and act through calmodulin binding or disulfide remodeling. To test this hypothesis, I screened for proteins that interact with AtCPSF30. For this, different approaches were performed. These screens led me to two proteins-one protein that is tyrosine-phosphorylated and whose phosphorylation state is modulated in response to ABA, which well-known ABA regulates guard cell turgor via a calcium-dependent pathway, and the other is ribosome protein L35(RPL35), which plays an important role in nuclear entry, translation activity, and endoplasmic reticulum(ER) docking. These results suggest that multiple calcium-dependent signaling mechanisms may converge on AtCPSF30, and AtCPSF30 might be directly interact with ribosome protein.
Digital Object Identifier (DOI)
https://doi.org/10.13023/ETD.2017.303
Recommended Citation
Hao, Guijie, "THE ROLE OF ALTERNATIVE POLYADENYLATION MEDIATED BY CPSF30 IN ARABIDOPSIS THALIANA" (2017). Theses and Dissertations--Plant and Soil Sciences. 90.
https://uknowledge.uky.edu/pss_etds/90
