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Abstract Competition Between Alternative Splicing ABSTRACT COMPETITION BETWEEN ALTERNATIVE SPLICING AND POLYADENYLATION DEFINES THE EXPRESSION OF THE OXT6 GENE ENCODING TWO PROTEINS INVOLVED IN MRNA PROCESSING by Zhaoyang Liu This thesis reports a study of the interaction between messenger RNA polyadenylation and splicing in the expression of an Arabidopsis gene OXT6. This gene encodes two proteins that may involve in both polyadenylation and splicing processes. Interestingly, alternative polyadenylation or splicing of intron-2 of the gene defines the expression ratio of the two transcripts. To reveal the relationship of these processing events, a set of mutations were introduced at the splice sites and polyadenylation signals within intron-2 and transformed into the oxt6 mutant plants. The splicing and polyadenylation events were monitored by quantitative RT-PCR, revealing a competition nature of alternative polyadenylation and splicing during the OXT6 transcript processing. ChIP (chromatin immunoprecipitation) analysis performed against RNA Polymerase II in splicing mutant lines suggests that Pol II binding may affect the switch between polyadenylation and splicing during transcript processing. This work is the first of its kind that exemplifies the interplay among transcription, splicing and polyadenylation that defines a gene’s expression outcome in plants. COMPETITION BETWEEN ALTERNATIVE SPLICING AND POLYADENYLATION DEFINES THE EXPRESSION OF THE OXT6 GENE ENCODING TWO PROTEINS INVOLVED IN MRNA PROCESSING A Thesis Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Master of Science Cell, Molecular and Structural Biology Graduate Program by Zhaoyang Liu Miami University Oxford, Ohio 2010 Advisor________________________ (Qingshun Quinn Li) Reader_________________________ (Chun Liang) Reader_________________________ (Nicholas P. Money) TABLE OF CONTENTS CHAPTER 1: INTRODUCTION............................................................................1 MULTIPLE LINKS BETWEEN MESSENGER RNA PROCESSING THROUGH POLYADENYLATION AND SPLICING ..........................................................................1 MESSENGER RNA POLYADENYLATION MECHANISM AND POLYADENYLATION FACTORS4 MESSENGER RNA SPLICING MECHANISMS AND SPLICING FACTORS ..........................6 INTERACTIONS BETWEEN POLYADENYLATION AND SPLICING .....................................8 THE OXT6 GENE AND ITS CONNECTION WITH ALTERNATIVE SPLICING AND POLYADENYLATION.............................................................................................10 CHAPTER 2: MATERIALS AND METHODS.....................................................13 ISOLATION OF THE OXT6 MUTANT.........................................................................13 DETECTION OF OXT6 POLYADENYLATION SIGNALS ...............................................13 CONSTRUCTION OF OXT6 SPLICING AND POLYADENYLATION MUTATIONS................13 PLANT TRANSFORMATIONS .................................................................................14 QUANTITATIVE RT-PCR ANALYSIS OF MUTANT GENE EXPRESSION.........................14 CHROMATIN IMMUNOPRECIPITATION (CHIP) ASSAYS.............................................15 CHAPTER 3: RESULTS.....................................................................................16 POLYADENYLATION AND SPLICING PROFILES OF THE OXT6 GENE...........................16 MUTAGENESIS OF THE SPLICE SITES AND POLY(A) SIGNALS ...................................16 ii POLYADENYLATION AND SPLICING PATTERNS OF OXT6 WERE ALTERED BY MUTATION ........................................................................................................................17 COMPETITIVE RELATIONSHIP BETWEEN SPLICING AND POLYADENYLATION IN INTRON-2 OF OXT6 ..........................................................................................................18 MUTATIONS ON SPLICE SITES CHANGE THE BINDING AFFINITY OF POL II ..................19 CHAPTER 4: DISCUSSION AND CONCLUDING REMARKS ..........................21 THE COMPETITION NATURE OF POLYADENYLATION AND SPLICING DURING PROCESSING OF THE OXT6 GENE ...........................................................................................21 THE REGULATORY ROLE OF POL II IN OXT6 GENE PROCESSING.............................23 FUTURE PERSPECTIVE .......................................................................................25 REFERENCES ...................................................................................................27 iii LIST OF TABLES TABLE 1. PRIMERS USED ................................................................................38 TABLE 2. OXT6 MUTATION CONSTRUCTS AND TRANSGENIC PLANTS USED..................................................................................................................41 iv LIST OF FIGURES FIGURE 1. THE STRUCTURE OF THE OXT6 GENE AND ITS RNA TRANSCRIPTS...................................................................................................42 FIGURE 2. POLY(A) SITES DETECTED IN ATCPSF30 AND ATC30Y TRANSCRIPTS...................................................................................................43 FIGURE 3. AN ILLUSTRATION OF THE SEVEN OXT6 MUTATION CONSTRUCTS...................................................................................................44 FIGURE 4. CRYPTIC POLY(A) SITES AND CRYPTIC SPLICE SITES DETECTED IN TRANSGENIC LINES. ...............................................................45 FIGURE 5. RATIO FOLD OF CHANGE OF THE ATCPSF30 AND ATC30Y TRANSCRIPTS IN THE OXT6 MUTATION CONSTRUCTS. .............................46 FIGURE 6. CHIP ASSAYS OF THE SPLICING MUTANT TRANSGENIC LINES. ............................................................................................................................47 v ACKNOWLEDGEMENT I would like to express my sincere gratitude to my advisor Dr. Qingshun Quinn Li for his constant encouragement and guidance. As an amazing advisor, he devoted great efforts on helping me to uncover the intriguing truth behind the involuted phenomena, and led me to overcome many crisis situations during my master’s research. I am also grateful to my committee members Dr. Chun Liang and Dr. Nicholas P. Money for their insightful comments, valuable suggestions and warm supports for both my study and research. I would like to thank all the fellow members in the Li lab who offered me great help. I acknowledge Dr. Man Liu and Dr. Denghui Xing, Yingjia Shen and Jun Zheng for their generous collaborations and helpful discussions. Thanks to student assistants who worked in the lab during the past two years. Particularly, I appreciate Daniel F. Comiskey for his assistance to my research. I would also like to thank the faculty, staff and students in the Department of Botany. I acknowledge Ms. Barb Wilson and Ms. Vickie Sandlin for their diligent work, and I thank Dr. John W. Hawes and Ms. Xiaoyun Deng at the Center of Bioinformatics and Functional Genomics, Miami University, for their excellent technical supports. Thanks to all my friends who have been so helpful and friendly to me. Without the support of my family, I would not able to complete my work. I sincerely thank my parents for their lasting love and understanding. Finally I would like to extend my gratitude to the NSF award and the Academic Challenge Program in the Department of Botany, Miami University, for funding support of this research. vi Chapter 1: Introduction Multiple links between messenger RNA processing through polyadenylation and splicing In eukaryotic cells, messenger RNA (mRNA) is transcribed by RNA polymerase II (Pol II), capped at 5’ end, spliced by the spliceosome and polyadenylated at the 3’ end to form a mature mRNA. Splicing is a process where introns are removed and exons are ligated together by a two-step transesterification reaction. Polyadenylation is a process with the cleavage at the polyadenylation site followed by the addition of a polymerization of an adenosine tail to the 3’-end of an mRNA molecule. Both splicing and polyadenylation serve many important biological functions including stability, translocation to the cytoplasm and translation (Coller et al., 1998; Huang and Carmichael, 1996; Kuhn and Wahle, 2004) and interact with multiple mRNA processing events (Zhao et al., 1999; Kornblihtt et al, 2004; Proudfoot, 2004; Gilmartin, 2005). The first function of mRNA polyadenylation is to protect the mature mRNA (Coller et al., 1998). In eukaryotic cells, mRNA is majorly degraded by two pathways that are both initiated by shortening of the 3’ polyadenosine [poly(A)] tail, followed by 3’-exonuclease or 5’-exonuclease digestion (Franks and Lykke-Andersen, 2008). Poly(A) tails can protect mature mRNA from unregulated digestion from both ends (Kuhn and Wahle, 2004). Secondly, poly(A) tail promotes the export of mRNA from the nucleus to the cytoplasm (Huang and Carmichael, 1996; Kessler et al., 1997). Both polyadenylation related proteins and poly(A) binding proteins are believed to be involved in mRNA exporting (Coller et al., 1998; Brodsky and Silver, 2000; Poon et al., 2000). In addition, poly(A) binding proteins also interact with the translational machinery, and highly increase translation efficiency through enhancement of translation initiation (Kuhn and Wahle, 2004; Siddiqui et al., 2007). More over, some polyadenylation related proteins also involved in other cellular processes such as the maturation of small nucleolar (sno-) RNAs 1 (Nedea et al., 2003; Morlando et al., 2004) and the formation of 3’-end of cell
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