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Open Feng Wang Dissertation.Pdf The Pennsylvania State University The Graduate School Intercollege Graduate Degree Program in Plant Biology GENOME-WIDE ANALYSIS OF PLANT HETEROCHROMATIC SHORT-INTERFERING RNAS A Dissertation in Plant Biology by Feng Wang © 2017 Feng Wang Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2017 The dissertation of Feng Wang was reviewed and approved* by the following: Michael J. Axtell Professor of Biology Dissertation Advisor Chair of Committee Charles T. Anderson Assistant Professor of Biology Surinder Chopra Professor of Agricultural Sciences Teh-hui Kao Distinguished Professor of Biochemistry and Molecular Biology Chair, Intercollege Graduate Degree Program in Plant Biology *Signatures are on file in the Graduate School ii ABSTRACT Small RNAs, usually 20-24 nt in length, are critical regulators of plant transcriptomes. They are loaded into ARGONAUTE (AGO) proteins, and mediate gene silencing by interacting with target transcripts through sequence-specific base pairing. In plants, small RNAs are classified into various different groups based on their biogenesis and function; microRNAs (miRNAs) and heterochromatic short-interfering RNAs (het-siRNAs) are the two most important types of small RNAs in flowering plants. Plant small RNAs are subject to various forms of modification. Despite intensive studies about miRNA modification, knowledge about het-siRNA modification is lacking. I systematically studied non-templated nucleotide patterns in plant small RNAs by analyzing small RNA sequencing (sRNA-seq) libraries from Arabidopsis, tomato, Medicago, rice, maize and Physcomitrella. Elevated rates of non-templated nucleotides were observed at the 3' end of plant small RNAs from wild-type specimens of all analyzed species. In all species I analyzed, 'off-sized' small RNAs, such as 25 nt and 23 nt siRNAs arising from het-siRNA loci, often had higher rates of non-templated nucleotides at the 3' end. In Arabidopsis, 23 nt siRNAs arising from het-siRNA clusters display a distinct pattern of 3'-non-templated nucleotides. This pattern of 3'-non- templated nucleotides in 23 nt siRNAs is not dependent on known terminal nucleotidyl transferases, and may result from modifications added to longer het-siRNA precursors. Het-siRNAs negatively regulate gene expression through the RNA-directed DNA methylation (RdDM) pathway. Biogenesis of most het-siRNAs depends on the plant- specific RNA polymerase IV (Pol IV), and AGO4 is the major effector protein of het- siRNAs. Through genome-wide analysis of sRNA-seq data sets, I found that AGO4 is required for the accumulation of a small subset of het-siRNAs in Arabidopsis thaliana. The accumulation of AGO4-dependent het-siRNAs also requires several other RdDM components, including RNA POLYMERASE V (Pol V), DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) and SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1). I also demonstrated that het-siRNA accumulation could not be fully recovered by a slicing-defective AGO4 from ago4 mutant plants. These data suggest that AGO4- dependent het-siRNAs are secondary het-siRNAs, whose biogenesis requires prior activities of RdDM at certain loci. In the current RdDM model, AGO4-bound het-siRNAs target Pol V transcripts through sequence-specific base pairing. However, the details of such interactions are largely unknown. Through crosslinking immunoprecipitation by an Arabidopsis AGO4 antibody and subsequent high-throughput sequencing, I identified a handful of het-siRNA:target interactions in Arabidopsis thaliana. These de novo identified het-siRNA:target interactions suggest that het-siRNAs act on both cis and trans loci. Successful interaction between a het-siRNA and its target(s) requires extensive base-pairing, and induces target cleavage between the 10th to 11th nucleotide counting from the 5' end of het-siRNAs. iii TABLE OF CONTENTS LIST OF FIGURES ........................................................................................................ vii LIST OF TABLES ........................................................................................................... ix ACKNOWLEDGEMENTS ............................................................................................. x Chapter 1 Introduction..................................................................................................... 1 1.1 Overview of Plant Small RNAs .............................................................................. 1 1.2 Plant small RNA biogenesis and classification ..................................................... 1 1.2.1 microRNAs ........................................................................................................ 2 1.2.2 Short-interfering RNA ....................................................................................... 3 1.3 ARGONAUTE Proteins in Arabidopsis ................................................................ 7 1.3.1 Function of ARGONAUTE proteins in Arabidopsis ......................................... 8 1.3.2 Catalytic ability of ARGONAUTEs in Arabidopsis ........................................ 10 1.4 Modification of plant small RNAs ....................................................................... 12 1.4.1 The 2'-O-methylation of small RNAs .............................................................. 13 1.4.2 Tailing of miRNAs .......................................................................................... 14 1.4.3 Trimming of miRNAs ...................................................................................... 15 1.4.4 miRNA editing ................................................................................................. 15 1.4.5 Heterochromatic siRNA modification ............................................................. 16 1.5 RNA-directed DNA methylation ......................................................................... 17 1.5.1 Canonical RdDM pathway ............................................................................... 17 1.5.2 Non-canonical RdDM pathway ....................................................................... 18 1.5.3 Biological roles of RdDM ................................................................................ 19 1.5.4 Unsolved issues in canonical RdDM ............................................................... 20 1.6 Objectives .............................................................................................................. 22 Chapter 2 Genome-Wide analysis of single non-templated nucleotides in plant endogenous siRNAs and miRNAs.................................................................................. 23 2.1 Introduction ........................................................................................................... 23 2.2 Materials and Methods ......................................................................................... 25 2.2.1 Small RNA sequencing library preparation ..................................................... 25 2.2.2 Source and processing of small RNA sequencing datasets ............................. 25 2.2.3 Preparation of Simulated sRNA-seq Libraries ................................................ 28 2.2.4 Analysis of non-templated nucleotides ............................................................ 28 2.3 Results .................................................................................................................... 29 2.3.1 Elevated mismatch rates at the 3' ends of genome-aligned plant miRNAs and siRNAs are due to non-templated nucleotides .......................................................... 29 2.3.2 'Off-sized' het-siRNAs and miRNAs often have higher rates of 3' end non- templated nucleotides................................................................................................ 36 2.3.3 Arabidopsis 23 nt siRNAs from the 24 nt-dominated clusters have a unique pattern of 3' non-templated nucleotides .................................................................... 38 iv 2.3.4 23 nt siRNAs with a 3' non-templated nucleotide are infrequently bound to AGO4 ........................................................................................................................ 45 2.3.5 23 nt siRNAs from the 24 nt-dominated clusters are mostly not 3'- or 5'- truncated variants of 24 nt siRNAs ........................................................................... 45 2.3.6 Further properties of 23 nt siRNAs with a 3'-most non-templated nucleotide 47 2.4 Discussion .............................................................................................................. 49 Chapter 3 AGO4 is specifically required for heterochromatic siRNA accumulation at Pol V-dependent loci in Arabidopsis thaliana ........................................................... 53 3.1 Introduction ........................................................................................................... 53 3.2 Materials and Methods ......................................................................................... 55 3.2.1 Plant materials and growth condition .............................................................. 55 3.2.2 Cloning of wild-type and slicing-defective AGO4 .......................................... 55 3.2.3 Plant transformation and transgenic plant selection ........................................ 55 3.2.4 sRNA-seq library preparation .......................................................................... 56 3.2.5 Differential expression analysis ......................................................................
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