Major portions of the genomes of most eukaryotes, including humans, are comprised of repetitive sequences which, if transcribed in an uncontrolled fashion, can have detrimental effects on the organism [1, 2]. Transcriptional gene silencing (TGS) is a pathway utilized by cells to control transcription of repetitive elements [1, 3-5]. A unique feature of this pathway is that the regions targeted for silencing are first transcribed int long, non-coding RNAs (lncRNAs) that recruit protein factors to establish a chromatin environment that is no longer conducive to transcription [1, 3-6]. The long term goal of my study is to develop a mechanistic understanding of how transcription and transcriptional products lead to silencing in certain contexts but not others. Specifically I propose to identify characteristicsof lncRNAs, including their sizes and end modifications, which distinguish lncRNAs from other RNAs in the cell. Using the model organism Arabidopsis thaliana. I will use specialized PCR techniques, including circular reverse-transcriptase polymerase chain reaction (cRT-PCR) to identify the transcript beginnings and ends, allowing me to deduce complete sequences of known lncRNAs. Sequencing will allow determination of whether lncRNAs possess a poly-adenosine tail or other modification, such as poly- uridine, on their 3' end. Likewise, enzymatic treatments prior to ligation and circular PCR can allow me to deduce if 5' ends possess a methyl-guanosine cap, triphosphate, or monophosphate chemical moiety. I also propose to characterize factors involved in lncRNA processing to determine how unique features of lncRNAs are produced. In A. thaliana, lncRNAs involved in TGS are produced by a specialized RNA polymerase, Pol V, which is evolutionarily related to the canonical polymerase, Pol II [7]. Pol II produced RNAs possess unique 5' and 3' end modifications that are added by proteins that interact with the carboxy-terminal domain (CTD) of the largest Pol II subunit. Intriguingly, the analogous Pol V CTD has diverged at the sequence level from Pol II. Whether the Pol V CTD has a role in RNA processing is unclear. I have preliminary evidence that the Pol V CTD interacts with an enzyme called RRP6L1. RRP6L1 is a predicted exonuclease and has previously been shown to interact with Pol V transcripts [8]. I propose to determine the effects of the Pol V CTD, RRP6L1, and Pol V-RRP6L1 interaction on lncRNA biogenesis. To do this, I will characterize the enzymatic properties of RRP6L1 and the effects of the Pol V CTD interaction on RRP6L1 activity in vitro. I will also characterize lncRNAs in vivo using techniques outlined above in rrp6L1 and targeted Pol V CTD mutant lines. The results from this study are expected to reveal how lncRNAs involved in TGS are processed and what factors are involved in their processing, which will help us understand how silencing RNAs are generated.
The majority of the human genome, as well as the genomes of other animals and plants, is comprised of heterochromatic sequence which must be maintained in a silent state for the proper function of cells. This project aims to increase our basic understanding of how silent regions are targeted by identifying functional characteristics of the specialized RNA molecules that mediate silencing.
| McKinlay, Anastasia; Podicheti, Ram; Wendte, Jered M et al. (2018) RNA polymerases IV and V influence the 3' boundaries of Polymerase II transcription units in Arabidopsis. RNA Biol 15:269-279 |
| Wendte, Jered M; Pikaard, Craig S (2017) The RNAs of RNA-directed DNA methylation. Biochim Biophys Acta Gene Regul Mech 1860:140-148 |
| Wendte, Jered M; Haag, Jeremy R; Singh, Jasleen et al. (2017) Functional Dissection of the Pol V Largest Subunit CTD in RNA-Directed DNA Methylation. Cell Rep 19:2796-2808 |
| Wendte, Jered M; Pikaard, Craig S (2016) Targeting Argonaute to chromatin. Genes Dev 30:2649-2650 |
