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Maria Louisa Vigh:
Mechanisms Underlying siRNA Amplification in Arabidopsis thaliana

Date: 20-02-2022    Supervisor: Peter Brodersen

The way that humans gain, spread and use knowledge about us and our surrounding nature has made us feed and grow into a dominating species on Earth. To overcome challenges such as climate changes, pandemics and overpopulation, we are relying on our ever-growing biological knowledge. For example, we need to understand the biology of our plants to keep feeding on them. We need to know how they germinate and develop, how they defend themselves against viruses and pests and how they deal with changes in external stimuli, such as temperature changes and drought. All these biological functions of our plants are possible due to gene expression regulation. Correct gene expression regulation is in fact a fundamental process for all organisms and various pathways to orchestrate gene expression exist. One such pathway is RNA silencing, wherein small RNA (sRNA) molecules are loaded into an RNA-induced silencing Complex (RISC) and once loaded, the sRNA guides RISC to messengerRNA (mRNA) complementary in sequence. RISC either degrades the mRNA or inhibits its translation.

Plants harbours a unique mechanism with which they can amplify a primary RNA silencing signal by generating a plethora of secondary sRNAs from RISC-targets instead of degrading them. The secondary sRNAs can be loaded into new RISCs and silence new mRNAs. This mechanism is called “siRNA amplification” and it is used by plants to defend themselves against viral pathogens but also to enter different developmental stages and maintain genome integrity. We were interested in how and when the plant cell chooses to employ siRNA amplification.

In this study we show that ectopic siRNA amplification can occur on endogenous RISC-targets upon mutation of various RNA metabolism factors. These factors include RNA exosomes, PELOTA1 involved in Non-Stop Decay and two uridyltransferases, HESO1 and URT1. We propose that all these factors hinder siRNA amplification in the same way; by shortening RISC dwell time on its targets. Furthermore, we show a novel role of HESO1 in promoting sRNA biogenesis from select RNA, which is considered dangerous for genome integrity. Our results suggest a possible role for HESO1 in defence signalling and moreover show that uridyltransferases can have contradictory functions in plant sRNA biogenesis. Finally, we describe the setup of a RISC-target identification methodology, which utilizes the catalytic domain of a deaminase from fruit flies. By fusion of the deaminase domain to ARGONAUTE1 (AGO1), the key effector protein in RISC, RISC-target are deaminated. The adenosine to inosine edits can be identified in highthroughput sequencing. We discuss the advantages and disadvantages of this method.