N6 Methyladenosine (m6A) and its writer mRNA adenosine methylase (MTA) are required for proper miRNA biogenesis in Arabidopsis thaliana”.

Wydział BiologiiW przedstawionej pracy doktorskiej wykazałem że enzym zwany metylazą adenozyny mRNA (MTA, ang. mRNA adenosine methylase) jest ważnym elementem w biogenezie mikroRNA Arabidopsis thaliana. Wykazałem, że MTA metyluje pri-miRNA i w przypadku braku tej metylacji pri-miRNA akumulują się w komórce, czemu towarzyszy obniżony poziom dojrzałych mikroRNA. Zmiany strukturalne pri-miRNA, spowodowane nieobecnością m6A, prowadzą do słabszego ich oddziaływania z białkiem HYL1 (ang. Hyponastic Leaves 1). Dodatkowo wykazałem, że białko MTA oddziałuje z polimerazą RNA II oraz białkiem TGH (ang. Tough). MTA oraz TGH są czynnikami niezbędnymi do prawidłowej budowy kompleksu Mikroprocesora. Ostatecznie wykazałem, że brak wrażliwości mutanta mta na obecność auksyn spowodowana jest obniżonym poziomem miR393b. Niższy poziom miR393b w mutancie wynika z braku m6A w prekursorze pri-miR393b.In this PhD thesis, I show that mRNA adenosine methylase (MTA) is a player in microRNA biogenesis in Arabidopsis thaliana. I show that MTA methylates pri-miRNAs and in the absence of this methylation pri-miRNAs tend to accumulate and miRNAs are downregulated. The structural changes caused by the absence of m6A lead to lower binding of Hyponastic Leaves 1 (HYL1) to pri-miRNAs. I also show that MTA interacts with RNA Polymerase II and miRNA biogenesis related protein TOUGH (TGH). MTA and TGH are both required for proper assembly of Microprocessor. Finally, I show that the auxin insensitivity seen in mta mutants is at least partially caused by the lower levels of miR393b; and the lower miR393b levels are in fact caused by the absence of m6A methylation of its precursor.1. The Polish National Science Centre grants PRELUDIUM (2017/27/N/NZ1/00202) and ETIUDA (2019/32/T/NZ1/00122) 2. The KNOW RNA Research Centre in Poznan (01/KNOW2/2014) 3. The European Union: Passport to the future - Interdisciplinary doctoral studies at the Faculty of Biology, Adam Mickiewicz University (POWR.03.02.00-00-I006/17-00

mRNA adenosine methylase (MTA) deposits m6A on pri-miRNAs to modulate miRNA biogenesis in Arabidopsis thaliana

Copyright © 2020 the Author(s). Published by PNAS. In Arabidopsis thaliana, the METTL3 homolog, mRNA adenosine methylase (MTA) introduces N6-methyladenosine (m6A) into various coding and noncoding RNAs of the plant transcriptome. Here, we show that an MTA-deficient mutant (mta) has decreased levels of microRNAs (miRNAs) but accumulates primary miRNA transcripts (pri-miRNAs). Moreover, pri-miRNAs are methylated by MTA, and RNA structure probing analysis reveals a decrease in secondary structure within stem-loop regions of these transcripts in mta mutant plants. We demonstrate interaction between MTA and both RNA Polymerase II and TOUGH (TGH), a plant protein needed for early steps of miRNA biogenesis. Both MTA and TGH are necessary for efficient colocalization of the Microprocessor components Dicer-like 1 (DCL1) and Hyponastic Leaves 1 (HYL1) with RNA Polymerase II. We propose that secondary structure of miRNA precursors induced by their MTA-dependent m6A methylation status, together with direct interactions between MTA and TGH, influence the recruitment of Microprocessor to plant pri-miRNAs. Therefore, the lack of MTA in mta mutant plants disturbs pri-miRNA processing and leads to the decrease in miRNA accumulation. Furthermore, our findings reveal that reduced miR393b levels likely contributes to the impaired auxin response phenotypes of mta mutant plants

N6 Methyladenosine (m6A) and its writer mRNA adenosine methylase (MTA) are required for proper miRNA biogenesis in Arabidopsis thaliana”.

Wydział BiologiiW przedstawionej pracy doktorskiej wykazałem że enzym zwany metylazą adenozyny mRNA (MTA, ang. mRNA adenosine methylase) jest ważnym elementem w biogenezie mikroRNA Arabidopsis thaliana. Wykazałem, że MTA metyluje pri-miRNA i w przypadku braku tej metylacji pri-miRNA akumulują się w komórce, czemu towarzyszy obniżony poziom dojrzałych mikroRNA. Zmiany strukturalne pri-miRNA, spowodowane nieobecnością m6A, prowadzą do słabszego ich oddziaływania z białkiem HYL1 (ang. Hyponastic Leaves 1). Dodatkowo wykazałem, że białko MTA oddziałuje z polimerazą RNA II oraz białkiem TGH (ang. Tough). MTA oraz TGH są czynnikami niezbędnymi do prawidłowej budowy kompleksu Mikroprocesora. Ostatecznie wykazałem, że brak wrażliwości mutanta mta na obecność auksyn spowodowana jest obniżonym poziomem miR393b. Niższy poziom miR393b w mutancie wynika z braku m6A w prekursorze pri-miR393b.In this PhD thesis, I show that mRNA adenosine methylase (MTA) is a player in microRNA biogenesis in Arabidopsis thaliana. I show that MTA methylates pri-miRNAs and in the absence of this methylation pri-miRNAs tend to accumulate and miRNAs are downregulated. The structural changes caused by the absence of m6A lead to lower binding of Hyponastic Leaves 1 (HYL1) to pri-miRNAs. I also show that MTA interacts with RNA Polymerase II and miRNA biogenesis related protein TOUGH (TGH). MTA and TGH are both required for proper assembly of Microprocessor. Finally, I show that the auxin insensitivity seen in mta mutants is at least partially caused by the lower levels of miR393b; and the lower miR393b levels are in fact caused by the absence of m6A methylation of its precursor.1. The Polish National Science Centre grants PRELUDIUM (2017/27/N/NZ1/00202) and ETIUDA (2019/32/T/NZ1/00122) 2. The KNOW RNA Research Centre in Poznan (01/KNOW2/2014) 3. The European Union: Passport to the future - Interdisciplinary doctoral studies at the Faculty of Biology, Adam Mickiewicz University (POWR.03.02.00-00-I006/17-00

<i>N</i>⁶-methyladenosine (m⁶A): Revisiting the Old with Focus on New, an <i>Arabidopsis thaliana</i> Centered Review

N6-methyladenosine (m6A) is known to occur in plant and animal messenger RNAs (mRNAs) since the 1970s. However, the scope and function of this modification remained un-explored till very recently. Since the beginning of this decade, owing to major technological breakthroughs, the interest in m6A has peaked again. Similar to animal mRNAs, plant mRNAs are also m6A methylated, within a specific sequence motif which is conserved across these kingdoms. m6A has been found to be pivotal for plant development and necessary for processes ranging from seed germination to floral development. A wide range of proteins involved in methylation of adenosine have been identified alongside proteins that remove or identify m6A. This review aims to put together the current knowledge regarding m6A in Arabidopsis thaliana