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

The role of the NEXT complex in microRNA biogenesis

Wydział BiologiiSERRATE (SE) jest białkiem ważny dla powstawania mikroRNA u Arabidopsis, jednakże jest również zaangażowane w inne procesy związane z RNA, między innymi: wycinanie intronów z pre-mRNA. W celu zrozumienia tej wielofunkcyjnej roli SE przeprowadzono jego koimmunoprecypitacje i zidentyfikowano partnerów białkowych SE z użyciem LC-MS/MS. Wśród zidentyfikowanych białek były między innymi wszystkie podjednostki kompleksu NEXT (ang. Nuclear Exosome Targeting): HEN2, ZCCHC8A, ZCCHC8B, RBM7. NEXT jest kompleksem wyszukującym RNA przeznaczone do degradacji przez egsozom RNA od strony 3’. Następnie z użyciem sekwencjonowania nowej generacji wykazano, że kompleks NEXT jest odpowiedzialny za degradacje pri-miRNA. Analiza podwójnych mutantów, w których poza zaburzoną biogenezą miRNA była również spowolniona degradacja pri-miRNA (hyl1-2 hen2-2, se-2 hen2-2) wykazała poprawę fenotypu roślina wraz z podniesionym poziomem miRNA. Dodatkowo zaobserwowano również bardzo wysoką akumulacje pri-miRNA. Dane te pokazują, że brak degradacji pri-miRNA skutkuje lepszą produkcją miRNA przez DCL1 mimo braku elementów go wspomagających. Zaproponowany na podstawie wyników tej rozprawy doktorskiej model zakłada, że białko SE jest zaangażowane w biogenezę miRNA w dwóch płaszczyznach. W jednej pomaga białku DCL1 w efektywnym i dokładnym cięciu pri-miRNA, natomiast w drugiej płaszczyźnie promuje degradacje nadmiaru pierwotnych prekursorów miRNA przez rekrutacje kompleksu NEXT.Abstract The SERRATE (SE) protein is very important factor in miRNA biogenesis in Arabidopsis thaliana. Moreover SE is involved in different processes connected with RNA, e.g. splicing of pre-mRNAs. In order to understand this multifunctional role of SERRATE, co-immunoprecipitation experiments were carried out, and SE partners were identified using LC-MS/MS. Analysis of the SE interactome revealed many new protein partners. Among proteins co-purified with SE, all subunits of the Nuclear Exosome Targeting (NEXT) complex: HEN2, ZCCHC8A, ZCCHC8B and RBM7, were found. NEXT is important for targeting RNA for degradation by the nuclear RNA exosome for 3’ end. Next, using new generation RNA sequencing it was shown that the NEXT complex is responsible for degradation of pri-miRNAs. The analysis of double mutants in which degradation of pri-miRNA was slowed down along with the disruption of miRNA biogenesis (hyl1-2 /hen2-2, se-2/ hen2-2) showed an improvement in the phenotype (as compared to single mutants) together with increased levels of miRNAs. In addition, very high pri-miRNA accumulation was also observed. These data show that the lack of degradation of pri-miRNAs results in more efficient production of miRNAs by DCL1 despite the lack of supportive proteins, HYL1 and SE. The model proposed on the basis of the results of this dissertation assumes that the SERRATE protein is involved in miRNA biogenesis in two different ways: it helps DCL1 in more efficient and accurate cleavage of pri-miRNAs and on the other hand it also promotes degradation of excess primary miRNA precursors by the recruitment of NEXT complex.Niniejsza praca powstała przy finansowym udziale: 1.Narodowego Centrum Nauki (granty: UMO-2013/10/A/NZ1/00557 (MAESTRO)dla Profesoradr. hab.Artura Jarmołowskiego; UMO-2014/13/N/NZ1/00049 (PRELUDIUM) dla Mateusza Bajczyka) 2.Fundacji na Rzecz Nauki Polskiej (stypendium w ramach subsydium Mistrz 3./2014, Laureat: Prof. dr hab. Artur Jarmołowski) 3.KNOW Poznańskie Konsorcjum RNA (01/KNOW2/2014). 4.Wydziału Biologii Uniwersytetu im. Adama Mickiewicza w Poznaniu (grant GDWB-01/2015 dla Mateusza Bajczyka).Autor uzyskał środki finansowe na przygotowanie rozprawy doktorskiej z Narodowego CentrumNauki(Program ETIUDA)w ramach finansowania stypendium doktorskiego na podstawie decyzji numer UMO-2018/28/T/NZ1/0039

The role of the NEXT complex in microRNA biogenesis

Wydział BiologiiSERRATE (SE) jest białkiem ważny dla powstawania mikroRNA u Arabidopsis, jednakże jest również zaangażowane w inne procesy związane z RNA, między innymi: wycinanie intronów z pre-mRNA. W celu zrozumienia tej wielofunkcyjnej roli SE przeprowadzono jego koimmunoprecypitacje i zidentyfikowano partnerów białkowych SE z użyciem LC-MS/MS. Wśród zidentyfikowanych białek były między innymi wszystkie podjednostki kompleksu NEXT (ang. Nuclear Exosome Targeting): HEN2, ZCCHC8A, ZCCHC8B, RBM7. NEXT jest kompleksem wyszukującym RNA przeznaczone do degradacji przez egsozom RNA od strony 3’. Następnie z użyciem sekwencjonowania nowej generacji wykazano, że kompleks NEXT jest odpowiedzialny za degradacje pri-miRNA. Analiza podwójnych mutantów, w których poza zaburzoną biogenezą miRNA była również spowolniona degradacja pri-miRNA (hyl1-2 hen2-2, se-2 hen2-2) wykazała poprawę fenotypu roślina wraz z podniesionym poziomem miRNA. Dodatkowo zaobserwowano również bardzo wysoką akumulacje pri-miRNA. Dane te pokazują, że brak degradacji pri-miRNA skutkuje lepszą produkcją miRNA przez DCL1 mimo braku elementów go wspomagających. Zaproponowany na podstawie wyników tej rozprawy doktorskiej model zakłada, że białko SE jest zaangażowane w biogenezę miRNA w dwóch płaszczyznach. W jednej pomaga białku DCL1 w efektywnym i dokładnym cięciu pri-miRNA, natomiast w drugiej płaszczyźnie promuje degradacje nadmiaru pierwotnych prekursorów miRNA przez rekrutacje kompleksu NEXT.Abstract The SERRATE (SE) protein is very important factor in miRNA biogenesis in Arabidopsis thaliana. Moreover SE is involved in different processes connected with RNA, e.g. splicing of pre-mRNAs. In order to understand this multifunctional role of SERRATE, co-immunoprecipitation experiments were carried out, and SE partners were identified using LC-MS/MS. Analysis of the SE interactome revealed many new protein partners. Among proteins co-purified with SE, all subunits of the Nuclear Exosome Targeting (NEXT) complex: HEN2, ZCCHC8A, ZCCHC8B and RBM7, were found. NEXT is important for targeting RNA for degradation by the nuclear RNA exosome for 3’ end. Next, using new generation RNA sequencing it was shown that the NEXT complex is responsible for degradation of pri-miRNAs. The analysis of double mutants in which degradation of pri-miRNA was slowed down along with the disruption of miRNA biogenesis (hyl1-2 /hen2-2, se-2/ hen2-2) showed an improvement in the phenotype (as compared to single mutants) together with increased levels of miRNAs. In addition, very high pri-miRNA accumulation was also observed. These data show that the lack of degradation of pri-miRNAs results in more efficient production of miRNAs by DCL1 despite the lack of supportive proteins, HYL1 and SE. The model proposed on the basis of the results of this dissertation assumes that the SERRATE protein is involved in miRNA biogenesis in two different ways: it helps DCL1 in more efficient and accurate cleavage of pri-miRNAs and on the other hand it also promotes degradation of excess primary miRNA precursors by the recruitment of NEXT complex.Niniejsza praca powstała przy finansowym udziale: 1.Narodowego Centrum Nauki (granty: UMO-2013/10/A/NZ1/00557 (MAESTRO)dla Profesoradr. hab.Artura Jarmołowskiego; UMO-2014/13/N/NZ1/00049 (PRELUDIUM) dla Mateusza Bajczyka) 2.Fundacji na Rzecz Nauki Polskiej (stypendium w ramach subsydium Mistrz 3./2014, Laureat: Prof. dr hab. Artur Jarmołowski) 3.KNOW Poznańskie Konsorcjum RNA (01/KNOW2/2014). 4.Wydziału Biologii Uniwersytetu im. Adama Mickiewicza w Poznaniu (grant GDWB-01/2015 dla Mateusza Bajczyka).Autor uzyskał środki finansowe na przygotowanie rozprawy doktorskiej z Narodowego CentrumNauki(Program ETIUDA)w ramach finansowania stypendium doktorskiego na podstawie decyzji numer UMO-2018/28/T/NZ1/0039

Regulation of Plant Microprocessor Function in Shaping microRNA Landscape

MicroRNAs are small molecules (∼21 nucleotides long) that are key regulators of gene expression. They originate from long stem–loop RNAs as a product of cleavage by a protein complex called Microprocessor. The core components of the plant Microprocessor are the RNase type III enzyme Dicer-Like 1 (DCL1), the zinc finger protein Serrate (SE), and the double-stranded RNA binding protein Hyponastic Leaves 1 (HYL1). Microprocessor assembly and its processing of microRNA precursors have been reported to occur in discrete nuclear bodies called Dicing bodies. The accessibility of and modifications to Microprocessor components affect microRNA levels and may have dramatic consequences in plant development. Currently, numerous lines of evidence indicate that plant Microprocessor activity is tightly regulated. The cellular localization of HYL1 is dependent on a specific KETCH1 importin, and the E3 ubiquitin ligase COP1 indirectly protects HYL1 from degradation in a light-dependent manner. Furthermore, proper localization of HYL1 in Dicing bodies is regulated by MOS2. On the other hand, the Dicing body localization of DCL1 is regulated by NOT2b, which also interacts with SE in the nucleus. Post-translational modifications are substantial factors that contribute to protein functional diversity and provide a fine-tuning system for the regulation of protein activity. The phosphorylation status of HYL1 is crucial for its activity/stability and is a result of the interplay between kinases (MPK3 and SnRK2) and phosphatases (CPL1 and PP4). Additionally, MPK3 and SnRK2 are known to phosphorylate SE. Several other proteins (e.g., TGH, CDF2, SIC, and RCF3) that interact with Microprocessor have been found to influence its RNA-binding and processing activities. In this minireview, recent findings on the various modes of Microprocessor activity regulation are discussed

Recent Insights into Plant miRNA Biogenesis: Multiple Layers of miRNA Level Regulation

MicroRNAs are small RNAs, 20–22 nt long, the main role of which is to downregulate gene expression at the level of mRNAs. MiRNAs are fundamental regulators of plant growth and development in response to internal signals as well as in response to abiotic and biotic factors. Therefore, the deficiency or excess of individual miRNAs is detrimental to particular aspects of a plant’s life. In consequence, the miRNA levels must be appropriately adjusted. To obtain proper expression of each miRNA, their biogenesis is controlled at multiple regulatory layers. Here, we addressed processes discovered to influence miRNA steady-state levels, such as MIR transcription, co-transcriptional pri-miRNA processing (including splicing, polyadenylation, microprocessor assembly and activity) and miRNA-encoded peptides synthesis. MiRNA stability, RISC formation and miRNA export out of the nucleus and out of the plant cell also define the levels of miRNAs in various plant tissues. Moreover, we show the evolutionary conservation of miRNA biogenesis core proteins across the plant kingdom

Table_1_Regulation of Plant Microprocessor Function in Shaping microRNA Landscape.PDF

<p>MicroRNAs are small molecules (∼21 nucleotides long) that are key regulators of gene expression. They originate from long stem–loop RNAs as a product of cleavage by a protein complex called Microprocessor. The core components of the plant Microprocessor are the RNase type III enzyme Dicer-Like 1 (DCL1), the zinc finger protein Serrate (SE), and the double-stranded RNA binding protein Hyponastic Leaves 1 (HYL1). Microprocessor assembly and its processing of microRNA precursors have been reported to occur in discrete nuclear bodies called Dicing bodies. The accessibility of and modifications to Microprocessor components affect microRNA levels and may have dramatic consequences in plant development. Currently, numerous lines of evidence indicate that plant Microprocessor activity is tightly regulated. The cellular localization of HYL1 is dependent on a specific KETCH1 importin, and the E3 ubiquitin ligase COP1 indirectly protects HYL1 from degradation in a light-dependent manner. Furthermore, proper localization of HYL1 in Dicing bodies is regulated by MOS2. On the other hand, the Dicing body localization of DCL1 is regulated by NOT2b, which also interacts with SE in the nucleus. Post-translational modifications are substantial factors that contribute to protein functional diversity and provide a fine-tuning system for the regulation of protein activity. The phosphorylation status of HYL1 is crucial for its activity/stability and is a result of the interplay between kinases (MPK3 and SnRK2) and phosphatases (CPL1 and PP4). Additionally, MPK3 and SnRK2 are known to phosphorylate SE. Several other proteins (e.g., TGH, CDF2, SIC, and RCF3) that interact with Microprocessor have been found to influence its RNA-binding and processing activities. In this minireview, recent findings on the various modes of Microprocessor activity regulation are discussed.</p