m6 A mRNA Modifcation as a New Layer of Gene Regulation in Plants

서필준 한국식물학회 2020 Journal of Plant Biology Vol.63 No.2

N6 -methyladenosine (m6 A) is the most prevalent mRNA modifcation in higher eukaryotes. Technical advances in m6 A detection and mapping have allowed a comprehensive understanding of transcriptome-wide distribution of the RNA modifcation and its biological impacts especially in model plant systems. The m6 A modifcation is reversibly catalyzed by antagonistic actions of m6 A writers and erasers and the mRNA modifcation is biologically interpreted by m6 A readers. In this review, we summarized the recent understanding of molecular mechanisms underlying m6 A deposition and m6 A-regulated biological functions in diverse aspects of plant development. We also illustrated the evolutionary history of m6 A modifcation components and discussed future perspectives of mRNA modifcation in plants.

MicroRNA biogenesis and function in higher plants

정재훈,서필준,박충모 한국식물생명공학회 2009 Plant biotechnology reports Vol.3 No.2

MicroRNAs (miRNAs) are endogenous, noncoding, small RNA molecules consisting of 21–24 nucleotides (nts) that regulate target genes at the posttranscriptional level in plants and animals. In plants, miRNAs negatively regulate target mRNAs containing a highly complementary sequence by either mRNA cleavage or translational repression. MiRNAs are processed from singlestranded precursors containing stem-loop structures by a Dicer-like enzyme and are loaded into silencing complexes, where they act on target mRNAs. Although plant miRNAs were first reported in Arabidopsis 10 years later than animal miRNAs, numerous miRNAs have since been identified from various land plants ranging from mosses to flowering plants, and their roles in diverse aspects of plant developmental processes have been characterized. Furthermore, most of the annotated plant miRNAs are evolutionarily conserved in various plants. In particular, recent functional studies using Arabidopsis mutants have contributed a great deal of information towards establishing a framework for understanding miRNA biogenesis and functional roles. Extensive appraisal of miRNA-directed regulation during a wide array of plant development and plant responses to environmental conditions has confirmed the versatile roles of miRNAs as a key component of plant molecular biology.

HiCORE: Hi-C Analysis for Identification of Core Chromatin Looping Regions with Higher Resolution

이홍우,서필준 한국분자세포생물학회 2021 Molecules and cells Vol.44 No.12

Genome-wide chromosome conformation capture (3C)- based high-throughput sequencing (Hi-C) has enabled identification of genome-wide chromatin loops. Because the Hi-C map with restriction fragment resolution is intrinsically associated with sparsity and stochastic noise, Hi-C data are usually binned at particular intervals; however, the binning method has limited reliability, especially at high resolution. Here, we describe a new method called HiCORE, which provides simple pipelines and algorithms to overcome the limitations of single-layered binning and predict core chromatin regions with three-dimensional physical interactions. In this approach, multiple layers of binning with slightly shifted genome coverage are generated, and interacting bins at each layer are integrated to infer narrower regions of chromatin interactions. HiCORE predicts chromatin looping regions with higher resolution, both in human and Arabidopsis genomes, and contributes to the identification of the precise positions of potential genomic elements in an unbiased manner.

Dependence and independence of the root clock on the shoot clock in Arabidopsis

이홍길,서필준 한국유전학회 2018 Genes & Genomics Vol.40 No.10

Temporal and spatial compartmentalization of biological processes is facilitated by tissue-specific uncoupled circadian clocks in plants. However, interactions among tissue-specific circadian clocks have not been well established. The primary objective of this study was to describe both organ-specific circadian behaviors and centralized actions of the root clock. We analyzed transcript accumulation of circadianly-oscillating genes in roots and shoots. Expression of many clock components was different in roots and shoots. In particular, evening-expressed clock components were highly expressed in roots and likely play important roles in oscillation of the root clock. Consistent with this, the root and shoot clocks responded differentially to circadian gene mutations. The root clock was even dampened in gi-2 mutant. Circadian clocks basically oscillate in an organ-specific manner in plants, but the root clock also requires shoot-derived signals for organism-level coordination of circadian activity.

Activation of a Mitochondrial ATPase Gene Induces Abnormal Seed Development in Arabidopsis

Kon Baek,서필준,박충모 한국분자세포생물학회 2011 Molecules and cells Vol.31 No.4

The ATPases associated with various cellular activities (AAA) proteins are widespread in living organisms. Some of the AAA-type ATPases possess metalloprotease activi-ties. Other members constitute the 26S proteasome com-plexes. In recent years, a few AAA members have been implicated in vesicle-mediated secretion, membrane fu-sion, cellular organelle biogenesis, and hypersensitive responses (HR) in plants. However, the physiological roles and biochemical activities of plant AAA proteins have not yet been defined at the molecular level, and regulatory mechanisms underlying their functions are largely unknown. In this study, we showed that overexpression of an Arabidopsis gene encoding a mitochondrial AAA protein, ATPase-in-Seed-Development (ASD), induces morphological and anatomical defects in seed maturation. The ASD gene is expressed at a high level during the seed maturation process and in mature seeds but is repressed rapidly in germinating seeds. Transgenic plants overexpressing the ASD gene are morphologically normal. However, seed formation is severely disrupted in the transgenic plants. The ASD gene is induced by abiotic stresses, such as low temperatures and high salinity, in an abscisic acid (ABA)-dependent manner. The ASD protein possesses ATPase activity and is localized into the mitochondria. Our observations suggest that ASD may play a role in seed maturation by influencing mitochondrial function under abiotic stress.

A Competitive Peptide Inhibitor KIDARI Negatively Regulates HFR1 by Forming Nonfunctional Heterodimers in Arabidopsis Photo-morphogenesis

홍신영,박충모,서필준,류재용,조신해,우제창 한국분자세포생물학회 2013 Molecules and cells Vol.35 No.1

Dynamic dimer formation is an elaborate means of modulating transcription factor activities in diverse cellular pro-cesses. The basic helix-loop-helix (bHLH) transcription factor LONG HYPOCOTYL IN FAR-RED 1 (HFR1), for example, plays a role in plant photomorphogenesis by forming non-DNA binding heterodimers with PHYTOCHROME-INTERACTING FACTORS (PIFs). Recent studies have shown that a small HLH protein KIDARI (KDR) negatively regulates the HFR1 activity in the process. However, molecular mechanisms underlying the KDR control of the HFR1 activity are unknown. Here, we demonstrate that KDR attenuates the HFR1 activity by competitively forming nonfunctional heterodimers, causing liberation of PIF4 from the transcriptionally inactive HFR1-PIF4 complex. Accordingly, the photomorphogenic hypocotyl growth of the HFR1-overexpres-sing plants can be suppressed by KDR coexpression, as observed in the HFR1-deficient hfr1-201 mutant. These results indicate that the PIF4 activity is modulated through a double layer of competitive inhibition by HFR1 and KDR, which could in turn ensure fine-tuning of the PIF4 activity under fluctuating light conditions.

The Floral Repressor BROTHER OF FT AND TFL1 (BFT) Modulates Flowering Initiation under High Salinity in Arabidopsis

Jae Yong Ryu,박충모,서필준 한국분자세포생물학회 2011 Molecules and cells Vol.32 No.3

Floral transition is coordinately regulated by both endo-genous and exogenous cues to ensure reproductive suc-cess under fluctuating environmental conditions. Abiotic stress conditions, including drought and high salinity, also have considerable influence on this developmental process. However, the signaling components and molecular mecha-nisms underlying the regulation of floral transition by environmental factors have not yet been defined. In this work, we show that the Arabidopsis BROTHER OF FT AND TFL1 (BFT) gene, which encodes a member of the FLOWERING LOCUS T (FT)/TERMINAL FLOWER 1 (TFL1) family, regulates floral transition under conditions of high salinity. The BFT gene was transcrip-tionally induced by high salinity in an abscisic acid (ABA)-dependent manner. Transgenic plants overexpressing the BFT gene (35S:BFT) and BFT-deficient mutant (bft-2) plants were phenotypically indistinguishable from Col-0 plants in seed germination and seedling growth under high salinity. In contrast, al-though the floral transition was delayed significantly in Col-0 plants under high salinity, that of the bft-2 mutant was not affected by high salinity. We also observed that expression of the APETALA1 (AP1) gene was suppressed to a lesser degree in the bft-2 mutant than in Col-0 plants. Taken together, our observations suggest that BFT me-diates salt stress-responsive flowering, providing an adaptive strategy that ensures reproductive success under unfavorable stress conditions.

개별 아미노산 처리에 따른 애기장대 유전자 발현 및 상추 생육

손주연(Ju-Yeon Son),서필준(Pil-Joon Seo),임상선(Sang-Sun Lim),함종현(Jong-Hyun Ham) 한국토양비료학회 2016 한국토양비료학회 학술발표회 초록집 Vol.2016 No.10

인공광 이용형 다목적 육묘 모듈의 개발

김성겸(Sung Kyeom Kim),서필준(Pil Joon Seo),전창후(Changhoo Chun) 한국원예학회 2004 한국원예학회 학술발표요지 Vol.2004 No.5

벼에서 CRISPR/Cas9 활용 고빈도 유전자 편집 방법

정유진,배상수,이긍주,서필준,조용구,강권규,Jung, Yu Jin,Bae, Sangsu,Lee, Geung-Joo,Seo, Pil Joon,Cho, Yong-Gu,Kang, Kwon Kyoo 한국식물생명공학회 2017 식물생명공학회지 Vol.44 No.1

CRISPR/Cas9 기술은 생명공학을 활용한 신품종 작물육성에 있어 패러다임 변혁을 가져다 줄 핵심 기반기술이다. 본 연구에서는 CRISPR/Cas9를 이용하여 유전자편집기술을 기존에 알려진 방법보다 쉽고 정확하게 실험 할 수 있도록 sgRNA 디자인, 벡터구축, 형질전환체 육성 및 분석 등을 자세히 기술하였다. sgRNA는 http://www.rgenome.net/ 사이트에서 NGG 영역을 중심으로 하여 target-up: 5'-ggcaGNNNNNNNNNNNNNNNNNNNN-3'과 target-down: 5'-aaacNNNNNNNNNNNNNNNNNNNNC-3'의 올리고를 디자인하였다. 식물형질전환용 벡터는 pPZP-Cas9-RGEN을 기본으로 하였으며, sgRNA의 프로모터는 OsU3를 이용하여 pPZP::35S::Cas9::PinII-OsU3::sgRNA::Bar-Gen 순으로 구축하였다. 형질전환체의 육성은 단기형질전환 Agrobacterium 법을 사용하였으며 재분화 식물체를 얻는데48일 정도 소요되었다. 형질전환체 유무는 genomic PCR 분석으로 single copy 선발은 TaqMan PCR로 분석하였다. 정밀유전자편집 식물체는 T1 세대에서 T-DNA 삽입되지 않은 식물체를 Bar-strip에 의해 선발하였다. 선발된 식물체의 sgRNA 영역의 염기배열 조사에 의해 유전자 편집 식물체를 육성하였다. 따라서 본 연구에서 CRISPR/Cas9 system에 의한 정밀유전자편집 기술을 이용하여 보다 빠르고 쉽고 경제적으로 유전자가 편집된 개체를 확보할 수 있었다. 본 실험에서 확립된 system은 상업용 식물 계통육성에 이용 가능하여 육종적 가치가 매우 클 것으로 사료된다. The CRISPR/Cas9 is a core technology that can result in a paradigm for breeding new varieties. This study describes in detail the sgRNA design, vector construction, and the development of a transgenic plant and its molecular analysis, and demonstrates how gene editing technology through the CRISPR/Cas9 system can be applied easily and accurately. CRISPR/Cas9 facilitates targeted gene editing through RNA-guided DNA cleavage, followed by cellular DNA repair mechanisms that introduce sequence changes at the site of cleavage. It also allows the generation of heritable-targeted gene mutations and corrections. Here, we present detailed procedures involved in the CRISPR/Cas9 system to acquire faster, easier and more cost-efficient gene edited transgenic rice. The protocol described here establishes the strategies and steps for the selection of targets, design of sgRNA, vector construction, and analysis of the transgenic lines. The same principles can be used to customize the versatile CRISPR/Cas9 system, for application to other plant species.