Crime scene reconstruction: determining time of RNA death by analysis of RNA degradation fragments
In addition to regulating RNA synthesis, increasing or decreasing RNA degradation is an alternative way to control gene expression. Moreover, RNA degradation plays an important role in plant defense against pathogens by directly attacking pathogen RNA or negatively regulating the expression of immune-related genes. Therefore, the study of RNA degradation mechanisms will increase understanding of crop defense mechanisms and lead to new approaches for gene expression control which will in turn provide new tools and research directions for the development of agricultural biotechnology. Scientists are currently able to use next generation sequencing technologies to globally detect RNA degradation fragments within cells. However, how to use RNA degradome data to determine the sequence features triggering RNA degradation, the proteins involved, as well as temporal understanding of RNA degradation occurrence remains obscure.
Dr. Ho-Ming Chen and her group at the Agricultural Biotechnology Research Center have developed novel approaches to analyze RNA degradome data. Their analyses have revealed that RNA-binding proteins can hinder RNA decay, leading to the accumulation of RNA degradation fragments at the binding sites of RNA-binding proteins which can be viewed as in vivo footprints of RNA-binding proteins. The properties of these RNA-binding proteins can be used to infer the state of RNA when degradation occurs.
In the recent Plant Cell paper published by Dr. Ho-Ming Chen’s group, they further demonstrate that the exon-junction complex (EJC) is also one of the RNA-binding proteins with the ability to stop RNA decay. Notably, EJCs are deposited onto RNA during RNA synthesis and maturation but are displaced by ribosomes in the pioneer round of translation. Therefore, EJC footprints in the RNA degradome can serve as a marker for RNA degradation before steady-state translation. EJC footprints were observed in the RNA degradomes of Arabidopsis, rice, worm and human cells. This finding will lead to novel approaches for identifying the RNAs that are rapidly degraded after synthesis and exploring the underlying mechanisms through RNA degradome analysis.
This paper was published and highlighted in the April issue of The Plant Cell in 2020. The first authors include Wen-Chi Lee, Bo-Han Hou, and Cheng-Yu Hou. This study was funded by the Agricultural Biotechnology Research Center, Academia Sinica.
Article link: http://www.plantcell.org/content/32/4/904
Size control study of microalga reveals secret of aberrant cell division!
Retinoblastoma (RB) is the first identified tumor suppressor. Mutation in RB not only causes retinoblastoma, its mutation is also associated with many types of cancer. Hence, it is important to study how the RB pathway regulates cell division. Unfortunately, RB null mutants often lead to embryonic lethality in mammalian model systems such as mouse. It makes study of the RB pathway difficult. Also, lack of the RB pathway in the eukaryotic single-cell system, budding yeast, makes it unsuitable for this study. RB gene is also present in plants and its function is important for plant growth and development.
Dr. Su-Chiung Fang's group at Agricultural Biotechnology Research Center and Biotechnology Center in Southern Taiwan, Academia Sinica, took the advantage of the powerful genetics of the unicellular microalga, Chlamydomonas reinhardtii (Chlamydomonas), to investigate how the RB pathway regulates cell division. Unlike other model systems, Chlamydomonas RB mutant is viable and divides supernumerously to generate tiny cells. The size suppression genetic screen has allowed them to identify a SMALL UBIQUITIN-LIKE MODIFIER (SUMO) protease as a novel downstream player of the RB. They further showed that the SUMO protease regulates SUMOylation of RIBOSOMAL PROTEIN L30 (RPL30) to control cell division. In RB mutant cells, the supernumerous cell division can be attenuated by increasing the amount of SUMOylated RPL30 protein (Figure 1). This finding reveals a novel mechanism through which SUMO conjugation regulates RPL30 to control cell division in RB mutant cells and suggests that RPL30 may be useful molecular target for anti-cancer drugs screening. This new insight is also fundamental for application of agricultural biotechnology.
The lead author for this work is Yen-Ling Lin, a PhD student in Microbial Genomics PhD program of Academia Sinica-National Chung Hsing University in the Fang Lab. This study was funded by Academia Sinica Agricultural Biotechnology Research Center and the Ministry of Science and Technology.
Article link：〈SUMO protease SMT7 modulates ribosomal protein L30 and regulates cell-size checkpoint function〉, http://www.plantcell.org/content/32/4/1285
Wen-Chi Lee, Bo-Han Hou, Cheng-Yu Hou, Shu-Ming Tsao, Ping Kao, Ho-Ming Chen* (2020) Widespread Exon Junction Complex Footprints in the RNA Degradome Mark mRNA Degradation before Steady State Translation. The Plant Cell, Vol. 32: 904–922.
The RNA degradome is composed of assorted RNA degradation intermediates derived from diverse RNA degradation pathway. In 2016, we reported that the plant RNA degradome contains in vivo ribosome footprints. In this study, we further demonstrate that exon junction complexes (EJCs) are able to protect mRNAs against 5'-3' degradation resulting in marked footprints in the RNA degradome. In addition to Arabidopsis, in vivo EJC footprints were also observed in the degradome datasets of rice, worm and human cells, indicating that this phenomenon is conserved across species. Because EJCs are deposited on mRNAs during splicing and displaced by ribosomes in the pioneer round of translation, EJC footprints can serve as markers for the mRNA degradation before steady-state translation. Predominant EJC footprints were observed in hundreds of Arabidopsis nonsense-mediated mRNA decay (NMD) targets which are presumed to be destroyed during the pioneer round of translation, validating this possibility. NMD is important in plant immune response and pathogen defense, but NMD targets were poorly defined due to the lack of suitable methodology. Here, we show that EJC footprints can be used to validate potential NMD substrates and inference of NMD activity. We also demonstrate that cleavage guided by some microRNAs (miRNAs) can result in EJC footprint accumulation on mRNAs, indicating that plant miRNAs can destabilize EJC-bound mRNAs prior to translation. Together, this is the first work reporting the presence of EJC footprints in the RNA degradome, and the analysis of EJC footprints in the RNA degradome revealed new insights into NMD and miRNA regulation in plants, demonstrating new applications of RNA degradome data.
Yen-Ling Lin, Chin-Lin Chung, Ming-Hui Chen, Chun-Han Chen, Su-Chiung Fang* (2020) SUMO protease SMT7 modulates ribosomal protein L30 to regulate cell-size checkpoint function. Plant Cell. 32(4): 1285-1307.
Proliferating cells actively coordinate growth and cell division to ensure cell-size homeostasis; however, the underlying mechanism through which size is controlled is poorly understood. Defect in a SUMO protease protein, suppressor of mat3 7 (SMT7), has been shown to reduce cell division number and increase cell size of the small-size mutant mating type locus 3-4 (mat3-4), which contains a defective retinoblastoma tumor suppressor-related protein of Chlamydomonas (Chlamydomonas reinhardtii). Here we describe development of an in vitro SUMOylation system using Chlamydomonas components and use it to provide evidence that SMT7 is a bona fide SUMO protease. We further demonstrate that the SUMO protease activity is required for supernumerous mitotic divisions of the mat3-4 cells. In addition, we identified RIBOSOMAL PROTEIN L30 (RPL30) as a prime SMT7 target and demonstrated that its SUMOylation is an important modulator of cell division in mat3-4 cells. Loss of SMT7 caused elevated SUMOylated RPL30 levels. Importantly, overexpression of the translational fusion version of RPL30-SUMO4, which mimics elevation of the SUMOylated RPL30 protein in mat3-4, caused a decrease in mitotic division and recapitulated the size-increasing phenotype of the smt7-1 mat3-4 cells. In summary, our study reveals a novel mechanism through which a SUMO protease regulates cell division in the mat3-4 mutant of Chlamydomonas and provides yet another important example of the role that protein SUMOylation can play in regulating key cellular processes, including cell division.
Apaya MK, Hsiao PW, Yang YC and Shyur LF* (2020). Deregulating the CYP2C19/epoxy-eicosatrienoic acid-associated FABP4/FABP5 signaling network as a therapeutic approach for metastatic triple-negative breast cancer Cancers 12(1):199. doi: 10.3390/cancers12010199.
Recurrence and metastasis are the main causes of triple-negative breast cancer (TNBC) mortality. We have previously demonstrated that arachidonic acid-derived epoxy-eicosatrienoic acids (EETs) are important metastasis drivers in TNBC tumors from that we hypothesized that understanding the interplay between fatty acid binding protein (FABP) and EET-driven metastatic progression may uncover a new opportunity for TNBC intervention. In this work, the biological and clinical relevance of FABP upregulation in the EET signaling axis was deciphered. Publicly available genomics and clinical datasets, shRNA-mediated gene knockdown, EET supplementation, cancer and stromal cell co-cultures, and an orthotopic and resection xenograft tumor mouse model were used to delineate mechanisms by which FABP/EET dynamics and levels were critical in TNBC metastatic transformation and stromal cell interactions. TNBC cell proliferation, migratory transformation, and distal metastasis priming were influenced by EET-associated nuclear translocation of FABP4 and FABP5 isoforms and nuclear accumulation of SREBP-2 or PPAR-γ. Most notably, this study uncovers novel bioefficacy and modes of action of the anticancer drug doxorubicin and a bioactive phytogalactolipid, 1,2-di-O-α-linolenoyl-3-O-β-galactopyranosyl-sn-glycerol (dLGG) identified from medicinal plant Crassocephalum rabens. This study introduces a novel approach to combating TNBC by targeting the FABP/EET/CYP-associated metastatic signaling network.
Yue JJ, Hong CY, Wei P, Tsai YC, Lin CS (2020) How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis. Rice 13:9.
The breakthrough CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome-editing technology has led to great progress in monocot research; however, several factors need to be considered for the efficient implementation of this technology. To generate genome-edited crops, single guide (sg)RNA and Cas9 DNA are delivered into plant cells and expressed, and the predicted position is targeted. Analyses of successful targeted mutations have revealed that the expression levels, expression timing, and variants of both sgRNA and Cas9 need to be sophisticatedly regulated; therefore, the promoters of these genes and the target site positions are the key factors for genome-editing efficiency. Currently, various vectors and online tools are available to aid sgRNA design. Furthermore, to reduce the sequence limitation of the protospacer adjacent motif (PAM) and for other purposes, many Cas protein variants and base editors can be used in plants. Before the stable transformation of a plant, the evaluation of vectors and target sites is therefore very important. Moreover, the delivery of Cas9-sgRNA ribonucleoproteins (RNPs) is one strategy that can be used to prevent transgene issues with the expression of sgRNA and Cas proteins. RNPs can be used to efficiently generate transgene-free genome-edited crops that can reduce transgene issues related to the generation of genetically modified organisms. In this review, we introduce new techniques for genome editing and identifying marker-free genome-edited mutants in monocot crops. Four topics are covered: the design and construction of plasmids for genome editing in monocots; alternatives to SpCas9; protoplasts and CRISPR; and screening for marker-free CRISPR/Cas9-induced mutants. We have aimed to encompass a full spectrum of information for genome editing in monocot crops.
S. Kailasam, S. Singh, M.-J. Liu, C.-C. Lin and K.-C. Yeh* (2020) A HemK class glutamine-methyltransferase is involved in the termination of translation and essential for iron homeostasis in Arabidopsis. New Phytologist doi.org/10.1111/nph.16440
Fe-deficiency induced transcriptional cascades controls Fe transport and utilization is important for plant to maintain homeostasis of this important element executing the biological function. So far, we know that many genes participating in these processes are transcriptionally controlled by Fe-status. By a genetical approach, we identified a novel component, HemK-like methyltransferase, that is involved in the termination of translation. With defect of this gene, plants lose ion homeostasis and are highly sensitive to Fe deficiency and possess other developmental flaws. We detected that many Fe-deficiency-inducible transcripts exhibit ribosome stalling implying translational failings in the mutant. In this study, a check-point that fine tunes the peptide release for growth and development in plants is uncovered. Our report sheds light on mechanisms of translation termination and its importance in preserving both molecular and physiological responses to Fe-starvation.
Yuh Tzean, Ho-Hsiung Chang, Tsui-Chin Tu, Bo-Han Hou, Ho-Ming Chen, Yi-Shu Chiu, Wei-Yi Chou, Li Chang, and Hsin-Hung Yeh* (2020) Engineering Plant Resistance to Tomato Yellow Leaf Curl Thailand Virus Using a Phloem-Specific Promoter Expressing Hairpin RNA https://apsjournals.apsnet.org/doi/10.1094/MPMI-06-19-0158-R (Selected for publication in MPMI Focus Issue on Cell Biology of Virus-Plant and Virus-Vector Interaction and commentary in the same issue).
Transgenic approaches employing RNA interference (RNAi) strategies have been successfully applied to generate desired traits in plants; however, variations between RNAi transgenic siblings and the ability to quickly apply RNAi resistance to diverse cultivars remain challenging. In this study, we assessed the promoter activity of a cauliflower mosaic virus 35S promoter (35S) and a phloem-specific promoter derived from rice tungro bacilliform virus (RTBV) and their efficacy to drive RNAi against the endogenous glutamate-1-semialdehyde aminotransferase gene (GSA) that acts as a RNAi marker, through chlorophyll synthesis inhibition, and against tomato yellow leaf curl Thailand virus (TYLCTHV), a begomovirus (family Geminiviridae) reported to be the prevalent cause of tomato yellow leaf curl disease (TYLCD) in Taiwan. Transgenic Nicotiana benthamiana expressing hairpin RNA of GSA driven by either the 35S or RTBV promoter revealed that RTBV::hpGSA induced stronger silencing along the vein and more uniformed silencing phenotype among its siblings than 35S::hpGSA. Analysis of transgenic N. benthamiana, 35S::hpTYLCTHV, and RTBV::hpTYLCTHV revealed that, although 35S::hpTYLCTHV generated a higher abundance of small RNA than RTBV::hpTYLCTHV, RTBV::hpTYLCTHV transgenic plants conferred better TYLCTHV resistance than 35S::hpTYLCTHV. Grafting of wild-type (WT) scions to TYLCTHV RNAi rootstocks allowed transferable TYLCTHV resistance to the scion. A TYLCTHV-inoculation assay showed that noninfected WT scions were only observed when grafted to RTBV::hpTYLCTHV rootstocks but not 35S::hpTYLCTHV nor WT rootstocks. Together, our findings demonstrate an approach that may be widely applied to efficiently confer TYLCD resistance.
Yamada M, Han X, Benfey PN (2020) RGF1 controls root meristem size through ROS signaling.
>Nature. 2020 Jan;577(7788):85-88. doi: 10.1038/s41586-019-1819-6. Epub 2019 Dec 4.
The stem cell niche and the size of the root meristem in plants are maintained by intercellular interactions and signalling networks involving a peptide hormone, root meristem growth factor 1 (RGF1). Understanding how RGF1 regulates the development of the root meristem is essential for understanding stem cell function. Although five receptors for RGF1 have been identified, the downstream signalling mechanism remains unknown. Here we report a series of signalling events that follow RGF1 activity. We find that the RGF1-receptor pathway controls the distribution of reactive oxygen species (ROS) along the developmental zones of the Arabidopsis root. We identify a previously uncharacterized transcription factor, RGF1-INDUCIBLE TRANSCRIPTION FACTOR 1 (RITF1) , that has a central role in mediating RGF1 signalling. Manipulating RITF1 expression leads to the redistribution of ROS along the root developmental zones. Changes in ROS distribution in turn enhance the stability of the PLETHORA2 protein, a master regulator of root stem cells. Our results thus clearly depict a signalling cascade that is initiated by RGF1, linking this peptide to mechanisms that regulate ROS.
Yang, Shu-Yi, Lu, Wen-Chien, Ko, Swee-Suak, Sun, Ching-Mei, Hung, Jo-Chi and Chiou, Tzyy-Jen* (2020) uORF and Phosphate-Regulated Expression of Rice OsNLA1 Controls Phosphate Transport and Reproduction. Plant Physiology: pp.01101.02019
Rice (Oryza sativa) OsNLA1 has been proposed to play a crucial role in regulating phosphate (Pi) acquisition in roots, similar to that of Arabidopsis AtNLA. However, unlike AtNLA, OsNLA1 is not a target of miR827, a Pi starvation-induced miRNA. It is, therefore, of interest to know whether the expression of OsNLA1 depends on Pi supply and how it is regulated. In this study, we provide evidence that OsNLA1 controls Pi acquisition by directing the degradation of several OsPHT1 Pi transporters (i.e., OsPT1/2/4/7/8/12). We further show that OsNLA1 has an additional function in reproduction and uncover the mechanism of its expression regulation. Analysis of mRNA levels, promoter-GUS (β-glucuronidase) activity and protoplast transient expression showed that the expression of OsNLA1.1, the most abundant transcript variant, is upregulated in response to increasing Pi supply. The OsNLA1 promoter region was found to contain an upstream open reading frame (uORF) that is required for Pi-responsive expression regulation. OsNLA1 promoter activity was observed in roots, ligules, leaves, sheaths, pollen grains, and surrounding the vascular tissues of anthers, suggesting that OsNLA1 is important throughout the development of rice. Disruption of OsNLA1 resulted in increased Pi uptake from roots as well as impaired pollen development and reduced grain production. In summary, our study reveals that Pi-induced OsNLA1 expression regulated by a unique mechanism functions in Pi acquisition, Pi translocation, and reproductive success.
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2020/09/28 11:00 AM
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