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ABSTRACT: Viral microRNAs regulate gene expression using either translational repression or mRNA cleavage and decay. Two microRNAs from infectious laryngotracheitis virus (ILTV), iltv-miR-I5 and iltv-miR-I6, map antisense to the ICP4 gene. Post-transcriptional repression by these microRNAs was tested against a portion of the ICP4 coding sequence cloned downstream of firefly luciferase. Luciferase activity was downregulated by approximately 60% with the iltv-miR-I5 mimic. Addition of an iltv-miR-I5 antagomiR or mutagenesis of the target seed sequence alleviated this effect. The iltv-miR-I5 mimic, when co-transfected with a plasmid expressing ICP4, reduced ICP4 transcript levels by approximately 50%, and inhibition was relieved by an iltv-miR-I5 antagomiR. In infected cells, iltv-miR-I5 mediated cleavage at the canonical site, as indicated by modified RACE analysis. Thus, in this system, iltv-miR-I5 decreased ILTV ICP4 mRNA levels via transcript cleavage and degradation. Downregulation of ICP4 could impact the balance between the lytic and latent states of the virus in vivo.
Virology 03/2011; 411(1):25-31. · 3.35 Impact Factor
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ABSTRACT: MicroRNAs (miRNAs) are small regulatory noncoding RNAs varying in length between 20 and 24 nucleotides. They play a key role during plant development by negatively regulating gene expression at the posttranscriptional level. Moreover, recent studies reported several miRNAs associated with abiotic stress responses. Small RNA cloning and high-throughput deep sequencing methods provide expression profiles of not only known miRNAs, but also novel miRNAs. In this chapter, we describe the methods used to identify and characterize abiotic stress-associated miRNAs and their target genes.
Methods in molecular biology (Clifton, N.J.) 01/2010; 592:203-30.
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ABSTRACT: We have developed a novel approach called parallel analysis of RNA ends (PARE) for high-throughput identification of microRNA (miRNA) targets and diverse applications for the study of the RNA degradome. The method described here comprises a modified 5'-rapid amplification of cDNA ends, deep sequencing of 3' cleavage products of mRNA and bioinformatic analysis. Following RNA extraction and isolation of polyadenylated RNA, a 5'-RNA adapter that includes an MmeI recognition site is ligated to 5'-monophosphorylated products, which contain mRNA fragments generated through miRNA-induced cleavage. The ligated products are reverse-transcribed, slightly amplified and cleaved with MmeI. The 5' equally-sized fragments are gel-selected, ligated to a 3' double-stranded DNA adapter and PCR-amplified. Following gel purification, the products are subjected to deep sequencing. The data are then matched to cDNAs and analyzed through bioinformatics filters. We describe the high-throughput protocol in detail and indicate alternative uses for PARE. The procedure presented here can be accomplished in 6-7 d.
Nature Protocol 02/2009; 4(3):356-62. · 8.36 Impact Factor
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Marcelo A German,
Manoj Pillay,
Dong-Hoon Jeong,
Amit Hetawal,
Shujun Luo,
Prakash Janardhanan,
Vimal Kannan,
Linda A Rymarquis,
Kan Nobuta,
Rana German,
Emanuele De Paoli,
Cheng Lu,
Gary Schroth,
Blake C Meyers,
Pamela J Green
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ABSTRACT: MicroRNAs (miRNAs) are important regulatory molecules in most eukaryotes and identification of their target mRNAs is essential for their functional analysis. Whereas conventional methods rely on computational prediction and subsequent experimental validation of target RNAs, we directly sequenced >28,000,000 signatures from the 5' ends of polyadenylated products of miRNA-mediated mRNA decay, isolated from inflorescence tissue of Arabidopsis thaliana, to discover novel miRNA-target RNA pairs. Within the set of approximately 27,000 transcripts included in the 8,000,000 nonredundant signatures, several previously predicted but nonvalidated targets of miRNAs were found. Like validated targets, most showed a single abundant signature at the miRNA cleavage site, particularly in libraries from a mutant deficient in the 5'-to-3' exonuclease AtXRN4. Although miRNAs in Arabidopsis have been extensively investigated, working in reverse from the cleaved targets resulted in the identification and validation of novel miRNAs. This versatile approach will affect the study of other aspects of RNA processing beyond miRNA-target RNA pairs.
Nature Biotechnology 07/2008; 26(8):941-6. · 29.50 Impact Factor
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ABSTRACT: Fructokinases catalyze the key step of fructose phosphorylation in plants. LeFRK2, the major fructokinase-encoding gene in tomato plants, is abundantly expressed in roots, stems, and fruits. To analyze the role of LeFRK2 in plant development, we analyzed transgenic tomato plants with sense and antisense expression of StFRK, the potato homolog of LeFRK2. Increased fructokinase activity had no effect. However, plants in which LeFRK2 was specifically suppressed, either via antisense suppression or via co-suppression, exhibited growth inhibition and wilting of young leaves at daytime. Grafting experiments indicated that a stem interstock of antisense plants was sufficient to inhibit growth and cause leaf wilting. Stem secondary xylem exhibited particular suppression of LeFRK2 and the area of active xylem, estimated by eosin uptake, was significantly smaller in antisense stem compared to that of wild-type plants. These results suggest that LeFRK2 might be required for proper development of xylem that affected growth and wilting.
The Plant Journal 07/2003; 34(6):837-46. · 6.16 Impact Factor
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ABSTRACT: Fructokinases catalyze the key step of fructose phosphorylation in plants. LeFRK2, the major fructokinase-encoding gene in tomato plants, is abundantly expressed in roots, stems, and fruits. To analyze the role of LeFRK2 in plant development, we analyzed transgenic tomato plants with sense and antisense expression of StFRK, the potato homolog of LeFRK2. Increased fructokinase activity had no effect. However, plants in which LeFRK2 was specifically suppressed, either via antisense suppression or via co-suppression, exhibited growth inhibition and wilting of young leaves at daytime. Grafting experiments indicated that a stem interstock of antisense plants was sufficient to inhibit growth and cause leaf wilting. Stem secondary xylem exhibited particular suppression of LeFRK2 and the area of active xylem, estimated by eosin uptake, was significantly smaller in antisense stem compared to that of wild-type plants. These results suggest that LeFRK2 might be required for proper development of xylem that affected growth and wilting.
The Plant Journal 06/2003; 34(6):837 - 846. · 6.16 Impact Factor
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ABSTRACT: The LeFRK2 gene product has been proposed to play a role in transient starch accumulation in developing tomato (Lycopersiconesculentum) fruits. To analyze the role of LeFRK2 in starch biosynthesis, we produced transgenic tomato plants with sense and antisense expression of StFRK, the potato homologue to LeFRK2. Fruits of homozygous plants expressing sense or antisense StFRK exhibited suppression of LeFRK2, concomitantly with specific elimination of the FKI isozyme, indicating that FKI is the gene product of LeFRK2. The activity of fructokinase was reduced in antisense and sense homozygous fruits and increased in fruits of sense hemizygous plants. The modified activities of fructokinase led to small but significant changes both in the steady state levels of sugars and in the level of phosphorylated sugars in fruits. However, fruits lacking FKI had increased rather than decreased starch content. We therefore concluded that LeFRK2 is not required for transient starch accumulation in tomato fruits. Rather, LeFRK2 might have a role in the regulation of sucrose import into tomato fruits.
Plant Science.
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ABSTRACT: A full-length cDNA encoding a novel fourth fructokinase, LeFRK3, was cloned from green tomato (Lycopersicon esculentum Mill.) fruits. The putative protein shares 70, 65.5 and 69% amino acid homology with the three previously identified tomato fructokinases encoded by LeFRK1, LeFRK2 and LeFRK4, respectively. This fourth fructokinase has signature patterns of the pfkB family of carbohydrate kinases as well as substrate recognition sites and an ATP-binding domain. Confirmation for its fructokinase activity was obtained by complementation of triple mutant yeast cells that are unable to phosphorylate or grow on either glucose or fructose as LeFRK3 complemented growth on fructose but not on glucose. Moreover, soluble crude protein extracts prepared from the transformed yeast cells revealed fructose but not glucose phosphorylation activity. In contrast to the LeFRK1 gene product which is inhibited neither by fructose nor by Mg, and to LeFRK2 gene product which is inhibited by both fructose and Mg, the LeFRK3 product is inhibited by fructose but not by Mg. Separation by HPLC-ion exchange chromatography pointed to the gene product of LeFRK3 as the protein responsible for the third peak of fructokinase activity (FKIII), sharing the same pattern of fructose inhibition previously identified with FKIII in tomato fruits. Mapping of tomato fructokinases indicated that LeFRK3 is located on chromosome 2, unlike LeFRK1 (chromosome 3), LeFRK2 (chromosome 6), and LeFRK4 (chromosome 10). The relative expression levels of the four known FRK genes in different tomato organs were analyzed by quantitative RT-PCR. LeFRK2 and LeFRK3 are the predominant genes expressed in all organs with LeFRK3 having the highest level of expression in leaves and apices. LeFRK4 is expressed only in stamens. This differential expression patterns combined with the different biochemical characteristics of the four FRK isozymes suggest that each plays a different role in plant development.
Plant Science.
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ABSTRACT: A full-length cDNA clone encoding a novel fructokinase, LeFRK4, was isolated from a tomato (Lycopersicon esculentum Mill.) flower cDNA library. The putative protein shares 62.6 and 55.5% amino acid identity with the two known tomato fructokinases, LeFRK1 and LeFRK2, respectively, and possesses three signature patterns of the pfkB family of carbohydrate kinases, two substrate recognition sites and an ATP-binding domain. The identification of LeFRK4 as a fructokinase was confirmed by complementation of mutant yeast cells unable to phosphorylate or grow on either glucose or fructose. LeFRK4 complemented growth on fructose but not on glucose. Non-soluble crude protein extracts prepared from the transformed yeast cells exhibited fructose but not glucose phosphorylation activity. Expression analysis demonstrated that LeFRK4 is specifically expressed in stamens. Using quantitative RT-PCR, we examined the relative expression of the three known FRK genes in different tomato organs. LeFRK2, encoding a substrate-inhibited fructokinase, was by far the predominantly expressed FRK gene in all organs, except for flowers in which it shared the same expression level with LeFRK4. The exclusive expression of LeFRK4 in stamens may point to a specific role of LeFRK4 in pollen development, anthesis, and perhaps pollination.
Plant Science.
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ABSTRACT: Original (T0) transgenic plants are usually heterozygous for the transgene. In routine analysis, homozygous transgenic plants are sought among first generation (T1) plants, the descendants of each independent transgenic plant, based on segregation analysis of the second-generation (T2) seedlings. This procedure requires the growth of T1 plants until seed production, which is not only time-consuming and laborious, but is often accompanied by the waste of the T1 generation. Here we suggest an easy, rapid method to accurately identify homozygous and heterozygous transgenic plants at the seedling stage of T1 plants. DNA of T1 tomato seedlings was extracted by a fast extraction method and used as a template in duplex quantitative real-time PCR reactions. An endogenous single copy gene served to normalize the differences in the initial DNA levels. The parents, T0 transgenic plants, were used to neutralize transgene copy number and to bypass the need of calibration with known heterozygous and homozygous plants. Using this assay, the zygosity of tens of T1 seedlings was rapidly identified with 100% fidelity. This is the first report showing the profitability of this method in determining zygosity in plants.
Plant Science.
