Genome-Wide Identification of Reverse Complementary microRNA Genes in Plants

MicroRNAs (miRNAs) are ∼21-nucleotide small RNAs (sRNAs) with essential regulatory roles in plants. They are generated from stem-loop-structured precursors through two sequential Dicer-like 1 (DCL1)-mediated cleavages. To date, hundreds of plant miRNAs have been uncovered. However, the question, whether the sequences reverse complementary (RC) to the miRNA precursors could form hairpin-like structures and produce sRNA duplexes similar to the miRNA/miRNA* pairs has not been solved yet. Here, we interrogated this possibility in 16 plant species based on sRNA high-throughput sequencing data and secondary structure prediction. A total of 59 RC sequences with great potential to form stem-loop structures and generate miRNA/miRNA*-like duplexes were identified in ten plants, which were named as RC-miRNA precursors. Unlike the canonical miRNAs, only a few cleavage targets of the RC-miRNAs were identified in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), and none in Soybean (Glycine max) based on degradome data. Surprisingly, the genomic regions surrounding some of the RC-miRNA target recognition sites were observed to be specifically methylated in both Arabidopsis and rice. Taken together, we reported a new class of miRNAs, called RC-miRNAs, which were generated from the antisense strands of the miRNA precursors. Based on the results, we speculated that the mature RC-miRNAs might have subtle regulatory activity through target cleavages, but might possess short interfering RNA-like activity by guiding sequence-specific DNA methylation.     

转录因子靶基因的鉴定

文章介绍了鉴定转录因子靶基因研究中常用的生物学方法,尤其是在基因组范围内转录因子靶位点的筛选方法,如染色质免疫沉淀、甲基化酶鉴定、pull-down、蛋白结合芯片和生物信息学方法等。     

Identification of MicroRNA Target Genes in Vivo

MicroRNAs (miRNAs) are short non-coding RNAs transcribed from intergenic or intronic sequences as long precursors that are sequentially processed by the endonucleases Drosha and Dicer into short double-stranded sequences. It is clear that miRNAs play essential roles in gene expression, development, and cell fate specification in animals. However, one of the barriers of miRNA research is how to find the target genes. In this study, we have developed a rapid and effective method to isolate miRNA target genes in vivo. MicroRNA was synthesized in vitro and labeled by biotin. After transfected into cells, the miRNA/mRNA complexes were isolated by streptavidin-coated magnetic beads. hsa-miR155 was taken as model to validate this method, which is a very important modulator in tumor development. It is useful for validation of targets predicted in silico, and, potentially, for discovery of previously uncharacterized targets.     

Genome-Wide Identification and Analysis of Genes Encoding Proteolytic Enzymes in Pineapple

Pineapple, Ananas comosus, is an economically important fruit crop. Recently its genome was completely sequenced and a total of 27,024 protein coding genes were predicted. Using a set of well evaluated bioinformatics tools we have predicted the protein subcellular locations and comparatively analyzed the protein conserved domains of the predicted proteomes in pineapple, Oryza sativa (rice), Sorghum bicolor (sorghum), and Brachypodium distachyson. Our analysis revealed that ~24–26 % of proteins were located in nucleus, 17–21 % in cytosol, 9–11 % in chloroplast, and 8–11 % proteins were secreted in these monocot plants. The secretomes in the four species were analyzed comparatively and a large number of secreted glycosyl hydrolases were identified. As pineapple proteolytic enzymes, knowns as bromelains, have been used for medical treatments, we focused on genome-wide identification and analysis of pineapple genes encoding proteases. A total of 512 pineapple genes encoding putative proteolytic enzymes were identified, with 152 secreted, 74 localized in cytosol, 67 in nucleus, 60 in chloroplast, 18 in mitochondria, and the remaining in other subcellular locations. The top large protease families in pineapple were papain family cysteine protease (62 genes), peptidase S8 family (56 genes), aspartyl protease family (38 genes), and serine carboxypeptidase (33 genes). Gene expression analysis revealed that among 512 protease genes 432 were expressed in various tissues and 72 genes were differentially expressed. The highly expressed protease genes were identified including 7 papain family cysteine proteases. The protease genes with the predicted protein subcellular locations will facilitate the efforts for examining their biological roles in pineapple growth and development and for expressing the recombinant proteases for medical use. The information of protein subcellular location of all plant species can be accessed at the PlantSecKB website (http://proteomics.ysu.edu/secretomes/plant.php).     

Genome-Wide Identification and Characterization of RBR Ubiquitin Ligase Genes in Soybean

RBR (RING1-IBR-RING2) proteins play an important role in protein ubiquitination and are involved in many cellular processes. Recent studies showed plant RBR genes were induced by abiotic and biotic stresses. However, detailed studies on RBR genes in the important oil crop, soybean (Glycine max (L.) Merr.), is still lacking. Here we performed a genome-wide search and identified 24 RBR domain-containing genes from the soybean genome sequence and cloned 11 of them. Most soybean RBR proteins contain a highly conserved RBR supra-domain. Phylogenetic analyses indicated all 24 soybean RBR proteins are most related to the RBR proteins from Phaseolus vulgaris, and could be classified into seven groups including Ariadne A, Ariadne B, ARA54, Plant IIA, Plant IIB, Plant IIC, and Helicase. Tandem duplication and block duplication were found among the Ariadne B and Plant IIC group of soybean RBR genes. Despite the conserved RBR supra-domain, there are extensive variations in the additional protein motifs and exon-intron structures between different groups, which indicate they might have diverse functions. Most soybean RBR proteins are predicted to localize in nucleus, and four of them were experimentally confirmed by GFP fusion proteins. Soybean RBR genes are broadly expressed in many tissue types with a little more abundant in the roots and flowers than leaves, stems, and seeds. The expression of GmRTRTP3 (Plant IIB) and GmRTRTP5 (Plant IIC) are induced by NaCl treatment, which suggests these RBR genes might be involved in soybean response to abiotic stresses.     

全基因组预测目标基因的新方法及其应用

运用隐马尔可夫模型, 利用Perl编程, 以几种模式生物的蛋白质数据库为基础, 构建了目标基因的全基因组预测的新方法。该方法具有高通量, 准确度高且操作简易等优点, 特别在多结构域蛋白家族预测上更显优势。应用该方法对几种模式生物的全基因组PPR和TPR蛋白家族进行了预测, 其中粳稻日本晴中含有536个PPR蛋白、199个TPR蛋白; 籼稻9311中含有519个PPR蛋白、177个TPR蛋白; 拟南芥中含有735个PPR蛋白、292个TPR蛋白; 红藻中6个PPR蛋白、32个TPR蛋白; 蓝细菌以及古细菌中没有PPR蛋白, 但蓝细菌含有10个TPR蛋白, 古细菌有4个TPR蛋白, 并对所得结果进行了进一步生物信息学分析。     

Identification of Target Genes in Their Native Cellular Context

Ewing’s sarcoma is the second most common tumor of bone in children and young adults, and requires highly intensive chemotherapy along with surgery and/or radiation for successful treatment. Because these therapies are associated with significant short- and long-term side effects, new therapeutic approaches are needed. Most cases of Ewing’s sarcoma contain somatic translocations between chromosomes 11 and 22 that result in the t(11;22)(q24;q12). This translocation encodes the EWS/FLI fusion protein. EWS/FLI formation appears to be the critical oncogenic event in the development of Ewing’s sarcoma. It is hoped that an in-depth understanding of the mechanism that EWS/FLI uses to cause oncogenic transformation will result in new therapies for this disease. Unfortunately, this hope has not been realized. One difficulty has been the lack of an appropriate model system in which to study the fusion oncoprotein. We recently described and validated the use of retroviral RNA interference approaches to study EWS/FLI in Ewing’s sarcoma cell lines. We now put this model into a historical context, and describe the benefits (both perceived and observed) of this model over previous approaches using heterologous cell types.     

miR-378促进脂质生成相关靶基因鉴定

旨为验证miR-378在脂肪细胞中的功能,及其脂质相关靶基因的筛选和鉴定.利用miR-378类似物转染3T3-L1细胞,验证miR-378在脂肪细胞中的功能;根据靶标位点的保守性以及促脂功能确定miR-378潜在靶基因;采用microRNA pulldown技术验证miR-378与靶基因的靶标关系;运用双荧光素报告基因...     

Genome-Wide Identification of Brassica napus PEN1-LIKE Genes and Their Expression Profiling in Insect-Susceptible and Resistant Cultivars

Recently, it has been reported that a gene (PEN1) in Arabidopsis thaliana is highly resistant to Plutella xylostella. We screened all the homologous genes of PEN1 in Arabidopsis thaliana and found that the motif of these genes was very conserved. At present, few insect resistance genes have been identified and characterized in Brassica napus. Therefore, we screened all the homologous genes containing this motif in the Brassica napus genome and systematically analyzed the basic information, conserved domain, evolutionary relationship, chromosomal localization and expression analysis of these genes. In this study, 12 PEN1 homologous genes were identified in the Brassica napus genome, which is more than the number in Arabidopsis thaliana. These genes are unevenly distributed on the 12 chromosomes in Brassica napus. Furthermore, all the PEN1 homologous genes contained light responsiveness elements, and most of the genes contained gibberellin-responsive elements, meJA-responsive elements and abscisic-acid-responsive elements. The results will provide a theoretical basis for screening insect resistance genes from the genome of Brassica napus and analyzing the molecular mechanism of insect resistance in Brassica napus.     

Genome-Wide Identification and Characterization of Nucleotide-Binding Site (NBS) Resistance Genes in Pineapple

Pineapple is a major tropical fruit and the most important crop processing CAM photosynthesis. It originated in southwest Brazil and northeast Paraguay and survived the harsh, semi-arid environment. Disease resistance genes have contributed to the survival and thriving of this species. The largest class of disease resistance (R) genes in plants consists of genes encoding nucleotide-binding site (NBS) domains. The sequenced genome of pineapple (Ananas comosus (L.) Merr.) provides a resource for analyzing the NBS-encoding genes in this species. A total of 177 NBS-encoding genes were identified using automated and manual analysis criteria, and these represent about 0.6 % of the total number of predicted pineapple genes. Five genes identified here contained the N-terminal Toll/Interleukin-l receptor (TIR) domain, and 46 genes carried the N-terminal Coiled-Coil (CC) motif. A majority of these NBS-encoding genes (84 %) contained a leucine-rich repeat (LRR) domain. A total of 130 of 177 (73 %) of these NBS-encoding genes were distributed across 20 pineapple linkage groups. The identification and characterization of NBS genes in pineapple yielded a valuable genomic resource and improved understanding of R genes in pineapple, which will facilitate the development of disease resistant pineapple cultivars.