RNA化学修饰在消化道肿瘤发生发展中的作用

生物体内大分子物质的特定化学修饰是调节分子结构与功能的高效性方法,DNA和蛋白质的修饰会影响下游信号通路的激活,RNA化学修饰则在基因选择性表达中发挥关键性调节作用.大自然中存在170多种不同类型的RNA化学修饰,它们分别参与编码和非编码RNA的修饰过程.RNA修饰的失调会影响多种疾病的发生发展.在这篇综述中,我们着重介绍了N6-甲基腺苷、5-甲基胞嘧啶、N1-甲基腺苷、7-甲基鸟嘌呤和Pseudouridine在内的多种RNA化学修饰,并对其在消化道肿瘤中的作用进行了归纳和总结.

Construction of Recombinant Retroviral Vector Containing HIV-1 Tat Gene and Functional Detection of Expressed Tat in Target Cells

Objective: To construct recombinant retroviral vector containing HIV-1 Tatgene and evaluate the junction of the expressed Tat in target cells. Methods: HIV-1 Tat_(101) genewas recovered from pEV plasmid by Hind Ⅲ digestion and cloned into expression plasmid LZESpBMN-Z toconstruct recombinant retroviral expression plasmid named LZRS-Tat_(101). Using the method ofcalcium phosphate, the construct of LZRS-Tat_(101) was then transfected into packaging cell linesPhoenix (ΦNX) which contained env and gal genes encoding structural proteins and pol gene codingfor 3 enzymes ( reverse transcriptase, protease and integrate) essential for retroviral integrationand replication . The stable transfected cell lines was obtained using puromycin to screen for morethan 3 days. Then, immunohistochemical (IHC ) staining was carried out to detect the expressionlevel of Tat_(101) protein in both transiently and stably trancfected ΦNX, respectively. Thesupematants containing recombinant virus collected from transient and stable transfected cells wereemployed to infect 293 cells, respectively, and the expressed Tat in 293 cells was tested by Westernblot. Meantime, the supematants of infected 293 cells was further added to HL3T1 cells which wereHela cell lines containing an HIV-1-LTR/CAT reporter construct to establish a co-culture system.After co-culture for 72 hours, the protein was extracted from HL3T1 cells and used for CAT activityassay. Results: After LZRS- Tat_(101) was transfected into ΦNX, the amount of expressed Tat intransient transfection cells was significantly higher than that in stable transfection cells; Tatcould be detected not only in 293 cells but also in the supematants from 293 cells culture, and Tatin the supematants could activate HIV-1 LTR promoter in HL3T1, resulting in high 'expression of CATlocated at the downstream of LTR. Conclusion: The construct of recombinant retrovirus LZRS-Tat_(101) could express Tat protein in target cells and the expressed Tat was functionally activeand can really exhibit the ability to activate transcription.

Characterizing and annotating the genome using RNA-seq data

Bioinformatics methods for various RNA-seq data analyses are in fast evolution with the improvement of sequencing technologies. However, many challenges still exist in how to efficiently process the RNA-seq data to obtain accurate and comprehensive results. Here we reviewed the strategies for improving diverse transcriptomic studies and the annotation of genetic variants based on RNA-seq data. Mapping RNA-seq reads to the genome and transcriptome represent two distinct methods for quantifying the expression of genes/transcripts. Besides the known genes annotated in current databases, many novel genes/transcripts(especially those long noncoding RNAs) still can be identified on the reference genome using RNA-seq. Moreover, owing to the incompleteness of current reference genomes, some novel genes are missing from them. Genome-guided and de novo transcriptome reconstruction are two effective and complementary strategies for identifying those novel genes/transcripts on or beyond the reference genome. In addition, integrating the genes of distinct databases to conduct transcriptomics and genetics studies can improve the results of corresponding analyses.

De novo transcriptome assembly of RNA-Seq reads with different strategies

De novo transcriptome assembly is an important approach in RNA-Seq data analysis and it can help us to reconstruct the transcriptome and investigate gene expression profiles without reference genome sequences.We carried out transcriptome assemblies with two RNA-Seq datasets generated from human brain and cell line,respectively.We then determined an efficient way to yield an optimal overall assembly using three different strategies.We first assembled brain and cell line transcriptome using a single k-mer length.Next we tested a range of values of k-mer length and coverage cutoff in assembling.Lastly,we combined the assembled contigs from a range of k values to generate a final assembly.By comparing these assembly results,we found that using only one k-mer value for assembly is not enough to generate good assembly results,but combining the contigs from different k-mer values could yield longer contigs and greatly improve the overall assembly.

Applications of next-generation sequencing to the study of biological invasions

Through the widespread implementation of next-generation sequencing （NGS）, analyses of the whole genome （the entire DNA content） and the whole transcriptome （the genes being expressed） are becoming commonplace. NGS enables the analysis of a vast amount of previously unattainable genetic information. Despite this potential, NGS has yet to be widely imple- mented in genetic studies of biological invasions. The study of the genomic causes and consequences of biological invasions al- lows a deeper understanding of the molecular mechanisms underpinning the invasion process. In this review, we present a brief introduction to NGS followed by a synthesis of current research in the genomics and transcriptomics of adaptation and coloniza- tion. We then highlight research opportunities in the field, including： （1） assembling genomes and transcriptomes of non-model organisms, （2） identifying genomic regions and candidate genes underlying evolutionary processes, and （3） studying the adaptive role of gene expression variation. In particular, because introduced species face a broad range of physiological and biotic chal- lenges when colonizing novel and variable environments, transcriptomics will enable the study of gene regulatory pathways that may be responsible for acclimation or adaptation. To conclude, we identify a number of research approaches that will aid our fu- ture understanding of biological invasions

Comparative transcriptome analysis of the lichen-forming fungus Endocarpon pusillum elucidates its drought adaptation mechanisms

The lichen-forming fungus was isolated from the desert lichen Endocarpon pusillum that is extremely drought resistant.To understand the molecular mechanisms of drought resistance in the fungus,we employed RNA-seq and quantitative real-time PCR to compare and characterize the differentially expressed genes in pure culture at two different water levels and with that in desiccated lichen.The comparative transcriptome analysis indicated that a total of 1781 genes were differentially expressed between samples cultured under normal and PEG-induced drought stress conditions.Similar to those in drought resistance plants and non-lichenized fungi,the common drought-resistant mechanisms were differentially expressed in E.pusillum.However,the expression change of genes involved in osmotic regulation in E.pusillum is different,which might be the evidence for the feature of drought adaptation.Interestingly,different from other organisms,some genes involved in drought adaption mechanisms showed significantly different expression patterns between the presence and absence of drought stress in E.pusillum.The expression of 23 candidate stress responsive genes was further confirmed by quantitative real-time PCR using dehydrated E.pusillum lichen thalli.This study provides a valuable resource for future research on lichen-forming fungi and shall facilitate future functional studies of the specific genes related to drought resistance.

Transcriptome analysis reveals long noncoding RNAs involved in fiber development in cotton (Gossypium arboreum)

Long noncoding RNAs （lncRNAs） play important roles in various biological regulatory processes in yeast, mammals, and plants. However, no systematic identification of lncRNAs has been reported in Gossypium arboreum. In this study, the strand-specific RNA sequencing （ssRNA-seq） of samples from cotton fibers and leaves was performed, and lncRNAs involved in fiber initiation and elongation processes were systematically identified and analyzed. We identified 5,996 lncRNAs, of which 3,510 and 2,486 can be classified as long intergenic noncoding RNAs （lincRNAs） and natural antisense transcripts （IncNAT）, respectively. LincRNAs and lncNATs are similar in many aspects, but have some differences in exon number, exon length, and transcript length. Expression analysis revealed that 51.9% of lincRNAs and 54.5% of lncNATs transcripts were preferentially expressed at one stage of fiber development, and were significantly highly expressed than protein-coding tran- scripts （21.7%）. During the fiber and rapid elongation stages, rapid and dynamic changes in lncRNAs may contribute to fiber development in cotton. This work describes a set of lncRNAs that are involved in fiber development. The characterization and expression analysis of lncRNAs will facilitate future studies on their roles in fiber development in cotton.

Overview of available methods for diverse RNA-Seq data analyses

RNA-Seq technology is becoming widely used in various transcriptomics studies;however,analyzing and interpreting the RNA-Seq data face serious challenges.With the development of high-throughput sequencing technologies,the sequencing cost is dropping dramatically with the sequencing output increasing sharply.However,the sequencing reads are still short in length and contain various sequencing errors.Moreover,the intricate transcriptome is always more complicated than we expect.These challenges proffer the urgent need of efficient bioinformatics algorithms to effectively handle the large amount of transcriptome sequencing data and carry out diverse related studies.This review summarizes a number of frequently-used applications of transcriptome sequencing and their related analyzing strategies,including short read mapping,exon-exon splice junction detection,gene or isoform expression quantification,differential expression analysis and transcriptome reconstruction.

De novo assembly and comparative analysis of root transcriptomes from different varieties of Panax ginseng C. A. Meyer grown in different environments

Panax ginseng C. A. Meyer is an important traditional herb in eastern Asia. It contains ginsenosides, which are primary bioactive compounds with medicinal properties. Although ginseng has been cultivated since at least the Ming dynasty to increase production, cultivated ginseng has lower quantities of ginsenosides and lower disease resistance than ginseng grown under natural conditions. We extracted root RNA from six varieties of fifth-year P. ginseng cultivars representing four different growth conditions, and performed Illumina paired-end sequencing. In total, 163,165,706 raw reads were obtained and used to generate a de novo transcriptome that consisted of 151,763 contigs(76,336 unigenes), of which 100,648 contigs(66.3%) were successfully annotated. Differential expression analysis revealed that most differentially expressed genes(DEGs) were upregulated(246 out of 258, 95.3%) in ginseng grown under natural conditions compared with that grown under artificial conditions. These DEGs were enriched in gene ontology(GO) terms including response to stimuli and localization. In particular, some key ginsenoside biosynthesis-related genes, including HMG-Co A synthase(HMGS), mevalonate kinase(MVK), and squalene epoxidase(SE), were upregulated in wild-grown ginseng. Moreover, a high proportion of disease resistance-related genes were upregulated in wild-grown ginseng. This study is the first transcriptome analysis to compare wild-grown and cultivated ginseng, and identifies genes that may produce higher ginsenoside content and better disease resistance in the wild; these genes may have the potential to improve cultivated ginseng grown in artificial environments.

Transcriptome profile of human neuroblastoma cells in the hypomagnetic field

Research has shown that the hypomagnetic field(HMF)can affect embryo development,cell proliferation,learning and memory,and in vitro tubulin assembly.In the present study,we aimed to elucidate the molecular mechanism by which the HMF exerts its effect,by comparing the transcriptome profiles of human neuroblastoma cells exposed to either the HMF or the geomagnetic field.A total of 2464 differentially expressed genes(DEGs)were identified,216 of which were up-regulated and2248 of which were down-regulated after exposure to the HMF.These DEGs were found to be significantly clustered into several key processes,namely macromolecule localization,protein transport,RNA processing,and brain function.Seventeen DEGs were verified by real-time quantitative PCR,and the expression levels of nine of these DEGs were measured every 6 h.Most notably,MAPK1 and CRY2,showed significant up-and down-regulation,respectively,during the first 6 h of HMF exposure,which suggests involvement of the MAPK pathway and cryptochrome in the early bio-HMF response.Our results provide insights into the molecular mechanisms underlying the observed biological effects of the HMF.

Transcriptome changes in Polygonum multiflorum Thunb. roots induced by methyl jasmonate

Transcriptome profiling has been widely used to analyze transcdptomic variation in plants subjected to abiotic or biotic stresses. Although gene expression changes induced by methyl jasmonate （MeJA） have been profiled in several plant species, no information is available on the MeJA-triggered transcriptome response of Polygonum multiflorum Thunb., a species with highly valuable medicinal properties. In this study, we used transcriptome profiling to investigate transcdptome changes in roots of P. mu/tiflorum seedlings subjected to a 0.25 mmol/L-MeJA rootirrigation treatment. A total of 18 677 differentially expressed genes （DEGs） were induced by MeJA treatment, of which 4535 were up-regulated and 14 142 were down-ragulated compared with controls. These DEGs were associated with 125 metabolic pathways. In addition to various common primary and secondary metabolic pathways, several sec- ondary metabolic pathways related to components with significant pharmacological effects were enriched by MeJA, including arachidonic acid metabolism, linoleic acid metabolism, and stilbenoid biosynthesis. The MeJA-induced transcdptome changes uncovered in this study provide a solid foundation for future study of functional genes controlling effective components in secondary metabolic pathways of P. multiflorum.

Identification and analysis of mouse non-coding RNA using transcriptome data

Transcripts are expressed spatially and temporally and they are very complicated, precise and specific; however, most studies are focused on protein-coding related genes. Recently, massively parallel c DNA sequencing(RNA-seq) has emerged to be a new and promising tool for transcriptome research, and numbers of non-coding RNAs, especially linc RNAs, have been widely identified and well characterized as important regulators of diverse biological processes. In this study, we used ultra-deep RNA-seq data from 15 mouse tissues to study the diversity and dynamic of non-coding RNAs in mouse. Using our own criteria, we identified totally 16,249 non-coding genes(21,569 non-coding RNAs) in mouse. We annotated these non-coding RNAs by diverse properties and found non-coding RNAs are generally shorter, have fewer exons, express in lower level and are more strikingly tissue-specific compared with protein-coding genes. Moreover, these non-coding RNAs show significant enrichment with transcriptional initiation and elongation signals including histone modifications(H3K4me3, H3K27me3 and H3K36me3), RNAPII binding sites and CAGE tags. The gene set enrichment analysis(GSEA) result revealed several sets of linc RNAs associated with diverse biological processes such as immune effector process, muscle development and sexual reproduction. Taken together, this study provides a more comprehensive annotation of mouse non-coding RNAs and gives an opportunity for future functional and evolutionary study of mouse non-coding RNAs.

Transcriptional and translational responses of rapeseed leaves to red and blue lights at the rosette stage

Under different red （R）：blue （B） photon flux ratios, the growth performance of rapeseed （Brassica napus L.） is significantly different. Rapeseed under high R ratios shows shade response, while under high B ratios it shows sun-type morphology. Rapeseed under monochromatic red or blue light is seriously stressed. Transcriptomic and proteomic methods were used to analyze the metabolic pathway change of rapeseed （cv. ＂Zhongshuang 11＂） leaves under different R：B photon flux ratios （including 100R：0B%, 75R：25B%, 25R：75B%, and 0R：100B%）, based on digital gene expression （DGE） and two-dimensional gel electrophoresis （2-DE）. For DGE analysis, 2054 differentially expressed transcripts （llog2（fold change）l〉1, q〈0.005） were detected among the treatments. High R ratios （100R：0B% and 75R：25B%） enhanced the expression of cellular structural components, mainly the cell wall and cell membrane. These components participated in plant epidermis development and anatomical structure morphogenesis. This might be related to the shade response induced by red light. High B ratios （25R：75B% and 0R：100B%） promoted the expression of chloroplast-related components, which might be involved in the formation of sun-type chloroplast induced by blue light. For 2-DE analysis, 37 protein spots showed more than a 2-fold difference in expression among the treatments. Monochromatic light （ML; 100R：0B% and OR： 100B%） stimulated accumulation of proteins associated with antioxidation, photosystem II （PSII）, DNA and ribosome repairs, while compound light （CL; 75R：25B% and 25R：75B%） accelerated accumulation of proteins associated with carbohydrate, nucleic acid, amino acid, vitamin, and xanthophyll metabolisms. These findings can be useful in understanding the response mechanisms of rapeseed leaves to different R：B photon flux ratios.