Role of alternative polyadenylation in epigenetic silencing and antisilencing

Epigenetic marks such as DNA methylation and histone modifications are widely involved in regulating different aspects of developmental and environmental responses (1). Meanwhile, DNA methylation and histone modification are also used constitutively to silence transposable elements and repeat elements (TREs) (2). Such TRE-mediated silencing should necessarily be limited to the intended targets only and not spread to adjacent genes and their regulatory elements. Higher eukaryotic organisms have evolved antisilencing mechanisms to keep the balance between silencing and antisilencing that is required for precise gene expression regulation.Research in the authors' laboratory is supported by US National Science Foundation Grant IOS–0817829 (to Q.Q.L.) and US National Institutes of Health Grant 1R15GM94732-1 A1 (to Q.Q.L.)

Genome-Wide Determination of Poly(A) Sites in Medicago Truncatula: Evolutionary Conservation of Alternative Poly(A) Site Choice

BACKGROUND: Alternative polyadenylation (APA) plays an important role in the post-transcriptional regulation of gene expression. Little is known about how APA sites may evolve in homologous genes in different plant species. To this end, comparative studies of APA sites in different organisms are needed. In this study, a collection of poly(A) sites in Medicago truncatula, a model system for legume plants, has been generated and compared with APA sites in Arabidopsis thaliana. RESULTS: The poly(A) tags from a deep-sequencing protocol were mapped to the annotated M. truncatula genome, and the identified poly(A) sites used to update the annotations of 14,203 genes. The results show that 64% of M. truncatula genes possess more than one poly(A) site, comparable to the percentages reported for Arabidopsis and rice. In addition, the poly(A) signals associated with M. truncatula genes were similar to those seen in Arabidopsis and other plants. The 3\u27-UTR lengths are correlated in pairs of orthologous genes between M. truncatula and Arabidopsis. Very little conservation of intronic poly(A) sites was found between Arabidopsis and M. truncatula, which suggests that such sites are likely to be species-specific in plants. In contrast, there is a greater conservation of CDS-localized poly(A) sites in these two species. A sizeable number of M. truncatula antisense poly(A) sites were found. A high percentage of the associated target genes possess Arabidopsis orthologs that are also associated with antisense sites. This is suggestive of important roles for antisense regulation of these target genes. CONCLUSIONS: Our results reveal some distinct patterns of sense and antisense poly(A) sites in Arabidopsis and M. truncatula. In so doing, this study lends insight into general evolutionary trends of alternative polyadenylation in plants

Genome-wide characterization of intergenic polyadenylation sites redefines gene spaces in Arabidopsis thaliana

Background:Messenger RNA polyadenylation is an essential step for the maturation of most eukaryotic mRNAs.Accurate determination of poly(A) sites helps define the 3’-ends of genes, which is important for genome annotation and gene function research. Genomic studies have revealed the presence of poly(A) sites in intergenic regions, which may be attributed to 3’-UTR extensions and novel transcript units. However, there is no systematically evaluation of intergenic poly(A) sites in plants. Results:Approximately 16,000 intergenic poly(A) site clusters (IPAC) in Arabidopsis thaliana were discovered and evaluated at the whole genome level. Based on the distributions of distance from IPACs to nearby sense and antisense genes, these IPACs were classified into three categories. About 70 % of them were from previously unannotated 3’-UTR extensions to known genes, which would extend 6985 transcripts of TAIR10 genome annotation beyond their 3’-ends, with a mean extension of 134 nucleotides. 1317 IPACs were originated from novel intergenic transcripts, 37 of which were likely to be associated with protein coding transcripts. 2957 IPACs corresponded to antisense transcripts for genes on the reverse strand, which might affect 2265 protein coding genes and 39 non-protein-coding genes, including long non-coding RNA genes. The rest of IPACs could be originated from transcriptional read-through or gene mis-annotations. Conclusions:The identified IPACs corresponding to novel transcripts, 3’-UTR extensions, and antisense transcription should be incorporated into current Arabidopsis genome annotation. Comprehensive characterization of IPACs from this study provides insights of alternative polyadenylation and antisense transcription in plants.Funding supports were in part from US National Science Foundation (No. 1541737 to QQL), the Hundred Talent Plans of Fujian Province and Xiamen City (to QQL). This project was also funded by the National Natural Science Foundation of China (Nos. 61201358 and 61174161), the Natural Science Foundation of Fujian Province of China (No. 2012J01154), and the specialized Research Fund for the Doctoral Program of Higher Education of China (Nos. 20120121120038 and 20130121130004), and the Fundamental Research Funds for the Central Universities in China (Xiamen University: Nos. 2013121025, 201412G009, and 2014X0234)

Role of FY in polyadenylation

APA是存在于真核生物中一种重要的基因表达调控机制，其关键步骤是mRNA前体的核苷酸序列的识别，直接决定了成熟mRNA转录本的序列。本项目研究人员全面探讨了FY不同结构域在poly(A)位点使用中的作用，同时阐明FY与AtCPSF30在polyA信号识别过程中的关系。进一步研究发现FY的WD40结构域的突变与PPLPP结构域的缺失对3’UTR区间polyA位点选择和信号使用有着相反的影响。该论文揭示了拟南芥开花因子FY在选择性多聚腺苷化（Alternative PolyAdenylation, APA）过程中的功能，为植物复杂的多聚腺苷化（polyA）信号识别机制提供遗传学证据，完善了植物polyA过程的分子机制，同时为深入理解通过APA调控植物生长发育和植物对环境响应的分子机制奠定基础。A crucial step for mRNA polyadenylation is poly(A) signal recognition by trans-acting factors. The mammalian cleavage and polyadenylation specificity factor (CPSF) complex components CPSF30 and WDR33 recognize the canonical AAUAAA signal for efficient polyadenylation. In Arabidopsis thaliana, the flowering time regulator FY is the homologue of WDR33. However, its role in mRNA polyadenylation is poorly understood. Using poly(A) tag sequencing, we found that over 50% of alternative polyadenylation (APA) events are altered in fy single mutants or double mutants with Atcpsf30, but mutation of the FY WD40-repeat has a stronger effect than deletion of the plant-unique PPLPP domain. fy mutations disrupt AAUAAA or AAUAAA-like poly(A) signal recognition.Notably, A-rich signal usage is suppressed in the WD40-repeat mutation, but promoted in Pro-Pro-Leu-Pro-Pro (PPLPP)-domain deficiency. However, fy mutations do not aggravate the alteration of signal usage in the Atcpsf30 null mutant. Furthermore, the WD40-repeat mutation shows a preference for 3'UTR shortening, but the PPLPP-domain deficiency shows a preference for lengthening. Interestingly, the WD40-repeat mutant exhibits shortened primary roots and late flowering with alteration of APA of related genes.Importantly, the long transcripts of two APA genes affected in fy are related to abiotic stress responses. These results reveal a conserved and specific role of FY in mRNA polyadenylation.This work was supported in part by a grant from the National Key R&D Project of China (2016YFE0108800), a grant from the U.S. NSF (IOS-154173), both to QQL, and grants (2017M620274, 2018T110649) from China Postdoctoral Science Foundation to JL

scDAPA: detection and visualization of dynamic alternative polyadenylation from single cell RNA-seq data

Motivation: Alternative polyadenylation (APA) plays a key post-transcriptional regulatory role in mRNA stability and functions in eukaryotes. Single cell RNA-seq (scRNA-seq) is a powerful tool to discover cellular heterogeneity at gene expression level. Given 30 enriched strategy in library construction, the most commonly used scRNA-seq protocol—10 Genomics enables us to improve the study resolution of APA to the single cell level. However, currently there is no computational tool available for investigating APA profiles from scRNA-seq data. Results: Here, we present a package scDAPA for detecting and visualizing dynamic APA from scRNA-seq data. Taking bam/sam files and cell cluster labels as inputs, scDAPA detects APA dynamics using a histogram-based method and the Wilcoxon rank-sum test, and visualizes candidate genes with dynamic APA. Benchmarking results demonstrated that scDAPA can effectively identify genes with dynamic APA among different cell groups from scRNA-seq data.This research was supported in part by the Fundamental Research Funds for the Central Universities in China [Xiamen University: 20720170076 and 20720190106], and the National Natural Science Foundation of China [61802323, 31801268 and 61573296]

洱海流域水生态分区

分区边界的确定是生态分区的重要步骤,但目前多数水生态分区的边界确定以定性分析、专家判断为主。本研究以洱海流域为例,建立了一套两级分区体系。该体系基于GIS技术,用子流域作为分区基本单元,并用相关分析法,定量筛选一、二级分区指标。其中,一级分区指标为高程、坡度和植被归一化指数(NDVI),二级分区指标为农田百分比和城镇百分比。通过指标图层的叠加和重分类,合并同质性子流域,从而将洱海流域划分为5个一级区和9个二级区。藻类群落分布的验证结果表明分区合理。本研究将定量分析和子流域边界应用于水生态分区,使分区边界的确定更科学,在实际管理中更具有可操作性。本研究结果为水生态分区研究提供了新的方法,为洱海流域水生态管理提供了基本管理单元

改良分子信标-实时PCR快速检测产单核李斯特菌

目的:建立改良分子信标-实时PCR检测产单核李斯特菌(LMO)的快速方法,应用于食品中LMO的污染状况调查及食物中毒快速诊断。方法:根据GenBank公布的LMO hlyA基因的保守序列,设计引物和改良分子信标探针,建立改良分子信标-实时PCR检测体系,应用于食品中LMO检测。结果:改良分子信标-实时PCR反应体系DNA灵敏度为110 fg,菌液灵敏度为99 cfu/m l或4 cfu/PCR反应体系,无交叉反应。以此反应体系检测28株LMO,均出现特异的荧光信号。上述方法可将检测时间由原来的至少4 d缩短至1 d。对228份食品进行LMO检测,8份增菌液LMO实时荧光PCR阳性,其中6份LMO细菌培养阳性。结论:改良分子信标-实时PCR反应体系快速、灵敏度高,特异性强,能提高LMO的检出率和准确性,可应用于LMO食品污染状况调查及食物中毒的快速诊断

食品污染产单核李斯特菌不同检测方法学评估

目的评价以不同方法检测食品中污染产单核李斯特菌(LMO)效果,并对其检测方法进行优化。方法同时用国标法、改良国标法、改良分子信标-实时荧光PCR法、免疫磁性分离法对深圳市2004～2006年食品样品进行LMO分离鉴定并进行比较。结果228份样品中8份增菌液LMO实时荧光PCR阳性,其中6份LMO细菌培养阳性。国标法、改良国标法,改良分子信标-实时荧光PCR法、免疫磁性分离法都能检出LMO,检出率分别为2.63%、2.63%、3.50%、2.63%。深圳市食品中LMO的污染率为2.63%(国标法),速食类冻品污染率为12.8%(国标法)。结论荧光PCR法检出率最高,将检测时间由原来的至少4～5d缩短至1d,可用于食品污染LMO状况调查的初筛和食物中毒的快速诊断;国标法与改良国标法的检出率相等,而后者的操作更方便。免疫磁性分离法的检出率最低

改良分子信标-实时PCR快速检测志贺菌

目的:建立改良分子信标-实时PCR检测志贺菌的快速方法,应用于志贺菌食物中毒的快速诊断和门诊肠道致病菌的检测。方法:根据GenBank公布志贺菌ipaH基因的保守序列,设计引物和改良分子信标探针,建立实时PCR-改良分子信标检测体系,应用于对志贺菌食物中毒的快速诊断和门诊肠道致病菌的检测。结果:改良分子信标-实时PCR反应体系DNA灵敏度为93 fgμ/l,菌液灵敏度为64 cfu/m l或2 cfu/PCR反应体系,无交叉反应。此反应体系检测67株志贺菌,均出现特异的荧光信号。对细菌性食物中毒样本等共657份样品进行志贺菌检测,42份志贺菌实时PCR阳性,其中41份志贺菌细菌培养阳性。从样品处理到检测结果仅需2 h~1 d时间。结论:改良分子信标-实时PCR检测体系快速、灵敏度高,特异性强,可用于志贺菌食物中毒的快速诊断,为食源性疾病的分子流行病学调查提供新的检测手段,对于提高肠道门诊的工作效率有重要意义

Progress in the studies of vivipary in mangrove plants

【中文摘要】植物胎生是指有性繁殖产生的后代在母体上直接萌发的现象, 在红树植物中最为常见。红树植物生长在热带亚热带海岸潮间带, 耐受高盐、高温、淹水缺氧和海浪冲击等复杂环境。胎生被认为是红树植物对这种特殊生境的重要适应方式。 该文从形态发育、生理生化、分子水平、生态适应4个层次讨论红树植物胎生现象对复杂生境的适应性, 并指出现有研究存 在的不足, 对将来的研究方向进行了展望。与非胎生胚胎发育相比, 红树植物胎生是一个遗传的程序, 在进化过程中形成了 一些特殊的结构。植物激素对胎生发育起关键的调控作用, 繁殖体发育过程中, 其盐离子的种类与浓度的动态变化则是对海 岸潮间带生境的重要适应特征。这种胎生繁殖体依靠在母体上完善的一系列功能性特征能更有效地适应落地后的滩涂环境。 然而, 红树植物胎生发育过程的分子机理及调控机制还有待研究。理解胎生这一特殊适应性现象的本质及其进化过程将为红 树林保护繁育、适应气候变化提供理论依据。 【Abstract】Vivipary in plants refers to a phenomenon that sexually reproduced offsprings germinate while still attached to the maternal bodies. This is mostly manifested in mangrove plants, which occur in tropical and subtropical intertidal zones and encounter harsh environmental conditions such as high salinity, high temperatures, waterlogging, hypoxia and tidal waves. Vivipary has long been recognized as one of the most important adaptive features under such a complex environment. Here we discuss four aspects of vivipary: morphological anatomy, physiology and biochemistry, molecular biology and ecological adaptation. We also discuss shortcomings in current studies and prospect of future directions. Differing from regular seed development, viviparous seeds in mangroves are evolved with many special structures, indicating a genetically based process. Hormones play an important role in regulating the process, whilst the dynamics of salt ion concentration during embryo and propagule development seems to be an adaptive feature. The ecological significance of vivipary is fully exhibited in the propagules that can effectively establish themselves on muddy tidal zones. Such a success heavily relies on sound functional features developed on the mother plants. However, the molecular mechanism and the regulation of viviparous seed development in mangroves remain elusive. Systematic studies of vivipary in mangroves not only help to understand the nature and evolutionary process of this distinct adaptive phenomenon, but also provide the foundation for mangrove forest restoration and protection in many parts of the world.福建省对外合作项目(2016I0013