miRNA对褐飞虱鞘脂质代谢基因表达的影响及沉默NlSPT1和Nl SMase4的small RNA分析

【背景】鞘脂质是第二大类膜脂,作为信号转导物质参与细胞生长发育、繁殖及凋亡等过程。鞘脂质代谢酶调控鞘脂质代谢以维持生物体内代谢的稳态。【目的】以重要水稻害虫褐飞虱(Nilaparvata lugens)为研究对象,通过RNA干扰(RNA interference,RNAi)技术检测微小RNA(microRNA,miRNA)生物合成通路核心组分NlAgo1、NlDicer1和NlDrosha沉默后褐飞虱鞘脂质代谢通路相关基因的相对转录水平,结合small RNA测序分析沉默丝氨酸棕榈酰转移酶1(serine palmitoyltransferase 1,SPT1)和鞘磷脂酶4(sphingomyelinase 4,SMase4)基因的差异miRNA,探究miRNA在褐飞虱鞘脂质代谢中的作用,为害虫防治提供新的分子靶标。【方法】利用RNAi技术,分别对羽化后第1天(1 PAE,post adult eclosion)的雌成虫NlAgo1、NlDicer1和NlDrosha进行dsRNA注射,以ds GFP为对照;分别解剖羽化后第5天的卵巢组织,以β-actin作为内参基因,采用实时荧光定量PCR(qRT-PCR)方法检测NlAgo1、NlDicer1和NlDrosha沉默后鞘脂质代谢通路相关基因的表达量变化;根据已有的小RNA文库联合miRNA-靶基因预测软件对可能调控NlSPT1和NlSMase4表达的miRNA进行预测;通过small RNA测序技术对沉默NlSPT1和NlSMase4的差异miRNA进行鉴定和靶基因富集性分析。【结果】与对照组相比,沉默NlAgo1、NlDicer1或NlDrosha显著上调卵巢中NlSPT1和NlSMase4等鞘脂质代谢通路相关基因的表达;靶基因预测结果显示,有6条miRNA能与NlSPT1结合,13条miRNA能与NlSMase4结合;沉默NlSPT1和NlSMase4的差异miRNA的靶基因显著富集在细胞核和蛋白质结合等生物学过程以及内吞作用、内质网加工、MAPK信号通路、TOR信号通路、凋亡、脂质代谢等代谢通路。【结论】NlAgo1、NlDicer1和NlDrosha依赖性的miRNA通过影响鞘脂质代谢相关基因的表达影响鞘脂质代谢。NlSPT1和NlSMase4沉默引起褐飞虱卵巢miRNA表达水平的改变。研究结果可为基于鞘脂质代谢基因为靶标的害虫防治提供理论依据。

Species-specific regulatory pathways of small RNAs play sophisticated roles in flower development in Dimocarpus longan Lour.

Flower development plays vital role in horticultural plants.Post-transcriptional regulation via small RNAs is important for plant flower development.To uncover post-transcriptional regulatory networks during the flower development in Dimocarpus longan Lour.‘Shixia’,an economically important fruit crop in subtropical regions,we collected and analyzed sRNA deep-sequencing datasets and degradome libraries Apart from identifying miRNAs and phased siRNA generating loci(PHAS loci),120 hairpin loci,producing abundant sRNAs,were identified by in-house protocols.Our results suggested that 56 miRNA-target pairs,2221-nt-PHAS loci,and 111 hairpin loci are involved in posttranscriptional gene silencing during longan reproductive development.Lineage-specific or species-specific post-transcriptional regulatory modules have been unveiled,including miR482-PHAS and miRN15.miR482-PHAS might be involved in longan flower development beyond their conserved roles in plant defense,and miRN15 is a novel miRNA likely associated with a hairpin locus(HPL-056)to regulate strigolactone receptor gene DWARF14(D14)and the biogenesis of phasiRNAs from D14.These small RNAs are enriched in flower buds,suggesting they are likely involved in post-transcriptional regulatory networks essential for longan flower development via the strigolactone signaling pathway.

用于苹果树病毒鉴定的small RNA深度测序数据集

病毒病是为害苹果的重要致病因子,开展病毒种类鉴定是防控苹果病毒病的首要任务。由于苹果长期通过嫁接的方式进行繁育,导致树体感染多种病毒,加之苹果病毒的浓度较低,序列变异较大,致使苹果病毒种类的鉴定尤为困难。此外,现有的分子检测技术主要为聚合酶链式反应和分子杂交技术,这些技术基于已知病毒核苷酸序列为基础开展鉴定,而对于未知病毒,由于其核苷酸序列尚未测定,则无法采用这些分子技术进行鉴定。针对这一瓶颈,该文提供了一个自然生长的苹果树的small RNA (sRNA)深度测序的数据集,通过结合生物信息学方法对原始数据进行过滤、sRNA组装及Blast比对,可准确地分析和判断自然条件下病毒的种类,为苹果病毒的鉴定带来了新的突破,同时也为苹果无毒苗木的判定提供技术支撑。

Advances in Small RNAs and Sexual Reproduction in Plants

Small RNAs are non-coding RNA molecules with 20-30 nucleotides （nt） in length that mainly play regulatory roles in gene expression at the post-transcription level by directly cutting target mRNA or inhibiting its translation. Small RNAs play regulatory roles in the growth and development process of plants at the core of gene regulatory networks, which has been widely studied and confirmed in sporophyte generation of plants. However, few researches have been conducted on small RNAs and gametophyte generation. It is reported that small RNAs play important roles in floral organ development, gametogenesis, fertilization, and early zygotic development of plants. In addition, various small RNAs also play roles in controlling genetic integrity, cell differentiation and functions during the sexual reproduction process of plants. However, most of the specific functions of small RNAs in the sexual reproduction process are unknown yet. This study mainly aimed to introduce small RNAs in plants, summarize the latest advances in researches of small RNAs and plant sexual reproduction, and make prospect on its future.

乳腺癌耐药细胞株外泌体中small RNA表达分析

目的:分析乳腺癌细胞株和乳腺癌耐药细胞株外泌体中small RNA的表达并比较其差异。方法:采用NextSeq CN500测序仪检测乳腺癌细胞株和乳腺癌耐药细胞株外泌体中small RNA的表达,DEGseq R软件包分析各small RNA表达差异。结果:与乳腺癌细胞株外泌体相比,乳腺癌耐药细胞株外泌体中960种miRNAs表达上调,29种miRNAs表达下调;44种piRNAs表达上调,60种piRNAs表达下调;93种tsRNAs表达上调,3种tsRNAs表达下调。结论:乳腺癌耐药细胞株外泌体与乳腺癌细胞外泌体间small RNA存在表达差异,可能是与乳腺癌细胞耐药有关。

不同MHC基因型哈萨克绵羊人工感染包虫病后小肠组织Small RNA的Solexa测序及分析

前期研究发现哈萨克绵羊的单倍型MHC-DRB1基因单倍型MvaⅠbc-SacⅡab-Hin1Ⅰab与细粒棘球蚴病(包虫病)抗性密切相关,但其作用机制尚不明确。为了研究其中可能存在的重要分子免疫调控机制,试验采用PCR-RFLP方法选择抗病和非抗病哈萨克绵羊,人工感染包虫病一定时间后分别取小肠组织进行Solexa深度测序及生物信息学分析。结果表明:试验组、对照组、空白对照组分别获得高质量reads 14 962 858,13 706 679,16 075 005条,其中长度22 nt的reads所占比例最多。联立分析发现各组之间公有序列分别占8.32%、10.98%和9.65%。miRNA分别为4 269,3 670,3 779条;KEGG分析发现差异表达miRNA主要涉及代谢途径、肿瘤通路、表皮细胞细菌入侵等通路。说明这些途径中可能存在与宿主抗病性和易感性密切相关的调控机制。

水稻干旱胁迫的small RNA转录组分析

small RNA在植物环境胁迫过程中起重要的调节作用。然而,参与干旱逆境响应复杂过程中的miRNAs及其调控机制仍有待于深入挖掘。对干旱胁迫处理不同时间的水稻根、茎和叶组织进行small RNA文库构建和转录组测序。所有样品共检测到403个miRNAs,其中已知miRNAs 352个,新预测miRNAs 51个。干旱处理后24 h的根、茎和叶中分别有37、35和56个差异表达miRNAs,而96 h的根、茎和叶中分别有35、62和65个差异表达miRNAs。15个miRNAs在干旱处理后两个时间点的根、茎和叶中均差异表达,其中,Osa-miR159f、Osa-miR164f、Osa-miR5082和Osa-miR5493受干旱诱导上调表达,是干旱响应中关键调控因子。预测到miRNAs靶基因4537个,并进行了功能注释和富集分析。这些结果为鉴定干旱响应miRNAs并解析其参与抗旱的分子调控机理提供了理论和数据资源。

Genome-wide interrogation of transfer RNA-derived small RNAs in a mouse model of traumatic brain injury

Transfer RNA(t RNA)-derived small RNAs(ts RNAs) are a recently established family of regulatory small non-coding RNAs that modulate diverse biological processes. Growing evidence indicates that ts RNAs are involved in neurological disorders and play a role in the pathogenesis of neurodegenerative disease. However, whether ts RNAs are involved in traumatic brain injury-induced secondary injury remains poorly understood. In this study, a mouse controlled cortical impact model of traumatic brain injury was established, and integrated ts RNA and messenger RNA(m RNA) transcriptome sequencing were used. The results revealed that 103 ts RNAs were differentially expressed in the mouse model of traumatic brain injury at 72 hours, of which 56 ts RNAs were upregulated and 47 ts RNAs were downregulated. Based on micro RNA-like seed matching and Pearson correlation analysis, 57 differentially expressed ts RNA-m RNA interaction pairs were identified, including 29 ts RNAs and 26 m RNAs. Moreover, Gene Ontology annotation of target genes revealed that the significantly enriched terms were primarily associated with inflammation and synaptic function. Collectively, our findings suggest that ts RNAs may be associated with traumatic brain injury-induced secondary brain injury, and are thus a potential therapeutic target for traumatic brain injury. The study was approved by the Beijing Neurosurgical Institute Animal Care and Use Committee(approval No. 20190411) on April 11, 2019.

Small RNA deep sequencing reveals the presence of multiple viral infections in cucurbit crops in Guangdong,China

Viral diseases are among the most critical damaging factors that impose a global threat to the cucurbit industry.China is the world’s leading country for the production and consumption of cucurbits.Guangdong,a province in southern China dominated by the tropical and subtropical climate,favors the survival of different plant viruses and their vectors.Five main cucurbit crops showing various disease symptoms were surveyed and collected to identify viruses infecting cucurbits in Guangdong during 2018–2020.In the field,the incidence ranged from 5-30%,or even 60-100% in the case of severely infected cucurbits.A total of 357 symptomatic samples were collected and subsequently screened for cucurbit viruses by small RNA deep sequencing and assembly(sRSA).Seventeen virus species belonging to 10 genera were identified in the five main cucurbit crops.The most common viruses were papaya ringspot virus(PRSV;Potyvirus),zucchini tigre mosaic virus(ZTMV;Potyvirus),zucchini yellow mosaic virus(ZYMV;Potyvirus),and watermelon silver mottle virus(WSMoV;Orthotospovirus),with infection rates of 24.4,19.0,17.1,and 14.3%,respectively.Notably,the most prevalent viruses were melon yellow spot orthotospovirus(MYSV)in cucumber,PRSV in squash,cucumber green mottle mosaic virus(CGMMV;Tobamovirus)in bottle gourd,WSMoV in white gourd,and ZYMV in luffa.Mixed infections were prevalent,and the types of mixed infections varied substantially in different cucurbit crops.Moreover,the full-length nucleotide sequences of watermelon green mottle mosaic virus(WGMMV),CGMMV,and watermelon virus A(WVA;Wamavirus)identified in bottle gourd were cloned and analyzed.This study is the first reporting WGMMV infecting bottle gourd in China mainland.In summary,the results demonstrate that in Guangdong,the most prevalent viruses belong to potyviruses,orthotospoviruses,and tobamoviruses groups.The findings will facilitate agricultural researchers and farmers to plan and implement effective disease control strategies aiming at timely detection and management of cucurbit-infecting viral pathogens.

Small RNA deep sequencing reveals full-length genome of Citrus yellow vein clearing virus in Chongqing,China

To identity the potential pathogen associated with the yellow vein clearing symptom on lemon trees, the profiles of virus-de- rived small interfering RNAs from citrus samples were obtained and analyzed by deep sequencing method in this study. Twenty-seven contigs almost cover the full length genome of Citrus yellow vein clearing virus （CYVCV） isolate YN were obtained using the small RNA deep sequencing technology. Analysis showed that this isolate CQ shared the highest nucleotide identity with isolate Y1 （JX040635） and YN （KP313242）, both of which belong to the genus Mandarivirus in the family Alphaflexiviridae. Mapping analysis of viral-derived siRNA （vsiRNA） profiles revealed an uneven distribution pattern of their generations along both positive and negative genome strands, and 22- and 21-nt vsiRNAs ranked the majority. BLAST against viroids and other viral databases confirmed that this sample was single-infected only by CYVCV, which indicated that CYVCV was the exact causal agent for the yellow clearing symptom occurring on lemon. This is the first CYVCV isolate detected in Chongqing and the second in China. This result could provide a molecular basis for the investigation of citrus viral diseases to protect citrus health in this region.

Role of nickel-regulated small RNA in modulation of Helicobacter pylori virulence factors

Helicobacter pylori(H.pylori)is a Gram-negative bacterium that infects about half of the world's population.H.pylori infection prevails by several mechanisms of adaptation of the bacteria and by its virulence factors including the cytotoxin associated antigen A(CagA).CagA is an oncoprotein that is the protagonist of gastric carcinogenesis associated with prolonged H.pylori infection.In this sense,small regulatory RNAs(sRNAs)are important macromolecules capable of inhibiting and activating gene expression.This function allows sRNAs to act in adjusting to unstable environmental conditions and in responding to cellular stresses in bacterial infections.Recent discoveries have shown that nickelregulated small RNA(NikS)is a post-transcriptional regulator of virulence properties of H.pylori,including the oncoprotein CagA.Notably,high concentrations of nickel cause the reduction of NikS expression and consequently this increases the levels of CagA.In addition,NikS expression appears to be lower in clinical isolates from patients with gastric cancer when compared to patients without.With that in mind,this minireview approaches,in an accessible way,the most important and current aspects about the role of NikS in the control of virulence factors of H.pylori and the potential clinical repercussions of this modulation.

Dynamics and Mechanism of A Quorum Sensing Network Regulated by Small RNAs in Vibrio Harveyi

Bacterial quorum sensing （QS） has attracted much interests and it is an important process of cell communication. Recently, Bassler et al. studied the phenomena of QS regulated by small RNAs and the experimental data showed that smafl RNAs played important role in the QS of Vibrio harveyi and it can permit the fine-tuning of gene regulation and mmntenance of homeostasis. According to Michaelis-Menten kinetics and mass action law in this paper, we construct a mathematical model to investigate the mechanism induced QS by coexist of small RNA and signal molecular （AI） and show that there are periodic oscillation when the time delay and Hill coefficient exceed a critical value and the periodic oscillation produces the change of concentration and induces QS. These results are fit to the experimental results. In the meanwhile, we also get some theoretical value of Hopf Bifurcation on time deday. In addition, we also find this network is robust against noise.

High-Level Accumulation of Exogenous Small RNAs Not Affecting Endogenous Small RNA Biogenesis and Function in Plants

RNA silencing is a fundamental plant defence and gene control mechanism in plants that are directed by 20-24 nucleotide （nt） small interfering RNA （siRNA） and microRNA （miRNA）. Infection of plants with viral pathogens or transformation of plants with RNA interference （RNAi） constructs is usually associated with high levels of exogenous siRNAs, but it is unclear if these siRNAs interfere with endogenous small RNA pathways and hence affect plant development. Here we provide evidence that viral satellite RNA （satRNA） infection does not affect siRNA and miRNA biogenesis or plant growth despite the extremely high level of satRNA-derived siRNAs. We generated transgenic Nicotiana benthamiana plants that no longer develop the specific yellowing symptoms generally associated with infection by Cucumber mosaic virus （CMV） Y-satellite RNA （Y-Sat）. We then used these plants to show that CMV Y-Sat infection did not cause any visible phenotypic changes in comparison to uninfected plants, despite the presence of high-level Y-Sat siRNAs. Furthermore, we showed that the accumulation of hairpin RNA （hpRNA）-derived siRNAs or miRNAs, and the level of siRNA-directed transgene silencing, are not significantly affected by CMV Y-Sat infection. Taken together, our results suggest that the high levels of exogenous siRNAs associated with viral infection or RNAi-inducing transgenes do not saturate the endogenous RNA silencing machineries and have no significant impact on normal plant development.

Epstein-Barr virus-encoded small RNAs in idiopathic orbital inflammatory pseudotumor tissues: a comparative case series

AIM： To investigate the positive rate and types of cells that express Epstein-Barr virus-encoded small RNAs （EBERs） and to determine the distribution of EBER-expressing cells in idiopathic orbital inflammatory pseudotumor （IOIP） tissues. METHODS： We retrospectively examined 40 archived paraffin specimens from two teaching hospitals in Southern China between January 2007 and January 2015 that were pathologically determined to exhibit IOIP. Eleven concurrent paraffin specimens of thyroid-associated ophthalmopathy （TAO） composed the control group. In situ hybridization was performed to detect EBERs. Immunohistochemistry was employed to detect CD3, CD20, Vimentin, and smooth muscle actin （SMA）, and the positive rate, types of positive cells, and distribution and location of EBERs were evaluated. RESULTS： The positive expression rate of EBERs was 47.5% （19/40） in the IOIP group, which was significantly higher than that in the TAO group [0 （0/11）, P=-0.011]. in the IOIP group, the lymphocyte infiltrative subtype, fibrotic subtype, and mixed subtype exhibited EBER-positive rates of 57.1% （12121）, 12.5% （118）, and 54.5% （6/11）, respectively, and no significant differences were found between these subtypes （P=0.085）. Positive signals of EBERs were mainly present in medium-small lymphocytes between or around follicles and in the nuclei of activated immunoblasts （14/19）. CONCLUSION： The positive rate, types, and distribution of EBER-expressing cells in IOIP have been documented.These findings are conducive for a better understanding of the underlying mechanisms of Epstein-Barr virus infection in IOIP pathogenesis.

Coherence resonance in a noise-driven gene network regulated by small RNA

Small RNA has recently drawn more and more attention. In this paper, we concentrate on the influence of noises on gene network regulated by small RNA using chemical Langevin equation. It shows that the noise can cause oscillation when the oscillate does not occur in the corresponding deterministic system. The coherence of the noise induced oscillation reaches a maximum for an optimal intensity of noise, and the coherence resonance appears accordingly. The findings imply probably omnipresent importance of noise in the functioning process of living organism.

Global Annotation of Small RNA and MicroRNA Mature Sequences from Developing Ovules of Gossypium hirsutum L.

The involvement of small RNAs in cotton fiber development is under explored.The objective of this work was to directly clone,annotate,and analyze small RNAs of developing ovules to reveal

Characterization and function of Tomato yellow leaf curl virus-derived small RNAs generated in tolerant and susceptible tomato varieties

supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China （20134320120013）;the Natural Science Foundation of Hunan Province, China （14JJ3095）

Exosome-based small RNA delivery:Progress and prospects

RNA interfering(RNAi), mediated by small interfering RNAs and microRNAs, is currently one of the most promising tools of gene therapy. Small RNAs are capable of inducing specific post-transcriptional gene silencing, providing a potentially effective platform for the treatment of a wide array of diseases. However, similar to other nucleic acid-based drugs,the major hurdle of RNAi therapy is lack of efficient and non-immunogenic delivery vehicles. Currently, viruses, synthetic polymers, and lipid-based carriers are among the most widely studied vehicles for small RNA delivery. However, many drawbacks are reported to be associated with these delivery vehicles. There is a pressing need to replace them with more efficient and better-tolerated approaches. Exosomes secreted from the endocytic compartment of live cells, are a subtype of endogenous extracellular vesicles that transfer genetic and biochemical information among different cells, thus playing an important role in cellcell communication. Recently, accumulating attention has been focused on harnessing exosomes as nanaocarriers for small RNAs delivery. Due to their natural role in shuttling endogenous nucleic acid in our body, exosomes may exhibit higher delivery efficiency, lower immunogenicity, and better compatibility than existing foreign RNA carriers. Importantly,exosomes own intrinsic homing capacity that can guide small RNAs across natural membranous barriers. Moreover, such a capacity can be further improved by adding appropriate targeting moieties. In this manuscript, we briefly review the progress and challenges of RNAi therapy, and discuss the potential of exosomes' applications in small RNA delivery with focus on the most recent advances in exosome-based small RNA delivery for disease therapy.

拟南芥六个新的small RNAs的克隆和功能预测

以模式植物拟南芥为例,建立了一种克隆small RNA分子的技术平台,为今后开展small RNA分子的生物学功能研究提供技术支撑。通过用抗病信号分子水杨酸(SA)处理拟南芥叶片后,进行small RNA分子群体的分离与接头连接、PCR扩增、T-载体克隆与检测、测序分析和生物信息学分析等一系列实验,成功地克隆了一些small RNAs,并对其表达和功能进行了分析。

Pathological significance of tRNA-derived small RNAs in neurological disorders

Non-coding RNAs(ncRNAs) are a type of RNA that is not translated into proteins. Transfer RNAs(tRNAs), a type of ncRNA, are the second most abundant type of RNA in cells. Recent studies have shown that tRNAs can be cleaved into a heterogeneous population of ncRNAs with lengths of 18–40 nucleotides, known as tRNA-derived small RNAs(tsRNAs). There are two main types of tsRNA, based on their length and the number of cleavage sites that they contain: tRNA-derived fragments and tRNA-derived stress-induced RNAs. These RNA species were first considered to be byproducts of tRNA random cleavage. However, mounting evidence has demonstrated their critical functional roles as regulatory factors in the pathophysiological processes of various diseases, including neurological diseases. However, the underlying mechanisms by which tsRNAs affect specific cellular processes are largely unknown. Therefore, this study comprehensively summarizes the following points:(1) The biogenetics of tsRNA, including their discovery, classification, formation, and the roles of key enzymes.(2) The main biological functions of tsRNA, including its miRNA-like roles in gene expression regulation, protein translation regulation, regulation of various cellular activities, immune mediation, and response to stress.(3) The potential mechanisms of pathophysiological changes in neurological diseases that are regulated by tsRNA, including neurodegeneration and neurotrauma.(4) The identification of the functional diversity of tsRNA may provide valuable information regarding the physiological and pathophysiological mechanisms of neurological disorders, thus providing a new reference for the clinical treatment of neurological diseases. Research into tsRNAs in neurological diseases also has the following challenges: potential function and mechanism studies, how to accurately quantify expression, and the exact relationship between tsRNA and miRNA. These challenges require future research efforts.