piRNA的生物学功能

非编码小RNA（non-coding RNA, ncRNA）主要有siRNA（small interfering RNA）、miRNA(microRNA)和piRNA (piwi-interacting RNA)三类,其中piRNA是近年来新发现的一类小RNA分子,特异性地同Argonuat蛋白家族中的Piwi亚家族蛋白结合,主要在生殖细胞系中表达,对维持生殖系DNA完整、抑制转座子转录、抑制翻译、参与异染色质的形成、执行表观遗传调控和生殖细胞发生等均有重要作用．piRNA基因几乎遍布于整个基因组,但呈高度不连续性分布,大部分定位于20～90 kb的染色体基因簇上．与来自于双链RNA的siRNA和发卡结构miRNA不同之处是piRNA来自长单链RNA前体,或者是两股非重叠的反向转录前体,其生成与Dicer无关．作为调节RNA（riboregulator）,piRNA和miRNA可能在动物起源早期就已经出现了,帮助生命进入了一个多细胞动物的时代,产生了今天的生物体复杂性和多样性．piRNA成为ncRNA的研究热点,进展飞快,有很多综述及时介绍piRNA的研究进展,本文结合siRNA、miRNA的特点介绍了关于piRNA的形成机制和作用的最新研究成果．     

小RNA分子与精子发生调控

近来研究发现小RNA(small RNAs)可作为转录后及翻译水平上基因表达调节的重要调节因子,利用小RNA来阐明调节精子发生的分子机制取得了显著进展。这些小RNA主要分为3类,即小干扰RNA(siRNA)、微小RNA(miRNA)以及与piwi蛋白相互作用的RNA(piRNA)。在减数分裂和精子发生过程中,小RNA具有多种生物学功能,如利用siRNA体外转染或体内注射来敲低特定基因从而研究该基因在精子发生过程中的作用;miRNA可能参与精子发生中有丝、减数及后减数分裂阶段的基因表达调节;piRNA主要参与调节雄性生殖细胞减数及后减数分裂的过程,在精子发生中起抑制反转录转座子(retrotransposons)的作用。文章对小RNAs合成、作用机制、功能及展望等最新进展进行了综述。     

非编码RNA与基因表达调控

近年来,随着对基因组的深入研究,发现真核生物中存在许多形态和功能各异的非编码RNA分子,这类RNA分子并不表达蛋白质,但它们在基因转录水平、转录后水平及翻译水平起了重要的调控作用。具有调控作用的RNA分子种类非常丰富,如长链非编码RNA(long non-coding RNA,lncRNA)、miRNA、PIWI相互作用RNA(PIWI-interacting RNA,piRNA)、内源性小干扰RNA(endogenous small interfering RNA,endo-siRNA)、竞争性内源RNA(competitive endogenous RNA,ceRNA)等,它们使基因表达过程更为丰富、严谨和有序。本文综述几类典型的非编码RNA对基因表达的调节作用,以助于理解细胞中RNA分子调节网络的功能和机制。     

小RNA的组成和功能——三类内源小RNA的研究概况

生物体中存在许多非编码的小RNA,可通过与靶mRNA完全或不完全的碱基互补配对,致使mRNA断裂或翻译抑制,从而达到调节基因表达的目的。这些小RNA组成了细胞中高度复杂的RNA调控网络,在生物的各项生理活动、生长发育过程中发挥着十分重要的作用。内源小RNA主要包括miRNA、siRNA和piRNA三类,概述这3种小RNA的生物发生,作用机制及功能等方面的研究进展。     

小RNA分子研究进展

在过去很长的一段时间内,RNA始终没有得到科学家足够的重视,直到非编码性小RNA（non—coding small RNA）的出现,使得研究者对RNA功能的传统观念发生了惊人的改变。生命体内存在着大量的miRNA、siRNA与piRNA,它们在不同水平上调节着基因的表达,影响生命活动。另外一些较长的非编码RNA广泛地参与到细胞内不同的生化过程中。发挥重要作用。随着小分子RNA的发现至今,RNA的研究领域进入炙手可热的状态,RNAi的机制在一步步被完善与深化．更多新类型的小分子RNA被发现。在应用方面,siRNA已经成为分子生物学实验中一种简单而有效的基因沉默工具,并且在人类疾病治疗方面初有成效。     

第三类小RNA和生殖细胞发育

果蝇中重复相关小干扰RNA(rasiRNA)和哺乳动物中Piwi相互作用RNA(piRNA)是最近发现的大小在30个核苷酸左右的小RNA,它们与已经发现的22个核苷酸左右的短干扰RNA(siRNA)和微小RNA(miRNA)有明显区别,因此命名为第三类小RNA。第三类小RNA可以与Piwi形成基因沉默复合体,并可能采取与经典RNA干扰不同的方式而影响特定基因的表达。目前这类小RNA主要在生殖细胞及干细胞中发现,尤其对生殖细胞中生理功能的全面研究,可能对RNA干扰现象有一个更为全面的理解。     

基因表达调控多面手——microRNA和siRNA的作用机制

miRNA(microRNA)是一类广泛存在于高等真核细胞中的长度约为21个碱基的小分子非编码RNA,参与调控三分之一以上基因的表达,并与多种人类疾病存在重要关联。而siRNA(small interfering RNA)是RNA干扰(RNA interference,RNAi)中的效应分子,其结构和作用机制与miRNA存在许多类似之处。由于miRNA和siRNA具有重要的生物学功能。因此,对它们作用机制的理解具有非常重要的理论意义和应用指导价值。该综述将对它们作用机制的研究进展做一总结和回顾。     

真核生物中的微小RNA及其功能研究进展

真核生物中存在两种主要的非编码RNA(non-coding RNA),在真核生物中发挥重要作用。一类为微小RNA(microRNA,miRNA),另一为小干扰RNA(siRNA)。miRNA大小为19～25nt,在体内与蛋白质形成核糖核蛋白复合体(miRNP),在真核基因的表达调控,生长发育中起重要作用。siRNA在RNA干扰(RNA地 interference,RNAi)途径中起定位特异mRNA的作用。miRNA与siRNA有联系也有区别。miRNA在真核生物中的调控机制具有保守性。     

油菜油份积累及胚胎发育相关small RNA的研究进展

越来越多的证据表明miRNA与siRNA这些小分子RNA在植物的生长发育和种子形成等过程中起着重要的调控作用。但目前在油料作物油菜中关于小分子RNA的研究还很少,尤其关于miRNA与siRNA在油菜胚胎发育过程和种子油份积累中表达与功能的研究更少。     

线虫prg-1基因对小RNA表达的影响

以秀丽隐杆线虫为材料发现,prg-1基因突变不仅影响piRNA的表达,还影响部分miRNA的表达,同时还发现ncRNA-like型小RNA,对ncRNA-like序列比较,认为ncRNA-like与piRNA或miRNA序列极为相似;对ncRNA-like与miRNA或piRNA的基因座比较,发现ncRNA-like与miRNA或piRNA基因座完全相同．推测这些ncRNA-like型小RNA可能与miRNA或piRNA有着相同的RNA前体来源．     

Small noncoding RNAs in the germline

Small noncoding RNAs have emerged as potent regulators of gene expression, especially in the germline. We review the biogenesis and regulatory function of three major small noncoding RNA pathways in the germline: The small interfering RNA (siRNA) pathway that leads to the degradation of target mRNAs, the microRNA (miRNA) pathway that mostly represses the translation of target mRNAs, and the newly discovered Piwi-interacting RNA (piRNA) pathway that appears to have diverse functions in epigenetic programming, transposon silencing, and the regulation of mRNA translation and stability. The siRNA and miRNA pathways are present in the germline as well as many somatic tissues, whereas the piRNA pathway is predominantly confined to the germline. Investigation of the three small RNA pathways has started to reveal a new dimension of gene regulation with defining roles in germline specification and development.     

一类新的基因沉默小RNA-piRNA（piwi-interacting RNA）

piRNA是单链非编码小分子RNA,长度约26-31nt,大部分集中在29-30nt,5’端具有尿嘧啶偏向性（约86％）,能够与Argonaute蛋白家族中的Piwi亚家族蛋白相互结合而产生作用。piRNA的功能主要是维持基因组中转座子的正常沉默状态,以防止基因组中转座子爆发而引起相应基因的改变。piRNA与siRNA及miRNA均是近些年发现的非编码小RNA,它们均可通过一套相应的机制进行RNA干扰,在转录、转录后甚至翻译水平对靶基因及蛋白进行调节,它们之间既有联系又有区别。piRNA数据库的建立将对这类小分子RNA的研究有很大的促进作用。     

Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot

The northern blot, or RNA gel blot, is a widely used method for the discovery, validation and expression analysis of small regulatory RNA such as small interfering RNA (siRNA), microRNA (miRNA) and piwi-interacting RNA (piRNA). Although it is straightforward and quantitative, the main disadvantage of a northern blot is that it detects such RNA less sensitively than most other approaches. We found that the standard dose of UV used in northern blots was not the most efficient at immobilizing small RNA of 20–40nt on nylon membranes. However, increasing the dose of UV reduced the detection of miRNA by hybridization in northern blotting experiments. We discovered that using the soluble carbodiimide, EDC, to cross-link RNA to nylon membranes greatly improved the detection of small RNA by hybridization. Compared to standard UV cross-linking procedures, EDC cross-linking provided a 25–50-fold increase in the sensitivity of detection of siRNA from plants and miRNA or piRNA from mammalian cells. All types of hybridization probes tested benefited from the new cross-linking procedure. Cross-linking was dependent on a terminal phosphate and so, should be applicable to other related categories of small RNA.     

Molecular functions of small regulatory noncoding RNA

Recently, using large-scale genomic sequencing, a great number of small noncoding RNAs (ncRNA) has been discovered. Short ncRNAs can be classified into three major classes — small interfering RNA (siRNA), microRNA (miRNA), and piwi-interacting RNA (piRNA). These short ncRNAs ranging from 20 to 300 nt in size are now recognized as a new paradigm of gene regulation for controlling many biological processes. In this paper, we review the biogenesis and recent research on the functions of small regulatory non-coding RNAs and aim at understanding their important functions in living organisms.     

Argonaute:The executor of small RNA function

The discovery ot small non-coding RNAs- microRNA(miRNA),short interfering RNA(siRNA) and PIWIinteracting RNA(piRNA)- represents one of the most exciting frontiers in biology specifically on the mechanism of gene regulation.In order to execute their functions,these small RNAs require physical interactions with their protein partners,the Argonaute(AGO) family proteins.Over the years,numerous studies have made tremendous progress on understanding the roles of AGO in gene silencing in various organisms.In this review,we summarize recent progress of AGO-mediated gene silencing and other cellular processes in which AGO proteins have been implicated with a particular focus on progress made in flies,humans and other model organisms as compliment.     

Lesser known relatives of miRNA

Since the discovery of RNAi (RNA interference) major attention has focused on studying miRNA (microRNA) and siRNA (small interfering RNA). However, within the last few years, several other small ncRNAs (non-coding RNAs) have been discovered and thus various newer acronyms representing these ‘other’ classes of small ncRNAs have populated the literature. Of these, piRNA (Piwi-interacting RNA) has been gaining importance because of its role as the guardian of the germline genome. Some of the other newly discovered small ncRNAs have been mostly reported in plants, and they are yet to be studied more comprehensively. Nevertheless, piRNA and the ‘other’ small ncRNAs deserve some discussion because they are members of the increasingly large family of small ncRNAs.     

Functional analysis of the RNAi response in ovary-derived silkmoth Bm5 cells

Experiments of dsRNA-mediated gene silencing in lepidopteran insects in vivo are characterized by high variability although lepidopteran cell cultures have shown an efficient response to RNAi in transfection experiments. In order to identify the core RNAi factors that regulate the RNAi response of Lepidoptera, we employed the silkmoth ovary-derived Bm5 cells as a test system since this cell line is known to respond potently in silencing after dsRNA transfection. Two parallel approaches were used; involving knock-down of the core RNAi genes or over-expression of the main siRNA pathway factors, in order to study possible inhibition or stimulation of the RNAi silencing response, respectively. Components from all three main small RNA pathways (BmAgo-1 for miRNA, BmAgo-2/BmDcr-2 for siRNA, and BmAgo-3 for piRNA) were found to be involved in the RNAi response that is triggered by dsRNA. Since BmAgo-3, a factor in the piRNA pathway that functions independent of Dicer in Drosophila, was identified as a limiting factor in the RNAi response, sense and antisense ssRNA was also tested to induce gene silencing but proved to be ineffective, suggesting a dsRNA-dependent role for BmAgo-3 in Bombyx mori. After efficient over-expression of the main siRNA factors, immunofluorescence staining revealed a predominant cytoplasmic localization in Bm5 cells. This is the first study in Lepidoptera to provide evidence for possible overlapping of all three known small RNA pathways in the regulation of the dsRNA-mediated silencing response using transfected B. mori-derived Bm5 cells as experimental system.     

siRNA与miRNA在生物基因调控中的沉默机制比较

siRNA和miRNA的沉默机制是生物基因调控的重要手段之一. 小干扰RNA（small interfering RNA,siRNA）是RNA干扰的引发物,激发与之互补的目标mRNA沉默. 非编码RNA中的微小RNA（microRNA,miRNA）,能够识别特定的目标mRNA,通过与mRNAs的3′ 非翻译区结合,影响该目标蛋白的翻译水平. siRNA和miRNA的基因调控机制对生物学研究及疾病的病因和治疗等有直接影响. 本文主要对siRNAs和miRNAs的生物起源及沉默机制进行比较性论述：提出Dicers酶蛋白、Ago蛋白以及20 nt~25 nt的双链RNAs的 3类大分子是RNA沉默的特征结构,并进行了说明性论述|总结性叙述了siRNA和miRNA的2类小分子经典沉默机制,并提出其异同点. 最后,本文根据近期研究进展,对siRNA和miRNA的生物起源及沉默机制提出了新的疑问.