Hamster PIWI proteins bind to piRNAs with stage-specific size variations during oocyte maturation

In animal gonads, transposable elements are actively repressed to preserve genome integrity through the PIWI-interacting RNA (piRNA) pathway. In mice, piRNAs are abundantly expressed in male germ cells, and form effector complexes with three distinct PIWIs. The depletion of individual Piwi genes causes male-specific sterility with no discernible phenotype in female mice. Unlike mice, most other mammals have four PIWI genes, some of which are expressed in the ovary. Here, purification of PIWI complexes from oocytes of the golden hamster revealed that the size of the PIWIL1-associated piRNAs changed during oocyte maturation. In contrast, PIWIL3, an ovary-specific PIWI in most mammals, associates with short piRNAs only in metaphase II oocytes, which coincides with intense phosphorylation of the protein. An improved high-quality genome assembly and annotation revealed that PIWIL1- and PIWIL3-associated piRNAs appear to share the 5′-ends of common piRNA precursors and are mostly derived from unannotated sequences with a diminished contribution from TE-derived sequences, most of which correspond to endogenous retroviruses. Our findings show the complex and dynamic nature of biogenesis of piRNAs in hamster oocytes, and together with the new genome sequence generated, serve as the foundation for developing useful models to study the piRNA pathway in mammalian oocytes.     

Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity

RNA silencing regulates gene expression through mRNA degradation, translation repression and chromatin remodelling. The fundamental engines of RNA silencing are RISC and RITS complexes, whose common components are 21-25 nt RNA and an Argonaute protein containing a PIWI domain of unknown function. The crystal structure of an archaeal Piwi protein (AfPiwi) is organised into two domains, one resembling the sugar-binding portion of the lac repressor and another with similarity to RNase H. Invariant residues and a coordinated metal ion lie in a pocket that surrounds the conserved C-terminus of the protein, defining a key functional region in the PIWI domain. Furthermore, two Asp residues, conserved in the majority of Argonaute sequences, align spatially with the catalytic Asp residues of RNase H-like catalytic sites, suggesting that in eukaryotic Argonaute proteins the RNase H-like domain may possess nuclease activity. The conserved region around the C-terminus of the PIWI domain, which is required for small interfering RNA (siRNA) binding to AfPiwi, may function as the receptor site for the obligatory 5' phosphate of siRNAs, thereby specifying the cleavage position of the target mRNA.     

piRNA/PIWI功能调控与精子发生

piRNAs(PIWI-interacting RNAs)是一类与PIWI相互作用的小非编码RNAs(small noncoding RNAs, sncRNAs),其长度介于24~32 nt,特异性地在动物生殖腺细胞中表达。近来研究表明piRNA/PIWI系统在动物生殖腺细胞的基因组转座元件沉默及转录后调控mRNAs方面具有重要功能。最近,中国科学院上海生物化学与细胞生物学研究所刘默芳课题组的一项研究表明,在人和小鼠的精子发生过程中,PIWI (鼠源同源蛋白MIWI、人源同源蛋白HIWI)的严格代谢调控至关重要。以此为契机,本文综述了piRNA/PIWI在哺乳动物(主要是小鼠和人)精子发生过程中调控功能的研究进展。     

Mice deficient for a small cluster of Piwi-interacting RNAs implicate Piwi-interacting RNAs in transposon control

The mammalian testis expresses a class of small noncoding RNAs that interact with mammalian PIWI proteins. In mice, the PIWI-interacting RNAs (piRNAs) partner with mammalian PIWI proteins, PIWIL1 and PIWIL2, also known as MIWI and MILI, to maintain transposon silencing in the germline genome. Here, we demonstrate that inactivation of Nct1/2, two noncoding RNAs encoding piRNAs, leads to derepression of LINE-1 (L1) but does not affect mouse viability, spermatogenesis, testicular gene expression, or fertility. These findings indicate that piRNAs from a cluster on chromosome 2 are necessary to maintain transposon silencing.     

piRNA和PIWI蛋白的功能机制研究进展

piRNA是2006年7月在动物生殖细胞中发现的一类新小分子非编码RNA。piRNA特异地与PIWI家族蛋白相互作用,因此,被命名为PIWI-interacting RNA,简称piRNA。这类长度在26～32核苷酸的小分子非编码RNA代表了一个生殖细胞转座子沉默的独特小RNA通路。它们可能通过与PIWI家族蛋白质相互作用,在表观遗传学水平和转录后水平沉默转座子等基因组自私性遗传元件,参与生殖干细胞自我维持和分化命运决定、减数分裂、精子形成等生殖相关事件。在piRNA发现后短短数年的时间,对其生物发生、功能及作用机制的研究都取得了诸多重大突破。该文就piRNA研究的最新研究进展作一简述。     

RNA干扰敲减PIWIL1表达对人结肠癌细胞SW620增殖，粘附及侵袭能力影响

PIWIL1为AGO蛋白(Argonaute proteins )PIWI亚家族的成员之一,在睾丸中特异表达. 其在干细胞自我更新、RNA干扰和翻译调节中起着重要的作用.本文采用实时PCR方法检测PIWI基因家族中PIWIL1、PIWIL2、PIWIL3、PIWIL4在人结肠癌SW620细胞中mRNA表达水平,首次证实了在SW620细胞中,PIWIL1相对PIWIL2、PIWIL3、PIWIL4,其表达水平最高（P﹤0.05）.构建表达针对PIWIL1的小发卡结构干扰RNA的重组质粒,并用Western 印迹验证其达到较高的干扰效率.用脂质体将重组质粒转染入SW620细胞中,通过MTT实验、集落形成实验、细胞聚集实验及细胞侵袭转移实验,分别观察到其可抑制SW620细胞的生长、增殖、侵袭转移能力,增强了SW620细胞的粘附能力.提示PIWIL1可能在结肠癌发生、发展过程中发挥作用.     

解读AGO蛋白结构及其功能

RNA沉默是由小RNA特异向导和RNA诱导的沉默复合物（RISC）切割或者抑制靶标mRNA翻译的一种调控系统. 作为RISC的核心成分,AGO蛋白(argonaute proteins)由N末端、PAZ、MID和PIWI 4个结构域组成. PAZ区能非序列特异性识别结合双链小RNA 3′末端悬垂的2个核苷酸,MID与PIWI界面处的“保守口袋”识别结合小RNA 5′端第1位核苷酸,PIWI区具有切割mRNA的催化中心. 根据系统进化学分析,AGO蛋白家族分为3个组：AGO like、PIWI-like和GROUP3. 拟南芥共编码10种AGO蛋白.目前已经证实,具有切割活性的为AtAGO1、AtAGO4和AtAGO7,三者参与的小RNA通路也已得到确认. 在拟南芥10种AGO蛋白中,AtAGO1与AtAGO10、AtAGO1与AtAGO7、AtAGO4与AtAGO6存在功能上的部分冗余.     

PIWI/piRNA“机器”与雄性生殖细胞发育

PIWI(P-element-induced wimpy testis)蛋白在动物生殖系细胞中特异性表达,为动物生殖细胞发育分化所必需。piRNA(PIWI-interacting RNAs)是最近在动物生殖系细胞中发现的一类非编码小分子RNA,这类小RNA特异性地与PIWI家族蛋白相互作用。PIWI/piRNA"机器"通过沉默转座元件和调控编码mRNA等方式在动物生殖细胞发育分化过程中发挥重要作用。本文围绕PIWI/piRNA"机器"的生物学功能及分子机制,对近期取得的相关研究进展进行了系统性总结。     

Epigenetic Disruption of the PIWI Pathway in Human Spermatogenic Disorders

Epigenetic changes are involved in a wide range of common human diseases. Although DNA methylation defects are known to be associated with male infertility in mice, their impact on human deficiency of sperm production has yet to be determined. We have assessed the global genomic DNA methylation profiles in human infertile male patients with spermatogenic disorders by using the Infinium Human Methylation27 BeadChip. Three populations were studied: conserved spermatogenesis, spermatogenic failure due to germ cell maturation defects, and Sertoli cell-only syndrome samples. A disease-associated DNA methylation profile, characterized by targeting members of the PIWI-associated RNA (piRNA) processing machinery, was obtained. Bisulfite genomic sequencing and pyrosequencing in a large cohort (n = 46) of samples validated the altered DNA methylation patterns observed in piRNA-processing genes. In particular, male infertility was associated with the promoter hypermethylation-associated silencing of PIWIL2 and TDRD1. The downstream effects mediated by the epigenetic inactivation of the PIWI pathway genes were a defective production of piRNAs and a hypomethylation of the LINE-1 repetitive sequence in the affected patients. Overall, our data suggest that DNA methylation, at least that affecting PIWIL2/TDRD1, has a role in the control of gene expression in spermatogenesis and its imbalance contributes to an unsuccessful germ cell development that might explain a group of male infertility disorders.     

Multifunctionality of PIWI proteins in control of germline stem cell fate

PIWI proteins interacting with specific type of small RNAs (piRNAs) repress transposable elements in animals. Besides, they have been shown to participate in various cellular processes: in the regulation of heterochromatin formation including telomere structures, in the control of translation and the cell cycle, and in DNA rearrangements. PIWI proteins were first identified by their roles in the self-renewal of germline stem cells. PIWI protein functions are not limited to gonadogenesis, but the role in determining the fate of stem cells is their specific feature conserved throughout the evolution of animals. Molecular mechanisms underlying these processes are far from being understood. This review focuses on the role of PIWI proteins in the control of maintenance and proliferation of germinal stem cells and its relation to the known function of PIWI in transposon repression.     

The Bombyx ovary-derived cell line endogenously expresses PIWI/PIWI-interacting RNA complexes

Genetic studies and large-scale sequencing experiments have revealed that the PIWI subfamily proteins and PIWI-interacting RNAs (piRNAs) play an important role in germ line development and transposon control. Biochemical studies in vitro have greatly contributed to the understanding of small interfering RNA (siRNA) and microRNA (miRNA) pathways. However, in vitro analyses of the piRNA pathway have been thus far quite challenging, because their expression is largely restricted to the germ line. Here we report that Bombyx mori ovary-derived cultured cell line, BmN4, endogenously expresses two PIWI subfamily proteins, silkworm Piwi (Siwi) and Ago3 (BmAgo3), and piRNAs associated with them. Siwi-bound piRNAs have a strong bias for uridine at their 5′ end and BmAgo3-bound piRNAs are enriched for adenine at position 10. In addition, Siwi preferentially binds antisense piRNAs, whereas BmAgo3 binds sense piRNAs. Moreover, we identified many pairs in which Siwi-bound antisense and BmAgo3-bound sense piRNAs are overlapped by precisely 10 nt at their 5′ ends. These signatures are known to be important for secondary piRNA biogenesis in other organisms. Taken together, BmN4 is a unique cell line in which both primary and secondary steps of piRNA biogenesis pathways are active. This cell line would provide useful tools for analysis of piRNA biogenesis and function.     

人X染色体长臂(Xq)和短臂(Xp)基因组学比较分析

X染色体发生X染色体失活 ,但是Xp基因有 30 %表现为逃逸 ,而Xq仅不到 3%。为了研究X染色体基因失活和表达逃逸发生和维持的分子机制 ,比较了Xq和XpDNA序列的RNA模拟结合强度。X染色体的核苷酸序列被分为 5 0kb一段 ,对每一段DNA做 7碱基 (7nt)字符串组合分析 (共有 4 7=16 384种组合 ) ,记录每段 5 0kbDNA中每种 7nt字符串的频率。选择生发中心B细胞中的 12 0个高表达基因 ,计算这些基因的内含子 7nt字符串的出现频率 ,称为intron 7nt,以此作为RNAs(RNA群 ,模拟细胞中RNA在小片段的总和 )。已知一段DNA序列的 7nt频率值和intron 7nt,即可以计算该DNA段与intron 7nt的结合强度。每段 5 0kbDNA与intron 7nt的结合强度取决于该DNA段与intron 7nt互补核苷酸的频率 ,互补的核苷酸序列越多 ,结合强度就越大。DNA段与intron 7nt的模拟结合强度称为RNA结合强度 ,试图模拟该段DNA可以结合的RNA小片段的总量。之所以采用 7nt字符串组合分析是考虑到连续 7个核苷酸互补则可以形成相对稳定的结合。研究发现 :1)Xp各DNA段的RNA结合强度均值显著大于Xq (P <0 0 0 1) ;2 )Xp上高结合RNA的DNA段数目显著高于Xq (P <0 0 0 1) ;3)RNA高结合DNA段形成的簇与X染色体基因表达逃逸区关联。有证据表明 ,RNA可以通过改变染色质     

The regulatory function of piRNA/PIWI complex in cancer and other human diseases: The role of DNA methylation

Piwi-interacting RNAs (piRNAs) are a class of short chain noncoding RNAs that are constituted by 26-30 nucleotides (nt) and can couple with PIWI protein family. piRNAs were initially described in germline cells and are believed to be critical regulators of the maintenance of reproductive line. Increasing evidence has extended our perspectives on the biological significance of piRNAs and indicated that they could still affect somatic gene expression through DNA methylation, chromatin modification and transposon silencing, etc. Many studies have revealed that the dysregulation of piRNAs might contribute to diverse diseases through epigenetic changes represented by DNA methylation and chromatin modification. In this review, we summarized piRNA/PIWI protein-mediated DNA methylation regulation mechanisms and methylation changes caused by piRNA/PIWI proteins in different diseases, especially cancers. Since DNA methylation and inhibitory chromatin marks represented by histone H3 lysine 9 (H3K9) methylation frequently cooperate to silence genomic regions, we also included methylation in chromatin modification within this discussion. Furthermore, we discussed the potential clinical applications of piRNAs as a new type promising biomarkers for cancer diagnosis, as well as the significance of piRNA/PIWI protein-associated methylation changes in treatment, providing disparate insights into the potential applications of them.