piRNA biogenesis during adult spermatogenesis in mice is independent of the ping-pong mechanism

piRNAs, a class of small non-coding RNAs associated with PIWI proteins, have broad functions in germline development, transposon silencing, and epigenetic regulation. In diverse organisms, a subset of piRNAs derived from repeat sequences are produced via the interplay between two PIWI proteins. This mechanism, termed “ping-pong” cycle, operates among the PIWI proteins of the primordial mouse testis; however, its involvement in postnatal testes remains elusive. Here we show that adult testicular piRNAs are produced independent of the ping-pong mechanism. We identified and characterized large populations of piRNAs in the adult and postnatal developing testes associated with MILI and MIWI, the only PIWI proteins detectable in these testes. No interaction between MILI and MIWI or sequence feature for the ping-pong mechanism among their piRNAs was detected in the adult testis. The majority of MILI- and MIWI-associated piRNAs originate from the same DNA strands within the same loci. Both populations of piRNAs are biased for 5′ Uracil but not for Adenine on the 10th nucleotide position, and display no complementarity. Furthermore, in Miwi mutants, MILI-associated piRNAs are not downregulated, but instead upregulated. These results indicate that the adult testicular piRNAs are predominantly, if not exclusively, produced by a primary processing mechanism instead of the ping-pong mechanism. In this primary pathway, biogenesis of MILI- and MIWI-associated piRNAs may compete for the same precursors; the types of piRNAs produced tend to be non-selectively dictated by the available precursors in the cell; and precursors with introns tend to be spliced before processed into piRNAs.     

MIWI/piRNA激活小鼠精子细胞mRNA翻译的新功能机制研究

哺乳动物减数分裂后期的精子发生(spermatogenesis),即精子形成(spermiogenesis),是一个剧烈的细胞形态变化过程。伴随精子细胞中细胞核压缩和染色质重构,基因转录活性将逐渐降低直至完全停止,那些为精子细胞后期阶段发育所需的基因都需要提前转录为信使核糖核酸(mRNA),然后以翻译抑制状态储存在精子细胞中,直到特定发育阶段再被激活翻译,以合成蛋白质发挥作用。这个现象被称为“转录–翻译解偶联”,是精子发生中基因表达调控的一个典型特征。然而,目前对于精子细胞中被抑制的mRNA是如何被翻译激活的还知之甚少。我们当前的这项研究发现,MIWI/piRNA通过与翻译起始因子eIF3f、RNA结合蛋白HuR等因子形成功能性翻译激活复合物,特异性地激活小鼠精子细胞中包含AU序列富含元件(AU-rich element,ARE)mRNA的翻译。此项研究揭示了PIWI/piRNA在精子细胞翻译激活中的新功能,并证明此功能为精子细胞发育和功能性精子生成所必需。     

The small RNA content of human sperm reveals pseudogene-derived piRNAs complementary to protein-coding genes

At the end of mammalian sperm development, sperm cells expel most of their cytoplasm and dispose of the majority of their RNA. Yet, hundreds of RNA molecules remain in mature sperm. The biological significance of the vast majority of these molecules is unclear. To better understand the processes that generate sperm small RNAs and what roles they may have, we sequenced and characterized the small RNA content of sperm samples from two human fertile individuals. We detected 182 microRNAs, some of which are highly abundant. The most abundant microRNA in sperm is miR-1246 with predicted targets among sperm-specific genes. The most abundant class of small noncoding RNAs in sperm are PIWI-interacting RNAs (piRNAs). Surprisingly, we found that human sperm cells contain piRNAs processed from pseudogenes. Clusters of piRNAs from human testes contain pseudogenes transcribed in the antisense strand and processed into small RNAs. Several human protein-coding genes contain antisense predicted targets of pseudogene-derived piRNAs in the male germline and these piRNAs are still found in mature sperm. Our study provides the most extensive data set and annotation of human sperm small RNAs to date and is a resource for further functional studies on the roles of sperm small RNAs. In addition, we propose that some of the pseudogene-derived human piRNAs may regulate expression of their parent gene in the male germline.     

新型非编码小RNA——Piwi-interacting RNA(piRNA)

piRNA(Piwi-interacting RNA)是最近从哺乳动物睾丸组织中发现的一类能与PIWI蛋白质相互作用,且长度分布在26～31nt的新型小分子单链RNA,主要综述piRNA的相关研究进展.     

piRNA——一种新型的小分子RNA

小分子RNA已成为全球生物学研究关注的热点之一.最近,在哺乳动物的生殖细胞中发现了一种新型的小分子RNA：piRNA,比以往发现的小分子RNA稍长些,大小在30个核苷酸左右,piRNA必须与哺乳动物的Piwi蛋白结合才能发挥作用.目前对piRNA的研究还处于初始阶段,但鉴于piRNA主要分布在包括人类等数种动物体睾丸的精原细胞内,科学家们推测这类小分子RNA可能与动物体内精子的发育和维持的功能相关.     

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。     

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。     

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。     

A comprehensive analysis of piRNAs from adult human testis and their relationship with genes and mobile elements

Background

Piwi-interacting RNAs (piRNAs) are a recently discovered class of small non-coding RNAs whose best-understood function is to repress mobile element (ME) activity in animal germline. To date, nearly all piRNA studies have been conducted in model organisms and little is known about piRNA diversity, target specificity and biological function in human.

Results

Here we performed high-throughput sequencing of piRNAs from three human adult testis samples. We found that more than 81% of the ~17 million putative piRNAs mapped to ~6,000 piRNA-producing genomic clusters using a relaxed definition of clusters. A set of human protein-coding genes produces a relatively large amount of putative piRNAs from their 3’UTRs, and are significantly enriched for certain biological processes, suggestive of non-random sampling by the piRNA biogenesis machinery. Up to 16% of putative piRNAs mapped to a few hundred annotated long non-coding RNA (lncRNA) genes, suggesting that some lncRNA genes can act as piRNA precursors. Among major ME families, young families of LTR and endogenous retroviruses have a greater association with putative piRNAs than other MEs. In addition, piRNAs preferentially mapped to specific regions in the consensus sequences of several ME (sub)families and some piRNA mapping peaks showed patterns consistent with the “ping-pong” cycle of piRNA targeting and amplification.

Conclusions

Overall our data provide a comprehensive analysis and improved annotation of human piRNAs in adult human testes and shed new light into the relationship of piRNAs with protein-coding genes, lncRNAs, and mobile genetic elements in human.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-545) contains supplementary material, which is available to authorized users.     

piRNA analysis framework from small RNA-Seq data by a novel cluster prediction tool - PILFER

With the increasing number of studies focusing on PIWI-interacting RNA (piRNAs), it is now pertinent to develop efficient tools dedicated towards piRNA analysis. We have developed a novel cluster prediction tool called PILFER (PIrna cLuster FindER), which can accurately predict piRNA clusters from small RNA sequencing data. PILFER is an open source, easy to use tool, and can be executed even on a personal computer with minimum resources. It uses a sliding-window mechanism by integrating the expression of the reads along with the spatial information to predict the piRNA clusters. We have additionally defined a piRNA analysis pipeline incorporating PILFER to detect and annotate piRNAs and their clusters from raw small RNA sequencing data and implemented it on publicly available data from healthy germline and somatic tissues. We compared PILFER with other existing piRNA cluster prediction tools and found it to be statistically more accurate and superior in many aspects such as the robustness of PILFER clusters is higher and memory efficiency is more. Overall, PILFER provides a fast and accurate solution to piRNA cluster prediction.     

Dicer independent small RNAs associate with telomeric heterochromatin

Small RNAs play important roles in the establishment and maintenance of heterochromatin structures. We show the presence of telomere specific small RNAs (tel-sRNAs) in mouse embryonic stem cells that are ∼24 nucleotides in length, Dicer-independent, and 2′-O-methylated at the 3′ terminus. The tel-sRNAs are asymmetric with specificity toward telomere G-rich strand, and evolutionarily conserved from protozoan to mammalian cells. Furthermore, tel-sRNAs are up-regulated in cells that carry null mutation of H3K4 methyltransferase MLL (Mll^(−/−)) and down-regulated in cells that carry null mutations of histone H3K9 methyltransferase SUV39H (Suv39h1/h2^(−/−)), suggesting that they are subject to epigenetic regulation. These results support that tel-sRNAs are heterochromatin associated pi-like small RNAs.     

线虫体内新piRNA研究

PIWI-interacting RNAs(piRNA)是一类內源性小RNA,负责抵御转座子和转基因对基因组的入侵.已发现1.6万多种piRNA,在piRNA上游存在保守序列,根据上游序列特征可以预测新的piRNA.将线虫同步化培养至L4时期,分别提取野生和prg-1突变样本中的小RNA,并对其进行高通量测序.基于piRNA上游保守序列特征,在野生线虫L4时期中,发现了967种新piRNA,这些新piRNA在prg-1突变后表达消失.新piRNA的基因座集中分布在四号染色体的2个piRNA簇内,首位碱基以U为主.与已发表的成虫发育时期的PRG-1免疫共沉淀数据比对,发现有153种piRNA存在于与PRG-1免疫沉淀的数据中.同时还发现一些只在野生线虫中表达的non-21nt小RNA,它们与已知piRNA的基因座相同,推测这些non-21nt小RNA可能是其piRNA前体加工的产物.总之,通过小RNA测序,在线虫中发现了一些新的piRNA.