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1.
Small RNAs regulate diverse biological processes by directing effector proteins called Argonautes to silence complementary mRNAs. Maturation of some classes of small RNAs involves terminal 2'-O-methylation to prevent degradation. This modification is catalyzed by members of the conserved HEN1 RNA methyltransferase family. In animals, Piwi-interacting RNAs (piRNAs) and some endogenous and exogenous small interfering RNAs (siRNAs) are methylated, whereas microRNAs are not. However, the mechanisms that determine animal HEN1 substrate specificity have yet to be fully resolved. In Caenorhabditis elegans, a HEN1 ortholog has not been studied, but there is evidence for methylation of piRNAs and some endogenous siRNAs. Here, we report that the worm HEN1 ortholog, HENN-1 (HEN of Nematode), is required for methylation of C. elegans small RNAs. Our results indicate that piRNAs are universally methylated by HENN-1. In contrast, 26G RNAs, a class of primary endogenous siRNAs, are methylated in female germline and embryo, but not in male germline. Intriguingly, the methylation pattern of 26G RNAs correlates with the expression of distinct male and female germline Argonautes. Moreover, loss of the female germline Argonaute results in loss of 26G RNA methylation altogether. These findings support a model wherein methylation status of a metazoan small RNA is dictated by the Argonaute to which it binds. Loss of henn-1 results in phenotypes that reflect destabilization of substrate small RNAs: dysregulation of target mRNAs, impaired fertility, and enhanced somatic RNAi. Additionally, the henn-1 mutant shows a weakened response to RNAi knockdown of germline genes, suggesting that HENN-1 may also function in canonical RNAi. Together, our results indicate a broad role for HENN-1 in both endogenous and exogenous gene silencing pathways and provide further insight into the mechanisms of HEN1 substrate discrimination and the diversity within the Argonaute family.  相似文献   

2.
Argonautes confront new small RNAs   总被引:1,自引:0,他引:1  
Argonaute is at the heart of all effector complexes in RNA interference. In the classical RNAi pathway Argonaute functions as the Slicer enzyme that cleaves an mRNA target directed by a complementary siRNA. Two recently described Argonaute protein subfamilies mediate distinct functions in RNAi. The Piwi subfamily functions in the germline through a novel class of small RNAs that are longer than Argonaute-specific siRNAs and miRNAs. Piwi-interacting RNAs (piRNAs) carry a 2'-O-methylation on their 3' end and appear to be synthesized by a Piwi Slicer dependent mechanism. Piwi/piRNA complexes in mammals and flies are directly linked to the control of transposable elements during germline development. Amplified RNAi in C. elegans is mediated by secondary siRNAs selectively bound to secondary Argonautes (SAGOs) that belong to a worm-specific Argonaute subfamily (WAGO). Secondary siRNAs are 5' triphosphorylated that may allow specific loading into SAGO complexes that are rate limiting for RNAi in C. elegans. Interestingly, SAGOs lack conserved Slicer amino acid residues and probably act in a Slicer-independent fashion.  相似文献   

3.
RNA interference (RNAi)-related pathways affect gene activity by sequence-specific recruitment of Ago proteins to mRNA target molecules. The sequence specificity of this process stems from small RNA (sRNA) co-factors bound by the Ago protein. Stability of sRNA molecules in some pathways is in part regulated by Hen1-mediated methylation of their 3' ends. Here we describe the effects of the Caenorhabditis elegans HEN1 RNA-methyl-transferase homolog, HENN-1, on the different RNAi pathways in this nematode. We reveal differential effects of HENN-1 on the two pathways that are known to employ methylated sRNA molecules: the 26G and 21U pathways. Surprisingly, in the germline, stability of 21U RNAs, the C. elegans piRNAs, is only mildly affected by loss of methylation; and introduction of artificial 21U target RNA does not further destabilize non-methylated 21U RNAs. In contrast, most 26G RNAs display reduced stability and respond to loss of HENN-1 by displaying increased 3'-uridylation frequencies. Within the 26G RNA class, we find that specifically ERGO-1-bound 26G RNAs are modified by HENN-1, while ALG-3/ALG-4-bound 26G RNAs are not. Global gene expression analysis of henn-1 mutants reveals mild effects, including down-regulation of many germline-expressed genes. Our data suggest that, apart from direct effects of reduced 26G RNA levels of henn-1 on gene expression, most effects on global gene expression are indirect. These studies further refine our understanding of endogenous RNAi in C. elegans and the roles for Hen1 like enzymes in these pathways.  相似文献   

4.
5.
Endogenous small interfering RNAs (siRNAs) are a class of naturally occuring regulatory RNAs found in fungi, plants, and animals. Some endogenous siRNAs are required to silence transposons or function in chromosome segregation; however, the specific roles of most endogenous siRNAs are unclear. The helicase gene eri-6/7 was identified in the nematode Caenorhabditis elegans by the enhanced response to exogenous double-stranded RNAs (dsRNAs) of the null mutant. eri-6/7 encodes a helicase homologous to small RNA factors Armitage in Drosophila, SDE3 in Arabidopsis, and Mov10 in humans. Here we show that eri-6/7 mutations cause the loss of 26-nucleotide (nt) endogenous siRNAs derived from genes and pseudogenes in oocytes and embryos, as well as deficiencies in somatic 22-nucleotide secondary siRNAs corresponding to the same loci. About 80 genes are eri-6/7 targets that generate the embryonic endogenous siRNAs that silence the corresponding mRNAs. These 80 genes share extensive nucleotide sequence homology and are poorly conserved, suggesting a role for these endogenous siRNAs in silencing of and thereby directing the fate of recently acquired, duplicated genes. Unlike most endogenous siRNAs in C. elegans, eri-6/7-dependent siRNAs require Dicer. We identify that the eri-6/7-dependent siRNAs have a passenger strand that is ~19 nt and is inset by ~3-4 nts from both ends of the 26 nt guide siRNA, suggesting non-canonical Dicer processing. Mutations in the Argonaute ERGO-1, which associates with eri-6/7-dependent 26 nt siRNAs, cause passenger strand stabilization, indicating that ERGO-1 is required to separate the siRNA duplex, presumably through endonucleolytic cleavage of the passenger strand. Thus, like several other siRNA-associated Argonautes with a conserved RNaseH motif, ERGO-1 appears to be required for siRNA maturation.  相似文献   

6.
The Argonaute proteins, which are the direct partners of the small RNAs involved in RNA interference mechanisms, can be divided into two subfamilies, the Argonautes and the Piwis. In animals, the Argonaute subfamily binds 21-22 nucleotide small interfering RNAs (siRNAs) and microRNAs (miRNAs), which direct cleavage and translational inhibition of their target RNAs respectively. The partners of the Piwi proteins are 24-30-nucleotide small RNAs called Piwi-interacting RNAs or piRNAs. In Drosophila, Piwi proteins and piRNAs protect the genome of the germline against selfish elements. Recent studies suggest that this function is conserved in mammals.La famille des Argonautes, les partenaires directs des petits ARNs dans les mécanismes d'interférence par l'ARN, se divise en deux sous-groupes : les Argonautes et les Piwis. Chez les animaux, le sous-groupe des Argonautes se lie aux petits ARNs interférents (siARNs) et aux microARNs (miARNs) qui mesurent 21-22 nucléotides et sont responsables du clivage et de l'inhibition traductionnelle des ARNs cibles respectivement. Les protéines Piwis ont pour partenaires de petits ARNs de 24-30 nucléotides appelés Piwi-interacting RNAs ou piARNs. Chez la drosophile, les protéines Piwi et les piARNs protègent le génome de la lignée germinale contre les éléments mobiles. Des analyses récentes suggèrent que cette fonction est conservée chez les mammifères.  相似文献   

7.
The Argonaute protein family   总被引:5,自引:0,他引:5  
  相似文献   

8.
The Piwi proteins of the Argonaute superfamily are required for normal germline development in Drosophila, zebrafish, and mice and associate with 24-30 nucleotide RNAs termed piRNAs. We identify a class of 21 nucleotide RNAs, previously named 21U-RNAs, as the piRNAs of C. elegans. Piwi and piRNA expression is restricted to the male and female germline and independent of many proteins in other small-RNA pathways, including DCR-1. We show that Piwi is specifically required to silence Tc3, but not other Tc/mariner DNA transposons. Tc3 excision rates in the germline are increased at least 100-fold in piwi mutants as compared to wild-type. We find no evidence for a Ping-Pong model for piRNA amplification in C. elegans. Instead, we demonstrate that Piwi acts upstream of an endogenous siRNA pathway in Tc3 silencing. These data might suggest a link between piRNA and siRNA function.  相似文献   

9.
Small silencing RNAs repress gene expression by a set of related mechanisms collectively called RNA-silencing pathways [1, 2]. In the RNA interference (RNAi) pathway [3], small interfering mRNA (siRNAs) defend cells from invasion by foreign nucleic acids, such as those produced by viruses. In contrast, microRNAs (miRNAs) sculpt endogenous mRNA expression [4]. A third class of small RNAs, Piwi-interacting RNAs (piRNAs), defends the genome from transposons [5-9]. Here, we report that Drosophila piRNAs contain a 2'-O-methyl group on their 3' termini; this is a modification previously reported for plant miRNAs and siRNAs [10] and mouse and rat piRNAs [11, 12, 13]. Plant small-RNA methylation is catalyzed by the protein HEN1 [10, 14, 15]. We find that DmHen1, the Drosophila homolog of HEN1, methylates the termini of siRNAs and piRNAs. Without DmHen1, the length and abundance of piRNAs are decreased, and piRNA function is perturbed. Unlike plant HEN1, DmHen1 acts on single strands, not duplexes, explaining how it can use as substrates both siRNAs-which derive from double-stranded precursors-and piRNAs-which do not [8, 13]. 2'-O-methylation of siRNAs may be the final step in assembly of the RNAi-enzyme complex, RISC, occurring after an Argonaute-bound siRNA duplex is converted to single-stranded RNA.  相似文献   

10.
11.
In metazoans, Piwi-related Argonaute proteins have been linked to germline maintenance, and to a class of germline-enriched small RNAs termed piRNAs. Here we show that an abundant class of 21 nucleotide small RNAs (21U-RNAs) are expressed in the C. elegans germline, interact with the C. elegans Piwi family member PRG-1, and depend on PRG-1 activity for their accumulation. The PRG-1 protein is expressed throughout development and localizes to nuage-like structures called P granules. Although 21U-RNA loci share a conserved upstream sequence motif, the mature 21U-RNAs are not conserved and, with few exceptions, fail to exhibit complementarity or evidence for direct regulation of other expressed sequences. Our findings demonstrate that 21U-RNAs are the piRNAs of C. elegans and link this class of small RNAs and their associated Piwi Argonaute to the maintenance of temperature-dependent fertility.  相似文献   

12.
Argonaute proteins: mediators of RNA silencing   总被引:10,自引:0,他引:10  
Peters L  Meister G 《Molecular cell》2007,26(5):611-623
Small regulatory RNAs such as short interfering RNAs (siRNAs), microRNAs (miRNAs), and Piwi interacting RNAs (piRNAs) have been discovered in the past, and it is becoming more and more apparent that these small molecules have key regulatory functions. Small RNAs are found in all higher eukaryotes and play important roles in cellular processes as diverse as development, stress response, or transposon silencing. Soon after the discovery of small regulatory RNAs, members of the Argonaute protein family were identified as their major cellular protein interactors. This review focuses on the various cellular functions of mammalian Argonaute proteins in conjunction with the different small RNA species that are known today.  相似文献   

13.
Sorting of Drosophila small silencing RNAs   总被引:3,自引:0,他引:3  
Tomari Y  Du T  Zamore PD 《Cell》2007,130(2):299-308
In Drosophila, small interfering RNAs (siRNAs), which direct RNA interference through the Argonaute protein Ago2, are produced by a biogenesis pathway distinct from microRNAs (miRNAs), which regulate endogenous mRNA expression as guides for Ago1. Here, we report that siRNAs and miRNAs are sorted into Ago1 and Ago2 by pathways independent from the processes that produce these two classes of small RNAs. Such small-RNA sorting reflects the structure of the double-stranded assembly intermediates--the miRNA/miRNA( *) and siRNA duplexes--from which Argonaute proteins are loaded. We find that the Dcr-2/R2D2 heterodimer acts as a gatekeeper for the assembly of Ago2 complexes, promoting the incorporation of siRNAs and disfavoring miRNAs as loading substrates for Drosophila Ago2. A separate mechanism acts in parallel to favor miRNA/miRNA( *) duplexes and exclude siRNAs from assembly into Ago1 complexes. Thus, in flies small-RNA duplexes are actively sorted into Argonaute-containing complexes according to their intrinsic structures.  相似文献   

14.
15.
piRNA的生物学功能   总被引:3,自引:0,他引:3  
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.  相似文献   

16.
Förstemann K  Horwich MD  Wee L  Tomari Y  Zamore PD 《Cell》2007,130(2):287-297
Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide distinct classes of RNA-induced silencing complexes (RISCs) to repress mRNA expression in biological processes ranging from development to antiviral defense. In Drosophila, separate but conceptually similar endonucleolytic pathways produce siRNAs and miRNAs. Here, we show that despite their distinct biogenesis, double-stranded miRNAs and siRNAs participate in a common sorting step that partitions them into Ago1- or Ago2-containing effector complexes. These distinct complexes silence their target RNAs by different mechanisms. miRNA-loaded Ago2-RISC mediates RNAi, but only Ago1 is able to repress an mRNA with central mismatches in its miRNA-binding sites. Conversely, Ago1 cannot mediate RNAi, because it is an inefficient nuclease whose catalytic rate is limited by the dissociation of its reaction products. Thus, the two members of the Drosophila Ago subclade of Argonaute proteins are functionally specialized, but specific small RNA classes are not restricted to associate with Ago1 or Ago2.  相似文献   

17.
In C. elegans, DCR-1 is required for the maturation of both short interfering RNAs (siRNAs) and microRNAs (miRNAs), which are subsequently loaded into different Argonaute proteins to mediate silencing via distinct mechanisms. We used in vivo analyses to show that precursors of small RNAs contain structural features that direct the small RNAs into the RNA interference (RNAi) pathway or the miRNA-processing pathway. Nucleotide changes in the pre-let-7 miRNA precursor that make its stem fully complementary cause the resulting small RNA to be recognized as siRNA and induce binding to RDE-1, which leads to RNAi. Mismatches of 1 to 3 nucleotides at various positions in the stem of the precursor restore direction into the miRNA pathway, as the largest portion of such small RNA variants is associated with ALG-1. The Argonaute proteins to which the small RNAs are bound determine the silencing mode, and no functional overlap between RDE-1 and ALG-1 was detected.  相似文献   

18.

Background

Argonaute proteins are key in RNA silencing. In Drosophila melanogaster, the five proteins of the Argonaute family participate in the pathways and mechanisms mediated by three types of small RNAs: piRNAs, miRNAs, and siRNAs. Two Argonaute proteins, Argonaute 1 (Ago1) and Argonaute 2 (Ago2), are associated with miRNA and siRNA mechanisms, which are the most thoroughly studied. The available data points to a sorting specialization of Ago1 for miRNAs and Ago2 for siRNAs. However, this has been demonstrated only in D. melanogaster, one of the most modified insects, which emerged some 100 million years ago. Thus, an important question is whether this association of Ago1 with miRNAs and Ago2 with siRNAs occurs generally in insects, or was a specific innovation in higher flies.

Methods

We addressed this question by using RNAi approaches and studying Ago1 and Ago2 functions in the German cockroach, Blattella germanica, a much less modified insect that emerged some 320 million years ago.

Results

The results showed that B. germanica does preferentially use Ago1 in the miRNA pathway, but can also use Ago2 in some cases. Conversely, Ago2 operates in the RNAi, in siRNA sorting, whereas Ago1 seems to have no relevant role in this process.

Conclusions and general significance

These basic associations are equivalent to those observed in D. melanogaster, implying that they have been evolutionary conserved from at least cockroach to flies, and possibly stem from the last common ancestor of extant insects.  相似文献   

19.
Dicer is a key enzyme involved in RNA interference (RNAi) and microRNA (miRNA) pathways. It is required for biogenesis of miRNAs and small interfering RNAs (siRNAs), and also has a role in the effector steps of RNA silencing. Apart from Argonautes, no proteins are known to associate with Dicer in mammalian cells. In this work, we describe the identification of TRBP (human immunodeficiency virus (HIV-1) transactivating response (TAR) RNA-binding protein) as a protein partner of human Dicer. We show that TRBP is required for optimal RNA silencing mediated by siRNAs and endogenous miRNAs, and that it facilitates cleavage of pre-miRNA in vitro. TRBP had previously been assigned several functions, including inhibition of the interferon-induced double-stranded RNA-regulated protein kinase PKR and modulation of HIV-1 gene expression by association with TAR. The TRBP-Dicer interaction shown raises interesting questions about the potential interplay between RNAi and interferon-PKR pathways.  相似文献   

20.
Among the three main categories of small silencing RNAs in insects and mammals-siRNAs, miRNAs, and piRNAs-siRNAs were thought to arise primarily from exogenous sources, whereas miRNAs and piRNAs arise from endogenous loci. Recent work in flies and mice reveals several classes of endogenous siRNAs (endo-siRNAs) that contribute to functions previously reserved for miRNAs and piRNAs, including gene regulation and transposon suppression.  相似文献   

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