Novel modes of protein-RNA recognition in the RNAi pathway

Gene silencing mediated by RNA interference (RNAi) depends on short interfering RNAs (siRNAs) and micro RNAs (miRNAs). These RNAs have unique features, namely a defined size of 19-21 base pairs, and characteristic two-nucleotide single-stranded 3' overhangs and 5' monophosphate groups. These molecular features of siRNAs and miRNAs are produced by RNase III enzymes, which are a hallmark of gene silencing induced by double-stranded RNA. Recent structural studies of components of the RNAi pathway, including PAZ, Piwi and RNase III domains, as well as full-length Argonaute and viral p19 proteins, have revealed distinct and novel modes of sequence-independent recognition of the characteristic features of siRNAs and miRNAs in the RNAi pathway.     

The Caenorhabditis elegans HEN1 ortholog, HENN-1, methylates and stabilizes select subclasses of germline small RNAs

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.     

PRG-1 and 21U-RNAs interact to form the piRNA complex required for fertility in C. elegans

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.     

The coming of age for Piwi proteins

Piwi proteins, a subfamily of Argonaute (Ago) proteins, have recently been shown to bind endogenous small RNAs. However, differences between Ago proteins (which bind microRNAs and small interfering RNAs) and Piwi proteins and Piwi-interacting RNAs (piRNAs) suggest novel functions for Piwi proteins. Here, we highlight the recent progress in understanding Piwi function and the implications for germline and stem cell development.     

Piwi and piRNAs act upstream of an endogenous siRNA pathway to suppress Tc3 transposon mobility in the Caenorhabditis elegans germline

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.     

On the role of RNA amplification in dsRNA-triggered gene silencing.

We have investigated the role of trigger RNA amplification during RNA interference (RNAi) in Caenorhabditis elegans. Analysis of small interfering RNAs (siRNAs) produced during RNAi in C. elegans revealed a substantial fraction that cannot derive directly from input dsRNA. Instead, a population of siRNAs (termed secondary siRNAs) appeared to derive from the action of a cellular RNA-directed RNA polymerase (RdRP) on mRNAs that are being targeted by the RNAi mechanism. The distribution of secondary siRNAs exhibited a distinct polarity (5'-->3' on the antisense strand), suggesting a cyclic amplification process in which RdRP is primed by existing siRNAs. This amplification mechanism substantially augments the potency of RNAi-based surveillance, while ensuring that the RNAi machinery will focus on expressed mRNAs.     

Function of the Trypanosome Argonaute 1 protein in RNA interference requires the N-terminal RGG domain and arginine 735 in the Piwi domain

Argonaute proteins are central components of RNA interference (RNAi) and related phenomena in a wide variety of eukaryotes, including the early diverging protozoan Trypanosoma brucei. The single T. brucei Argonaute protein (TbAGO1) is in a complex with small interfering RNAs (siRNAs), and a fraction of this ribonucleoprotein particle is associated with polyribosomes. In this study, we generated a panel of insertion, deletion, and single point mutants of TbAGO1 and assayed them in vivo for their function in RNAi. In addition to the signature domains of Argonaute proteins, PAZ and Piwi, TbAGO1 has an N-terminal domain with a high abundance of RGG repeats. Deletion of the N-terminal domain blocked association of AGO1 with polyribosomes and severely affected mRNA cleavage. Nevertheless, the mutant protein was in a complex with siRNAs. In contrast, deletion of the Piwi domain led to a loss of siRNAs but did not abolish polyribosome association. Site-directed mutagenesis of conserved amino acids in the Piwi domain identified arginine 735 as essential for RNAi. Although the R735A mutant bound siRNAs and associated with polyribosomes, it displayed a severe defect in the cleavage of target mRNA.     

Small RNAs in the animal gonad: Guarding genomes and guiding development

Germ cells must safeguard, apportion, package, and deliver their genomes with exquisite precision to ensure proper reproduction and embryonic development. Classical genetic approaches have identified many genes controlling animal germ cell development, but only recently have some of these genes been linked to the RNA interference (RNAi) pathway, a gene silencing mechanism centered on small regulatory RNAs. Germ cells contain microRNAs (miRNAs), endogenous siRNAs (endo-siRNAs), and Piwi-interacting RNAs (piRNAs); these are bound by members of the Piwi/Argonaute protein family. piwi genes were known to specify germ cell development, but we now understand that mutations disrupting germline development can also affect small RNA accumulation. Small RNA studies in germ cells have revealed a surprising diversity of regulatory mechanisms and a unifying function for germline genes in controlling the spread of transposable elements. Future challenges will be to understand the production of germline small RNAs and to identify the full breadth of gene regulation by these RNAs. Progress in this area will likely impact biomedical goals of manipulating stem cells and preventing diseases caused by the transposition of mobile DNA elements.     

Mighty Piwis defend the germline against genome intruders

Piwis are a germline-specific subclass of the Argonaute family of RNA interference (RNAi) effector proteins that are associated with a recently discovered group of small RNAs (piRNAs). Recent studies in Drosophila and zebrafish directly implicate Piwi proteins in piRNA biogenesis to maintain transposon silencing in the germline genome (Brennecke et al., 2007; Gunawardane et al., 2007; Houwing et al., 2007). This function may be conserved in mice as loss of Miwi2, a mouse Piwi homolog, leads to germline stem cell and meiotic defects correlated with increased transposon activity (Carmell et al., 2007).     

PIWI associated siRNAs and piRNAs specifically require the Caenorhabditis elegans HEN1 ortholog henn-1

Small RNAs--including piRNAs, miRNAs, and endogenous siRNAs--bind Argonaute proteins to form RNA silencing complexes that target coding genes, transposons, and aberrant RNAs. To assess the requirements for endogenous siRNA formation and activity in Caenorhabditis elegans, we developed a GFP-based sensor for the endogenous siRNA 22G siR-1, one of a set of abundant siRNAs processed from a precursor RNA mapping to the X chromosome, the X-cluster. Silencing of the sensor is also dependent on the partially complementary, unlinked 26G siR-O7 siRNA. We show that 26G siR-O7 acts in trans to initiate 22G siRNA formation from the X-cluster. The presence of several mispairs between 26G siR-O7 and the X-cluster mRNA, as well as mutagenesis of the siRNA sensor, indicates that siRNA target recognition is permissive to a degree of mispairing. From a candidate reverse genetic screen, we identified several factors required for 22G siR-1 activity, including the chromatin factors mes-4 and gfl-1, the Argonaute ergo-1, and the 3' methyltransferase henn-1. Quantitative RT-PCR of small RNAs in a henn-1 mutant and deep sequencing of methylated small RNAs indicate that siRNAs and piRNAs that associate with PIWI clade Argonautes are methylated by HENN-1, while siRNAs and miRNAs that associate with non-PIWI clade Argonautes are not. Thus, PIWI-class Argonaute proteins are specifically adapted to associate with methylated small RNAs in C. elegans.     

The role of piRNAs in mouse spermatogenesis.

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.     

Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi

Argonaute (AGO) proteins interact with small RNAs to mediate gene silencing. C. elegans contains 27 AGO genes, raising the question of what roles these genes play in RNAi and related gene-silencing pathways. Here we describe 31 deletion alleles representing all of the previously uncharacterized AGO genes. Analysis of single- and multiple-AGO mutant strains reveals functions in several pathways, including (1) chromosome segregation, (2) fertility, and (3) at least two separate steps in the RNAi pathway. We show that RDE-1 interacts with trigger-derived sense and antisense RNAs to initiate RNAi, while several other AGO proteins interact with amplified siRNAs to mediate downstream silencing. Overexpression of downstream AGOs enhances silencing, suggesting that these proteins are limiting for RNAi. Interestingly, these AGO proteins lack key residues required for mRNA cleavage. Our findings support a two-step model for RNAi, in which functionally and structurally distinct AGOs act sequentially to direct gene silencing.     

Structural features of small RNA precursors determine Argonaute loading in Caenorhabditis elegans

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.     

Argonaute proteins: mediators of RNA silencing

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.