Double-stranded RNA Precursors Research Articles

Selection of RNA Oligonucleotides That Can Modulate Human Dicer ActivityIn Vitro

Human ribonuclease Dicer is an enzyme that excises small regulatory RNAs from perfectly or partially double-stranded RNA precursors. Although Dicer substrates and products have already been quite well characterized, our knowledge about cellular factors regulating the activity of this enzyme is still limited. To learn more about this problem, we attempted to determine whether RNA could function not only as a Dicer substrate but also as its regulator. To this end, we applied an in vitro selection method. We identified 120 RNA oligomers binding human Dicer. Sixteen of them were subjected to more detailed in vitro studies. We found that 6 out of 16 oligomers affected Dicer ability to digest pre-microRNAs (miRNAs), although most of them were cleaved by this enzyme. For the 6 most active oligomers the putative mechanism of Dicer inhibition was determined. Three oligomers were classified as typical competitive inhibitors and one as an allosteric inhibitor. The remaining 2 oligomers acted as selective inhibitors. They affected the production of 1 miRNA, whereas the formation of other miRNAs was hardly influenced. In general, the data obtained suggest that one can modulate the generation of specific miRNAs by using RNA oligomers. Moreover, we found that sequences similar to those of the selected oligomers can be found within the molecules composing human transcriptome.

Male germ cells express abundant endogenous siRNAs

In mammals, endogenous siRNAs (endo-siRNAs) have only been reported in murine oocytes and embryonic stem cells. Here, we show that murine spermatogenic cells express numerous endo-siRNAs, which are likely to be derived from naturally occurring double-stranded RNA (dsRNA) precursors. The biogenesis of these testicular endo-siRNAs is DROSHA independent, but DICER dependent. These male germ cell endo-siRNAs can potentially target hundreds of transcripts or thousands of DNA regions in the genome. Overall, our work has unveiled another hidden layer of regulation imposed by small noncoding RNAs during male germ cell development.

Dicer recognizes the 5′ end of RNA for efficient and accurate processing

A hallmark of RNA silencing is a class of approximately 22-nucleotide RNAs that are processed from double-stranded RNA precursors by Dicer. Accurate processing by Dicer is crucial for the functionality of microRNAs (miRNAs). The current model posits that Dicer selects cleavage sites by measuring a set distance from the 3' overhang of the double-stranded RNA terminus. Here we report that human Dicer anchors not only the 3' end but also the 5' end, with the cleavage site determined mainly by the distance (∼22 nucleotides) from the 5' end (5' counting rule). This cleavage requires a 5'-terminal phosphate group. Further, we identify a novel basic motif (5' pocket) in human Dicer that recognizes the 5'-phosphorylated end. The 5' counting rule and the 5' anchoring residues are conserved in Drosophila Dicer-1, but not in Giardia Dicer. Mutations in the 5' pocket reduce processing efficiency and alter cleavage sites in vitro. Consistently, miRNA biogenesis is perturbed in vivo when Dicer-null embryonic stem cells are replenished with the 5'-pocket mutant. Thus, 5'-end recognition by Dicer is important for precise and effective biogenesis of miRNAs. Insights from this study should also afford practical benefits to the design of small hairpin RNAs.

Loqs-PD and R2D2 define independent pathways for RISC generation in Drosophila

In Drosophila, siRNAs are classified as endo- or exo-siRNAs based on their origin. Both are processed from double-stranded RNA precursors by Dcr-2 and then loaded into the Argonaute protein Ago2. While exo-siRNAs serve to defend the cell against viruses, endo-siRNAs restrict the spread of selfish DNA in somatic cells, analogous to piRNAs in the germ line. Endo- and exo-siRNAs display a differential requirement for double-stranded RNA binding domain proteins (dsRBPs): R2D2 is needed to load exo-siRNAs into Ago2 while the PD isoform of Loquacious (Loqs-PD) stimulates Dcr-2 during the nucleolytic processing of hairpin-derived endo-siRNAs. In cell culture assays, R2D2 antagonizes Loqs-PD in endo-siRNA silencing and Loqs-PD is an inhibitor of RNA interference. Loqs-PD can interact via the C-terminus unique to this isoform with the DExH/D-helicase domain of Drosophila Dcr-2, where binding of R2D2 has also been localized. Separation of the two pathways is not complete; rather, the dicing and Ago2-loading steps appear uncoupled, analogous to the corresponding steps in miRNA biogenesis. Analysis of deep sequencing data further demonstrates that in r2d2 mutant flies, siRNAs can be loaded into Ago2 but not all siRNA classes are equally proficient for this. Thus, the canonical Ago2-RISC loading complex can be bypassed under certain circumstances.

Hen1 is required for oocyte development and piRNA stability in zebrafish

Piwi-interacting RNAs (piRNAs) are germ line-specific small RNA molecules that have a function in genome defence and germ cell development. They associate with a specific class of Argonaute proteins, named Piwi, and function through an RNA interference-like mechanism. piRNAs carry a 2'-O-methyl modification at their 3' end, which is added by the Hen1 enzyme. We show that zebrafish hen1 is specifically expressed in germ cells and is essential for maintaining a female germ line, whereas it is dispensable in the testis. Hen1 protein localizes to nuage through its C-terminal domain, but is not required for nuage formation. In hen1 mutant testes, piRNAs become uridylated and adenylated. Uridylation frequency is highest on retro-transposon-derived piRNAs and is accompanied by decreased piRNA levels and mild derepression of transposon transcripts. Altogether, our data suggest the existence of a uridylation-mediated 3'-5' exonuclease activity acting on piRNAs in zebrafish germ cells, which is counteracted by nuage-bound Hen1 protein. This system discriminates between piRNA targets and is required for ovary development and fully efficient transposon silencing.

MicroRNAs, the epigenetic memory and climatic adaptation in Norway spruce

*Norway spruce expresses a temperature-dependent epigenetic memory from the time of embryo development, which thereafter influences the timing bud phenology. MicroRNAs (miRNAs)are endogenous small RNAs, exerting epigenetic gene regulatory impacts. We have tested for their presence and differential expression. *We prepared concatemerized small RNA libraries from seedlings of two full-sib families, originated from seeds developed in a cold and warm environment. One family expressed distinct epigenetic effects while the other not. We used available plant miRNA query sequences to search for conserved miRNAs and from the sequencing we found novel ones; the miRNAs were monitored using relative real time-PCR. *Sequencing identified 24 novel and four conserved miRNAs. Further screening of the conserved miRNAs confirmed the presence of 16 additional miRNAs. Most of the miRNAs were targeted to unknown genes. The expression of seven conserved and nine novel miRNAs showed significant differences in transcript levels in the full-sib family showing distinct epigenetic difference in bud set, but not in the nonresponding full-sib family. Putative miRNA targets were studied. *Norway spruce contains a set of conserved miRNAs as well as a large proportion of novel nonconserved miRNAs. The differentially expression of specific miRNAs indicate their putative participation in the epigenetic regulation.

In Vitro Assembly of Plant RNA-Induced Silencing Complexes Facilitated by Molecular Chaperone HSP90

RNA-induced silencing complexes (RISCs) play central roles in posttranscriptional gene silencing. In plants, the mechanism of RISC assembly has remained elusive due to the lack of cell-free systems that recapitulate the process. In this report, we demonstrate that plant AGO1 protein synthesized by in vitro translation using an extract of evacuolated tobacco protoplasts incorporates synthetic small interfering RNA (siRNA) and microRNA (miRNA) duplexes to form RISCs that sequester the single-stranded siRNA guide strand and miRNA strand, respectively. The formed RISCs were able to recognize and cleave the complementary target RNAs. In this system, the siRNA duplex was incorporated into HSP90-bound AGO1, and subsequent removal of the passenger strand was triggered by ATP hydrolysis by HSP90. Removal of the siRNA passenger strand required the ribonuclease activity of AGO1, while that of the miRNA star strand did not. Based on these results, the mechanism of plant RISC formation is discussed.

RETRACTED: Small RNA duplexes function as mobile silencing signals between plant cells.

In the plant RNA interference (RNAi) pathway, 21-nucleotide duplexes of small interfering RNA (siRNA) are processed from longer double-stranded RNA precursors by the RNaseIII Dicer-like 4 (DCL4). Single-stranded siRNAs then guide Argonaute 1 (AGO1) to execute posttranscriptional silencing of complementary target RNAs. RNAi is not cell-autonomous in higher plants, but the nature of the mobile nucleic acid(s) signal remains unknown. Using cell-specific rescue of DCL4 function and cell-specific inhibition of RNAi movement, we genetically establish that exogenous and endogenous siRNAs, as opposed to their precursor molecules, act as mobile silencing signals between plant cells. We further demonstrate physical movement of mechanically delivered, labeled siRNA duplexes that functionally recapitulate transgenic RNAi spread. Cell-to-cell movement is unlikely to involve AGO1-bound siRNA single strands, but instead likely involves siRNA duplexes.

Evidence for large complex networks of plant short silencing RNAs.

BackgroundIn plants and animals there are many classes of short RNAs that carry out a wide range of functions within the cell; short silencing RNAs (ssRNAs) of 21–25 nucleotides in length are produced from double-stranded RNA precursors by the protein Dicer and guide nucleases and other proteins to their RNA targets through base pairing interactions. The consequence of this process is degradation of the targeted RNA, suppression of its translation or initiation of secondary ssRNA production. The secondary ssRNAs in turn could then initiate further layers of ssRNA production to form extensive cascades and networks of interacting RNA [1]. Previous empirical analysis in plants established the existence of small secondary ssRNA cascade [2], in which a single instance of this event occurred but it was not known whether there are other more extensive networks of secondary sRNA production.Methodology/Principal FindingsWe generated a network by predicting targets of ssRNA populations obtained from high-throughput sequencing experiments. The topology of the network shows it to have power law connectivity distribution, to be dissortative, highly clustered and composed of multiple components. We also identify protein families, PPR and ULP1, that act as hubs within the network. Comparison of the repetition of genomic sub-sequences of ssRNA length between Arabidopsis and E.coli suggest that the network structure is made possible by the underlying repetitiveness in the genome sequence.Conclusions/SignificanceTogether our results provide good evidence for the existence of a large, robust ssRNA interaction network with distinct regulatory function. Such a network could have a massive effect on the regulation of gene expression via mediation of transcript levels.

The endogenous siRNA pathway is involved in heterochromatin formation in Drosophila

A new class of small RNAs (endo-siRNAs) produced from endogenous double-stranded RNA (dsRNA) precursors was recently shown to mediate transposable element (TE) silencing in the Drosophila soma. These endo-siRNAs might play a role in heterochromatin formation, as has been shown in S. pombe for siRNAs derived from repetitive sequences in chromosome pericentromeres. To address this possibility, we used the viral suppressors of RNA silencing B2 and P19. These proteins normally counteract the RNAi host defense by blocking the biogenesis or activity of virus-derived siRNAs. We hypothesized that both proteins would similarly block endo-siRNA processing or function, thereby revealing the contribution of endo-siRNA to heterochromatin formation. Accordingly, P19 as well as a nuclear form of P19 expressed in Drosophila somatic cells were found to sequester TE-derived siRNAs whereas B2 predominantly bound their longer precursors. Strikingly, B2 or the nuclear form of P19, but not P19, suppressed silencing of heterochromatin gene markers in adult flies, and altered histone H3-K9 methylation as well as chromosomal distribution of histone methyl transferase Su(var)3-9 and Heterochromatin Protein 1 in larvae. Similar effects were observed in dcr2, r2d2, and ago2 mutants. Our findings provide evidence that a nuclear pool of TE-derived endo-siRNAs is involved in heterochromatin formation in somatic tissues in Drosophila.

X chromosome inactivation in the absence of Dicer

Dicer is central to the RNA interference (RNAi) pathway, because it is required for processing of double-stranded RNA (dsRNA) precursors into small RNA effector molecules. In principle, any long dsRNA could serve as a substrate for Dicer. The X inactive specific transcript (Xist) is an untranslated RNA that is required for dosage compensation in mammals. It coats and silences 1 of the 2 X chromosomes in female cells and initiates a chromosomewide change in chromatin structure that includes the recruitment of Polycomb proteins, but it is largely unknown how Xist RNA mediates these processes. To investigate a potential link between the RNAi pathway and X inactivation, we generated and analyzed Dicer-deficient embryonic stem (ES) cells. In the absence of Dicer, coating by Xist RNA, initiation of silencing, and recruitment of Polycomb proteins occur normally. Dicer ablation had modest effects on the steady-state levels of spliced Xist RNA. Together our data indicate that the RNAi machinery is not essential for the initiation of X inactivation.

Functional analysis of dicer-2 missense mutations in the siRNA pathway of Drosophila

The Drosophila RNase III enzyme Dicer-2 processes double-stranded RNA (dsRNA) precursors into small interfering RNAs (siRNAs). It also interacts with the siRNA product and R2D2 protein to facilitate the assembly of an RNA-induced silencing complex (RISC) that mediates RNA interference. Here, we characterized six independent missense mutations in the dicer-2 gene. Four mutations (P8S, L188F, R269W, and P365L) in the DExH helicase domain reduced dsRNA processing activity. Two mutations were located within an RNase III domain. P1496L caused a loss of dsRNA processing activity comparable to a null dicer-2 mutation. A1453T strongly reduced both dsRNA processing and RISC activity, and decreased the levels of Dicer-2 and R2D2 proteins, suggesting that this mutation destabilizes Dicer-2. We also found that the carboxyl-terminal region of R2D2 is essential for Dicer-2 binding. These results provide further insight into the structure–function relationship of Dicer, which plays a critical role in the siRNA pathway.

Characterization of craniofacial morphology in a conditional dicer mutant mouse

Recent studies have shown that small non‐coding RNAs, including small interfering RNAs (siRNAs) and highly conserved microRNAs (miRNAs), are actively involved in the regulation of gene expression at multiple levels including chromatin architecture, transcription, RNA editing, RNA stability, and translation. Biogenesis of functional siRNAs and miRNAs from double‐stranded RNA precursors requires the action of Dicer, an RNase III family member. Mutations in the Dicer allele have been shown to cause several developmental defects in plants and animals and more recently has been linked to several different types of human diseases, including cancers and HIV. In the mouse, mutation of Dicer leads to early embryonic lethality. In order to examine the role of Dicer and small RNAs in craniofacial morphogenesis, a tissue‐specific conditional Dicer knockout mouse was generated. Mutant mice live to only embryonic day (E) 17.5, but exhibit marked craniofacial dimorphisms. A comparison of mutant mice to wild type (WT) liter mates reveals gross morphological defects in the craniofacial region. Phenotypic features of mutant embryos at E17.5 included agenesis of the frontal bones, midface retrognathia, and apparent disruption in palatogenesis. These preliminary results suggest a prominent role for Dicer and small RNAs in craniofacial development.Financial Support: This work was supported by a faculty development grant (SMS Rocha‐Sanchez) from HFF* Correspondent author

A three-dimensional view of the molecular machinery of RNA interference

In eukaryotes, small non-coding RNAs regulate gene expression, helping to control cellular metabolism, growth and differentiation, to maintain genome integrity, and to combat viruses and mobile genetic elements. These pathways involve two specialized ribonucleases that control the production and function of small regulatory RNAs. The enzyme Dicer cleaves double-stranded RNA precursors, generating short interfering RNAs and microRNAs in the cytoplasm. These small RNAs are transferred to Argonaute proteins, which guide the sequence-specific silencing of messenger RNAs that contain complementary sequences by either enzymatically cleaving the mRNA or repressing its translation. The molecular structures of Dicer and the Argonaute proteins, free and bound to small RNAs, have offered exciting insights into the molecular mechanisms that are central to RNA silencing pathways.

Biogenesis and germline functions of piRNAs

Small interfering RNAs and microRNAs are generated from double-stranded RNA precursors by the Dicer endonucleases, and function with Argonaute-family proteins to target transcript destruction or to silence translation. A distinct class of 24- to 30-nucleotide-long RNAs, produced by a Dicer-independent mechanism, associates with Piwi-class Argonaute proteins. Studies in flies, fish and mice implicate these Piwi-associated RNAs (piRNAs) in germline development, silencing of selfish DNA elements, and in maintaining germline DNA integrity. However, whether piRNAs primarily control chromatin organization, gene transcription, RNA stability or RNA translation is not well understood, neither is piRNA biogenesis. Here, we review recent studies of piRNA production and function, and discuss unanswered questions about this intriguing new class of small RNAs.

The Piwi-piRNA Pathway Provides an Adaptive Defense in the Transposon Arms Race

Increasingly complex networks of small RNAs act through RNA-interference (RNAi) pathways to regulate gene expression, to mediate antiviral responses, to organize chromosomal domains, and to restrain the spread of selfish genetic elements. Historically, RNAi has been defined as a response to double-stranded RNA. However, some small RNA species may not arise from double-stranded RNA precursors. Yet, like microRNAs and small interfering RNAs, such species guide Argonaute proteins to silencing targets through complementary base-pairing. Silencing can be achieved by corecruitment of accessory factors or through the activity of Argonaute itself, which often has endonucleolytic activity. As a specific and adaptive regulatory system, RNAi is used throughout eukarya, which indicates a long evolutionary history. A likely function of RNAi throughout that history is to protect the genome from both pathogenic and parasitic invaders.

Short Silencing RNA: The Dark Matter of Genetics?

Plants and animals have single-stranded silencing RNAs (sRNAs) of 21-25 nucleotides in length that are derived from a double-stranded (ds)RNA precursor by Dicer (DCL) processing. These RNAs are the guide RNA for nucleases of the AGO class that cleave targeted RNA in a nucleotide sequence-specific manner. The cleaved RNAs are then degraded further or they are the template for an RNA-dependent RNA polymerase (RDR) that generates a dsRNA. In this paper, I discuss the possibility that this RDR-generated dsRNA initiates a cascade in which there are multiple rounds of secondary sRNA production. I propose that these secondary sRNAs feature in mechanisms that can either buffer mRNA populations against change or, in certain circumstances, mediate extensive changes in mRNA populations. The RNA cascades may also have RNA-mediated epigenetic characteristics in addition to the DNA and chromatin transcriptional silencing potential that has been previously linked with RNA silencing.

An RNA-Dependent RNA Polymerase Prevents Meristem Invasion by Potato Virus X and Is Required for the Activity But Not the Production of a Systemic Silencing Signal

One of the functions of RNA silencing in plants is antiviral defense. A hallmark of RNA silencing is spreading of the silenced state through the plant. Little is known about the nature of the systemic silencing signal and the proteins required for its production, transport, and reception in plant tissues. Here, we show that the RNA-dependent RNA polymerase RDR6 in Nicotiana benthamiana is involved in defense against potato virus X at the level of systemic spreading and in exclusion of the virus from the apical growing point. It has no effect on primary replication and cell-to-cell movement of the virus and does not contribute significantly to the formation of virus-derived small interfering (si) RNA in a fully established potato virus X infection. In grafting experiments, the RDR6 homolog was required for the ability of a cell to respond to, but not to produce or translocate, the systemic silencing signal. Taking these findings together, we suggest a model of virus defense in which RDR6 uses incoming silencing signal to generate double-stranded RNA precursors of secondary siRNA. According to this idea, the secondary siRNAs mediate RNA silencing as an immediate response that slows down the systemic spreading of the virus into the growing point and newly emerging leaves.

T cell lineage choice and differentiation in the absence of the RNase III enzyme Dicer

The ribonuclease III enzyme Dicer is essential for the processing of micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) from double-stranded RNA precursors. miRNAs and siRNAs regulate chromatin structure, gene transcription, mRNA stability, and translation in a wide range of organisms. To provide a model system to explore the role of Dicer-generated RNAs in the differentiation of mammalian cells in vivo, we have generated a conditional Dicer allele. Deletion of Dicer at an early stage of T cell development compromised the survival of αβ lineage cells, whereas the numbers of γδ-expressing thymocytes were not affected. In developing thymocytes, Dicer was not required for the maintenance of transcriptional silencing at pericentromeric satellite sequences (constitutive heterochromatin), the maintenance of DNA methylation and X chromosome inactivation in female cells (facultative heterochromatin), and the stable shutdown of a developmentally regulated gene (developmentally regulated gene silencing). Most remarkably, given that one third of mammalian mRNAs are putative miRNA targets, Dicer seems to be dispensable for CD4/8 lineage commitment, a process in which epigenetic regulation of lineage choice has been well documented. Thus, although Dicer seems to be critical for the development of the early embryo, it may have limited impact on the implementation of some lineage-specific gene expression programs.

Mammalian microRNAs derived from genomic repeats

In this article, we show that a subset of conventional mammalian microRNAs is derived from LINE-2 transposable elements and other genome repeats. These repeat-derived microRNAs arise from conventional precursor hairpins and are distinct from the rasiRNAs, which appear to be processed from long double-stranded RNA precursors. The insertion of transposable elements into new genomic sites appears to be one of the driving-forces that create new microRNAs during mammalian evolution. Two of the LINE-2-derived microRNAs exhibit perfect complementarity to a large family of mRNA and EST transcripts that contain portions of MIR and other LINE-2 elements in their 3'-untranslated regions.