Short interfering RNA strand selection is independent of dsRNA processing polarity during RNAi in Drosophila

Short interfering RNAs (siRNAs) guide mRNA cleavage during RNA interference (RNAi). Only one siRNA strand assembles into the RNA-induced silencing complex (RISC), with preference given to the strand whose 5' terminus has lower base-pairing stability. In Drosophila, Dcr-2/R2D2 processes siRNAs from longer double-stranded RNAs (dsRNAs) and also nucleates RISC assembly, suggesting that nascent siRNAs could remain bound to Dcr-2/R2D2. In vitro, Dcr-2/R2D2 senses base-pairing asymmetry of synthetic siRNAs and dictates strand selection by asymmetric binding to the duplex ends. During dsRNA processing, Dicer (Dcr) liberates siRNAs from dsRNA ends in a manner dictated by asymmetric enzyme-substrate interactions. Because Dcr-2/R2D2 is unlikely to sense base-pairing asymmetry of an siRNA that is embedded within a precursor, it is not clear whether processed siRNAs strictly follow the thermodynamic asymmetry rules or whether processing polarity can affect strand selection. We use a Drosophila in vitro system in which defined siRNAs with known asymmetry can be generated from longer dsRNA precursors. These dsRNAs permit processing specifically from either the 5' or the 3' end of the thermodynamically favored strand of the incipient siRNA. Combined dsRNA-processing/mRNA-cleavage assays indicate that siRNA strand selection is independent of dsRNA processing polarity during Drosophila RISC assembly in vitro.     

An NMD pathway in yeast involving accelerated deadenylation and exosome-mediated 3'-->5' degradation

Eukaryotic mRNAs containing premature termination codons are subjected to accelerated turnover, known as nonsense-mediated decay (NMD). Recognition of translation termination events as premature requires a surveillance complex, which includes the RNA helicase Upf1p. In Saccharomyces cerevisiae, NMD provokes rapid decapping followed by 5'-->3' exonucleolytic decay. Here we report an alternative, decapping-independent NMD pathway involving deadenylation and subsequent 3'-->5' exonucleolytic decay. Accelerated turnover via this pathway required Upf1p and was blocked by the translation inhibitor cycloheximide. Degradation of the deadenylated mRNA required the Rrp4p and Ski7p components of the cytoplasmic exosome complex, as well as the putative RNA helicase Ski2p. We conclude that recognition of NMD substrates by the Upf surveillance complex can target mRNAs to rapid deadenylation and exosome-mediated degradation.     

Uncoupling of RNAi from active translation in mammalian cells

Small inhibitory RNAs (siRNAs) are produced from longer RNA duplexes by the RNAse III family member Dicer. The siRNAs function as sequence-specific guides for RNA cleavage or translational inhibition. The precise mechanism by which siRNAs direct the RNA-induced silencing complex (RISC) to find the complementary target mRNA remains a mystery. Some biochemical evidence connects RNAi with translation making attractive the hypothesis that RISC is coupled with the translational apparatus for scanning mRNAs. Such coupling would facilitate rapid alignment of the siRNA antisense with the complementary target sequence. To test this hypothesis we took advantage of a well-characterized translational switch afforded by the ferritin IRE-IRP to analyze RNAi mediated cleavage of a target mRNA in the presence and absence of translation. Our results demonstrate that neither active translation nor unidirectional scanning is required for siRNA mediated target degradation. Our findings demonstrate that nontranslated mRNAs are highly susceptible to RNAi, and blocking scanning from both the 5' and 3' ends of an mRNA does not impede RNAi. Interestingly, RNAi is about threefold more active in the absence of translation.     

Mechanism of induction and suppression of antiviral immunity directed by virus-derived small RNAs in Drosophila

The small RNA-directed viral immunity pathway in plants and invertebrates begins with the production by Dicer nuclease of virus-derived siRNAs (viRNAs), which guide specific antiviral silencing by Argonaute protein in an RNA-induced silencing complex (RISC). Molecular identity of the viral RNA precursor of viRNAs remains a matter of debate. Using Flock house virus (FHV) infection of Drosophila as a model, we show that replication of FHV positive-strand RNA genome produces an approximately 400 bp dsRNA from its 5' terminus that serves as the major Dicer-2 substrate. ViRNAs thus generated are loaded in Argonaute-2 and methylated at their 3' ends. Notably, FHV-encoded RNAi suppressor B2 protein interacts with both viral dsRNA and RNA replicase and inhibits production of the 5'-terminal viRNAs. Our findings, therefore, provide a model in which small RNA-directed viral immunity is induced during the initiation of viral progeny (+)RNA synthesis and suppressed by B2 inside the viral RNA replication complex.     

The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes

RISC, the RNA-induced silencing complex, uses short interfering RNAs (siRNAs) or micro RNAs (miRNAs) to select its targets in a sequence-dependent manner. Key RISC components are Argonaute proteins, which contain two characteristic domains, PAZ and PIWI. PAZ is highly conserved and is found only in Argonaute proteins and Dicer. We have solved the crystal structure of the PAZ domain of Drosophila Argonaute2. The PAZ domain contains a variant of the OB fold, a module that often binds single-stranded nucleic acids. PAZ domains show low-affinity nucleic acid binding, probably interacting with the 3' ends of single-stranded regions of RNA. PAZ can bind the characteristic two-base 3' overhangs of siRNAs, indicating that although PAZ may not be a primary nucleic acid binding site in Dicer or RISC, it may contribute to the specific and productive incorporation of siRNAs and miRNAs into the RNAi pathway.     

mRNA translation is not a prerequisite for small interfering RNA-mediated mRNA cleavage

RNA interference constitutes a major means of eliminating mRNAs, yet how the small interfering RNAs (siRNA) within the RNA-induced silencing complex (RISC) finds its homologous target in the cell remains unknown. An attractive hypothesis is that RNA interference is linked to translation which allows RISC ready access to every translated mRNA. To test whether translation could direct siRNAs to mRNAs, chemical and biological inhibitors of translation and their effects on mRNA cleavage were tested. Our results show that mRNA degradation by siRNAs is not dependent on mRNA translation.     

Cytoplasmic degradation of splice-defective pre-mRNAs and intermediates

Specific systems of nuclear RNA degradation appear to target and degrade aberrant pre-mRNA molecules. In this work we report on a Dbr1p-dependent RNA decay pathway that limits the accumulation of splice-defective lariat intermediates stalled at the second step of splicing. In this pathway, splice-defective lariat intermediates are debranched by Dbr1p and subsequently degraded 5' to 3' primarily by the cytoplasmic exonuclease, Xrn1p. When debranching is blocked, these splicing intermediates can be degraded in a 3' to 5' direction in a manner dependent on Ski2p, a cofactor for the cytoplasmic exosome. In that Xrn1p and Ski2p are cytoplasmic and Dbr1p localizes to both the nucleus and the cytoplasm, these data suggest that this decay pathway occurs within the cytoplasm. Furthermore, the finding that lariat intermediates accumulate in the dbr1Delta strain suggests that this pathway also functions as an inherent quality control mechanism for the process of pre-mRNA splicing.     

SID-1 is a dsRNA-selective dsRNA-gated channel

Systemic RNAi in Caenorhabditis elegans requires the widely conserved transmembrane protein SID-1 to transport RNAi silencing signals between cells. When expressed in Drosophila S2 cells, C. elegans SID-1 enables passive dsRNA uptake from the culture medium, suggesting that SID-1 functions as a channel for the transport of double-stranded RNA (dsRNA). Here we show that nucleic acid transport by SID-1 is specific for dsRNA and that addition of dsRNA to SID-1 expressing cells results in changes in membrane conductance, which indicate that SID-1 is a dsRNA gated channel protein. Consistent with passive bidirectional transport, we find that the RNA induced silencing complex (RISC) is required to prevent the export of imported dsRNA and that retention of dsRNA by RISC does not seem to involve processing of retained dsRNA into siRNAs. Finally, we show that mimics of natural molecules that contain both single- and double-stranded dsRNA, such as hairpin RNA and pre-microRNA, can be transported by SID-1. These findings provide insight into the nature of potential endogenous RNA signaling molecules in animals.     

Function of the ski4p (Csl4p) and Ski7p proteins in 3'-to-5' degradation of mRNA

One of two general pathways of mRNA decay in the yeast Saccharomyces cerevisiae occurs by deadenylation followed by 3'-to-5' degradation of the mRNA body. Previous results have shown that this degradation requires components of the exosome and the Ski2p, Ski3p, and Ski8p proteins, which were originally identified due to their superkiller phenotype. In this work, we demonstrate that deletion of the SKI7 gene, which encodes a putative GTPase, also causes a defect in 3'-to-5' degradation of mRNA. Deletion of SKI7, like deletion of SKI2, SKI3, or SKI8, does not affect various RNA-processing reactions of the exosome. In addition, we show that a mutation in the SKI4 gene also causes a defect in 3'-to-5' mRNA degradation. We show that the SKI4 gene is identical to the CSL4 gene, which encodes a core component of the exosome. Interestingly, the ski4-1 allele contains a point mutation resulting in a mutation in the putative RNA binding domain of the Csl4p protein. This point mutation strongly affects mRNA degradation without affecting exosome function in rRNA or snRNA processing, 5' externally transcribed spacer (ETS) degradation, or viability. In contrast, the csl4-1 allele of the same gene affects rRNA processing but not 3'-to-5' mRNA degradation. We identify csl4-1 as resulting from a partial-loss-of-function mutation in the promoter of the CSL4 gene. These data indicate that the distinct functions of the exosome can be separated genetically and suggest that the RNA binding domain of Csl4p may have a specific function in mRNA degradation.     

In C. elegans,High Levels of dsRNA Allow RNAi in the Absence of RDE-4

C. elegans Dicer requires an accessory double-stranded RNA binding protein, RDE-4, to enact the first step of RNA interference, the cleavage of dsRNA to produce siRNA. While RDE-4 is typically essential for RNAi, we report that in the presence of high concentrations of trigger dsRNA, rde-4 deficient animals are capable of silencing a transgene. By multiple criteria the silencing occurs by the canonical RNAi pathway. For example, silencing is RDE-1 dependent and exhibits a decrease in the targeted mRNA in response to an increase in siRNA. We also find that high concentrations of dsRNA trigger lead to increased accumulation of primary siRNAs, consistent with the existence of a rate-limiting step during the conversion of primary to secondary siRNAs. Our studies also revealed that transgene silencing occurs at low levels in the soma, even in the presence of ADARs, and that at least some siRNAs accumulate in a temperature-dependent manner. We conclude that an RNAi response varies with different conditions, and this may allow an organism to tailor a response to specific environmental signals.     

Analysis of the 3(')-hydroxyl group in Drosophila siRNA function

Members of the RNA-dependent RNA polymerase (RdRP) gene family have been shown to be essential for dsRNA-mediated gene silencing based on genetic screens in a variety of organisms, including Caenorhabditis elegans, Arabidopsis, Neurospora, and Dictyostelium. A hallmark of this process is the formation of small 21- to 25-bp dsRNAs, termed siRNAs for small interfering RNAs, which are derived from the dsRNA that initiates gene silencing. We have developed methods to demonstrate that these siRNAs produced in Drosophila embryo extract can be uniformly incorporated into dsRNA in a template-specific manner that is subsequently degraded by RNase III-related enzyme activity to create a second generation of siRNAs. SiRNA function in dsRNA synthesis and mRNA degradation depends upon the integrity of the 3'-hydroxyl of the siRNA, consistent with the interpretation that siRNAs serve as primers for RdRP activity in the formation of dsRNA. This process of siRNA incorporation into dsRNA followed by degradation and the formation of new siRNAs has been termed "degradative PCR" and the proposed mechanism is consistent with the genetic and biochemical data derived from studies in C. elegans, Arabidopsis, Drosophila, and Dictyostelium. The methods used to study the function of both natural and synthetic siRNAs in RNA interference in Drosophila embryo extracts are detailed. The importance of the 3'-hydroxyl group for siRNA function and its incorporation into dsRNA is emphasized and the results support a model that places RNA-dependent RNA polymerase as a key mediator in the RNA interference mechanism in Drosophila.