Viral RNA recognition by the Drosophila small interfering RNA pathway

Viral RNA is a common activator of antiviral responses. In this review, we dissect the mechanism of viral RNA recognition by the small interfering RNA pathway in Drosophila melanogaster. This antiviral response in fruit flies can help understand general principles of nucleic acid recognition.     

Accurate transcriptome-wide prediction of microRNA targets and small interfering RNA off-targets with MIRZA-G

Small interfering RNA (siRNA)-mediated knock-down is a widely used experimental approach to characterizing gene function. Although siRNAs are designed to guide the cleavage of perfectly complementary mRNA targets, acting similarly to microRNAs (miRNAs), siRNAs down-regulate the expression of hundreds of genes to which they have only partial complementarity. Prediction of these siRNA ‘off-targets’ remains difficult, due to the incomplete understanding of siRNA/miRNA–target interactions. Combining a biophysical model of miRNA–target interaction with structure and sequence features of putative target sites we developed a suite of algorithms, MIRZA-G, for the prediction of miRNA targets and siRNA off-targets on a genome-wide scale. The MIRZA-G variant that uses evolutionary conservation performs better than currently available methods in predicting canonical miRNA target sites and in addition, it predicts non-canonical miRNA target sites with similarly high accuracy. Furthermore, MIRZA-G variants predict siRNA off-target sites with an accuracy unmatched by currently available programs. Thus, MIRZA-G may prove instrumental in the analysis of data resulting from large-scale siRNA screens.     

ATP requirements and small interfering RNA structure in the RNA interference pathway.

We examined the role of ATP in the RNA interference (RNAi) pathway. Our data reveal two ATP-dependent steps and suggest that the RNAi reaction comprises at least four sequential steps: ATP-dependent processing of double-stranded RNA into small interfering RNAs (siRNAs), incorporation of siRNAs into an inactive approximately 360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, and ATP-independent recognition and cleavage of the RNA target. Furthermore, ATP is used to maintain 5' phosphates on siRNAs. A 5' phosphate on the target-complementary strand of the siRNA duplex is required for siRNA function, suggesting that cells check the authenticity of siRNAs and license only bona fide siRNAs to direct target RNA destruction.     

Gene silencing in chick embryos with a vector-based small interfering RNA system

In this paper, the use of vector-based RNA interference (RNAi) to specifically interfere with gene expression in chick embryos is reported. In ovo electroporation was carried out to transfer a small interfering RNA (siRNA) expression vector into chick embryos. En2 was chosen for the target gene because the family gene, En1, is expressed in a similar pattern. Four sets of 19-mer sequences were designed with the En2 open reading frame region connected to a sequence of short hairpin RNA (shRNA), which exerts siRNA effects after being transcribed, and inserted into pSilencer U6-1.0 vector. En2 and En1 expression were suppressed by the siRNA whose sequence completely matched En2 and En1. Suppression occurred when the siRNA sequence differed by up to two nucleotides from the target sequence. The sequence that differed by four nucleotides from the target gene did not show siRNA effects. One set that completely matched the En2 target did not show siRNA effects, which may be due to location of the siRNA in the target gene. Thus, multiple sets of shRNA must be prepared if we are to consider. This system will greatly contribute to the analysis of function of genes of interest, because the target gene can be silenced in a locally and temporally desired manner.     

Loss of function of OsDCL1 affects microRNA accumulation and causes developmental defects in rice

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are two types of noncoding RNAs involved in developmental regulation, genome maintenance, and defense in eukaryotes. The activity of Dicer or Dicer-like (DCL) proteins is required for the maturation of miRNAs and siRNAs. In this study, we cloned and sequenced 66 candidate rice (Oryza sativa) miRNAs out of 1,650 small RNA sequences (19 to approximately 25 nt), and they could be further grouped into 21 families, 12 of which are newly identified and three of which, OsmiR528, OsmiR529, and OsmiR530, have been confirmed by northern blot. To study the function of rice DCL proteins (OsDCLs) in the biogenesis of miRNAs and siRNAs, we searched genome databases and identified four OsDCLs. An RNA interference approach was applied to knock down two OsDCLs, OsDCL1 and OsDCL4, respectively. Strong loss of function of OsDCL1IR transformants that expressed inverted repeats of OsDCL1 resulted in developmental arrest at the seedling stage, and weak loss of function of OsDCL1IR transformants caused pleiotropic developmental defects. Moreover, all miRNAs tested were greatly reduced in OsDCL1IR but not OsDCL4IR transformants, indicating that OsDCL1 plays a critical role in miRNA processing in rice. In contrast, the production of siRNA from transgenic inverted repeats and endogenous CentO regions were not affected in either OsDCL1IR or OsDCL4IR transformants, suggesting that the production of miRNAs and siRNAs is via distinct OsDCLs.     

Ebolavirus proteins suppress the effects of small interfering RNA by direct interaction with the mammalian RNA interference pathway

Cellular RNA interference (RNAi) provides a natural response against viral infection, but some viruses have evolved mechanisms to antagonize this form of antiviral immunity. To determine whether Ebolavirus (EBOV) counters RNAi by encoding suppressors of RNA silencing (SRSs), we screened all EBOV proteins using an RNAi assay initiated by exogenously delivered small interfering RNAs (siRNAs) against either an EBOV or a reporter gene. In addition to viral protein 35 (VP35), we found that VP30 and VP40 independently act as SRSs. Here, we present the molecular mechanisms of VP30 and VP35. VP30 interacts with Dicer independently of siRNA and with one Dicer partner, TRBP, only in the presence of siRNA. VP35 directly interacts with Dicer partners TRBP and PACT in an siRNA-independent fashion and in the absence of effects on interferon (IFN). Taken together, our findings elucidate a new mechanism of RNAi suppression that extends beyond the role of SRSs in double-stranded RNA (dsRNA) binding and IFN antagonism. The presence of three suppressors highlights the relevance of host RNAi-dependent antiviral immunity in EBOV infection and illustrates the importance of RNAi in shaping the evolution of RNA viruses.     

Transgene overexpression with cognate small interfering RNA in tobacco

Small interfering RNAs (siRNAs) are a key component of RNA silencing, including cosuppression. Here, we show an example in which siRNA does not serve in the downregulation of target genes. A tobacco endoplasmic reticulum omega-3 fatty acid desaturase (NtFAD3) catalyzes the formation of alpha-linolenate (18:3). Introduction of the NtFAD3 gene into tobacco plants caused strong reduction of 18:3 content in leaf tissues, which is associated with the production of the NtFAD3 siRNAs. However, this silencing effect was lacking in the root tissues. Both the introduced NtFAD3 and endogenous NtFAD3 genes were expressed successfully, and the roots showed increased 18:3 phenotype. Surprisingly, the NtFAD3 siRNAs were produced even in the root tissues. Expression of a hairpin double-stranded RNA against the NtFAD3 gene caused efficient reduction of 18:3 content in root tissues. Therefore, cosuppression of the NtFAD3 gene in tobacco appears to include an as yet unidentified developmental stage and tissue-specific mechanism of regulation of siRNA function.     

Genome reprogramming and small interfering RNA in the Arabidopsis germline

The movement of mobile small RNA signals between cells has garnered much interest over the last few years, and has recently been extended to germ cells during gamete development. Focusing on plants, we review mobile RNA signals that arise following reprogramming in the germline, and their effect on transposable element silencing on the one hand and on meiotic and apomictic germ cell fate on the other. A potential role for reprogramming and small RNA in hybrid formation and speciation is proposed.     

The hub protein loquacious connects the microRNA and short interfering RNA pathways in mosquitoes

Aedes aegypti mosquitoes vector several arboviruses of global health significance, including dengue viruses and chikungunya virus. RNA interference (RNAi) plays an important role in antiviral immunity, gene regulation and protection from transposable elements. Double-stranded RNA binding proteins (dsRBPs) are important for efficient RNAi; in Drosophila functional specialization of the miRNA, endo-siRNA and exo-siRNA pathway is aided by the dsRBPs Loquacious (Loqs-PB, Loqs-PD) and R2D2, respectively. However, this functional specialization has not been investigated in other dipterans. We were unable to detect Loqs-PD in Ae. aegypti; analysis of other dipteran genomes demonstrated that this isoform is not conserved outside of Drosophila. Overexpression experiments and small RNA sequencing following depletion of each dsRBP revealed that R2D2 and Loqs-PA cooperate non-redundantly in siRNA production, and that these proteins exhibit an inhibitory effect on miRNA levels. Conversely, Loqs-PB alone interacted with mosquito dicer-1 and was essential for full miRNA production. Mosquito Loqs interacted with both argonaute 1 and 2 in a manner independent of its interactions with dicer. We conclude that the functional specialization of Loqs-PD in Drosophila is a recently derived trait, and that in other dipterans, including the medically important mosquitoes, Loqs-PA participates in both the miRNA and endo-siRNA based pathways.     

Sorting of Drosophila small silencing RNAs partitions microRNA* strands into the RNA interference pathway

In flies, small silencing RNAs are sorted between Argonaute1 (Ago1), the central protein component of the microRNA (miRNA) pathway, and Argonaute2 (Ago2), which mediates RNA interference. Extensive double-stranded character—as is found in small interfering RNAs (siRNAs)—directs duplexes into Ago2, whereas central mismatches, like those found in miRNA/miRNA* duplexes, direct duplexes into Ago1. Central to this sorting decision is the affinity of the small RNA duplex for the Dcr-2/R2D2 heterodimer, which loads small RNAs into Ago2. Here, we show that while most Drosophila miRNAs are bound to Ago1, miRNA* strands accumulate bound to Ago2. Like siRNA loading, efficient loading of miRNA* strands in Ago2 favors duplexes with a paired central region and requires both Dcr-2 and R2D2. Those miRNA and miRNA* sequences bound to Ago2, like siRNAs diced in vivo from long double-stranded RNA, typically begin with cytidine, whereas Ago1-bound miRNA and miRNA* disproportionately begin with uridine. Consequently, some pre-miRNA generate two or more isoforms from the same side of the stem that differentially partition between Ago1 and Ago2. Our findings provide the first genome-wide test for the idea that Drosophila small RNAs are sorted between Ago1 and Ago2 according to their duplex structure and the identity of their first nucleotide.     

RNA interference and chemically modified small interfering RNAs

RNA interference (RNAi) is a powerful biological process for specific silencing of gene expression in diversified eukaryotic cells and has tremendous potential for functional genomics, drug discovery through in vivo target validation, and development of novel gene-specific medicine. The future success of this technology relies on identifying appropriate chemical modifications to improve stability, potency and in vivo cellular delivery. The present review summarizes the role of the chemist's toolbox in this emerging technology.