RNA interference: biology, mechanism, and applications

Abstract

Double-stranded RNA-mediated interference (RNAi) is a simple and rapid method of silencing gene expression in a range of organisms. The silencing of a gene is a consequence of degradation of RNA into short RNAs that activate ribonucleases to target homologous mRNA. The resulting phenotypes either are identical to those of genetic null mutants or resemble an allelic series of mutants. Specific gene silencing has been shown to be related to two ancient processes, cosuppression in plants and quelling in fungi, and has also been associated with regulatory processes such as transposon silencing, antiviral defense mechanisms, gene regulation, and chromosomal modification. Extensive genetic and biochemical analysis revealed a two-step mechanism of RNAi-induced gene silencing. The first step involves degradation of dsRNA into small interfering RNAs (siRNAs), 21 to 25 nucleotides long, by an RNase III-like activity. In the second step, the siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the cognate mRNA and degrades it. Several key components such as Dicer, RNA-dependent RNA polymerase, helicases, and dsRNA endonucleases have been identified in different organisms for their roles in RNAi. Some of these components also control the development of many organisms by processing many noncoding RNAs, called micro-RNAs. The biogenesis and function of micro-RNAs resemble RNAi activities to a large extent. Recent studies indicate that in the context of RNAi, the genome also undergoes alterations in the form of DNA methylation, heterochromatin formation, and programmed DNA elimination. As a result of these changes, the silencing effect of gene functions is exercised as tightly as possible. Because of its exquisite specificity and efficiency, RNAi is being considered as an important tool not only for functional genomics, but also for gene-specific therapeutic activities that target the mRNAs of disease-related genes.

FIG. 1.

Schematic illustration of systemic viral spread as well as RNAi and subsequent viral recovery in plants. Green and red indicate the presence and loss of GFP fluorescence, respectively, and orange denotes the presence of both colors. The red dots on leaves show viral lesions. The bold arrows indicate the stages of plant growth, and the leaves are numbered accordingly. An arrow with a thin line shows a newly emerged leaf recovered from viral attack.

FIG. 2.

Two-step model for the mechanism of gene silencing induced by double-stranded RNA. In step I, dsRNA is cleaved by the Dicer enzyme to produce siRNAs. A putative kinase seems to maintain 5′ phosphorylation at this step. The siRNAs have also been proposed to be responsible for nuclear DNA methylation (•) and systemic spread of silencing. Amplification might occur due to the presence of RdRP (▴). In step II, the siRNAs generated in step I bind to the nuclease complex (RISC). A helicase present in the complex might activate RISC by unwinding the siRNAs. The antisense component of siRNA in the RISC guides the complex towards the cognate mRNA (—), resulting in endonucleolytic cleavage (↓) of the mRNA. RdDM, RNA-dependent DNA methylation.

FIG. 2.

Two-step model for the mechanism of gene silencing induced by double-stranded RNA. In step I, dsRNA is cleaved by the Dicer enzyme to produce siRNAs. A putative kinase seems to maintain 5′ phosphorylation at this step. The siRNAs have also been proposed to be responsible for nuclear DNA methylation (•) and systemic spread of silencing. Amplification might occur due to the presence of RdRP (▴). In step II, the siRNAs generated in step I bind to the nuclease complex (RISC). A helicase present in the complex might activate RISC by unwinding the siRNAs. The antisense component of siRNA in the RISC guides the complex towards the cognate mRNA (—), resulting in endonucleolytic cleavage (↓) of the mRNA. RdDM, RNA-dependent DNA methylation.

FIG. 2.

Two-step model for the mechanism of gene silencing induced by double-stranded RNA. In step I, dsRNA is cleaved by the Dicer enzyme to produce siRNAs. A putative kinase seems to maintain 5′ phosphorylation at this step. The siRNAs have also been proposed to be responsible for nuclear DNA methylation (•) and systemic spread of silencing. Amplification might occur due to the presence of RdRP (▴). In step II, the siRNAs generated in step I bind to the nuclease complex (RISC). A helicase present in the complex might activate RISC by unwinding the siRNAs. The antisense component of siRNA in the RISC guides the complex towards the cognate mRNA (—), resulting in endonucleolytic cleavage (↓) of the mRNA. RdDM, RNA-dependent DNA methylation.

FIG. 3.

Role of the RNAi process in heterochromatinization of nuclear DNA. The methylated (H3-K9) histone and many chromatin components are involved in the cross talk in several epigenetic regulatory pathways within the nucleus. The centromeric region (black oval) of the chromatin (thick purple line) might be responsible for the production of dsRNA transcripts (continuous red line and broken blue line). The transcript coming out of the DNA strand subjected to heterochromatinization is represented by broken blue lines. The siRNAs of the nucleus join the complex, of which the histone methyltransferase is a constituent. The siRNA binding the chromodomain (CHR), methyltransferase (MTase), and the SET domain of the methyltransferase are indicated.