Question

Why would RNAi be a less efficient mechanism for regulating the expression of specific genes if Dicer hydrolyzed double-stranded RNA every 11 bp rather than every 22 bp?

Answer 1

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 the 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.

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 the 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.

During the occurrence of RNAi/PTGS, double-stranded RNA (dsRNA) molecules, which cleave the inducer molecules into smaller pieces first (16) and eventually destroy the cellular or viral cognate mRNA molecules (called the target) (17) act as inducers or activators of this process. As a result, the target mRNAs cannot accumulate in the cytosol, although they remain detectable by nuclear run-on assays (73). In certain instances, the DNA expressing the target mRNA also undergoes methylation as a by-product of the degradation process (226).

The natural functions of RNAi and its related processes seem to be the protection of the genome against invasion by mobile genetic elements such as viruses and transposons as well as orchestrated functioning of the developmental programs of eukaryotic organisms. There are several excellent recent reviews which deal with different aspects of RNAi separately (95, 191). Here, we have put together the various aspects of the RNAi process known to date, identified the mechanistic similarities and differences operating in various forms of eukaryotic life, and focused on the experimental results that have led to conceptual advancements in this field.

RNA interference (RNAi) is the process by which the expression of a target gene is effectively silenced or knocked down by the selective inactivation of its corresponding mRNA by double-stranded RNA (dsRNA). RNAi is activated by dsRNA species delivered to the cytoplasm of cells. The silencing mechanisms can either lead to the degradation of a target mRNA, as induced by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), or the suppression of translation of specific mRNAs, as induced by microRNA (miRNA). The focus of this review will be how shRNAs and siRNAs lead to protein knockdown. Through the activity of several proteins (discussed below), targeting of a cellular mRNA by short, anti-sense nucleic acids (siRNAs and shRNAs) results in its subsequent degradation. This, in turn, blocks further expression/accumulation of the proteins, leading to a decrease in its levels, and eventual knockdown