Possible Role for RNA-dependent RNA Polymerase
Genetic screens in Neurospora, C. elegans, and Arabidopsis have identified several genes that appear to be crucial for PTGS and RNAi. Several of these, including Neurospora qde-1, Arabidopsis SDE-1/SGS-2 and C. elegans ego-1, appear to encode RNA-dependent RNA polymerases (RdRPs). At first glance, it might be assumed that this is proof that an RdRP activity is required for RNAi. Certainly the existence of an RdRP might explain the remarkable efficiency of dsRNA-induced silencing if it amplifed either the dsRNA prior to cleavage or the siRNAs directly. But mutants of these genes have varying phenotypes, which makes the role of RdRP in RNAi difficult to discern (1, 3, 17, 18).

In C. elegans ego-1 mutants ("ego" stands for "enhancer of glp-1"), RNAi functions normally in somatic cells, but is defective in germline cells where ego-1 is primarily expressed. In Arabidopsis SDE-1/SGS-2 mutants ("SGS" stands for suppressor of gene silencing), siRNAs are produced when dsRNA is introduced via an endogenously replicating RNA virus, but not when introduced by a transgene. It has been proposed that perhaps the viral RdRP is substituting for the Arabidopsis enzyme in these mutants (1, 3, 17, 18). Although no homolog of an RdRP has been found in flies or humans, an RdRP activity has recently been reported in Drosophila embryo lysates (30). One model of amplification, termed the "random degradative PCR" model, suggests that an RdRP uses the guide strand of an siRNA as a primer for the target mRNA, generating a dsRNA substrate for Dicer and thus more siRNAs (27, 30). Evidence supporting this model has been found in worms, whereas experimental results refuting the model have been obtained from Drosophila embryo lysates (26, 27).

RNAi Initiators
Two C. elegans genes, rde-1 and rde-4 ("rde" stands for "RNAi deficient"), are believed to be involved in the initiation step of RNAi. Mutants of these genes produce animals that are resistant to silencing by injection of dsRNA, but silencing can be effected in these animals by the transmission of siRNA from heterozygous parents that are not silencing deficient. The C. elegans rde-1 gene is a member of a large family of genes and is homologous to the Neurospora qde-2 ("qde" stands for "quelling deficient") and the Arabidopsis AGO1 genes ("AGO" stands for "argonaute"; AGO1 was previously identified to be involved in Arabidopsis development). Although the function of these genes in PTGS is unclear, a mammalian member of the RDE-1 family has been identified as a translation initiation factor. Interestingly, Arabidopsis mutants of AGO1, which are defective for cosuppression, also exhibit defects in leaf development. Thus some processes or enzymes involved in PTGS may also be involved in development (1, 3, 17, 18).

RNAi Effectors
Important genes for the effector step of PTGS include the C. elegans rde-2 and mut-7 genes. These genes were initially identified from heterozygous mutant worms that were unable to transmit RNAi to their homozygous offspring (16). Worms with mutated rde-2 or mut-7 genes exhibit defective RNAi, but interestingly, they also demonstrate increased levels of transposon activity. Thus, silencing of transposons appears to occur by a mechanism related to RNAi and PTGS. Although the rde-2 gene product has not yet been identified, the mut-7 gene encodes a protein with homology to the nuclease domains of RNase D and a protein implicated in Werner syndrome (a rapid aging disease) in humans (1, 3, 17, 18, 31). Perhaps this protein is a candidate for the nuclease activity required for target RNA degradation.

PTGS Has Ancient Roots
Discoveries from both genetic and biochemical approaches point to the fact that PTGS has deep evolutionary roots. Proposals have been put forth that PTGS evolved as a defense mechanism against transposons or RNA viruses, perhaps before plants and animals diverged (1, 3, 17, 18).

Interestingly, it was noted by many researchers that disruption of genes required for RNAi often causes severe developmental defects. This observation suggested a link between RNAi and at least one developmental pathway.

A group of small RNA molecules, known as small temporal RNAs (stRNAs), regulates C. elegans developmental timing through translational repression of target transcripts. Research indicates that the C. elegans lin-4 and let-7 stRNAs are generated from 70-nt transcripts following the folding of these longer transcripts into a stem-loop structure. The folded RNA molecules are cleaved to produce 22-nt stRNAs by the enzyme Dicer (called DCR-1 in C. elegans). Thus Dicer generates both siRNAs and stRNAs, and represents an intersection point for the RNAi and stRNA pathways (32-34).

Recently, nearly 100 additional ~22 nt RNA molecules, termed microRNAs (miRNAs), were identified in Drosophila, C. elegans, and HeLa cells (35-38). Much like lin-4 and let-7, these miRNAs are formed from precursor RNA molecules that fold into a stem-loop secondary structure. The newly discovered ~22 nt miRNAs are believed to play a role in regulation of gene expression, and at least two of them are known to require Dicer for their production (37). It appears that the use of small RNAs for both gene regulation and RNAi is a common theme throughout evolution.

Non-specific Gene Silencing by Long dsRNAs
While the natural presence of RNAi had been observed in a variety of organisms (plants, protozoa, insects, and nematodes), evidence for the existence of RNAi in mammalian cells took longer to establish. Transfection of long dsRNA molecules (>30 nt) into most mammalian cells causes nonspecific suppression of gene expression, as opposed to the gene-specific suppression seen in other organisms. This suppression has been attributed to an antiviral response, which takes place through one of two pathways.

In one pathway, long dsRNAs activate a protein kinase, PKR. Activated PKR, in turn phoshorylates and inactivates the translation initiation factor, eIF2a, leading to repression of translation. (39) In the other pathway, long dsRNAs activate RNase L, which leads to nonspecific RNA degradation (40).

A number of groups have shown that the dsRNA-induced antiviral response is absent from mouse embryonic stem (ES) cells and at least one cell line of embryonic origin. (41, 42) It is therefore possible to use long dsRNAs to silence specific genes in these specific mammalian cells. However, the antiviral response precludes the use of long dsRNAs to induce RNAi in most other mammalian cell types.

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