Keywords: double stranded RNA, post-transcriptional gene silencing, RNA interference, transcriptional gene silencing.

RNA silencing is a remarkable type of gene regulation. This process has been found to occur in many different organisms such as plants (co-suppression), fungi (quelling), and animals (RNA interference; RNAi). Double-stranded RNA (dsRNA) is a potent trigger in RNA silencing mechanisms operating in a wide range of organisms.RNA silencing was first discovered in transgenic plants where it was termed co-suppression or post-transcriptional gene silencing (PTGS). At the genome level, RNA can induce the epigenetic modification of homologous DNA sequences through the process of RNA-directed DNA methylation (RdDM), that has only been demonstrated in plants.Two distinct gene silencing phenomena are observed in plants: transcriptional gene silencing (TGS), which involves decreased RNA synthesis because of promoter methylationand post transcriptional gene silencing (PTGS), which involves sequence specific RNA degradation.

RNA-directed DNA methylation (RdDM) and post-transcriptional gene silencing/RNA interference (PTGS/RNAi) are both triggered by dsRNAs that are cleaved by RNAseIII-type enzymes (i.e. Dicer, Caf) into small interfering RNAs (siRNAs), probably in both the nucleus and the cytoplasm. In the cytoplasm, the siRNAs serve as guides for endonucleolytic cleavage of homologous mRNA in association with the RNA-induced silencing complex (RISC). DsRNA can trigger RdDM.

RNA-dependent DNA methylation (RdDM) was first discovered with viroids, which are plant pathogens consisting of non-coding, highly base paired, rod shaped RNA several hundred nucleotides in length. RdDM results in dense methylation at most symmetrical and non-symmetrical cytosines within the region of homology between the inducing RNA and the target DNA. DNA targets as short as 30 bp can be modified. RdDM provides an alternate means to induce the sequence specific methylation observed in both PTGS and TGS.

The mechanism of RdDM is unknown but is assumed to involved RNA-DNA interactions based on sequence homology. The minimal DNA target size for RdDM of 30 bp opens the possibility that the 21-25 nucleotides (nt) RNA degradation products of dsRNA could be responsible for directing de novo methylation. The methylation of promoter sequences usually results in promoter inactivation, probably by histone deacetylation and chromatin condensation. RNA interference is a conserved process in which double-stranded RNA is processed into 21–25 nucleotide siRNAs that trigger posttranscriptional gene silencing. In addition, plants display a phenomenon termed RNA-directed DNA methylation (RdDM) in which DNA with sequence identity to silenced RNA is de novo methylated at its cytosine residues.

Post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals: are responses to various types of foreign nucleic acid including viruses, transposons, transgenes and dsRNA. These processes represent natural defense mechanisms against viruses and transposons. The silencing effect can be transmitted systemically from a silenced regions (stock) to unsilenced region (scions) in plants and so that the silencing signal can travel over a long distance through the plant’s vascular system. A remarkable characteristic of PTGS in plants and fungi is that diffusible trans-acting molecules mediate it, which is able to mediate gene silencing between nuclei in heterokaryotic strains.

The cloning of genes coding for cellular components of the PTGS machinery in different system such as Neurospora crassa, Caenorhabditis elegans and Arabidopsis thaliana has demonstrated the existence of a common genetic basis PTGS. RdRP plays a key role in PTGS . Initial models proposed that copy antisense RNA (cRNA) could be produced by an RdRP on a sense transgenic mRNA, usually referred to as aberrant RNA (aRNA) and that is able to mediate gene silencing in plants and fungi through diffusible signal.

RNA based silencing mechanisms which are effective at the genome level and in the cytoplasm, are able to combat parasitic sequences that have an RNA genome (RNA viruses) or a dsRNA replication intermediate. The host defence function of RNA-mediated silencing is demonstrated by the increased sensitivity of Arabidopsis PTGS mutants to some viruses and the mobilization of transposons in RNAi mutants  of C. elegans and in the Mut-6 mutant of Chlamydomonas. The native function of PTGS is most likely in providing resistance to virus infection. It has been found that many viruses are potent inducers of PTGS and viruses encode factors that inhibit this response of the plant . RNA silencing is an evolutionarily conserved surveillance systemthat occurs in a broad range of eukaryotic organisms. In plants,RNA silencing acts as an antiviral system; thus, successfulvirus infection requires suppression of gene silencing . In plants, PTGS has been strictly linked to RNA virus resistance mechanisms.  Plant RNA viruses are in fact both triggers and targets for PTGS, and display increased sensitivity to viral infections.

The involvement of dsRNA and DNA methylation in both TGS and PTGS  type of silencing is increasingly recognized. RNA helicases are potential candidate for the RdDM machinery as it is likely that RNA-DNA paring requires a ssRNA that is complementary to target DNA. We do not know the details of the silencing mechanism, especially not of the way transposons feed into it. It seems that copy number is bad news. We are proposing that the dsRNA may be a red signal for multicopy DNA. As soon as multiple copies are found in the genome, there is bound to be transcription of one strand here and of another strand there, allowing formation of dsRNA. This field is still in its romantic phase. Several questions will remain. Is the silencing effect replicated, and how? How does the guide RNA can find its target and ear mark it for degradation? How does the trigger RNA migrate in plants and animals? How does the mechanism feed back into the nucleus to silence gene transcription? How does an epigenetic modification influence the initiation and maintenance of PTGS and RdDM mediated TGS? Thus, genetics as well as biochemistry will need to be more exercised to resolve all these questions.

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