siRNA Pathway in Nematode and Mammal
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siRNA Pathway in Nematode and Mammal
Introduction of dsRNA (double-stranded RNA), that are homologous in sequence to a gene, has proven to suppress that gene’s expression through a process known as RNAi (RNA interference). The mechanism of RNAi involves the breaking of a dsRNA matching a specific gene sequence into siRNA (Small interfering RNAs). These siRNAs are 21-23nt dsRNA duplexes with symmetric 2-3nt 3 overhangs and 5-phosphate and 3-hydroxyl groups trigger the degradation of mRNA that match its sequence (Ref.1). Interference of gene expression by siRNA is now recognized as a naturally occurring biological strategy for silencing alleles during development in plants, invertebrates, and vertebrates. siRNAs appears to suppress gene expression without producing a non-specific cytotoxic response. It is believed that the small size of the siRNAs, as compared with dsDNA, prevents activation of the dsRNA-inducible interferon system in mammalian cells. This avoids the non-specific phenotypes normally produced by dsRNA (>30 base pairs) (Ref.2).

RNA silencing occurs in a broad range of eukaryotic organisms including fungi (quelling), animals (RNAi), and plants (Post-Transcriptional Gene Silencing [PTGS]). In all these organisms, the process is triggered by dsRNA and requires a conserved set of gene products (Ref.1). In some organisms, such as Caenorhabditis elegans, Arabidopsis, and Neurospora, RNAi requires a target RNA copying step, without which siRNAs fail to reach sufficient concentration to accomplish target mRNA cleavage. A family of RdRPs (RNA-dependent RNA Polymerases) is essential to amplify the original silencing signal, generating secondary siRNAs, thus allowing a more sustained RNAi response from the siRNAs. In C. elegans, the dsRNA precursor of siRNA is processed into the functional siRNA by the rde-4, which recruits Dicer Ribonuclease-III) to generate primary siRNAs. The primary siRNA is then passed to downstream components of the RNAi pathway through rde-1 (Ref.3). The siRNAs can now travel through two pathways; 1) a strand of the siRNA can be processed into aberrant RNA by RISC, then RdRP initiates primer-independent copying at the 3 end of the aberrant RNA, converting it into dsRNA; 2) the siRNA can also bypass RISC processing and can be directly converted into dsRNA by RdRP which uses one of the siRNA strands as a primer. The dsRNA resulting from RdRP copying of an aberrant transcript is then converted by Dicer into secondary siRNAs, which, as part of a RISC Complex, could destroy additional aberrant RNA from the transgene, as well as transcripts from an endogenous gene of corresponding sequence, leading to the silencing of both transgene and endogenous gene.

Endogenously expressed siRNAs have not been found in mammals. However, the related miRNAs (microRNAs) have been cloned from various organisms and cell types. These short RNA species (~22nt) are produced by Dicer cleavage of longer (~70nt) endogenous precursors with imperfect hairpin RNA structures. These miRNA precursor hairpin or long dsRNA lead to a single-stranded 21-23nt RNA that is associated with the miRNP (microribonucleoprotein)-RISC Complex. This complex directs either mRNA translational repression or mRNA target cleavage, depending on the degree of complementarity between the 21-23nt RNA and the mRNA. Many of the miRNAs and siRNAs might reside in the same silencing complex that contains the human Argonaute protein, eIF2C2 (a eukaryotic translation Initiation Factor), Gemin3 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 20) and Gemin4 (Gem (nuclear organelle) associated protein4) (Ref.4).

RNA silencing, which is active at different levels of gene expression in the cytoplasm and the nucleus, appears to have evolved to counter the proliferation of foreign sequences, such as TEs (Transposable Elements) and viruses, many of which produce dsRNAs during replication. RNA silencing is proving to be useful for the study of functional genomics in invertebrates and plants. It is likely that gene vectors that encode siRNAs or miRNAs will become widely accepted as gene-knockout tools. It is also foreseeable that these new RNAi constructs could be coupled to lentivirus, adenovirus or other delivery vectors that are used in gene therapy, which paves the way for a new wave of therapeutic molecules in the years to come. It is certain that the ability of siRNAs to silence specific genes, either when transfected directly as siRNAs or when generated from DNA vectors, will transform future studies of cellular systems and biology in mammalian cells (Ref.5).