Institute of Medical Biochemistry
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Medical Biochemistry, II. year, 2002/2003
What Is SiRNA
 
RNA interference

In order to understand what is siRNA, the phenomenon of RNA interference, originally described in plants, the fly Drosophila, and the worm Caenorhabditis elegans, must be considered first. It is a post-transcriptional silencing of gene expression that occurs if a double-stranded RNA homologous with the gene appears in the cell.

The mechanism of RNA interference is following:
  1. The double-stranded is first cleaved by an RNase called Dicer to short fragments of dsRNA 21-23 bp, with 2-3 overhangs at 3´ ends:
  2. These fragments, designated small interfering RNA (siRNA), subsequently interact with a large (cca 500 kDa) multiprotein complex called RNA-inducing silencing complex (RISC).
  3. Helicase activity of RISC complex unwinds the siRNA double helix and only one strand remains associated with the complex.
  4. This siRNA strand then guides the whole complex to the target mRNA with which the siRNA strand is complementary, nuclease activity of RISC complex cleaves the mRNA and so begins its degradation by cellular RNases.

The result is a sequence-specific degradation of a particular mRNA. The original molecule of siRNA is not consumed, it can even be amplified when bound to mRNA. Cellular RNA-dependent RNA polymerases may use the siRNA strand as a primer and continue synthesis of double stranded RNA, which is then cleaved by the nuclease Dicer to further, secondary siRNA. It is an explanation for a high efficiency and long-lasting nature RNA interference.
Double-stranded RNA does not occur in the normal process of expression of eukaryotic genetic expression; however, it is a key intermediate in the genomic replication of many viruses. All higher eukaryotic cells respond to presence of double-stranded RNA; and the primary function of RNA interference in plants and invertebrates appears to be a defence against activity of viruses and transposons.
In mammalian cells the major defence, triggered by the dsRNA longer than cca 30 bp, lies in induction of interferon synthesis, which causes a non-specific degradation of RNA, inhibition of proteosynthesis, and ultimately apoptosis of infected cells. Nevertheless, the mammalian cells are also equipped with the RNase Dicer, and transfection by synthetic siRNA is able to induce the RNA interference, albeit without the amplification by RNA-dependent RNA polymerases.

StRNA and MiRNA

Small temporal RNA (stRNA) denotes products of two genes that in the worm C. elegans control timing of larval development. Transcripts of these genes code no proteins, but RNAs, which fold into a stem-loop ("hair-pin") structures. From them, the mentioned RNase Dicer cuts out the final fragments of stRNA 21-22 bp long, i.e. just as the siRNA, but single-stranded. These small temporal RNA (stRNA) subsequently interact with a protein complex analogous to RISC, and hybridise with homologous sequences in the 3´ untranslated ends of the target regulated mRNA. Unlike siRNA the result is not degradation of mRNA, but only inhibition of translation.
Further investigation revealed even in human cells virtually dozens of such small RNA. For the present called microRNA (miRNA) and with no known function, but it is likely they regulate gene expression in tissue development and other cellular functions. The originally described RNA interference, thus, may be just a tip of an iceberg of numerous activities of small RNAs.

Utilisation of siRNA

What is clear already now, however, is the fact that possibility of selective and specific gene silencing in mammalian cells by introducing a synthetic siRNA may be extremely useful. This technique seems to be much more efficient and easier than the previously developed approaches such as gene knock-outs, anti-sense oligonucleotides and ribozymes. Especially promising is the possibility to infect the cell with a plasmide or viral vector bearing DNA encoding RNA of the hair-pin structure, from which the cellular Dicer cuts out the effector fragment just as the endogenous stRNA and miRNA.
In the functional genomics usage of siRNA enables targeted inactivation of particular genes, yielding a valuable information on their function.
Next, siRNA can prove most useful in the antiviral therapy; it is especially considered as a new strategy against the HIV virus. The target need not be only the rapidly mutating RNA genome of the virus or its transcripts, but also the cell surface receptors for HIV virus, such as CD4 nebo CC chemokine receptor 5, or protein rev that is required for export of viral RNA from the nucleus. The strategy can work for other viruses as well or in gene therapy in general, and so it appears that the ancient mechanism of RNA interference hides a tremendous therapeutic potential.

Literature:

Kitabwalla M, Ruprecht RM: RNA interference - a new weapon against HIV and beyond. N. Eng. J. Med. 2002; 347 (17): 1364-7.
McManus MT, Sharp PA: Gene silencing in mammals by small interfering RNAs. Nat. Rev. Genet. 2002; 3 (10): 737-47.
Cullen BR: RNA interference: antiviral defense and genetic tool. Nat. Immunol. 2002; 3 (7): 597-9.
Ahlquist P: RNA-dependent RNA polymerases, viruses, and RNA silencing. Science 2002; 296 (5571): 1270-3.
Moss EG: RNA interference: it's a small RNA world. Curr. Biol. 2001; 11 (19): R772-5.

MUDr. Jan Pláteník, PhD.
 
 
 
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