RNAi Screening
The starting point for RNA interference was discovered by Guo and Kemphues in 1995 whilst attempting to use antisense RNA to block gene expression in the maternal germ cell line. During their experiments, they came accross the suprising development that both antisense and sense RNA could silence the Par1 gene in C. Elegans.
Work on this principle continued until, in 2006, the Nobel prize in Physiology or Medicine was awarded to Andrew Fire and Craig Mello for 'the discovery that double-stranded RNA triggers suppression of gene activity in a homology-dependent manner', which they termed RNA interference.
RNA interference works using the following mechanism:
- Long (around 200 base pairs), double stranded RNA (dsRNA) which matches the target gene is introduced to the target cell.
- A specialised 'Dicer' enzyme then cuts the long dsRNA down to molecules of around 21 to 25 base pairs. These are known as 'short interfering RNA' or siRNA.
- These siRNAs form a complex with other proteins and become what is known as a RNA-induced silencing complex (RISC), which activates when the siRNA component is unzipped by ATP driven helicase.
- The RISC is then able to recognise and bind to target mRNA and cleave it using the protein subunits.
- The mRNA is then degraded, preventing protein production.
An extremely useful ability of these RNAs in C. elegans is that they are systemic - they can cross cell boundaries, like the epithelial boundary of the gut. This ability is not conserved in flies or other mammals which makes C. elegans an extremely valuable model organism.
Research into dilute RNAs suggest that the perfect target tissue for injections is the intestine, even if the target gene is expressed in a tissue in a different area of the body, for instance muscle tissue. It gives researchers the ability to perform high volume loss-of-function analyses of gene function.
A further advantage is that C. elegans can now be 'fed' these dsRNAs via a plasmid, in E. coli for example, which is much quicker than injection and much more cost effective.
RNAi is used as a tool for directly relating the action of a gene to a specific function, using its knock-out ability to determine a genes effects. These attributes enable scientists to research a huge array of disease causing genes, using the model of C. elegans.
Recently, large-scale RNAi screens have lead to the discovery of hundreds of new longevity genes in the roundworm Caenorhabditis elegans, which could help in the research into aging and senescence in humans.
References:
Genetic Requirements for Inheritance of RNAi in C. elegans
Genome-wide RNAi screens in Caenorhabditis elegans: impact on cancer research
RNAi screens to identify components of gene networks that modulate aging in Caenorhabditis elegans
RNAi in C. elegans: Soaking in the Genome Sequence
The Nobel Prize in Physiology or Medicine 2006 Andrew Z. Fire, Craig C. Mello
par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed.
