RNA technologies including RNA interference
RNA Interference
1980s it was discovered that single-stranded RNA molecules in the bacterium Escherichia coli
can bind to mRNA.
This inactivates mRNA so the protein encoded by the mRNA is no longer formed.
Around 1990, plant scientists were trying to intensify the color of red petunias.
Inserted a gene that controls formation of red pigment – but in some cases the result was that
the flowers lost their color altogether.
mRNA for the pigment had disappeared.
Andrew Fire and Craig Mello published their discovery of RNA interference in 1998.
Double-stranded RNA (dsRNA) plays an active part in regulating gene activity.
These RNA molecules control degradation of mRNA molecules by biochemical machinery.
The expression of the gene for a specific protein can be abolished: the gene is silenced.
The production of the protein is turned off.
RNA interference is a natural mechanism of great importance in regulation of the activity of our
genes.
It is a highly useful research tool.
In basic research, RNA interference is primarily used to study the function of specific genes.
RNA interference may be used to treat diseases.
Genes are silenced when mRNA is eliminated
Together, sense RNA and the corresponding antisense RNA make up double-stranded RNA, and
it seemed as if this could silence the gene that encoded the muscle protein.
With a special staining technique they could show mRNA for genes that were active in
nematode embryos.
When the gene was active, the embryo could be stained because its cells contained the mRNA.
Antisense RNA binding to the mRNA for this gene reduced the staining somewhat.
But doublestranded RNA (sense RNA plus antisense RNA) eliminated the staining entirely – the
mRNA had disappeared and the gene had been silenced.
Double-stranded RNA extinguished the gene it had been patterned on.
This stopped the formation of the protein encoded by the gene.
Double-stranded RNA could interfere with the expression of genes; thus the phenomenon was
called RNA interference.
Fire’s and Mello’s conclusions
• Gene silencing was highly effective when doublestranded RNA was injected but weak or nonexistent after injection of single-stranded RNA (sense RNA or antisense RNA for the gene).
• The mRNA affected by the double-stranded RNA disappeared – it was apparently broken
down and eliminated.
• The double-stranded RNA injected must match the mature, “trimmed” mRNA sequence for
the gene.
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Interference could not be elicited by intron sequences. This implies that the interference takes
place after transcription, probably in the cytoplasm rather than in the cell nucleus.
• Only the mRNA corresponding to the sense RNA strand of the double-stranded RNA was
silenced – no other mRNA in the cells was affected. RNA interference was specific for the gene
with a code corresponding to that of the mRNA molecule.
• Just a few double-stranded RNA molecules were needed to silence a gene completely. The
effect was so strong that Fire and Mello suggested that enzymes were involved in the process.
• The effect of double-stranded RNA could spread from cell to cell and from tissue to tissue and
could even be passed on to offspring.
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http://www.youtube.com/watch?v=Vh3-NHdjnyQ
Regulation of gene expression
Several hundred genes in our genome encode small RNA molecules collectively called
microRNA, which contain segments of codes from other genes.
Precursors of these microRNA molecules form hairpin structures of double-stranded RNA.
It is these molecules that activate the RNA interference machinery.
The result is that mRNA from a gene with a similar coding sequence is broken down or blocked
so that the protein cannot be produced.
Use of microRNA molecules to regulate gene expression has turned out to be fundamental for
eukaryote cells – from single-cell organisms to humans. RNA interference plays an important
role in switching off genes during an organism’s development and to control cellular function.
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MicroRNAs
MicroRNAs (miRNAs) are small non-coding RNAs that regulate various target genes and play
important roles in cell proliferation, apoptosis, and differentiation.
One of the important mechanisms of miRNA expression is epigenetic alteration such as DNA
methylation and histone modification.
The CCCTC- binding factor, CTCF, is known to bind insulators and exhibits an enhancer-blocking
and barrier function, and more recently, it also contributes to the three-dimensional
organization of the genome.
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RNA and Exosomes
Ribonucleic acid (RNA) was once thought to exist in a stable form only inside cells, where it
served as an intermediate in the translation from genes to proteins.
However, recent research has shown that RNAs can play a role in a variety of complex cellular
functions, including newly discovered mechanisms of cell-to-cell communication.
RNA can be exported from cells in extracellular vesicles or bound to lipids or proteins, to
circulate through the body and affect cells at a great distance.
These extracellular RNAs, or “exRNAs,” may also be absorbed from food, the microbes that live
in our bodies, or the environment, producing a variety of biological responses.
However, the actual impact of these exRNAs is not known.
An opportunity exists to find out about intercellular and inter-species information exchange
based on the release, transport, uptake, and regulatory role of exRNAs.
Here is a video about
Unlocking the Mysteries of Extracellular RNA Communication
http://www.youtube.com/watch?v=bfMg3RhxNm8
What are exosomes?
https://www.youtube.com/watch?v=sE2krsErbwI
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Exosomes are small membrane bound vesicles sharing similar topology to the plasma
membrane.
- contain mRNA and miRNA, and many different proteins depending on their host cell.
- generally all enriched in proteins involved in MVB formation, tetraspanins, membrane
transport and fusion and a number of cytosolic proteins.
In addition to these generic proteins, proteins associated with neurodegenerative diseases such
as Alzheimer’s, Prion disease, and Parkinson’s disease have been identified in exosomes.
Video : History and promise of exosomes https://www.youtube.com/watch?v=Ry9kL-R-7E8
Video : Exosomes in cancer research https://www.youtube.com/watch?v=SZZ_-fnkEVc
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