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[SANET-MG] small RNA the new genetics
April 5, 2006
Prof. Joe Cummins
Small RNA regulates genes in a large way
For many years the view has been that plant and animal genes are
regulated by small gene switches called promoters which are attached to
the structural genes that specify proteins. The promoter switches
respond to proteins produced from controlling genes called repressors
which down regulate the target gene and inducers which trigger and up
regulate the target gene. The regulatory genes are most frequently
constitutive meaning that the genes have a constant low level production
of regulatory protein. Until recently promoters and regulatory genes
dominated investigations in gene regulation, only facing refinement
following discovery of split genes which separated genes into exons
specifying domains of protein activity and introns code sequences
separating exons that are split out of the pre message RNA sequences for
the protein. Intron splitting and messenger RNA splicing takes place in
the nucleus and that process regulates release of messenger RNA to the
cytoplasm where it is translated into protein.
The fundamental model for gene regulation involving regulatory genes
specifying proteins that controlled activity of structural genes that
specified cellular constituents and the development of tissues and
organs was essentially the only genetic model for cellular processes
until the early 1990s. In the 1990s when very small control sense RNA
(control sense means a code sequence that does not specify protein but
does regulate genes) oligonucleotides (RNA chains around 21 to 25
nucleotides long) were found to reduce expression of specific genes in
fungi, plants and worms. Later the vertebrate animals were found to
employ small control sense RNA (RNAi). It is now clear that RNAi is a
universal mechanism for controlling gene action including bacteria. Gene
silencing controlled by RNAi regulates basic biological processes ,
including transition from one stage of development to another.
Furthermore, RNAi is used as a form of immunity to protect the cell from
invasion by foreign nucleic acids introduced by mobile genetic elements
and transposons. RNAi has begun to impact genetic engineering and direct
RNA therapy to treat disease(1).
*RNAi has several pathways for gene silencing*. All of the pathways use
small double stranded molecules made up of RNA chains 21 to 25
nucleotides long, with 2 to 3 nucleotide single strand at the tail (3’)
end. The RNAi chains are cut from long double stranded RNA molecules
using a RNase 111 enzyme (dicer) in the cell cytoplasm. The RNAi
molecules are joined to a nucleoprotein complex called RNA induced
silencing complex (RISC). The antisense (guide) strand of RNAi directs
the RNA cutting activity of RISC to the homologous (target) site on the
RNA message to be inactivated by cleavage(1).RNAi belongs to a large
class of small RNAs called micro RNA (miRNA) many miRNAs have not yet
been assigned a function. In plants there are three RNA silencing
pathjways.The first is post transcriptional gene silencing using RNAi
cut from large double stranded RNA from replicating plant viruses,
transgenic inverted repeats and products of RNA dependent RNA
polymerase. The second is a class of miRNA cleaved from miRNA genes that
target specific RNA messages leading either to degradation of the mRNA
or to posttranscriptional gene silencing. The third pathway is
transcriptional gene silencing with RNAi directed chromatin re-modeling
and DNA and histone methylation (2).
*RNAi provides a defense against viruses and transposable elements in
both plants and animals.* The main defense against viruses is the
recognition and silencing of double stranded RNA replication
intermediates. Some plant viruses defend against RNAi bu producing
proteins that recognize and inactivate RNAi. Pathogenic viroids
(infectious RNAs that do not code for any protein) cause disease by
suppressing host gene expression. Transposable elements are found in
most organisms and make up as much as 40% (as in human) or more of the
genome. Once activated these genetic elements cuase extensive mutation
as they move about the genome. The RNAi provides a sequence specific
defense against the mobile genetic element. RNAi has begun to enter the
control of viruses in plants and animals alike. The main approaches
involve gene therapy and the use of stabilized RNAi to treat infected
animals Monkeys treated with RNAi regulating apolipoprotein B
ecapsulated in nucleic acid lipid particles responded quickly to the
treatment reducing the regulated protein 90%. The treatment was stronger
and longer lasting than had been expected from rodent studies (1,3,4,5).
*RNAi is a key to development *. Micro RNA and RNAi have been shown to
govern transition between developmental stages in plants in a manner
similar but not identical to animals. RNAi plays a direct role in
development and as a regulator of regulators such as the transcription
factors. The micro RNA and RNAi regulatory pathways in plants and a
comparison of the pathways in plants and animals is described in
references (6) and (7). Synthetic designer plantRNAi genes have been
used to “knock down” specific genes or to knock down a whole gene family
simultaneously and to be active in Arabidopsis, tomato and tobacco (8).
It is presently unclear how close transgenic RNAi crops are to approval.
Field test releases in the United States do not overtly identify RNAi
anti-virus crops but many field test releases of virus resistant crops
designate the gene tested to be confidential business information.
However, it is clear that a flood of crops modified with RNAi genes will
soon appear.
Engineered RNAi interference based resistance to dengue virus type 2 was
achieved in genetically modified mosquitoes (Aedes aegypti ).The
modification was achieved using a mariner transposon vector Fuller
genetic modification will be required to achieve full resistance to the
virus. Dengue virus infects an estimated 50 million people world wide
each year. It may be possible to replace most of the virulent mosquitoes
with pathogen resistant vectors. (9). Two problems are evident in this
development, the first is the appearance of resistant virus similar to
the RNAi resistant plant viruses which employ RNAi suppressors. The
promoters of anti-dengue RNAi discounted suppressors without considering
experimental verification that suppressors will not appear in the
mosquito. The second problem is the insertion of anti-dengue RNAi into
insect bites. The response of mammals including human are presently
untested. If the RNAi does not provoke harmful side effects it may prove
immediately useful for treating those infected with the dengue virus.
Discovery of RNAi provides a major discovery in genetics. The deployment
of that discovery in human and animal therapy along with its use in
developing enhanced food crops should be done with care and human
exposure to the synthetic genes should be done only after adequate
testing of the products . Exposure of the public in feed , food or in
therapy should be accompanied by full disclosures. Deployment of such
powerful genes should not be done on the cover of confidential business
information. Suddenly, everything changed in genetics, we can never go
back to where it was so we have to get used to it and to insure that the
old problems related too much secrecy and too little testing are not
repeated in the brave new world.
References
1. Dykxhoorn DM and Lieberman J. The silent revolution: RNA
interference as basic biology, research tool, and therapeutic.
Annu Rev Med. 2005,56:401-23
2. Qi Y and Hannon GJ. Uncovering RNAi mechanisms in plants:
biochemistry enters the foray. FEBS Lett. 2005 Oct 1;579(26):5899-903
3. Buchon N and Vaury C. RNAi: a defensive RNA-silencing against
viruses and transposable elements. Heredity. 2006 Feb;96(2):195-202
4. Leonard JN and Schaffer DV. Antiviral RNAi therapy: emerging
approaches for hitting a moving target. Gene Ther. 2006
Mar;13(6):532-40
5. Zimmermann TS, Lee AC, Akinc A, Bramlage B, Bumcrot D, Fedoruk MN,
Harborth J, Heyes JA, Jeffs LB, John M, Judge AD, Lam K,
McClintock K, Nechev LV, Palmer LR, Racie T, Rohl I, Seiffert S,
Shanmugam S, Sood V, Soutschek J, Toudjarska I, Wheat AJ, Yaworski
E, Zedalis W, Koteliansky V, Manoharan M, Vornlocher HP and
Maclachlan I. RNAi-mediated gene silencing in non-human primates.
Nature advance online publication26 March 2006 |
doi:10.1038/nature04688
6. Jones-Rhoades,M,Bartel,D and Bartel,B. MicroRNAs and their
regulatory role in plants AnnuRev. Plant Biol. 2006,57,19-53
7. Herr,A. Pathwaythrough the small RNA world of plants FEBS letters
2005,579, 5879-88
8. Bangham,J. RNA world: Designer plant miRNAs meet their targets
Nature Reviews Genetics 2006,7, 334-335 | doi:10.1038/nrg1865
9. Franz AW, Sanchez-Vargas I, Adelman ZN, Blair CD, Beaty BJ, James
AA and Olson KE. Engineering RNA interference-based resistance to
dengue virus type 2 in genetically modified Aedes aegypti. Proc
Natl Acad Sci U S A. 2006 Mar 14;103(11):4198-203
n genetics
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