Small RNA regulates genes in a large way
By Prof. Joe Cummins
April 5, 2006
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, 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 in 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 strands 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 pathways. 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 post-transcriptional gene silencing. The third pathway is transcriptional gene silencing with RNAi directed chromatin re-modeling and DNA and histone methylation (2).
RNAi provides a defence against viruses and transposable elements in both plants and animals. The main defence against viruses is the recognition and silencing of double-stranded RNA replication intermediates. Some plant viruses defend against RNAi by 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 cause extensive mutation as they move about the genome. The RNAi provides a sequence-specific defence 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 encapsulated in nucleic acid lipid particles responded quickly to the treatment reducing the regulated protein by 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 are described in references (6) and (7).
Synthetic designer plant RNAi 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 worldwide 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 viruses 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 under the cover of confidential business information. Suddenly, everything has changed in genetics, and we have to ensure that old problems related to 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. Pathway through 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