RNA interference (RNAi)

SIGNIFICANCE: The term RNAi stands for RNA interference, a gene regulatory mechanism that downregulates gene expression in a sequence-specific manner by acting at the post-transcriptional level. This has undoubtedly been one of the biggest advances in genetics in decades, since it has added another perspective to the role of RNA in cells.

Background

In the late 1980s to early 1990s, plant biologist Richard Jorgensen was trying to make intense purple petunias by introducing extra copies of the purple pigment gene (Chalcone synthase chsA). He was quite surprised when the effort yielded instead white and variegated petunias. It seemed that somehow the introduced gene (the transgenes) had blocked (silenced) its own expression as well as the expression of the endogenous purple pigment genes (termed cosuppression). Analysis of transcription rates in these cells using nuclear run-on assays did not indicate a decrease, implying that the phenomenon should be renamed as post-transcriptional (PTGS). Following soon thereafter, were reports of quelling observed in the fungi Neurospora crassa. An effort to boost the orange phenotype(al1+) shown by (wt) Neurospora by transforming it with a plasmid containing a 1500 bp fragment of the coding sequence of the al1+ gene, generated Neurospora. Once again, in these al1+quelled strains, it was the level of the mature messenger RNA (mRNA) that was affected in a homology-dependent manner. These findings were better understood when in 1998 Andrew Fire and Craig Mello reported that introduction of double-stranded RNA (dsRNA) corresponding to the unc-22 gene into a roundworm (Caenorhabditis elegans) specifically disrupted the expression of the endogenous unc-22 gene. unc-22 in C. elegans codes for a nonessential myofilament protein, and a reduction in unc-22 activity produced a twitching in these worms. If the dsRNA was introduced into the worm gonads, the phenotype was heritable, since the progeny too demonstrated the classic twitching phenotype. This demonstration offered an easy method to generate loss-of-function mutants since the silencing mechanism in C. elegans could be initiated simply by soaking the worms in solutions containing dsRNA or by feeding the worms E. coli that expressed the dsRNA. The silencing of a functional gene by introduction of exogenous dsRNA, to the target gene was renamed RNA (RNAi). Since then RNAi-related events have been identified in a multitude of eukaryotes ranging from protozoa, parasites, and fruit flies to mouse- and human-derived cell lines.

Molecular Mechanism

One of the earliest clues into the molecular basis of RNAi was provided by the discovery of short (20–25 nucleotides long) RNAs in the plant cells that matched the silencing target. Around the same time, in vitro RNAi assays using fruit-fly (Drosophila melanogaster) extracts revealed that the long dsRNA was being cleaved into 21–23 base pairs (bp) long RNA duplexes with 2 base overhangs at each end. These RNA species were called short interfering RNA (siRNA), and the enzyme responsible for producing these siRNAs from the dsRNA was called Dicer. Soon thereafter, the “sense” strand (sequence is identical to the target mRNA) is destroyed and the fate of the “antisense” strand thereon depends upon the organism. In mammals and fruit flies, the antisense strand is incorporated into the RISC (RNA-induced silencing complex), which is made up of RNA-binding proteins and RNA nucleases such as the Argonaute family of proteins found in the flowering plant Arabidopsis thaliana. The activated RISC (with the antisense strand of RNA) can now bind to the target mRNA and cleave it, thereby initiating sequence-specific mRNA degradation. This cleaving activity, whereby the RISC cleaves mRNA as directed by siRNA, is called the “Slicer” activity. In worms and plants, the next set of events following destruction of the siRNA sense strand are somewhat different even though the final outcome—RNA destruction—is similar. In these organisms the antisense strand pairs up with the complementary target mRNA, thus initiating synthesis of a long dsRNA by the enzyme RdRP (RNA dependent RNA polymerase). Then Dicer cleaves the long dsRNA and generates siRNAs which act via the RISC to target mRNAs for destruction, as described earlier.

Function

Over the years, substantial evidence has been collected that indicates a general role for PTGS (RNAi) in silencing parasitic genes such as viruses, transposons (“jumping genes”), and repetitive genes (including transgenes). In plant viruses, the double-stranded RNA intermediate in RNA virus has been shown to initiate PTGS/RNAi. These findings were supported by the discovery of virus encoded genes that suppress PTGS by a variety of mechanisms such as reducing target mRNA degradation and counteracting the systemic spread of RNA silencing. In 2005, Courtney Wilkins and her team working with a C. elegans infection model (utilizing mammalian vesicular stomatitis virus or VSV) have shown that VSV infection is more potent in RNAi defective worms (rde-1 and rde-4 mutants) and vice versa. protection assays to investigate whether RNAi is indeed induced upon viral infection revealed the presence of virus-specific siRNAs only in the virus-infected wild-type cells. Transposon duplication and insertion has long been known to create “junk DNA” and thus destabilize the genome. Experiments wherein worms with mutations in the RNAi pathway genes were shown to be incapable of silencing transposons lead to the idea that RNAi has evolved as a cellular defense mechanism that silences transposable elements to maintain genome stability. RNAi has also been shown to limit in a dosage-dependent manner in plants as well as fruit flies. In both of these instances, gene expression is enhanced by adding extra copies of the particular gene but only up to a point. Beyond that point, gene expression (especially transcription) is drastically reduced by modifying proteins that help to package DNA.

Impact

Over the years, experiments in Arabidopsis, C. elegans, and Drosophila have shown that besides their usual functions such as maintaining genome stability (by blocking transposition), the proteins involved in the RNAi machinery also play a role in the development of the organism. Research has shown that these genomically encoded short, 21- to 28-nucleotide RNAs that regulate temporal development are in fact members of the microRNA (miRNA) family, whose members have been identified across species as diverse as plants, flies, worms, and humans. Although miRNAs, like the siRNAs, are derived by Dicer activity on the dsRNA precursor and are found to be associated with RISC and their target mRNAs, unlike siRNAs, miRNAs are single-stranded and seem capable of affecting previously unknown, post-transcriptional steps such as RNA splicing, mRNA localization, and RNA turnover. An in-depth understanding of the siRNA-miRNA kinship could provide the key to several technological advancements from understanding gene functions by specific gene silencing (akin to genetic knockouts) to exploiting the therapeutic potential of this innate cellular defense mechanism to treat diseases like macular degeneration that are caused by overexpression of certain genes. Some RNA-targeting therapeutic programs have successfully treated diseases of the liver.

Since the mid-nineties, researchers have used gene silencing in numerous applications; one of the most notable is to genetically modify crops. Using gene silencing, scientists have been able to alter crops to resist disease, certain insects, and other environmental stresses. There is also the potential—and much ongoing research—for RNAi to be used in gene therapies to cure various diseases in humans.

Bibliography

Agarwal, Neema, et al. “RNA Interference: Biology, Mechanism, and Applications.” Microbiology and Molecular Biology Reviews, December 2003, pp. 657-685.

Bharathi, Jothi Kanmani, et al. "Recent Trends and Advances of RNA Interference (RNAi) to Improve Agricultural Crops and Enhance Their Resilience to Biotic and Abiotic Stresses." Plant Physiology and Biochemistry, vol. 194, 2023, pp. 600-618, doi.org/10.1016/j.plaphy.2022.11.035. Accessed 10 Sept. 2024.

Germain, Noelle D., Wendy K. Chung, and Patrick D. Sarmiere. "RNA Interference (RNAi)-Based Therapeutics for Treatment of Rare Neurologic Diseases." Molecular Aspects of Medicine, vol. 91, 2023, doi.org/10.1016/j.mam.2022.101148. Accessed 10 Sept. 2024.

Montgomery, Mary K., SiQun Xu, and Andrew Fire. “RNA as a Target of Double-Stranded RNA Mediated Genetics Interference in C. elegans.” Proceedings of the National Academy of Sciences 95 (December 1998): 15502–07. Print.

Novina, Carl D., and Philip Sharp. “The RNAi Revolution.” Nature, 430 (July 8, 2004): 161–164. Print.

"Pocket K No. 34: RNAi for Crop Improvement." International Service for the Acquisition of Agri-Biotech Applications. ISAAA, 2015. Web. 30 Nov. 2015.

Wilkins, Courtney, et al. “RNA Interference Is an Antiviral Defence Mechanism in Caenorhabditis elegans.” Nature 436 (August 18, 2005): 1044-1047.

Younis, Adnan, Muhammad Irfan Siddique, Chang-Kil Kim, and Ki-Byung Lim. "RNA Interference (RNAi) Induced Gene Silencing: A Promising Approach of Hi-Tech Plant Breeding." International Journal of Biological Sciences 10.10 (2014): 1150–58. Print.