RNA Interference (RNAi) – Principles, Mechanism, Application
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is a conserved biological response to double-stranded RNA that mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes. This natural mechanism for sequence-specific gene silencing promises to revolutionize experimental biology and may have important practical applications in functional genomics, therapeutic intervention, agriculture and other areas.
RNA interference (RNAi) is one of the pathways, collectively
named RNA silencing pathways, that employ small RNAs as guides for
sequence-specific silencing. RNAi was discovered in C. elegans and defined as
sequence-specific mRNA degradation induced by long double-stranded RNA (dsRNA).
Although some authors use the term RNAi as a synonym for RNA silencing, this
review will adhere to the original definition as formulated by Fire et al.
The primary aim of this contribution is to provide an
overview of RNA interference mechanism with focus on selected aspects concerning
RNAi targeting and off-targeting in animals as these would be most relevant
features for discussing the use of RNAi for pest control. Therefore, I will
purposefully not go into the details. Interested readers should check out
referenced reviews or original articles. For a thorough overview of RNAi,
readers are welcome to refer to a comprehensive compilation of information on
RNAi and related pathways in different animal taxons and plants, which we
assembled with colleagues for the European Food and Safety Authority.
RNA Interference Definition
- RNA interference is the process by which RNA molecules suppress gene expression by neutralising the targeted messenger RNA molecules.
- RNA interference is an evolutionarily conserved mechanism that is triggered by double-stranded RNA and employs the gene’s own DNA sequence to silence it. This is referred to as gene silencing.
- It is a gene regulatory system that restricts transcript levels in two ways.
- Restricting transcription (transcriptional gene silence) and degrading RNA production (post-transcriptional gene silencing)
- Andrew Z. Fire and Craig C. Mello, two American scientists, found the mechanism in C.elegans cells. They blocked the expression of specific genes by introducing short lengths of double-stranded RNA into the C. elegans cells.
Principles of RNA Silencing
Some kind of RNA silencing pathway exists in almost every
eukaryotic organism with some notable exceptions among fungi and protists. RNA
silencing pathways utilize 20-30 nucleotide long RNAs loaded on Argonaute
proteins, which guide sequence-specific repression through basepairing with
target RNAs. RNA silencing pathways differ in the origin and biogenesis of
small RNAs, mechanisms leading to target repression, and biological roles
RNA substrates giving rise to small RNA guides in RNA
silencing pathways vary in structure. They include double-stranded RNA (dsRNA)
with blunt ends, small and long RNA hairpins with perfect and less-than-perfect
complementarity, sense and antisense RNA (basepaired or not), or
single-stranded “aberrant” RNA that would be converted to dsRNA by an
RNA-dependent RNA polymerases (RdRP) or converted directly to small RNAs.
Substrates can be converted to a small RNA either by Dicer, an RNase III
cleaving dsRNA and/or canonical microRNA (miRNA) precursors, or by some
Dicer-independent mechanism.
Target repression can be post-transcriptional or
transcriptional. Post-transcriptional RNA silencing could have a form of
endonucleolytic cleavage of cognate RNA (traditionally associated with RNAi),
or translational repression coupled with mRNA destabilization (historically
associated with animal miRNAs). Transcriptional RNA silencing is common in
plants but rare among animals. It may involve de novo DNA methylation or
transcriptionally repressive histone modifications.
Common biological roles of RNA silencing pathways include
regulation of endogenous gene expression, antiviral immunity, and genome
protection against transposable elements. During evolution, RNA silencing could
evolve into a complex system of interconnected pathways [exemplified by plants,
reviewed for example in or into a relatively simple set up (mammalian soma).
The following text will focus on RNAi but includes also the miRNA pathway
because of its close mechanistic relationship to RNAi.
RNAi Pathway
The canonical RNAi pathway is initiated by cleavage of long
dsRNA into small interfering RNAs (siRNAs). One siRNA strand then becomes
loaded onto an Argonaute protein possessing endonucleolytic activity (e.g.,
AGO2 in vertebrates and arthropods). A complementary mRNA is cleaved by the
Argonaute in the middle of the siRNA:mRNA duplex. In some taxons (e.g., plants
or C. elegans), RNAi pathways employ the above-mentioned RdRPs, which can
provide an amplification loop synthesizing small RNAs or dsRNA on targeted RNA
templates. C. elegans employs so-called “transitive RNAi” where RdRP produces
secondary siRNAs extending upstream of the targeted sequence Plants also
exhibit transitive silencing; the transitivity may even spread downstream of
the targeted sequence.
Canonical RNAi is traditionally viewed as a defense pathway
providing antiviral innate immunity in invertebrates and plants against viruses
that produce dsRNA. However, RNAi could evolve additional roles, such as
maintenance of genome integrity through suppression of transposable elements or
control of gene expression. In plants, for example, the basic RNAi mechanism
has been integrated into a complex pathway system of post-transcriptional and
transcriptional silencing, which employs multiple Dicer, Argonaute and RdRP
proteins and functions in antiviral defense, protection of genome integrity,
and regulation of gene expression In C. elegans. RNAi exists as a complex of
antiviral RNAi, endo-RNAi controlling endogenous genes, and exo-RNAi responding
to dsRNA in the environment. RNAi is functional in insects and other arthropod
subphyla, including Chelicerata [ticks and mites; genomic data suggest that
Myriapoda arthropods also have functional RNAi. In vertebrates, the RNAi
pathway has become vestigial; protein factors for siRNA biogenesis and target
repression serve the miRNA pathway. This is presumably a consequence of the
innate immunity system evolving an array of protein sensors detecting pathogen
markers such as dsRNA, which trigger the so-called interferon response. An
important limiting factor for functional RNAi in somatic mammalian cells seems
to be inefficient siRNA production due to the low processivity of mammalian
Dicer, which is adapted for non-processive miRNA biogenesis.
miRNA Pathway
While the miRNA pathway can share some components with the
RNAi pathway, it differs in several fundamental aspects. miRNAs are
genome-encoded repressors of gene expression with defined sequences (i.e., can
be precisely annotated). While RNAi employs a population of siRNAs
stochastically generated from dsRNA to destroy a pool of RNAs with the
complementary sequence, one specific miRNA sequence can guide repression of
many different mRNAs through imperfect miRNA:mRNA basepairing.
Animal miRNA biogenesis starts with a primary miRNA
(pri-miRNA), a long Pol II transcript carrying one or more local hairpins,
which can be cut out from the pri-miRNA by RNase III activity of the nuclear
Microprocessor complex. The resulting miRNA precursor (pre-miRNA) is
transported to the cytoplasm, where it is cleaved by Dicer. One strand of the
resulting duplex is loaded onto an AGO protein similarly to the RNAi pathway.
Vertebrates have usually four AGO paralogs; teleost fish acquired an additional
AGO3 paralogue through a fish-specific genome duplication event. All four
mammalian AGO proteins accommodate miRNAs equally well, including AGO2, which
is the only one with “slicing” endonucleolytic activity. All four mouse AGO
proteins seem to be functionally redundant in the miRNA pathway, as shown by
rescue experiments in embryonic stem cells lacking all four Ago genes.
Typical miRNA:mRNA interaction in animals occurs with
partial complementarity (described in detail further below) and results in
translational repression, which is associated with substantial mRNA
degradation. Plant miRNA biogenesis [reviewed in employs one of the Dicer
paralogs (DCL1), which processes both pri-miRNA and pre miRNA. Plant miRNAs
often have higher sequence complementarity resulting in RNAi-like cleavage of
their targets but also frequently repress translation. In animals, miRNAs can
also mediate RNAi-like cleavage, as demonstrated by reporters designed to have
full complementarity to a specific miRNA, but naturally occurring RNAi-like
endonucleolytic cleavage of targets is rare. The experimental approach to
knocking down gene expression in mammalian cells by delivering a siRNA (either
as an in vitro synthesized RNA or expressed from a plasmid vector) is commonly
called RNAi. Mechanistically, however, the approach hijacks the miRNA pathway
and its aforementioned ability to produce RNAi-like cleavage.
Co-Existence of RNAi and miRNA Pathways
While there is an apparent mechanistic overlap, there is
functional divergence of RNAi and miRNA pathways, which likely influenced the
co-existence of the two pathways in different model systems during evolution.
One is represented by Drosophila, where both pathways genetically diverged such
that each pathway has a dedicated Dicer and AGO protein, while the crosstalk
between the two pathways is minimal. Dicer in the RNAi pathway is
phylogenetically more derived, which would be consistent with its engagement in
dsRNA-based antiviral defense and host-pathogen evolutionary arms race. C.
elegans employs a single Dicer in production of miRNAs and siRNAs, but has a
complex system of Argonaute proteins and RdRP amplification, which contributes
to the separation of the pathways. Mammals have a single Dicer mainly serving
for miRNA biogenesis; canonical RNAi was functionally replaced by the interferon
response, which allows for sensing more structural features of replicating RNA
viruses. Functional RNAi in mammalian cells requires high Dicer activity,
enough dsRNA substrate, and suppression of the interferon response. However,
these three conditions are rarely met—a unique example occurs in the mouse
oocyte.
Interestingly, in one of the plant RNA silencing mechanisms,
RNAi essentially serves as an amplifier of miRNA silencing where miRNA-mediated
cleavage of mRNA targets is followed by RdRP-mediated production of long dsRNA,
which is processed by Dicer into so-called phased siRNAs (phasiRNA). PhasiRNAs
themselves are a complex small RNA category as they can be generated by
different Dicers and mediate target cleavage as well as transcriptional silencing.
RNA Interference Mechanism
- With the aid of an enzyme called Dicer, long double-stranded RNA is cut into minute bits. These bits are known as small interfering RNA or siRNA.
- Through the RNA-induced silencing complex, the siRNAs pass. The duplex unwinds, activating the RNA. These complexes impede translation and increase RNA breakdown.
- The siRNA binds to the Argonaute protein and removes one of the double-stranded strands. The strand that remains binds to mRNA target sequences. Either the Argonaute protein cleaves the mRNA or recruits other components to control the target sequence.
Why RNAi as a genetic tool?
- Synthetic dsRNA complementary to our target mRNA is designed and injected into the cell line using expression vectors, much like siRNA.
- Once it has been correctly introduced into a cell, the next steps are carried out by the cell’s RNA interference system.
- The dicer identifies the external dsRNA and cleaves it into 21 to 23nt dsRNA fragments.
- It is processed by Dicer and transferred to the cytoplasmic RISC, where the Ago2 protein binds to the siRNA fragments.
- The passenger strand of the siRNA fragments, which is identical to the mRNA, is eliminated, leaving the directed strand in the complex.
- The RISC then migrates the mRNA to its complementary mRNA, attaches to it, and destroys it.
- The inability of mRNA to be translated into protein reduces gene expression.
- The siRNA is a crucial component of in vitro RNAi research; its length is typically 21 nucleotides and is referred to as 21mer.
- The 21mer siRNA is more specific and performs exceptionally well in experiments; however, recent studies imply that the 27mer siRNA is more efficient.
- The 27mer is correctly cleaved by the dicer, resulting in a 2 nucleotide overhang at its 3′ terminus.
- This siRNA resembles the endogenous microRNA more closely. Consult our article on siRNA: Small Interfering RNA (siRNA): Structure And Function
- In in vitro RNA interference investigations, another molecule known as short hairpin RNA (shRNA) is also utilised.
- Chemical modification can improve the efficacy and specificity of the siRNA in the RNA interference (RNAi) process by enhancing the sequence specificity and subsequently reducing the capacity for cross-hybridization.
- Several features must be present in the chosen synthetic nucleic acid (siRNA or shRNA) for RNAi to respond more correctly.
RNA Interference Applications
- The usage of synthetic dsRNA molecules triggers the RNA interference response of a cell and regulates the expression of genes.
- Thus, artificially induced RNA interference has a wide range of applications in the clinical, medicinal, and other research sectors.
- It is now commonly utilised in gene knockout research.
- Additionally, it is utilised in genomics research and investigations. It is currently utilised therapeutically against viral infections, cancer, and neurological illnesses, with researchers intending to use it as a safer treatment for curing ailments.
- RNAi therapeutics can also be utilised in personalised medicine and gene therapy with a specific target.
- In recent years, RNAi technology has become increasingly prevalent in plant research and crop enhancement.
- Scientists are currently utilising RNA interference and antisense RNA in crop development. Using the current technologies, new plant characteristics and disease-resistant plant species are being developed.
- In addition, it is utilised for pest control and crop enhancement. Flvr Savr tomato, decaffeinated coffee, and nicotine-free tobacco are a few of the most notable examples of plant species created via RNAi technology.
- RNAi is also utilised for disease and pathogen resistance, male sterility development, and functional genomic research in plants. Using RNA interference (RNAi), virus-resistant plant species against Banana Bract Mosaic Virus, Rice Tungro Bacilliform Virus, Tobacco Mosaic Virus, and Cucumber Mosaic Virus are produced.
- Artificially designed dsRNA complementary to viral RNA is inserted into the plant genome, mimicking the natural si/miRNA and destroying viral dsRNA whenever it attacks.
References
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10.1016/B978-0-444-64046-8.00282-2. Epub 2019 Jul 31. PMCID: PMC7152241.
Kim, D. H., & Rossi, J. J. (2008). RNAi mechanisms and
applications. BioTechniques, 44(5), 613–616. doi:10.2144/000112792
Agrawal, N., Dasaradhi, P. V. N., Mohmmed, A., Malhotra, P.,
Bhatnagar, R. K., & Mukherjee, S. K. (2003). RNA Interference: Biology,
Mechanism, and Applications. Microbiology and Molecular Biology Reviews, 67(4),
657–685. doi:10.1128/mmbr.67.4.657-685.2003
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