The discovery of RNA interference (RNAi) and its utilization in downregulation of specific target transcripts have revolutionized gene function analysis and elucidation of many key biochemical/genetic pathways. The insights into gene function, combined with a technology that made silencing of gene function possible using the potent, highly specific and selective RNAi approaches, provided the solution to longstanding complex obstacles in targeted crop improvementsfor agriculture, and disease therapies for medicine. In this introductory chapter, I aim briefly to cover the basics and peculiarities of RNAi and the advances made in understanding the mechanisms, components, function, evolution, application, safety and risk assessment of RNAi, while at the same time highlighting the related chapters of this book.
- Gene silencing
- RNA interference
- RNAi inducers and delivery
- RNAi-based disease therapy
The “central dogma” of genetics as first presented by Francis Crick is that genes, packed inside the cell as the deoxyribonucleic acid (DNA) molecule, are transcribed into messenger ribonucleic acids (mRNA), which are subsequently translated into proteins (or enzymes). These final protein products provide all life functions, and together with DNA and RNA, constitute the molecules of life. Therefore, if there is a disruption (interference) of a gene function, messenger RNA synthesis, or protein translation, normal life processes get altered or even stopped. “No gene-no messenger”, or “no messenger-no protein”, has been the basis of understanding biological processes. One of the easy-to-access points in cellular processes is messenger RNA due to its cytoplasmic location, “naked” structure, comparatively short half-life, and temporal existence between transcription and translation. Further, mRNA is in between the chain of events from DNA to protein; it has the universal chemical structure, consisting of only four nucleotides, regardless of the encoded message. In contrast, proteins are chemically much more variable, consisting of combinations of 21 different amino acids, with side chains that vary from very hydrophilic to highly hydrophobic. If mRNA is altered or eliminated before translation, there is no functional gene product, which results in changing the cellular process from the native state. This is the entire rationale of RNA interference (RNAi).
RNA interference is a process in eukaryotic cells in which double stranded endogenous or exogenous RNA molecules trigger a cytoplasmic response, which involves sequence specific target identification and destruction. This may include native messenger RNAs (mRNAs) that code vitally important proteins . Any type of double-stranded RNA (dsRNA) molecules can activate RNAi where long dsRNAs, microRNAs (miRNAs) and small interfering RNAs (siRNAs) and their various forms and modifications are considered the main players/inducers . Let us take a look at RNAi discovery history.
Plant scientists in the 1990s first used targeted gene silencing by introducing an antisense gene into plants. The first example was silencing of a nopaline synthase (NOS) gene, for which the silencing was only visible by loss of a band on a Norther blot and loss of NOS activity . The second antisense gene used in plants targeted the petunia chalcone synthase (CHS) gene, encoding the first step in floral pigment production, and the result was visible in the loss of petal pigmentation . Curiously, attempts to create dark pigmented petunia flowers by overexpression of the same CHS gene resulted in similar colorless petunia petals [4, 5]. It was thought that such a phenotype was “
Following these seminal discoveries, similar phenomena were discovered in other organisms including the nematode (
In plants, the suppression of targeted genes during viral infections was discovered  and subsequently developed into a system by which plant gene function may be studied through inhibition by infection with viruses bearing a short sequence targeted against plant mRNAs . This phenomenon was termed as "
Over the past decade, RNAi has been demonstrated in many eukaryotes including humans as well as some prokaryotic life forms  and has been recognized to form an integral part of many gene regulatory networks during development. This revolutionary breakthrough in biological science has become a valuable
RNAi research has rapidly advanced and expanded over the past decade, evidenced by increasing numbers of publications, research projects, and practical applications in both agriculture and medicine. For example, searching
2. Components, mechanism, and function
The principle mechanism of RNAi is complex, but very straightforward and easy to understand. RNAi is induced by the introduction of specific exogenous dsRNA either by virus genome RNAs, injection of synthetic dsRNAs or, in plants, is mediated by Agrobacterium. RNAi is also part of the normal development and dsRNAs are produced by endogenous genes encoding miRNA precursors or other long dsRNA molecules. In either case, the dsRNAs are recognized by the enzyme dicer and cleaved into short, double-stranded fragments of ~19-25 base pair long siRNAs . These siRNAs are separated into two single-stranded RNAs (ssRNAs), which are referred to as the “
It is not clear as yet how the
Although the pathways toward RNAi from exogenous and endogenous dsRNA converge at the RISC and use the same downstream RNAi machinery, there are also some clear differences in their processing and handling . Endogenous dsRNAs cleaved by dicer (
The main biological function of RNAi is regulation of gene activity of cells at the post-transcriptional level (PTGS) either by the inhibition of translation of mRNA or by direct degradation of the mRNA. In addition to PTGS, RNAi pathway components may contribute to maintenance of genome organization and structure, mediated by RNA-induced histone modification. Histone modification in turn affects heterochromatin formation and may silence gene activity at the pre-transcriptional level . This process is referred to as “RNA-induced gene silencing (RITS) and requires dicer, siRNA and RISC component proteins such as AGO and R2D2 . In addition, RNAi components and inducers (siRNA/dicer/AGO) may also possibly upregulate expression of genes in binding into a promoter region and through histone demethylation, a process dubbed RNA activation [37, 38].
Because of sequence-specific recognition, regulatory properties, and the possibility of systemic spreading of dsRNAs, RNAi is the key “sterilizing agent” of cells and tissues, and it functions as potent immune response against foreign nucleic acids from viruses, transposons, or transformation events which can invade and harm the genome and its stability . The chapters presented in Section 2 of this book have a more detailed coverage of the history of the RNAi discovery, mechanism, and functional components and on the biological role of RNAi including natural small RNAs/microRNAs as well as long noncoding RNAs in gene regulations.
3. Differences among organisms
Although the RNAi pathway is a universal process in eukaryotic cells, and it consists of similar component(s), mechanisms, and functions as described above, there are some variations among organisms in both up-take of exogenous dsRNAs and induction of RNAi. First, RNAi is systemic and heritable in plants and
RNAi is not found in some eukaryotic protozoa (e.g.,
Studies on components, mechanisms, and functions of RNAi have demonstrated variations among organisms, differences in eukaryotes and prokaryotes, and indicate that RNAi is derived from an ancestral immune defense function against transcripts of transposons and viruses [50, 51]. Although some eukaryotes might have lost RNAi components or, even, the entire pathway following the emergence of the Eukaryota, parsimony-based phylogenetic analyses suggest that an ancestral lineage of all eukaryotes possibly had a primitive RNAi capability including relevant components for some key functions such as histone modification . Phylogenetic studies also indicate that miRNAs of plants and animals may have evolved independently, but the conservation of some key proteins involved in RNAi also indicate that the last common ancestor of modern eukaryotes already possessed an siRNA-based gene silencing system. The RNAi-like defense system of prokaryotes is functionally similar, but structurally distinct from the eukaryotic RNAi system . It seems likely that a proto-RNAi system possessed at least some form of dicer-like, AGO, PIWI, and RdRP proteins. These basic components were shared by major eukaryotic lineages and functioned within an RNA degradation exosome complex .
Being an important component of an antiviral innate immune defense system in eukaryotes, RNAi components and various interaction/regulatory mechanisms, including the miRNA pathway, evolved later but at faster rates under strong directional selection . This could have been a means of generating an improved response to the evolutionary arms race with viral genes. Correspondingly, some plant viruses have evolved the means to suppress the RNAi response in their host cells . Extensive studies reported that an ancient duplication of RNAi components followed by species-specific gene duplications and losses provided evolutionary diversification, specificity and adaptation of the RNAi system in many organisms . Chapter(s) presented in Section 2 has covered some evolutionary aspects of RNAi.
Since its first discovery as anti-sense gene suppression,
The results of the functional genomics studies, advances in the understanding of the RNAi mechanism, improved design of trait-specific RNAi inducers (such as miRNAs), selection of target gene sequences combined with the development of proper delivery systems, as well as screens for “off-target” and cross reactivity have brought the practical applications of RNAi far beyond its initial experimental reach.
Agricultural application of RNAi through tissue culture-derived genetic modifications and transgenic research in a wide range of technical, food, and horticulture crops have been particularly successful and have solved many problems. Examples include, but are not limited to, crop yield and quality improvements [15, 58], food/nutrient quality improvements and fortification [59–62], decreasing the harmful precursors and carcinogens [63, 64], and improvement of plant pest and disease resistance [65–66]. Many of these applications are now evaluated for commercialization or are already in commercial production . In this context, targeting far red (FR) photoreceptor gene (
Therapeutic application of RNAi has also been successful in medicine and molecular pharmacology with examples in inflammatory and infectious disease [68-71], cancer [72-75], as well as hereditary and neurodegenerative diseases . Indeed, for many other disorders RNAi may have great potential. To highlight advances made on this field, in Section 4 of this book, we present several relevant chapters on advances of RNAi application in key human diseases of blood, ocular, nervous, kidney, and oncogenic origin. In addition, Section 5 chapters discuss RNAi utilization in various immune and infectious diseases. Section 6 chapters present the latest advances of RNAi application in studies of insects and parasitic pests such as ticks. All of these chapters highlight various aspects of RNAi and add interesting insights to the present RNAi discussions.
6. Safety and risk assessment
Manipulation of the organisms’ own genetic sequence signature(s) (cis-genesis) is usually considered safer compared to “trans-genesis” that utilizes “foreign” genetic material to create genetically modified (GM) crops and its products . However, for RNAi, when broken down to ~21 nucleotides this quickly may lose its meaning, as a trans-RNAi will only work if it has sufficient homology to an endogenous target transcript. Chemically, RNA is “
Safety concerns about RNAi-based drugs are exemplified by the lethality of 23 out of 49 distinct RNAi therapy experiments in mice because of potential "off-target" effects that could shut down non-targeted gene(s) with sequence similarity to therapeutic RNAi inducer . This observed lethality, however, could be due to “oversaturation" of the dsRNA pathway and delivery issues of short hairpin RNAs  that needs to be optimized for harmless therapeutic applications. There are several suggested approaches to minimize or eliminate such “off-target”, “oversaturation” or delivery issues, in particular through the use of (1) comprehensive
There may also be concerns about the uptake of intact plant miRNA by consumers through plant diet. Plant microRNAs and some long dsRNA molecules, with sequence complimentary and perfect matches to endogenous human genes, were demonstrated to survive the digestive tract of humans and can freely and routinely enter the blood system [67, 81].
Risk assessment and available protocols/guidelines are in the early stages of development. Some suggest that dsRNA-derived products must be subject to risk assessment studies . Other findings indirectly support the safety of RNAi [81, 82], provided its use is within specific dosage ranges, the correct delivery system is in place and RNAi inducers without possible off-target effects, unintended gene silencing and secondary dsRNA production can be designed. However, it is always advisable to admit to possible risks of any novel genomic technology, including RNAi, and consider potential biohazards and evaluate risks for environmental health, before release of a new product [58, 81–85]. To accomplish this, Heinemann et al.  proposed the following five-step guidance: (1) to perform detailed
7. Conclusions and future perspectives
Thus, being a revolutionizing discovery in genome biology to characterize functions of any desired unknown genetic sequences, the discovery of RNAi has significantly widened our knowledge on core cellular processes. This knowledge has created opportunities and solutions to longstanding obstacles in conventional agriculture and medicine, offering a bright future to curing complex human and animal diseases, improve crop production and protection, and a sustained global food security through proper manipulation of key genes with agricultural or medicinal importance. Although key issues on specificity, selectivity, and delivery of RNAi inducing structures still exist, and some safety risks associated with the use of RNAi products have been recognized, the general believe is that RNAi is a safer technology than trans-genomics utilizing “foreign” genetic information. Safe applications, however, require proper designing, dosage and delivery of RNAi inducers, and before its delivery for wide consumer market, the safety risks should be assessed. Addressing the advances made over the past three decades in RNAi research and commercialization, in this book, we have compiled and presented a diverse collection of chapters contributed by the science research communities. We all believe that RNAi, in combination with the rapidly expanding genomic information in key organisms and novel genome editing tools, will become even more powerful and efficient, and that we will all enjoy its benefits far into the future.
I thank the Academy of Sciences of Uzbekistan and Committee for Coordination Science and Technology Development of Uzbekistan for basic science FA-F5-T030; and several applied (FA-A6-T081 and FA-A6-T085) and innovation (I-2015-6-15/2 and I5-FQ-0-89-870) research grants. I am particularly grateful and thank the Office of International Research Programs (OIRP) of the United States Department of Agriculture (USDA) – Agricultural Research Service (ARS) and U.S. Civilian Research & Development Foundation (CRDF) for international cooperative grants P121, P121B, and UZB-TA-2292, which are devoted to study, development, application, risk assessment, and commercialization of RNAi cotton cultivars and their products. I sincerely thank Dr. Alexander R. van der Krol, Waginengen University, Netherlands, and Dr. Eric J. Devor, Iowa State University, the USA, for their critical reading and suggestions to this chapter manuscript.