Hubbry Logo
RNA silencingRNA silencingMain
Open search
RNA silencing
Community hub
RNA silencing
logo
7 pages, 0 posts
0 subscribers
Be the first to start a discussion here.
Be the first to start a discussion here.
RNA silencing
RNA silencing
RNA silencing
View on Wikipedia
from Wikipedia

RNA silencing or RNA interference refers to a family of gene silencing effects by which gene expression is negatively regulated by non-coding RNAs such as microRNAs. RNA silencing may also be defined as sequence-specific regulation of gene expression triggered by double-stranded RNA (dsRNA).[1] RNA silencing mechanisms are conserved among most eukaryotes.[2] The most common and well-studied example is RNA interference (RNAi), in which endogenously expressed microRNA (miRNA) or exogenously derived small interfering RNA (siRNA) induces the degradation of complementary messenger RNA. Other classes of small RNA have been identified, including piwi-interacting RNA (piRNA)[3] and its subspecies repeat associated small interfering RNA (rasiRNA).[4]

Background

[edit]

RNA silencing describes several mechanistically related pathways which are involved in controlling and regulating gene expression.[5][6][7] RNA silencing pathways are associated with the regulatory activity of small non-coding RNAs (approximately 20–30 nucleotides in length) that function as factors involved in inactivating homologous sequences, promoting endonuclease activity, translational arrest, and/or chromatic or DNA modification.[8][9][10] In the context in which the phenomenon was first studied, small RNA was found to play an important role in defending plants against viruses. For example, these studies demonstrated that enzymes detect double-stranded RNA (dsRNA) not normally found in cells and digest it into small pieces that are not able to cause disease.[11][12][13][14][2]

While some functions of RNA silencing and its machinery are understood, many are not. For example, RNA silencing has been shown to be important in the regulation of development and in the control of transposition events.[15] RNA silencing has been shown to play a role in antiviral protection in plants as well as insects.[16] Also in yeast, RNA silencing has been shown to maintain heterochromatin structure.[17] However, the varied and nuanced role of RNA silencing in the regulation of gene expression remains an ongoing scientific inquiry. A range of diverse functions have been proposed for a growing number of characterized small RNA sequences—e.g., regulation of developmental, neuronal cell fate, cell death, proliferation, fat storage, haematopoietic cell fate, insulin secretion.[18]

RNA silencing functions by repressing translation or by cleaving messenger RNA (mRNA), depending on the amount of complementarity of base-pairing. RNA has been largely investigated within its role as an intermediary in the translation of genes into proteins.[19] More active regulatory functions, however, only began to be addressed by researchers beginning in the late-1990s.[20] The landmark study providing an understanding of the first identified mechanism was published in 1998 by Fire et al.,[1] demonstrating that double-stranded RNA could act as a trigger for gene silencing.[20] Since then, various other classes of RNA silencing have been identified and characterized.[5] Presently, the therapeutic potential of these discoveries is being explored, for example, in the context of targeted gene therapy.[21][22]

While RNA silencing is an evolving class of mechanisms, a common theme is the fundamental relationship between small RNAs and gene expression.[9] It has also been observed that the major RNA silencing pathways currently identified have mechanisms of action which may involve both post-transcriptional gene silencing (PTGS)[23] as well as chromatin-dependent gene silencing (CDGS) pathways.[5] CDGS involves the assembly of small RNA complexes on nascent transcripts and is regarded as encompassing mechanisms of action which implicate transcriptional gene silencing (TGS) and co-transcriptional gene silencing (CTGS) events.[24] This is significant at least because the evidence suggests that small RNAs play a role in the modulation of chromatin structure and TGS.[25][26]

Despite early focus in the literature on RNA interference (RNAi) as a core mechanism which occurs at the level of messenger RNA translation, others have since been identified in the broader family of conserved RNA silencing pathways acting at the DNA and chromatin level.[27] RNA silencing refers to the silencing activity of a range of small RNAs and is generally regarded as a broader category than RNAi. While the terms have sometimes been used interchangeably in the literature, RNAi is generally regarded as a branch of RNA silencing. To the extent it is useful to craft a distinction between these related concepts, RNA silencing may be thought of as referring to the broader scheme of small RNA related controls involved in gene expression and the protection of the genome against mobile repetitive DNA sequences, retroelements, and transposons to the extent that these can induce mutations.[28] The molecular mechanisms for RNA silencing were initially studied in plants[13] but have since broadened to cover a variety of subjects, from fungi to mammals, providing strong evidence that these pathways are highly conserved.[29]

At least three primary classes of small RNA have currently been identified, namely: small interfering RNA (siRNA), microRNA (miRNA), and piwi-interacting RNA (piRNA).

small interfering RNA (siRNA)

[edit]
Main article: Small interfering RNA

siRNAs act in the nucleus and the cytoplasm and are involved in RNAi as well as CDGS.[5] siRNAs come from long dsRNA precursors derived from a variety of single-stranded RNA (ssRNA) precursors, such as sense and antisense RNAs. siRNAs also come from hairpin RNAs derived from transcription of inverted repeat regions. siRNAs may also arise enzymatically from non-coding RNA precursors.[30] The volume of literature on siRNA within the framework of RNAi is extensive. One of the potent applications of siRNAs is the ability to distinguish the target versus non-target sequence with a single-nucleotide difference. This approach has been considered as therapeutically crucial for the silencing dominant gain-of-function (GOF) disorders, where mutant allele causing disease is differed from wt-allele by a single nucleotide (nt). This type of siRNAs with capability to distinguish a single-nt difference are termed as allele-specific siRNAs.[31]

microRNA (miRNA)

[edit]
Main article: MicroRNA

The majority of miRNAs act in the cytoplasm and mediate mRNA degradation or translational arrest.[32] However, some plant miRNAs have been shown to act directly to promote DNA methylation.[33] miRNAs come from hairpin precursors generated by the RNaseIII enzymes Drosha and Dicer.[34] Both miRNA and siRNA form either the RNA-induced silencing complex (RISC) or the nuclear form of RISC known as RNA-induced transcriptional silencing complex (RITS).[35] The volume of literature on miRNA within the framework of RNAi is extensive.

Three prime untranslated regions and microRNAs

[edit]
Main article: Three prime untranslated region

Three prime untranslated regions (3'UTRs) of messenger RNAs (mRNAs) often contain regulatory sequences that post-transcriptionally cause RNA interference. Such 3'-UTRs often contain both binding sites for microRNAs (miRNAs) as well as for regulatory proteins. By binding to specific sites within the 3'-UTR, miRNAs can decrease gene expression of various mRNAs by either inhibiting translation or directly causing degradation of the transcript. The 3'-UTR also may have silencer regions that bind repressor proteins that inhibit the expression of a mRNA.

The 3'-UTR often contains microRNA response elements (MREs). MREs are sequences to which miRNAs bind. These are prevalent motifs within 3'-UTRs. Among all regulatory motifs within the 3'-UTRs (e.g. including silencer regions), MREs make up about half of the motifs.

As of 2014, the miRBase web site,[36] an archive of miRNA sequences and annotations, listed 28,645 entries in 233 biologic species. Of these, 1,881 miRNAs were in annotated human miRNA loci. miRNAs were predicted to have an average of about four hundred target mRNAs (affecting expression of several hundred genes).[37] Freidman et al.[37] estimate that >45,000 miRNA target sites within human mRNA 3'UTRs are conserved above background levels, and >60% of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs.

Direct experiments show that a single miRNA can reduce the stability of hundreds of unique mRNAs.[38] Other experiments show that a single miRNA may repress the production of hundreds of proteins, but that this repression often is relatively mild (less than 2-fold).[39][40]

The effects of miRNA dysregulation of gene expression seem to be important in cancer.[41] For instance, in gastrointestinal cancers, nine miRNAs have been identified as epigenetically altered and effective in down regulating DNA repair enzymes.[42]

The effects of miRNA dysregulation of gene expression also seem to be important in neuropsychiatric disorders, such as schizophrenia, bipolar disorder, major depression, Parkinson's disease, Alzheimer's disease and autism spectrum disorders.[43][44][45]

piwi-interacting RNA (piRNA)

[edit]
Main article: Piwi-interacting RNA

piRNAs represent the largest class of small non-coding RNA molecules expressed in animal cells, deriving from a large variety of sources, including repetitive DNA and transposons.[46] However, the biogenesis of piRNAs is also the least well understood.[47] piRNAs appear to act both at the post-transcriptional and chromatin levels. They are distinct from miRNA due to at least an increase in terms of size and complexity. Repeat associated small interfering RNA (rasiRNAs) are considered to be a subspecies of piRNA.[4]

Mechanism

[edit]
MiRNA processing

The most basic mechanistic flow for RNA Silencing is as follows: (For a more detailed explanation of the mechanism, refer to the RNAi:Cellular mechanism article.)

1: RNA with inverted repeats hairpin/panhandle constructs --> 2: dsRNA --> 3: miRNAs/siRNAs --> 4: RISC --> 5: Destruction of target mRNA

  1. It has been discovered that the best precursor to good RNA silencing is to have single stranded antisense RNA with inverted repeats which, in turn, build small hairpin RNA and panhandle constructs.[7] The hairpin or panhandle constructs exist so that the RNA can remain independent and not anneal with other RNA strands.
  2. These small hairpin RNAs and/or panhandles then get transported from the nucleus to the cytosol through the nuclear export receptor called exportin-5, and then get transformed into a dsRNA, a double stranded RNA, which, like DNA, is a double stranded series of nucleotides. If the mechanism didn't use dsRNAs, but only single strands, there would be a higher chance for it to hybridize to other "good" mRNAs. As a double strand, it can be kept on call for when it is needed.
  3. The dsRNA then gets cut up by a Dicer into small (21-28 nt = nucleotides long) strands of miRNAs (microRNAs) or siRNAs (short interfering RNAs.) A Dicer is an endoribonuclease RNase, which is a complex of a protein mixed with strand(s) of RNA.
  4. Lastly, the double stranded miRNAs/siRNAs separate into single strands; the antisense RNA strand of the two will combine with another endoribonuclease enzyme complex called RISC (RNA-induced silencing complex), which includes the catalytic component Argonaute, and will guide the RISC to break up the "perfectly complementary" target mRNA or viral genomic RNA so that it can be destroyed.[2][7]
  5. It means that based on a short sequence specific area, a corresponding mRNA will be cut. To make sure, it will be cut in many other places as well. (If the mechanism only worked with a long stretch, then there would be higher chance that it would not have time to match to its complementary long mRNA.) It has also been shown that the repeated-associated short interference RNAs (rasiRNA) have a role in guiding chromatin modification.[2]

Biological functions

[edit]

Immunity against viruses or transposons

[edit]

RNA silencing is the mechanism that our cells (and cells from all kingdoms) use to fight RNA viruses and transposons (which originate from our own cells as well as from other vehicles).[2] In the case of RNA viruses, these get destroyed immediately by the mechanism cited above. In the case of transposons, it's a little more indirect. Since transposons are located in different parts of the genome, the different transcriptions from the different promoters produce complementary mRNAs that can hybridize with each other. When this happens, the RNAi machinery goes into action, debilitating the mRNAs of the proteins that would be required to move the transposons themselves.[48]

Down-regulation of genes

[edit]

For a detailed explanation of the down-regulation of genes, see RNAi:downregulation of genes

Up-regulation of genes

[edit]

For a detailed explanation of the up-regulation of genes, see RNAi:upregulation of genes

RNA silencing also gets regulated

[edit]

The same way that RNA silencing regulates downstream target mRNAs, RNA silencing itself is regulated. For example, silencing signals get spread between cells by a group of enzymes called RdRPs (RNA-dependent RNA polymerases) or RDRs.[2]

Practical applications

[edit]

Growing understanding of small RNA gene-silencing mechanisms involving dsRNA-mediated sequence-specific mRNA degradation has directly impacted the fields of functional genomics, biomedicine, and experimental biology. The following section describes various applications involving the effects of RNA silencing. These include uses in biotechnology, therapeutics, and laboratory research. Bioinformatics techniques are also being applied to identify and characterize large numbers of small RNAs and their targets.

Biotechnology

[edit]

Artificial introduction of long dsRNAs or siRNAs has been adopted as a tool to inactivate gene expression, both in cultured cells and in living organisms.[2] Structural and functional resolution of small RNAs as the effectors of RNA silencing has had a direct impact on experimental biology. For example, dsRNA may be synthesized to have a specific sequence complementary to a gene of interest. Once introduced into a cell or biological system, it is recognized as exogenous genetic material and activates the corresponding RNA silencing pathway. This mechanism can be used to effect decreases in gene expression with respect to the target, useful for investigating loss of function for genes relative to a phenotype. That is, studying the phenotypic and/or physiologic effects of expression decreases can reveal the role of a gene product. The observable effects can be nuanced, such that some methods can distinguish between “knockdown” (decrease expression) and “knockout” (eliminate expression) of a gene.[49] RNA interference technologies have been noted recently as one of the most widely utilized techniques in functional genomics.[50] Screens developed using small RNAs have been used to identify genes involved in fundamental processes such as cell division, apoptosis and fat regulation.

Biomedicine

[edit]

Since at least the mid-2000s, there has been intensifying interest in developing short interfering RNAs for biomedical and therapeutic applications.[51] Bolstering this interest is a growing number of experiments which have successfully demonstrated the clinical potential and safety of small RNAs for combatting diseases ranging from viral infections to cancer as well as neurodegenerative disorders.[52] In 2004, the first Investigational New Drug applications for siRNA were filed in the United States with the Food and Drug Administration; it was intended as a therapy for age-related macular degeneration.[50] RNA silencing in vitro and in vivo has been accomplished by creating triggers (nucleic acids that induce RNAi) either via expression in viruses or synthesis of oligonucleotides.[53] Optimistically many studies indicate that small RNA-based therapies may offer novel and potent weapons against pathogens and diseases where small molecule/pharmacologic and vaccine/biologic treatments have failed or proved less effective in the past.[51] However, it is also warned that the design and delivery of small RNA effector molecules should be carefully considered in order to ensure safety and efficacy.

The role of RNA silencing in therapeutics, clinical medicine, and diagnostics is a fast developing area and it is expected that in the next few years some of the compounds using this technology will reach market approval. A report has been summarized below to highlight the many clinical domains in which RNA silencing is playing an increasingly important role, chief among them are ocular and retinal disorders, cancer, kidney disorders, LDL lowering, and antiviral.[53] The following table displays a listing of RNAi based therapy currently in various phases of clinical trials. The status of these trials can be monitored on the ClinicalTrials.gov website, a service of the National Institutes of Health (NIH).[54] Of note are treatments in development for ocular and retinal disorders, that were among the first compounds to reach clinical development. AGN211745 (sirna027) (Allergan) and bevasiranib (Cand5) (Opko) underwent clinical development for the treatment of age-related macular degeneration, but trials were terminated before the compounds reached the market. Other compounds in development for ocular conditions include SYL040012 (Sylentis) and QPI-007 (Quark). SYL040012 (bamosinan) is a drug candidate under clinical development for glaucoma, a progressive optic neurdegeneration frequently associated to increased intraocular pressure; QPI-007 is a candidate for the treatment of angle-closure glaucoma and Non-arteritic anterior ischaemic optic neuropathy; both compounds are currently undergoing phase II clinical trials. Several compounds are also under development for conditions such as cancer and rare diseases.

Clinical domain Drug Indication Target
Ocular and retinal disorders TD101 Pachyonychia congenita Keratin 6A N171K mutant
Ocular and retinal disorders QPI-1007 Non-arteritic anterior ischaemic optic neuropathy Caspase 2
Ocular and retinal disorders AGN211745 Age-related macular degeneration, choroidal neovascularization VEGFR1
Ocular and retinal disorders PF-655 Diabetic macular oedema, age-related macular degeneration RTP801
Ocular and retinal disorders SYL040012 Glaucoma β2 adrenergic receptor
Ocular and retinal disorders Bevasiranib Diabetic macular oedema VEGF
Ocular and retinal disorders Bevasiranib Macular degeneration VEGF
Cancer CEQ508 Familial adenomatous polyposis β-catenin
Cancer ALN-PLK1 Liver tumor PLK1
Cancer FANG Solid tumor Furin
Cancer CALAA-01 Solid tumor RRM2
Cancer SPC2996 chronic lymphocytic leukemia BCL-2
Cancer ALN-VSP02 Solid tumor VEGF, kinesin spindle protein
Cancer NCT00672542 Metastatic melanoma LMP2, LMP7, and MECL1
Cancer Atu027 Solid malignancies PKN3
Kidney disorders QPI-1002/I5NP Acute kidney injury p53
Kidney disorders QPI-1002/I5NP Graft dysfunction kidney transplant p53
Kidney disorders QPI-1002/I5NP Kidney injury acute renal failure p53
LDL lowering TKM-ApoB Hypercholesterolaemia APOB
LDL lowering PRO-040,201 Hypercholesterolaemia APOB
Antiviral miravirsen Hepatitis C virus miR-122
Antiviral pHIV7-shI-TAR-CCR5RZ HIV HIV Tat protein, HIV TAR RNA, human CCR5
Antiviral ALN-RSV01 RSV RSV nucleocapsid
Antiviral ALN-RSV01 RSV in lung transplant patients RSV nucleocapsid

Main challenge

[edit]

As with conventional manufactured drugs, the main challenge in developing successful offshoots of the RNAi-based drugs is the precise delivery of the RNAi triggers to where they are needed in the body. The reason that the ocular macular degeneration antidote was successful sooner than the antidote with other diseases is that the eyeball is almost a closed system, and the serum can be injected with a needle exactly where it needs to be. The future successful drugs will be the ones who are able to land where needed, probably with the help of nanobots. Below is a rendition of a table[53] that shows the existing means of delivery of the RNAi triggers.

Species/formulation Packaging capacity Applications and considerations
Viral vector
Adenovirus Usually < 10 Kb dsDNA vector with large packaging capacity, transient expression, highly immunogenic
Adeno-associated virus (AAV) ~4.5Kb ssDNA vector, small packaging capacity, mildly immunogenic, lasting expression in non-dividing cells, capsid pseudotyping/engineering facilitates specific cell-targeting
Lentivirus Up to 13.5 Kb RNA vector, integration competent and incompetent forms available, less immunogenic than adenovirus or AAV, envelope pseudo typing facilitates cell targeting, clinical production more difficult than for adenovirus or AAV
Herpes simplexvirus 150 Kb DNA vector, episomal, lasting expression, immunogenic
Bacterial vector species (bacterial minicells can carry plasmids, siRNAs or drugs)
Escherichis coli, S. Typhymurium Delivery of short hairpin RNA or siRNA to gut tissue
Non-viral formulations
Nanoparticle Self-assembling, may target specific receptors, requires technical expertise to prepare
Stable nucleic acid lipid particle (SNALP) Stable for systemic delivery, broad cell-type delivery
Aptamer Targeting of specific receptors, requires sophisticated screening to develop
Cholesterol Stable for systemic delivery, broad cell-type delivery

Laboratory

[edit]

The scientific community has been quick to harness RNA silencing as a research tool. The strategic targeting of mRNA can provide a large amount of information about gene function and its ability to be turned on and off. Induced RNA silencing can serve as a controlled method for suppressing gene expression. Since the machinery is conserved across most eukaryotes, these experiments scale well to a range of model organisms.[55] In practice, expressing synthetic short hairpin RNAs can be used to reach stable knock-down.[56] If promoters can be made to express these designer short hairpin RNAs, the result is often potent, stable, and controlled gene knock-down in both in vitro and in vivo contexts.[57] Short hairpin RNA vector systems can be seen as roughly analogous in scope to using cDNA overexpression systems.[58] Overall, synthetic and natural small RNAs have proven to be an important tool for studying gene function in cells as well as animals.[59]

Bioinformatics approaches to identify small RNAs and their targets have returned several hundred, if not thousands of, small RNA candidates predicted to affect gene expression in plants, C. elegans, D. melanogaster, zebrafish, mouse, rat, and human.[60] These methods are largely directed to identifying small RNA candidates for knock-out experiments but may have broader applications. One bioinformatics approach evaluated sequence conservation criteria by filtering seed complementary target-binding sites. The cited study predicted that approximately one third of mammalian genes were to be regulated by, in this case, miRNAs.[61]

Ethics & Risk-Benefit Analysis

[edit]

One aspect of RNA silencing to consider is its possible off-target affects, toxicity, and delivery methods. If RNA silencing is to become a conventional drug, it must first pass the typical ethical issues of biomedicine.[62] Using risk-benefit analysis, researchers can determine whether RNA silencing conforms to ethical ideologies such as nonmaleficence, beneficence, and autonomy.[63]

There is a risk of creating infection-competent viruses that could infect non-consenting people.[64] There is also a risk of affecting future generations based on these treatments. These two scenarios, in respect to autonomy, is possible unethical. At this moment, unsafe delivery methods and unintended aspects of vector viruses add to the argument against RNA silencing.[63]

In terms of off-target effects, siRNA can induce innate interferon responses, inhibit endogenous miRNAs through saturation, and may have complementary sequences to other non-target mRNAs. These off-targets could also have target up-regulations such as oncogenes and antiapoptotic genes. The toxicity of RNA silencing is still under review as there are conflicting reports.[63][64][65]

Number of RNAi publications since 1998

RNA silencing is quickly developing, because of that, the ethical issues need to be discussed further. With the knowledge of general ethical principles, we must continuously perform risk-benefit analysis.[63]

See also

[edit]

References

[edit]
Revisions and contributorsEdit on WikipediaRead on Wikipedia

RNA silencing

View on Grokipedia
from Grokipedia
RNA silencing, also known as (RNAi), is a conserved gene regulatory mechanism in eukaryotes, involving both post-transcriptional and, in some cases, transcriptional silencing, that uses small non-coding RNAs, such as small interfering RNAs (siRNAs) and microRNAs (miRNAs), to target and degrade specific messenger RNAs (mRNAs) or inhibit their translation through base-pairing complementarity, thereby silencing expression. This process is mediated by the (RISC), which incorporates an (AGO) protein loaded with the guide RNA to recognize and cleave target transcripts. Discovered in the late 1990s through studies on Caenorhabditis elegans and plants, RNA silencing was first demonstrated as a potent tool by introducing double-stranded RNA (dsRNA), leading to its recognition with the 2006 in or awarded to and . The core mechanism begins with the processing of dsRNA precursors by enzymes (or Dicer-like proteins in ) into 21–25 small RNAs, which are then incorporated into RISC for target recognition and silencing. In animals, miRNAs typically repress without mRNA cleavage, while siRNAs induce direct degradation; in , additional RNA-dependent RNA polymerases (RDRs) amplify the silencing signal, enabling systemic spread and even transcriptional silencing via RNA-directed . Key components include AGO family proteins (e.g., AGO2 in mammals for slicing activity), , and accessory factors like HEN1 for RNA stabilization in . Biologically, RNA silencing plays critical roles in development, genome stability, and defense against viruses and transposons by targeting invasive nucleic acids, with viruses often countering it through suppressor proteins. In plants, it operates through distinct pathways for post-transcriptional gene silencing (PTGS), transcriptional gene silencing (TGS), and heterochromatin formation, ensuring precise gene regulation and antiviral immunity. Therapeutically, RNAi has matured into a clinical tool, with six FDA-approved siRNA therapeutics (e.g., patisiran for hereditary transthyretin-mediated amyloidosis) as of 2025, primarily targeting liver-expressed genes, though challenges like delivery to extra-hepatic tissues persist.

Introduction

Definition and Scope

RNA silencing, also known as (RNAi), is a conserved post-transcriptional mechanism prevalent in eukaryotes, where double-stranded RNA (dsRNA) triggers the degradation of target messenger RNAs (mRNAs) or repress their translation, thereby inhibiting . This process is mediated by small non-coding RNAs, typically 20-25 nucleotides in length, that serve as guides within the (RISC), whose core effector is an protein family member responsible for target recognition and cleavage or repression. Unlike transcriptional , which prevents mRNA synthesis through modifications and , RNA silencing acts on mature transcripts in the , providing a rapid and sequence-specific means of control. The scope of RNA silencing extends across diverse eukaryotic kingdoms, including , animals, and fungi, where it functions in development, defense against viruses and transposons, and maintenance of stability. Its evolutionary conservation is evident in the shared core machinery, such as Dicer-like enzymes for processing and proteins in RISC, tracing back to an ancient eukaryotic , with key insights emerging from discoveries in the early that highlighted its universality. The primary classes of s involved include small interfering RNAs (siRNAs), which typically derive from exogenous dsRNA and mediate precise mRNA cleavage; microRNAs (miRNAs), endogenously encoded regulators that often repress ; and Piwi-interacting RNAs (piRNAs), which primarily safeguard genomes from transposons. Early observations of RNA silencing phenomena include cosuppression in petunia plants, reported in 1990 when transgenic introduction of a chalcone synthase gene unexpectedly silenced both endogenous and transgene expression, leading to altered pigmentation. In animals, the mechanism was elucidated in 1998 through experiments in Caenorhabditis elegans, where dsRNA injection potently and specifically interfered with gene function, establishing dsRNA as the key trigger. Reflecting its prevalence, the human genome encodes approximately 2,300 mature miRNAs, underscoring the broad impact of RNA silencing on regulating over half of protein-coding genes.

Historical Development

The phenomenon of RNA was first observed in the late 1980s and early 1990s through unexpected gene suppression effects in transgenic organisms. In 1990, researchers attempting to overexpress chalcone synthase to enhance purple pigmentation in flowers instead observed uniform white flowers due to co-suppression of both the transgene and endogenous gene, marking the initial documentation of posttranscriptional gene in plants. Similarly, in 1992, quelling was identified in the filamentous fungus , where introduction of transgenes led to sequence-specific of homologous genes, providing early evidence of a conserved mechanism across eukaryotes. These findings, though puzzling at the time, laid the groundwork for recognizing RNA-mediated regulation beyond simple overexpression artifacts. A major breakthrough came in 1993 with the discovery of the first microRNA (miRNA) in Caenorhabditis elegans. The lin-4 gene was found to encode small non-coding RNAs that negatively regulated the lin-14 mRNA through antisense complementarity, temporally controlling developmental transitions without altering DNA. This was overshadowed until 1998, when Andrew Fire and Craig Mello demonstrated that double-stranded RNA (dsRNA), rather than single-stranded, potently and specifically silenced genes in C. elegans, naming the process RNA interference (RNAi); their seminal work earned the 2006 Nobel Prize in Physiology or Medicine. Building on this, in 1999, small antisense RNAs (~25 nucleotides) were identified in plants undergoing posttranscriptional gene silencing, linking dsRNA triggers to the production of these guiding molecules. By 2001, the RNase III family enzyme Dicer was characterized as the key processor that cleaves dsRNA into ~21-22 nucleotide small interfering RNAs (siRNAs), initiating the RNAi pathway in Drosophila. The field expanded rapidly in the mid-2000s, with piwi-interacting RNAs (piRNAs) identified in 2006 as a distinct class of ~24-30 RNAs bound to proteins in animal germlines, primarily silencing transposons to safeguard genome integrity. RNAi mechanisms were soon integrated with , as shown in fission yeast where siRNAs directed and formation at centromeres around 2002-2005, revealing RNA's role in transcriptional silencing. Post-2010, synthetic siRNAs advanced toward therapeutics, with milestones including the 2018 FDA approval of (Onpattro) for hereditary transthyretin-mediated , the first RNAi-based drug, followed by givosiran in 2019, lumasiran in 2020, in 2021, in 2022, and nedosiran in 2023; by 2025, six RNAi therapeutics are FDA-approved, with additional approvals expected. By the 2020s, hybrid CRISPR-RNAi tools emerged, combining Cas9-guided delivery with RNAi for enhanced spatiotemporal in research and potential therapies, as demonstrated in mammalian cells and applications up to 2025.

Small RNA Molecules

Small Interfering RNAs (siRNAs)

Small interfering RNAs (siRNAs) are short double-stranded molecules, typically 20-25 in length, that serve as key effectors in RNA silencing pathways. These molecules consist of a sense and an antisense strand forming a duplex with 19 base-paired and characteristic 2-nucleotide 3' overhangs on both ends, which are essential for their recognition and processing by cellular machinery. Unlike microRNAs, siRNAs exhibit perfect base-pairing complementarity to their target mRNAs, enabling precise endonucleolytic cleavage rather than translational repression or deadenylation. This structural specificity ensures high efficiency in targeting exogenous or endogenous nucleic acids for degradation. The biogenesis of siRNAs begins with the introduction or generation of long double-stranded (ds) precursors, which can arise from , transposon activity, or cellular transcription. These precursors are cleaved by the endoribonuclease, an RNase III family enzyme, into siRNA duplexes of approximately 21-23 nucleotides—the optimal length for maximal silencing potency in mammalian systems. The resulting duplexes are then loaded into the (RISC), where the passenger strand is discarded, and the guide strand is incorporated into Argonaute-2 (Ago2), the slicer component of RISC responsible for target mRNA recognition and cleavage. This process is highly conserved across eukaryotes, with length specificity (21-23 nt) dictating efficient processing and RISC activation. siRNAs exist in both endogenous and synthetic forms, with distinct yet overlapping roles in cellular defense and . Endogenous siRNAs are derived from transposons, viral elements, or naturally occurring dsRNAs such as cis-natural antisense transcripts and pseudogene-gene pairs, particularly in reproductive tissues like oocytes where they suppress transposon activity to safeguard genomic integrity during early development. These siRNAs contribute to antiviral defense by targeting viral dsRNA intermediates for degradation, thereby limiting pathogen spread, and to transposon silencing by repressing that could disrupt expression. Synthetic siRNAs, chemically designed to mimic endogenous duplexes, were first demonstrated to mediate in cultured mammalian cells in , revolutionizing and therapeutic applications by allowing targeted knockdown of specific s without triggering responses. In mammals, endogenous siRNAs in oocytes regulate transcripts involved in processes like microtubule organization, highlighting their broader role in developmental control.

MicroRNAs (miRNAs)

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play a central role in RNA silencing by regulating primarily through post-transcriptional mechanisms, including translational repression and mRNA destabilization. These molecules, typically 21-23 nucleotides in length, are processed from hairpin-structured precursor transcripts and exhibit imperfect base-pairing complementarity to the 3' untranslated regions (3' UTRs) of target messenger RNAs (mRNAs). Unlike small interfering RNAs (siRNAs), miRNAs generally do not trigger mRNA cleavage but instead fine-tune by modulating protein output from multiple targets, influencing processes such as development, cell differentiation, and . The discovery of miRNAs began with the identification of lin-4 in in 1993, which was found to encode small RNAs with antisense complementarity to the 3' UTR of the lin-14 mRNA, establishing the paradigm for miRNA-mediated regulation. The biogenesis of miRNAs involves a multi-step pathway that ensures precise maturation and incorporation into effector complexes. Primary miRNAs (pri-miRNAs) are transcribed from genomic loci by , often as independent transcription units or embedded within introns of protein-coding genes. In the nucleus, the Drosha-DGCR8 complex cleaves the pri-miRNA at the base of the stem-loop structure to generate a precursor miRNA (pre-miRNA) of approximately 60-70 . The pre-miRNA is then exported to the by Exportin-5 in a Ran-GTP-dependent manner, where it is further processed by the Dicer-TRBP complex near the terminal loop, yielding a miRNA duplex. One strand of this duplex, the mature miRNA, is preferentially loaded into the protein within the (RISC) to guide target recognition. miRNAs are encoded predominantly in intergenic regions or introns, with some arising from exons, and their allows for coordinated expression with host genes in the case of intronic miRNAs. A single miRNA can target hundreds of mRNAs through short, imperfect interactions, primarily via a conserved "" sequence spanning 2-8 at the miRNA 5' end, which binds to complementary motifs in the 3' UTRs of targets to inhibit or promote deadenylation and decay. In humans, databases such as miRBase annotate over 1,900 distinct miRNAs as of 2023, reflecting their extensive regulatory potential across tissues and developmental stages. These molecules contribute to fine-tuning rather than complete shutdown, enabling subtle control over cellular responses. Beyond basic regulation, miRNAs have emerged as promising biomarkers in cancer diagnostics; for instance, circulating miRNA profiles have shown utility in early detection of and colorectal cancers through non-invasive biopsies since 2020.

Piwi-interacting RNAs (piRNAs)

Piwi-interacting RNAs (piRNAs) are a class of germline-specific small non-coding RNAs, typically 24-31 nucleotides in length, that play a crucial role in maintaining genome stability by silencing transposable elements (TEs) in animal gonads. These single-stranded RNAs associate exclusively with proteins from the Piwi clade of Argonaute proteins, distinguishing them from other small RNA pathways that utilize Ago or Ago-like proteins. piRNAs were first identified in 2006 through deep sequencing of small RNAs from Drosophila melanogaster ovaries and mouse testes, revealing their abundance and specificity to reproductive tissues. A hallmark of piRNA structure is a strong for at the 5' terminal position (1U ) in primary piRNAs and at the 10th position (10A ) in secondary piRNAs, which arises from their biogenesis mechanism and facilitates target recognition. Unlike miRNAs or siRNAs, piRNAs are processed independently of enzymes; instead, primary piRNAs are transcribed from discrete genomic loci known as piRNA clusters—often repetitive regions enriched in TE sequences—and loaded directly onto proteins in the nucleus or . Secondary piRNAs are generated through an amplification mechanism called the ping-pong cycle, where Aubergine (Aub)-bound sense piRNAs slice complementary TE transcripts, producing 5' ends for new antisense piRNAs that are then loaded onto 3 (Ago3); this reciprocal slicing amplifies the piRNA population and ensures robust TE targeting. In gonads, s are highly abundant, with millions of unique sequences (over 1.5 million) detected in ovarian tissues, predominantly mapping to TEs and preventing their mobilization during to safeguard integrity across generations. This silencing is essential for , as disruptions in the piRNA pathway lead to TE derepression, DNA damage, and sterility. In humans, piRNAs similarly associate with PIWIL proteins (homologs of ), and mutations in PIWIL genes, such as PIWIL1, have been linked to defective and due to impaired piRNA-mediated TE control. Recent studies from the have further elucidated piRNAs' roles beyond transposon defense, including contributions to epigenetic inheritance by directing heritable patterns in the and maintaining pluripotency through in gonadal stem cells.

Mechanisms of Action

Core RNA Interference Pathway

The core (RNAi) pathway represents the central biochemical mechanism by which small RNAs mediate in eukaryotes, conserved from to humans. This pathway is initiated when double-stranded (dsRNA) precursors, such as those derived from viral infections or endogenous transcripts, are recognized and processed into duplexes approximately 21-25 nucleotides long. These duplexes serve as guides for targeting complementary RNAs (mRNAs), leading to their degradation or translational repression. The first key step involves the RNase III family enzyme , which binds to dsRNA via its and PAZ domains, then cleaves it in an ATP-dependent manner to generate the duplexes. In , Dicer-2 processes exogenous dsRNA into small interfering RNAs (siRNAs), while Dicer-1 processes (miRNA) precursors; in mammals, a single Dicer processes both types of substrates. These duplexes include 2-nucleotide 3' overhangs that facilitate subsequent loading. Recent cryo-electron (cryo-EM) structures have revealed the dynamic architecture of Dicer heterodimers with accessory proteins like TRBP or , showing how they position the dsRNA for precise cleavage and ensure directionality. Following processing, the duplex is loaded into the (RISC), a multiprotein assembly whose core is an (Ago) protein. During RISC assembly, the duplex is unwound in an ATP-dependent process mediated by chaperones like Hsc70/, and the passenger strand is ejected, leaving the thermodynamically stable guide strand (antisense to the target) bound in Ago's central cleft. In mammals, Ago2 is the primary slicer-competent isoform, with its domain harboring the catalytic residues for endonucleolytic activity. Cryo-EM studies from 2022 have provided high-resolution insights into human Ago proteins, elucidating conformational changes in the MID and domains that enable guide strand selection and stabilize the complex. Once assembled, RISC scans target mRNAs through base-pairing between the guide RNA and the target, primarily involving the region ( 2-8 of the guide) for initial recognition. For siRNAs with perfect or near-perfect complementarity, Ago2's activity cleaves the target mRNA between 10 and 11 relative to the guide's 5' end, leading to endonucleolytic degradation and rapid clearance of the transcript. In contrast, miRNAs often exhibit partial complementarity, particularly relying on , which represses , promotes deadenylation via of the CCR4-NOT complex, and ultimately triggers mRNA decay without direct cleavage. This pathway is highly conserved across species, enabling precise . A notable feature of the core pathway is its susceptibility to off-target effects, where partial complementarity—especially in the seed region—can lead to unintended silencing of non-cognate transcripts, mimicking miRNA-like repression even with siRNA triggers. These effects arise from the guide strand's ability to bind multiple partially matched sites, highlighting the pathway's sensitivity to sequence specificity.

Integration with Gene Regulation

RNA silencing pathways intersect with broader gene regulatory networks by influencing both post-transcriptional and transcriptional control, enabling precise modulation of . In addition to cytoplasmic mRNA degradation, small RNAs can direct chromatin modifications that lead to transcriptional repression. For instance, in the fission yeast , siRNAs generated from centromeric transcripts are incorporated into the RNA-induced transcriptional silencing (RITS) complex, which recruits methyltransferases to deposit H3K9me marks, promoting formation and at pericentromeric regions.00881-0) This nuclear RNAi mechanism, first elucidated in the early , exemplifies how RNA silencing extends beyond translation to enforce epigenetic silencing, preventing spurious transcription from repetitive elements. At the post-transcriptional level, miRNAs primarily down-regulate target genes through mRNA destabilization. A key step involves the GW182 protein family, which interacts with Argonaute-loaded miRNAs in the (RISC) to recruit the CCR4-NOT deadenylase complex. This interaction promotes rapid poly(A) tail shortening of target mRNAs, followed by and exonucleolytic decay, thereby reducing protein output without direct cleavage. In and mammalian systems, GW182's conserved motifs, such as the PAM2 domain, mediate this deadenylation, highlighting a conserved mechanism for miRNA-mediated repression that integrates with cellular mRNA turnover pathways. Although predominantly repressive, RNA silencing can occasionally lead to gene up-regulation through indirect mechanisms. For example, miR-373 activates E-cadherin expression by binding to complementary sequences in its promoter, competing with repressive transcription factors or recruiting activating complexes, thus enhancing transcription in a sequence-specific manner. Rare instances of mRNA stabilization also occur when miRNAs target destabilizing elements in 3' UTRs, preventing decay factors from binding, though such cases are less common than repressive outcomes. Regulatory networks further incorporate RNA silencing via competitive endogenous RNAs (ceRNAs), including circular RNAs (circRNAs), which act as miRNA sponges to titrate miRNAs away from canonical targets. The circRNA ciRS-7 (also known as CDR1as), highly expressed in mammalian brains, contains over 70 binding sites for miR-7, sequestering it and derepressing miR-7 targets involved in neuronal function. This sponging reduces miRNA availability, effectively up-regulating and integrating RNA silencing into a broader ceRNA network that fine-tunes developmental and stress responses. Recent studies have revealed that RNA silencing components localize to phase-separated biomolecular condensates, enhancing pathway efficiency through spatial organization. In plants, Dicer-like proteins and Argonautes form liquid-like dicing bodies that concentrate siRNA precursors, accelerating processing and silencing of viral RNAs. Similarly, in animal cells, Argonaute-miRNA complexes participate in cytoplasmic , which exhibit properties to compartmentalize mRNA decay, linking RNA silencing to condensate-mediated regulation observed in 2024 investigations.

Regulation of the Silencing Machinery

The RNA silencing machinery is tightly regulated by endogenous microRNAs (miRNAs) that control the expression of key components such as and proteins. For instance, the let-7 miRNA family directly targets mRNA within its coding sequence, reducing its expression and thereby modulating miRNA biogenesis in a feedback manner; reduced let-7 levels lead to overexpression in contexts like . Similarly, other miRNAs, such as miR-99a, post-transcriptionally repress 2 (AGO2) expression, coupling AGO2 protein stability to overall miRNA abundance to prevent excess silencing activity. In , miR162 targets Dicer-like 1 (DCL1), establishing a negative feedback loop that fine-tunes miRNA production. Post-translational modifications, particularly , further modulate function. at specific residues, such as tyrosine 529 in the small RNA-binding pocket of human AGO proteins, alters binding affinity and slicer activity, thereby adjusting the efficiency of target mRNA cleavage. Target engagement triggers hierarchical of AGO2, including at serine 387 by p38 MAPK, which enhances miRNA-mediated repression while an opposing cycle maintains dynamic control. kinases 1 and 2 phosphorylate AGO proteins within a conserved cluster, promoting miRNA-induced silencing complex (miRISC) binding to targets. Feedback loops ensure homeostasis in the silencing pathway. Autoregulation occurs through siRNAs or miRNAs targeting biogenesis genes; in , small RNAs maintain optimal protein levels via a feedback loop involving AGO-associated slicing activity on their own transcripts. Environmental cues, such as stress, enhance miRNA-mediated without altering miRNA expression levels, as observed in where stress boosts the activity of the (RISC). The LIN-28 provides another layer of control by binding the terminal loop of let-7 precursors, recruiting terminal uridylyl transferases to inhibit their maturation and processing by . Protein modifications like ubiquitination regulate RISC turnover and stability. Endogenous ubiquitination targets Argonaute proteins for proteasomal degradation, preventing accumulation and ensuring pathway responsiveness; this is complemented by viral mechanisms, such as HIV-1 Tat protein, which acts as a suppressor of RNA interference (RNAi) by enhancing AGO degradation or inhibiting RISC assembly. Subcellular localization to processing bodies (P-bodies) facilitates storage and decay of silenced mRNAs; Argonaute proteins and GW182 localize to P-bodies, where miRISC components are sequestered for regulated release or degradation, distinct from stress granules under cellular stress. Epitrascriptomic modifications, including N6-methyladenosine (m6A), influence pri-miRNA processing. The m6A mark on primary miRNA transcripts recruits the microprocessor complex (Drosha-DGCR8), promoting efficient cleavage and maturation; disruption of this modification impairs miRNA biogenesis and silencing efficiency. These regulatory mechanisms collectively fine-tune RNA silencing to respond to developmental, environmental, and pathological signals.

Biological Roles

Defense Against Viruses and Transposons

RNA silencing serves as a primary innate immune mechanism in many eukaryotes, targeting viral double-stranded RNA (dsRNA) for degradation to limit infection spread. In plants and insects, viral dsRNA is processed by Dicer enzymes into small interfering RNAs (siRNAs), which are incorporated into the RNA-induced silencing complex (RISC) containing Argonaute proteins. The RISC then uses these virus-derived siRNAs (vsiRNAs) to recognize and cleave complementary viral RNAs, thereby inhibiting replication. This pathway is highly conserved and effective against a range of RNA viruses, such as those infecting , where Dicer-2 initiates the antiviral response.00977-4.pdf) Experimental evidence underscores the protective role of RNAi against viruses in model organisms. In Caenorhabditis elegans, mutations in core RNAi components like rde-1 (encoding an Argonaute protein) or dcr-1 (Dicer) lead to increased viral loads and higher susceptibility to infection by viruses such as Orsay virus, demonstrating that RNAi restricts viral propagation in nematodes. Similarly, in plants, virus-induced gene silencing (VIGS) exploits this pathway, where viral vectors carrying host gene fragments trigger endogenous siRNA production to silence both viral and targeted host transcripts, confirming the mechanism's dual role in defense and functional genomics. Human immunodeficiency virus (HIV-1) can evade RNAi targeting its trans-activation response (TAR) element through mutations in the LTR promoter that upregulate viral transcription, allowing persistent replication despite siRNA-mediated suppression.30062-5) Beyond viruses, RNA silencing defends against transposons—mobile genetic elements that can cause genomic instability—primarily through piwi-interacting RNAs (piRNAs) in the and siRNAs in somatic tissues. In the germline, piRNAs, typically 24-31 long and bound to proteins, guide transposon silencing to prevent insertions that could lead to mutations during . Somatic transposon control in involves both piRNA and siRNA pathways, where siRNAs target repetitive transposon transcripts to maintain integrity in non-germline cells. In mice, piRNA pathway mutants, such as those lacking Mili or Henmt1, exhibit transposon derepression—a phenomenon known as transposon "bloom"—resulting in DNA damage, meiotic arrest, and male sterility due to unchecked transposon activity. Emerging evidence suggests that antiviral RNAi also operates in mammals, albeit in a context overshadowed by responses. Studies from 2021-2025 have detected vsiRNAs in mammalian cells infected with alphaviruses and other RNA viruses, indicating an active RNAi pathway that processes viral dsRNA. For , infection remodels the host small landscape, including siRNA-like molecules that target viral sequences, supporting a role for endogenous RNAi in restricting replication in mammalian somatic cells.00452-7)

Post-Transcriptional Gene Silencing

Post-transcriptional gene silencing (PTGS) represents a core function of RNA silencing in which microRNAs (miRNAs) suppress the expression of endogenous to maintain cellular and coordinate developmental processes. Unlike siRNA-mediated silencing that often cleaves perfectly complementary targets, miRNA-directed PTGS typically involves imperfect base-pairing, leading to fine-tuned of protein output without complete mRNA elimination in many cases. This mechanism affects a substantial portion of the , with estimates indicating that miRNAs regulate approximately 60% of human protein-coding through coordinated networks that integrate with broader gene regulatory circuits. The primary mechanisms of miRNA-mediated PTGS include translational repression, where the miRNA-loaded RNA-induced silencing complex (RISC) inhibits ribosome recruitment or elongation on target mRNAs, and mRNA decay, which involves deadenylation followed by decapping and exonucleolytic degradation. Recent studies have clarified that translational repression often precedes and triggers poly(A) tail shortening, with the miRISC recruiting deadenylases such as the CCR4-NOT complex to remove the poly(A) tail, thereby destabilizing the mRNA. This successive process ensures rapid and efficient silencing, as evidenced in mammalian systems where miRNA binding to the 3' untranslated region (UTR) of targets initiates both outcomes. Additionally, miRNAs function as regulatory hubs within signaling pathways, coordinately targeting multiple components—such as receptors, kinases, and transcription factors—to modulate pathway outputs and prevent aberrant activation. Illustrative examples highlight PTGS's role in precise biological control. In Caenorhabditis elegans, the miRNA lin-4 was the first identified regulator of developmental timing, where it binds to the 3' UTR of lin-14 mRNA to repress its translation, ensuring timely progression from larval stages by reducing LIN-14 protein levels post-hatching. Similarly, the let-7 miRNA family controls progression by targeting oncogenes like RAS and HMGA2, thereby acting as tumor suppressors that inhibit proliferation in models; for instance, enforced let-7 expression arrests cells in and promotes . These examples underscore how individual miRNAs can orchestrate temporal and proliferative control through PTGS. miRNA networks amplify PTGS's impact through familial clustering and coordinated targeting. The let-7 family, comprising multiple paralogous members often encoded in genomic clusters (e.g., let-7a-1/let-7f-2 cluster on human ), exhibits redundant yet synergistic regulation of shared targets, enhancing robustness in pathways like and differentiation. In sex chromosome dosage compensation, sex-biased miRNA expression—such as elevated X-linked miRNAs in females—helps balance by preferentially repressing autosomal targets, mitigating imbalances between XX and XY cells in mammals. Furthermore, specific miRNAs like miR-21 exemplify PTGS in , where it targets pro-apoptotic genes such as PDCD4 to inhibit and promote survival in cancer contexts. Post-2018 single-cell RNA sequencing studies have revealed miRNA expression gradients across tissue cells, illustrating spatially resolved heterogeneity that refines PTGS at the organismal level, as seen in developmental tissues where miRNA levels vary to establish morphogen-like gradients.

Roles in Development and Epigenetics

RNA silencing plays a pivotal role in orchestrating developmental timing and progression across diverse organisms. In Caenorhabditis elegans, the heterochronic pathway exemplifies this control, where microRNAs such as lin-4 and let-7 regulate the temporal sequence of cell fate decisions during post-embryonic development. The lin-4 miRNA, discovered as the first known miRNA, post-transcriptionally represses the lin-14 mRNA to promote progression from early to late larval stages, preventing precocious differentiation; mutations in lin-4 lead to reiteration of early cell divisions at the expense of later ones. Similarly, let-7 miRNA targets lin-41 and lin-28 to fine-tune the transition to adult fates, ensuring coordinated developmental progression.00801-9) These miRNAs integrate with broader gene regulatory networks to maintain temporal precision, highlighting RNA silencing's essential function in heterochrony. In animal embryogenesis, miRNAs facilitate critical transitions, such as maternal mRNA clearance and cell proliferation-apoptosis balance. During early development, miR-430 is zygotically expressed to deadenylate and degrade hundreds of maternal transcripts, enabling the maternal-to-zygotic transition and clearing regulatory impediments to embryogenesis; its absence results in developmental delays and morphological defects. In Drosophila melanogaster, the bantam miRNA promotes tissue growth by inhibiting through repression of the pro-apoptotic gene hid, while simultaneously stimulating proliferation, thus coordinating organ size and patterning during imaginal disc development.00231-9) Dysregulation of such miRNAs, as seen with miR-124 in mammalian neurodevelopment, contributes to disorders like autism spectrum disorder, where reduced miR-124 expression impairs neuronal differentiation and in the . In plants, miRNAs like miR-172 regulate floral development by specifying organ identity and morphology. In Arabidopsis thaliana, miR-172 targets APETALA2 (AP2) transcripts to promote the transition from vegetative to reproductive phases and ensure proper petal and stamen formation; loss of miR-172 activity leads to carpel-like petals and sepalloid stamens, disrupting flower architecture. Recent studies in C. elegans have also uncovered intergenerational effects of RNA silencing, where small RNAs mediate heritable gene repression across generations, influencing developmental phenotypes like stress responses and germline integrity in response to environmental cues.30930-2) Epigenetically, RNA silencing induces heritable modifications through siRNA- and piRNA-directed mechanisms. In plants, the RNA-directed DNA methylation (RdDM) pathway uses 24-nucleotide siRNAs to recruit DNA methyltransferases, establishing and maintaining methylation at transposons and repetitive elements to silence genes involved in development and stress responses; this process, canonically involving IV and V, ensures epigenetic stability across cell divisions. In animals, piRNAs facilitate paramutation-like inheritance, as observed in , where maternally transmitted piRNAs from a paramutagenic locus epigenetically convert a naive at an unlinked site, leading to stable, heritable repression without DNA sequence changes. piRNAs also drive germline reprogramming in mammals by guiding de novo of transposons during , protecting the genome from mutagenic insertions and enabling epigenetic inheritance in the next generation.

Applications and Challenges

Therapeutic and Biomedical Uses

RNA silencing technologies, particularly small interfering RNAs (siRNAs) and (miRNA) modulators, have revolutionized therapeutic strategies for various diseases by enabling precise . The landmark approval of (Onpattro) by the FDA in August 2018 marked the first siRNA-based drug for clinical use, targeting hereditary transthyretin-mediated (hATTR) with in adults. , delivered via lipid nanoparticles, silences the (TTR) gene to reduce toxic protein aggregates, demonstrating significant improvements in neuropathy scores and in phase III trials. Subsequent siRNA approvals, such as givosiran for acute hepatic in 2019, have expanded this class, highlighting RNAi therapeutics' potential in rare genetic disorders. In March 2025, the FDA approved an expanded indication for (Amvuttra) for transthyretin amyloid , demonstrating reduced cardiovascular death, hospitalizations, and urgent visits in phase III trials. miRNA-based approaches have also advanced into therapeutics, with inhibitors and mimics addressing dysregulated . Miravirsen, a locked nucleic acid-modified antisense targeting miR-122, has been evaluated in phase II trials for chronic hepatitis C virus (HCV) genotype 1 infection, achieving prolonged, dose-dependent reductions in HCV levels without evidence of viral resistance or significant adverse effects. This strategy exploits miR-122's role in stabilizing HCV , offering a non-mutagenic alternative to direct antivirals. miRNA mimics, such as MRX34 (a liposomal miR-34a mimic), entered clinical testing for cancer, with phase I trials in patients with advanced solid tumors showing partial responses and stable disease in subsets. However, despite premedication with dexamethasone to mitigate immune activation, the trial was halted in 2016 due to severe adverse events including . These efforts underscore miR-34a's tumor-suppressive functions in inhibiting and across and other malignancies, while highlighting delivery and safety challenges for miRNA mimics. In , RNA silencing supports for neurodegenerative disorders like , where short hairpin RNAs (shRNAs) delivered via (AAV) vectors target the mutant gene to lower toxic protein levels. Preclinical studies using AAV-shRNA constructs have demonstrated up to 50% reduction in mutant huntingtin without affecting wild-type alleles, paving the way for clinical translation. Ongoing phase I/II trials, such as uniQure's AMT-130 (an AAV-miRNA therapy with shRNA-like silencing), report slowed disease progression by 75% at three years, informing shRNA-optimized designs for durable, one-time administration. However, in November 2025, uniQure announced that it and the FDA were no longer aligned on the approval pathway, with the agency indicating insufficient clinical data for current plans. For cancer, RNAi targets oncogenes like and EGFR; siRNAs conjugated to nanoparticles have shown preclinical efficacy in silencing these drivers, enhancing sensitivity in lung and pancreatic models. Effective delivery remains pivotal, with lipid nanoparticles (LNPs) enabling liver-specific siRNA uptake, as exemplified by patisiran's ionizable lipid formulation that protects against degradation and facilitates endosomal escape. AAV vectors provide long-term shRNA expression in non-dividing cells like neurons, though limits dosing. Stability challenges are addressed through chemical modifications, such as 2'-O-methyl (2'-OMe) substitutions on ribose sugars, which enhance serum half-life, reduce immune stimulation, and improve binding affinity without compromising potency. These modifications, often combined with phosphorothioate backbones, have been integral to approved drugs and trial candidates. As of 2025, over 38 new RNA interference trials have initiated globally, focusing on (26%) and rare diseases, with expanded pipelines from companies like Alnylam and targeting cardiovascular and metabolic indications. In infectious diseases, RNAi therapeutics for variants continue to progress; siRNA-LNP formulations targeting conserved viral regions have demonstrated broad-spectrum antiviral activity in preclinical models, offering adjunctive potential alongside mRNA vaccines by suppressing viral replication and enhancing immune responses. Clinical data from phase II trials of broad-spectrum siRNAs like SNS812 show rapid viral clearance in patients, supporting variant-proof strategies.

Biotechnological and Agricultural Applications

RNA silencing has emerged as a powerful tool in for high-throughput and . siRNA libraries enable systematic screening of functions by silencing thousands of targets simultaneously, facilitating the identification of novel therapeutic candidates in various disease models. For instance, genome-wide siRNA screens have been instrumental in uncovering pathways involved in cancer progression and , accelerating target validation in pharmaceutical pipelines. In , RNAi circuits in yeast, such as , allow for programmable regulation, including Boolean logic gates that process inputs to control cellular behaviors like metabolic flux. These circuits, constructed using dsRNA triggers, enable dynamic pathway engineering, such as optimizing production by fine-tuning enzyme expression levels. In , RNA silencing underpins the development of virus-resistant crops, exemplified by the transgenic papaya introduced in 1998, which expresses the coat protein gene of (PRSV) to confer resistance via post-transcriptional gene silencing. This innovation rescued Hawaii's papaya industry from near collapse due to PRSV outbreaks, with the cultivar demonstrating sustained efficacy against local strains over decades. Similarly, hairpin RNAi transgenics, where constructs produce self-complementary dsRNAs, have been widely adopted to silence endogenous plant genes for trait improvement, such as enhancing nutrient uptake or reducing allergenicity in crops like and . Pest management has been revolutionized by RNAi-based strategies, including enhancements to Bt toxin efficacy through miRNA targeting of insect immune responses. Transgenic plants overexpressing insect-specific miRNAs, like those targeting chitin synthase in lepidopteran pests, synergize with Bt proteins to delay resistance development and increase mortality rates in species such as . In the 2020s, sprayable dsRNA formulations have gained traction as non-transgenic biopesticides; for example, Ledprona, approved by the U.S. EPA in 2023, targets the Colorado potato beetle by silencing essential genes upon foliar application, offering species-specific control with minimal impact on non-target organisms. Monsanto's MON 87411 corn, approved in 2017, incorporates an RNAi construct against the western corn rootworm's DvSnf7 gene, reducing larval damage and supporting in fields. Virus-induced gene silencing (VIGS) serves as a rapid, non-transgenic tool for dissecting plant gene functions in , particularly in recalcitrant like and , by exploiting viral vectors to deliver silencing constructs. In , RNAi applications extend to enhancing production traits in salmon farming; for instance, transient dsRNA delivery silences growth-regulating genes in (Salmo salar), promoting faster somatic growth and improving feed efficiency in controlled settings. Recent advances in miRNA editing, using CRISPR-Cas9 to modify miRNA loci, have yielded climate-resilient crops by 2023–2025, such as drought-tolerant varieties where edited miR156 enhances tillering and yield under water stress, addressing projections of increasing abiotic pressures from .

Technical Limitations and Ethical Issues

One significant technical limitation of RNA silencing, particularly with small interfering RNAs (siRNAs), is off-target silencing, where partial complementarity in the seed region (positions 2–8 of the guide strand) allows unintended mRNA targets to be cleaved or translationally repressed by the (RISC). Early siRNA applications in the 2000s showed off-target effects affecting 10–30% of screened genes, often due to shared seed sequences across the , though design algorithms developed in the 2020s, such as those incorporating for sequence optimization, have reduced these rates to below 5% in many cases by prioritizing unique targeting and chemical modifications like 2'-O-methyl groups. Another barrier is the activation of innate immune responses, as double-stranded RNAs longer than 30 base pairs or certain siRNA structures are recognized by endosomal Toll-like receptor 3 (TLR3), triggering type I interferon production, release, and potential toxicity, which has limited in therapeutics. Chemical modifications, such as incorporation of 2'-fluoro or locked nucleic acids, mitigate this by evading TLR3 binding while preserving silencing efficacy. siRNAs also exhibit instability , rapidly degrading via serum nucleases and renal clearance, with half-lives often under an hour without protection, necessitating lipid nanoparticle encapsulation or conjugation to moieties like for prolonged circulation. Delivery challenges further hinder RNA silencing applications, with endosomal entrapment representing a primary bottleneck: following cellular uptake, over 99% of siRNA-laden nanoparticles remain sequestered in endosomes and lysosomes, preventing escape to the where RISC loading occurs, and leading to lysosomal degradation. Strategies like pH-sensitive or histidine-rich peptides promote disruption for escape, but efficiency remains below 2–5% in non-hepatic tissues. Tissue specificity compounds this, as effective delivery is largely confined to the liver via receptor-mediated uptake, while achieving targeted silencing in the , lungs, or tumors requires overcoming blood-brain or barriers. In agricultural contexts, scalability of exogenous RNAi sprays for faces issues with environmental degradation by UV light and nucleases, uneven foliar absorption, and variable efficacy across crop species, limiting large-scale deployment. Ethically, the deployment of RNAi-based gene drives in ecosystems, such as those engineered in mosquitoes to suppress transmission by biasing inheritance of sterility genes, poses risks of unintended ecological disruption, including drive spread to non-target , loss of , and irreversible alterations to food webs if containment fails. Equity concerns arise in therapeutic access, as RNAi drugs' high development and production costs—often $400,000–$600,000 per patient annually—disproportionately burden low-income populations and regions, potentially widening global health disparities despite their potential for rare disease treatment. The dual-use potential of RNAi technologies raises issues, as sequence-specific silencing could be repurposed to design agents disrupting essential genes in pathogens or cells, though current limitations in delivery and specificity make weaponization challenging compared to other tools. In , European Union regulations under Directive 2001/18/EC and Regulation (EU) No 503/2013 classify RNAi-based as requiring comprehensive risk assessments for environmental release, focusing on off-target effects in non-target organisms and long-term ecological impacts, with approvals in the 2010s emphasizing case-by-case evaluations by the . Recent discussions on heritable editing via piRNA pathways, which silence transposons across generations, underscore concerns over for future descendants, unintended epigenetic consequences, and equitable , as highlighted in 2024 reviews calling for international moratoriums until safety is assured.

References

  1. https://www.cell.com/molecular-therapy-family/nucleic-acids/fulltext/S2162-2531(23)00191-9
  2. https://www.annualreviews.org/doi/10.1146/annurev-virology-091919-064218
  3. https://www.nature.com/articles/nature02874
  4. https://www.ncbi.nlm.nih.gov/books/NBK580472/
  5. https://www.nature.com/articles/35888
  6. https://pmc.ncbi.nlm.nih.gov/articles/PMC2709356/
  7. https://pmc.ncbi.nlm.nih.gov/articles/PMC3017132/
  8. https://journals.asm.org/doi/10.1128/microbiolspec.funk-0008-2016
  9. https://www.sciencedirect.com/science/article/abs/pii/S0169534708002437
  10. https://pmc.ncbi.nlm.nih.gov/articles/PMC2724769/
  11. https://pubmed.ncbi.nlm.nih.gov/12354959/
  12. https://pmc.ncbi.nlm.nih.gov/articles/PMC6468295/
  13. https://pubmed.ncbi.nlm.nih.gov/8252621/
  14. https://www.science.org/doi/10.1126/science.286.5441.950
  15. https://www.nature.com/articles/35053110
  16. https://www.sciencedirect.com/science/article/pii/S2214574525001233
  17. https://www.nature.com/articles/35078107
  18. https://www.embopress.org/doi/full/10.1093/emboj/20.23.6877
  19. https://pmc.ncbi.nlm.nih.gov/articles/PMC5542916/
  20. https://pmc.ncbi.nlm.nih.gov/articles/PMC2729316/
  21. https://www.nature.com/articles/nature07007
  22. https://doi.org/10.1016/j.cell.2018.03.006
  23. https://doi.org/10.1038/nsmb1293
  24. https://doi.org/10.1016/j.cell.2007.06.028
  25. https://doi.org/10.1038/ng865
  26. https://www.sciencedirect.com/science/article/pii/S1097276524001242
  27. https://www.nature.com/articles/s41388-024-03076-3
  28. https://doi.org/10.1016/j.cell.2007.02.057
  29. https://wires.onlinelibrary.wiley.com/doi/10.1002/wsbm.1572
  30. https://pmc.ncbi.nlm.nih.gov/articles/PMC6974405/
  31. https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2024.1495035/full
  32. https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2020.01237/full
  33. https://pmc.ncbi.nlm.nih.gov/articles/PMC8138440/
  34. https://www.cell.com/fulltext/S0092-8674(13)01228-2
  35. https://www.nature.com/articles/s41586-022-04790-2
  36. https://www.sciencedirect.com/science/article/pii/S1097276521010285
  37. https://academic.oup.com/nar/article/50/12/6618/6613925
  38. https://bartellab.wi.mit.edu/publication_reprints/Bartel_Cell09.pdf
  39. https://pmc.ncbi.nlm.nih.gov/articles/PMC7876455/
  40. https://pmc.ncbi.nlm.nih.gov/articles/PMC2234192/
  41. https://www.pnas.org/doi/10.1073/pnas.0803230105
  42. https://www.nature.com/articles/oncsis201411
  43. https://www.nature.com/articles/6800789
  44. https://www.nature.com/articles/srep27598
  45. https://www.sciencedirect.com/science/article/pii/S0960982217301392
  46. https://journals.asm.org/doi/10.1128/mbio.00264-17
  47. https://pmc.ncbi.nlm.nih.gov/articles/PMC2742160/
  48. https://rupress.org/jcb/article/191/5/905/36156/piRNAs-transposon-silencing-and-Drosophila
  49. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021882
  50. https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1005620
  51. https://www.sciencedirect.com/science/article/pii/S1074761321003988
  52. https://www.nature.com/articles/s41540-024-00367-z
  53. https://www.nature.com/articles/s41598-017-13470-5
  54. https://febs.onlinelibrary.wiley.com/doi/10.1111/febs.16422
  55. https://www.sciencedirect.com/science/article/pii/S0092867404000455
  56. https://elifesciences.org/articles/03032
  57. https://www.nature.com/articles/srep17957
  58. https://www.pnas.org/doi/10.1073/pnas.0712321105
  59. https://pmc.ncbi.nlm.nih.gov/articles/PMC4742387/
  60. https://pmc.ncbi.nlm.nih.gov/articles/PMC5741053/
  61. https://www.nature.com/articles/cddis201447
  62. https://www.embopress.org/doi/10.15252/embj.2018100754
  63. https://pmc.ncbi.nlm.nih.gov/articles/PMC5223506/
  64. https://pubmed.ncbi.nlm.nih.gov/36059036/
  65. https://pmc.ncbi.nlm.nih.gov/articles/PMC4412961/
  66. https://www.accessdata.fda.gov/drugsatfda_docs/nda/2018/210922Orig1s000Approv.pdf
  67. https://www.accessdata.fda.gov/drugsatfda_docs/label/2018/210922s000lbl.pdf
  68. https://investors.alnylam.com/press-release?id=28831
  69. https://www.nejm.org/doi/full/10.1056/NEJMoa1209026
  70. https://pmc.ncbi.nlm.nih.gov/articles/PMC9938166/
  71. https://pubmed.ncbi.nlm.nih.gov/32238921/
  72. https://translationalneurodegeneration.biomedcentral.com/articles/10.1186/s40035-017-0101-9
  73. https://www.uniqure.com/programs-pipeline/phase-1-2-clinical-trial-of-amt-130
  74. https://uniqure.gcs-web.com/news-releases/news-release-details/uniqure-provides-regulatory-update-amt-130-huntingtons-disease-0
  75. https://www.cas.org/resources/cas-insights/sirna-therapeutics
  76. https://www.nature.com/articles/s41576-021-00439-4
  77. https://www.creative-biolabs.com/gene-therapy/aav-vector-design-service-rnai-delivery.htm
  78. https://www.nature.com/articles/s41392-020-0207-x
  79. https://pubs.acs.org/doi/10.1021/acsomega.7b00291
  80. https://www.asgct.org/uploads/files/general/Landscape-Report-2025-Q2.pdf
  81. https://www.atsjournals.org/doi/abs/10.1164/ajrccm.2025.211.Abstracts.A4987
  82. https://www.nature.com/articles/nmeth.2958
  83. https://pmc.ncbi.nlm.nih.gov/articles/PMC8377577/
  84. https://www.jci.org/articles/view/38475
  85. https://pmc.ncbi.nlm.nih.gov/articles/PMC7600125/
  86. https://pmc.ncbi.nlm.nih.gov/articles/PMC9595607/
  87. https://www.pnas.org/doi/10.1073/pnas.2307800120
  88. https://journals.biologists.com/jcs/article/123/8/1183/31591/Breaking-down-the-barriers-siRNA-delivery-and
  89. https://www.nature.com/articles/s12276-023-00998-y
  90. https://enveurope.springeropen.com/articles/10.1186/s12302-025-01052-6
  91. https://www.nature.com/articles/s41434-024-00468-8
  92. https://bmcmedethics.biomedcentral.com/articles/10.1186/s12910-021-00588-5
  93. https://www.forbes.com/sites/matthewherper/2018/08/10/alnylam-prices-breakthrough-drug-at-450000-per-patient-but-offers-money-back-guarantee/
  94. https://pmc.ncbi.nlm.nih.gov/articles/PMC2443345/
  95. https://www.efsa.europa.eu/en/efsajournal/pub/9321
  96. https://pmc.ncbi.nlm.nih.gov/articles/PMC7186845/
  97. https://pubmed.ncbi.nlm.nih.gov/37581898/
Add your contribution
Related Hubs
User Avatar
No comments yet.