Gene Silencing | Botany Optional for UPSC PDF Download

Introduction

• Gene silencing refers to the control of gene expression within a cell to prevent the activation of a specific gene. This regulatory process can take place during either transcription or translation and has become a valuable tool in scientific research. Researchers are increasingly utilizing gene silencing techniques to develop therapeutic approaches for addressing various conditions, including cancer, infectious diseases, and neurodegenerative disorders.
• It's worth noting that gene silencing is often used interchangeably with the term "gene knockdown." When genes are silenced, their expression is diminished, whereas gene knockout involves completely removing a gene from an organism's genome, resulting in no expression. Gene silencing is considered a form of gene knockdown because the methods employed, such as RNAi (RNA interference), CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), or siRNA (small interfering RNA), typically reduce gene expression by at least 70%, without completely erasing it. Gene silencing approaches are preferred over gene knockouts since they enable the study of essential genes that are vital for the survival of animal models and cannot be deleted. Moreover, they offer a more comprehensive understanding of disease development, as diseases often involve genes with reduced expression levels.

Research Methods

Antisense Oligonucleotides

• Antisense oligonucleotides, discovered in 1978 by Paul Zamecnik and Mary Stephenson, are short fragments of nucleic acids that can bind to complementary target mRNA molecules within cells. These oligonucleotides, typically 13–25 nucleotides long and made of single-stranded DNA or RNA, can regulate gene expression through two mechanisms: RNase H-dependent and steric blocking. 
• RNase H-dependent oligonucleotides lead to the degradation of target mRNA molecules, while steric-blocker oligonucleotides hinder mRNA translation. Most antisense drugs function through the RNase H-dependent mechanism, wherein RNase H degrades the RNA strand of the DNA/RNA heteroduplex.

Ribozymes

• Ribozymes, catalytic RNA molecules, inhibit gene expression by cleaving mRNA molecules, effectively silencing the genes responsible for producing those mRNA molecules. Sidney Altman and Thomas Cech discovered catalytic RNA molecules, including RNase P and group II intron ribozymes, in 1989. 
• Ribozymes come in various motifs, such as hammerhead, hairpin, hepatitis delta virus, group I, group II, and RNase P ribozymes. They cleave RNA molecules by attacking a specific phosphodiester bond on the mRNA, resulting in the formation of cleaved products with a 2'3'-cyclic phosphate and a 5' hydroxyl terminal end. Researchers are exploring the use of ribozymes to develop gene silencing therapeutics for disease treatment.

RNA Interference (RNAi)

• RNA interference is a natural cellular process that regulates gene expression. Discovered in 1998 by Andrew Fire and Craig Mello, RNAi begins when double-stranded RNA (dsRNA) enters a cell, initiating the RNAi pathway. An enzyme called Dicer cleaves the dsRNA into small fragments, including small interfering RNAs (siRNA) and microRNA (miRNA). 
• These fragments integrate into the RNA-induced silencing complex (RISC), containing Argonaute proteins, which are essential components of the RNAi pathway. One strand, the guide strand, binds to RISC, directing sequence-specific silencing of target mRNA molecules. RNAi can silence genes through endonucleatic cleavage or translational repression of the mRNA molecules.

Three Prime Untranslated Regions and MicroRNAs

• The three prime untranslated regions (3'UTRs) of mRNA molecules often contain regulatory sequences that post-transcriptionally induce gene silencing. These 3'UTRs can include binding sites for microRNAs (miRNAs) and regulatory proteins. MiRNAs, in particular, play a significant role in decreasing gene expression by binding to specific sites within the 3'UTR. 
• Additionally, the 3'UTR may contain silencer regions that bind repressor proteins to inhibit mRNA expression. MiRNAs have thousands of target mRNAs, causing gene silencing for multiple genes. Dysregulation of miRNAs can be relevant in various diseases, including cancer and neuropsychiatric disorders. MiRNAs have also been found to affect the stability and production of numerous proteins and can influence various biological processes.

Applications

Medical Research

• Gene silencing techniques have found extensive applications in medical research, particularly in studying genes associated with various disorders such as cancer, infectious diseases, respiratory diseases, and neurodegenerative disorders. 
• These techniques are also instrumental in drug discovery processes, including synthetic lethality, high-throughput screening, and miniaturized RNAi screens.

Cancer

• Gene silencing, specifically RNA interference (RNAi), has been employed to target genes linked to various cancers. In the case of chronic myelogenous leukemia (CML), siRNA was used to cleave the BCR-ABL fusion protein, enhancing the sensitivity of cancer cells to drugs like Gleevec. SiRNA has also been utilized to selectively target mutated tumor suppressor genes, for instance, p53. 
• Additionally, receptors involved in mitogenic pathways contributing to cancer cell proliferation, such as chemokine receptor 4 (CXCR4), have been targeted using siRNA. SiRNA molecules have been used to regulate the expression of cancer-related genes like antiapoptotic proteins, clusterin, and survivin, thus increasing cancer cell sensitivity to chemotherapy. In vivo studies have explored the potential of siRNA in cancer therapeutics, demonstrating improved survival rates in mice implanted with cancer cells when treated with siRNAs targeting specific genes.

Infectious Disease

• Gene silencing techniques have been applied to combat various viral diseases. For instance, siRNA was used to silence the primary HIV receptor, CCR5, preventing the virus from entering host cells. Similar strategies have been employed to reduce the replication of hepatitis B and C viruses. 
• RNAi has been a part of commercial strategies to control plant virus diseases for over two decades. SiRNA has also been successful in targeting other viruses, including human papillomavirus, West Nile virus, and Tulane virus. SiRNA molecules have been used to prevent the replication of the Tulane virus by targeting both its structural and non-structural genes, offering insights into potential therapies for human norovirus, a common cause of gastroenteritis.

Bacteria

• Unlike viruses, bacteria are less susceptible to direct siRNA silencing due to their replication process occurring outside host cells. However, siRNA can still impact bacterial infections by targeting host genes involved in the immune response or bacterial entry into cells. 
• For instance, siRNA has been used to reduce pro-inflammatory cytokines in mice treated with lipopolysaccharide (LPS), decreasing septic shock. SiRNA has also prevented bacterial invasion of murine lung epithelial cells by targeting the caveolin-2 (CAV2) gene.

Respiratory Diseases

• Ribozymes, antisense oligonucleotides, and RNAi have been applied to target mRNA molecules involved in respiratory conditions such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. 
• SiRNA targeting transforming growth factor TGF-α reduced mucus secretion in human airway epithelial cells. Additionally, siRNA targeting transforming growth factor TGF-β has been used to improve fibrosis of lung tissue associated with COPD and asthma.

Neurodegenerative Disorders

• In the context of Huntington's disease (HD), gene silencing has been explored as a potential therapy. Allele-specific gene silencing using antisense oligonucleotides has been employed to target single nucleotide polymorphisms (SNPs) associated with the mutant huntingtin protein, showing promise in reducing mutant protein levels. 
• Non-allele-specific gene silencing via siRNA has also been used to target both normal and mutant huntingtin proteins, resulting in improved motor control and survival rates in mice. For amyotrophic lateral sclerosis (ALS), siRNA has been used to target the Cu/Zn superoxide dismutase (SOD1) gene mutations linked to the disease.

Therapeutics Challenges

• Challenges in gene silencing therapies include effective delivery to target cells, especially in neurological disorders, where the blood-brain barrier restricts access. Delivery methods such as direct injection or implantation of pumps have been explored. 
• Furthermore, maintaining specificity for the targeted cells is crucial, as molecules like antisense oligonucleotides and siRNA may inadvertently bind to unintended mRNA molecules. Researchers are actively seeking improved delivery methods and specific gene silencing therapeutics that are both safe and effective.

Food

• Gene silencing techniques have been applied to create nonbrowning apples known as Arctic Apples. This trait is achieved by reducing the expression of polyphenol oxidase (PPO) through gene silencing, resulting in apples that resist browning. 
• Arctic Apples represent one of the first approved food products to utilize gene silencing for this purpose.

Short history of gene silencing

1990 - Jorgensen

• Jorgensen conducted experiments on petunias to deepen their pigmentation by introducing transgenes homologous to endogenous genes. However, this often led to plants where both genes were suppressed, a phenomenon known as co-suppression.
• Co-suppression resulted in the degradation of both endogenous and transgene mRNA.

1995 - Guo and Kemphues

• Guo and Kemphues experimented with C. elegans by injecting either antisense or sense RNAs into the germline of the worms. Surprisingly, both approaches were equally effective at silencing homologous target genes.

1998 - Mello and Fire

• Building upon previous experiments, Mello and Fire extended their studies by combining sense and antisense RNAs, creating double-stranded RNA (dsRNA). They found that dsRNA was ten times more effective than using single-stranded RNA.

HOW does it works?

• The groundbreaking discovery of the RNA interference (RNAi) mechanism by Professor Andrew Fire and Professor Craig Mello in 1998 earned them the Nobel Prize in 2006.
• RNAi works by binding a specific RNA strand to an existing mRNA strand.
• When mRNA creates a copy of the DNA strand, binding RNA to the mRNA prevents replication of that DNA portion.
• Specific genes can be targeted and prevented from replicating into new DNA strands.

Types of Gene silencing

• Genes are controlled through regulation at two different stages: transcription and post-transcription. Consequently, gene silencing can be initiated either during transcription or after transcription has occurred.
• There are primarily two categories of gene silencing:
  • Transcriptional Gene Silencing
  • Post-Transcriptional Gene Silencing
    

Transcriptional gene silencing

• Transcriptional gene silencing results from histone modifications that create a chromatin environment, known as heterochromatin, around a gene. This chromatin structure makes the gene inaccessible to transcriptional machinery, such as RNA polymerase and transcription factors.
• Genomic imprinting is a genetic phenomenon in which specific genes are expressed in a manner that depends on their parental origin. It operates independently of classical Mendelian inheritance and has been observed in insects, mammals, and flowering plants.
• Paramutation is a genetic interaction between two alleles at the same locus, leading to a heritable change in one allele induced by the other. This phenomenon was initially discovered through its effect on the color of corn kernels in maize plants.
• Position effect refers to changes in gene expression when a gene's location on a chromosome is altered, often due to translocation events. In Drosophila, this phenomenon, known as position effect variegation (PEV), is well-documented and affects eye color.
• RNA-Directed DNA Methylation is an epigenetic process observed in plants, particularly in Arabidopsis thaliana, where small double-stranded RNAs guide DNA methylation at complementary DNA loci.
• Transposon silencing is a form of transcriptional gene silencing that targets transposons, which are DNA sequences capable of moving within the genome. This silencing mechanism helps prevent genomic instability caused by transposon activity and the associated deleterious mutations. Transposon insertions have been linked to various diseases, including hemophilia, severe combined immunodeficiency (SCID), and a predisposition to cancer.
• Transgene silencing occurs when a transgene is inserted into a transcriptionally inactive region of the genome. In such cases, the transgene does not exhibit the desired activity due to its instability. An example is the slow fruit-softening tomato, where reduced expression of the polygalacturonase enzyme results from transgene silencing.

Post transcriptional gene silencing

• Gene silencing, also known as RNA interference (RNAi), is a process where exogenous or endogenous RNA molecules suppress the expression of specific genes corresponding to their mRNA sequences. RNAi is triggered by the introduction of double-stranded RNA (dsRNA) molecules, leading to gene silencing in a sequence-specific manner.
  • The initial evidence for RNAi came from studies in the nematode Caenorhabditis elegans and was further explored in the fruit fly Drosophila melanogaster. RNAi is also referred to as post-transcriptional gene silencing, co-suppression, or quelling.
  • In RNAi, dsRNA molecules are processed into short interfering RNAs (siRNAs) by an enzyme called Dicer. These siRNAs then base-pair with target mRNA in a dsRNA-enzyme complex, leading to mRNA degradation. RNAi is a highly specific and potent process observed primarily in eukaryotes, regulating approximately 30% of the genome.
  • RNAi involves several components, including siRNA (small interfering RNA), which plays a central role in post-transcriptional gene silencing. SiRNAs are short (21-25 nucleotides) RNA fragments that bind to complementary regions of target mRNA, marking it for degradation. Even a single base pair difference between siRNA and target mRNA can block the process.
• Micro RNA (miRNA) is another type of RNA involved in post-transcriptional gene regulation. MiRNAs originate from capped and polyadenylated full-length precursors (pri-miRNA) and undergo processing into mature miRNAs, which are typically around 22 nucleotides long. MiRNAs regulate gene expression post-transcriptionally, often through partial complementarity with target mRNA. They also play a role in the induction of heterochromatin formation, contributing to pre-transcriptional gene silencing.
• Dicer is an essential enzyme in RNAi, functioning as an RNAse III-like dsRNA-specific ribonuclease. It processes dsRNA into uniformly sized small RNAs, mainly siRNAs. Dicer proteins are found in various organisms, including C. elegans, Drosophila, yeast, and humans.
• RISC (RNA Inducing Silencing Complex) is a large RNA-protein complex that mediates mRNA degradation in response to siRNA. It involves endonucleases called argonaute proteins, which cleave the target mRNA strand.
  • The RNAi pathway consists of several steps, starting with the slicing of dsRNA into siRNAs by Dicer. These siRNAs are then transferred to the RISC complex, where they guide the complex to the target mRNA, resulting in its degradation.
• Nonsense-Mediated Decay (NMD) is another cellular mechanism that detects nonsense mutations and prevents the expression of truncated or erroneous proteins. NMD is triggered by exon junction complexes (EJCs) deposited during pre-mRNA processing.
• Antisense RNA technology is used to block mRNA activity in a stoichiometric manner. Antisense RNA has a complementary sequence to mRNA and can form stable duplexes with it, interfering with gene expression at the RNA processing or translation levels. This technology is commonly used in plants for gene inhibition.

Advantages of gene silencing

• Simplified Knockout Analysis: Gene silencing downregulates gene expression, making knockout analysis simpler.
• Effectiveness Compared to Antisense Oligonucleotides: SiRNA is more effective and sensitive at lower concentrations than antisense oligonucleotides.
• Cost-Effective: Gene silencing techniques are cost-effective.
• High Specificity: SiRNA is highly specific, with the middle region (nucleotides 9-14) being the most sensitive.
• Versatility: Researchers can perform experiments with SiRNA in various cell types simultaneously.
• Labeling Possibility: SiRNA can be labeled for tracking purposes.
• Easy Transfection: SiRNA can be easily transfected into cells using vectors.
• Blocking Undesirable Gene Expression: Gene silencing can block the expression of unwanted genes and substances.
• Inducing Viral Resistance: It can be used to induce resistance against viruses.
• Analyzing Unknown Genes: Gene silencing is a powerful tool for analyzing unknown genes in sequenced genomes.
• Potential for Gene Therapy: It holds promise as an approach in future gene therapy.
• Quick Manufacturing: Oligonucleotides for gene silencing can be manufactured rapidly, often within a week, requiring only the mRNA sequence.

Disadvantages of gene silencing

• Injection Damage: Techniques like "high-pressure injection" and electroporation used for gene silencing can damage normal tissues and organs, limiting their clinical application.
• Toxicity of Delivery Methods: Liposomes and cationic encapsulated SiRNA can be toxic to the host and trigger severe immune responses.
• Emerging Strategies: Emerging strategies, such as chemical modification of SiRNA molecules and encapsulation with different molecules, are in the early stages of development and need thorough investigation before being used in therapeutic applications.

Applications of Gene Silencing

• Specific Gene Silencing in Cell Culture: Gene silencing using RNAi can be applied to silence specific genes in cell culture.
• Cancer Treatments: RNA interference has been employed in cancer treatments.
• Biotechnology in Food Engineering: RNAi has been utilized in biotechnology to engineer food plants that produce reduced levels of natural plant toxins. This technology takes advantage of the stable and heritable RNAi phenotype in plant stocks, such as cotton seeds.
• Modulation of HIV-1 Replication: RNAi has been used to modulate the replication of HIV-1, the virus responsible for AIDS.
• Small RNA Applications in Andrology and Urology: Small RNA molecules have applications in andrology and urology.
• Development of Epigenomic Analysis Technologies: Gene silencing technologies are being developed for epigenomic analysis and clinical applications in molecular diagnosis.
• Oligonucleotide Drugs: There are currently at least six oligonucleotide drugs utilizing RNAi for various illnesses, including cancer.

Conclusion

Gene silencing is an important epigenetic mechanism for regulating gene expression and finds wide-ranging applications in agriculture and biotechnology. Among various gene silencing mechanisms, RNA interference (RNAi) stands out as a crucial post-transcriptional gene silencing process.