RNA Silencing

In this tutorial, you will learn the fundamentals of RNA Silencing, including the roles of miRNAs and siRNAs in gene regulation. You will explore how key complexes like RISC and RITS function in silencing mechanisms and examine the therapeutic applications of antisense technology. Additionally, you will gain insights into how miRNAs and siRNAs work together to modulate gene expression and discover the various research and agricultural applications of RNA silencing.

Contents:

1. What is RNA Silencing?
2. MicroRNAs (miRNAs)
3. Small Interfering RNAs (siRNAs)
4. RNA-Induced Silencing Complex (RISC)
5. RNA-Induced Transcriptional Silencing Complex (RITS)
6. Therapeutic Applications of Antisense Technology
7. Combined Action of miRNAs and siRNAs in Gene Silencing
8. Applications of RNA Silencing

What is RNA Silencing?

RNA silencing, also known as RNA interference (RNAi), is a biological process in which small RNA molecules suppress gene expression by targeting specific messenger RNA (mRNA) molecules for degradation or translational repression. This is a key regulatory mechanism in controlling gene expression, playing a vital role in various cellular processes such as development, defense against viruses, and genome stability.

MicroRNAs (miRNAs)

miRNAs are small, single-stranded non-coding RNAs that regulate gene expression post-transcriptionally. They are transcribed from the cell’s genome and form stem-loop structures within the nucleus. These precursors are processed and exported to the cytoplasm, where they play an essential role in RNA silencing mechanisms.

Synthesis and Mechanism:

• MiRNA’s are single stranded RNAs, derived from ss RNA’s that are transcribed within nucleus from cells own genome and that contain double stranded (ds) stem loop structure.
• Nuclease enzyme within nucleus recognizes these stem loop structures and cleave them from longer single stranded RNA’s. Stem loop RNA fragments are then exported from nucleus to cytoplasm where DICER acts on them and converts them into short, linear ds miRNAs.
• These miRNAs in turn gets converted into RNA induced transcriptional silencing (RITS) complex and helps in chromatin alteration.

Small Interfering RNAs (siRNAs)

siRNAs are short, double-stranded RNA molecules that play a critical role in defending cells against viral infections and regulating gene expression. siRNAs arise from longer double-stranded RNAs, often introduced by viruses or through experimental methods in research.

Synthesis and Mechanism:

• These are derived from longer RNA molecules. siRNAs are double strander, linear molecules located in cell cytoplasm. siRNAs precursors arise as a result of viral infection i.e., virus synthesizes these as a part of their life cycle.
• RNA interference (RNAi) is a method by which cell organizes these double stranded RNAs and inactivates them and protects organism from assaults.
• ds RNAs can also be introduced in cells for research and therapeutic purpose which are recognized by DICER (Enzyme complex) in cytoplasm and are cleaved by Dicer into siRNAs, which further gets converted into RNA induced silencing complex (RISC).

RNA-Induced Silencing Complex (RISC)

RISC is a key effector in RNA interference (RNAi), mediating gene silencing through its association with miRNAs or siRNAs.

Mechanism:

• First miRNA or siRNA associates with RNA induced silencing complex (RISC complex). RNA-RISC complex becomes functional by being a specific agent of RNAi. It seeks out mRNA molecules that are complementary to antisense RNA in RISC.
• Now RNA interference (RNAi) can take one of the two pathways for gene silencing. If antisense RNA is exactly complementary to mRNA, RISC will cleave mRNA. Cleaved mRNA is then degraded by ribonucleases.
• If antisense RNA in RNA induced gene silencing complex (RISC Complex) is not exactly complementary to mRNA, then RISC will stay bound to mRNA and interfere with the ability of ribosomes to translate mRNA.

RNA-Induced Transcriptional Silencing Complex (RITS)

RITS is involved in transcriptional gene silencing and chromatin modification. It acts by recruiting chromatin-modifying enzymes to specific gene promoters or larger chromatin regions, leading to transcriptional repression.

Mechanism of action of RITS:

• RITS is transcriptional silencing mechanism different from RISC. miRNAs or siRNAs associates with RITS complex (RNA induced transcriptional silencing complex).
• Antisense RNA in RITS targets RITS complex to some specific gene promoters or the larger regions of chromatin. RITS then recruits chromatin modifying enzymes to these regions.
• These chromatin modifying enzymes in turn methylate histones and Deoxyribonuclease (DNA), resulting in heterochromatin formation and subsequent transcriptional silencing. This is called chromatin alteration.

Therapeutic Applications of Antisense Technology

Therapeutic applications of Antisense technology are the applications that are applied in treating human diseases such as Inflammatory, respiratory, renal, and cardiovascular diseases. They also play a vital role in case of viral infections, cancer chemotherapy.

Three therapeutic applications of Antisense technology:

• Renal and cardiovascular diseases: Antisense oligonucleotides (AS ODNs) are used to treat specific components in blood vessels that influence pathophysiological mechanism. Example- Systemic delivery of human tissue kallikrein gene results in decreasing blood pressure. It may also less intensify glomerular sclerosis.
• Respiratory diseases: Respirable antisense oligonucleotides (RASONS) are used for treatment of respiratory tract diseases like asthma, pneumonia, lung cancer etc. RASONS can deliver medicines even to deep lungs by interacting with unique pulmonary surfactant of alveolar epithelial cells, resulting in enhanced cellular uptake of oligonucleotides.
• Inflammatory diseases: In inflammation, antisense technology can be used to demonstrate relative importance of signaling components at molecular level in a controlled manner. This includes defining role of particular mediator in inflammation and screening of potential anti-inflammatory drugs.

Combined Action of miRNAs and siRNAs in Gene Silencing

Although miRNAs and siRNAs have distinct origins and structures, they both play vital roles in gene silencing through RNA interference mechanisms. Their combined action is often seen in research and therapeutic applications, where both molecules can be utilized to enhance gene silencing.

• Dicer Processing: Both miRNA and siRNA precursors are processed by the Dicer enzyme into their active forms.
• Involvement in RISC/RITS: These molecules are loaded into RISC for post-transcriptional gene silencing or into RITS for transcriptional gene silencing.
• Silencing Mechanisms: miRNAs and siRNAs ultimately lead to mRNA degradation, translational repression, or chromatin modification, resulting in gene silencing and altered gene expression.

Applications of RNA Silencing

• Therapeutic Potential: RNAi-based therapies are being explored for the treatment of diseases such as cancer, viral infections, and genetic disorders. By targeting specific disease-causing genes, siRNAs and miRNAs can reduce the production of harmful proteins.
• Agricultural Applications: In plants, RNA silencing can be used to create crops resistant to viruses, pests, and other stresses. Genetically modified crops can be engineered to produce specific siRNAs that silence genes involved in pathogen attacks.