RNA Interference Overview

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Questions and Answers

What is a primary role of miRNA in biological systems?

Stimulating protein synthesis

Regulating RNA interference

Facilitating brain and heart development (correct)

Promoting cell division

Which method is NOT used to analyze the phenotypic effects of RNA interference?

Morphological analysis

Centrifuge-based purification (correct)

Northern blotting

Growth curve analysis

What is a significant limitation of using dsRNA for gene knockdown?

It results in complete gene deletion

Not all cells effectively uptake the dsRNA (correct)

It can induce mutations in other genes

It has a slow degradation rate

What does the tetracycline operon achieve in the context of dsRNA production?

Generates RNA polymerase for transcription control Signup and view all the answers

Which of the following methods can be used to confirm the loss of target mRNA?

QRT-PCR Signup and view all the answers

Which phenomenon may occur if there is a lack of control in dsRNA production?

Unintentional knockdown of off-target genes Signup and view all the answers

What is the purpose of using stem-loop structures in RNA interference?

To enhance transcription efficiency from a single promoter Signup and view all the answers

What does vector-based RNAi utilize for generating siRNAs?

Plasmids or viral vectors for shRNA expression Signup and view all the answers

What is the initial indication of RNA interference (RNAi) significance derived from?

The overexpression of the chalcone synthase gene in plants Signup and view all the answers

Who are credited with the discovery of RNA interference?

Andrew Fire and Craig Mello Signup and view all the answers

What is the primary purpose of RNA interference in gene silencing?

To degrade specific mRNA sequences Signup and view all the answers

What role does Dicer play in the RNAi pathway?

It cleaves long dsRNA into smaller siRNA fragments Signup and view all the answers

What is the primary function of the RNA-Induced Silencing Complex (RISC)?

To degrade target mRNA using siRNA Signup and view all the answers

Which method is typically used to assess the knockdown efficiency in an RNAi experiment?

qRT-PCR Signup and view all the answers

Which characteristic is true about small interfering RNAs (siRNAs)?

They typically have 2-nucleotide 3' overhangs and are 21-23 nucleotides long Signup and view all the answers

What distinguishes vector-based RNAi from siRNA-based approaches?

Vector-based RNAi involves the expression of shRNAs Signup and view all the answers

What distinguishes miRNA from siRNA?

miRNA is typically single-stranded and can form hairpin structures Signup and view all the answers

What is the role of the T7 polymerase in opposing T7 RNAi?

It generates dsRNA from sense and antisense strands Signup and view all the answers

Why is RNA interference (RNAi) considered useful in research?

It enables specific gene knockdown for functional studies Signup and view all the answers

How does conditional RNAi, particularly the Tet on/off system, function?

It allows for regulated transcription of RNAi constructs in response to tetracycline Signup and view all the answers

Why are lentiviral vectors used for delivering RNAi constructs in mammalian cells?

They facilitate stable integration and long-term gene silencing Signup and view all the answers

What are the typical features of small interfering RNAs (siRNAs)?

Short fragments of approximately 21-23 nucleotides with 2-nucleotide 3' overhangs Signup and view all the answers

Which of the following describes hairpin-based RNAi?

It involves a single promoter driving hairpin RNA synthesis Signup and view all the answers

What is a common application of RNA interference technology?

Functional genomics and gene knockdown studies Signup and view all the answers

What makes ZFN technology advantageous despite its longer production time?

It allows for gene editing with high efficiency. Signup and view all the answers

What is a characteristic of Non-Homologous End Joining (NHEJ)?

It directly joins the broken ends, often resulting in errors. Signup and view all the answers

How do TALENs compare to ZFNs in terms of production time?

TALENs can be produced more quickly than ZFNs. Signup and view all the answers

What is the purpose of using a repair template in Homology-Directed Repair (HDR)?

To enable precise insertion, deletion, or modification of DNA. Signup and view all the answers

What is a significant challenge in genome editing related to delivery methods?

Nanoparticles can induce an immune response. Signup and view all the answers

What is one of the primary treatments for sickle cell disease and β-thalassemia?

Bone marrow transplant Signup and view all the answers

What role do helper plasmids play in AAV production?

They provide genes from adenovirus necessary for viral assembly. Signup and view all the answers

Which of the following describes the functionality of CRISPR-Cas9 in DNA insertion?

It uses guide DNA to bind to specific sites for cleavage. Signup and view all the answers

What is a consequence of iron overload in sickle cell disease?

Formation of long chains in red blood cells Signup and view all the answers

What is the primary function of antisense DNA in treating Duchenne Muscular Dystrophy (DMD)?

To skip mutated exons and enable functional protein production Signup and view all the answers

How are AAV vectors classified once produced?

As delivery systems for gene therapy Signup and view all the answers

Which mechanism is involved in immune recognition of AAV?

PRR detecting virus and RNA Signup and view all the answers

What is a significant mutation characteristic of Becker Muscular Dystrophy?

A truncated protein due to an exon deletion Signup and view all the answers

Study Notes

RNA Interference (RNAi)

Classical RNAi discovery: Plant studies showed that excessive RNA expression of the chalcone synthase gene unexpectedly reduced color pigment. Leading researchers to understand that overlapping RNA sequences formed dsRNA. Cleavage of dsRNA into siRNAs led to mRNA degradation and gene knockdown.
Key Players in RNAi: Andrew Fire and Craig Mello, who contributed significantly to understanding RNAi, demonstrating its ability to silence genes using dsRNA in nematodes.
Dicer: An RNase III endonuclease that cleaves long dsRNA into siRNA fragments (21-23 nucleotides) with 2-nucleotide 3' overhangs.
RISC (RNA-Induced Silencing Complex): A multiprotein complex that incorporates one strand of the siRNA (guide strand) and facilitates base pairing with complementary target mRNA. Argonaute within RISC mediates the degradation of the target mRNA.
Small Interfering RNA (siRNA): 21-23 nucleotides long with 2-nucleotide 3' overhangs, derived from dsRNA cleavage. Essential for sequence-specific degradation of target mRNA.
miRNA vs. siRNA:
- miRNA (microRNA): Noncoding regulatory RNAs, usually single-stranded with hairpin structures. Processed by Dicer and play roles in regulating gene expression.
- siRNA (small interfering RNA): Derived from long dsRNA, primarily involved in the degradation of complementary mRNA.
Key Features and Applications of RNAi:
- Sequencespecific gene silencing
- Target and degrade specific mRNA
- Cross cell barrier effects
- Potential therapeutic applications
- Functional genomics
- Gene knockdown studies
- Development of RNAi-based therapies
Performing an RNAi Experiment:
- Design and synthesize specific siRNAs targeting the gene of interest.
- Introduce siRNAs into cells using transfection methods.
- Assess:
  - mRNA levels (qRT-PCR)
  - Protein levels (Western blotting or ELISA)
Vector-Based vs. siRNA-Based RNAi:
- Vector-based: Uses plasmids or viral vectors to express shRNAs, which are processed into siRNAs within the cell.
- siRNA-based: Direct introduction of chemically synthesized double-stranded RNA molecules that can immediately integrate into RISC.
Opposing T7 vs. Hairpin-Based RNAi:
- Opposing T7: Two promoter regions (T7) in opposite directions generate dsRNA from sense and antisense strands.
- Hairpin-based: Single promoter drives transcription of an RNA construct that forms a hairpin structure, which is processed by Dicer.
Conditional RNAi, Tet on/off System:
- Regulates the expression of RNAi constructs.
- Tetracycline operator regulates T7 polymerase activity, controlling transcription of the target RNA in response to tetracycline presence.
Lentivirus and RNAi in Mammals:
- Lentiviral vectors deliver RNAi constructs into mammalian cells for stable, long-term gene silencing.
- They infect both dividing and non-dividing cells, making them promising tools for RNAi-based therapies.

Genome Editing

AAV (Adeno-Associated Virus) Transduction: Enters cells in the same way as B cells encounter an antigen. It evades lysosomes and enters the nucleus for self-replication and integrates its DNA into the host cell.
AAV Production:
- Plasmid Transfection: Three plasmids are introduced into HEK293 cells:
  - Helper Plasmid: Contains adenovirus genes for viral functioning.
  - Rep/Cap Plasmid: Encodes AAV replication (Rep) and capsid (Cap) proteins for viral assembly.
  - Transgene Plasmid: Contains the gene of interest flanked by AAV sequences.
- HEK293 cells are co-transfected with the plasmids.
- Harvesting AAV: AAV is separated by centrifugation in a 60% Iodixanol solution with varying densities. AAV supernatant will divide based on size and weight, allowing for collection of the virus.
AAV Receptor Binding: Naturally occurring or designed receptors can enhance viral engulfment.
AAV Immune Response: PRRs (Pattern Recognition Receptors) can recognize the virus, initiating an immune response to kill the virus or infected cells. (e.g., TLR, RLR)
AAV Applications:
- Chimpanzee AAVs used in COVID-19 vaccines to carry the spike protein.
- Gene therapy for muscular dystrophy, like using antisense DNA to skip mutated exons in DMD.
Genome Editing Tools:
- CRIPR-Cas9: Guide DNA binds to a specific site, and Cas9 cleaves the DNA. Quick but prone to off-target binding.
- ZFN (Zinc Finger Nucleases): Engineered proteins with zinc finger domains for specific DNA sequence binding and a FokI nuclease domain to cut DNA. Accurate but slow to produce.
- TALEN (Transcription Activator-Like Effector Nucleases): Use TALE proteins for DNA sequence recognition and a FokI nuclease for cutting. More precise and quicker to develop than ZFNs.
DNA Repair Mechanisms in Genome Editing:
- NHEJ (Non-Homologous End Joining): Directly joins broken DNA ends, often resulting in small insertions or deletions.
- HDR (Homology-Directed Repair): Uses a repair template with similar sequences to repair the break precisely.
First CRISPR-Cas9 Gene Editing Therapy: Leber congenital amaurosis type 10, a rare blindness caused by a CEP290 gene mutation.
- Editas and Allergen developed EDIT-101 (AGN-151187) gene therapy using an AAV5 vector to deliver Cas9 and guide RNAs, aiming to restore CEP290 protein production in photoreceptors.
Barriers to Genome Editing:
- Delivery Challenges: NPs (nanoparticles) containing proteins, RNA, and DNA for delivery can be susceptible to phagocytosis, degradation, and extraction. They can also trigger immune responses.
- Delivery Methods:
  - In vitro nucleus cell injection (cells in culture)
  - Injection into embryos followed by electroporation.

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Description

This quiz explores the mechanisms and key players involved in RNA interference (RNAi), including the discovery of RNAi, the function of Dicer, and the role of RISC in gene silencing. Delve into the molecular biology of small interfering RNAs (siRNAs) and their critical impact on gene regulation.