Case 3 - BBS2042
Case 3 - RNA as a Signaling Molecule
1. Learning Goals
Evolution of Knowledge of RNAi
Production, Structure and Function of RNAi
miRNA
shRNA
siRNA
Difference between Endogenous and Synthetic RNAi Production
Gene Silencing through RNAi
Involvement of components such as DICER, RISK, etc.
PKR Reaction with RNA
miRNA as an Extracellular Signaling Molecule
Local and distant communication between cells
2. Timeline
2.1 Pre-RNAi Era: Puzzling Gene Silencing (1980s–Early 1990s)
1980s - Antisense RNA concept
Researchers utilized antisense RNA to block gene expression by hybridizing to mRNA.
Results indicated unexpected effects: stronger than anticipated, inconsistent silencing, noted even with introduction of sense RNA.
Findings attributed to experimental artifacts due to a lack of understanding at that time.
Perspective at the Time
Gene regulation primarily believed to occur at transcription level; RNA was regarded as passive.
1990 - Co-suppression in Plants by Napoli, Lemieux & Jorgensen
Introduction of extra copies of a pigment gene in petunia led to unexpected silencing of both the transgene and endogenous gene, termed co-suppression.
Key insight retroactively recognized:
Silencing necessitated high sequence similarity and occurred post-transcriptionally.
Shift in perspective: Possible that RNA could actively regulate gene expression, albeit mechanics remained unclear.
2.2 Discovery of RNAi as a Distinct Mechanism (1995–1998)
1995–1997 - Quelling in Fungi by Romano & Macino
Gene silencing observed during transgene integration in Neurospora crassa.
Phenomenon termed quelling, indicating similarities observed across kingdoms (plants, fungi, animals).
1998 - Formal Discovery of RNAi by Andrew Fire & Craig Mello in C. elegans
Double-stranded RNA (dsRNA) identified as the actual silencing trigger.
dsRNA demonstrated far greater potency than sense or antisense RNA alone.
Characteristics of silencing involved:
Sequence-specificity
Systemic silencing
Heritability across cell divisions
Paper officially coined the term “RNA interference (RNAi)”.
Perspective Revolution
Recognized dsRNA as a biological signal rather than merely a lab artifact.
2.3 Mechanistic Dissection (1999–2002)
1999–2000 - Identification of Small RNAs
Silencing observed to correlate with small RNAs that are ~21–25 nucleotides long, now recognized as small interfering RNAs (siRNAs).
Key Concept
dsRNA does not act directly but gets processed.
2001 - Discovery of Dicer
RNase III enzyme Dicer identified and shown to cleave dsRNA into siRNAs, establishing RNAi as a multi-step enzymatic pathway.
2001–2002 - RISC and Argonaute Discovery
RNA-induced silencing complex (RISC) identified, where Argonaute proteins bind siRNAs and mediate target cleavage.
Notably, one strand of siRNA is retained as the guide strand.
Perspective Shift
RNAi defined as a programmable, enzyme-driven silencing system.
2.4 Expansion: Endogenous RNAi Pathways (2001–2005)
2001 - Discovery of MicroRNAs (miRNAs)
Small regulatory RNAs; previously discovered types lin-4 and let-7 recognized as part of RNAi-related pathways.
miRNAs regulate gene expression via:
Translational repression
mRNA destabilization
Major realization: RNAi extends beyond defense mechanisms; it serves a role in normal gene regulation.
Early 2000s
Recognized as conserved across eukaryotes.
RNAi machinery found in:
Plants
Fungi
Invertebrates
Vertebrates (with variations)
Perspective Change
RNA-based regulation depicted as an evolutionarily ancient and central phenomenon.
2.5 RNAi as Genome Defense (2003–2007)
Transposon Silencing
RNAi demonstrated to suppress:
Transposable elements
Repetitive sequences
Viral RNAs
piRNAs Discovered (2006)
piwi-interacting RNAs active in germline cells, protecting genome integrity during gametogenesis.
Perspective Shift
RNAi posited to have evolved as a genome's immune response.
2.6 RNAi as a Research Tool (2001–2010)
Development of Gene Knockdown Technology
Synthetic siRNAs employed for reverse genetics.
Genome-wide RNAi screens conducted in:
C. elegans
Drosophila
Mammalian cells
2006 - Fire & Mello Awarded the Nobel Prize
Recognition of RNAi as a powerful experimental tool beyond mere biological curiosity.
2.7 Therapeutic RNAi: Promise and Challenges (2005–2015)
Initial Enthusiasm
RNAi proposed as a method to silence genes responsible for diseases.
Major Hurdles Faced
Delivery challenges
Stability issues
Off-target effects
Immune activation responses
Chemical Modifications
Modified siRNAs enhances:
Stability
Specificity
Reduction in immunogenicity
Perspective Adjustment
Feasibility of RNAi therapy recognized, yet complex in biological and technical factors.
2.8 Clinical Success and Integration (2018–Present)
2018 - First FDA-Approved RNAi Drug: Patisiran
Treats hereditary transthyretin amyloidosis.
Delivered via lipid nanoparticles.
Expansion of RNA-based Therapeutics
Introduction of GalNAc-conjugated siRNAs for liver targeting.
Various RNAi drugs approved since initial successes.
Modern Perspective
RNAi viewed as a validated therapeutic modality.
3. RNAi
All RNAi-related small RNAs exhibit:
Length ranging from ~20–25 nucleotides (functional form)
Function through Argonaute proteins in RISC
Utilize base pairing to target RNAs, resulting in:
mRNA cleavage
Translational repression
mRNA destabilization
3.1 MicroRNA (miRNA) Structure
Precursor Forms of miRNA:
pri-miRNA - Several hundred to thousands of nucleotides, containing one or more imperfect hairpins.
pre-miRNA - Approximately 70 nucleotides in hairpin form, with stem-loop featuring mismatches and bulges.
Mature miRNA - ~21–25 nucleotides, typically possessing imperfect complementarity to targets.
Binding Site
miRNAs often bind the 3′ UTR of mRNAs.
3.2 miRNA Biogenesis (Endogenous, Canonical Pathway)
Transcription of pri-miRNA
miRNA genes are primarily transcribed by RNA polymerase II, resulting in primary miRNA transcript that can contain multiple stem-loop structures.
Similar to mRNAs, pri-miRNAs are usually 5′ capped with 7-methylguanosine and polyadenylated, facilitating interaction with processing machinery.
Recognition and Cropping by the Microprocessor Complex
The pri-miRNA is identified in the nucleus by Microprocessor complex, composed of:
Drosha - RNase III endonuclease.
DGCR8 - Binds dsRNA–ssRNA junction.
DGCR8 measures about 11 bp from the junction, positioning Drosha for precise cleavage to produce ~70 nt precursor miRNA (pre-miRNA) with a 2-nt 3′ overhang.
Significance: This step serves as a critical regulatory checkpoint in miRNA expression.
Nuclear Export of Pre-miRNA
Recognized by Exportin-5, which binds the 2-nt 3′ overhang in a Ran-GTP-dependent manner and transports the pre-miRNA through the nuclear pore complex into the cytoplasm.
Cytoplasmic Processing by Dicer
Pre-miRNA encounters Dicer in the cytoplasm; functions aided by accessory proteins such as:
TRBP (TAR RNA-binding protein)
PACT
Components of Dicer
ATPase/RNA-helicase, DOF (domain of unknown function), PAZ (binds the 2 bp 3′ overhang), RNase III domains (A, B, C that cut RNA).
Dicer measures distance from pre-miRNA loop, producing a ~22 nt miRNA duplex with 3′ overhangs.
RISC Loading and Strand Selection
miRNA duplex transferred to Argonaute (AGO) protein for forming RISC.
Strand selection influenced by:
Thermodynamic asymmetry of duplex
5′ nucleotide identity (often U or A preferred)
Passenger strand (miRNA*) discarded while the guide strand retains association with AGO.
Final Outcome
miRNA-AGO complex binds to target mRNAs, typically resulting in translational repression and deadenylation via recruitment of GW182/TNRC6, CCR4–NOT deadenylase complex, and DCP1/2 decapping enzymes.
3.3 Small Interfering RNA (siRNA) Structure
Characteristics:
Perfectly base-paired duplex
Length of ~21–23 nucleotides
2 nt 3′ overhangs
Derived from long dsRNA, shorter effects compared to shRNA.
3.4 siRNA Biogenesis (Endogenous Pathway)
Generation of Long dsRNA
Endogenous siRNAs stem from long double-stranded RNA, which can arise due to:
Viral replication intermediates
Bidirectional transcription
Read-through transcription of inverted repeats
Transposable element activity
dsRNA typically accumulates in the cytoplasm, with nuclear origins also present in plants and fungi.
Dicer-Mediated Processing
Dicer recognizes long dsRNA substrates and, with aid from TRBP or R2D2 (insects), cleaves them into 21–23 nt siRNA duplexes.
Contrarily to miRNAs, siRNA duplexes display perfect complementarity.
RISC Assembly and Activation
siRNA duplex loaded into AGO protein (often AGO2, the only human Argonaute with slicer activity).
The passenger strand is cleaved by AGO2 itself (slicer-dependent unwinding) to form catalytically active RISC.
Target Recognition and Cleavage
siRNA-loaded RISC associates with target mRNA showing high complementarity, initiating endonucleolytic cleavage between nucleotides 10 and 11 relative to the guide strand.
Cleaved mRNA fragments undergo degradation by:
XRN1 (5′→3′ exonuclease)
Exosome Complex (3′→5′)
Synthetic siRNA Pathway (Bypassing Early Steps)
Synthetic siRNAs get introduced into cytoplasm through lipid nanoparticles, electroporation, or transfection.
These pre-formed ~21 nt duplexes with 3′ overhangs can:
Bypass the Drosha and Dicer steps
Directly load into AGO
Chemical Modifications
2′-O-methyl ribose and phosphorothioate linkages improve stability and minimize innate immune activation by recruiting PKR, TLR3, RIG-I, and MDA5.
3.5 Short Hairpin RNA (shRNA) Structure
Composition
Artificial hairpin RNA structure, featuring a stem (~19–29 bp) topped with a loop mimicking pre-miRNA.
Generally delivered through a plasmid via bacterial or viral vectors; longer effect compared to siRNA.
3.6 shRNA Biogenesis (Synthetic but Endogenous-like)
Nuclear Transcription
Encoded on DNA vectors and transcribed in the nucleus, typically by RNA polymerase III (U6 or H1 promoters), occasionally by RNA Pol II for regulated expression.
The transcribed RNA folds into a short hairpin, mimicking pre-miRNA.
Export and Cytoplasmic Processing
Exported by Exportin-5 and processed by Dicer, generating an siRNA-like duplex entering RISC.
Note: Unlike miRNAs, Drosha is not involved in processing, and complementarity is usually perfect.
4. RNAi Loading Pathways
RNAi can silence genes at multiple levels:
Post-transcriptional Gene Silencing (PTGS)
Inducing mRNA cleavage, destabilization, or preventing translation.
Transcriptional Gene Silencing (TGS)
Primarily seen in plants, fungi, and some animals, involves chromatin modification and heterochromatin formation.
Co-transcriptional Silencing (Nascent RNA Targeting)
Dominant Mechanism in Animals
Post-transcriptional silencing is the most established pathway, elaborated below.
4.1 Post-transcriptional Loading of siRNA
Step 1: Formation of Catalytically Active RISC
After siRNA processing and strand selection, the guide strand remains bound to AGO (commonly AGO2 in mammals).
AGO2 forms the core of the RNA-induced silencing complex (RISC), positioning the guide strand such that nucleotides 2–8 (the seed region) are accessible for target recognition.
Key Components
AGO2 (PIWI domain = RNase H-like slicer), guide siRNA, and accessory proteins (HSP90, RISC-loading complex).
Step 2: Target Recognition and Binding
RISC scans cellular mRNAs for sequence complementarity with the guide strand.
Mutual recognition starts with the seed region.
Full duplex formation stabilizes RISC–mRNA interaction and depends on factors like:
mRNA secondary structure
RNA-binding proteins
Subcellular localization (e.g., P-bodies).
Step 3: Endonucleolytic Cleavage
On achieving perfect base pairing, AGO2 cleaves the target mRNA between nucleotides 10 and 11 relative to the guide strand.
Cleavage outcome produces:
A 5′ fragment lacking a poly(A) tail.
A 3′ fragment lacking a 5′ cap.
This cleavage is irreversible, committing the mRNA to degradation.
Step 4: mRNA Degradation
Cleaved mRNA fragments yield rapid degradation through general RNA decay machinery:
XRN1 degrades the 5′ fragment in a 5′→3′ direction.
Exosome complex degrades the 3′ fragment in a 3′→5′ direction.
RISC is recycled for targeting additional transcripts.
Efficiency: One siRNA-loaded RISC can silence multiple mRNAs.
4.2 Post-transcriptional Loading of miRNA
Step 1: miRNA-RISC Formation
miRNA guide strand loads into AGO1–4 (AGO2 may or may not slice).
The miRNA seed region (nucleotides 2–8) is significant.
Typically, binding occurs within the 3′ UTR of mRNAs.
Step 2: Imperfect Target Pairing
Unlike siRNA, miRNA–mRNA binding is often imperfect; mismatches and bulges are common.
Perfect pairing generally confined to the seed region, preventing AGO2 slicer activation.
Step 3: Recruitment of Silencing Cofactors
miRNA-associated RISC engages GW182/TNRC6 proteins acting as scaffolds to organize suppression machinery:
Deadenylation:
Uses CCR4–NOT complex and PAN2–PAN3 to shorten the poly(A) tail.
Decapping:
Involves DCP1/DCP2 for the removal of the 5′ cap.
Translational Repression:
Disrupts initiation by interfering with eIF4F and blocking ribosome recruitment.
Step 4: mRNA Fate
mRNAs may either:
Be temporarily stored.
Be degraded within processing bodies (P-bodies).
Repression can be reversible in specific environments: This allows fine-tuning of expression rather than binary on/off control.
4.3 shRNA Loading
shRNA-derived siRNAs follow the same route as siRNAs post-Dicer processing:
Loaded into AGO2, inducing endonucleolytic cleavage, leading to significant knockdown.
However, Caveat:
Overexpression may saturate Exportin-5 or AGO, potentially disrupting fatty endogenous miRNA pathways.
5. What is PKR?
PKR (Protein Kinase R) is:
A cytoplasmic serine/threonine kinase.
Induced by type I interferons.
Part of the innate antiviral immune response.
Structural Features
Contains two dsRNA-binding motifs (dsRBMs) at the N-terminus.
Possesses a C-terminal kinase domain.
Inactive State
PKR exists in a monomeric, inactive form until binding dsRNA triggers activation.
5.1 PKR Activation Pathway (the “PKR Reaction”)
Step 1: Recognition of dsRNA
PKR binds to long double-stranded RNA (generally >30 bp) such as:
Viral replication intermediates.
Synthetic long dsRNA (e.g., long shRNA or dsRNA transfection).
Short siRNAs (~21 nt) are usually below the activation threshold.
Step 2: PKR Dimerization and Autophosphorylation
Binding of dsRNA brings two PKR molecules into close proximity allowing trans-autophosphorylation at threonine residues (notably Thr446 and Thr451) converting PKR into an active kinase form.
Step 3: Phosphorylation of eIF2α
Activated PKR phosphorylates the α-subunit of eukaryotic initiation factor 2 (eIF2α) at Ser51, marking the central effector step.
Step 4: Global Translation Inhibition
Phosphorylated eIF2α sequesters eIF2B (a guanine nucleotide exchange factor), thereby preventing GDP → GTP exchange on eIF2, obstructing the assembly of the translation initiation complex.
Result: Global inhibition of protein synthesis.
Step 5: Downstream Stress Responses
Activation of PKR also triggers:
Formation of stress granules.
Activation of NF-κB.
Induction of apoptosis (via pathways involving FADD and caspases).
Amplification of interferon signaling.
5.2 How PKR Interferes with RNAi
Competition for dsRNA
Both PKR and Dicer bind to dsRNA.
PKR can sequester long dsRNA, preventing Dicer from accessing the substrate and subsequently reducing siRNA production.
Global Translation Shutdown
Typically, RNAi is measured as reduced protein levels.
PKR activation inhibits translation independently of RNAi, which complicates distinguishing between sequence-specific silencing and non-specific translational arrest.
Notable Issue: Identification of experimental confounders.
Inhibition of RISC Function
PKR-induced stress responses impact P-body and stress granule dynamics, modify Argonaute localization, and reduce effective RISC–mRNA interactions.
5.3 How RNAi Strategies Avoid PKR Activation
Use of Short siRNAs
Typically 21–23 nt duplexes, often below PKR activation threshold.
Chemical Modifications of siRNAs
Applications of 2′-O-methyl and phosphorothioate linkages reduce recognition by PKR, TLR3, RIG-I.
shRNA Design Optimizations
Shortened stem lengths and miRNA-based scaffolds (shRNAmir).
Lower expression levels to mitigate risk.
Delivery Strategies
Utilization of lipid nanoparticles and targeted conjugates (e.g., GalNAc).
6. Conceptual Shift: miRNA Beyond Intracellular Regulation
6.1 Why Extracellular miRNA Signaling is Possible
Free RNA generally exhibits instability, yet extracellular miRNAs are preserved through:
Extracellular Vesicles (EVs)
Exosomes (30–150 nm)
Microvesicles
RNA-Binding Proteins
Argonaute 2 (AGO2)
Nucleophosmin (NPM1)
Lipoprotein Particles
HDL
LDL (less frequent).
These carriers protect miRNAs from RNases while enabling transport.
6.2 Biogenesis and Selective Loading of miRNAs into Vesicles
Step 1: miRNA Availability in Donor Cells
miRNAs generated via canonical biogenesis, though only a selective subset is exported.
Step 2: Active Sorting into Extracellular Vesicles
miRNA loading is a non-random process incorporating:
RNA-binding proteins that specifically bind miRNAs and target them to EVs.
Post-transcriptional modifications, such as 3′ end uridylation/adenylation, influencing sorting efficiency.
ESCRT machinery (for exosomal loading), where ribonucleoproteins bind to exomotives in RNA and facilitate loading into exosomes.
Step 3: Vesicle Release
Multivesicular bodies (MVBs) merge with the plasma membrane, allowing exosomes to be discharged into the extracellular space.
6.3 Modes of Extracellular miRNA Signaling
Canonical RNAi in Recipient Cells
Mechanism of Action (Step-by-step)
Vesicle Binding
Mediated via integrins and tetraspanins (CD9, CD63, CD81).
Uptake
Accomplished through endocytosis, membrane fusion, or phagocytosis.
miRNA Release into Cytoplasm
Loading into Recipient-cell Argonaute
Post-transcriptional Gene Silencing
Noteworthy: This represents the most substantiated operative mechanism.
miRNAs Bound to Lipoproteins
HDL-associated miRNAs can be taken up via SR-BI receptors with observed functional gene regulation in recipient cells.