Non-coding RNA & RNA Interference – Vocabulary Flashcards

Central Dogma & Overview of Gene Expression

• DNA → (transcription) → RNA → (translation) → Protein.

  • Visual reminder: \text{DNA} \xrightarrow{\text{RNA polymerase}} \text{RNA} \xrightarrow{\text{ribosome}} \text{Protein}

• Gene expression can be regulated at every arrow and even after the protein is made.

Layers of Gene Regulation

• Transcriptional control

  • Promoters, enhancers, transcription‐factor binding, chromatin (histone modifications), DNA methylation.

• Translational control

  • Ribosome function, initiation factors; conversion of nucleotide language to peptide language.

• Post-translational control

  • Covalent modifications (phosphorylation, ubiquitination, etc.) that alter activity or half-life of proteins.

Historical Background of RNA Silencing

• 1963: Synthetic antisense RNA blocks translation in vitro (Singer, Jones, Nirenberg).

• 1981–1990s: Natural antisense transcription (NAT) discovered in bacteria and eukaryotes (cis & trans).

• 1990: “Co-suppression” in petunias (Napoli & Jorgensen) – purple pigment gene (CHS) introduced → white flowers; 50-fold drop in endogenous + transgene mRNA.

• 1998: Fire & Mello demonstrate that double-stranded RNA (dsRNA) is a potent silencer in C. elegans.

  • Nobel Prize in Physiology/Medicine 2006.

• 2001: Bernstein et al. identify Dicer as bidentate RNase launching RNAi.

• 2024: Ambros & Ruvkun awarded Nobel Prize for discovery of microRNA (miRNA) and post-transcriptional regulation.

Fundamentals of RNA Interference (RNAi)

• Naturally occurring pathway that destroys or represses specific mRNAs.

• Requires dsRNA → processed to small RNAs (≈21\text{ nt}).

• Outcome: No translation ⇒ no protein, i.e. gene silencing at mRNA stage.

Molecular Players: Dicer and RISC

• Dicer

  • RNase III family; bidentate—measures and cleaves dsRNA into \sim21\text{ nt} duplexes.

  • Works with cofactors (e.g., TRBP).

• RISC (RNA-Induced Silencing Complex)

  • Multi-protein endonuclease complex.

  • Core = Argonaute 2 (AGO2) + guide RNA (one strand of siRNA/miRNA).

  • Function: Base-pair with target mRNA and cleave or repress translation.

Mechanistic Steps of RNAi

1. Formation or introduction of dsRNA.

2. Dicer cleavage → small interfering RNA (siRNA) duplex.

3. Strand selection: “guide” strand incorporated into RISC; “passenger” strand discarded.

4. RISC scans the cytoplasm; guide RNA base-pairs with complementary mRNA.

5. AGO2 endonuclease activity cuts mRNA at the centre of the duplex.

6. mRNA fragments degraded → \text{Protein}_{\text{target}} = 0.

Diagrammatic pipeline:
\text{dsRNA} \xrightarrow{\text{Dicer}} \text{siRNA} \xrightarrow{\text{RISC loading}} \text{mRNA cleavage}

MicroRNA (miRNA)

• Encoded in the genome, transcribed as primary miRNA (pri-miRNA) ➔ hairpin.

• Nuclear processing (Drosha) → pre-miRNA; exported to cytoplasm.

• Dicer trims hairpin → mature miRNA duplex; loaded into RISC.

• Binds mainly 3′ UTRs with partial complementarity → translational repression or destabilisation.

• Classic example: lin-4 miRNA (22 nt) down-regulates lin-14 mRNA in C. elegans development.

  • Genome size context: \approx100{\,}000{\,}000 bp.

Small Interfering RNA (siRNA)

• Perfectly complementary 21–23 nt duplex (synthetic or viral/TE origin).

• Triggers AGO2-mediated slicing (cleavage) at a single site.

• Widely exploited in research & therapy.

Argonaute Family & RISC Function

• AGO proteins = endonucleases (“slicers”).

• Guide RNA directs AGO to target via Watson–Crick base pairing.

• Catalytic triad (DDH motif) performs phosphodiester bond break.

Experimental & Therapeutic Applications

• Laboratory knock-down (“What does gene X do?”) → loss-of-function.

  • Synthetic antisense (15–20 nt) or siRNA.

• Therapeutic targeting of pathogenic transcripts (oncogenes, viral RNAs, inflammatory mediators).

• Delivery formats: lipid nanoparticles (LNP), GalNAc conjugates (hepatocyte targeting), viral vectors (shRNA).

Commercial Availability of siRNA

• Companies (e.g., Horizon Discovery) sell:

  • ON-TARGETplus (reduced off-target), Accell (self-delivering), siGENOME, Lincode (lncRNA-specific), custom designs.

• Guarantees ≥75\% knock-down; off-target reduction up to 90\%.

Clinical Translation & Approved Drugs

• 2018 FDA: Patisiran (first RNAi drug) for hereditary transthyretin amyloidosis.

• 2020 NICE (UK): Givosiran approved for acute intermittent porphyria.

  • Targets hepatic ALAS1 mRNA.

  • Phase 3 ENVISION trial demonstrated efficacy (Balwani et al., NEJM 2020).

• Ongoing trials (Nature Rev Drug Discov 2019):

  • Fitusiran (antithrombin, haemophilia) – Phase III.

  • Inclisiran (PCSK9, hypercholesterolaemia) – Phase III.

  • VIR-2218 (HBV) – Phase I/II, etc.

• Modalities: GalNAc-ESC siRNA (subcutaneous), LNP (intravenous), shRNA-modified CD34⁺ HSCs (ex vivo).

Long Non-Coding RNAs & Other ncRNA Classes

• lncRNA (>200\text{ nt})

  • Diverse nuclear & cytoplasmic functions: chromatin modification, scaffolding, sponging miRNA.

  • Less conserved; often cell-type specific.

• snoRNA (60–300 nt): guide rRNA modification.

• snRNA (≈150\text{ nt}): spliceosome components.

• piRNA (26–31 nt): associate with PIWI protein; silence transposons in germ line.

Key Terms & Definitions

• \textbf{Dicer}: RNase III enzyme that cuts dsRNA → ~21 nt fragments.

• \textbf{RISC}: RNA-Induced Silencing Complex; executes gene silencing.

• \textbf{siRNA}: Small interfering RNA, perfectly complementary duplex guiding mRNA cleavage.

• \textbf{miRNA}: MicroRNA encoded in genome, imperfect complementarity, translational repression.

• \textbf{lncRNA}: Long non-coding RNA (>200 nt) with regulatory roles.

Numerical & Statistical Highlights

• lin-4 miRNA length: 22\text{ nt}.

• Dicer products: \sim21\text{ nt}.

• CHS transcript reduction in petunia: 50\times lower than wild-type.

• siRNA guarantees: \ge75\% knock-down; off-target reduction \le10\% residual.

• Inclisiran lowers LDL-C up to 52.6\% at 180 days (ORION-1).

• piRNA size range: 26\text{–}31\text{ nt}.

Ethical, Practical & Future Considerations

• Off-target effects: partial complementarity can silence unintended genes; design algorithms + chemical modifications mitigate risk.

• Delivery challenges: tissue specificity vs systemic exposure; immune activation by dsRNA.

• Gene-specific therapy prompts questions of equity, cost (rare diseases, orphan drugs).

• Expansion beyond post-transcriptional silencing: CRISPR Cas13, RNA editing (ADAR recruitment) may complement classical RNAi.

Learning Objective Checklist (Self-Test)

• Can you list at least four different RNA classes and their sizes/functions?

• Explain how miRNA leads to reduced protein without perfect complementarity.

• Define Dicer and outline its catalytic product.

• Sketch the RNAi pathway including RISC and AGO2.

• Describe how lncRNAs differ from miRNAs with one functional example.