Regulatory RNAs – Comprehensive Study Notes

Overview & Historical Context

• Jacob & Monod (Nobel 1965) demonstrated that gene expression is controlled by dedicated regulatory elements; originally considered both proteins and RNAs.
• Early plant example: Petunia over-expression of a second chalcone-synthase gene caused complete loss of violet pigment → first hint of RNA-triggered "gene silencing".
• C. elegans RNAi discovery: injecting/feeding double-stranded RNA (dsRNA) switches specific genes OFF.
• Leukemia model: over-expression of a single microRNA in mouse immune cells transforms normal liver tissue → illustrates oncogenic potential of small RNAs.

Coding vs Non-Coding RNAs

• Coding RNAs: mRNAs.
• Non-coding RNAs (ncRNAs) fulfill other tasks:
  • rRNA & tRNA – translation machinery.
  • snRNA – splicing.
  • gRNA – mitochondrial RNA editing.
  • 7SL RNA – co-translational ER targeting.
  • Small regulatory RNAs – focus of this lecture.

Length Benchmarks

• Bacterial sRNAs: $\approx 80{-}100\,\text{nt}$.
• 6S RNA (E. coli): $184\,\text{nt}$.
• Typical mi/siRNAs: $21{-}23\,\text{nt}$ guide strand produced from $\approx 70\,\text{nt}$ hairpins or long dsRNA.
• Drosha measures $\sim 11\,\text{bp}$ basal stem; Dicer measures $\sim 22\,\text{bp}$ upper stem.

Regulatory RNAs in Bacteria

• trans-acting elements
  • 6S RNA
  – Binds $\sigma^{70}$ RNA polymerase holo-enzyme, mimics open promoter DNA.
  – Accumulates in stationary phase → shuts down $\sigma^{70}$ dependent promoters; release upon nutritional up-shift when NTPs trigger 6S-templated "pRNA" synthesis → structural change → complex dissociates.
  • sRNAs (small RNAs)
  – No processing; immediately active.
  – Need RNA-chaperone Hfq to stabilize RNA–RNA interactions.
  – Two major functions:
  ▪ Translation control at Ribosome Binding Site (RBS).
  • Activator mode: sRNA binds mRNA upstream sequestration region → exposes RBS.
  • Repressor mode: sRNA base-pairs over RBS → masks it.
  ▪ RNA stability control together with RNase E (e.g., RyhB controls iron metabolism; OxyS during oxidative stress).

• cis-acting elements
  • Riboswitches
  – Located in the 5′ UTR of the same mRNA they regulate.
  – Consist of an Aptamer (ligand-binding) + Expression Platform (structural switch).
  – Ligands: purines (guanine, adenine), SAM, FMN, vitamin B$_{12}$, thiamine-PP, etc.
  – Outcomes:
  ▪ Translation ON/OFF by exposing or hiding RBS (SAM example).
  ▪ Premature transcription termination by forming intrinsic terminator hairpin (guanine riboswitch, SAM-I/II).
  – Energetically economical – no protein synthesis required; viewed as relic of an "RNA world".
  • Attenuation
  – Leader peptides containing multiple codons of the product amino acid (e.g., Trp). Ribosome speed senses charged-tRNA availability.
  – Two mutually exclusive RNA structures: Terminator (regions 3–4) vs Anti-terminator (regions 2–3).
  – High amino acid → fast ribosome → terminator forms → transcription halts.
  – Low amino acid → stalled ribosome → anti-terminator → operon transcribed.
  – Provides $\approx 10\text{-fold}$ fine-tuning; main on/off switch still provided by repressor proteins.

Regulatory RNAs in Eukaryotes

• RNA Interference (RNAi)
  • Core concept: small $\approx 22\,\text{nt}$ RNAs guide Argonaut proteins to complementary RNA targets.
  • Consequences
  – Perfect/near-perfect match → Argonaut RNase ("Slicer") cleaves mRNA.
  – Partial match → translational repression, poly(A) shortening, localization to P-bodies.
  – Some organisms (plants, worms, fungi) use RNA-dependent RNA polymerase (RdRP) to amplify the signal, generating secondary siRNAs.

• Classes
  • siRNA – processed from long exogenous or endogenous dsRNA by Dicer.
  • miRNA – encoded in the genome, transcribed as primary miRNA (pri-miRNA); folded hairpins processed by Drosha → pre-miRNA, exported to cytoplasm → Dicer → mature duplex.

• Biogenesis details
  • Microprocessor (Drosha + DGCR8/Pasha) in nucleus performs "Cropping"; leaves $\sim2\,\text{nt 3' overhang}$.
  • Exportin-5 transports pre-miRNA.
  • Cytoplasmic Dicer "Dicing" creates $\sim22\,\text{nt}$ duplex; PAZ domain grips 3′ overhang, RNase III domains cut.
  • Duplex loading into Argonaut; passenger strand discarded, guide strand retained.

• RISC vs RITS
  • RISC (RNA-Induced Silencing Complex) – cytoplasmic, post-transcriptional gene silencing.
  • RITS (RNA-Induced Transcriptional Silencing) – nuclear, recruits histone methyltransferase Clr4, HP1 homolog Swi6 → H3K9me heterochromatin (shown in S. pombe centromeres).

Disease Connections & Examples

• Cancer
  • miR-15a / miR-16-1 normally repress BCL-2; deletion → chronic lymphocytic leukemia.
  • let-7 family targets RAS oncogene; down-regulation increases RAS.
  • miR-17–92 cluster over-expressed → oncogenic; targets tumor suppressors PTEN, RB2.
• Therapeutic/diagnostic companies: Asuragen, Crogen Pharmaceuticals, Miragen, Regulus, Rosetta Genomics explore miRNA-based products (cancer, cardiovascular, viral diseases).

Key Protein Machines

• Dicer – length-measuring RNase III; PAZ domain (binds 3′ overhang), RNase III A & B cut both strands → $\text{\sim22 nt} products.
• Drosha – nuclear RNase III; defines pre-miRNA hairpin base with DGCR8.
• Argonaut – endonuclease (PIWI domain) + PAZ; binds 5′ phosphate of guide RNA.
• Hfq – bacterial hexameric chaperone, RNA annealing facilitator.

Chromosome-Scale Regulation: X-Inactivation (Mammals)

• Females (XX) equalize gene dosage with males (XY) by inactivating one X.
• X-inactivation center (Xic) encodes:
  • Xist RNA – coats its own chromosome in cis; recruits chromatin silencing machinery → hypoacetylation, H3K27me3, DNA methylation.
  • Tsix RNA – antisense to Xist; represses Xist on the active X.
• Choice is random at $32{-}64$ cell stage → females are mosaics (e.g., tortoiseshell cats with black/orange patches).

Evolutionary Perspective

• RNAi assumed ancient immune system against mobile genetic elements & viruses.
  • Up to $45\%$ of human genome = transposon remnants; often packaged in heterochromatin via RNAi.
  • Budding yeast (S. cerevisiae) lost RNAi; fission yeast (S. pombe) retained it (centromere silencing) but lacks canonical miRNAs.
• Fungi Neurospora crassa
  • Quelling – vegetative RNAi of duplicated DNA.
  • RIP – repeat-induced point mutation during sexual cycle.
  • MSUD – meiotic silencing by unpaired DNA.
• Dosage compensation strategies: mammals inactivate one X; Drosophila up-regulates male X via different ncRNAs.

Industrial & Clinical Applications

• Diagnostic signatures: circulating miRNAs as non-invasive cancer biomarkers.
• Therapeutics: antagomiRs (miRNA inhibitors), miRNA mimics, siRNA drugs (e.g., for viral infections, hypercholesterolemia).

Conceptual Connections & Take-Home Messages

• Regulatory RNAs provide multilayered control: transcriptional, post-transcriptional, translational, chromatin.
• They act faster and more economically than protein regulators, can form complex networks (one miRNA targets many mRNAs & vice versa).
• Many mechanisms (riboswitches, attenuation) illustrate RNA’s pre-protein regulatory potential, supporting the RNA-world hypothesis.