W4 L11: Non-coding RNAs 

Non-coding RNA = doesn’t code for proteins

1st non-coding RNA

• Alanine tRNA in baker’s yeast
• Cloverleaf structure was solved in 1974

tRNA-Phe

• tRNA decode the mRNA sequence during protein synthesis at ribosome

  

Ribosomal RNA

• 80% of total RNA
• Transcribed by Pol I (except 5S by Pol III)
• Humans: 300-400 rDNA repeats on diff. chromosomes (13-15, 21, 22)
• Eukaryotic ribosomes (2 subunits)
  • 40S subunit > 18S rRNA
  • 60S subunit > 5S, 5.8S, 28S
• 5.8S, 18S & 28S are made from a single transcript (45S precursor) > rRNA processing in nucleolus

Total RNA isolated from human cells

• 28s is biggest, 5.8s is smallest

  

  

Small nuclear RNAs - snRNAs

• U-rich sequence involved in splicing (U1, U2, U4, U5, U6)

• Base-pairing w/ pre-mRNAs defines splice-sites

• From 107- 210 nts long, each associated w/ 6-10 proteins

  • small nuclear ribonucleoprotein particles (snRNPs)

• Highly expressed (1 mio snRNPs) & evolutionarily conserved

  

Small nucleolar RNAs - snoRNAs

• 200 diff species in mammals, 60-150 nts length
• Most snoRNAs are encoded w/in introns of Pol II transcribed genes
• Assemble w/ proteins to form small nucleolar ribonucleoproteins (snoRNPs)
• Guide specific RNA modifications in i.e. rRNA by base-pairing
  • C/D box > directs 2’-O-ribose methylation by recruiting methyl transferase enzyme
  • H/ACA box > recruits an enzyme that converts uridine to pseudouridine
• Main function takes place in nucleolus (>rRNA processing)
• In C/D box → hybridise/ anneal w/ complementary seq. in rRNA → guide methylation of bases in rRNA
• In H/ACA box → modify uridine base & form pseudouridine

miRNAs & siRNAs

• Small non-coding RNAs affect translation or decay of mRNAs in cytoplasm

• 20-22 nucleotides long RNA molecules

• Specifically bind to complementary sequences locates in 3’-UTR regions of mRNAs (in complex w/ RNA-binding proteins (i.e. Ago)

• Repress mRNA expression by promoting decay &/or inhibit translation

  • miRNAs → promoting deadenylation, translation, repression, decay

  • siRNA → cleavage of mRNA & exosome-mediated degradation

    

MicroRNAs repress translation by ‘imperfect’ hybridisation w/ target mRNAs in cytoplasm

siRNAs cleave mRNAs upon ‘perfect’ hybridisation

• Established as defence mechanism against invading dsRNA viruses & unwanted actions of transposons/repetitive elements expressed from genome

• “invading” dsRNA processed into siRNA that targets invader mRNAs for degradation

• siRNA mediated defence mechanism are crucial in plants, worms & insects - less in mammals where a protein based system to fight viruses has taken over

  

How are miRNAs/siRNAs generated in cells?

Developmental defects in inducible 𝘋𝘪𝘤𝘦𝘳 gene knock-out

• ‘Stable’ 𝘋𝘪𝘤𝘦𝘳 gene knock-out eliminates generation of miRNA in mammals & is embryonic lethal

• ‘Conditional’ 𝘋𝘪𝘤𝘦𝘳 knock-out in limb primordia leads to defects in tissue morphogenesis/ development

  

miRNA & disease

• ~5,000 diff miRNA regulate expression of almost every gene (>80%)
• miRNA combinatorial control genes w/ crucial functions in cell proliferation, development, inflammation, ageing
• Many miRNAs have been linked to disease (e.g. cancer oncogenes or tumour suppressor)
  • 1,100 miRNAa linked to more than 850 diseases
• Represents a novel target for diagnostics (biomarkers) & drug development
  • discovery of extracellular miRNAs detectable in blood

miRNAs expression signatures classify human cancers

• Profiling of miRNA expression in diff issues (tumours) enables to establish disease (tumour) specific ‘cancer markers’ for diagnostics

• Many cancer-related miRNAs specifically target mRNAs coding for proteins w/ key functions in cell proliferation, migration & immune-response

  Heat-map representing expression of miRNAs in 6 solid tumours - expression signature

  

Long non-coding RNAs (lncRNAs)

• Human: 16,000 lncRNA genes annotated (GENCODE v26) - giving raise to ~30,000 diff transcripts
• Mainly transcribed by Pol II & sharing similarities w/ mRNAs → most lncRNAs contain a 5’ cap & a poly(A) tail at 3’end
• Not translated into proteins but functional mols
• Tissue/cell type specific expression - many of them v. low abundance (1-2 copies/cell)
• Involved in many cellular processes e.g. gene imprinting, cell differentiation & development, antiviral response etc.
• Function of thousands of incRNAs is unknown

LncRNAs can interact w/ proteins, RNA & DNA to execute regulatory functions in nucleus & cytoplasm

Nuclear lncRNAs control chromatin structure/transcription in cis or trans

XIST - 1st lncRNA discovered controls mammalian dosage compensation

XIST: X inactivation specific transcript

• Xist is a large (17kb) cis-acting regulatory lncRNA

• XIST associates w/ X-chromosome that is was expressed from (cis regulation)

• Initiates histone modifications (methylation, deacetylation) →results in heterochromatin formation

• Deletion of Xist gene abolishes X inactivation

  • Xa: active X chromosome

  • Xi: inactivated X chromosome

    

Cytoplasmic lncRNA have diverse functions

LncRNAs can regulate mRNA stability & translation (examples)

mRNA stability

• lncRNA 𝘛𝘐𝘕𝘊𝘙 interacts w/ complementary sequences in a target mRNA & recruits RNA-binding protein (STAU1) → promotes stability of mRNA

  

Translational control

• Under stress conditions, lncRNA antisense to Uchl1 moves from nucleus to cytoplasm & binds the end of Uchl1 mRNA to promote cap-independant translation

  

lncRNAs (NORAD) can act as decoy for RNA-binding proteins

• NORAD is lncRNAs activated by DNA damage
• NORAD IS ~5,3 kb polyadenylated transcript predominantly localised in cytoplasm
• NORAD sequesters PUMILO RNA-binding proteins - acting as -ve regulator by limiting their availability to interact w/ mRNA targets
• Involved in control of cell mitosis

Circular RNAs (circRNAs) can act as decoys for miRNAs/RBPs

• 100s of circular RNAs (e.g. CDR1as) have recently been discovered in eukaryotes
• CDRs are generated via back-splicing mechanisms by joining 5’ & 3’ end of linear RNA molecules originating from protein coding genes
• CircRNAs lack cap/poly(A) tails - highly abundant & stable
• Originally thought to be non-coding, but recent data shows circRNAs can be translated into proteins ( not really non-coding RNA)

SUMMARY

• tRNAs and rRNAs are most abundant ncRNAs w/ fundamental functions in protein synthesis & ribosome architecture, respectively.
• Small nuclear (snRNAs) are required for splicing
• Small nucleolar RNAs (snoRNAs) guide site-specific modification of rRNA
• Regulatory small ncRNAs such as miRNAs/siRNAs control gene expression post-transcriptionally by annealing to sequences in the 3’UTRs of mRNA targets.
• Processing of miRNAs/siRNAs involves Drosha, Dicer & other proteins to finally form the RISC complex that assembles on mRNA target.
• 1000s of lncRNAs (>200 nts) exist in eukaryotes that have nuclear & cytoplasmic functions in gene expression control.
• Circular RNAs (circRNAs) are highly abundant & stable; produced by a back-splicing mechanism in eukaryotes.