W4 L11: Non-coding RNAs

Non-coding RNA = doesn’t code for proteins

tRNA-Phe

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

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

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

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

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

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

~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

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

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

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

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

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.