Life Of RNA Flashcards
What are the Big picture steps of Eukaryotic RNA processing
5’ capping, Splicing and 3’end processing
Nuclear export
Processes occur co-transcriptionally
How is Transcription started
Phos of CTD of RNA pol 2 key in activating RNA pol 2
How does 5’capping happen and its effect on Transcription
Instantaneous after 20-25 nts transcribed.
Needs methyltransferase enzyme
And
Guanyltransferase and RNA triphosphate
This done by Bifunctional capping enzyme in eukaryotic cells that binds to Ser 5 phos of RNA pol II CTD
Accelerates Transcription by phosphorylation C-Terminal
What is the function of capping
Protect mRNA from 5’-3’ exonucleases
Interacts with small ribosome 40s via interact with eiF4F and eiF3
Splicing Process steps detailed
E Complex– U1 binds to GU sequence at 5’ splice site
A complex– U2 binds to branch site and hydrolysed ATP
B1 complex– U4/5/6 trimmer binds. U5 binds to exons at 5’site and U6 binds to U2
B2 Complex– U1 released, U5 moves from exon to intron and U6 binds to 5’splice site
C1 Complex– U4 released. U6/U2 catalysed transesterification making 5’ end of introns ligate to A site and form lariat
U5 binds exon at 3’ splice site and cleaves 5’site for lariat formation
C2 Complex– U2/5/6 remain bound to lariat and 3 site is cleaved and exon lighted using ATP hydrolysis
What is Alternatice Splicing
Splicing factors speed up RNA pol allowing alternate splicing by speeding over weak splice sites
How does elongation effect Splicing
High elongation rate allow simultaneous presentation to Splicing machinery of strong and suboptimal 3’ splice sites- so favour strong skipping weak.
SC35 interacts with RNA pol II and P-TElongation factor B via pho CTD to stimulate transcriptional elongation.
What is left post Splicing
EJC- used for proof-reading by NMD on every exon/exon junction
hTREX– Complex of 17 proteins for nuclear export
The process of 3’ end processing
- CPSF binds to AU rich poly A signal
- CstF binds to downstream GU or U rich sequence stabilise complex with CF1 and CF2
- PAP binds and stimulates 10-35 nucleotide cleavage at poly A site
- CF released and downstream cleavage part degrades rapidly
- Bound PAP adds 12 A residues at slow rate to 3’OH end
- PAB2 binds to accelerate PAP adding A to poly A tail
- After 200-250 A residues PAB2 signals to PAP to stop polymerisation
How does Transcription Terminate?
- Poly(A) site recognises CTD change in elongation complex
- Anti- terminators released upon passage through Poly(A) site
- Xrn2 is recruited to the EC
- After cleavage Xrn2 degrade downstream RNA till it reaches RNAPII and aid of SETX terminate Transcription releasing RNAPII from the template DNA
How does Xrn2 work
Chews up Junk RNA till reaches RNA pol and stops
How is 3’ end Errors controlled
Aberrant Poly(a) tails have short tail
Recognised by TRAMP and binds to short tail intimating RNA digestion by exsosome
Detail Nuclear Export
1 ALY binds 5’ cap and ATPase
2. ALY recruits TAP + p15 that interacts with NPC
3. TAP binds to NPC and pulls RNP complex through pore forming dumb bell like structure
Squeezes through then reassembles in spherical from in cytoplasm
4. Ribosome asssociates as soon as RNP enters cytoplasm
5 hTREX dissociates
How does EJC work?
For mRNA quality control in cytoplasm
Pioneer translation is when ribosome checks every exon - exon junction to identify premature stop codons and if does degrades RNA by recruiting SURF complex resulting in Nonsense Mediated Decay
If no error ribosome removes EJC
How is mRNA regulated
5’ Cap is in control of mRNA.
De-capping by Dcp1p/Dcp2p enzyme causes 5’-3’ degradation via Xrn1p
Cycled between P-bodies and Stress Granules
What are P-bodies
De-capped mRNA targeted to here
Xrn1 concentrated here and recapping enzymes
As translation repressed P-bodies increase in size and number due aggregates forming
xrn1 deletion shows this as causes P-bodies grow in size
Nutrient starvation causes P-bodies to reduce as RNA recycled
Explain Stress Granules
Second cytoplasmic foci for untranslated mRNA
mRNA recapped here
How are RNA epigenetics studied
Total RNA extraction
100nts Fragments of mRNA
Precipitate out using Ab vs specific modification looking for
Transcriptome wide profiling via NGS
Where do most RNA modifications occur?
In nucleus
Example
Modifications
methyltransferases
Acetylation
What is Retinitis Pigmentosa
Genetic mutation in PRP31
Causes disease progresses from Night blindness to Tunnel vision to complete blindness
Caused by globular formations in blood vessels supplying retinal pigment epithelium (RPE)
Causing progressive loss of rods .
What is Spinal Muscular Atrophy?
How could it be treated?
Caused by mutation in SMN1 gene.
Disrupts exon recognition so majority of SMN2 transcripts lack exon 7
Treat with with bifunctional oligont with functional ESE so promotes exon recognition and incorporation into mature transcript so normal protein translated
What is Cystic Fibrosis
Caused by mutation in huge CFTR gene
Could be fixed by SMaRT introduce correct version of C-terminal RNA to bind and be incorporated in place of deficient one
Known as Trans-Splicing
What is KSHV
Huge DNA virus that picks up host genes to enhance viral replication
Can encode ORF57 inhibitor as drug target as it gets hTREX and EJC
What are miRNAs?
Endogenously expressed non-coding RNAs
22nt in length
Modulate gene expression lost Transcription binding to complementary signals in mRNA 3’UTR
Where were miRNAs first discovered
First discovered in 1993 in C.elegans
Not appreciated till found to be conserved in eukaryotes
Overview of miRNA production
Pre-miRNA synthesised from RNA Pol 11
Nuclear processing
Nuclear export
Cytoplasm processing
Load onto argonaute proteins
Carries out repression or degradation function
How are miRNA found in genome
Intronic or intergenic
Intronic within introns they regulate sharing promoters and regulatory elements, often expressed at same time
Via pol11 some times pol111
Nuclear processing for miRNA
- Primary microRNA is cleaved at stem of hairpin structure to give pre-miRNA
- Cleavage performed by Drosha and DiGeorge syndrome critical region gene 8 protein. DGCR8
- DGCR8 binds ssRNA segment of miRNA and guide DROSHA to slice RNA, due to RNAse 3 activity
- DROSHA cleaves RNA 11bp from stem loop junction give a 5’-P group and a 3’ overhang
Argonatue loading of miRNA
miRNA-RISC complex formed
One strand of miRNA degraded by Ago proteins leaving only guide strand then guides complex to complementary mRNA and elicits translational repression or mRNA cleavage
How does miRNA carry out Translational repression
When many mismatches
Repress elongation step
Targets mRNA to P-bodies
How does mRNA carry out translational degradation
Cleavage by Ago2 proteins
Cleaves by recruits xrn1p
Ago2 recruits 3’-5’ expnucleases
Decapping enzymes
