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Flashcards in Life Of RNA Deck (31):

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

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

1. CPSF binds to AU rich poly A signal

2. CstF binds to downstream GU or U rich sequence stabilise complex with CF1 and CF2

3.PAP binds and stimulates 10-35 nucleotide cleavage at poly A site

4. CF released and downstream cleavage part degrades rapidly

5. Bound PAP adds 12 A residues at slow rate to 3'OH end

6. PAB2 binds to accelerate PAP adding A to poly A tail

7. After 200-250 A residues PAB2 signals to PAP to stop polymerisation


How does Transcription Terminate?

1. Poly(A) site recognises CTD change in elongation complex

2. Anti- terminators released upon passage through Poly(A) site

3. Xrn2 is recruited to the EC

4. 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




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

1. Primary microRNA is cleaved at stem of hairpin structure to give pre-miRNA

2.Cleavage performed by Drosha and DiGeorge syndrome critical region gene 8 protein. DGCR8

3.DGCR8 binds ssRNA segment of miRNA and guide DROSHA to slice RNA, due to RNAse 3 activity

4. 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