Lectures 7-8: Post-transcriptional control of gene expression (pt1) Flashcards
What are the 4 main controls during protein synthesis?
1 - transcription control
2 - RNA processing control
3 - translational control
4 - protein activity control
What are the problems with the different protein synthesis control processes?
Genetic diseases are caused by defects in these events
Viruses alter or use these events to their advantage
Do transcription levels effect the amount of mRNA produced?
No
When are the Cap and poly A tail added?
Added post-transcriptionally (not encoded in the genome)
Why is the poly A tail added?
For stability
What are the different primary transcript processes?
Capping, Splicing, Polyadenylation and editing
Why does primary transcript processing take place and how?
They are coupled to transcription via the RNA polymerase II CTD (C-terminal domain) which acts as a landing pad
What is the structure and synthesis of the m7G cap?
Takes place on all RNA polymerase II RNAs
RNA initially contains triphosphate at 5’ end
2 step event: GpppN structure then methylation
Methylation alters chemical behaviour of the base
What are the functions of the m7G cap?
Protects mRNA from degradation by 5’-3’ nucleases
Facilitates splicing
Facilitates export from the nucleus
Critical for translation of most mRNAs
Functions mediated through protein binding (CBP80/20 nucleus and eIF4 cytoplasm)
Switches function based on location
How are exons presented in the pre-mRNA sequence?
They are not a continuous coding stream
What happens to mRNA if introns are not spliced out?
A truncated protein forms that can be degraded or have a detrimental effect
What is the gene with the largest mRNA and introns?
Dystrophin - gene linked to duchenne muscular dystrophy
What are the different conserved sequences in introns?
5’ splice site
3’ splice site
Branch site
What is the function of the conserved sequences in introns?
They define the limits of exons and introns
Recruit splicing machinery required to remove introns and join exons
What are the 2 steps of splicing introns?
Step 1: cut at 5’ splice site
Creation of bond between 5’ end of intron and branch site
Step 2: cut at 3’ slice site to release intron lariat
Ligation of 2 exons
What are the reactions that take place when splicing exons?
2 trans-esterification reactions (chemical)
What is the spliceosome?
An enzymatic complex that catalyses the removal of introns
Requiring ATP
>200 proteins that assemble onto each intron
Proteins: RNA-binding proteins, ATPases, GTPases
Contain several snRNPs (U1, U2, U4, U5 & U6)
What are snRNPs?
Small nuclear ribonucleoprotein particles
Stable RNA - protein complexes in the nucleus
Do not code for protein
Base-pair wit conserved sequences in the intron
(splicing catalysed by snRNAs NOT snRNPs)
What structural properties do snRNPs have?
Main: Sm ring
Sm binding site - conserved sequence on all molecules
Allows the binding of subset proteins
How was the function of snRNP ring determined?
Anti-Sm and Lupus
Anti-Sm antibodies react against the Sm proteins
Anti-Sm antibodies are very rare unless you have systemic lupus erythematosis (SLE)
How is splicing catalysed?
By RNA
How is the proteome expanded in alternative splicing?
Different genes can be mixed and matched to produce different isoforms meaning different proteins are produced
What are the different types of alternative splicing?
Exon skipping
Intron retention
Mutually exclusive exons
Alternative 5’ splice site
Alternative 3’ splice site
How do mutations impact alternative splicing?
It can lead to less isoforms of genes
How is alternative splicing regulated?
Using activators- intronic and exonic splicing enhancers (ISE) (ESE)
Using repressors - intronic and exonic splicing silencers (ISS) (ESS)
How do mutations effect splicing?
Loss of normal function or only one protien isoform
What are examples of diseases that contain mutations causing defects in splicing?
Spinal muscular atrophy
Retinitis pigmentosa
Myotonic dystrophy
Where does polyadenylation take place?
It takes place at a site downstream from a conserved AAUAAA 10-35 nucleotide sequence
G/U or U rich tract just downstream of poly(A) site
U-rich upstream ‘USE’
What proteins are required for polyadenylation?
Cleavage and polyadenylation specificity factor (CPSF) - binds AAUAAA
Cleavage stimulatory factor (CstF) - binds G/U
Poly(A) polymerase
How does the conserved sequence impact polyadenylation?
A signal is received to cleave endonuclease and adds the As by polyA polymerase
Why is the poly A tail significant?
All mRNAs have 3’ poly(A) tail (not histones etc)
Approx 250 nucleotides long
Bound by poly A-binding protein
Enhances export of RNA
Stabilises the 3’ end of the mRNA
Enhances translation of mRNA
What is RNA editing?
Nucleotide alterations which result in different or additional nucleotides in the mature RNA
Where does RNA editing occur?
mRNA, tRNA and rRNA
What are the 2 types of RNA editing?
Insertion/deletion
Modification (point)
What is the significance of RNA editing in medicine and development?
Disease - Atherosclerosis
Brain function - human (depression)
Development - Drosophila
Parasites - Typanosoma Leishmania and Trypanosoma mitochondria (drug target)
What is a marked nucleotide base modification?
A reversible process where a methyl group is added which binds different proteins
What is altered identity base modification?
Flipping of nucleotides, purine to different purine and pyrimidine to pyrimidine
What are the effects of mRNA editing?
Creation of start codons (insertion and flipping)
Creating of new ORFs by nucleotide insertion (insertion and flipping)
Creation of stop codons (insertion and flipping)
How does deamination take place?
Using deaminase enzyme
What is the change from adenosine to inosine the same as?
Adenosine to guanosine (equivalent of A to G)
What is cytidine deamination?
apoB mRNA editing using the APOBEC-1 enzyme
Both forms of apoB-100 & 48 circulate in the blood and have different functions
The long form of very low density lipoproteins linked to atherosclerosis
What happens when Adenosine is changed to inosine in the Q/R site of glutamate receptors?
L-glutamate excitatory neurotransmitter
Editing decrease Ca2+ permeability of channels containing the R version
Editing varies in different parts of body
Editing carried out by ADAR2
Mutations in ADAR2 gene in mice lead to seizures, post-natal death, neurodegeneration in hippocampus
What are properties of the nuclear pore?
Hydrophobic
Contain cytoplasmic filaments
Cage-like structure
How is tRNA export mediated?
Using Exp-t and ran GTP
How is miRNA export mediated?
Exp-5 and ran GTP
How is snRNA export mediated?
CRN1, ran GTP, CBC and PHAX
How is mRNA export mediated?
TAP/Mex67, p15/Mtr2, CBC and ALY/Yra1
How is rRNA export mediated?
CRM1, Arx1, Mex67, Mtr2, ran GTP and Nmd3
Why is mRNA localised?
For protein synthesis
Generates cell polarity
Prevents expression in wrong place
Promotes efficiency of subsequent protein targeting
Local control of translation
What are examples of localised protein synthesis generating cell polarity?
Drosophila embryo - bicoid, nanos
Drosophila - neuroblasts
Fibroblasts - β-actin
Yeast mRNA - Ash1
How does diffusion-based localisation occur?
mRNAs freely diffuse in the cytoplasm and are locally entrapped by anchor proteins
How does active transport based-localisation occur?
mRNA recognised by specific trans-acting factors in the nucleus
Cytoplasmic factors ensure transport along a polarised cytoskeleton
