Lecture 17 - Cytoplasmic mechanisms of post-transcriptional control of gene expression Flashcards
To what extent can we regulate gene expression by regulating movement through NPC
Movement through NPC NOT a way of regulating gene expression
3 cytoplasmic mechanisms of post-transcriptional control of gene expression
1) Translation regulation
2) RNA degradation
3) mRNA localization
Translation regulation : Exemple of where it occurs
(Xenopus) Oocyte in prep. for embryogenesis has many transcripts but waits for fertilization to express them
Translation regulation : What is found ON the translationally dormant transcript (4)
1) 5’ cap
2) Coding-region of gene
3) CPE -> U-rich signal UUUUAU
4) Poly(A) SIGNAL (AAUAAA)
Translation regulation : How translationally dormant mRNAs are activated (in given example)
By cytoplasmic polyadenylation
Translation regulation : What are CPE and Poly(A) signal
CPE : U-rich signal
Poly(A) signal : AAUAAA downstream of CPE
Translation regulation : Why fully ready transcript is translationally dormant
eIF4E on 5’ cap is bound to maskin so can’t interact w/ other translation factors.
Translation regulation : What is maskin also bound to
CPEB, which is bound to CPE signal
Translation regulation : How is CPEB regulated and how is this linked to fertilization
By phosphorylation. After fertilization, activation of many phosphatases and kinases
Translation regulation : What happens to CPEB after fertilization and what this leads to (next event)
Is phosphorylated so maskin leaves and PAP (and CPSF) are recruited -> Poly(A) tail extended
Translation regulation : What elongation of Poly(A) tail allows
PABPC1 interacts with eIF4G which interacts with eIF4E
Translation regulation : What length of Poly(A) influences
Stability of the mRNA. More Poly(A) tail = more degradation by Poly(A) exonucleases, more contact between PAP and translation factors
Translation regulation : What mRNAs that don’t have CPE signal do
Also have enough Poly(A), depending on their stretch of Poly(A) tail to maintain contact with PABPC1 (which also interacts with translation initiation factors that recruit 40 S to 5’ cap)
Translation regulation : What process allows faster translation
Circular structure of the mRNA (due to PABPC1/eIF4G interaction)
Translation regulation : Why circular structure allows more translation
40S and 60S falling apart at 3’ end after end of 1 translation are immediately recruited at the 5’ cap -> HIGHLY EXPRESSED mRNA
Translation regulation : Other type of mRNA translation regulation
Iron-dependent regulation
First mRNA going through iron-dependent regulation and what its protein does
Ferritin mRNA. Ferritin binds iron ions so is required in high presence of iron
Regions on ferritin mRNA necessary for its regulation and where they are
Loop structures near 5’ end (on 5’ UTR) called IREs (iron responsive elements)
What happens to ferritin mRNA when iron level is low
IRE-BP (IRE binding protein) changes to an active conformation and binds to ferritin mRNA IREs -> Block ribosome scanning
What happens to ferritin mRNA when iron level is high
IRE-BP is inactive so doesn’t bind IREs. Ferritin can be produced
Second mRNA going through iron-dependent regulation and what its protein does
TfR mRNA. Protein is transferrin receptor, a protein required for iron intake in the cell. Required in low iron level
Regions on TfR mRNA necessary for its regulation and where they are
Loops at its 3’ end with AU-rich region in their helix called IREs (again)
What AU-rich regions in 3’ IREs helix of TfR mRNA do
Target the mRNA for exonucleases and endonucleases
What happens to TfR mRNA s when iron level is high
IRE-BP is inactive and doesn’t bind their IREs to protect them
What happens to TfR mRNA s when iron level is low
IRE-BP is active so it binds the IREs (and their AU-rich regions) on the 3’ and protects them from exo/endonucleases targeting
3 mRNA regulated degradation pathways
1) Decapping pathway (Deadenylation independent)
2) Deadenylation-dependent pathway
3) Endonucleolytic pathway
Decapping pathway (Deadenylation independent) explanation (2 steps)
1) mRNA is decapped before being deadenylated
2) degraded 5’->3’ by exonucleases
Deadenylation-dependent pathway explanation (2 steps)
1) Poly(A) shortened to less than 20 residues
2) Decapped and exonucleolytic digestion 5’->3’ or 3’->5’ exonucleolytic decay by exosome
Side effect of Poly(A) shortening in deadenylation-dependent pathway
Less interaction with PABPC1, loss of interaction with 5’ Cap and eIFs
Endonucleolytic pathway explanation (2 steps)
1) Endonuclease cuts in middle
2) 3’->5’ decay with exosome
Explanation of mRNA degradation by shortening its half-life (destabilizing it)
Addition of AU rich sequence AUUUA in 3’ UTR of mRNAs reduce their stability
Example of gene where addition of AU rich sequence in 3’ UTR was shown to reduce stability
Beta-globin gene
Why addition of AU rich sequence in 3’ UTR destabilizes eukaryotic mRNAs
AU-rich element recruits deadenylating enzyme and the exosome (3’->5’ decay) to degrade the mRNA
2 types of regulatory RNAs length + what they do
21 nts approx. bind 3’ UTR sequences of certain mRNAs
1) miRNAs (micro RNAs) 2) siRNAs (silencing RNAs)
How miRNA obtained : 2 steps
1) 70-nt precursor RNA forms a hairpin w/ few mismatches in stem
2) Dicer (a ribonuclease) produces mature miRNAs from precursor
How well miRNAs base pair w/ their target mRNA+ effect of their base-pairing
Don’t base pair perfectly. Repress translation
How siRNAs produced
From dsRNA through Dicer-mediated cleavage
How well siRNAs base pair w/ their target mRNA + effect
Base pair PERFECTLY. Induce cleavage/degradation
Which one of miRNA/siRNA has more flexibility
miRNA cause since it doesn’t base pair perfectly, it can base pair w/ different RNAs
Where siRNAs come from
Double stranded RNA from viruses or transposable elements
What RNAP transcribes miRNAs
RNAP II
Where miRNA/siRNAs go and how many
In the RNA-induced silencing complex (RISC). ONE ssRNA of either miRNA or siRNA
What RISC does to RNAs that are IMprecisely complementary to a miRNA it contains
DOES NOT degrade the RNA but translationally represses it. Complexes bind to 3’ to block translational machinery binding at 5’ end.
What RISC does to RNAs that are precisely complementary to a siRNA it contains
Degrades the RNA : ENDOnuclease function
REGULATION of mRNA localization principle
Exclude mRNA from a certain region of the cytoplasm -> Localize it at the site where proteins it encodes are required
What often directs localization of mRNAs
3’ UTR elements
