Lecture 17 - Cytoplasmic mechanisms of post-transcriptional control of gene expression

Question 1

To what extent can we regulate gene expression by regulating movement through NPC

Answer 1

Movement through NPC NOT a way of regulating gene expression

Question 2

3 cytoplasmic mechanisms of post-transcriptional control of gene expression

Answer 2

1) Translation regulation
2) RNA degradation
3) mRNA localization

Question 3

Translation regulation : Exemple of where it occurs

Answer 3

(Xenopus) Oocyte in prep. for embryogenesis has many transcripts but waits for fertilization to express them

Question 4

Translation regulation : What is found ON the translationally dormant transcript (4)

Answer 4

1) 5’ cap
2) Coding-region of gene
3) CPE -> U-rich signal UUUUAU
4) Poly(A) SIGNAL (AAUAAA)

Question 5

Translation regulation : How translationally dormant mRNAs are activated (in given example)

Answer 5

By cytoplasmic polyadenylation

Question 6

Translation regulation : What are CPE and Poly(A) signal

Answer 6

CPE : U-rich signal

Poly(A) signal : AAUAAA downstream of CPE

Question 7

Translation regulation : Why fully ready transcript is translationally dormant

Answer 7

eIF4E on 5’ cap is bound to maskin so can’t interact w/ other translation factors.

Question 8

Translation regulation : What is maskin also bound to

Answer 8

CPEB, which is bound to CPE signal

Question 9

Translation regulation : How is CPEB regulated and how is this linked to fertilization

Answer 9

By phosphorylation. After fertilization, activation of many phosphatases and kinases

Question 10

Translation regulation : What happens to CPEB after fertilization and what this leads to (next event)

Answer 10

Is phosphorylated so maskin leaves and PAP (and CPSF) are recruited -> Poly(A) tail extended

Question 11

Translation regulation : What elongation of Poly(A) tail allows

Answer 11

PABPC1 interacts with eIF4G which interacts with eIF4E

Question 12

Translation regulation : What length of Poly(A) influences

Answer 12

Stability of the mRNA. More Poly(A) tail = more degradation by Poly(A) exonucleases, more contact between PAP and translation factors

Question 13

Translation regulation : What mRNAs that don’t have CPE signal do

Answer 13

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)

Question 14

Translation regulation : What process allows faster translation

Answer 14

Circular structure of the mRNA (due to PABPC1/eIF4G interaction)

Question 15

Translation regulation : Why circular structure allows more translation

Answer 15

40S and 60S falling apart at 3’ end after end of 1 translation are immediately recruited at the 5’ cap -> HIGHLY EXPRESSED mRNA

Question 16

Translation regulation : Other type of mRNA translation regulation

Answer 16

Iron-dependent regulation

Question 17

First mRNA going through iron-dependent regulation and what its protein does

Answer 17

Ferritin mRNA. Ferritin binds iron ions so is required in high presence of iron

Question 18

Regions on ferritin mRNA necessary for its regulation and where they are

Answer 18

Loop structures near 5’ end (on 5’ UTR) called IREs (iron responsive elements)

Question 19

What happens to ferritin mRNA when iron level is low

Answer 19

IRE-BP (IRE binding protein) changes to an active conformation and binds to ferritin mRNA IREs -> Block ribosome scanning

Question 20

What happens to ferritin mRNA when iron level is high

Answer 20

IRE-BP is inactive so doesn’t bind IREs. Ferritin can be produced

Question 21

Second mRNA going through iron-dependent regulation and what its protein does

Answer 21

TfR mRNA. Protein is transferrin receptor, a protein required for iron intake in the cell. Required in low iron level

Question 22

Regions on TfR mRNA necessary for its regulation and where they are

Answer 22

Loops at its 3’ end with AU-rich region in their helix called IREs (again)

Question 23

What AU-rich regions in 3’ IREs helix of TfR mRNA do

Answer 23

Target the mRNA for exonucleases and endonucleases

Question 24

What happens to TfR mRNA s when iron level is high

Answer 24

IRE-BP is inactive and doesn’t bind their IREs to protect them

Question 25

What happens to TfR mRNA s when iron level is low

Answer 25

IRE-BP is active so it binds the IREs (and their AU-rich regions) on the 3’ and protects them from exo/endonucleases targeting

Question 26

3 mRNA regulated degradation pathways

Answer 26

1) Decapping pathway (Deadenylation independent) 2) Deadenylation-dependent pathway 3) Endonucleolytic pathway

Question 27

Decapping pathway (Deadenylation independent) explanation (2 steps)

Answer 27

1) mRNA is decapped before being deadenylated | 2) degraded 5’->3’ by exonucleases

Question 28

Deadenylation-dependent pathway explanation (2 steps)

Answer 28

1) Poly(A) shortened to less than 20 residues | 2) Decapped and exonucleolytic digestion 5’->3’ or 3’->5’ exonucleolytic decay by exosome

Question 29

Side effect of Poly(A) shortening in deadenylation-dependent pathway

Answer 29

Less interaction with PABPC1, loss of interaction with 5’ Cap and eIFs

Question 30

Endonucleolytic pathway explanation (2 steps)

Answer 30

1) Endonuclease cuts in middle | 2) 3’->5’ decay with exosome

Question 31

Explanation of mRNA degradation by shortening its half-life (destabilizing it)

Answer 31

Addition of AU rich sequence AUUUA in 3’ UTR of mRNAs reduce their stability

Question 32

Example of gene where addition of AU rich sequence in 3’ UTR was shown to reduce stability

Answer 32

Beta-globin gene

Question 33

Why addition of AU rich sequence in 3’ UTR destabilizes eukaryotic mRNAs

Answer 33

AU-rich element recruits deadenylating enzyme and the exosome (3’->5’ decay) to degrade the mRNA

Question 34

2 types of regulatory RNAs length + what they do

Answer 34

21 nts approx. bind 3’ UTR sequences of certain mRNAs | 1) miRNAs (micro RNAs) 2) siRNAs (silencing RNAs)

Question 35

How miRNA obtained : 2 steps

Answer 35

1) 70-nt precursor RNA forms a hairpin w/ few mismatches in stem 2) Dicer (a ribonuclease) produces mature miRNAs from precursor

Question 36

How well miRNAs base pair w/ their target mRNA+ effect of their base-pairing

Answer 36

Don’t base pair perfectly. Repress translation

Question 37

How siRNAs produced

Answer 37

From dsRNA through Dicer-mediated cleavage

Question 38

How well siRNAs base pair w/ their target mRNA + effect

Answer 38

Base pair PERFECTLY. Induce cleavage/degradation

Question 39

Which one of miRNA/siRNA has more flexibility

Answer 39

miRNA cause since it doesn’t base pair perfectly, it can base pair w/ different RNAs

Question 40

Where siRNAs come from

Answer 40

Double stranded RNA from viruses or transposable elements

Question 41

What RNAP transcribes miRNAs

Answer 41

RNAP II

Question 42

Where miRNA/siRNAs go and how many

Answer 42

In the RNA-induced silencing complex (RISC). ONE ssRNA of either miRNA or siRNA

Question 43

What RISC does to RNAs that are IMprecisely complementary to a miRNA it contains

Answer 43

DOES NOT degrade the RNA but translationally represses it. Complexes bind to 3’ to block translational machinery binding at 5’ end.

Question 44

What RISC does to RNAs that are precisely complementary to a siRNA it contains

Answer 44

Degrades the RNA : ENDOnuclease function

Question 45

REGULATION of mRNA localization principle

Answer 45

Exclude mRNA from a certain region of the cytoplasm -> Localize it at the site where proteins it encodes are required

Question 46

What often directs localization of mRNAs

Answer 46

3’ UTR elements