L10 - Post-transcriptional control of gene expression IIII

Question 1

When do we need RNA degradation?

Answer 1

Damaged mRNA

Incorrectly transcribed/processed mRNA

To control gene expression – can control abundance

Like proteins, RNAs have a half-life which can range from minutes to days

Question 2

Why is turnover important in controlling mRNA levels?

Answer 2

Turnover is important, as if we are primarily controlling a gene through transcription, if the RNA has a long half-life, then it’s not going to stop protein expression because the mRNA is still there

Need to be able to regulate how the RNA is produced and degraded, to control what RNA you have in the cell

Question 3

Casein mRNA

Answer 3

Expressed in mammary gland mRNA increases ~70-fold on stimulation by prolactin (hormone promoting milk production)
BUT transcription increases only ~2-fold

How?
– Half-life increases dramatically (~40-fold) in response to prolactin
– Poly(A) tail length INCREASED – indication of mRNA being stabilised
– 3’ UTR of RNA binds proteins which aid in this stabilisation

Sounds wasteful, but allows for a very rapid change in mRNA levels, so, being able to degrade allows for a very rapid change in protein production

Question 4

Why are eukaryotic mRNAs circular during translation?

Answer 4

Monitors mRNA integrity – it will not be circular if it has lost cap or poly (A)

Brings ribosomes ending translation close to the AUG (it is suggested that ribosomes “recycle” onto the 5’ end)

Question 5

Circular mRNAs & degradation

Answer 5

Most degradation starts at the cap or the poly A tail, so the closed loop needs to be broken first before exonucleases can gain access

Most of the RNA sequence is degraded by exonucleases, so if you don’t have a free end that they can access then you can’t degrade the RNA

To degrade an RNA, you start by either removing the polyA tail and/or the cap to make it accessible for exonucleases

Question 6

mRNA degradation - phase I

What happens?

Answer 6

The first phases is removing either cap or polyA tail

Endonucleases cut the middle of the RNA but they’re not as common, the most common way is by removing the polyA tail and cap

But some cases you can split the RNA in the middle, they still have the polyA and cap but have a free end

These processes start the degradation process

Question 7

mRNA degradation - phase I

How is the cap removed?

Answer 7

Cap removed by decapping enzymes either DCP1 or 2

Question 8

mRNA degradation - phase I

How is the polyA tail removed?

Answer 8

Deadenylases remove the polyA tail by the Ccr/Not complex

Question 9

mRNA degradation - phase 2

Answer 9

• Now we degrade with exonucleases
These are progressive enzymes that will degrade through the RNAs

Exonucleases are digressive enzymes

Enzymes can only work one way either 5’ to 3’ or 3’ to 5’

There’s a directionality, they either work from one end or the other, can’t do both

Question 10

DEGRADATION

What is the exosome?

Answer 10

The exosome is the main 3’ to 5’ exonuclease in the cell

Involved in RNA turnover and processing

Multi-subunit complex

Question 11

DEGRADATION

What is XRN1?

Answer 11

5’ to 3’ exonuclease

Involved in RNA turnover and processing

Also involved in transcription termination

Functions after decapping of the mRNA

Question 12

DEGRADATION

What is deadenylation-dependent decay?

Answer 12

Most degradation of an RNA is deadenylation-dependent decay, so we have to take of the polyA tail first or shorten it

The first thing we do is deadenylation by the CCR4-NOT complex, which either removes or shortens the tail

Then we can have one of two things:
– Further degradation by the exosome
– Or we can start degradation from the other, so shorten polyA tail, then we get decapping, we then degrade the RNA with XRN1

Question 13

DEGRADATION

What does the length of the polyA tail tell us?

Answer 13

Generally with an RNA the length of its polyA tail, is indicating the length of time it’s going to survive

The shorter the more likely its going to be degraded

Question 14

DEGRADATION

Why is decapping the mRNA important?

Answer 14

Efficiency of translation is reduced as soon as cap is removed & translation initiation is stopped – this is therefore an important step

Decapping the RNA is a good way of removing the RNA from the pool of RNAs

Question 15

DEGRADATION

What causes deadenylation-dependent decay?

Answer 15

Varies from mRNA to mRNA

There’s a number of elements within the RNA that target and promote deadenylation:
• AU-rich elements (ARE)
• Nonsense codons 
• C-fos 
• miRNA 

All mRNAs gradually remove their polyA tails, these elements speed up the process

Question 16

DEGRADATION

How do AU-rich elements (ARE) promote deadenylation?

Answer 16

Sequences present in the 3’ UTR that target RNA for degradation

So RNA can have own sequences in them that make them a target for degradation

Question 17

DEGRADATION

How do nonsense codons promote deadenylation?

Answer 17

Mutation that introduces a stop codon in the sequence, this cause deadenylation and degradation of the mRNA

Question 18

DEGRADATION

How does C-fos promote deadenylation?

Answer 18

A sequence in the coding sequence that promotes deadenylation and degradation

Question 19

DEGRADATION

How does miRNApromote deadenylation?

Answer 19

Short RNAs that bind 3’ UTR of mRNAs and their binding promotes deadenylation and degradation

Question 20

DEGRADATION

Autoregulation

Answer 20

In some cases, we get proteins that bind their own message - target themselves for degradation

Negative feedback

For example:
• Rps28B binds its own message
• The binding recruits Edc3, which recruits the decapping complex, and starts degradation, this happens independent of shortening of polyA tail
• Edc3 is one of several activators of decapping enzymes in the cell

Question 21

DEGRADATION

What is nonsense-mediated decay (NMD)?

Answer 21

When we get an early stop codon, either by a mutation or by errors

We don’t want mRNAs that code for truncated proteins as these can be toxic for the cell, so they need to be degraded

Question 22

DEGRADATION

What are premature termination/stop codons (PTCs) a result of errors in?

Answer 22

Transcription 
Splicing 
Editing 
Polyadenylation 
Mutations 

One third of all inherited disorders are caused by nonsense or frameshift mutations that introduce PTCs

Question 23

DEGRADATION

When is an mRNA degraded by NMD?

Answer 23

Nearly all properly spliced mRNAs have the STOP codon in the final exon

If the stop codon is too far away from the exon junction (>55) then the mRNA is targeted for NMD

It its <55 then its not targeted for degradation

Question 24

DEGRADATION

How does the cell know where the stop codon is?

Answer 24

In each round on splicing the EJC is placed just upstream of each splice site

EJC complex is just in front of each splice junction

In the first round of translation, EJCs are removed from the mRNA by the ribosome

When ribosomes reach PTC, EJC remains downstream, so the mRNA is targeted for degradation

Question 25

DEGRADATION

What is the EJC?

Answer 25

Exon junction complex

They’re linked to splicing, mRNA export, localisation & translation

They’re also key to NMD

Question 26

DEGRADATION

How does NMD act as a mechanism to monitor mRNA?

Answer 26

Does it have the correct structure?
Does it have the right organisation of stop & start codons?

The ones that don’t fit the rules are targeted for degradation

Is a process known as surveillance – also happens with proteins & other types of RNA

Question 27

What is RNAi?

Answer 27

RNA interference

Question 28

What is siRNA?

Answer 28

Small inhibitory RNA

Question 29

What is miRNA?

Answer 29

Micro RNA

Question 30

What is RISC?

Answer 30

RNA induced silencing complex

Question 31

How are siRNA & miRNA similar?

Answer 31

They both base pair to mRNA

If miRNA can bind perfect to RNA then it will act as siRNA and vice versa

They are both produced in the cell

Question 32

siRNA structure & function

Answer 32

21-23 nucleotide RNAs

Perfect complimentary to target RNA

Thought to be mainly a viral defence mechanism

Leads to the degradation of target RNA, leads to the cleavage of RNA

Question 33

miRNA structure & function

Answer 33

21-23 nucleotide RNAs

Imperfect complimentary to target RNA

Key gene regulatory mechanism in the cell

Leads to block in translation, might lead to degradation, but primary function is to block translation

Question 34

Mechanism of siRNA/miRNA production

Answer 34

1. Mainly miRNA in human cells – we have gene for this in our genome
2. Often genes that produce multiple RNAs join together in one precursor RNA
3. This is then processed by an enzyme called Drosha into individual pre-miRNA
4. They’re then exported to the cytoplasm by exportin 5
5. Then cut by an enzyme called Dicer to produce miRNA
6. siRNA produce from dsRNA which are also cut by Dicer

Question 35

What does dicer do when producing siRNA/miRNA?

Answer 35

Dicer will take an RNA and cut it into chunks of 21-23 nucleotides, acts as a molecular ruler

Question 36

Mechanism of siRNA/miRNA function

Answer 36

1. RNAs get unwound and get introduced into the RISC complex
2. The RISC complex is the complex that mediates the function of the miRNA
3. The miRNA doesn’t cut the RNA or stall the translation themselves; they function as guides to bring in factors that do these jobs
4. We’ve got RISC which includes a protein called argonuate
5. miRNA bind the 3’ UTR of miRNA and block translation, stop translation initiation and stop translation of the RNA, and indirectly leads to degradation of RNA
6. If we have siRNA with perfect binding, then we have cleaving and the argonuate cleaves the protein

Question 37

Can the length of 3’ UTRs change?

Answer 37

YES

During embryonic development 3’ UTRs frequently get LONGER

mRNAs in proliferating cells tend to have SHORTER 3’ UTRs

This process can be regulated

Question 38

Why is it important for 3’ UTRs to be different lengths?

Answer 38

Lots of miRNA are tissue specific or they’re developmental specific – expressed during certain phases in development

There’s a simple way of regulating miRNA function - by changing the 3’ UTR length

All genes have multiple polyA signals, so can have different length 3’ UTR

They can use different PolyA signals under different conditions

Question 39

How does changing the length of 3’ UTRs help regulate processes?

Answer 39

If we have a long 3’ UTR we can have multiple miRNA binding sites

So by changing the length of the sequence, we can change how many miRNA can bind, so can change how the miRNA can affect the mRNA by changing with polyA signal is used

So, cell can switch polyA site use depending on how well cells are growing

Question 40

Why do we want long 3’ UTRs in embryonic development?

Answer 40

During embryonic development we need very tight regulation on gene expression, so we would go for longer 3’ UTR and more miRNA regulation

Question 41

Why do we want short 3’ UTRs in fast proliferating cells?

Answer 41

don’t need that much regulation, just want things produced faster