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BIOC0020 > L10 & 11 : Local mRNA translation and degradation > Flashcards

L10 & 11 : Local mRNA translation and degradation Flashcards

1
Q

What is RNA degradation?

A

Cleavage of inter-nucleotide phosphodiester bond

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2
Q

What key roles foes RNA degradation play in metabolism and regulation?

A
  1. mRNA turnover
    - Recycling of mRNA, regulation of gene expression
  2. RNA processing
    - Trimming and general processing of mRNAs (and ncRNAs)
  3. mRNA quality control
    - Eliminate non functional and harmful mRNAs
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3
Q

How is the half life of mRNA controlled? Example in humans?

A

Number of mRNA copies and of encoded proteins depends on both rates of synthesis and decay
Eg. Average human mRNA T1/2 = ~8 hr with variation of 15 m (c-fos) and 17 h (beta-globin)

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4
Q

What is the stability of different mRNAs and in different organisms? How does it change with growth rate?

A

Different mRNAs have different stability
Average mRNA stability differs between organisms

Average mRNA T1/2 generally scales with cell growth rates

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5
Q

Describe the two pathways of de-adenylation mediated mRNA degradation?

A
  1. mRNA is first deadenylated

2a. De-adenylation can be followed by mRNA de-capping and 5’ to 3’ degradation by XNR1
2b. Alternatively, can be followed by 3’ to 5’ degradation by exosome

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6
Q

What are the steps leading to 5’ to 3’ mRNA degradation?

A
  1. mRNA polyA trimmed by Pan2/3 de-adenylation complex
  2. Short polyA then degraded by CCR4-NOT complex
  3. De-adenylation followed by de-capping by DCP1-DCP2 complex
  4. De-capping followed by 5’ to 3’ degradation by XNR1
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7
Q

Describe the structure and role of PolA nuclease (PAN) in polyA trimming?

A

Pan2/Pan3 complex
- Pan2 = catalytic
- Pan3 = tethering/ scaffolding

Pan2/3 starts deadenylation process
Recruited to polyA via interaction between Pam2 motif and PABP

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8
Q

Describe the structure and role of CCR4-NOT complex in de-adenylation?

A

General multicomponent machine for cellular mRNA de-adenylation
- NOT1 = scaffolding protein
- CCR4 + CAF1 = catalytic
- PPI modules
- NOT1 can also directly interact with other protions (eg. DDX6)

CCR4-NOT complex can take over mRNA degradation from Pan2/3 or initiate de novo

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9
Q

Describe the structure and role of DCP1/2 + XNR1 in mRNA decapping and 5’ to 3’ degradation?

A

Decapping:
- Decapping proteins 1/2 mediate process
- DCP2 binds RNA and has catalytic activity
- DCP1 functions as activator (with additional co-activators)
- Complex recognises capped RNA >20 nt
- DCP2 contains conserved Nudix motif that recognises m7Gppp cap structure
- Cleaves bond between beta and gamma phosphates of cap
- Generates RNA with 5’ monophosphate end

Degradation:
- 5’ to 3’ exonuclease XRN1 degrades mRNA

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10
Q

How are deadenylation and decapping of mRNA linked>

A
  • CCR4-NOT complex interacts directly with helicase DDX6
  • DDX6 helps recruit DCP1/2 complex
  • SM complex binds to shortened polyA tails and assists in recruiting DCP1/2
  • Deadenylation leads to translation repression and causes release of cap-binding proteins (eg. eIF4E), enables decapping
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11
Q

How is miRNA mediated mRNA degradation linked to translation repression?

A
  • miRNA mediates repression reshapes cap and polyA complexes
  • Accessible to deadenylation and decapping enzymes
  • Leads to mRNA destabilisation and 5’ to 3’ degradation
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12
Q

How is specificity achieved for mRNA degradation in different regulatory pathways?

A

Recruitment of machinery is regulated by range of adaptors
These ensure regulatory specificity

Note: Adaptors can be individual proteins or pre-assembled protein-RNA complexes

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13
Q

How does miRNA loaded RISC complex interaction with deadenylation machinery?

A

RISC-miRNA recruits deadenylation machinery via GW182
Acts as a scaffold linking RISC to downstream repressors such as CCR4-NOT (main deadenylase complex in cytoplasm)

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14
Q

What allows GW182 to interact flexibly with multiple partners?

A

Uses short tryptophan based motifs along a long and flexible protein chain
- Flexibility allows it to adjust to different positioning of miRNAs
- Multiplicity of motifs favours multiple, combinatorial interactions for efficient repression

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15
Q

What is the structure of the exosome complex?

A

Complex macromolecular machine:
- Common core of 9 subunits organised in 2 stacked rings around cavity
- Core is catalytically inactive and acts as scaffold

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16
Q

What is the function of the exosome complex?

A

Represents scaffold where different adaptors, RNA chaperones and exonucleases dock
Function to degrade RNA 3’ to 5’

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17
Q

Give an example of an exosomal nuclease for the cytoplasm and nucleus?

A

Cytoplasm: Rpr44
Nucleus: Rpr6

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18
Q

How do mechanisms of RNA degradation differ between pro and euk exosomes?

A

Eukaryotic
RNA threads into exosomal cavity to reach and be degraded by Rpr44 nuclease

Prokaryotic (bacterial)
mRNA degradation occurs inside cavity itself

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19
Q

How is the exosome structure conserved and diversified across evolution?

A

Core structure of exosome conserved between pro and euk
Eukaryotic exosomes have evolved to include additional components to perform more complex and regulated RNA processing and degradation functions

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20
Q

How do adaptors and chaperones assist in degradation of structured RNAs? Example?

A

Unfold structured RNAs and deliver to exosome for degradation

Eg. Main cytoplasmic complex is SKY

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21
Q

What are the competitive roles of AREBP in mRNA turnover?

A

Stability elements (eg. AU-rich regions) in the 3’ UTR of mRNAs
Recognised by AREBP (AU-rich element binding proteins)

Depending on the specific AREBP:
- AREBPs (eg. AUF1) recruit deadenylase to these mRNAs, leading to degradation
- AREBPS of ELAV family (eg. HuR) can bind competitively and instead protect from degradation

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22
Q

How is AREBP activity linked to cellular signalling networks?

A

Activity of AREBPs regulated by kinases through phosphorylation
Extends the signalling networks to RNA regulation, influencing stability and turnover of mRNA

23
Q

Describe the concept of pre-assembled complexes in mRNA degradation?

A

Requires sequential action of multiple degradation complexes, which are pre-assembled

24
Q

Describe the concept of selectivity and signalling in mRNA degradation?

A

Selectivity:
- Provided by protein and RNA adaptors recruiting individual mRNAs
Signalling:
- Activity of adaptors linked to signalling pathways by PTMs (eg. phosphorylation) and sometimes RNAs

25
How is mRNA transport, translation and degradation regulated?
Coordinated fashion Influences - Physical location of RNPs - Length of polyA influences dynamic equilibrium between translation and degradation - Connections between regulatory processes allowed by adaptors - Translation regulated temporally and spatially
26
List the key enzymes and functions involved?
1. PAN2/3 complex: deadenylase 2. CCR4-NOT complex: main deadenylase in cytoplasm 3. DCP1/2: mRNA decapping 4. DDX6: helicase linked deadenylation and decapping 5. XRN1: 5' to 3' exonuclease 6. Exosome complex: 3' to 5' exonuclease complex 7. SKY complex: delivers mRNA to exosome 8. AUF1/HuR: AREBP adaptors, mediating mRNA decay
27
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28
Why localise mRNA?
- Local protein synthesis is efficient (rather than post translational protein translocation) - Prevents proteins to act in wrong place during transport - Allows local and fast control of protein synthesis in response to stimuli
29
2 examples of how mRNA transport and localisation is important in metazoa?
In drosophila embryo - >70% mRNAs differentially distributed In humans: - Many mRNAs necessary for axonal development and synaptic plasticity transported within granules
30
Briefly describe the main mechanism for mRNA transport?
1. mRNA transcribed in nucleus and packaged into RNPs 2. RNP remodelling then allows connection with molecular motor 3. Transported along cytoskeleton to final destination 4. Selectivity defined by mRNA sequence/structure
31
What is another way fast mRNA transport achieved in axons?
Ribonucleoproteins (RNPs) associated with vesicles (eg. lysosomes) Allows them to move efficiently along axon
32
Describe the study of mRNA localisation in 4 different model systems? Why use them?
Facilitates global understanding Budding yeast: - Simple system - Allows genetic, biochemical and visual characterisation of functional mRNA localisation Drosophila embyro: - More complex system - Allows to connect localisation of different mRNAs with organismal development Chicken fibroblasts: - Robust system - Connect mRNA localisation with function in semi-specialised cells Mice: - Allow to study protein function in mammalian brain development - molecule function in mammalian cells
33
What is understood about the role of RNPs in mRNA transport and local translation?
- mRNAs transported in protein-RNA granules - Composition of different RNP classes only partially defined - Only some interactions have been characterised (in drosophila and humans)
34
What is the morphology of neurons?
- Cell body: contains nucleus and most organelles - Axon: transmits signals away from cell body - Axon terminals: form synapses with target cells - Dendrites: branched projections that receive signals (increase SA for input)
35
How is mRNA transport and local translation essential for neuronal development?
- Create local micro environments to maintain system - Key for axon guidance, maintenance, repair - Key to synaptic activation - Assist in guiding connection formation in specific neuronal layers during development
36
Explain the neurite transcriptome for local function?
- Transcriptome includes thousands of mRNAs - Large share are enriched (localised/ not evenly distributed) in neurites - Many codify for proteins important in signal transduction, synaptic morphology, mRNA transport and translation, intracellular signalling
37
How are mRNAs distributed along dendrites? Experiment?
Differential distribution is mRNA specific - In some cases, only a few copies are present in the distal end Experiment: single molecule FISH - can label mRNA fluorophores and monitor localisation
38
Describe regulation of localisation during development in retinal neurons?
mRNA enriched and translated in retinal cell bodies vs connecting axons change during development - An increase in total number of mRNAs correlates with changes in morphology - mRNA enrichment of axons increases during branching and decreases during pruning
39
How is selectivity provided in local mRNA translation?
Via mRNA recognition by RNA binding proteins RNA binding proteins recruit specific mRNAs to transport complexes
40
How does the domain structure of RNA BPs allow differential binding to mRNAs?
Domain structure of proteins reveal multiple RNA binding domains - Individual domains can recognise separate RNA motifs/ structures - Domains can cooperate to recognise the same RNA element - Domains can cooperate to re-arrange the RNA structure
41
What is IMP1, its roles, and importance?
Zipcode binding protein 1/ IGF2 mRNA binding protein 1 RNA binding protein important regulating transport, stability, translation - Necessary for embryonic development, particularly of nervous system - Regulates cell polarity, adhesion, migration - In tumours, correlates with early dissemination and unfavourable prognosis
42
Describe the conservation of IMP1 across metazoa?
IMP1 family is highly conserved Drosophila, xenopus, chick, mouse, human IMP are mRNA binding proteins involved in mRNA localisation
43
What is an important role of IMP1 in mammals and drosophila?
Important for local translation of mRNAs linked to axonal development, pathfinding and maintenance
44
Compare domain structure of IMP1 in mammals and drosophila?
- Different protein-interaction modules in mammal and drosophila - Both possess 4 KH domains as core for RNA recognition - Drosophila mRNA possess Q-rich region, linked to mediating granules formation - Human IMP1 has additional protein/RNA recognition domain (RRM)
45
Describe the paralogues of the IMP family and how they expand function?
3 family members - Share same domain structure and are highly conserved - Sequence specificity of paralogues subtly different - IMP1 and IMP3 expressed mainly in embryonic stages, with little or no expression in adult somatic cells - IMP1 also in some somatic cells in adult
46
How does IMP1 mediate the transport of B-actin mRNA in transitionally repressed state?
47
What is the role of the 2 RRM domains of IMP1?
2 IMP1 RRM domains mediate interaction with molecular motor Kinesin 11
48
What is the role of the 4 KH domains of IMP1?
Key to mRNA binding, but role is target dependent - While KH3 and KH4 are key domains for recognition of B-actin mRNA - All 4 domains important to recognise IGF2 mRNA
49
How does KH3 and KH4 recognise and bind the B-actin zipcode?
1. KH34 binds B-actin mRNA 3'UTR (zipcode) by 2 short RNA motifs separated by 13 nucleotides 2. KH domains bind RNA using conserved GxxG loop and hydrophobic groove 3. Reaction is driven by the binding of the second domain, which is concentration independent 4. KH3 and KH4 form intramolecular psuedo dimer as RNA molecule must undergo 180 degree change of direction 5. KH34 binds and structurally rearranges B-actin mRNA
50
Describe the binding affinity of KH34?
Much higher than KH12 and RRM12 KH34 binding affinity and pattern very similar to one of the full length IMP protein
51
What is the current hypothesis for how IMP1 represses translation? Any exceptions?
IMP1 sequesters mRNA within larger protein-RNA granules in packaged form - Prevents access to ribosome machinery Also proposed that in some cases, IMP1 mediates mRNA packaging to protect from degradation
52
Explain phosphorylation of IMP1 by Src kinase and the effect?
- Phosphorylation of IMP1 at Y396 impairs B-actin mRNA binding - Activated Src regulates actions of IMP1 to localise and repress translation during transport - Phosphorylation causes IMP1 to release mRNA, allowing local translation at destination
53
Describe phosphorylation of IMP1 in a neuron developmental context? Role of BDNF?
IMP1 function regulated by external signals in axonal development Brain derived neurotrophic factor (BDNF): - Neurogenic protein - Activates Tyrosine kinase receptor B (TrkB) - Phosphorylates kinase Src - Activated Src then phosphorylates IMP1 Other mRNA binding proteins also contribute to local translation in neurons and neuronal development
54
What is the dysfunction of mRNA local translation linked to?
Number of severe neuropathologies Examples: - Amyotrophic lateral sclerosis (ALS) - Frontotemporal dementia (FTD)

BIOC0020 (13 decks)

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