NEUR3001 Lecture Notes - Lecture 7: Thalamus, Critical Role, Myelin Basic Protein
RNA Trafficking in the Brain
1. Basis of RNA Trafficking
1.1. Why Traffic in CNS?
• Major cell types in CNS are highly polarised cells with long processes which require transporting molecules
• Dorsal root ganglion (DRG) axon carries sensory info from body → brain
o From toe to brainstem
o Extremely long
o RNA trafficking helps make protein and RNA transport to synapses efficient
1.2. Localising mRNA Advantages (Instead of Protein Transport)
I. ↑ energy cost effectiveness → production of multiple protein copies from single localised mRNA molecules
II. Prevents proteins from acting ectopically during translocation
III. Facilitates assembly of macromolecular protein complexes
IV. Decentralise control of gene expression → permit local control of translation in response to extrinsic cues
1.3. Major Steps of mRNA Trafficking **
1. Nuclear Export: Nucleo-cytoplasmic transport
2. Remodelling: Trafficking granule assembly
3. Suppression of Translation
4. Granule Transport: Cytoplasmic Trafficking
5. Reactivation of Translation & Attachment of Protein
2. Trafficked mRNA Granule Packaging
• Granules contain RNA & molecules for protein synthesis, regulation of translation & transport proteins
• 2-component system
o Cis-acting RNA elements (zipcodes bind to trans)
o Trans-acting protein factors
• Β-actin mRNA zipcode → zipcode-binding protein (ZBP)
• A2RE mRNA → hnRNP A/B
2.1. Identifying RNA cis-elements via EMSA
- Electrophoresis mobility shift assay with zipcode
Nuclear export
B actin mRNA as probe with
5’ proximal motif and 3’
distal motif
2.2. Identifying trans-acting Proteins: ZBP1 & β-actin mRNA
- Colocalisation of ZBP1-GFP & β-actin mRNA observed in differentiated NG cells
- ZBP1 associates with β-actin mRNA @ zipcode
o Β-actin mRNA localisation requires ZBP1 which binds to zipcode (conserved 54-nucleotide element in
3’ untranslated region of β-actin mRNA)
o ZBP1 promotes translocation of β-actin transcript to actin-rich protrusions in primary fibroblasts &
neurons
- Regulated by Src phosphorylation
o ↑ FRET intensity @ filopodia & growth gone
o FRET demonstrates proximity of ZBP and Src
location → colocalization
o Phos-ZBP1 lower affinity for zipcode cis-
element → Src phosphorylation of ZBP1
abolishes translational repression
2.3. Identifying trans-acting Proteins: Huntingtin (Htt) & β-actin mRNA
- Colocalisation of Htt & mRNA observed in rat neurons
- Htt mediates dendritic transport of β-actin mRNA
o Htt KO ↓ β-actin mRNA levels → accumulation of mRNA in cell body
o Motor proteins kinesin & dynein involved → bind to Htt interaction
Filopodia
Growth cone
Amt of β-actin
c
↓ in Htt-1 but completely
↓ transport of β-actin
mRNA in Htt-2
Document Summary
Rna trafficking in the brain: basis of rna trafficking. Localising mrna advantages (instead of protein transport: energy cost effectiveness production of multiple protein copies from single localised mrna molecules. Decentralise control of gene expression permit local control of translation in response to extrinsic cues. Major steps of mrna trafficking *: nuclear export: nucleo-cytoplasmic transport, remodelling: trafficking granule assembly, suppression of translation, granule transport: cytoplasmic trafficking, reactivation of translation & attachment of protein. Colocalisation of zbp1-gfp & -actin mrna observed in differentiated ng cells. Zbp1 associates with -actin mrna @ zipcode: -actin mrna localisation requires zbp1 which binds to zipcode (conserved 54-nucleotide element in. 3" u(cid:374)tra(cid:374)slated regio(cid:374) of -actin mrna: zbp1 pro(cid:373)otes tra(cid:374)slo(cid:272)atio(cid:374) of -actin transcript to actin-rich protrusions in primary fibroblasts & neurons. Filopodia: fret intensity @ filopodia & growth gone, fret demonstrates proximity of zbp and src location colocalization, phos-zbp1 lower affinity for zipcode cis- element src phosphorylation of zbp1 abolishes translational repression.
