RNA localization
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RNA localization. mRNA can be localized to subcellular compartments by actin or tubulin -dependent processes. Examples :. Xenopus : Vg1 mRNA (TGF b ) to vegetal pole. Drosophila : nanos, oskar mRNA (posterior) and bicoid (anterior). (requires mRNA binding protein staufen).

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RNA localization

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RNA localization

mRNA can be localized to subcellular compartments

by actin or tubulin-dependent processes

Examples:

Xenopus: Vg1 mRNA (TGFb) to vegetal pole

Drosophila: nanos, oskar mRNA (posterior) and bicoid (anterior)

(requires mRNA binding protein staufen)

prospero (into ganglion of mother cells; neuroblast TF)

(requires staufen and miranda)

Yeast: Ash1 mRNA to daughter cell


lamellipodia staining

perinuclear staining in myotubes


Bertrand et al., Mol Cell (98) 2:437-445


SUMMARY 2

I.mRNA decay

- regulated and non-regulated turn-over

- ordered pathway (deadenylation, decapping, exonucleolytic

degradation)

- NMD: recognition of premature stop codons

II.Cytoplasmic mRNA localization

- ZIP code in 3’ UTR

- both actin and tubulin-mediated

- yeast mating type switch as a model: Ash1 mRNA localization

(via 3’ UTR, She2/3, Myo4 and actin cables)


ER translocation & vesicular transport


3min

7min

37min

117min

from:

Jamieson and Palade


In vitro reconstitution of ER translocation:

- Sec61 complex: conserved translocation channel

Sec61 subunits (a, b, g)

Sec62/63

TRAM (translocating chain-assoc. membrane protein)

- phospholipids (proteoliposomes) and luminal chaperones (BIP)

- SRP/SRP receptor only required for co-translational translocation

not for post-translational translocation (e.g pre-pro-alpha factor).

- energetics of translocation: protein conducting channel (cotranslational)

molecular ratcheting (posttranslational)


Probing of translocation intermediates with fluorescent peptides

From: Liao and Johnson Cell (97)


The Sec61 complex forms a channel

Menetret et al. Mol Cell (2000)6:1219


From: Beckmann et al. Cell (2001)Vol 107, 361-372


From: Beckmann et al. Cell (2001)Vol 107, 361-372


From: Van den Berg et al. Nature (2004)427, 36-44


From: Van den Berg et al. Nature (2004)427, 36-44


Topology of membrane-spanning proteins


Type I membrane proteins have a cleavable signal sequence


Type II membrane proteins have internal signal sequence


Type III membrane proteins have internal signal sequence


Type II+III membrane proteins have internal signal sequences


From: Beckmann et al. Cell (2001)Vol 107, 361-372


Translocation of proteins with multiple membrane

spanning domains


From: Van den Berg et al. Nature (2004)427, 36-44


Formation of a glycosylphosphatidylinositol (GPI)-anchor


ER function

- Proper folding of proteins (chaperones, lectins,

petidyl-prolyl-isomerases)

- Formation of disulfide bonds (PDI)

GSH prevents oxidation in cytosol

GS-SG + NADPH + H+<=> 2 GSH + NADP+

- Proteolytic cleavages

- Addition & processing of carbohydrates

- Assembly into multimeric proteins

- Ca2+ storage

- Lipid synthesis

- Detoxification (liver!)


Folding of Influenza hemagglutinin (HA)


Ser/Thr


Summary

ER translocation: SRP-dependent and -independent pathways; translocation occurs through Sec61 complex; topogenic sequences determine overall orientation.

ER function

Compartmental identity: maturation versus fixed compartments

Identification of components: combination of genetics, biochemistry...

Vesicular coats: COPI ~ retrograde: Golgi->ER

COPII ~anterograde: ER->Golgi

CCV post-Golgi, various adaptors


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