RNA maturation transport & localization

RNA export to the cytoplasm: model systems

RNA degradation

Links between RNA processing, transport, degradationRNA localization in the cytoplasm

RNA processing: brief overview

Evidence for checkpoints?mRNA

transport

All eukaryotic mRNAs are processed

Specific process for Pol II transcriptsnuclear CBC binds to m7G-cap: role in splicing, transport, stability (exchanged in cytoplasm)

Function in transport, stability, translation

1. Capping

CPSF: cleavage and polyadenylation specificity factor

CStF: cleavage stimulatory factor

CF: cleavage factor

PAP: polyA polymerase

PAB: polyA binding protein

Function in transport, stability, translation

2. Polyadenylation

3. Splicing

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3. Splicing

Splicing is mediated by snRNPs

Compartments inside the nucleus

1. Nucleolus

2. Perichromatin granule clusters

Carter et al., Science (1993) 259: 1330

Balbiani Rings (Chironomus tentans)

BR RNP maturation

BR RNP translocation

BR RNP translocation

Retroviruses as mRNA export models

Gene location is influenced by gene activity

Casolari et al., 2004

SUMMARY

I. Multiple steps of mRNA biosynthesis are tightly coupled

II. Mex67/TAP is one major mRNA export factorBinding to mRNA may already occur at the site of transcription

III. Many questions remain- How are mRNA substrates released in the cytoplasm? - Are there multiple mRNA export pathways?- How is processing and transport mechanistically “coupled”?- Is mRNA export regulated?etc.

RNA half-lives vary greatly but are highly coordinated

RNA half-lives vary greatly but are highly coordinated

Wang et al., PNAS 2002

AUUUA element regulates half-life

A.

B.Casein mRNA prolactin

+

-

30,000 mRNA/cell

300 mRNA/cell

No change in transcription

Examples of regulated mRNA turnover

mRNA DEGRADATION

mRNA DECAY NMD

‘turnover’ ‘surveillance’

mRNA DECAY

AAAm7Gppp

poly A shortening Deadenylase complex

AAAAAAAAAAAAAAAAm7Gppp

Decapitation Decapping enzyme (DCP1 complex)

AAA

5’-3’ exonucleolytic cleavage Xrn1 complex

Decay factors localize to cytoplasmic processing bodies (P bodies)

Sheth et al. Science 2003

mRNA activity is regulated by multiple factors

Active mRNAs Inactive mRNAs

Storage

Decay

Transport

Translation

From Neu-Yilik et al. (2001) EMBO 20:532-540

Position of nonsense codon affects mRNA amounts

m7Gppp

Stop in penultimate exon/5’ of splicing mark

AAAAAAAAAAAAAAAA

Decapitation Decapping enzyme (DCP1 complex)

5’-3’ exonucleolytic cleavage Xrn1 complex

Nonsense Mediated Decay

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RNA localizationmRNA can be localized to subcellular compartmentsby actin or tubulin-dependent processesExamples:

Xenopus: Vg1 mRNA (TGFb) to vegetal pole

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

(requires mRNA binding protein staufen)

(requires staufen and miranda)

prospero (into ganglion of mother cells; neuroblast TF)

Yeast: Ash1 mRNA to daughter cell

Examples of localized mRNAs in various systems

lamellipodia staining perinuclear staining in myotubes

3’ UTR determins localization of many mRNAs

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

Ash1 mRNA specifically localizes to new daughter cells

Mechanism of Ash1 mRNA localization

SUMMARY

I. mRNA decay- regulated and non-regulated turn-over but apparently coordinated- ordered pathways (e.g. deadenylation, decapping, exonucleolytic degradation)- cross-talk between translation and turnover- important regulation via non-coding RNAs- turnover occurs in specific cytoplasmic compartments- 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)