1. RNA maturation transport & localization RNA export to the cytoplasm: model systems RNA degradation Links between RNA processing, transport, degradation RNA localization in the cytoplasm RNA processing: brief overview

2. Evidence for checkpoints? mRNA transport All eukaryotic mRNAs are processed

3. Specific process for Pol II transcripts nuclear CBC binds to m 7 G-cap: role in splicing, transport, stability (exchanged in cytoplasm) Function in transport, stability, translation 1. Capping

4. 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

5. 3. Splicing

7. Splicing is mediated by snRNPs

8. Compartments inside the nucleus 1. Nucleolus

9. 2. Perichromatin granule clusters Carter et al., Science (1993) 259: 1330

10. Balbiani Rings (Chironomus tentans)

11. BR RNP maturation

12. BR RNP translocation

14. Retroviruses as mRNA export models

15. Gene location is influenced by gene activity Casolari et al., 2004

17. SUMMARY I.Multiple steps of mRNA biosynthesis are tightly coupled II.Mex67/TAP is one major mRNA export factor Binding 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.

18. RNA half-lives vary greatly but are highly coordinated

19. Wang et al., PNAS 2002

20. AUUUA element regulates half-life

21. A. B. Casein mRNA prolactin + - 30,000 mRNA/cell 300 mRNA/cell No change in transcription Examples of regulated mRNA turnover

22. mRNA DEGRADATION mRNA DECAY NMD ‘turnover’‘surveillance’

23. mRNA DECAY AAA m7Gppp poly A shorteningDeadenylase complex AAAAAAAAAAAAAAAA m7Gppp DecapitationDecapping enzyme (DCP1 complex) AAA 5’-3’ exonucleolytic cleavageXrn1 complex

24. Decay factors localize to cytoplasmic processing bodies (P bodies) Sheth et al. Science 2003

25. mRNA activity is regulated by multiple factors Active mRNAsInactive mRNAs Storage Decay Transport Translation

26. From Neu-Yilik et al. (2001) EMBO 20:532-540 Position of nonsense codon affects mRNA amounts

27. m7Gppp Stop in penultimate exon/ 5’ of splicing mark AAAAAAAAAAAAAAAA DecapitationDecapping enzyme (DCP1 complex) 5’-3’ exonucleolytic cleavageXrn1 complex Nonsense Mediated Decay AAAAAAAAAAAAAAAA

28. 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) (requires staufen and miranda) prospero (into ganglion of mother cells; neuroblast TF) Yeast: Ash1 mRNA to daughter cell

29. Examples of localized mRNAs in various systems

30. lamellipodia stainingperinuclear staining in myotubes 3’ UTR determins localization of many mRNAs

31. Bertrand et al., Mol Cell (98) 2:437-445 Ash1 mRNA specifically localizes to new daughter cells

32. Mechanism of Ash1 mRNA localization

33. 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)