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RNA maturation transport & localization

RNA maturation transport & localization. RNA processing: brief overview. RNA export to the cytoplasm: model systems. RNA degradation. Links between RNA processing, transport, degradation. RNA localization in the cytoplasm. All eukaryotic mRNAs are processed. Evidence for checkpoints?.

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RNA maturation transport & localization

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

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

  3. 1. Capping Specific process for Pol II transcripts nuclear CBC binds to m7G-cap: role in splicing, transport, stability (exchanged in cytoplasm) Function in transport, stability, translation

  4. 2. Polyadenylation 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

  5. 3. Splicing

  6. 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

  13. BR RNP translocation

  14. Retroviruses as mRNA export models

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

  16. 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.

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

  18. RNA half-lives vary greatly but are highly coordinated Wang et al., PNAS 2002

  19. AUUUA element regulates half-life

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

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

  22. mRNA DECAY AAAAAAAAAAAAAAAA m7Gppp poly A shortening Deadenylase complex AAA m7Gppp Decapitation Decapping enzyme (DCP1 complex) AAA 5’-3’ exonucleolytic cleavage Xrn1 complex

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

  24. mRNA activity is regulated by multiple factors Active mRNAs Inactive mRNAs Storage Decay Transport Translation

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

  26. Nonsense Mediated Decay Stop in penultimate exon/ 5’ of splicing mark m7Gppp AAAAAAAAAAAAAAAA Decapitation Decapping enzyme (DCP1 complex) AAAAAAAAAAAAAAAA 5’-3’ exonucleolytic cleavage Xrn1 complex

  27. 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

  28. Examples of localized mRNAs in various systems

  29. 3’ UTR determins localization of many mRNAs lamellipodia staining perinuclear staining in myotubes

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

  31. Mechanism of Ash1 mRNA localization

  32. 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)

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