Bio 402/502 Section II, Lecture 5

1. Bio 402/502Section II, Lecture 5 mRNA export, RNA surveillance, and Transport of proteins in and out of the nucleus Dr. Michael C. Yu

2. mRNA export - formation of an export competent mRNP Balbiani Rings (Chironomus tentans) • Why export as a protein/DNA complex? RNAs are too big and lack the signals to interact w/ nuclear export receptors Sees formation of mRNP as transcription commences • Specific “adaptor” proteins must first bind to the RNA and chaperone this molecule to the export receptor, which, in turn, guides the RNA across the NPC

3. There are other types of RNA transport in a cell

4. Factors involved in mRNA export are co-transcriptionally recruited Model from yeast: • THO complex: major role in transcriptional elongation and recruitment of mRNA export factors • Yra1 - mRNA export factor, interacts with Mex67 • Mex67 - promotes translocation across NPC (Stutz & Izaurralde,2003)

5. Proteins involved in the nuclear export of mRNAs (Sub2p) (Mex67p) (Yra1p) (Mtr2p) (Cullen, 2003) (yeast homolog is indicated in parentheses)

6. Path of transporting mRNA to the nuclear pore complex • Sub2, Yra1p and hnRNP proteins such as Npl3p associate co-transcriptionally with the mRNA in yeast. • In mammalian cells, Aly/REF(Yra1) and UAP56(Sub2) are part of the exon-junction complex (EJC) on the spliced mRNA (not shown). UAP56 is replaced by the TAP-p15 (Mex67-Mtr2 in yeast) heterodimers • The Mex67-Mtr2 heterodimers mediate the interaction of the mRNP with components of the nuclear pore complex (NPC). • The DEAD box protein Dbp5p is required for release of mRNP on the cytoplasmic side of the NPC. • DEAD box-mediated ATPase activities important for mRNA export are indicated by stars. (Linder & Stutz, 2001)

7. Genetic approach to identify genes involved in mRNA export process Non-essential genes RNA FISH w/ oligo dT Mutagenized cells or collection of non-essential gene KOs (Lei et al, 2003) essential genes RNA FISH w/ oligo dT Growth at permissive temperature Shift to non-permissive temperature

8. Mex67(yeast) and NXF1(Drosophila) are essential genes involved in mRNA export Nuclear mRNA accumulation is observed after shifting mex67 TS mutant to the restrictive temperature (37°C) (Stutz & Izaurralde, 2003) Visualization of poly(A) mRNA is accomplished by in situ using fluorescently-labeled oligo-dT probe

9. Linking mRNA biogenesis with mRNA export: Mlp proteins Mlp proteins: filamentous proteins on the nuclear side of NPC v • Yra1p and Nab2p are essential for mRNP docking to the Mlp export gate at the nuclear periphery. • mRNP complexes produced in the GFP-yra1-8 mutant strain are retained by the Mlp selective filter. • mRNP stalling negatively feeds back on mRNA synthesis. • Loss of Mlp1p or Mlp2p alleviates the negative effect on mRNA synthesis and allows a fraction of transcripts to reach the cytoplasm. (Vinciguerra et al., 2005)

10. Mlp proteins act as selective filters at NPC entrance • The perinuclear Mlp1p protein contributes to mRNP surveillance by retaining unspliced transcripts within the nucleus • This is achieved possibly via recognition of a component associated with the 5´ splice site. (Vinciguerra & Stutz, 2004)

11. Nab2 is responsible for the docking of mRNPs to Mlp • Nab2p, a shuttling mRNA binding protein involved in polyA tail length regulation, directly interacts with Mlp proteins. • Possible mechanism: by signaling proper 3´ end formation. (Vinciguerra & Stutz, 2004)

12. Mlp1 is involved in the nuclear retention of unspliced mRNA • An intact 5’ splice site and branchpoint are • required for nuclear retention of pre-mRNAs • Numerous splicing factors, including U1 • snRNA and branchpoint binding protein • (BBP/SF1), have been found to affect nuclear • retention of pre-mRNAs • In yeast, perinuclearly located Mlp1 physically • retains improperly spliced pre-mRNAs but • does not affect the splicing process itself • Thus, it appears that Mlp1 retains pre-mRNAs • that assemble into a spliceosome but fail to • proceed through splicing before reaching the • nuclear pore complex (Galy, V. et al, 2004)

13. What happens to produced messages? pathways for mRNA degradation mRNA DEGRADATION mRNA DECAY NMD ‘turnover’ ‘surveillance’

14. NMD: Nonsense Mediated Decay Nonsense codon: a cause for pre-mature termination, usually by a point mutation that creates a stop codon at the wrong place Stop in penultimate exon/ 5’ of splicing mark m7Gppp AAAAAAAAAAAAAAAA Decapitation Decapping enzyme (DCP1 complex) AAAAAAAAAAAAAAAA 5’-3’ exonucleolytic cleavage Xrn1 complex

15. mRNAs are checked for possible defects during its synthesis (Wagner, E. and Lykke-Anderson, J., 2002)

16. Exosome Exosome Nuclear mRNA surveillance checks many different kinds of wrong mRNAs (Vasudevan and Peltz, 2003)

17. The TRAMP complex is responsible for RNA degradation • TRAMP complex: • Trf4p: poly(A) polymerase • Air2p: RNA binding protein • Mtr4p: ATP-dependant RNA helicase • Trf5p is highly related to Trf4p • Air1p is highly related to Air2p (Anderson, 2005)

18. Molecular mechanism by which TRAMP complex degrades mRNA (LaCava et al., 2005)) • - The TRAMP complex interacts with RNAs or RNP complexes, making them targets for degradation. • The zinc finger domains of Air2p might be involved in substrate binding. The RNA is then polyadenylated by Trf4p. • Exosome recruitment and activation requires the intact TRAMP complex. The activated exosome then rapidly deadenylates the RNA and can penetrate into regions of stable structure. • Helicase activity of Mtr4p is important for dissociation or remodeling of stable RNP structures to allow passage of the exosome.

19. What happens to messages made? pathways for mRNA degradation mRNA DEGRADATION mRNA DECAY NMD ‘turnover’ ‘surveillance’

20. Normal decay of mRNAs AAAAAAAAAAAAAAAA m7Gppp poly A shortening Deadenylase complex AAA m7Gppp Decapitation Decapping enzyme (DCP1 complex) AAA 5’-3’ exonucleolytic cleavage Xrn1 complex Decay of mRNA Avg. mRNA half-lives: E. coli: 4 min (2-10 min) Yeast: 22 min (4-40 min) Humans: 10 hours (0.5-24 hours)

21. Regulation of mRNA activity by multiple trans-factors mRNAs that are inactive for translation Mature mRNAs for translation Possible fate for these mRNAs: storage, decay, transport

22. Factors involved in mRNA decay are localized to P-bodies in the cytoplasm (Sheth et al, 2003)

23. Summary: molecular mechanisms for mRNA turnover • 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 RNA • turnover occurs in specific cytoplasmic compartments • NMD: recognition of premature stop codons (Wagner, E. and Lykke-Anderson, J. 2002)

24. ~2000Å ~150Å Ribosome The Nuclear Pore Complex NPC: all traffic between nucleus & cytoplasm in higher eukaryotes occurs via NPC (exception: mitosis) Cytoplasmic filament Cytoplasmic ring Cytoplasm Nucleus Inner ring Basket Distal ring

25. More on the nuclear pore complex NPC size: diameter = 120nm, 8-fold rotational symmetry, MW = 125 million daltons, composed of 50-100 different proteins called nucleoporins (Nups) NPC extension is thought to be the initial cargo docking site

26. The nuclear pores on the membrane Nuclear basket

27. Small molecules can diffuse freely through the nuclear pore However, large molecular requires active transport

28. Soluable nuclear transport receptors facilitates the transport • Karyopherins: nuclear transport receptors that bind to Nups. • Different factors are utilized for export (exportin) and import (importin) • Nuclear transport factors bind to cargo (or adaptors that bind cargo) that contains either nuclear localization sequence (NLS) or nuclear export signal (NES) • Types of cargo: proteins and RNAs (J. Lingappa, 2003)

29. Summary of factors involved in nuclear protein import cycle (Stewart, 2007)

30. Nuclear localization sequence is required for protein nuclear import NLS: consists of either one or two stretches of basic amino acids Functional NLS is required for nuclear localization (mutation in NLS results in cytosolic localization)

31. The nuclear import/export cycle 1. In the cytoplasm, NLS-containing cargo is bound by the heterodimeric import receptor (importin / (Lei and Silver, 2004)

32. The nuclear import/export cycle 2. Ran-GTP binds to importin, causing conformational change in the importin, which releases the cargo (Lei and Silver, 2004)

33. The nuclear import/export cycle 3. If cargo is phosphorylated, it can interact with exportins and Ran-GTP, allowing the complex to exit the nucleus (Lei and Silver, 2004)

34. The nuclear import/export cycle 3. In the cytoplasm, hydrolysis of Ran-GTP to Ran-GDP causes dissociation of the export complex (Lei and Silver, 2004)

35. The molecular machineries behind nuclear protein transport Nucleoporins (known as Nups): • Make up the cytoplasmic filaments, nuclear basket, and line the pore • Contain different types of FG (Phe/Gly) repeats • Nuclear transport factors make multiple sequential contacts with distinct Nups, resulting in docking and translocation of nuclear transport complexes • Exact mechanism by which Nups mediate translocation of complexes remains unclear. but may involve sequential association with of receptor plus cargo with FG repeats lining pore (Bayliss, R. et al. 2000)

36. RanGTP/GDP exchange: driving force behind cargo exchange • Ran-GTP: • A small GTPase that gives directionality to nuclear transport, see below • Members of the Ras oncogene family (like Rab, which regulates vesicle fusion) • Binds to nuclear transport receptors (Matsurra and Stewart, 2004)

37. How does the Ran GTPase cycle work? • Ran GTPase has little hydrolysis or exchange activity on its own. • Regulated by GTPase-activating protein (RanGAP) that promotes hydrolysis that is predominantly in cytoplasm and guanine exchange factor (RanGEF) that promotes GTP exchange that is predominantly in the nucleus • Ran exists in inactive GDP-bound form in the cytoplasm • In the nucleus, however, Ran is largely bound to GTP • Ran gradient is critical for most nuclear transport, except bulk mRNA transport which is mediated by factors that are not karyopherins Ran gradient is created by having RanGAP (which promotes GTP hydrolysis) only in cytoplasm and RanGEF (which promotes loading of Ran with GTP) only in nucleus (Nalkieny and Dreyfus, 1999)

38. Mechanism of proteins that shuttles between nucleus and cytoplasm 1. Proteins contain both nuclear localization and export signals (NLS + NES). 2. These proteins shuttle back and forth between nucleus and cytosol. 3. Rate of export and import determines in which compartment it resides. 4. Export/ import of shuttling proteins can be regulated, i.e. by phosphorylation-dephosphorylation of residues adjacent to NLS or NES signals resulting in blockade/exposure of signals.

39. Experimental approaches studying nuclear trafficking Heterokaryon assay - determines if a nuclear protein shuttles in/out of nucleus 1. Nuclei transfected/injected to express tagged protein (+ marker protein that stays in "donor" nucleus) 2. Fuse cells to another set of cells using PEG. 3. Examine whether tagged protein shows up in cytoplasm alone (export), or in recipient nucleus as well (export followed by import, implying shuttling) using IF Heterokaryon: solid arrow HeLa cell = arrowead Xenopus cell: dotted arrow Green: nuclear restriced Red: shuttling

40. Experimental approaches studying nuclear trafficking Immunofluorescent tags • Transfect cells with proteins tagged with GFP, RFP, YFP, etc. Assess nuclear vs. cytoplasmic location by IF (immunofluorescence) • Or, you can transfect cells which are epitope tagged and use antibodies conjugated with fluorescently-tag to perform IF. Confocal microscopy Combined image SC35: nuclear SRPK1: cytoplasm (Ding et al, 2006)