RNA transport and the nuclear pore complex to: Cytoplasm 5‘ Florian Krapp, Seminar Molecular Machines and RNA Biology 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

nucleo-cytoplasmatic transport mRNA tRNA pre−miRNA pre−ribosomes ribosomal proteins snRNA snRNP nuclear proteins 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

Contents 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

1. The nuclear pore complex (NPC) nuclear pore complexes of Xenopus laevis oocyte (SFM, Uni Münster) 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

The nuclear envelope outer nuclear membrane perinuclear space inner nuclear membrane nuclear lamina ... there are 2.000 NPCs per nucleus! 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

What should an NPC be able to do? integration in the nuclear envelope outer nuclear membrane inner nuclear membrane nuclear lamina passive transduction of small molecules interaction with nuclear export/import signals selective directional transport 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

Some data on the NPC molecular mass structural proteins size 50 MDa (S. cerevisiae) 120 MDa (Vertebrates) (ribosome: 4 MDa) structural proteins 30 (S. cerevisiae) 50-100 (Vertebrates) size 120 nm diameter (opening ~60 nm) 200 nm deep 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

What does an NPC look like? Cryo-EM pictures Bagley, S. et al 3D structure of Dyctiostelium discoideum NPC Cryo-electron tomography (Beck, M. et al) 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

Components of the NPC cytoplasmatic filaments nucleoplasmatic envelope cytoplasmatic cytoplasmatic filaments cytoplasmatic coaxial ring thin ring star ring spoke ring transporter nucleoplasmatic coaxial ring NPC basket Bagley, S. et al 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

cross section and envelope integration cyt. coaxial ring thin ring star ring spoke ring nuc. coaxial ring NPC basket cytoplasm outer nuclear membrane perinuclear space inner nuclear membrane nucleoplasm Bagley, S. et al 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

Nucleoporins (Nups) structural proteins the NPC is built of mostly symmetrical on cytoplasmatic and nuclear sides FG-nucleoporins containing repeats (phenylalanine and glycine) 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

Localization of Nups Rout, M. P. et al 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

specificity of Nups the NPC is no static structure each Nup may have specific interaction partners Nups also interact with each other 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

Canis lupus familiaris Ran with bound GDP (Steward, M. et al.) 2. RanGTP pathway Canis lupus familiaris Ran with bound GDP (Steward, M. et al.) 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

What is Ran? small GTPase (Ras superfamily) exists in 2 forms GTP−bound: predominantly in the nucleoplasm GDP−bound: predominantly in the cytoplasm transition mediated by 2 proteins RanGEF (Ran guanine exchange factor) in the nucleoplasm RanGAP (GTPase activating protein) in the cytoplasm 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

The Ran cycle Cytoplasm Nucleoplasm PI RanGAP Ran GTP Ran GDP Export NTF2 GDP GTP Ran GTP Ran GDP RanGEF 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

Karyopherins, NES and NLS Karyopherins (importin−β−superfamily) Importins (e. g. importin−α and importin−β) Exportins NES: nuclear export signal short amino acid sequence (5−6 amino acids) NLS: nuclear localization signal ... −Pro−Pro−Lys−Lys−Lys−Arg−Lys−Val-... (Simian Virus 40) 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

Ran-controlled export Export cargo NES Export cargo Exportin Exportin NES Ran GTP Ran GDP RanGAP PI Cytoplasm Nucleoplasm Export cargo NES Export cargo Exportin Exportin NES Ran GTP Ran GTP 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

Ran-controlled import Import cargo NLS Import cargo Importin NLS Importin Cytoplasm Nucleoplasm Import cargo Import cargo  NLS Importin NLS Importin Ran GTP Ran GTP 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

Nuclear transport: an overview Export Import Export cargo Import cargo NES NLS Export cargo Exportin Exportin NES PI Import cargo Importin Importin NLS Ran GTP Ran GDP RanGAP NTF2 Cytoplasm Nucleoplasm Ran GDP GTP RanGEF Export cargo Export cargo Import cargo Import cargo NES Exportin NES GDP Importin NLS NLS  Exportin Ran GTP Ran GTP Importin 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

non-protein transport by Ran pre-miRNA Exportin−5 snRNA cap, CRM1 (Exportin−1), PHAX snRNP Importin−β ribosomal subunits CRM1 (Exportin−1) 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

Summary Ran NES and NLS define destination of Proteins karyopherins mediate transport through the NPC the Ran cycle controls and supports protein transport necessary for transport of: pre-miRNA snRNA / snRNP ribosomal subunits tRNA Proteins 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

3. Transport of mRNA crystal structure of S. cerevisiae Mex67:Mtr2 heterodimer (Fribourg, S. et al.) 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

Why do we need another transport system? numerous processing events must occur before export 5‘ capping 3‘ poly−A attachment splicing immature mRNA must not be delivered to cytoplasm! mRNA export is linked to transcription processing translation 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

mRNA and mRNP mRNA is not „naked“ 5‘ cap and cap binding proteins 3‘ poly−A tail and poly−A binding proteins splicing factors general RNA binding factors other factors involved in pre−mRNA processing 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

conservation of mRNP transport most of the involved proteins have orthologs... Yeast Vertebrate Mex67p NXF1/TAP Mtr2 p15 Yra1p Aly/REF Sub2p UAP56/HEL THO THO TREX TREX − EJC Dbp5p Dbp5 ... ... 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

3 steps of mRNP transport Assembly of mRNP export complexes - quality control Translocation through NPC Disassembly of mRNP export complexes 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

Mex67p and Mtr2p Mex67p Mtr2p both form a receptor heterodimer associated with NPCs capable of shuttling cargo needs heterodimerization with... Mtr2p both form a receptor heterodimer involved in 75% of all mRNA transports 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

3 steps of mRNP transport translation 3. mRNP disassembly dissociate Mex67 mature mRNP Cytoplasm Nucleoplasm 2. translocation mature mRNP attach Mex67 processing 1. mRNP assembly splicing transcription gene 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

1. mRNP assembly 5‘•••5‘ AAAAAAAAA Dbp5 x Mex67p x Mex67p CBC x Mex67p Pab1 Mtr2 Mtr2 Pab1 AAAAAAAAA Mtr2 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

2. translocation: mechanism weak interactions between Nups and Mex67p:Mtr2p simple diffusion prevention of the return of mRNPs 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

2. translocation: transfer of Dbp5 Gle1 x AAAAAAAAA Pab1 Mex67p Mtr2 5‘•••5‘ CBC Dbp5 Gle1 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

2. translocation: transfer of Dbp5 Gle1 Dbp5 x Mex67p x Mex67p CBC x Mex67p 5‘•••5‘ Pab1 Mtr2 Mtr2 Pab1 AAAAAAAAA Mtr2 Gle1 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

2. translocation: transfer of Dbp5 Gle1 Dbp5  x Mex67p x Mex67p CBC x Mex67p 5‘•••5‘ Pab1 Mtr2 Mtr2 Pab1 AAAAAAAAA Mtr2 Gle1 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

2. translocation: transfer of Dbp5 Gle1 Dbp5  x Mex67p x CBC x Mex67p 5‘•••5‘ Pab1 Mtr2 Pab1 AAAAAAAAA Mtr2 Gle1 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

3. mRNP disassembly ...is almost done! 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

Literature and Pictures Papers and Articles Erkmann, J.A. and U. Kutay, Nuclear export of mRNA: from the site of transcription to the cytoplasm. Exp Cell Res, 2004. 296(1): p. 12-20. Cole, C.N. and J.J. Scarcelli, Transport of messenger RNA from the nucleus to the cytoplasm. Curr Opin Cell Biol, 2006. 18(3): p. 299-306. Stewart, M., Ratcheting mRNA out of the nucleus. Mol Cell, 2007. 25(3): p. 327-30. Bagley, S. et al., The nuclear pore complex. J Cell Sci, 2000. 113(22): p. 3885-3886. Beck, M. et al., Nuclear Core Complex Structure and Dynamics revealed by Cryoelectron Tomography. Science, 2004. 306(5700): p. 1387-1390. Rout, M. P. et al., The Yeast Nuclear Pore Complex: Composition, Architecture, and Transport Mechanism. The Journal of Cell Biology, 2000. 148: p. 635-651. Rout, M. P. et al., Virtual gating and nuclear transport: the hole picture TRENDS in Cell Biology, 2003. 13(12): p. 622-628. Corbett, A. et al., Hot trends erupting in the nuclear transport field. EMBO reports, 2004. 5(5): p. 453-458. Lund, E. et al, Nuclear Export of MicroRNA Precursors. Science, 2004. 303(5654): p. 95-98. Hakata, Y. et al., A Multifunctional Domain in Human CRM1 (Exportin 1) Mediates RanBP3 Binding and Multimerization of Human T-Cell Leukemia Virus Type 1 Rex Protein. Mol Cell Biol, 2003. 23(23): p. 8751-8761. Ohno, M. et al., PHAX, a mediator of U snRNA nuclear export whose activity is regulated by phosphorylation. Cell, 2000. 101(2): p. 187-198. Internet sources http://celldynamics.uni-muenster.de/Projects/ProA6Img/A6figure1.jpg http://www.unige.ch/sciences/biologie/bicel/stutz/research.html http://www.ncbi.nlm.nih.gov/Structure/index.shtml http://wikipedia.org/ http://electron.mit.edu/~gsteele/mirrors/elchem.kaist.ac.kr/jhkwak/TopometrixWeb/images/nucpor.jpg 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA

Thank you for listening! 1. The nuclear pore complex (NPC) 2. RanGTP pathway 3. Transport of mRNA