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

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

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

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

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

6. 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) RanGTP pathway Transport of mRNA

7. 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)
(Vertebrates)
size
120 nm diameter (opening ~60 nm)
200 nm deep
1. The nuclear pore complex (NPC) RanGTP pathway Transport of mRNA

8. 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) RanGTP pathway Transport of mRNA

9. 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) RanGTP pathway Transport of mRNA

10. 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) RanGTP pathway Transport of mRNA

11. 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) RanGTP pathway Transport of mRNA

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

13. 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) RanGTP pathway Transport of mRNA

14. 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) RanGTP pathway Transport of mRNA

15. 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) RanGTP pathway Transport of mRNA

16. 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) RanGTP pathway Transport of mRNA

17. 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) RanGTP pathway Transport of mRNA

18. 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) RanGTP pathway Transport of mRNA

19. 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) RanGTP pathway Transport of mRNA

20. 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) RanGTP pathway Transport of mRNA

21. 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) RanGTP pathway Transport of mRNA

22. 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) RanGTP pathway Transport of mRNA

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

24. 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) RanGTP pathway Transport of mRNA

25. 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) RanGTP pathway Transport of mRNA

26. 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) RanGTP pathway Transport of mRNA

27. 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) RanGTP pathway Transport of mRNA

28. 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) RanGTP pathway Transport of mRNA

29. 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) RanGTP pathway Transport of mRNA

30. 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) RanGTP pathway Transport of mRNA

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

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

33. 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) RanGTP pathway Transport of mRNA

34. 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) RanGTP pathway Transport of mRNA

35. 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) RanGTP pathway Transport of mRNA

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

37. 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, (1): p
Cole, C.N. and J.J. Scarcelli, Transport of messenger RNA from the nucleus to the cytoplasm.
Curr Opin Cell Biol, (3): p
Stewart, M., Ratcheting mRNA out of the nucleus.
Mol Cell, (3): p
Bagley, S. et al., The nuclear pore complex.
J Cell Sci, (22): p
Beck, M. et al., Nuclear Core Complex Structure and Dynamics revealed by Cryoelectron Tomography.
Science, (5700): p
Rout, M. P. et al., The Yeast Nuclear Pore Complex: Composition, Architecture, and Transport Mechanism.
The Journal of Cell Biology, : p
Rout, M. P. et al., Virtual gating and nuclear transport: the hole picture
TRENDS in Cell Biology, (12): p
Corbett, A. et al., Hot trends erupting in the nuclear transport field.
EMBO reports, (5): p
Lund, E. et al, Nuclear Export of MicroRNA Precursors.
Science, (5654): p
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, (23): p
Ohno, M. et al., PHAX, a mediator of U snRNA nuclear export whose activity is regulated by phosphorylation.
Cell, (2): p
Internet sources
1. The nuclear pore complex (NPC) RanGTP pathway Transport of mRNA

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