Download 1. The nuclear pore complex (NPC)

RNA transport and the nuclear pore complex
Florian Krapp, Seminar
Molecular
Machines
2. RanGTP
pathway
3. Transport ofand
mRNARNA Biology
1. The nuclear pore complex (NPC)
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
– 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
3D structure of Dyctiostelium discoideum NPC
Bagley, S. et al
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
cytoplasmatic filaments
cytoplasmatic coaxial ring
thin ring
envelope
spoke ring
nucleoplasmatic
star ring
nucleoplasmatic coaxial ring
transporter
NPC basket
1. The nuclear pore complex (NPC)
Bagley, S. et al
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
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:
– GDP−bound:
predominantly in the nucleoplasm
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
PI
RanGAP
Ran
Ran
GTP
GDP
Export
Cytoplasm
NTF2
Nucleoplasm
GDP
Ran
GTP
Ran
GTP
RanGEF
1. The nuclear pore complex (NPC)
2. RanGTP pathway
3. Transport of mRNA
GDP
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
NES
Exportin
Ran
Exportin
GTP
Ran
GDP
RanGAP
PI
Cytoplasm
Nucleoplasm
Export cargo
NES
Exportin
Export cargo
NES
Exportin
Ran
Ran
GTP
GTP
1. The nuclear pore complex (NPC)
2. RanGTP pathway
3. Transport of mRNA
Ran-controlled import
Import cargo
NLS
Import cargo
NLS
Importin
Importin
Cytoplasm
Nucleoplasm
Import cargo
NLS
Importin
Ran
GTP
Import cargo
NLS
Importin
Ran
1. The nuclear pore complex (NPC)
2. RanGTP pathway
GTP
3. Transport of mRNA
Nuclear transport: an overview
Export
Export cargo
NES
Exportin
Export cargo
NES
Exportin
Ran
Import
Import cargo
NLS
PI
GTP
Ran
Import cargo
NLS
Importin
GDP
Importin
RanGAP
NTF2
Cytoplasm
Nucleoplasm
Ran
GDP
GTP
Export cargo
NES
Export cargo
NES
Exportin
Ran
GTP
RanGEF
GDP
Ran
Import cargo
NLS
Importin
GTP
Exportin
1. The nuclear pore complex (NPC)
Import cargo
NLS
Importin
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
Mtr2
Yra1p
Sub2p
THO
TREX
−
Dbp5p
...
NXF1/TAP
p15
Aly/REF
UAP56/HEL
THO
TREX
EJC
Dbp5
...
1. The nuclear pore complex (NPC)
2. RanGTP pathway
3. Transport of mRNA
3 steps of mRNP transport
1. Assembly of mRNP export complexes
- quality control
2. Translocation through NPC
3. Disassembly of mRNP export complexes
1. The nuclear pore complex (NPC)
2. RanGTP pathway
3. Transport of mRNA
Mex67p and Mtr2p
• Mex67p
– 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
2. translocation
Nucleoplasm
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
Dbp5
x
x
Mex67p
Mex67p
Mtr2
Mtr2
1. The nuclear pore complex (NPC)
2. RanGTP pathway
x
Mex67p
Mtr2
3. Transport of mRNA
CBC
5‘•••5‘
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
Dbp5
x
x
Mex67p
x
Mex67p
Mtr2
Mtr2
Mex67p
CBC
5‘•••5‘
Mtr2
Gle1
1. The nuclear pore complex (NPC)
2. RanGTP pathway
3. Transport of mRNA
2. translocation: transfer of Dbp5
Gle1
Dbp5
x
x
Mex67p
x
Mex67p
Mtr2
Mtr2
Mex67p
CBC
5‘•••5‘
Mtr2
Gle1
1. The nuclear pore complex (NPC)
2. RanGTP pathway
3. Transport of mRNA
2. translocation: transfer of Dbp5
Gle1
Dbp5

x
x
Mex67p
x
Mex67p
Mtr2
Mtr2
Mex67p
CBC
5‘•••5‘
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

x
CBC
5‘•••5‘
Mex67p
Mtr2
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
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1. The nuclear pore complex (NPC)
2. RanGTP pathway
3. Transport of mRNA