Transcript 560k ppt - UCLA.edu

Transport across NPCs is driven by diffusion.
The importin -cargo complex diffuses from the
cytoplasmic side of the NPC to the nuclear side of
the NPC because its concentration is higher in the
cytoplasm where the complex forms and lower in
the nucleus where the complex is dissociated when
Ran•GTP binds the importin .
The Ran•GTP- importin  complex formed in the
nucleus diffuses into the cytoplasm where its
concentration is low because Ran-GAP in the NPC
cytoplasmic filaments converts Ran•GTP to
Ran•GDP, dissociating the Ran•GTP- importin 
complex.
The same arguments apply to the transport of the
NTF2- Ran•GDP complex from the cytoplasm into
the nucleus and of free NTF2 from the nucleus into
the cytoplasm.
Cell fusion experiments revealed that several
hnRNP proteins that appear to be confined to
nuclei actually shuttle between the nucleus and
cytoplasm.
•Cultured Xenopus and human cells were fused to
each other using polyethylene glycol.
•Cycloheximide was added to prevent further
protein synthesis.
•After 2 h, the cells were fixed and stained with
fluorescent antibodies specific for human hnRNP A
or human hnRNP C.
Construction of several hnRNP A deletion
mutants and analysis of the ability of these
mutants to shuttle between the nucleus and
cytoplasm using cell fusion experiments
revealed that deletion of an ~10 aa region
caused the mutant hnRNP A to be retained in
nuclei and not shuttle into the cytoplasm.
The sequence required for shuttling into the
cytoplasm is called a nuclear export
sequence or NES.
Several NESs have been defined in different
shutlling hnRNP proteins.
NESs are bound by proteins that are homologous to
importin . These proteins required for nuclear export
are called exportins.
The importin -related proteins, different importins and
exportins, are referred to as karyopharins.
All of these proteins have the ability to interact with the
FG-domains of FG-nucleoporins and consequently
can diffuse through the molecular meshwork that fills
the central transporter of NPCs.
The direction of transport is dependent on the
localization of Ran-GAP in the cytoplasm and
Ran-GEF in the nucleus.
This maintains Ran in the GTP form in the
nucleus and in the GDP form in the cytoplasm.
Ran-GAP is bound to the cytoplasmic filaments
of the NPC.
Ran-GEF is bound to chromosomes.
The export of most mRNPs requires a heterodimeric
mRNA transporter. The small subunit of the mRNA
transporter and the middle and C-terminal domains
of the large subunit interact with FG-repeats of the
FG-nucleoporins.
The large subunit binds to mRNAs cooperatively
with SR-proteins bound to exonic enhancers.
Thus, SR-proteins both specify exons during RNAsplicing and assist in transporting the resulting
mRNA into the cytoplasm, distinguishing exons from
other RNAs in the nucleus.
Other proteins that assist in binding of the mRNA
exporter to mRNAs include:
•The nuclear cap binding protein.
•PABPII bound to the polyA tail.
•Proteins retained at spliced exon junctions following
RNA splicing that form exon-junction complexes.
Thus, mRNP export depends on the additive effects
of multiple weak protein-RNA and protein-protein
interactions that bind to mRNAs cooperatively
through interactions with structures and sequences
that distinguish mRNAs including the 5’ cap, 3’ polyA
tail, and exonic splicing enhancers.
Fig. 12-25
Fig. 12-25
The association of snRNPs involved in
RNA-splicing with a pre-mRNA prevents
its nuclear export.
This prevents the export of an mRNA
until all of its introns have been spliced
out.
Fig. 12-26