RR10: Nucleo-Cytoplasmic Transport

Question 1

Where are mRNAs formed?

Answer 1

In the nucleus.

Question 2

Where are proteins formed?

Answer 2

In the ribosome.

Question 3

Where do we find ribosomes?

Answer 3

In the cytoplasm.

Question 4

What’s the general thing that mRNAs have to do in order to get translated into proteins by the ribosome?

Answer 4

mRNAs are formed in the nucleus.
Ribosome translate mRNAs into proteins, but they are found in the cytoplasm.
mRNAs need to get out of the nucleus and into the cytoplasm.

Question 5

Why might it be hard for mRNAs to go from the nucleus to the cytoplasm?

Answer 5

Because the nucleus is surrounded by an envelope that blocks most macromolecules from passing through it.

Question 6

What’s the nuclear pore complex?

Answer 6

It’s a highly ordered structure.
125 megadaltons (30x bigger than a ribosome)
Composed of 50 (yeast) or 100 (vertebrates) different proteins.
It holds nuclear pores.
Small molecules of 40-60kDa can diffuse freely, but bigger molecules need RNPs to be transported.

Question 7

What makes up the pores in the nuclear pore complex?

Answer 7

FG-nucleoporins make up the pore. Rich in phenylalanine and glycine. The proteins have FG repeats and they interact with each other, which forms a gel.
FG-nucleoporins are hydrophobic and possess disordered domains.

Question 8

Why are FG-nucleoporins important?

Answer 8

They play a role in regulating the movement through the pore.
Proteins that have domains that permit them to interact with FG repeats will be able to pass.

Question 9

What sequences allow proteins to pass the nuclear pore complex from the cytoplasm to the nucleus?

Answer 9

Nuclear Localization Signals (NLS)

Question 10

What are Nuclear Localization Signals?

Answer 10

They are signals used by proteins synthesized in the cytoplasm but need to be imported into the nucleus.
They are formed of amino acids that are present on specific factors that need to go into the nucleus

Question 11

How did we find out about Nuclear Localization Signals?

Answer 11

Studying the virus SV40.
SV40 couldn’t go into the nucleus because the T antigen used by SV40 had a mutation.
They took the stretch of amino acid from T antigene and put it in another protein that is usually in the cytoplasm.
With that new sequence (NLS), the protein was observed to go into the nucleus.

Question 12

Is the sequence of Nuclear Localization Signal enough for a protein to go into the nucleus?

Answer 12

Yes. By adding nuclear localization signal to a cytoplasmic protein is enough for that protein to be able to make its way into the nucleus. (but there are other factors involved outside that protein)

Question 13

What is Ran?

Answer 13

Ran is a monomeric G protein that exist in 2 conformations:
1 bound to GTP (on)
1 bound to GDP (off)
Interacts with GTP and GDP.
They have GTPase activity.
It’s a required protein to get proteins into the nucleus via NLS.

Question 14

What are nuclear transport receptors?

Answer 14

They are also called importins.
They are proteins that bind to NLS domains present in cargo proteins to facilitate transport through the pore by associating with FG repeats on the nucleoporins.

Question 15

What are cargo proteins?

Answer 15

A protein that is carried for nuclear transport.

Question 16

Describe the mechanism for nuclear import.

Answer 16

1. Cargo protein with NLS
2. Importin recognizes the NLS and interacts with it specifically
3. Complex Importin + Cargo
4. Makes its way through the nuclear pore because Importin can interact with FG repeats on FG-nucleoporins.
5. In the nucleus
6. Ran GTP interacts specifically with the importin and changes the conformation of importin by binding it.
7. Importin lets go of cargo
8. Cargo protein does whatever function it has to do in the nucleus.
9. Ran GTP still bound to Importin will leave the nucleus through the nuclear pore (concentration gradient) because Importin can interact with FG repeats.
10. Back in the cytoplasm, GAP protein will bind the Importin/RanGTP complex and hydrolyzes it into its GDP conformation.
11. GAP changes the conformation of the complex and Importin is released to be able to bind to another cargo protein and start the mechanism again.

Question 17

During the mechanism for nuclear import, what makes the Importin and Ran GTP bound together leave the nucleus?

Answer 17

A concentration gradient. Because the concentration of Ran GTP and Importin complex is much higher in the nucleus than in the cytoplasm.

Question 18

What’s the role of GAP protein?

Answer 18

It enhances the GTPase function.
Turns GTP to GDP.

Question 19

How are concentration gradients maintained in the nucleus and the cytoplasm?

Answer 19

They are maintained by the energy associated with:
- the cytoplasmic side
- the hydrolysis of Ran GTP
- the generation of Ran GTP in the nucleus by its associated GEF

Question 20

What’s GEF?

Answer 20

Guanine nucleotide Exchange Factor.
They turn RanGDP to RanGTP.

Question 21

What are the roles of Ran GEF and Ran GAP?

Answer 21

They make sure we have the right conformation of Ran in the nucleus and in the cytoplasm.
Ran GAP: in the cytoplasm (Ran GTP to Ran GDP)
Ran GEF: in the nucleus (Ran GDP to Ran GTP)

Question 22

What’s the mechanism of nuclear export?

Answer 22

1. Start in the nucleus, we want to get out.
2. Exportin recognizes NES on cargo
3. Recruitment of Ran GTP forming a ternary complex
4. The complex goes through the nuclear pore because exportin interacts with FG-nucleoporins.
5. In the cytoplasm, Ran GAP interacts with Ran GTP to hydrolyze it into GDP.
6. This changes the conformation of the complex and cargo is released.
7. Exportin can make its way back into the nucleus by itself through gradient concentration.
8. GDP makes its way back into the nucleus by itself
9. The cycle can start again.

Question 23

What is Exportin t and what does it do?

Answer 23

It’s used to export tRNAs from the nucleus to the cytoplasm.
Exportin t binds to tRNAs and RanGTP and passes through the nuclear pore complex.
The complex dissociate when ti interacts with Ran GAP in the cytoplasm.

Question 24

What RNA molecules use a RAN dependent process to get exported?

Answer 24

tRNAs.
ribosomal subunits
mRNAs that associate with hnRNP proteins (HIV Rev)

Question 25

Which type of RNA molecules are mostly exported through a RAN-independent process?

Answer 25

mRNAs

Question 26

What does mRNP mean?

Answer 26

It means messenger RiboNucleoProtein.
They are non-translating mRNAs, they regulate mRNA translation, localization and turn-over.

Question 27

How does the mRNP export work?

Answer 27

It’s a RAN-independent process.
1. Mature mRNA interacts with 2 subunits of the mRNA exporter, NXF1 and NXT1
2. NXF1 and NXT1 interact with proteins on the mRNA, like senior proteins, proteins that are associated with mature mRNA.
3. The complex formed can interact with FG repeats on nucleoporins.
4. The mRNA exporter will make its way through the nuclear pore complex into the cytoplasm.

Question 28

What are hnRNPs?

Answer 28

RNA binding proteins that bind to mRNA to protect it and assemble into bundles.

Question 29

How does the transport of mRNA work in Chironomous tenlans?

Answer 29

RAN-independent
mRNA and hnRNP form bundles that will go through the nuclear pore complex.
Those bundles are a mix of mRNA and proteins forming mRNPs.
The complex can go through the nuclear pore complex because it recognizes that it’s a mature mRNA going through it.
As soon as the bundle goes through the nuclear pore complex, the ribosome with the mRNA, which means that the 5’ end must be the first to go in and out of the nuclear pore complex.

Question 30

What does the RNA helicase do on the cytoplasmic side when the mRNP goes through the nuclear pore complex from the nucleus to the cytoplasm?

Answer 30

It takes out all of the protein that were bound to the mRNA that recognized that it was a mature mRNA.

Question 31

What is cytoplasmic remodelling?

Answer 31

It’s the removal of all proteins on the mRNA after going through the nuclear pore complex from the nucleus to the cytoplasm.
It’s done by RNA helicase as soon as the mRNA passes through the NPC.

Question 32

What proteins are removed of mRNAs after going through the NPC?

Answer 32

NXF1
NXT1
PABPN (protein that interacts with the poly A tail)

Question 33

Why do we do cytoplasmic remodelling?

Answer 33

Because all the proteins that were binding to mRNA are replaced by cytoplasmic factors.
The mRNA has to have a cytoplasmic coat to be ready to be translated.

Question 34

What does the protein eIF4E do in the cytoplasm?

Answer 34

It binds the 5’ end of the mRNA at the cap.

Question 35

What does the C protein do in the cytoplasm?

Answer 35

It’s a poly binding protein that binds to the poly A tail.

Question 36

What do we do with all of the binding proteins that were taken off the mRNA by RNA helicase as soon as they got into the cytoplasm?

Answer 36

They’re sent back into the nucleus to bind to another mRNA.

Question 37

Are mRNAs naked?

Answer 37

No, they’ re always associated with proteins.