RR10: Nucleo Cytoplasmic Transport

Question 1

where do the mRNAs need to go after theyve been transcribed and processed?

Answer 1

need to get out of the nucleus and into the cytoplasm where they will be met by chromosomes to be translated

Question 1

what surrounds the nucleus?

Answer 1

it has a double enveloppe dotted with nuclear pores

Question 2

what is the structure of the nuclear pore?

Answer 2

held up by structures called nuclear pore complexes (NPCs)
* On the nucleoplasmic side you have the nuclear basket, a channel that goes through and filaments that are hanging off
* It is a giant structure (125 megadaltons) but it ensures and regulates traffic through the pore complex from cytoplasm to nucleus and vice versa

Question 3

what type of molecules can freely move through the NPC?

Answer 3

small molecules and proteins (40-60 kilodaltons) can move in and out freely but larger complexes require help

Question 4

what makes up the pore per se?

Answer 4

• There is a class of proteins called nucleoporins, and the ones that are rich in phenylalanine and glycine are referred to as FG nucleoporins, which are hydrophobic and poses disordered domains
• those FG nucleoporins are the ones that make up the pore in the middle where the traffic is going to move and they regulate the movement through the pore
• the FG repeats present on these proteins interact with one another and have a very disordered state and form a gel-like interphase (not liquid nor solid)

Question 5

what do proteins need to move freely through the pore?

Answer 5

need domains that can interact with the FG repeats of the pore

Question 6

what is the nuclear localisation signal? (NLS)

Answer 6

a specific sequence on a nuclear protein synthesised in the cytoplasm that will confer movement through the nuclear pore
stretch of amino acids that is present on specific factors that have to make their way through the pore

Question 7

where are nuclear proteins synthesised?

Answer 7

in the cytoplasm, and they are then imported through NPCs

Question 8

how was this NLS investigated in the SV40 virus?

Answer 8

• The virus uses the t-antigen which causes havoc in a lot of nuclear processes
• There are mutations in T-antigen that somehow didn’t allow it to get into the nucleus which affects its ability to host grief in host cells
• Were able to identify stretch of amino acids, generally rich in lysine and arginine that if you took it out of its context in the T-antigen and put it into a non nuclear protein, the protein would go into the nucleus
• when you put the NLS onto a pyruvate kinase, all of it makes it into the nucleus —> just having the NLS is enough to confer nuclear localisation

Question 9

what are the critical factors required to interact with the NLS and get proteins into the nucleus?

Answer 9

The proteins are a G protein called RAN and a family of nuclear transport receptors, also called importins (one of the families of these receptors)

Question 10

what is the mechanism for importing cargo proteins into the nucleus

Answer 10

• how does importin work? With a cycle
• The cargo has an NLS and that NLS is recognised by the importin which interacts with it specifically
• It forms a complex with the cargo and by virtue of its ability to interact with those FG repeats on the FG nuclear porins it will make its way through the nuclear pore till it arrive in the nucleus, where it is greeted by Ran
• G proteins interact with GTP and GDP and they have an intrinsic GTP-ase activity that can greatly be enhanced by other factors
• Ran greets the cargo importin complex in the nucleoplasm especially when its in its GTP bound form (Ran-GTP)
• It will interact with the importin and change its conformation upon its binding
• The importin lets go of its cargo and the cargo can diffuse through the nucleoplasm and carry out its function
• Ran-GTP bound to the importin will make its way out through the nuclear pore, driven by a concentration gradient (from high to low)
  The Ran-GTP importin complex on the cytoplasmic side will be met by a Ran-GAP protein, a GAP is what enhances the GTP-ase function, so by interaction with Ran-GAP, GTP within Ran is rapidly hydrolysed to GDP
• this leads to a conformational change that releases the importin so it can interact with another cargo
• Ran-GDP makes its way back into the nucleus by virtue of a concentration gradient (with importin or something else)

Question 11

what drives the movement across the nucleus?

Answer 11

driven by concentration gradients

Question 12

what maintains those concentration gradients?

Answer 12

the cytoplasmic side, the hydrolysis of the GTP component of Ran GTP and generation of RanGTP in the nucleus by its associated GEF (guanine nucleotide exchange factor)
* GEF is always present, often bound to the chromosomes or the chromatin in the nucleus
* Ran GAP on the cytoplasmic side and Ran GEF on nucleoplasmic side that ensures you have ran in the appropriate GTP or GDP bound form on the correct side of the nuclear envelope

Question 13

how does the exporting mechanism work?

Answer 13

• same thing is true for exploring nuclear factors from the nucleus into the cytoplasm
• Instead of using an importin you use an exportin
• The exportins recognise their targets within the nucleus
• accompanied by the recruitment of RanGTP forming a ternary complex (cargo+ exportin + RanGTP)
• This complex makes its way through the nuclear pore on the cytoplasmic side
• the RanGAP will interact with RanGTP and hydrolyse to GDP, releasing the cargo free in the cytoplasm and the exporter makes its way back to the nucleus through the nuclear pore
• Drive by concentration gradients and driven by RanGEF and RanGAP

Question 14

what does exportin t do?

Answer 14

plays a role in exporting tRNAs from nucleus to cytoplasm so they can participate in protein synthesis

Question 15

what do ribosomal units require?

Answer 15

also require Ran

Question 16

what do most mRNAs have however?

Answer 16

most have a Ran independent process and have their own mRNA exporter

Question 17

in what way does mRNA go through the NPC in an Ran independent manner?

Answer 17

• MRNA interacts with two subunits of the exporter called NXF1 and NXT1
• Interact with proteins that have decorated the mRNA as its been processed, including SR proteins and then bring them to NPC so that they can make their way through that pore and make its way out on the cytoplasm side

Question 18

how does mRNP export work?

Answer 18

• we can visualise this in micrographs that are formed or prepared on specific genes that are required to generate the mRNA that will encode a protein that’s involved in the glue of a midge, present in the salivary glands
• The insects have to prepare a lot of this protein, so they transcribed this one gene a lot so they can stick themselves or the eggs onto the leaves
• The micrograph shows the transcription and incorporation of those mRNAs into packages that then have to make their way to the nucleus pores and then through
• All the bundles are mRNAs that are being assembles with heterogeneous nuclear ribonuclear proteins or HNRNPs (proteins that interact with the mRNA)
• These HNRNPs are a spectrum of RNA binding proteins that bind to the mRNA to protect it and help it to assemble into little croissants
• The messenger RNA ribonuclear proteins: mRNPs
• MRNP that has been bundled up and assembled in all the appropriate factors involved in maturation now makes its way to the NPC
• There there are some Gatekeepers that ensure that only matured mRNAs go through (we dont know how that works)
• the mRNA gets threaded through by the mRNA exporter
• The moment the mRNA gets through to the cytoplasm its met with ribosomes, which indicates that it goes through in a very polarized manner: 5’ end needs to go through first so it can interact with ribosomes

Question 19

what happens to the proteins associated with mRNA on the cytoplasmic side?

Answer 19

they are removed
that is called cytoplasmic remodelling

Question 20

what removes those proteins?

Answer 20

RNA helicase which is associated with those cytoplasmic filaments

Question 21

what are examples of proteins that are removed as the protein comes out on the cytoplasmic side?

Answer 21

the nuclear cap binding complex that recognises the A 5’ end will be taken off
PABPN1 (protein that interacts with poly A tail) is also removed
the exporter is also removed

Question 22

what are examples of some of those cytoplasmic factors that bind to the same structures as nuclear factors?

Answer 22

• there is a cap binding protein EIF4E that binds to the 5’ end at the cap
• New poly binding protein called C for cytoplasmic that bind to the poly A tail and then a lot of other proteins that will interact with the mRNA on the cytoplasmic site

Question 23

where do nuclear proteins go?

Answer 23

sent back again to the nucleus where they belong, and the mRNA is now in a cytoplasmic coat ready to be translated