Lecture 16 : NPC and RNA export

Question 1

2 important structures of NPC on the membrane level

Answer 1

Membrane nucleoporins and Structural nucleoporins (Y-complex)

Question 2

Structure (1) on cytoplasmic face and structure (1) on nucleoplasmic face of NPC

Answer 2

Cyto : Cytoplasmic filaments

Nucleo : Nuclear basket

Question 3

Structure in the middle of the NPC, what proteins form it

Answer 3

Mesh formed by FG-nucleoporins. Proteins in the middle of the hole.

Question 4

How FG-nucleoporins form the mesh in the hole in the middle of the NPC (what characteristics do they have)

Answer 4

Many hydrophilic regions separated by very hydrophobic ones (rich in phenylalanine - F and glycine - G). Hydrophobic regions stick together

Question 5

2 ways of crossing the NPC and main factor that determines which way will be used

Answer 5

1) Free diffusion (up to 40kDa)

2) Interaction with the FG-nucleoporins

Question 6

4 important mechanisms of import/export discussed

Answer 6

1) NLS proteins import
2) Export through exportin 1
3) Export through exportin t
4) Export through mRNA exporter

Question 7

NLS proteins import : Why need protein import

Answer 7

All proteins synthesized in cytoplasm, even nuclear ones so have to go back (histones, DNA repair and replication enzymes, etc.)

Question 8

NLS proteins import : What is found on proteins that are imported in the nucleus and what does it serve for

Answer 8

NLS : Nuclear localizing signal. Helps these proteins to be recognized by another proteins that will import them

Question 9

NLS proteins import : Protein 3D structure as they are imported in the nucleoplasm

Answer 9

Folded state

Question 10

NLS proteins import : Which protein was used to prove that NLS is enough to drive import

Answer 10

Pyruvate kinase

Question 11

NLS proteins import : Where pyruvate kinase NORMALLY found in virus where it was taken from and in cell where experiment was done

Answer 11

In the virus = in the nucleoplasm (has a NLS)

In the wild-type cell of experiment = in cytoplasm

Question 12

NLS proteins import : What was done to show that NLS enough to drive import

Answer 12

NLS sequence identified from virus and introduced in pyruvate kinase gene in wild-type cell : Expressed pyruvate kinase all went to the nucleoplasm

Question 13

NLS proteins import : Can we add same NLS sequence to gene of any protein to make it go in the nucleoplasm ? Why

Answer 13

No cause NLS sequences are specific to certain proteins

Question 14

Key protein for : 1) NLS proteins import

2) Export through exportin 1
3) Export through exportin t

Answer 14

Ran

Question 15

Ran description + its 2 states + what drives its function

Answer 15

GTPase (can hydrolyze GTP). When GTP-> GDP +Pi, becomes inactive (one conformation). When GDP+Pi -> GTP becomes active (other conformation)

Question 16

How ran switches from binding GTP to binding GDP and how the opposite happens

Answer 16

GTP to GDP : Hydrolizes GTP

GDP to GTP : Exchanges a GDP for a GTP

Question 17

What can accelerate a GTPase (Ran included) switch from GTP to GDP and from GTP to GDP

Answer 17

GTP to GDP : GAP (GTPase activating protein) - accelerates the process to make it INACTIVE
GDP to GTP : GEF (Guanidine nucleotide Exchange Factor) - accelerates the process to make it ACTIVE

Question 18

NLS proteins import : Name of best understood protein necessary for proteins import

Answer 18

Importin alpha and beta

Question 19

NLS proteins import : What is importin alpha and beta, how it recognizes proteins to be imported in the nucleoplasm

Answer 19

A heterodimeric nuclear-import receptor. Alpha subunit binds NLS region. Beta subunit binds FG-nucleoporins.

Question 20

NLS proteins import : General working scheme of proteins importing other proteins in the nucleus

Answer 20

Whether they are monomers or heterodimers, bind NLS with one region and bind FG-nucleoporins with another region

Question 21

NLS proteins import : Where are importin alpha/beta (or other such proteins) present in a higher concentration and why

Answer 21

In the cytoplasm, so that the concentration gradient works in favor of the import

Question 22

NLS proteins import : What happens in cytoplasm and through NPC when going to nucleoplasm - first step

Answer 22

Importin binds to cargo protein (with alpha subunit) and goes through NPC (with beta subunit)

Question 23

NLS proteins import : What happens in nucleoplasm - step 2

Answer 23

Ran GDP -> Ran GTP (w/ GEF help) and Ran-GTP binds to importin. Cargo is released in nucleoplasm

Question 24

NLS proteins import : What happens to Ran-GTP/Importin complex - step 3

Answer 24

Go back to cytoplasm through NPC

Question 25

NLS proteins import : What happens to Ran-GTP/Importin complex in the NPC, on its way to the cytoplasm - step 4

Answer 25

a Ran-GAP associated with the cytoplasmic filaments inactivates Ran-GTP (becomes Ran-GDP) and importin is released

Question 26

Export through exportin 1 : What exportin 1 does that is similar to importin alpha/beta

Answer 26

Interacts with the cargo and with FG-nucleoporins in the NPC

Question 27

Export through exportin 1 : What happens in the nucleoplasm - first step

Answer 27

Exportin 1 interacts with a Ran-GTP and the cargo protein (trimolecular complex) - Ran-GTP now part of the complex

Question 28

Export through exportin 1 : What happens through trimolecular complex as it crosses the NPC to the cytoplasm - step 2

Answer 28

a Ran-GAP associated with the cytoplasmic filaments inactivates Ran-GTP (becomes Ran-GDP) and exportin is released. Exportin 1 then releases cargo protein

Question 29

Export through exportin 1 : What happens to separated exportin 1 and Ran-GDP after cargo is delivered and WHAT THIS INDICATES** - step 3

Answer 29

Must go back to the nucleoplasm **so they must have a NLS**

Question 30

Export through exportin t : What it serves for

Answer 30

Export tRNAs to the cytoplasm

Question 31

Export through exportin t : What happens in the nucleoplasm - first step

Answer 31

LIKE W/ EXPORTIN 1 : Exportin t binds mature tRNA and GTPase

Question 32

Export through exportin t : What happens on the way of the Ran-GTP/exportin t/tRNA complex on its way to the cytoplasm, through the NPC

Answer 32

LIKE W/ EXPORTIN 1 : Ran-GAP on cytoplasmic filaments inactivates Ran-GTP. Ran-GDP released. Exportin t releases tRNA

Question 33

2 other molecules/complexes of our interest that need to go through NPC using Ran

Answer 33

Ribosomal subunits and some specific mRNAs

Question 34

Export through mRNA exporter : Name of complex being exported

Answer 34

messenger ribonucleoprotein complex (mRNP complex)

Question 35

Export through mRNA exporter : What can show to the cell that an mRNA is mature (3)

Answer 35

5' cap, 3' Poly(A) tail and NOT associated with splicing factors

Question 36

Export through mRNA exporter : What other proteins bind mRNA in the nucleoplasm and when

Answer 36

When spliceosome leaves, SR proteins bind mRNA and recruit NXF1 and NXT1 (2 subunits)

Question 37

Export through mRNA exporter : Difference of this transport through NPC (compared to other ones seen)

Answer 37

mRNP export through NPC is Ran independent

Question 38

Export through mRNA exporter : How can an immature mRNA be recognized and excluded from export

Answer 38

Splicing factors still hanging on to it

Question 39

Export through mRNA exporter : What happens to mRNP (mRNA with with 5' cap, poly A tail, NXF1, NXT1) crossing to the cytoplasm through the NPC

Answer 39

Enzyme Dbp5 associated with cytoplasmic filaments is not a GAP but an RNA helicase. It uses ATP to remove NXF1/NXT1 and remodel the mRNP complex

Question 40

Export through mRNA exporter : What happens to NXF1 and NXT1 once separated from mRNP in the cytoplasm and how

Answer 40

Put back in the nucleoplasm using Ran-dependent import

Question 41

Export through mRNA exporter : Shape of the mRNP (mRNA w/ proteins) as it is being transcribed and how this was visualized

Answer 41

Larva-like structure. Proteins of mRNP were dark-stained and seen on EM micrographs

Question 42

Export through mRNA exporter : In what direction does the mRNP get through the NPC. Why + consequence

Answer 42

Always 5' because of cap that is leading. Therefore, can start being translated as it is still crossing the NPC

Question 43

What proteins (2) form the mRNA exporter and what do they do that is similar to all other import/export proteins that take cargos

Answer 43

NFX1/NXT1 dimers binding to mRNA and mRNP proteins. Form a domain that interacts with FG repeats

Question 44

Export through mRNA exporter : Other proteins that assist in mRNA export (4)

Answer 44

1) SR proteins 2) Exon-junction complex proteins (REF) 3) Nuclear cap-binding complex (CAP) 4) Poly(A) binding proteins

Question 45

Export through mRNA exporter : What SR proteins do

Answer 45

Stimulate binding of mRNA exporter (recruit NXF1/NXT1)

Question 46

Export through mRNA exporter : What Exon-junction complex proteins (REF) do do

Answer 46

Binds mRNA exporter and binds upstream of exon junctions

Question 47

Export through mRNA exporter : What Nuclear cap-binding complex (CAP) does

Answer 47

Leads 5' end to go out first + Targets mRNP to the NPC

Question 48

Export through mRNA exporter : What Poly(A) binding proteins do + their name

Answer 48

Required for export. (PABPN1)

Question 49

Changes that occur during mRNP remodelling

Answer 49

1) Cap-binding complex changed for eIF4E (a translation initiation factor) 2) PABPN1 changed to PABPC1 3) REFs, mRNA exporter (NFX1 and NXT1) and SR proteins leave

Question 50

Export through mRNA exporter : T/F -> All proteins leave the mRNP in the cytoplasm

Answer 50

False : Some leave the mRNP in the nucleoplasm, some in the cytoplasm

Question 51

Export through mRNA exporter : Important function of all proteins binding the mRNP with respect to the splicing process

Answer 51

Delineate the progress of the splicing process. Will bind as splicing is being completed to move RNA to the cytoplasm

Question 52

Export through mRNA exporter : How immature/not fully spliced mRNAs/pre-mRNAs are (thought to be) excluded from the export system

Answer 52

A nucleoporin associated protein blocks pre-mRNAs associated w/ snRNPs from leaving the nucleus