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Biol 200 > Lecture 16 : NPC and RNA export > Flashcards

Lecture 16 : NPC and RNA export Flashcards

1
Q

2 important structures of NPC on the membrane level

A

Membrane nucleoporins and Structural nucleoporins (Y-complex)

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2
Q

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

A

Cyto : Cytoplasmic filaments

Nucleo : Nuclear basket

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3
Q

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

A

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

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4
Q

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

A

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

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5
Q

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

A

1) Free diffusion (up to 40kDa)

2) Interaction with the FG-nucleoporins

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6
Q

4 important mechanisms of import/export discussed

A

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

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7
Q

NLS proteins import : Why need protein import

A

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

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8
Q

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

A

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

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9
Q

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

A

Folded state

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10
Q

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

A

Pyruvate kinase

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11
Q

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

A

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

In the wild-type cell of experiment = in cytoplasm

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12
Q

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

A

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

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13
Q

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

A

No cause NLS sequences are specific to certain proteins

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14
Q

Key protein for : 1) NLS proteins import

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

A

Ran

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15
Q

Ran description + its 2 states + what drives its function

A

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

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16
Q

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

A

GTP to GDP : Hydrolizes GTP

GDP to GTP : Exchanges a GDP for a GTP

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17
Q

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

A

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

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18
Q

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

A

Importin alpha and beta

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19
Q

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

A

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

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20
Q

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

A

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

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21
Q

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

A

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

22
Q

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

A

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

23
Q

NLS proteins import : What happens in nucleoplasm - step 2

A

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

24
Q

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

A

Go back to cytoplasm through NPC

25
Q

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

A

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

26
Q

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

A

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

27
Q

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

A

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

28
Q

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

A

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

29
Q

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

A

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

30
Q

Export through exportin t : What it serves for

A

Export tRNAs to the cytoplasm

31
Q

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

A

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

32
Q

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

A

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

33
Q

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

A

Ribosomal subunits and some specific mRNAs

34
Q

Export through mRNA exporter : Name of complex being exported

A

messenger ribonucleoprotein complex (mRNP complex)

35
Q

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

A

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

36
Q

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

A

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

37
Q

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

A

mRNP export through NPC is Ran independent

38
Q

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

A

Splicing factors still hanging on to it

39
Q

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

A

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

40
Q

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

A

Put back in the nucleoplasm using Ran-dependent import

41
Q

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

A

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

42
Q

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

A

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

43
Q

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

A

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

44
Q

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

A

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

45
Q

Export through mRNA exporter : What SR proteins do

A

Stimulate binding of mRNA exporter (recruit NXF1/NXT1)

46
Q

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

A

Binds mRNA exporter and binds upstream of exon junctions

47
Q

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

A

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

48
Q

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

A

Required for export. (PABPN1)

49
Q

Changes that occur during mRNP remodelling

A

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

50
Q

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

A

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

51
Q

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

A

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

52
Q

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

A

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

Biol 200 (30 decks)

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