Lecture 16 : NPC and RNA export Flashcards
2 important structures of NPC on the membrane level
Membrane nucleoporins and Structural nucleoporins (Y-complex)
Structure (1) on cytoplasmic face and structure (1) on nucleoplasmic face of NPC
Cyto : Cytoplasmic filaments
Nucleo : Nuclear basket
Structure in the middle of the NPC, what proteins form it
Mesh formed by FG-nucleoporins. Proteins in the middle of the hole.
How FG-nucleoporins form the mesh in the hole in the middle of the NPC (what characteristics do they have)
Many hydrophilic regions separated by very hydrophobic ones (rich in phenylalanine - F and glycine - G). Hydrophobic regions stick together
2 ways of crossing the NPC and main factor that determines which way will be used
1) Free diffusion (up to 40kDa)
2) Interaction with the FG-nucleoporins
4 important mechanisms of import/export discussed
1) NLS proteins import
2) Export through exportin 1
3) Export through exportin t
4) Export through mRNA exporter
NLS proteins import : Why need protein import
All proteins synthesized in cytoplasm, even nuclear ones so have to go back (histones, DNA repair and replication enzymes, etc.)
NLS proteins import : What is found on proteins that are imported in the nucleus and what does it serve for
NLS : Nuclear localizing signal. Helps these proteins to be recognized by another proteins that will import them
NLS proteins import : Protein 3D structure as they are imported in the nucleoplasm
Folded state
NLS proteins import : Which protein was used to prove that NLS is enough to drive import
Pyruvate kinase
NLS proteins import : Where pyruvate kinase NORMALLY found in virus where it was taken from and in cell where experiment was done
In the virus = in the nucleoplasm (has a NLS)
In the wild-type cell of experiment = in cytoplasm
NLS proteins import : What was done to show that NLS enough to drive import
NLS sequence identified from virus and introduced in pyruvate kinase gene in wild-type cell : Expressed pyruvate kinase all went to the nucleoplasm
NLS proteins import : Can we add same NLS sequence to gene of any protein to make it go in the nucleoplasm ? Why
No cause NLS sequences are specific to certain proteins
Key protein for : 1) NLS proteins import
2) Export through exportin 1
3) Export through exportin t
Ran
Ran description + its 2 states + what drives its function
GTPase (can hydrolyze GTP). When GTP-> GDP +Pi, becomes inactive (one conformation). When GDP+Pi -> GTP becomes active (other conformation)
How ran switches from binding GTP to binding GDP and how the opposite happens
GTP to GDP : Hydrolizes GTP
GDP to GTP : Exchanges a GDP for a GTP
What can accelerate a GTPase (Ran included) switch from GTP to GDP and from GTP to GDP
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
NLS proteins import : Name of best understood protein necessary for proteins import
Importin alpha and beta
NLS proteins import : What is importin alpha and beta, how it recognizes proteins to be imported in the nucleoplasm
A heterodimeric nuclear-import receptor. Alpha subunit binds NLS region. Beta subunit binds FG-nucleoporins.
NLS proteins import : General working scheme of proteins importing other proteins in the nucleus
Whether they are monomers or heterodimers, bind NLS with one region and bind FG-nucleoporins with another region
NLS proteins import : Where are importin alpha/beta (or other such proteins) present in a higher concentration and why
In the cytoplasm, so that the concentration gradient works in favor of the import
NLS proteins import : What happens in cytoplasm and through NPC when going to nucleoplasm - first step
Importin binds to cargo protein (with alpha subunit) and goes through NPC (with beta subunit)
NLS proteins import : What happens in nucleoplasm - step 2
Ran GDP -> Ran GTP (w/ GEF help) and Ran-GTP binds to importin. Cargo is released in nucleoplasm
NLS proteins import : What happens to Ran-GTP/Importin complex - step 3
Go back to cytoplasm through NPC
NLS proteins import : What happens to Ran-GTP/Importin complex in the NPC, on its way to the cytoplasm - step 4
a Ran-GAP associated with the cytoplasmic filaments inactivates Ran-GTP (becomes Ran-GDP) and importin is released
Export through exportin 1 : What exportin 1 does that is similar to importin alpha/beta
Interacts with the cargo and with FG-nucleoporins in the NPC
Export through exportin 1 : What happens in the nucleoplasm - first step
Exportin 1 interacts with a Ran-GTP and the cargo protein (trimolecular complex) - Ran-GTP now part of the complex
Export through exportin 1 : What happens through trimolecular complex as it crosses the NPC to the cytoplasm - step 2
a Ran-GAP associated with the cytoplasmic filaments inactivates Ran-GTP (becomes Ran-GDP) and exportin is released. Exportin 1 then releases cargo protein
Export through exportin 1 : What happens to separated exportin 1 and Ran-GDP after cargo is delivered and WHAT THIS INDICATES** - step 3
Must go back to the nucleoplasm so they must have a NLS
Export through exportin t : What it serves for
Export tRNAs to the cytoplasm
Export through exportin t : What happens in the nucleoplasm - first step
LIKE W/ EXPORTIN 1 : Exportin t binds mature tRNA and GTPase
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
LIKE W/ EXPORTIN 1 : Ran-GAP on cytoplasmic filaments inactivates Ran-GTP. Ran-GDP released. Exportin t releases tRNA
2 other molecules/complexes of our interest that need to go through NPC using Ran
Ribosomal subunits and some specific mRNAs
Export through mRNA exporter : Name of complex being exported
messenger ribonucleoprotein complex (mRNP complex)
Export through mRNA exporter : What can show to the cell that an mRNA is mature (3)
5’ cap, 3’ Poly(A) tail and NOT associated with splicing factors
Export through mRNA exporter : What other proteins bind mRNA in the nucleoplasm and when
When spliceosome leaves, SR proteins bind mRNA and recruit NXF1 and NXT1 (2 subunits)
Export through mRNA exporter : Difference of this transport through NPC (compared to other ones seen)
mRNP export through NPC is Ran independent
Export through mRNA exporter : How can an immature mRNA be recognized and excluded from export
Splicing factors still hanging on to it
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
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
Export through mRNA exporter : What happens to NXF1 and NXT1 once separated from mRNP in the cytoplasm and how
Put back in the nucleoplasm using Ran-dependent import
Export through mRNA exporter : Shape of the mRNP (mRNA w/ proteins) as it is being transcribed and how this was visualized
Larva-like structure. Proteins of mRNP were dark-stained and seen on EM micrographs
Export through mRNA exporter : In what direction does the mRNP get through the NPC. Why + consequence
Always 5’ because of cap that is leading. Therefore, can start being translated as it is still crossing the NPC
What proteins (2) form the mRNA exporter and what do they do that is similar to all other import/export proteins that take cargos
NFX1/NXT1 dimers binding to mRNA and mRNP proteins. Form a domain that interacts with FG repeats
Export through mRNA exporter : Other proteins that assist in mRNA export (4)
1) SR proteins
2) Exon-junction complex proteins (REF)
3) Nuclear cap-binding complex (CAP)
4) Poly(A) binding proteins
Export through mRNA exporter : What SR proteins do
Stimulate binding of mRNA exporter (recruit NXF1/NXT1)
Export through mRNA exporter : What Exon-junction complex proteins (REF) do do
Binds mRNA exporter and binds upstream of exon junctions
Export through mRNA exporter : What Nuclear cap-binding complex (CAP) does
Leads 5’ end to go out first + Targets mRNP to the NPC
Export through mRNA exporter : What Poly(A) binding proteins do + their name
Required for export. (PABPN1)
Changes that occur during mRNP remodelling
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
Export through mRNA exporter : T/F -> All proteins leave the mRNP in the cytoplasm
False : Some leave the mRNP in the nucleoplasm, some in the cytoplasm
Export through mRNA exporter : Important function of all proteins binding the mRNP with respect to the splicing process
Delineate the progress of the splicing process. Will bind as splicing is being completed to move RNA to the cytoplasm
Export through mRNA exporter : How immature/not fully spliced mRNAs/pre-mRNAs are (thought to be) excluded from the export system
A nucleoporin associated protein blocks pre-mRNAs associated w/ snRNPs from leaving the nucleus
