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PHGY 351 > Stochaj > Flashcards

Stochaj Flashcards

1
Q

What does formation of a biomolecular condensate involve?

A

Liquid-liquid phase separation

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2
Q

What are the different activities of biomolecular condensates?

A
  1. Activation / inactivation
  2. Buffering
  3. Force generation (droplet bending)
  4. Filtration (< NPC) → regulate trafficking
  5. Localization
  6. Sensing (formation of smaller/bigger condensates)
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3
Q

What is the role of the nucleoli ?

A

Biomolecular condensate inside the nucleus → involved in assembly and maturation of ribosomes (transcription of ribosomal RNA)

Phase separation required for efficiency

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4
Q

What possibilities for formation of biomolecular condensates?

A

They can be inducible or constitutive

Ex: inducible stress granules, constitutive → complex of protein+mRNA for transport out of the nucleus

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5
Q

What is the size of the nucleus?
What is the structure of the nuclear envelope?

A

Size of the nucleus ~ size of RBC ~ 5-10 um in diameter

  1. Nuclear lamina → structural support, connected to chromatin
  2. Inner nuclear membrane
  3. Perinuclear space → contiguous with the ER
  4. Outer nuclear membrane → similar composition as ER membrane/contiguous

*NPC found at the junction of inner and outer nuclear membrane → phase separation controls transport

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6
Q

Name 3 different condensates in the cell.

A
  1. Nuclear pore → transport
  2. Stress granule → storage, transcription regulation
  3. Transport granule → intracellular transport
  4. Centrosome → cell division
  5. Signalling cluster → cell-cell signaling
  6. Germ granule → Germ cell specification, storage of RNA and proteins
  7. Heterochromatin → gene regulation
  8. Nucleolus → Ribosomal synthesis
  9. Transcription foci → gene regulation
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7
Q

What are the components of the nuclear interior?

A
  1. Nucleolus → formed by phase separation
  2. Chromatin → phase separation regulates transcription
  3. Nuclear bodies → formed by phase separation
  4. Nuclear matrix
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8
Q

Where is lamina in the nucleus? What is it connected to?

A

Lamina is just under the inner nuclear envelop

Connected to proteins and chromatin → regulates communication to control cytoskeleton structure

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9
Q

How is transport from the nucleus and the cytosol different than transport from the cytosol to other compartments?

A

It goes both ways: cytosol ↔ nucleus

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10
Q

What is the structure/characteristics of the nuclear pore complex?

A

NPC ~ 125 MDA (huge) → 30 different proteins (at least 8 copies of each)
*8 identical subunits

Cytosol
- Cytosolic filaments (cytosolic fibrils)
- Central gated channel (ring subunit + annular subunit + column subunit + ring subunit)
- Nuclear basket (nuclear fibrils)
Nucleus

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11
Q

What are FXF and GLFG repeats?

A

Both found in FG repeat domains of Nucleoporins:
FXF repeats → ex: Nup358, Nup62, Nup153 → mediate phase separation, hydrophobic barrier by F

GLFG → Nup98
GLFG stands for Glycine-Leucine-Phenylalanine-Glycine → role in export from the nucleus to the cytoplasm

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12
Q

What are the 2 types of movement across the NPC?

A

Passive diffusion:
- smaller than the diffusion channel of NPC ~ 8 nm / < 40 kDa
- Proteins larger than 70 kDa are excluded form the nucleus if they have no import signal
- no energy required
- no transport apparatus
- movement depending on concentration gradient

Active transport:
- Particles > 70 kDa
- Particles up to ~ 45 nm can be translocated if they have proper signal
- Requires energy → RanGTP
- Specialized transport apparatus required (soluble transport factors and nucleoporins)
- 2-way movement = shuttling (ex: RNA binding proteins to help mRNA come out for translation)

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13
Q

How does liquid-liquid phase separation occur at the NPC?

A

FG-Nucleoporins form mesh like hydrogel blocker inside the channel → large molecules are blocked unless they have proper signal

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14
Q

What is required in an NLS?

A
  • Permanent signal, not cleaved after transport
  • Can be located anywhere within the polypeptide chaine
  • mediate post-translational import/modifications (import after complete folding)
  • Stretch of positively charged amino acids

Simple/monopartite:
KKKRK → can’t be reversed or changed

Bipartite:
avKRpaatkkagaKKK → not reversed, its another signal when bipartite

*1st discovered in SV40

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15
Q

What are the steps for import of a protein from the cytosol to the nucleus through the NPC?

A
  1. NLS receptors (importin alpha) binds to NLS on cargo protein
  2. importin beta binds to NPC (no energy, nor heat needed)
  3. Translocation of the whole complex across the NPC → importin beta interacts with FXF repeats (requires energy, does not occur at low temperatures)
  4. Dissociation of the complex → recycling of importins to the cytoplasm (requires energy)
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16
Q

How does the RanGTPase cycle occur?

A

In nucleus → RCC1 bound to chromatin = Ran GEF → RanGTP&raquo_space; RanGDP

In cytoplasm → Cytosolic filament-RanBP1/2-RanGAP → RanGDP&raquo_space; RanGTP

*RanGTP binds to the beta subunit of the importin complex → dissociation of the complex → recycling to the cytoplasm (bound to RanGTP)

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17
Q

What are the nuclear export signals (NES)?

A

NES are recognized by Crm1 (exportin protein involved in export of ribosomal subunits with GTP)
Ex: PKI (PKA inhibitor) and HIV protein Rev

Leucine-rich hydrophobic sequences

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18
Q

What proteins are involved in nuclear export ?

A

Nucleus → Exportin-cargo complex is only stable when bound to RanGTP (on exportin)

RanGTP-Exportin-Cargo → through NPC

Cytoplasm → Ran hydrolized to GDP → dissociation of the complex → exportin by itself is recycled to the nucleus (concentration gradient)

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19
Q

How does mRNA export occur?

A

Through the NPC, but NOT Ran-dependent, NO exportins

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20
Q

Are importins alpha always required to bind cargo?

A

No, they are adaptors, but some importins beta can direclty recognize cargos

*Different importin-betas fro different cargos → all RanGTP-dependent

*Transport signals are recognized by carriers, not by the NPC

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21
Q

How is nuclear transport sensitive to stress?

A

Some transport protein end up stuck in stress granules → reduction of transport

Importin-beta family → involved in stress granules, protein disaggregation, chaperone function
Importin-alpha → involved in stress granules

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22
Q

How does aging affect nuclear transport?
How about diseased neuron?

A

Aging cells become more leaky:
- Reduction in nucleocytoplasmic compartmentalization
- Deterioration of nuclear pore complex
- Reduced RanPB17

Compared to a diseased neuron:
- partial dislocation of NPC
- increase mRNA retention in the nucleus
- Some nuclear transporters make aggregates

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23
Q

Where do RNA granules form?

A

In the nucleus AND in the cytoplasm
*In constant interaction with other condensates → difficult to study isolated

Different cytoplasmic RNA granules form in different conditions (normal/stress):
- P-bodies, U-bodies increase size in stress
- IMP1 granules contain insulin-like GF2 mRNA-binding proteins → becomes composite of IMP1 + SG under stress

24
Q

What induces droplet formation from larger RNA granules ? REVIEW

A
  • Temperature, concentration, pH, salinity, osmolarity, binding partners, PTMs
  • The 2-phase/phase-separation allows conformational flexibility, accessibility to PTMs, sequence simplicity, mutlvalency, modularity

Intrincally disordered regions come together into droplets

25
Q

What are different stages of stress granules?
What induces changes between different stages?

A
  1. Monomers
  2. Liquid-like drops (reversible)
  3. Solid-like condensates (reversible)
  4. Solid-like fibrils (not reversible)

Transitions between phases due to:
- Changing physical and chemical conditions
- PTMs
- Ligands

Chaperones and Helicases can lead to reverse back / reduction of stress

*When stress decreases, phase disipates → 1 homogenous phase, when increases → stress granule formation

26
Q

What happens to the polysome (mRNA + ribosomes) when stress is induced?

A
  1. Ribosomes are detached from the mRNA (stop translation initiation)
  2. Specific proteins bind mRNA (G3BP1/2) → sequestered
  3. Liquid condensate
  4. Caprin1 + RBP enter the liquid condensate → mature stress Granule
    OR
  5. If stress persists → Misfolded proteins enter the condensate → solid-like condensate

*protects mRNA, sequesters pre-apoptotic factors
Stress granules = RNA + proteins (in the cytoplasm)

27
Q

What are the dimension of Stress Granules?

A

3-5 um in diameter → depends on stress

(or 0.1 - 2um)

28
Q

What different proteins are found in stress granules?

A

40S subunit
HuR
eIF4G
eIF4A
Importin-a (sometimes) → and othe nuclear transport factors → disrupt nucleoplasmic transport

29
Q

What is the role of HuR?
What gene is it encoded by?

A

RNA-binding protein encoded by the gene ELAVL1:
- ELAVL1 is ubiquitously expressed
- Loss of HuR is embryonic lethal in mice

HuR shuttles between nucleus and cytoplasm:
In nucleus → affects splicing
In cytoplasm → controls stability of numerous transcripts (cell cycle regulators, muscle dev. erythropoiesis, immune cells differentiation)

Cytoplasmic localization of HuR → proinflammatory response, can accumulate in cytoplasmic stress granules

30
Q

What are the characteristics of the structure of HuR?

A

~ 35 kDa → 326 AA
- 3x conserved RNA recognition motifs (RRM)
- hinge region → nucleocytoplasmic shuttling signal
- RRM1, RRM2 recognize AU rich elements (ARE) of target mRNA → stability
- RRM3 binds poly(A)-tails
- RRM3 facilitates HuR multimerization on target mRNA

31
Q

What is the effect of cell senescence on SG formation?

A

Cell senescence impairs SG formation → changing RNA binding sites, less proteins made, etc. → smaller, more numerous and divided granules (Aging cells)

32
Q

Give 3 examples of how Stress Granules are involved in human diseases.

A
  1. Cancer cells protect themselves by making stress granules when exposed to chemotherapy
  2. Persistent granules → solid-like fibrils → neurodegeneration
  3. Impaired stress granule formation → enhances infectivity
33
Q

What determines stress granules composition?

A
  • Cell-type
  • Type of stress
  • Age of the cell
    *SG form from pre-existing protein interactions

Stress granules vary in composition, in size

34
Q

How can Optodroplets be formed?

A

Make protein with Cry2 + sticky disordered regions (FUS)
Cry2 = light activatible domain

*Light inducible phase separation
When increased concentration formation of more droplets, when activated by light (optogenics) → more permanent stress granule formation because of dimerization of Cry2

Use fluorescent reporter to visualize
Granules are dynamic when less stress, more solid when more stress

35
Q

What happens to stress granules when stress goes away?

A

If they didn’t form into solid-like fibrils → they dissipate and becomes as before

If chronic stress, after ~5x → SG don’t dissipate as well (more permanent)
*Stress → change in protein conformation → different interactions

36
Q

How can we find out if diffusion of a small protein is done passively or actively through the NPC? (ex: we are interested in His1)

A

By Ran depleting the cell from GTP → Ran activity can’t occur → can’t have active transport

Than make a reporter gene with suspected His1NLS-GFP (small so can diffuse)

37
Q

When is it a good idea to use a GFP fusion protein with GFP+protein of interest to investigate protein transport through the NPC?

A

When we want to increase its size to eliminate the case of possible diffusion
GFP ~ 25 kDa

(increased size → less probability of free diffusion)

Then staing with DAPI to see co-localization or not with the nucleus

38
Q

What does it indicate if the protein is small enough to diffuse from the cytoplasm to the nucleus, but doesn’t?

A

There might be a CRS
*There might also be a cytoplasmic localization signal which helps the proteins to be targetted to the cytoplasm, but then we would still maybe see some in the nucleus

With a retention signal, when the proteins are in the target compartment they don’t move, but if they don’t start in the adequate compartment and can’t move, they won’t necessarily be transported to the target compartment

39
Q

What is the effect of cycloheximide?

A

It blocks protein synthesis

40
Q

What is a heterokaryon?

A

It is 1 cell that is the result of the fusion of 2+ different cell types from different genetic backgrounds together → has 2+ different nucleus

41
Q

What is Crm1?

A

An exportin protein
- It promotes Nup solubility

42
Q

What are the functions of the nuclear basket?

What is the importance of the nuclear basket in DNA damage?

A
  • Chromatin remodelling
  • mRNP binding site
43
Q

What is the importance of laminins and of the nuclear lamina ?

A
  • Chromatin organization
  • Gene regulation
  • Nuclear shape and size
  • Organization and anchoring of the NPC
44
Q

How do we achieve directionality in nuclear trafficking?

A

Through the RanGTPase cycle

45
Q

Why would some NPCs don’t have baskets in yeasts?

A

NPCs next to the nucleolus → makes shift from nucleolus to cytoplasm easier

46
Q

What RNAs? are encoded by RNA pol I, II and III?

A

RNA pol I → rRNA, except 5S rRNA, in the nucleolus
RNA pol II → mRNA (hnRNA), snRNA, miRNA
RNA pol III → 5S rRNA, tRNA, in the normal nucleus

47
Q

In what directions are different RNAs transported?

A

mRNA: N → C (TAP, Ran-independent)
tRNA: N → C (Exportin-t, requires Ran)
rRNA: N →C (as part of ribosomal subunit, Crm1, requires Ran)
snRNA: N → C, after binding to proteins and modification in the cytoplasm, C → N, requires Ran for both

48
Q

What are Karyopherins?

A

Subfamily of importin-beta importins → mediate nuclear RNA export

49
Q

What 3 components are present on RNA shuttling proteins?

A

NES, NLS, RNA-binding domain
*NES, NLS are 2 separate sequences

50
Q

How does nuclear export of mRNA occur?

A

hnRNP C1 bound to mRNA stay in the nucleus → detached before transport
hnRNP A1 bound to mRNA during transport, shuttling proteins → exchanged for mRNPs in the cytoplasm

hnRNP = heterogenous nuclear RNP
mRNP = mature RNP
*hnRNP has NES

51
Q

What are the reuqirements for mRNA nuclear export?

A

Signals:
- 5’ cap → leads mRNA out
- polyA-tail
- absence of introns → splicing factors have NRS to prevent export of non-spliced RNA

Energy (RNA helicase) → on cytoplasmic filaments for remodelling (Dbp5p, DDX3)

Transport factors (soluble and nucleoporins)
- TAPs (bind to mRNA) → interact with nucleoporins
- RNA helicase
*No need for Ran

52
Q

Which RNA modifications specifically help mRNA to be exported to the nucleus?

A

methylation of adenosin on m6 (m6A) → export, degradation, localization, etc.

53
Q

What could be an effect of mutations in mRNA export mediators?

A

Malformation of limbs in the fetus

54
Q

How does nuclear export of unspliced or partially spliced HIV-1 mRNA occur?
(Should not bc of presence on introns)

A

HIV-1 mRNA has a Rev protein binding sequence

Rev protein → very strong NES (overpowers the splicing factor NRS) → bound by Crm1

*Could inhibit Crm1? but all cells would dye because also involved in important processes

55
Q

What proteins are downregulated in cell senescence/aging?

A
  • G3BP1
  • HuR
  • hnRNP K
    → impairs stress granule formation

PHGY 351 (3 decks)

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