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Nuclear Domains Flashcards

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1
Q

Nuclear envelope basic

A

double membrane
outer membrane joined w ER

nuclear lamina supporting inside, located just beneath envelope

numerous perforations - nuclear pore complexes
made from curving of membrane
act as gatekeepers

allows nuclear proteins to be concentrated inside

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2
Q

nuclear traffic basic

A

import:
proteins required for nuclear structures
-histones
-DNA/RNA pol
-TFs

Export:
transcription and splicing occur outside of nucleus at external ribosomes
gives extra level of transcriptional control
-mRNA
-tRNA
-rRNA
-Ribosomal particles

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3
Q

Nuclear import/export selectivity - Ribosome Biogenesis

A

mRNA encoding ribosomal proteins transcribed in nucleus

the mRNA is shuttled out of the nucleus

translation of ribosomal proteins outside of nucleus

ribosome assembly occurs inside nucleus
ribosomal proteins imported back in
find rRNA in nucleus - assemble

final ribosomes then exported again

so entails export -> import -> export

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4
Q

Nuclear pore complex structure basic

A

large 125MDa
50 distinct proteins

multimerise into 8 fold rotational symmetrical structure
form:
-nuclear basket: involved in interactions inside the nucleus (eg chromatin regulation)
-central core
-cytoplasmic fibrils on cytoplasmic side

have diff layers of proteins forming rings to generate the hole
diff rings diff purposes
have cytoplasmic filaments projecting out the spoke proteins

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5
Q

cytoplasmic filament purposes

A

important in determining traffic

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6
Q

NPC division into structurally similar modules

A

Halves:
-cytoplasmic
-nuclear

Spokes:
-8 radial spokes

columns:
-16 radial columns

Rings:
-Outer
-inner
-outer
outer and inner rings make up core scaffold

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7
Q

NPC membrane ring

A

interacts with nuclear membrane

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8
Q

Nuclear pore flexibility

A

mRNA unwinds itself to pass through

but there is decent flexibility in what NPC can let through
pore can flexibly change in size?

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9
Q

nuclear import speed in S phase

A

nuclear import generally rapid

during S phase need to import many histone molecules to wrap newly replicated DNA

works out to 2-3 histones imported per second over 8hr period of S-phase

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10
Q

nuclear membrane transport types

A

Passive:
diffusion
no energy
-metabolites and other small molecules/proteins

Active:
requires energy and transport proteins
-large proteins
-protein complexes
-mRNA
-tRNA
-ribosomal particles

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11
Q

experiemnt for passive transport size limitations

A

coat dextran sugar w fluorescent probe
inject it into xenopus oocyte
watch how long it takws for diff sized dextrans to enter nucleus

~60-80kDa limit

larger proteins require nuclear localisation signal for import

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12
Q

Nuclear localisation signal

A

NLS:
Peptide - Stretch of basic Amino Acids
-Lysines/Arginines (K/R)

can tag on to other proteins - cause them to be imported
mutation of it prevents import

can be at beginning, middle, end of protein as long as it is exposed
so can be attached to any protein of interest
E.g. GFP (normally ends up in cytoplasm)
NLS directs their import from cytoplasm

coating with this signal is sufficient for import of a large gold particle

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13
Q

Nuclear export signals

A

NES
many are a leucine (L) rich sequence
can function autonomously like NLS
certain RNA sequences/structures also work as NES

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14
Q

cytosolic proteins necessary for nuclear import

A

Nuclear import receptors - Karyopherins

soluble cytosolic components

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15
Q

Experiment for testing cytosolic protein necessity for import

A

treat cell w Digitonin
permeabilises cell
removes cytoplasmic medium - leaves just some cytoskeleton

take NLS fused fluorescent protein tags
add ATP + cytosolic lysate
or just ATP

when Just ATP no import happened
need cytosolic components

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16
Q

Karyopherin binding

A

directly bind cargo
or via an adapter protein which binds cargo
binds the NLS

also bind the Nucleoporins (NUPS) in the nuclear pore complex
interact with the Phenylalanine-Glycine (FG) repeats in the NUPs

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17
Q

FG repeat importance in nucleoporins

A

FG/Phe-Gly repeat containing nucleoporins constitute the PERMEABILITY BARRIER of nuclear transport

form a Phe-Gly meshwork in the NPC core
once you bind and enter the meshwork can then pass through pore

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18
Q

Karyopherin/Import receptor interaction with FG repeats

A

Cargo binds Karyopherin

Karyopherin has FG binding sites

Binds the FG repeats on the NUPs

Cargo/Karyopherin complex can pass through

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19
Q

Importin-Beta

A

founding member of karyopherin import receptor family

has ~19 HEAT repeats

one part binds NPC FG repeats
another binds cargo
another binds Importin-Alpha - an adapter that binds cargo

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20
Q

Ran-GTP mediated nuclear IMPORT

A

High Ran-GTP in nucleus
high Ran-GDP outside

Protein binds import receptor

imported thru pore

Ran-GTP competes w receptor for cargo - kicks it off inside nucleus

Ran-GTP/Karyopherin exits nucleus

Ran-GTP is hydrolysed outside of nucleus
loses affinity fot import receptor

receptor now free for more cargo to bind

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21
Q

Ran-GTP mediated nuclear EXPORT

A

Exportins
Binds Ran-GTP inside nucleus
this allows it to bind cargo

leaves nucleus
Ran-GTP hydrolysed by Ran-GAP
kicks off Ran-GDP and cargo outside nucleus

Exportins can also bind importin alpha
helps the adaters to get out of nucleus

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22
Q

Establishing the Ran-GTP concentration gradient inside/outside

A

Ran-GEFs inside nucleus (RCC1)
Guanine nucleotide Exchange Factor
Ran-GDP + Pi => RanGTP

Ran-GAPs in cytoplasm
GTPase activating protein
Ran is slow GTPase
GAPs promote GTP hydrolysis

Keeps GTP form high inside nucleus
and GDP form high in cytoplasm

RanGDP and Pi freely diffuse i guess

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23
Q

Nucleus Mechanosensing

A

Nucleus and rest of cell are mechanocoupled via the cytoskeleton
pulling the actin filaments changes nucleus shape

Cells respond to mechanical forces in their environment (eg in the ECM)
Cells of most soft connective tissues need to do this to maintain the ECM during development, remodelling, repair

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24
Q

Mechanotransduction

A

Transduction of extracellular mechanical stimuli into signals
triggering a cellular response through gene expression eg
controls:
-cell motility
-nuclear motility
-gene expression

communication btwn cells and environment
issues can lead to disease

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25
Q

molecular players in mechanotransduction

A

Nuclear envelope TM proteins

Cytoplasmic players:
-cytoskeleton: Actin, IF, MT
connect to Nesprin family of nuclear envelope outer membrane proteins

Nesprins interact with inner membrane SUN domain proteins (SUN1/2)

then have other proteins embedded in inner membrane:
-Lamin B receptor
-LAP2Beta
These connect directly with IF lamins and with chromatin

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26
Q

Nesprins

A

Outer membrane TM proteins
connect nuclear and cytoplasmic cytoskeletal networks together

-Nesprin 1/2 - to actin
-Nesprin 3 to Plectin (an IF)
-Nesprin 4 - to MTs, motors, and centrosome

all contain KASH domains - embedded in outer nuclear membrane

Their C-terminus Interacts with SUN domains embedded in the INM (stick out in space between the ONM/INM)

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27
Q

SUN proteins

A

interact w nesprins
embedded in INM

domains located in the periplasmic space

then interact w proteins lying just underneath the nuclear envelope (Emerin, Lamins)

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28
Q

LINC complex

A

Nesprins
+SUN proteins
+Emerin

called the LINC complex
linker of Nucleoskeleton and cytoskeleton
-facilitates mechanitransduction
-positions nucleus within cell
-tehter centrosomes to nuclear envelope
-have other domains eg Spectrin repeats and Calpolin Homology (CH) domain (which in Nesprin 1/2 bind actin CS)

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29
Q

Nucleus mobility

A

nucleus can take different locations in cell depending on context
eg Epithelial cells
-in culture: is in middle of cell
-in tissue: locate towards the basal membrane

eg Myotubes(polynucleate)
-in culture: many nuclei randomly distributed in cell
-in tissue: nuclei found exactly where the synapses contact the cell

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30
Q

Nesprin mediation of nuclear migration -Klarsicht

A

In drosophila:
Klarsicht (a nesprin) can connect to motor proteins (Dyenin) on the MTs and allow nuclear migration on the MTs

relevant in photoreception
MT and dyenin align the nucleus towards the apical side

in mutants
migration perturbed
many stay towards basal side

this migration failure results in eye morphology/function defects

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31
Q

Human brain neural celll migration dependent on nuclear migration

A

scaffold of radial Glial cell bodies
other cells migrate on this from ventricular zone to primitive cortex (brain surface)

2 types of movement in these neurons
1. rapid movement by leading neurite
2. followed by second phase which is the cell body following along and also the nucleus (NUCLEOKINESIS)

general neural cell migration dependent on ability to perform nucleokinesis

mutation in eg Dyenin compromises nucleokinesis, can lead to human classical lissencephaly (smooth brain)

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32
Q

neuromuscular junction nuclear localisation

A

enrichment of myotube nuclei at neuromuscular junction is dependent on Nesprins

1 WT allele sufficient for WT phenotype
need -/- to disrupt
nuclei scatteres not directly below surface junction

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33
Q

SUN domain protein/Nesprin centrosome tethering

A

centrosome= cytoplasmic organelle
but is v close to nuclear envelope

Mediated by Nesprin 4 (ONM)
connected to SUN domain
connected to Emerin

Centrosome connects to Nesprin 4 tethering it to the nucleus

emerin mutation breaks this connection

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34
Q

INM proteins

A

-Lamin B receptor!
-Lap1!
-LAP2!
-Man-1
-Emerin!

can make contact to proteins on
outside
and connect to important nuclear structures:
-Lamins (IF)
-Chromatin
many INM proteins that interact w these can affect gene expression
allows mechanical stimulus to transduce signal all the way to chromatin

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35
Q

Nuclear lamina

A

intermediate filaments
just underneath envelope
meshwork of filaments

nuclear lamins regulate size, shape, mechanical integrity of nucelus

breaks down at onset of mitosis (phosphorylated lamins)

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36
Q

Lamins

A

Type V intermediate filaments
mutations linked to many diseases

genomic locus:
V large
many exons
typical structure:
-Domain at N-terminal half - Alpha helical rod domain - stiff part of filament
-followed by flexible part - globular domain - the Ig fold, important for contacts inside nucleus (eg w chromatin)

Have 2-6 separate genes
get Lamin A and C from alt splicing
and B1 and B2 isoforms

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37
Q

arrangement of monomers in lamin filaments

A

alpha helical stiff rod domains connected in filament
globular Ig domains stick out of side

periodicity of Ig domains (connected by flexible linker to rod shaped N terminus domain) are about 20nm apart

relatively short persistence length - measure of stiffness
allows it to be bent in diff directions

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38
Q

Assembly of lamin filaments

A

Lamin dimer
coiled coil from rod domains
(cross section = 2 a-helices in coiled coil)
both Ig domains toward C-terminus - both at same side of dimer

dimers assemble in staggered head to tail longitudinal assembly
come together overlapping in small region -giving the staggered tetramer
head to tail polymer
can be staggered or half staggered lateral assembly (parallel or antiparallel)
tetrameric cross section at overlap
dimeric at non-overlap

then form protofilament
hexameric overlap region
tetrameric region where not overlapped just 2

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39
Q

Processing steps for mature functional lamins

A

CAAX region at end is modified
-farnesylation
-endopeptidase
-carboxyl methyltransfer

then important step:
Have Pre-Lamin A:
protease cuts this region away producing mature lamin A

Likewise get mature Lamin B1 and B2 in diff pathways which dont undergo proteolysis - instead undergo farnesylation that remains there

if this important step goes wrong (mutations, protease cleavage region absence)
end up with progerin molecule

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40
Q

Lamin A mutations

A

Linked to laminopathies

hutchison-gilford progeria - advanced aging in children

also
-in coiled coil a-helical regions: cardiomyopathies
-in globular domain - Associated with HGP

41
Q

Processing of Progerin protein

A

eg in HGP
15AA deletion
all the other modifications take place
EXCEPT
protease cannot recognise the proteolysis site

end up with just Prelamin A (Progerins)
has modifications on the c-terminus that is usually cleaved

in HGP imparis nuclear mechanics leading to altered gene expression
leading to premature aging

42
Q

Genome interaction with nuclear lamina

A

there are dense heterochromatin regions next to the nuclear envelope
silenced regions

regions of heterochromatin away from lamina are often around the nucleolus

Progeria patient -reorganisation of heterochromatin
all the HC normally under membrane is interior - leading to massive change in gene expression

43
Q

Mapping Nuclear lamina - chromatin interactions via DamID

A

DNA methyltransferase from bacteria (Dam)
methylates the Adenosine in GATC sequence

fuse Dam enzyme to Lamin A protein
methylates and hence gives way to ID interacting with the Lamins
this methylation not normally present in Eukaryotes i guess

have one with Lamin A/Dam fusion
and one with just general Dam methylation for control

digest GATC using Dpn1 enzyme
size fractionate cut regions
label Experimental and control diff lables (Cy3, Cy5)

then hybridise them to microarray with wells containing probes for known regions covering the entire genome
colour assay tells us which genomic regions interact with lamins

saw large regions mapped to genome that were enriched for GATC methylation

LAMIN ASSOCIATED DOMAINS
LADs

44
Q

LADs in diff cell types

A

look in cells where differentiation was induced and cells in more polypotents state

regions were LADs were not currently in the more polypotent
would then form in later differentiated cells

association w nuclear envelope way to regulate gene expression
solidifying silenced state

TFs are more important so may be extra way to solidify silenced state ON TOP of other regulatory elements

45
Q

LADs dynamicity

A

LADs can change in differentiation
Breakdown of Nuclear envelope in mitosis provides opportunity to rearrange envelope-chromatin interactions

lamins phosphorylated by kinases
NE breakdown

after mitosis
membranes assemble around chromosomes in daughter
lamins start connecting to those chormosomes again
Nuclear envelope fragments surround them and then eventually fuse to make new NE
this is stage where breakup can disrupt former LADs and make new ones
-also some heterochromatin regions associated to envelope become associated to nucleolus

46
Q

Chromatin fibre hierarchical organisation

A

DNA double helix

7-11x compaction: nucleosome beads on string

30nm fibre - 35x compaction: nucleosomes packed into 30nm fibre (not in humans but in urchins)

higher order organisation

47
Q

Higher order organisation in interphase

A

loops around a scaffold
proteins form and organise loops anchored to scaffold
70x compaction

loops DNA into Topologically Associated Domains TADs

48
Q

Higher order organisation in mitosis

A

highest compaction
driven by SMC, motor proteins, nucleosomes, histones

gives rise to mitotic chromosome shape

49
Q

Chromosome scaffold

A

purify chromosomes
add salt to spread out DNA
2 things seen:
1. loops of DNA
2. Separated scaffold proteins

isoltate this scaffold structure and see if anything important for chormosome formation:
-Topoisomerase II
-condensin

50
Q

Cohesin in mitotic chormosomes

A

required for their organisation

SMC = structural maintenance of chromosomes

2 coiled coil proteins
Smc1
Smc3
(are motor proteins)
held together by Scc1
Scc3 bound to Scc1

cohesin complex
forms ring complex that holds the 2 sister chromatids together in mitosis
(can promote loops in interphase?)
-Smc1 and 3 connext with Scc1 to form ring
-Scc3 bound to Scc1

Sisters line up - each to one pole
once properly connected to poles Scc complex begins to degrade Securin (inhibits Separase)
then Separase can cleave Scc1
frees up sisters

51
Q

Condensin in chromosomes

A

v similar to cohesin
Smc2 and Smc4 motor proteins and CAP-H form ring
CAP-G/G2 and Cap-D1/2 bound to CAP-H

this ring complex maintains chromosome folding and loop formation

52
Q

condensin in maintaining Metaphase chromosomes

A

maintains the folding and loop formation of metaphase chromosomes

add EDTA
chelates Mg
gives amorphous structure of chromosomes, removes their normal structure

SMC2 WT: remove magnesium - chromosomes disappear, add Mg back-chromosomes restore and reappear
can cycle this many times

SMC2 OFF: gets rid of condensin, chromosomes start off fuzzy to begin with
and when remove magnesium chromosome structures completely disappear
adding Mg back gives some compaction but chromo shape is completely absent

meaning:
condensin is important for Establishing AND maintaining the shape of the chromosome

53
Q

Structural memory of chromosome

A

helps chromosomes recover their morphology after repeated disruption/packaging

dependent on condensin complex
and non-histone proteins

anchors keep memory intact
get rid of condensin - no way of knowing whate sites of scaffold to go back to
memory lost so chromosomes not restored in restoring Mg buffer

54
Q

Mitotic chromosome formation Pathway

A

used Chromosome conformation capture
and highly synced cells

prophase: condensins mediate formation of arrays and consecutive loops

Prometaphase: chromosomes - now a spiral staircase (helically arranged axial scaffold of condensin II at base of loop)
condensin I helps further compaction into clusters of smaller nested loops

gives final 10,000x compaction

55
Q

Condensin II and I action

A

localises to chromatin forming large loops

then Condensin I comes in and extrudes nested loops within these larger Condensin II formed loops

56
Q

arrangement of interphase eu-/heterochromatin

A

separate regions of dense heterochromatin and less dense euchromatin

57
Q

Rabl orientation

A

centromeres located at one side of nucleus
telomeres to other
this configuration seen in embryogenesis during cleavage divisions - no G phases in cell cycle

chromosome territories

58
Q

Interphase chromosome organisation
chromosome Territories experiment

A

experiment:
local UV radiation damage part of nucleus
if chromosomes in territories then fewer would be damaged than if randomly intermingled

only 1 or 2 were so supports territories

59
Q

3d organisation of interphase chromosomes

A

use multicolour FISH
target diff chromos with diff colours based on sequence
visualises 3d organisation

each chromosome occupies distinct territory within nucleus
proportional to chromosome size

some chromosomes have extensive contact with the nuclear envelope
-located externally
-more heterochromatin - more repressed regions/inactive genes in this cell type

some chromosomes are positioned internally
-will have more neighbours
can interact with more
may explain why certain chromosomal translocations occur with more frequency than others in cancer cells
eg btwn 4, 5, 10 - close vicinity

60
Q

position of interphase chromosome and gene expression

A

peripheral:
gene poor
less transcription
more hetrochromatin

internal
gene rich
more transcription
more euchromatin

61
Q

measuring dynamicity of chromatin in interphase nucleus

A

photoactivated GFP tags
activate in stripes

in mouse ES cells - lines disappeared completely - has dynamicity in interphase nucleus

in fibroblast no movement of lines - so more stable

62
Q

Global chromosome position transmitted in mitosis

A

fluorescence in nucleus
photobleach part
position of bleaching pattern in mother cell reappears in daughter

patterning is inherited and is not randomly established

63
Q

Chromosome positioning in diff tissues

A

diff cells use diff sets of genes
position relates to transcriptional activity

chromosome positioning changes in differentiation

64
Q

Gene migration out of territories

A

inactive genes located by periphery
if want to activate expression of gene in periphery
can loop it out

can probe certain genes and watch as they migrate out of territory during differentiation

65
Q

Transcription factories and territories

A

RNA pol II distrubution is not homogeneous in nucleus
localises together in transcription factories

can cluster these together and loop out certain genes
ensure all components for transcription are localised together

another layer of gene expression regulation from internal chromosome organisation

66
Q

long range interactions and chromatin loops

A

enhancer can be distal from promoter of gene (several kb)
but still affect expression

cohesins bind DNA and extrude loop
can bring the distal enhancer near to the promoter

useful for regulating expression and isolating DNA elements away from each other too

67
Q

Chromosome Coformation Capture (3C)

A

Can show Distal enhancer and promoter in the sequence are actually together in cell
gives idea of what sequences are interacting

add cross linker
cross links DNA in close proximity (ONLY these close sites)
then digest with restriction enzyme
can ligate these two cross linked pieces together
use known primers of candidate regions to amplify
then sequence the new sequence this ligation created from these interacting sequences

can show promoter regiosn and enhancers that are together and apart in diff cell types

68
Q

High-C (3C method)

A

instead of primers to amplify
biotinylate
idk how this works though

69
Q

diff sequence detection methods for 3C

A

PCR amplification
-one vs one
-known primers to check if candidate region is interacting
-if not interacting then cannot PCR between bound primers

High throughput sequencing:
-one vs all

Microarray:
-linker mediated amplification
-compare many vs many

biotin fill in/blunt end ligation
-compare all vs all

70
Q

Activity dependent subnuclear compartments

A

4 nuclear processes
-replcation
-transcription
-splicing
-DNA repair

can detect subdomains when stain for these particular activities
clusters of certain protein activity

are Membrane-less organelles
and can self organise

71
Q

DNA replication staining

A

PCNA stain
clamp protein that loads replication factors
stain it: shows clusters of it

no. of replication forks needed to replicate DNA is more than the no. of PCNA clusters
so the replication factories’ existence depends on replication activity

72
Q

Transcription cluster staining

A

stain for RNA pol II
see transcription structures

73
Q

DNA repair staining

A

stain for pATM
then gamma irradiate cell to induce double strand breaks

stain shows where activity happens

74
Q

interchromosomal compartments

A

site of transcription and splicing
occupied by splicing speckles - storage compartments for splicing factors

couples transcription of new mRNA that needs splicing and the splicing process
so splicing occurs co-transcriptionally

75
Q

splicing speckles dynamics

A

are depots for splicing factors
splicing factors move in and out all the time

76
Q

visualising interchromosomal space

A

interchromatin compartment

GFP-H2B fusion protein expressing cells
expose to hyper-osmolar medium
undergo chromatin condensation
and expansion of interchromosomal space
amplifies these regions making them easier to see

these are the regions in between DNA occupied by diff subnuclear compartments

77
Q

nuclear components and compartment dynamicity

A

unlike cytoplasmic ones where protein enters and stays
either in or out

in nucleus
no membranes
so more accessible
lots of fluidity

compartments are self organised and have their own integrity
but allow for this fluidity of components

78
Q

Proteinaceous Nuclear Bodies (NBs)

A

Nucleolus
Speckles
Cajal bodies
PML bodies

distinguished by unique subset of proteins in each
can be sites of activity
OR just storage structures - no meanignful activity within

79
Q

Nucleolus

A

site of ribosome biogenesis
-transcriptional processing of rDNA
-rDNA transcribed by RNA pol I in nucleus
-~80% of RNA in growing cells is rRNA

nucleolus also acts as storage compartment for proteins
eg Cdc14 phosphatase - needed for regulation of cell cycle exit of mitosis into interphase
during this it is in the nucleolus

80
Q

Speckles

A

concentration of splicing factors
not clear if just storage or provide extra activity

81
Q

PML bodies

A

mysterious
may have function or could also be storage depot for several types of proteins

also act to sequester different factors to affects expression

82
Q

finding out if certain protein localises to a certain nuclear body?

A

stain that protein
stain protein that is known to be in that NB

see if they co-localise in nucleus

use Ab (paul Mcloughlin shaking in his boots)
or GFP

or can use immunoprecipitation + mass spec to see if protein of interest interacts with proteins in that NB

83
Q

layers of the Nucleolus

A

3 distinct layers

inside - lots of rRNA genes - Fibrilar Centre

surrounding nucleolus - Dense Fibrilar componenrs - where pre-rRNA transcripts mature
several proteins break them into pieces so can be targeted to diff ribosome subunits

outside - granular components - site of assembly of pre-ribosome particles into Ribosomes
appears granular during this process

mammal cells have 1-5 nucleoli per cell

84
Q

Nucleolus Dynamicity

A

fuse Fibrallarin to GFP

use FRAP (fluorescence recovery after photobleaching)
assesses mobility of molecule

photobleach area of fibrillarin on nucleolus
look for recovery speed of GFP fluorescence

fibrallarin highly diffulible
nucleoplasm has low affinity Fibrillarin sites
nucleolus has high affinity sites
establishes an equilibrium of Fibrillarin in the nucleolus

similar mechanism in many nuclear compartments
-core affinity holds it together
-but many components move in and out dynamically

85
Q

Nucleolar Organiser regions (NORs)

A

regions of multiple DNA encoding rRNA genes
found clustered together on few chromosomes
always on edge of Acrocentric chromosome (on P arm i think)

need many rDNA genes as necessary for production of lots of ribosomes in stages where cells are growing and translationally active

NORs where rRNA genes are clustered is site of nucleolus formation

86
Q

Nucleolus formation on NORs

A

fusion of multiple NORs

nucleolus disassemble at start of mitosis
at end of Mitosis (G1 onset) - NORs organise together into one or a few Foci

these Foci fuse togetehr in G1 - can end up with 1 or multiple

not all NORs engage in forming nucleolus
some are inactive transcriptionally
-stain all rRNA
-some will be found outside these fused NORs

87
Q

Cajal Body purpose

A

processing of snRNAs and maturation of snRNPs (sn = small nuclear)

these are required for SPLICING

contain mature splicing factors stored here
supply them to ready to go splicing machinery during transcription

keep these in diff subcompartments
because in stress conditions
need to generate certain types of machinery
better to have them clustered in close vicinity for higher efficiency

88
Q

maturation of snRNAs

A

go into nucleus
to cajal body
some level of assembly here

89
Q

PML bodies

A

contain PML proteins and SUMO
-Promyelocytic leukemia protein
-Small ubiquitin-like modifier

90
Q

PML in acute myeloid leukemia

A

affected by a chromosomal translocation
15:17 translocations between PML and retinoic acid receptor (RAR)

RAR recruits proteins that repress transcription
by virtue of fusing to PML protein causes it to repress Tumour suppressor genes

retinoic acid can alleviate this repression
treating the cancer caused by it

91
Q

Post-translational modification of proteins in PML

A

proteins in PML bodies are SUMOylated
SUMO motif added on Lysine (K65, K160, K490)

92
Q

SUMOylation pathway

A

SUMO is small protein added on
similar to ubiquitin process (hence the U)

– SUMO E3 ligase brings together SUMO E2 conjugating enzyme
– bringing of SUMO E3 and E2 to target/substrate causes SUMO to transfer from E2 to target

SUMOylation changes activity of protein

93
Q

diff SUMO species

A

can be mono or poly SUMOylated
on lysine residues

carboxy terminal Glycine residue of SUMO forms an isopeptide bond w the amino group of lysine residue

94
Q

PML body struture

A

PML constitues part of outer structure of PML body, mono-SUMOylated there

inside of PML body is the poly(2/3)-SUMOylated proteins

many proteins are transiently recruited there
wehre they become SUMOylated
attachment of SUMO changes their properties

SUMO can act like a glue between SUMOylated proteins

95
Q

PML body protein diffusion

A

proteins can diffuse in and out

Nuclear localisation signal fused with GFP
photobleach the GFP specifically in PML body
quickly recovers inside

shows high dynamicity in proteins diffusing in and out

96
Q

Function of PML bodies

A

increase in size and number under cellular stress conditions or senescence conditions

many proteins sequestered/recruited there to be SUMOylated

sequesteration of transcriptional repressers/activators affects gene expression

97
Q

PMLbody sequestration of Daxx

A

cell stress
activates SUMOylation machenery
increase in PML-SUMO

SUMOylated PML interacts with Daxx and sequesters is to PML body

Daxx sequestration leads to Derepression of Pro-apoptotic genes

98
Q

Formation of heterochromatin domains

A

active euchromatin has H3K9 acetylation
at some point can be deacetylated
Suvar3-9 methylation comes and methylates H3K9
methylation interacts with HP1 protein via its chromodomain

HP1 recruits more Suvar3-9
cycle
propagates heterochromatin domains

99
Q

heterochromatin domain formation
-Liquid phase separation

A

diff proteins can separate based on characteristics
HP1 does this
associate with each other
form droplets

these HP1 droplets fuse together fomring larger droplets forming these domaisn

the droplet domains exclude other proteins incl. TFs - this is how thees domains are maintained
involved in making heterochromatin nuclear subdomains

Molecular Cell Biology (5 decks)

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