Sub-Nuclear Structures

Question 1

What are sub-nuclear structures?

Answer 1

Contains many functionally specialised regions of sub-structure that form and maintain themselves in the absence of membranes (non-membrane bound)

Question 2

Describe the nucleolus?

Answer 2

Largest non-membrane bound structure in the nucleus
Takes up to about 25% of the nuclear volume
It forms around tandem arrays of ribosomal gene repeats - nucleolar organising regions (NORs) on acrosomal chromosomes
A large multi-protein complex
It is held together without a cell membrane via protein-protein interactions
These interactions evolve around central hub proteins
Interactome map: shows the protein-protein interactions

Composed of proteins and nucleic acids
Fibrillar centers (FC) - site of rDNA genes being actively transcribed
Dense fibrillar components (DFC) - maturation of pre-mRNA transcripts (processing)
Granular components (GC) - assembly of pre-ribosomal particles
Peri-nucleolar region is at the ‘edge’

Question 3

What is the function of the nucleolus?

Answer 3

Pivotal role of the nucleolus is ribosome biogenesis
Site of rRNA synthesis/processing and assembly of ribosomal subunits

Nucleoli are formed around specific genetic loci called nucleolar organizing regions (NORs) - they comprise tandem repeats of rRNA genes
Low doses of actinomycin D which inhibit RNA Pol I transcription and during times of stress the nucleolus will downregulating the synthesis of rRNA and ribosome biogenesis
Stresses: hypoxia, pH fluctuation and redox stress

Regulates the level of the tumour suppressor protein p53

Question 4

Describe ribosomal biogenesis in the nucleosome?

Answer 4

1. rRNA genes are transcribed as a single unit by RNA pol I or III
2. Transcription yields a long precursor molecule, 45S pre-rRNA
   ]3. Further processing generates the 18S RNA, 5.8S, 5S and 28S RNA molecules
3. rRNAs are modified by guide RNAs, small nucleolar RNAs (snoRNAs) and small-nucleolar-ribonucleoproteins (snoRNPs)
4. Association and maturation of rRNA and r-proteins result in the formation of the 40S (small) and 60S (large) subunits of the complete ribosome
   These are exported through the NPC to the cytoplasm

Question 5

What are ribosomopathies?

Answer 5

Disorders in which genetic abnormalities cause impaired ribosome biogenesis/function, resulting in specific clinical phenotypes

Diamond-Blackfan anaemia (DBA) - mutation in ribosomal protein S19, (RPS19)
]low red blood cell counts , leads to variety of congenital abnormalities: craniofacial malformations, thumb or upper limb abnormalities and cardiac defects

Dyskeratosis congenita - mutation in DKC1, which encodes dyskerin (nucleolar protein associated with the snoRNPs)
Disorder that resembles premature aging skin pigmentation, premature greying, dystrophy of the nails and predisposition to cancer

Treacher Collins syndrome (TCS) - mutation in TCOF1 genes, which encodes a nucleolar protein called Treacle
Craniofacial deformities, such as absent cheekbones, a small lower jaw and malformed or absent ears

Question 6

What is specific about the nucleolus?

Answer 6

This is a very dynamic process - it is not a steady state structure
Proteins are moving in/out of the nucleolus all the time

3 types of nucleolar protein:
Mainly always in the nucleolus e.g. fibrillarin and nucleolin
Part time in the nucleolus e.g. ribosomal proteins.
Time/condition dependent e.g. p68 and cell cycle factors, Blooms in S phase

Question 7

Describe the perinuclear compartment?

Answer 7

Associated with the surface of nucleoli - compact perinuclear heterochromatin
Thought to play a role in RNA metabolism
]Contains a series of small RNAs - transcribed by RNA pol III, RNA BPs including (PTB) and heterogeneous nuclear ribonucleoprotein (hnRNP) complex
Perinuclear association constrains the movement of DNA sequences at different sites in multiple chromosomes and function in the maintenance of silencing of non-ribosomal genomic regions
It harbours nuclear bodies with unknown functions

Question 8

What is the nucleolus assosiation with disease?

Answer 8

Morphological changes in the nucleolus now observed in various disease states
Many of these involve either:
1. Changes in ribosome biogenesis
2. Apparent sequestration or loss of proteins to the nucleolus

3 main issues with the nucleolus - auto-immunity, viruses and cancer

Question 9

What does the nucleolus link to?

Answer 9

Biological characteristics of cancer cells influence both nucleolar size and the clinical outcome of the disease -> larger/darker/misshapen nucleoli
Increases ribosome biogenesis – increased protein translation – satisfies increased biosynthetic demand during proliferation
Proto-oncogenes can upregulate ribosomes biogenesis
Deficiencies in ribosome function – alterations in protein translation of specific proteins involved in regulating transformation
People with inherited
The nucleolus could be a drug target for cancer -> that selectively block ribosome biogenesis

Question 10

How is a virus linked to the nucleolus?

Answer 10

67 viral proteins from 37 different viruses representing each of the 7 Baltimore classes of viruses have been observed to show nucleolar localisation
Viruses target the nucleolus to redistribute cellular proteins in order to: utilise nucleolar proteins to enhance virus replication and/or subvert anti-viral pathways
e.g. coronaviruses and herpes

Question 11

What are some other sub-nuclear structures?

Answer 11

Nuclear speckles, Cajal bodies, gems, PcG bodies and PML bodies

Question 12

What is involved in splicing?

Answer 12

Cajal bodies, gems and nuclear speckles
The spliceosome consists of U1, U2, U4, U5 and U6 small nuclear RNPs (snRNPs) in conjunction with a large number of proteins (~125)
Highly dynamic and complex structure
3 distinct subnuclear structures involved in assembly/maturation

Question 13

Describe Cajal bodies?

Answer 13

Spherical sub-organelles of 0.3-1.0 µm in diameter
Usually 3-5 per cell
Contain proteins that participate in biogenesis of mRNA Eg Coilin and SMN proteins
Involved in the maturation and assembly of spliceosomal sub complexes, particularly snRNPs and snoRNPs
The spliceosome proteins U1 etc… Move through the Cajal body en route to nuclear speckles
Essential for the efficient macromolecular assembly of snRNPs in zebrafish
The CB also harbours a class of CB-specific RNAs (scaRNAs) that are involved in the posttranscriptional modification of snRNAs

Question 14

Describe nuclear gems?

Answer 14

Gemini of coiled bodies, or gems - name comes from Gemini twin as it’s in a relationship with Cajal bodies - often adjacent
Gems are similar in size and shape to CBs, and in fact are virtually indistinguishable under the microscope
Unlike CBs, gems do not contain small nuclear ribonucleoproteins (snRNPs), but do contain a the SMN protein, which functions in snRNP biogenesis
Therefore gems are believed to assist CBs in snRNP biogenesis

Question 15

What is spinal muscular atrophy?

Answer 15

Incurable disease caused by a genetic defect in the SMN1 gene which codes SMN, a major Cajal body and Gem protein
]SMN is essential for snRNP assembly (spliceosomal machinery)
Mutations cause widespread splicing defects, especially in spinal motor neurons, resulting in death of neuronal cells in the anterior horn of spinal cord and subsequent system-wide muscle wasting (atrophy).
SMA manifests in various degrees of severity but all have general muscle wasting and mobility impairment
SMA is the most common genetic cause of infant death

Question 16

Describe nuclear speckles?

Answer 16

Structures enriched in pre-mRNA splicing factors
Irregular, punctate structures that vary in size and shape
Human interphase nucleus contains 20-50 NSs - very stable in this phase
They can vary based on cellular ATP levels, phosphorylation and activated genes
During mitosis nuclear speckles dissemble into mitotic interchromatin granules
Speckles are dynamic structures and both their protein and RNA-protein components can cycle continuously between speckles and other nuclear locations

Question 17

What do nuclear speckles do?

Answer 17

Function : storage and modification compartment for pre-mRNA splicing factors, including snRNPS and SR proteins
Speckles are always in close proximity to active genes - proposed to be hubs for enhances metabolic activity involved in mRNA maturation and export
NS proteins are involved as ‘hubs’ in multiple nuclear gene expression regulation steps, such as epigenetic regulation, transcription activator and repressor functions, transcription elongation and termination, splicing, 3 end processing, mRNA PTM and mRNA packing and export
They are diffused throughout the nucleoplasm and larger ones correspond to interchromatin granule clusters

Question 18

What are some mutations involved in splicing factors?

Answer 18

Retinitis pigmentosa a genetic eye condition in which the vision progresses from night blindness to tunnel vision to complete blindness
Retinal pigment epithelium (RPE) is the pigment cell sheet that feeds the retinal cells. The RPE is attached to an intricate system of blood vessels
Genetically linked to mutations in splicing factor, PRP31
Unknown why it only affects diseases of the ey

Question 19

Describe polycomb bodies?

Answer 19

Polycomb bodies are hubs for gene repression, usually associated with heterochromatin
Contain a variety of polycomb group proteins which form at least 2 classes of Polycomb repressive complexes
PcGs remodel chromatin inducing epigenetic silencing
PcGs bind directly to specific DNA sequences and facilitate recruitment of the other complexes, both of which then modify local chromatin structure
Overexpression of PcG proteins linked with variety of cancers as aberrantly repress tumour suppressor gene expression
They have been observed associating with pericentromeric heterochromatin

Question 20

Describe PML bodies?

Answer 20

PML nuclear bodies (NBs) are spheres of 0.1–1.0 µm in diameter – 10-30 per cell
Stable insoluble scaffold - devoid of nucleic acids - assembly driven by oxidative stress
Unlike other nuclear sub-structures PML-NBs are functionally promiscuous - Implicated in the regulation of diverse cellular function
PML-NBs are dynamic structures that favour the sequestration/release of proteins and mediate their post-translational modifications
Promote specific nuclear events in response to various cellular stresses
Also called ND10 bodies

Question 21

How are PML bodies formed?

Answer 21

PML proteins first dimerize and then multimerize to nucleate NBs.
PML sumoylation then leads to organization in spherical body.
SIM-containing (SUMO interactive motif) or sumoylated partners (or both) are recruited by the SUMO or SIM of PML into the inner core of the body

Question 22

What is acute promyelocytic leukaemia?

Answer 22

Caused by mutations in PML protein
In 95% of cases of APL, PML protein forms a reciprocal translocation with the retinoic acid receptor-alpha (RARα) gene
]This results in loss of PML Bodies and also creates a hybrid protein with altered functions
This fusion protein binds which blocks transcription and differentiation of granulocytes
There is an abnormal accumulation of immature granulocytes which show characteristic hypergranular morphology
This results in loss of granulocytes, neutrophils etc

Question 23

What is the role of PML bodies?

Answer 23

Thought to regulate several diverse cellular functions: DNA-damage responses, apoptosis, cellular senescence and angiogenesis
The biochemical means used by PML bodies to regulate so many functions are varied and can be categorized into three main groups:
a. nuclear storage for the accumulation of proteins and release them when necessary
b. ‘catalytic surfaces’ where proteins accumulate to be post-translationally modified
c. active sites for specific nuclear functions such as transcriptional and chromatin regulation

Question 24

What do PML bodies regulate?

Answer 24

PML bodies regulate p53-dependent apoptotic and cellular senescence (cells stop dividing) pathways induced by cellular stress
PML activates p53 by several means
This is achieved by PML recruiting a host of cell proteins to PML bodies which can then post-translationally modify and activate p53
P-53 is a major tumour suppressant protein

Question 25

How does PML link to virus infection?

Answer 25

PML bodies forms part of the interferon induced host innate immune defence
Inhibits the lytic infection of DNA viruses
Some viruses have evolved mechanisms to disrupt PML bodies therefore stopping the antiviral response

Question 26

Describe cleavage bodies?

Answer 26

Involved in the cleavage and polyadenylation steps of pre-mRNA processing
0.3-1.0 micro m
They wither overlap with or are adjacent to Cajal bodies