Membraneless Organelles Flashcards
Give an overview of membraneless organelles?
Do not have a phospholipid boundary
Each membraneless compartment contains many molecular components
Comprises of protein and RNA (RNP - ribosome bound to a protein)
Concentrate specific components that enter and leave (sec timescale)
Dynamic, coherent assemblies that play key roles in biology - due to diverse physical properties
What are some types of membraneless organelles and their functions?
Nucleolus – Ribosome biogenesis
Paraspeckle – Regulation of gene expression
Nuclear speckle – Storage of splicing factors
Cajal bodies – Regulation of snRNP maturation
PML bodies – Regulation of transcription and protein storage
P bodies – mRNA processing and decay
Purinosomes - purine biosynthesis
Histone locus bodies - mRNA processing
Stress granules – storage of translationally stalled mRNA and translation machinery proteins
Germ granules – Regulation of mRNA translation in cytoplasm of germ cells
Balbiani body – protection of organelles during oocyte dormancy
Describe some properties of membraneless organelles?
They behave like liquid like droplets (think lava lamp) - that coexist with the cytoplasm
It is essentially like a liquid that stays separate from the ‘liquid’ cytoplasm by phase separation as it is driven by the physical interactions between the molecules (liquid-liquid demixing)
This leads to cellular subcompartmentalisation
P granules “drip” around the nucleus in germ line cells
They flow and deform around surfaces (as well as undergoing fission)
They can: undergo fusion, grow in size over time (nucleation) and the molecules dynamically reorganise - seen in FRAP
Remain stable for minutes to hours but exchange with its environment most seconds
Exhibit ‘wetting’ behaviour on a hydrophobic surface
Describe some general functions of membraneless organelles?
Reaction crucible - within the droplets have a high concentration of proteins = increase rate of reaction (regulating the biochemical reactions)
Sequestration - they can sequester molecules to prevent signalling activity - not floating around in the cell
Organisational hub - organise their internal space
Role - organising cells and controlling biological function in the cell
They act to determine cell fate - they all move to one side of the cell and the daughter cell with the P bodies will become the progenitor germ cell (gamete) and the other a somatic cell
How are the droplets formed?
This is shown using liquid-liquid phase separation (LLPS)
Protein molecules become concentrated
There is still exchange within and outside the droplets
This is moving to a higher entropy - unfavourable
Therefore enthalpy must be favourable
If the enery of heterotypic interactions (2 different liquids) is large, the demixed state is favoured
Describe the forces that drive the demixing of heterotypic interactions?
Phase separation - the creation of two distinct phases from a single homogeneous mixture
At concentrations above Ccritical, a protein will form droplets
Regulated by: posttranslational modifications, temperature and ionic strength can modulate Ccritical
Droplets allow diffusion within the compartment and exchange of molecules with the dilute phase
How is phase separation driven in cells?
Phase separation in cells can be promoted by:
Changes in concentration, temperature, pH, ionic strength
Increasing valency e.g. by addition of a third component - decreases entropy
Decreasing solubility (e.g. by oligomerisation, binding to partners)
What other components/modifications can tune/drive LLPS?
PTMs
Important for how phase separation is important in disease
Lys acetylation – disrupts cation-pi interactions
Lys ribosylation – increases multivalency
Arg Methylation – alters hydrophobicity and H-bonding
*Phosphorylation – alters electrostatic interactions
Arg citrullination – alters charge-charge interactions
RNA modifications and structure:
RNA can act as a scaffold for LLPS
Modifications recognised by ’reader’ proteins
RNA modifications alter structure/interactions
Increased RNA length can promote LLPS
What are phase transitions within cells mainly driven by?
The process of forming membraneless organelles can be driven by RNA or protein
Increasing either protein-protein or RNA-RNA interactions can promote assembly
Depleting proteins responsible prevents assembly
Describe how liquid to solid phase transitions in cells are associated with disease?
The prevalence of RNA in repeat expansion disease = an example of a toxic RNA being produced that has the potential for multiple intermolecular interactions and therefore formation of a multimeric RNA assembly in the cell
The fibrils can lead to motor neurone disease and dementia
Describe the general structure of membaneless structures?
A key set of proteins/RNA drive membraneless organelle formation
This “scaffold” specifically recruits client proteins/RNAs
Protein clients:
Strongly recruited - multivalent RNA binding + IDR (intrinsically disordered region) with proper sequence or charge
Weakly recruited - multivalent RNA binding and RNA binding + IDR with semi-compatible sequence Not recruited - monovalent RNA binding and RNA binding + IDR with incompatible sequence or charge
Nucleic Acid Clients:
Recruited - RNA with proper structure and/or multiple protein recognition sites
Not recruited - RNA with no interacting structure and one or no protein recognition sites
Multivalent interactions between proteins (ordered domains and IDRs) and RNA drive LLPS
What are some features of proteins driving RNP granue formation?
4 different types of protein-protein interactions that promotes this:
Stereospecific interactions between well-folded domains
Specific interactions between local structures in IDRs
Interactions of SLiMs and well-folded domains
Promiscuous interactions between IDRs
Assembly is promoted by Longer RNA length High local concentrations RNAs with increased ability to interact Multivalent RNA-binding proteins
Give the definitions of IDR and SLiMs?
• Intrinsically disordered region (IDR): A functional protein region without a unique structure (enriched with polar/charged residues)
Short Linear Motifs (SLiMs): Short stretches of protein sequences that mediate protein–protein interactions
Give the definitions of LARKs, multivalent and RNP?
Low-complexity Aromatic-Rich Kinked Segments (LARKs): Short stretches of protein sequences that can bind weakly to each other by forming a pair of kinked β-sheets
Multivalent: Comprising multiple binding sites for a ligand/protein partner
Ribonucleoprotein (RNP): A complex of RNA and RNA-binding protein (RBP)
Describe stree granules?
Contains:
mRNAs stalled in translation initiation (pre-initiation complexes)
Various translation initiation factors
RNA-binding proteins, and many non-RNA-binding proteins
Controls the utilization of mRNA during stress
Implicated in diseases, e.g. cancer, neurodegeneration, inflammatory disorders and viral infections
Describe the assembly of stress granules?
Puromycin promotes SG formation
mRNA-40S complex associates with RNA binding proteins that nucleate SG formation
Active mRNA-ribosomal particles
When the 60S falls off - leaving the 40S bound to the RNA
This then recruits RBP nucleation particles and all proteins needed for forming stress granules
Describe the regulation of stress granule assembly?
Formation of pre-initiation complexes and mRNA translation under basal conditions (normal)
Multi-step process to recruit both the 40S and 60S subunit
mRNA is recognised by the eIF4F complex
mRNA-eIF4F is joined by eIF2 ternary complex
This recruits the initiating tRNA - to start translation
This forms the pre-initiation complex - where the 40S ribosomal subunit is bound and then goes on to recruit the 60S subunit
What happens to stress granules under stress?
By inhibiting translation initiation - promotes stress granule formation
mTOR inhibition, phosphorylation or chemical interference
By using these cell signalling pathways we allow the PIC to accumulate and binds the recruiting proteins it drops the 60S subunit = promotes stress
Therefore stress granules increase in number under stress
What are the phases of cycle of the stress granule?
- Basal RNA metabolism
- Cytoplasmic translocation
- Stress granule nucleation
- Stress granule maturation
- Stress granule dispersal or removal and nuclear import
What diseases are stress granules associated with?
Liquid-to-solid transition of stress granules proteins are associated with disease
Basal state, acute stress, chronic stress (from amyloid fibrils) = neurodegenerative disease
Amyloid fibrils form due to toxic aggregates
Can cause Alzheimer’s disease, Frontotemporal lobar dementia, Motor Neurone Disease
What is a key player in pathology of neurodegenerative diseases?
TDP-43
It undergoes phosphorylation of serine residues to form these diseases
Comprises a folded N-terminal domain (NTD) nuclear localisation sequence (NLS), nuclear export sequence (NES), 2x RNA recognition motifs (RRMs) and an unstructured C-terminal tail
In what ways does phase separation play a role in many diseases states?
Due to high protein concentration it can trigger aggregation processes leading to solid gels or crystals = wrong environment for chemical reactions
Neurodegeneration RNP granule dynamicity and function Trapping of cellular factors Forms mutations/repeat expansions, abnormal PTMS, altered localisation Cancer
Cellular signalling
Forms mutations and chromosomal translocations
Infectious diseases Virus replication Cellular stress response Innate immune response Dormancy of bacteria/fungi Forms viral IDRs or proteases
What are viral factories?
Membraneless organelles of viral proteins, host proteins and RNA
Evolve in structure throughout viral infection - dynamic
Concentrate the components required for virus replication
Escape innate immune response
Assembly of virus particles occurs in the factories
In this confocal image, the viral factory comprises empty and full capsids
Give some examples of viral proteins being used in membraneless organelles - viral factories?
Measles:
Virus replication machinery comprises:
Nucleoprotein (N) - encapsidates the viral genome
RNA-dependent RNA polymerase (L) and its cofactor the phosphoprotein (P)
SARS-CoV-2
Nucleocapsid protein (N) undergoes phase separation with RNA
Small molecules that bind N modulate droplet properties
We can identify small molecules which bind to the protein component of SARS that forms the viral factories
These molecules change the droplet/liquid like properties = changing how dynamic these properties are
