Comprehensive Notes on the Nucleus, Nuclear Transport, and Chromatin
Nuclear Pore Complexes and Nucleo-Cytoplasmic Transport
Nuclear pore complexes (NPCs) perforate the nuclear envelope (NE) to mediate bi-directional, controlled exchange of molecules between the cytoplasm and the nucleus.
Proteins are imported into the nucleus.
Proteins, RNAs, and RNPs are exported out.
NPCs anchor chromatin and are involved in RNA metabolism (e.g., splicing).
Nucleoporins are located on the kinetochore during mitosis.
NPCs are large multimeric protein complexes.
Up to 500 proteins.
Up to 125 MDa in size.
There are approximately 3000-4000 NPCs in each nucleus, depending on cell type and cell cycle stage.
NPC protein components are called nucleoporins (Nups).
Approximately 30 different types, highly conserved in eukaryotes.
Multiple copies of each nucleoporin.
Nup subcomplexes are present.
Cytoplasmic ring with cytosolic fibrils on the cytoplasmic face.
Nuclear basket on the nuclear side; the main Nup is Tpr.
Membrane Nups (GP210, NDC1, Poms) anchor the NPC into the membrane.
Scaffold Nups bend the membrane and stabilize membrane deformation, allowing them to slide against each other for flexibility (e.g., Nup145/46).
Channel/central pore Nups contain repeat FG (phenylalanine, glycine) sequences, which fill the pore, restricting the size of molecules that can pass through (FG barrier).
NPCs have a huge transport capacity, approximately 1000 molecules/sec/NPC in both directions simultaneously.
Molecules require regulated import and/or export, some up to 200 KDa.
The pore diameter is approximately 40 nm.
Key parts of nucleo-cytoplasmic transport include:
Nuclear localization signals (NLS) and nuclear export signals (NES).
Transport proteins/karyopherins (importins and exportins).
The Ran cycle determines directionality.
Import of Proteins
Various nuclear localization signals (NLS) are rich in lysine and basic amino acid residues (e.g., arginine).
Classical NLS:
Monopartite (PKKKRKV in NLS of SV40 virus T-antigen from polyomavirus).
Bipartite NLS (KR[PAATKKAGQA]KKKK is NLS of nucleoplasmin).
Non-classical NLS.
NLS forms a binding domain for importin (a type of transport protein/karyopherin).
Once cargo is bound, importins traverse the pore by interacting with FG repeats in the pore.
In the nucleoplasm, RanGTP binds the complex, cargo dissociates, and importins+RanGTP are shuttled back outside.
Import adaptor protein = importin-α, which binds to classical NLS; then importin-β binds, forming a trimeric import complex.
Importin-β = transport protein (karyopherin).
Non-classical NLS binds importin-β directly.
Export of Proteins and RNAs
Variety of nuclear export signals on proteins and RNAs, which bind to exportins (karyopherin).
Exportins also interact with FG repeats; the export complex dissociates in the cytoplasm, and exportins are shuttled back in.
Regulation is by Ran GTPase.
The export complex consists of a cargo protein, exportin, and Ran-GTP.
Exportins and importins are related.
tRNAs and snRNAs are exported like proteins, using exportins and RanGTPase, and have specific karyopherins.
tRNAs use exportin-t.
snRNAs use Crm1.
Ribosomal subunits are also facilitated by RanGTPase and karyopherin-like proteins.
mRNA is exported as messenger ribonucleoprotein (mRNP), including mRNA processing enzymes (over 100).
Export uses Nxf1-Nxt dimers, not related to karyopherins.
Uses ATP to drive export.
The nuclear basket and cytosolic fibrils are involved.
The Ran Cycle
Ran is a monomeric GTPase regulated by RanGAP (GTPase activating protein) and RanGEF (Guanine exchange factor).
In the cytoplasm, there is a large concentration of Ran-GDP; cytoplasmic RanGAP is anchored to the NPC.
In the nucleoplasm, there is a large concentration of Ran-GTP; nucleoplasmic RanGEF is bound to chromatin.
Ran-GDP is imported again.
The Ran cycle drives transport through the pore and determines directionality.
Only RanGTP can bind to the transport receptor.
RanGTP binding to the import complex causes dissociation of the complex.
RanGTP binding to exportin promotes cargo binding and export of the export complex.
In the cytoplasm, the export complex dissociates because of RanGTP hydrolysis.
Ran-GDP is imported again.
Transport through the NPC is controlled by signaling to allow access to the transport machinery.
For example, NLS is not accessible in a protein before a signal; a transcription factor is tethered to the cytoskeleton or organelle away from the NPC and released upon a signal.
Summary of NPCs and Nucleo-Cytoplasmic Transport
NPCs are large complexes consisting of nucleoporins arranged into subcomplexes.
NPCs facilitate the import and export of proteins, RNAs, and RNPs.
Import and export are regulated by NLS/NES, karyopherins, and the Ran cycle (except mRNPs).
The Nuclear Envelope and Nuclear Periphery
The nuclear envelope (NE) is a barrier separating the nucleoplasm and chromatin from the cytoplasm, controlling nuclear processes and organization.
The NE consists of a double membrane: the outer nuclear membrane (ONM) and the inner nuclear membrane (INM).
The NE is continuous with the ER and has ribosomes, perforated by nuclear pore complexes (NPCs).
The NE is a permeable barrier.
Chromatin is in the nucleoplasm.
Nuclear structures include the nucleolus.
The NE forms invaginations called intranuclear strands.
The nucleus has a complex organization, including the peripheral nucleoskeleton (lamina), nuclear structures, and chromatin organization.
The nucleus is malleable and mobile.
Neutrophils migrate from the bloodstream.
Nuclear shape is dependent on cell type (e.g., Arabidopsis root hair and root tip).
Nuclei movement responds to stimuli, important in development, cellular function, and stress responses such as mycorrhizal symbiotic signaling.
The ONM and perinuclear space have ER functions due to continuity; however, the ONM also has specific proteins linked to INM proteins.
The INM and ONM are continuous but also have specific protein composition.
The perinuclear space exists between the ONM and INM.
Over 100 specific INM (and ONM) proteins exist, with some being tissue-specific NET (nuclear envelope transmembrane) proteins.
Most NETs are lamin-associated proteins (LAPs), which are metazoan-specific (animals); SUN and KASH proteins are the only ones conserved in animals, fungi, and plants.
Some INM proteins tether silenced chromatin to the NE, thus the NE is involved in chromatin organization and gene regulation.
Function and binding of INM proteins are regulated by phosphorylation.
The NE itself is involved in signaling by being a Ca^{2+} store, with Ca^{2+} pumps and ion channels in the INM and ONM.
In mitosis, the NE breaks down and re-assembles in telophase, with INM proteins and lamins responsible for this process.
One class of ONM-specific proteins is Klarsich/Anc1/Syne1 homology (KASH) proteins.
KASH proteins are anchored to the ONM by binding to INM proteins called Sad1/Unc84 (SUN) domain proteins.
KASH-SUN interactions form physical bridges across the NE, creating nucleo-cytoskeletal bridging complexes = Linker of Nucleoskeleton and Cytoskeleton (LINC) complex.
KASH proteins are poorly conserved, but SUN proteins are highly conserved in eukaryotes.
3 SUN proteins anchor three KASH proteins.
KASH proteins bind to cytoskeletal components: microtubules, actin, and intermediate filaments.
SUN proteins bind to the nucleoskeleton - lamina.
Functions of LINC Complexes
Distance maintenance between INM and ONM.
Force transmission leading to mechanotransduction.
Movement and shape control of nuclei.
Movement of chromosomes.
Anchorage of RanGAP in plants.
The Lamina
The lamina is a nucleoskeletal structure underneath the INM.
Lamins in animals include lamin A/C and lamin B.
Lamin-like proteins CRWN/NMCP are found in plants.
They separately evolved but have similar structure and function.
Lamin-binding proteins are in the INM, keeping them in the INM; some lamins are nucleoplasmic.
Long coiled-coil domain for dimerization; short N-terminal head and longer C-terminal tail domain for interactions and filament formation.
Farnesylation and isoprenylation attach lamin A/C and lamin B to the INM; lamin A/C is cleaved, while lamin B remains attached.
Various INM lamin-binding proteins include SUN proteins and (Lap, Emerin, Man) LEM domain proteins, and soluble proteins like histones and transcription factors.
Lamins directly (Lamin associated domains [LAPs]) and indirectly associate with chromatin, influencing chromatin organization, gene expression, and DNA repair.
They maintain the structure of the nucleus by shaping it and bearing force, and anchoring NPCs.
Cell and tissue-specific expression and interactions occur.
Laminopathies are mutations in lamins and lamin-binding INM proteins, causing dystrophies and early-onset aging (progeria).
Lamin A has over 400 mutations, making it the most mutated gene known, leading to cancer and aging.
Summary of the Nuclear Envelope and Lamina
The NE consists of the INM and ONM, separating chromatin and involved in nuclear processes and nucleic acid metabolism.
Nucleo-cytoskeletal bridging complexes, composed of SUN and KASH proteins, transmit mechanical forces.
The lamina is a nucleoskeletal layer at the NE periphery, consisting of filamentous proteins, with structural functions and involvement in nuclear processes.
The Nucleolus and Other Nuclear Bodies
Sub-nuclear, non-membrane-bound organelles.
Distinct foci (light and electron microscope); dynamic hubs/reaction sites/modification sites containing core protein components specific to their functions.
Nuclear bodies are steady-state structures, kept together by protein-protein and protein-RNA interactions.
The nucleolus contains rDNA involved in the transcription of ribosomal RNA (rRNA) and assembly of ribosomal subunits.
It’s the accumulation site of various rRNAs, processing enzymes, and ribosomal proteins and particles.
Functions include:
RNA processing (precursor v mature RNA, including rRNA, snRNA, snoRNA and tRNA
Assembly of ribonucleoproteins (RNPs; e.g., signal recognition particle RNA, telomerase, splicing factor U6 snRNP).
Lamin B1 scaffolds the nucleolus externally in animals.
Cajal Bodies (CBs) are involved in the final assembly and modification of small nuclear and small nucleolar ribonucleoproteins (snRNPs and snoRNPs), and RNA recycling.
Nuclear speckles are storage and modification sites of mRNA splicing factors; e.g., snRNPs, kinases & phosphatase regulating splicing factors; hubs for gene activation/transcription and RNA maturation.
Polycomb bodies are hubs for gene repression/silencing and consist of Polycomb group (PcG) proteins.
Chromatin Structure, Function, and Epigenetics
DNA in a human cell is 2 meters long but needs to fit into a sphere of 6µm – packaging of DNA → organisation of DNA into chromatin.
Chromatin = DNA plus protein; proteins are either histones or non-histone chromosomal proteins.
Non-histone chromosomal proteins are involved in DNA repair, replication, transcription, and chromatin modifications.
Histones organize the basic structure of eukaryotic chromatin, forming the nucleosome.
The nucleosome contains 8 core histones: 2 of each H2A, H2B, H3, and H4; H3-H4 dimerize and then tetramerize, H2A-H2B dimerize, and come together as an octamer with 1.7 turns.
Negatively charged DNA backbone associates with positively charged histones - approx. 142 hydrogen bonds.
1 nucleosome approx. every 200 nt (147nt on 1 nucleosome + linker DNA)
N-terminals tail for modifications
Histone variants
Chromatin organisation
Partial or complete removal and replacement of histone core; approx. ever 1-2h (e.g. histone variant).
Nucleosomes stacked on top of each other to compact DNA further into 30nm chromatin fiber.
Core histone tails and linker histone H1 involved in compacting.
Histone – DNA association dynamic – DNA unwraps approx. 4 times a second – important for access to DNA.
ATP-dependent chromatin remodeling complexes associate with histones and DNA of nucleosome and lead to temporary structural change → weaken DNA-histone binding.
Complexes at least 10 subunits; many different types.
Access to DNA; with chaperons replacement of histones.
Two types of chromatin:
Heterochromatin (highly condensed, repressed, gene-poor).
Euchromatin (less condensed, active, gene-rich).
Heterochromatin includes centromeres and telomeres; some of it located at the nuclear periphery; few genes or inactive genes.
Position effect – euchromatin next to hetero- chromatin can be silenced (translocation).
Some heterochromatin is present for the life of the cell, while some is only present for short periods depending on development and cell type.
Chromatin changes (heterochromatin ↔ euchromatin) occur through histone modifications, histone variants, Reader-Writer complexes, and barrier action; specialised chromatin structures.
Chromatin changes and structures can be inherited → epigenetics.
Histone Modifications
Common histone modifications are methylation, phosphorylation, and acetylation.
Modifications are commonly on arginine, serine, and lysine.
Specific enzymes add (writer enzymes) and remove modifications (eraser enzyme) (e.g., histone acetyl transferase and histone deacetylase).
Recruitment is dependent on transcription factors; the modification can stay in place and be inherited.
Modification either changes the DNA-histone interaction in the nucleosome or recruits proteins to alter chromatin structure or effect transcription.
For example, trimethylation of H3 lysine recruits H1 → further compaction of DNA and spread of heterochromatin.
Protein complexes recognizing specific markings are reader complexes.
Writer enzymes that add modifications can interact with reader complexes in the same protein complex, forming reader-writer complexes → spreading of the mark.
Multiple readers and writers exist for multiple markings; they can interact with each other.
ATP-dependent chromatin remodeling complexes are often associated.
They can either decondense (make DNA/genes accessible → gene activation or DNA repair) or condense (silencing, heterochromatin).
Eraser enzymes can also be part of reader complexes.
Barrier sequences stop the spread of heterochromatin markings.
Linked to barrier proteins; prevent access of reader- writer to nucleosome; can be linked to physical features (NPC, INM proteins, and lamins).
Barrier proteins can also be writer complexes that add strong markings opposing the spread or contain eraser complexes that delete signals for spread.
Examples of Writer and Eraser Enzymes
Writer enzymes:
Kinase
Histone Acetyltransferase
Histone methyltransferase
Eraser enzymes:
Phosphatase
Histone Deacetylase
Histone Demethylase
Histone variants and modifications act together, forming a highly diverse system for “marking” DNA → histone code.
Specific markings elicit specific responses, e.g., newly replicated DNA, damaged DNA, change in transcription.
Alternative splicing leads to histone variants; there are no variants for H4; variants are less well conserved and fewer are present than core histones; modifications on variants can be long-lasting.
Centromeres are an example of specialized chromatin structures.
They have a repeat sequence-rich DNA.
Histone H3 variant CENP-A is present.
In yeast, there is just one nucleosome and 125nt to make a centromere; in animals and plants, it is much larger (thousands of nts and nucleosomes).
A chromosome is one chromatin fiber; it is present in a highly condensed form during cell division and a relaxed state in interphase.
In interphase, chromosomes are folded into chromatin loops.
In interphase, chromosomes occupy chromosome territories but are dynamic.
Interphase chromatin folding into loops and coils is dynamic/changing.
Molecular mechanisms are unclear, but nuclear matrix proteins are involved that form a scaffold.
Loops decondense to allow access to DNA → transcription.
Heterochromatin is at the periphery, and active genes are more central.
Structural features of nuclei are involved in spatial chromosome organisation (NE, NPC, nucleolus), creating distinct biochemical environments by concentrating proteins/enzymes.
Repair factories and replication factories are organised by soluble lamins.
Summary of Chromatin
NE delineates the nucleus and is a permeable barrier; NPC mediate exchange of molecules.
Active transport requires RanGTPase, karyopherins, and NLS/NES.
The NE contains nucleo-cytoskeletal bridging complexes, involved in nuclei shape, movement, chromosome movement; the lamina is a structural feature associated with the NE.
Sub-nuclear organelles of various sizes, numbers, and functions are involved in RNA and DNA metabolism.
Nuclear structures (NE, NPC, lamina, nuclear organelles) are involved in chromatin organisation.
Chromatin = DNA+protein; histones package DNA into nucleosomes, histone code effects chromatin structure and gene expression.
Chromatin is packaged into chromosomes, organised into loops in interphase, occupies territories and is dynamic.
Histone code/chromatin structures can be inherited.
H3-H4 tetramere passed on to sister DNA helices in replication fork.
Markings are retained and spread by reader-writer complexes.