Exam 3- Ch. 10 The Nucleus

The Nucleus

Nuclear Envelope and Traffic

  • The nuclear envelope regulates traffic between the nucleus and cytoplasm.

Chromosome Organization

  • The nucleus organizes chromosomes for DNA replication and segregation.

Nuclear Bodies

  • Nuclear bodies are structures within the nucleus involved in RNA processing, gene regulation, and ribosome assembly.

Introduction to the Nucleus

  • The nucleus differentiates eukaryotic from prokaryotic cells.
  • It serves as the repository of genetic information and the cell’s control center.
  • DNA replication, transcription, and RNA processing occur within the nucleus.
  • Translation, the final stage of gene expression, occurs in the cytoplasm.

Nuclear Functions

  • The nuclear envelope separates the genome from the cytoplasm.
  • It allows unique regulation mechanisms in eukaryotes.

Regulation of Gene Expression

  • Prokaryotic mRNAs are translated while transcription is ongoing.
  • Eukaryotic mRNAs undergo posttranscriptional processing before transport to the cytoplasm.
Posttranscriptional Processing
  • Gene expression in eukaryotes is regulated by posttranscriptional mechanisms (e.g., alternative splicing).
  • The nuclear envelope limits access of selected proteins to genetic material.
Importance of Nucleus in Gene Expression Regulation
  • The nuclear envelope provides opportunities for transcriptional control.
  • Regulated transport of transcription factors from the cytoplasm to the nucleus is an example.

Structure of the Nucleus

  • The nucleus consists of: nuclear membrane (karyotheca), nuclear sap/karyolymph/nucleoplasm, chromatin network or fibers, and the nucleolus.
  • The inner nuclear membrane is lined by the nuclear lamina (major component of the nucleoskeleton), which serves as an attachment site for chromatin.

Structure and Components of the Nuclear Envelope

Nuclear Envelope
  • Double membrane barrier.
  • Enriched in membrane proteins binding the cytoskeleton.
  • The outer membrane is continuous with the rough ER.
  • The inner membrane contains about 60 specific integral membrane proteins and is enriched by the nuclear lamina.
Nuclear Pore Complexes (NPCs)
  • Large protein channels for selective transport via importin and exportin.
  • Regulate import/export of proteins, RNA, and other molecules.
Nucleoplasm
  • Gel-like substance inside the nucleus.
  • Contains enzymes, ions, nucleotides, and the nuclear matrix.
Chromatin
  • DNA + histone proteins.
  • Exists as euchromatin (active) and heterochromatin (inactive).
Nucleolus
  • Dense region for rRNA synthesis and ribosome assembly.

Structure of the Nuclear Envelope Components

  • Two nuclear membranes, nuclear lamina, and nuclear pore complexes constitute the structure.
Concentric Membranes
  • Two concentric membranes: inner and outer nuclear membranes.
  • The outer nuclear membrane is continuous with the endoplasmic reticulum.
Nuclear Pore Complexes
  • Sole channels allowing small polar molecules and macromolecules to pass.
Nuclear Lamina
  • Underlying the inner nuclear membrane, composed of lamin proteins (60-80 kDa) forming filaments.
  • Provides structural support to the nucleus.
  • Lamins are intermediate filament proteins that associate to form filaments via coiled-coil structures.
  • Connected to the inner nuclear membrane and chromatin.
  • Lamin assembly is facilitated by lipid addition (prenylation) and binding to specific proteins.
  • LINC complexes span inner and outer nuclear membranes, connecting to the cytoskeleton.
  • Lamins and lamin-associated proteins bind to chromatin.

Lamin filaments

  • The nuclear lamina of mouse fibroblasts visualized by cryo-electron tomography.
  • Lamin filaments are shown in yellow, and nuclear pore complexes in red.

Lamin assembly

  • Lamin polypeptides form dimers in which the central α-helical regions of two polypeptide chains are wound around each other.
  • Dimers associate to form tetramers, which then associate end-to-end to form filaments.

Lamins and Nuclear Connections

  • Lamins bind inner nuclear membrane proteins (e.g., emerin, lamin B receptor).
  • Connect the nucleus to the cytoskeleton via LINC complexes.
  • Interact directly with chromatin to help organize DNA.

Role of Lamins

Structural Support
  • Provides structural support to the nucleus and maintains nuclear integrity.
Chromatin Interaction
  • Lamins and associated proteins bind to chromatin.
  • Important for localizing nontranscribed heterochromatin to the periphery of the nucleus.
Regulation of Nuclear Shape
  • Lamins contribute to nuclear shape regulation and are essential for proper cellular function.
Connection to Cellular Processes
  • Lamins influence cellular processes by connecting the nucleus to the cytoskeleton.

Mutation on Lamin

Hutchinson-Gilford Progeria Syndrome (HGPS)
  • Caused by a mutation in the LMNALMNA gene, resulting in the production of a mutant form of Lamin A called progerin.
  • Progerin leads to premature aging, affecting various tissues and causing cardiovascular problems.
Emery-Dreifuss Muscular Dystrophy (EDMD)
  • Often caused by mutations in the LMNALMNA gene, resulting in muscle weakness and wasting, joint deformities, and cardiac conduction defects.
  • Mutations may affect the interaction of lamins with the inner nuclear membrane and chromatin.
Charcot-Marie-Tooth Disease (CMT)
  • Some forms of CMT are associated with mutations in the LMNALMNA gene, leading to peripheral neuropathy affecting the peripheral nerves.

Nuclear pore

  • Main gateway for exchange between the nucleus and cytoplasm.
  • Large structure (~120 million daltons in humans), built from ~30 nucleoporins (NUPs).
  • Allows passive diffusion for small molecules (<40kDa<40 kDa) and facilitated transport for larger proteins and RNAs (via transport receptors).
  • Regulates selective molecular traffic.

Molecular traffic through nuclear pore complexes

Physiological Role
  • RNAs must be transported from the nucleus to the cytoplasm.
  • Nuclear proteins (e.g., transcription factors) need transport to the nucleus.
Transport Mechanisms
  • Passive diffusion: Small molecules/proteins (<40kDa<40 kDa).
  • Selective transport: Larger proteins and RNAs, guided by specific signals.

Structural organization - Nuclear pore complexes

  • Electron microscopy reveals an eightfold symmetrical structure organized around a large central channel.

The nuclear pore complex

  • Made of eight spokes connected to rings on the cytoplasmic and nuclear sides.
  • The spoke–ring structure surrounds a central transport channel.
  • Cytoplasmic filaments extend from the cytoplasmic ring, and nuclear basket filaments extend from the nuclear ring.
  • The central channel is lined by FG-NUPs (rich in phenylalanine and glycine) to regulate transport.

Selective transport of proteins to and from the nucleus

  • Millions of macromolecules cross the nuclear envelope every minute.
  • Proteins Responsible for Genome Function: include histones, DNA polymerases, RNA polymerases, transcription factors, splicing factors, etc.
  • Targeted to the nucleus by nuclear localization signals (NLS).
Selective Molecular Traffic
  • Alan Smith and colleagues identified the nuclear localization signal in simian virus 40 (SV40) T antigen in 1984.
  • Mutation of a lysine residue affected nuclear import.
Characteristics of NLS
  • Short stretches rich in basic amino acids (lysine and arginine).
  • Bipartite NLS example in nucleoplasmin.
Transport Receptors: Key Features
  • Importins recognize classical NLS and facilitate nuclear import.
  • GTP-binding protein Ran controls directionality through the nuclear pore.
  • Transport is selective, signal-dependent, and energy-driven.
  • Essential for regulation of gene expression, cell cycle, and stress.

Nuclear import and export

Import into the Nucleus
  • Nuclear Localization Signal (NLS): Short amino acid sequence on cargo proteins.
  • Importins: Recognize NLS and transport proteins into the nucleus.
  • Transport driven by the Ran-GTP cycle.
Export from the Nucleus
  • Nuclear Export Signal (NES): Specific sequence on proteins or RNA.
  • Exportins: Bind NES-tagged molecules for export.
  • Export also regulated by Ran-GTP.

Protein import through the nuclear pore complex

  • The Ran-GAP promotes hydrolysis of GTP by Ran.
  • The Ran-GEF promotes the exchange of GDP for GTP on Ran.
  • The Ran-GAP is cytoplasmic, whereas the Ran-GEF is located in the nucleus.

Nuclear export

  • Cargo proteins with Nuclear Export Signals (NES) bind to exportins and Ran-GTP in the nucleus.
  • The complex moves through the nuclear pore complex (NPC).
  • In the cytoplasm, RanGAP triggers GTP hydrolysis → Ran-GDP.
  • Cargo and exportin are released into the cytoplasm.
  • Ran-GDP and exportin return to the nucleus.
  • Ran-GTP is regenerated by RanGEF bound to chromatin.

Transport of RNAs: mRNA Export

  • Exported as messenger ribonucleoprotein complexes (mRNPs).
  • Mediated by the NXF1/TAP transport pathway, with energy provided by RNA helicases.
tRNA and miRNA Export
  • Exportin-t → transports tRNAs.
  • Exportin-5 → transports miRNAs; Requires Ran-GTP.
rRNA Export
  • Ribosomal RNAs combine with proteins to form 40S and 60S subunits.
  • Exported via Exportin Crm1, with nuclear export signals (NES) on ribosomal proteins aiding export.
  • RNA export is selective, signal-dependent, and energy-driven.

mRNA Export

  • Distinct mechanism independent of karyopherins and Ran.
  • Pre-mRNAs associated with a complex of at least 20 proteins.
  • mRNA forms mRNPs (messenger ribonucleoproteins).
  • Exported via NXF1/TAP pathway through nuclear pores, requiring energy and RNA helicases.
  • Only processed mRNAs (capped, spliced, poly-A) are exported.

Transport of snRNAs between nucleus and cytoplasm

  • snRNAs are exported to the cytoplasm by Exportin Crm1.
  • In the cytoplasm, they assemble with proteins to form snRNPs.
  • snRNPs are recognized by Snurportin and imported back into the nucleus.
  • Final maturation happens in Cajal bodies; Transport is Ran-GTP dependent.

Regulation of nuclear protein transport

  • Both protein import and export are regulated.
  • Cells use nuclear transport to regulate functions, including transit through the cell cycle and response to external stimuli.
  • The movement of the transcription factor NF-κB illustrates how nuclear transport is regulated.

Regulation of nuclear import of transcription factors

  • NF-kB, IKB, NLS, Importin, Pho4.
  • Involves phosphorylation and proteolysis of lκB.
  • Involves dephosphorylation.

Review on Chromosome Organization

Introduction
  • DNA is packaged into chromosomes inside the nucleus.
  • Humans have 46 chromosomes (23 pairs).
  • Chromosomes ensure accurate DNA replication and segregation.
Levels of Chromosome Organization
  • DNA Double Helix (2 nm)
  • Nucleosomes (10 nm): DNA wrapped around histone proteins.
  • 30 nm Fiber: Nucleosomes coiled into a thicker fiber.
  • Chromatin Loops (300 nm): Looped domains attached to scaffold proteins.
  • Condensed Chromosome (700 nm per chromatid during mitosis).
Chromatin Types
  • Euchromatin: Lightly packed, transcriptionally active.
  • Heterochromatin: Densely packed, transcriptionally inactive; found at centromeres and telomeres.

Chromosome Territories

  • Each chromosome occupies a specific region (territory) within the nucleus.
  • Territories are organized to prevent entanglement and facilitate gene regulation.
  • Active genes are often located at the edges of territories for easier access.
  • Chromosome positioning influences transcription, replication, and repair.

Chromosome organization

  • Chromosomes occupy distinct territories, with centromeres and telomeres anchored to opposite sides of the nuclear envelope.

Chromatin localization and transcriptional activity

  • Euchromatin (active) is positioned toward the nuclear interior.
  • Heterochromatin (inactive) is found near the nuclear envelope or nucleolus.
  • Inactive heterochromatin binds to lamins and inner nuclear membrane proteins.
  • The nucleolus is surrounded by heterochromatin.
  • Spatial chromatin organization supports gene regulation, replication, and DNA repair.
  • Euchromatin is toward the interior, while heterochromatin is near the periphery, influencing gene expression, DNA replication, and repair.

Heterochromatin in interphase nuclei

  • During interphase, heterochromatin remains highly condensed and is transcriptionally inactive.
  • The euchromatin is decondensed and distributed throughout the nucleus.

Organization of Drosophila chromosomes

  • A model of the nucleus, showing the five chromosome arms in different colors.
  • The positions of telomeres and centromeres are indicated.
  • The two arms of chromosome 3 are shown to illustrate the topological separation between chromosomes.

Organization of chromosomes in the mammalian nucleus

  • Copies of chromosome 4.

Fluorescence in situ hybridization to transcriptionally active and inactive chromosomes

  • Fluorescence in situ hybridization with probes for the gene-rich chromosome 19 (red) and the gene-poor chromosome 18 (green).
  • Hybridization with probes spanning the entire mouse chromosome 2 (green) or only the exon sequences of that chromosome (red).

Replication and transcription factories

Replication factories
  • Discrete sites where DNA replication occurs, with multiple replication forks grouped together.
  • Contain DNA polymerases, helicases, and other replication factors.
Transcription factories
  • Specific nuclear regions with high concentrations of RNA polymerases and transcription factors.
  • Actively transcribing multiple genes at once, which helps coordinate gene expression efficiently.

Distribution of transcriptionally active and inactive chromatin

Chromatin Domains in the Nucleus
  • Inactive heterochromatin loops associate with the nuclear lamina (LADs) or nucleolus (NADs).
  • Active chromatin domains are located in the nuclear interior.
  • Domain boundaries are organized by CTCF and cohesin.

Genome organizational domains

Nucleolus-Associated Domains (NAD)
  • Chromatin regions associated with the nucleolus.
  • Gene-poor regions involved in ribosomal RNA production, important for ribosome biogenesis.
Lamin-Associated Domains (LAD)
  • Chromatin regions associated with the nuclear lamina.
  • Typically contain repressed or inactive genes.
  • Involved in maintaining heterochromatin structure and gene silencing.
Topologically Associating Domains (TAD)
  • Large chromatin regions where DNA regions within the domain interact more frequently.
  • Boundaries defined by proteins like CTCF, playing a key role in gene regulation and organization.

Replication factories

  • Newly replicated DNA was labeled by a brief exposure of cells to bromodeoxyuridine (BrdU), which is incorporated into DNA in place of thymidine. This substitution allows detection of newly synthesized DNA by immunofluorescence following staining with an antibody against BrdU. Note that the newly replicated DNA (red) is present in discrete clusters distributed throughout the nucleus.

Nuclear Bodies

  • Nuclear bodies are membrane-less, dynamic structures found inside the nucleus, formed and maintained by protein-protein and protein-RNA interactions.
  • They organize specific nuclear activities like RNA processing, gene regulation, and ribosome assembly.

Nucleus and Nuclear bodies

  • Includes list of several nuclear bodies such as Cajal Body, Fibrillar Center, Dense Fibrillar Component, Granular Component, Perinucleolar Chromatin, Perinucleolar Compartment, Nucleolus, Polycomb Body, Paraspeckle, Nuclear Speckle, PML Nuclear Body, Clastosome, Nuclear Lamina, Histone Locus Body, Nuclear Membrane, Chromosome Territory, Nuclear Pore Complex.

Examples of Nuclear Bodies

Nucleolus
  • Function: rRNA transcription, processing, and ribosome assembly
  • Number per nucleus: 1-4
Cajal body
  • Function: snRNP assembly
  • Number per nucleus: 0-10
Clastosome
  • Function: Proteasomal proteolysis
  • Number per nucleus: 0-3
Histone locus body
  • Function: Transcription and processing of histone pre-mRNAs
  • Number per nucleus: 2-4
Speckle
  • Function: Storage of pre-mRNA splicing factors
  • Number per nucleus: 20-50
PML body
  • Function: Transcriptional regulation, DNA repair
  • Number per nucleus: 10-30
Polycomb body
  • Function: Gene silencing
  • Number per nucleus: 10-20

The Nucleolus and rRNA

  • The nucleolus is a non-membrane-bound structure composed of proteins and nucleic acids found within the nucleus.
  • The nucleolus is associated with the genes for ribosomal RNAs and is the site of rRNA transcription, rRNA processing, and ribosome assembly.
  • Processing of pre-rRNA is mediated by small nucleolar RNAs (snoRNAs).

Structure of the nucleolus

  • Organized into three main regions:
Fibrillar Center (FC)
  • Contains inactive rRNA genes
Dense Fibrillar Component (DFC)
  • Site of early rRNA transcription and processing
Granular Component (GC)
  • Site of late-stage rRNA processing and ribosome subunit assembly

Ribosome Assembly

  • Transcription of rRNA genes by RNA Polymerase I (28S, 18S, and 5.8S rRNAs).
  • Processing and modification of pre-rRNA into mature rRNA.
  • Ribosomal proteins are imported into the nucleolus and assemble on pre-rRNA before cleavage.
  • As pre-rRNA is processed, more ribosomal proteins and 5S rRNA join to form pre-ribosomal particles.
  • Formation of pre-ribosomal subunits (40S and 60S).
  • Export to the cytoplasm for final maturation

Ribosome assembly

  • Ribosomal proteins, rDNA, Nucleolus, Pre-rRNA, 40S subunit, 60S subunit, Pre-ribosomal particles, 5S rRNA.

Ribosomal RNA gene

  • In higher eukaryotes, the primary transcript of rRNA genes is the 45S pre-rRNA.
  • The pre-rRNA is processed via a series of cleavages, which is similar in all eukaryotes.

Types of Eukaryotic Ribosomal RNA (rRNA)

  • Eukaryotic ribosomes consist of 5S, 5.8S, 18S, and 28S rRNAs.
  • The 5.8S, 18S, and 28S rRNAs are transcribed together within the nucleolus by RNA polymerase I, generating a 45S ribosomal precursor RNA.
  • The 45S pre-rRNA undergoes processing to yield the 18S rRNA of the 40S ribosomal subunit and the 5.8S and 28S rRNAs of the 60S ribosomal subunit.
  • The 5S rRNA, also part of the 60S subunit, is transcribed outside the nucleolus by RNA polymerase III.
  • Cells contain multiple copies of rRNA genes, with the human genome having approximately 200 copies for 5.8S, 18S, and 28S rRNAs and around 2000 copies for 5S rRNA.

Polycomb bodies: Centers of transcriptional repression

Polycomb Proteins and Transcriptional Repression
  • Responsible for gene repression via histone H3 lysine 27 methylation.
  • Two complexes: one binds to methylated H3 lysine 27, the other methylates adjacent residues.
Methylation Spreading
  • Methylation of H3 lysine 27 spreads to adjacent nucleosomes, creating repressed chromatin domains.
Polycomb Protein Localization
  • Concentrated in specific nuclear domains known as Polycomb bodies, revealed by immunofluorescence.

Polycomb bodies

  • Polycomb bodies are often associated with heterochromatin, aligning with their gene repression role.

Cajal bodies and Nuclear speckles

Cajal Bodies
  • Function: RNA processing (snRNA, snoRNA, snRNP assembly, RNA splicing, and telomere maintenance).
  • Structure: Membrane-less bodies with small nucleolar RNAs (snoRNAs) and proteins.
  • Location: Found in the nucleus near active transcription sites.
  • Key Role: Essential for spliceosome assembly and RNA modification.
Nuclear Speckles
  • Function: Store and regulate pre-mRNA splicing factors.
  • Structure: Dense regions containing splicing-related proteins and snRNPs.
  • Location: Dispersed in the nucleoplasm, often near active genes.
  • Role: Play a role in RNA splicing and gene expression regulation.

Cajal bodies

  • Small nuclear bodies involved in snRNP and RNA-protein complex assembly.
  • snRNAs exported to the cytoplasm, form snRNPs, and return to the nucleus.
  • Cajal bodies concentrate snRNPs for final maturation stages.
  • Maturation involves ribose methylation, pseudouridylation, and scaRNAs.
  • Fibrillarin, responsible for RNA methylation, concentrated in Cajal bodies and nucleoli.
  • Cajal bodies contain Cajal body-specific RNAs (scaRNAs), which guide ribose methylation and pseudouridylation of snRNAs.

Cajal bodies in the nucleus

  • Differential interference contrast microscope image of the nucleus of a human cell.
  • Immunofluorescent staining of the same nucleus with antibodies to the proteins coilin (green) and fibrillarin (red).
  • Fibrillarin (the protein responsible for methylation of RNAs) is present in both the nucleoli and in the Cajal bodies.
  • Coilin (a major Cajal body protein) is detectable only in the Cajal bodies.

Nuclear speckles

  • Staining with immunofluorescence antibodies indicates that splicing factors are concentrated in discrete bodies within the nucleus, termed nuclear speckles.

Nuclear speckles

  • Cajal bodies may aid in telomerase assembly.
  • Telomerase is crucial for replicating chromosomal DNA ends.
  • After assembly in Cajal bodies, snRNPs transfer to speckles.
  • Speckles are discrete nuclear bodies with mRNA splicing machinery components.
  • Speckles concentrate splicing factors and snRNPs and are storage sites for splicing components, recruited to actively transcribed genes for pre-mRNA processing.
  • Cajal bodies may facilitate assembly and delivery of the telomerase complex.

CRISPR CAS9 - Genome editing tool

  • CRISPR is a Genome editing tool.

CRISPR vs RNAi vs TALENS

  • Diagram comparing CRISPR, RNAi, and TALENS.

Principle of CRISPR-Cas9

  • CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas9 (CRISPR-associated protein 9) are part of a bacterial defense mechanism.
  • Cas9 is an endonuclease that cuts DNA at a specific location, guided by a custom RNA sequence (guide RNA or gRNA).
  • Guide RNA (gRNA) directs Cas9 to the target DNA sequence.
  • DNA cleavage: Cas9 induces a double-strand break, which is repaired by the cell’s DNA repair machinery, often leading to gene disruption or modification.