Sessions #68-69: The Nucleus - Structure, Organization, and Transport

What are the specific genes contained in the chromosomal regions within the nucleolus?

(1) 5.8S rRNA (2) 18S rRNA (3) 28S rRNA

What are the four types of RNAs found in the eukaryotic ribosomes?

(1) 5S (2) 5.8S rRNA (3) 18S rRNA (4) 28S rRNA

How are snRNAs exported out of the nucleus?

*Cm1* binds to the snRNA to transport it from the nucleus to the cytoplasm via the nuclear pore

What is the difference between importins and exportins?

*Importins:* - Move cargo into the nucleus - Uses Ran/GTP to detach from the cargo *Exportins:* - Move cargo outside of the cell - Uses Ran/GTP to attach the cargo

What is the difference between the inner and outer nuclear membranes?

*Inner* - Adjacent to the nuclear lamina, proteins specific to the nucleus *Outer* - Continuous with the endoplasmic reticulum, has ribosomes

How does Ran-GDP get back into the nucleus?

*NTF2* binds to Ran/GDP in the cytoplasm and transports it to the nucleus through the nuclear pores.

How are snRNAs imported into the nucleus?

*Snurportin* binds to the snRNP (a complex of snRNA with binding proteins) and transports it from the cytoplasm to the nucleus via the nuclear pore

CaaX box

- (Cysteine-Aliphatic-X) removed from lamin A post-translationally by proteolysis to form - Helps create lipid anchor to nuclear envelope

Pho4

- A transcription factor found in yeasts that exists in the cytoplasm in a phosphorylated (inactive) state. - The phosphate group blocks the nuclear localization sequence (NLS)

NF-kB

- A transcription factor that maintains in the cytoplasm as an inactive complex with IkB. - Controls and regulates the immune responses

How is NF-kB activated?

- By *phosphorylation of IkB* which becomes ubiquitinated and degraded in the proteasome - This unmasks the nuclear localization sequence (NLS) on NF-kB, allowing the import to the nucleus

What is the structure of the nuclear pore complex?

- Eight spokes around a central channel - Spokes attached to rings on the cytoplasmic and nuclear side of the nuclear envelope - Cytoplasmic filaments extend from the cytoplasmic ring - Filaments forming the nuclear basket extend from the nuclear ring

Lamin B

- Expressed in virtually all somatic cells - Remains associated with nuclear envelope throughout the cell cycle.

NTF2 (Nuclear Transport Factor 2)

- Import receptor for Ran/GDP - Binds Ran/GDP and transports it to the nucleus

IkB

- Inhibitor of NF-kB - Masks the nuclear localization sequence (NLS) in the cytoplasm

What is the function of the nuclear membrane?

- To act as a barrier that separates the contents of the nucleus from the cytoplasm. - Segregates transcription and translation machinery

Describe the process of nuclear import through the nuclear pore complex.

1. An *importin* binds to the *nuclear localization signal (NLS)* of a cargo protein in the cytoplasm 2. The *cargo/importin complex* binds to specific nuclear pore proteins in the *cytoplasmic filaments* 3. The complex is translocated through the nuclear pore by binding to more interior nuclear pore proteins. 4. The cargo and the importin dissociate, and the cargo is released into the nucleus 5. The importin binds to the Ran/GTP and is exported through the nuclear pore. 6. The GTPase-activating protein (*Ran GAP*) in the cytoplasmic hydrolyzes the GTP on Ran to GDP, releasing the importin so that it can bind to a new cargo protein.

What is the function of snoRNAs?

1. Cleavage of pre-rRNA 2. Direct the specific base modifications of pre-rRNA

What are the two types of heterochromatin?

1. Constitutive 2. Faculative

What are two of the inner nuclear membrane proteins that lamins bind to?

1. Emerin 2. Lamin B receptor

What are the three regions of the nucleolus?

1. Fibrillar Center 2. Dense Fibrillar Component 3. Granular Component

What are the components of the nuclear envelope?

1. Inner membrane 2. Outer membrane 3. Nuclear Lamina 4. Nuclear Pore Complexes

What are three modifications described for lamins?

1. Isoprenylation 2. Carboxyl methylation 3. Phosphorylation

What are two of the base modifications involved in the processing of pre-rRNA?

1. Methylation 2. Pseudouridylation

What are the two mechanisms that molecules can travel through the nuclear pore complex?

1. Passive diffusion 2. Active transport

What maintains the distribution of Ran/GTP across the nuclear envelope?

1. Ran GTPase-Activating Protein (Ran GAP) 2. Ran Guanine Nucleotide Exchange Factor (Ran GEF)

Describe the process of nuclear export through the nuclear pore complex.

1. The *exportin* binds to the *nuclear export signals (NES)* of a cargo protein in the nucleus 2. *Ran/GTP* then binds to the exportin-cargo complex and then this complex is exported via the nuclear pore complex 3. *Ran GAP* stimulates the hydrolysis of bound GTP, leading to the formation of Ran/GDP, and the release of the cargo protein and exportin into the cytoplasm 4. Exportin is then transported back into the nucleus.

What is 45S pre-rRNA spliced into?

1. The 18S rRNA in the 40S ribosomal subunit 2. The 5.8S and 28S rRNA in the 60S ribosomal subunit

What does the transcription of 5.8S, 18S, and 28S rRNA form?

45S ribosomal precursor RNA

Nuclear Envelope

A barrier that separates the contents of the nucleus from the cytoplasm and provides the structural framework of the nucleus.

Nucleolar Organizer Region (NOR)

A chromosomal region containing the genes for ribosomal RNA (rRNA); most often found in physical association with the nucleolus.

Ribonucleoprotein Complex (RNP)

A complex of ribonucleic acid and RNA-binding protein

Nuclear Lamina

A fibrous meshwork that provides structural support to the nucleus.

Exportin

A karyopherin that recognizes nuclear export signals and directs transport from the nucleus to the cytosol.

Importin

A karyopherin that recognizes nuclear localization signals and directs nuclear import.

Nuclear Pore Complex

A large structure forming a transport channel through the nuclear envelope.

Karyopherin

A nuclear transport protein

Ran GTPase-Activating Protein (Ran GAP)

A protein in the cytoplasm that helps maintain the unequal distribution of Ran/GTP across the nuclear envelope. It stimulates the hydrolysis of GTP bound to Ran.

Ran Guanine Nucleotide Exchange Factor (Ran GEF)

A protein in the nucleus that helps maintain the unequal distribution of Ran/GTP across the nuclear envelope. It stimulates the exchange of GDP for GTP.

Nuclear Transport Receptors

A protein that recognizes nuclear localization signals and mediates transport across the nuclear envelope.

Ran

A small GTP-binding protein involved in nuclear import and export

Rev Protein (HIV)

A transregulating protein that involves the export of unspliced and incompletely spliced mRNAs out of the nucleus.

Inner Nuclear Membrane

Adjacent to the nuclear lamina

Nuclear Localization Signals

An amino acid sequence that targets proteins for transportation from the cytoplasm to the nucleus.

Nuclear Export Signals

An amino acid sequence that targets proteins for transportation from the nucleus to the cytoplasm.

Cm1

An exportin with leucine-rich nuclear export signal that transports snRNA from the nucleus to the cytoplasm.

Snurportin

An importin that brings snRNPs from the cytoplasm to the nucleus.

What types of amino acids are commonly found in the nuclear localization sequence?

Basic - arginine and lysine

How does Ran regulate the movement of nuclear transport of cargo proteins?

By controlling the activity of the nuclear transport receptors.

Constitutive Chromatin

Chromatin that contains DNA sequences that are *generally not transcribed*.

Faculative Heterochromatin

Chromatin that contains DNA sequences that are not transcribed in the cell being examines, but are transcribed in other cell types.

Fibrillar Center (FC)

Contains the genes (rDNA) required to make rRNA

Outer Nuclear Membrane

Continuous with the endoplasmic reticulum

What stimulates the fusion of the nuclear envelope to form a nuclear membrane during telophase?

Dephosphorylation of nuclear pore proteins and lamins A/B/C.

Lamin A

Encodes for lamin A and spliced lamin C without the CaaX box

What is the nuclear lamina composed of?

Fibrous proteins called lamins (intermediate filaments)

Dense Fibrillar Component (DFC)

Harbors pre-rRNA processing machinery (snoRNPs)

During mitosis, what is the state of the chromatin?

Heterochromatin

During interphase, what is the state of the chromatin?

Heterochromatin + Euchromatin

Heterochromatin

Highly condensed chromatin that is transcriptionally inactive.

What types of amino acids are commonly found in the nuclear export signal?

Hydrophobic ex. leucine

What is the protein family that functions as nuclear transport receptors?

Karyopherin

What component of the nucleus do lamins bind to?

Lamins bind to chromatin via histones H2A and H2B

What types of molecules pass through the nuclear pores by active transport?

Large molecules, such as proteins and RNA

Where are the enzymes for Ran that stimulate GTP hydrolysis to GDP?

Localized on the cytoplasmic side of the nuclear envelope

Where are the enzymes for Ran that exchange GDP for GTP?

Localized to the nuclear side of the nuclear envelope

Granular Component (GC)

Location of ribosomal assembly

Euchromatin

Loosely packed chromatin that is transcriptionally active.

Nuclear Membranes

Membranes forming the nuclear envelope 1. Inner 2. Outer

What is the purpose of 45 pre-rRNA after mitosis?

Newly transcribed 45S pre-rRNA acts as an organizing site for formation of new nucleoli after mitosis

Does the nucleolus have a membrane?

No

Does nuclear import require conformational changes (ex. folding) of substrate for translocation?

No, nuclear import does not require any conformational changes of the substrate for translocation.

Is the NLS sequence cleaved after import into the nucleus?

No, the NLS sequence is nos cleaved after import into the nucleus.

Do the amino acids that form the nuclear localization signals have to be immediately adjacent to each other?

No, the amino acids that form the nuclear localization signal are close together, but not immediately adjacent to each other.

What are the only channels in the nuclear envelope?

Nuclear Pores

Where does RNA polymerase transcribe 5.8S, 18S, and 28.S rRNA?

Nucleolus

Where does RNA polymerase transcribe 5S rRNA?

Outside the nucleolus

How is Pho4 activated?

Pho4 is dephosphorylated, and then binds to an importin to enter the nucleus.

What is the nuclear membrane made of?

Phospholipid bilayers

What stimulates the breakdown of the nuclear envelope in prophase?

Phosphorylation of nuclear pore proteins and lamins A/B/C.

What facilitates the association of lamins with the inner nuclear membrane?

Post-translational addition of lipid (prenylation of C-terminal cysteine residues)

Nucleoporin

Proteins that form the nuclear pore complex (8 subunits)

Which RNA polymerase transcribes 5.8S, 18S, and 28.S rRNA?

RNA Polymerase I

Which RNA polymerase transcribes 5S rRNA?

RNA Polymerase III

Rev Response Element (RRE)

Regions of introns of the virus' mRNA, in which *Rev protein* binds to.

Lamina-Associated Domains (LADs)

Regions where heterochromatin associates with nuclear lamins

Nucleolus-Associated Domains (NADs)

Regions where heterochromatin associates with periphery of nucleoli

Small Nucleolar RNAs (snoRNAs)

Small RNAs present in the nucleolus that function in pre-rRNA processing.

What types of molecules pass through the nuclear pores by passive diffusion?

Small molecules

Lamin C

Spliced version of lamin A

What happens when nuclear lamins are dephosphorylated?

The nuclear envelope fragments reform the nuclear membrane

What happens when nuclear lamins are phosphorylated?

The nuclear membrane breaks down

What is the function of the nuclear pore complex?

The nuclear pore complex is responsible for the selective traffic of proteins and RNAs between the nucleus and the cytoplasm.

Nucleolus

The nuclear site of rRNA transcription, processing, and ribosome assembly.

Chromosome Territory

The region within a nucleus occupied by a particular chromosome during interphase.

How does the distribution of Ran/GTP affect nuclear transport?

There is a high concentration of Ran/GTP in the nucleus and this determines the directionality of nuclear transport of cargo proteins.

What is the distribution of Ran/GTP and Ran/GDP across the nuclear envelope?

There is a high concentration of Ran/GTP in the nucleus. There is a high concentration of Ran/GDP in the cytoplasm.

What is the purpose of Rev protein attaching to the Rev Response Element (RRE)?

This attachment hides the introns of the HIV mRNA and allows it to be transported outside of the nucleus.

Lamins

Type V intermediate filaments proteins that form the nuclear lamina.

What is a bipartite nuclear localization sequence?

When the nuclear localization sequence is composed of two separate elements.