Chap 10: The NUCLEUS

What processes go in the nucleus?

DNA replication, Transcription, Splicing / RNA processing

What is transported out to the nucleus?

mRNA, miRNA, nucleasRNA, smRNA, ribosomes (made in the nucleolus)

What does the nuclear envelope separate?

Nuclear contents from the cytoplasm

How are the inner and outer membranes of the inner envelop joined at?

Nuclear Pore Complexes (NPCS)

What happens if nuclear pore complexes are mutated?

Progeria

Perinuclear space

Space between the inner and outer nuclear membranes

What is the perinuclear space directly connected with?

Lumen of the ER 

What are the fibrous proteins called in the nuclear lamina?

Lamins

2 lamins form what?

a dimer that extend to a alpha helical region = coiled coil

What are the types of lamins that animal cells have?

A and B 

What is A type lamins?

only expressed in differentiated cells, determine stiffness of nuclear nevelope.

• high levels = stiff nuclear envelope

• lower levels = softer & flexible nuclear envelope

What are lamins B1 and B2 encoded by?

LMNB1 & LMNB2

What is A type lamins encoded by?

LMNA gene

• lamin c has shorter c-terminus due to splicing

What does emerin bind to?

Lamin B receptor

• directly connected to cytoskeleton by protein complex LINC 

• - inner and outer nuclearmembranes 

Do lamins and lamin associated proteins bind to…

chromatin

What passes through the nuclear pore complexes channels

small polar, ions and macromolecules

• proteins and rnas

In vertebrates, the NPC is composed of 

30 proteins

• Nucleoporins 

Lamin A gives …

structural support to the nuclear lamina

Link complex is…

SUN and KASH

How are proteins transported to and from the nucleus?

Through nuclear localization signals

What are nuclear localization signals recognized and bound by?

Nuclear transport receptors

• direct protein transport through the nuclear pore complex

Nuclear localization signal is composed of 2 separated elements it is called..

bipartite 

What is monopartite

nuclear localization signal that is composed of a single, uninterrupted stretch of amino acids

NLS is… 

biparite

What are importins?

Proteins with NLS and bound by nuclear transport receptors

• importing protein into the nucleus from the cytoplasm through the NPC

What is RAN

protein-protein interactions with G nucleotide biding protein

• gtp bidning prtein and controls direction of movement

RAN GAP is what

gtpase activating protein that is found in the cytoplasm that hydrolyzes gtp to gdp

Where is there more RanGTP ?

IN the nucleus

Where is there more RAN GDP

In the cytoplasm

RanGEF is only found where?

This changes gdp to gtp,

only found in the nucleus

What is Nuclear Export Signals right in?

Hydrophobic amino acids, Leucine

What are nuclear export signals recognized by?

Receptor within the nucleus, EXPORTINS

Importins and exportins are part of the family of nuclear transport receptors known as..

karyopherins

NUCLEAR EXPORT OF PROTEINS:  What is exported out?

RAN GAP stimulates the hydrolysis of GTP

• forming RAN GDP 

• releases cargo protein and exporting out into the cytoplasm 

WHAT COMES BACK AFTER NUCLEAR EXPORT OF PROTEINS

Ran GDP and the exportin are then returned to the nucleus

• RAN GTP regenerated by RANGEF bound to chromatin

What are RNAs involved in?

Protein Synthesis

when mRNAs are transported out of the nucleus, do they involve karyopherins?

No they do not involve karyopherins, and is independent of RAN

mRNA export: mRNAs are bound by a group of proteins called…

exporter complex

• mediates their transport through the nuclear pore complex

• HELICASE ASSOCIATED WITH cytoplasmic face of the NPC 

Where do mRNAS, miRNAs, tRNAs, rRNAs function in?

the CYTOPLASM 

In what are snoRNAs involved in?

in RNA processing

What are snRNAs transported from the nucleus to the cytoplasm?

by the EXPORTING Crm1

In the Cytoplasm what do snRNAs associate?

with proteins to form snRNPs

Sequences present on the snRNP proteins are recognized by importin called..

snurportin

• transport back to the nucleus

What is a mechanism of regulation?

transcription factors associate with cytoplasmic proteins that mask their NLS;

EXAMPLE: NF-xb

Transcription factor NF-xB is activated in what type of cells?

Mammalian cells in response to a variety of extracellular signals 

If there is no signals in mammalian cells NFXB is a what type of complex?

inactive complex with inhibitory protein IXB in the cytoplasm

When IxB protein binds to the NF-xb nuclear localization signal what happens?

It masks it, preventing NF-xB from being transported to the nucleus

When there is extracellular triggers what happens to IxB?

it phosphorylates IxB

• when it is phosphorylated, the protein is recognized and ubiquinated by E3 ligase

• IXB protein gets degradated by the proteasom 

What happens when IxB (protein) gets lost through proteasome degradation?

NF-xB nuclear localization signal is accessible for recognition by IMPORTIN

=

nuclear import of NF-xB = activation transcription

What is the yeast transcription factor?

Pho4

What does Pho4 do?

Regulates genes that enable yeast cells to adapt when there are low levels of phosphate

What happens when phosphate levels are there adequately?

Activate a kinase that phosphoylates Pho4 at serine residue adjacent = inhibits recognition and binding by importin

What happens if there are sufficient phosphate levels (Pho4)

Pho4 is kept in the cytoplasm

If phosphate levels drop in yeast what happens to Pho4?

Dephosphorylation and nuclear import, where it activates target genes

Is Chromatin in random organization?

No, they have clusters

• territorial

What was the basic model studied in?

Polytene chromosomes in D. melanogaster

Region of chromosomes is called

Chromosome territory

What is FISH

Flourescence in situ hybridization

• labeled oligonucleotides complementary to repeated sequences on the chromosome

Interactions between different regions of chromosomes in cells have been analyzed by what

3C

What is chromatin in interphase cells called?

Euchromatin

• decondensed and active

What is Heterochromatin

highly condensed 

• not transcribed 

  • for stuctual integrity support

Where is Heterochromatin found?

around the nucleolus and below the nuclear envelope ; lamina pore ocmplex 

Where are the gene rich chromosomes located?

In the center of the nucleus

What is constitutive Heterochromatin

It is never transcribed, the telomeres are the protection of the chromosomes and the centromeres are meant to keep the sis chromatids togehter

What is facultative heterochromatin

Regions of the genome that become condensed depending where the genes are needed

What are TADs?

Topologically associated domain

• regions of dna within a chromosome that are maintained in association with itself

Boundaries of TADs contain what

binding sites for cohesin and CTCF

Heterochromatin is loaclized to the nuclear lamin by..

binding lamins and lamin b receptors

what are LADS

Laima associated domains

• region of heterochromatin associated with the nuclear lamina

• heterochromatin is inactive and is closer to the nucleolus

What is NAD: 

Nucleolus  associated domain 

• regions of chromatin associated with the nucleolus

Are replications random or concentrated?

They are concentrated at one spot

What do replication factories contain?

Clustered sites of DNA replication +proteins and enzymes

What are Transcription hubs?

They are in clustered sites 

• new synthesized RNA and highly active RNA polymerases and transcription factors 

What is a nucleolus?

Prominient nuclear body

• site for rRNA trancription

• transcribed RNA is processed

• RRNA PROCESSING AND RIBOSOME ASSEMBLY

Nucleolus is a what..

ribosome production factory W

Eukaryotic ribosomes have 4 RNA molecules?

5s

5.8s

18s

28s

rRNAs

Where are 5.8s, 18s, and 28s rRNAs transcribed?

In the nucleolus by RNA poly 1

making 45s precursor RNA

Where is the 45 pre-rRNA processed?

to the 18s rRNA of the 40s small ribosomal subunit

&

5.8s and 28s rRNAs of the 60S

Where is the 5s rRNA found?

in the 60S ribosomal subunit

Where does transcription of 5s rRNA take place?

Outside the nucleolus and catalyzed by RNA poly 3

Nucleolus is organized around the chromosomal regions that contain genes for.. 5.8s, 18s, and 28s, rRNAs

NUCLEOLAR ORGANIZING REGIONS

What are the 3 regions within the nucleolus?

Fibrillar center

dense fibrillar component

granular component

Genes encoding rRNA are localized where?

Fibrillar center

Where are the genes encoding rRNA transcribed at?

dense fibrillar component — where pre-rRNA is processed

Where does the assembly of ribosomal subunits take place?

Granular component of the nucleolus

What do small nucleolar rnas do? 

snoRNAs

function in pre-rRNA cleavage, ribose methylation, and pseudouridylation

Genes that encode ribosomal proteins are transcribed outside the nucleolus by..

RNA POLY 2 = creating mRNAs that are translated on the cytoplasmic ribosomes

Ribosomal proteins are transported from the cytoplasm to the nucleus where they are assembled with rRNAs to form…

pre-ribosomal particles

Where are genes for 5s rRNA transcribed?

Outside the nucleolus by RNA POLY 3 

• assembled into pre-ribosomal particles within the nucleolus 

Final stage of ribosomal subunit maturation would be..

exportation of pre-ribosomal particles to the cytoplasm creating

ACTIVE 40S AND 60S SUBUNITS

How is Ribosome Assembled?

1. Ribosomal RNA genes are transcribed at the interface between fibrillar center and dense fibrillar component of the nucleolus

2. Creating pre-rRNAs

3. Ribosomal proteins are imported to the nucleolus from the cytoplasm

4. Begin to assmble on pre-rRNA during their processing

5. Pre-rRNA is processed

6. 5srRNA assemble to form pre-ribosomal particles

7. Final step: exportation of pre-ribosomal particles to the cytoplasm

8. CREATING 40S AND 60S RIBOSOMAL SUBUNIts

What is involved in the 60s Ribosomal subunits?

28, 5.8, 5 s

What is involved in the 40s subunit?

18s

40s Subunit does what?

decodes the genetic message from mRNA

What does the 60s subunit do?

Catalyzes the formation of peptide bonds to create protein

Protein synthesis is what subunits?

40+60 = 80s ribosome

Nucleoli fuse to form what?

Nucleolus 

What is FG-NUPS?

The central channel is lined with FG-nups of the NUCLEAR PORE COMPLEXES

• phenylalanine + glycine repeats - disordered regions

• selective barrier that controls movement of macromolecules between nucleus and cytoplasm

• form filaments that extend from the cytoplasmic and nuclear rings

What does the nuclear pore complex consist of?

Assembly of 8 spokes (around a central channel) 

• connected to rings at the nuclear and cytoplasmic surfaces - spoke “ring assembly” is anchored within the nuclear envelope sites of fusion between inner and outer nuclear membranes 

• PROTEINS form filaments that extend both the cytoplasmic and nuclear rings = distinct basketlike structure on the nuclear side