5. The Nucleus and Nuclear Transport

location of chromatin

inside nucleus

nucleolus

dense region in nucleus that is important for rRNA synthesis and ribosome assembly

nuclear envelope

formed by two membranes, inner and outer

nuclear pore complex

where the inner and outer membrane of the nuclear envelope join

what is the outer membrane of the nuclear envelope continuous with?

the ER

nuclear lamina

layer around the inside of the nuclear envelope composed of intermediate filaments that provides structure and support to the nucleus

proteins that associate the nuclear lamina with the nuclear envelope

proteins emerin and laminB receptor (LBR)

function of lamin A gene

this mutation is important for nuclear envelope stability

outcome of a mutation in lama A gene

Hutchinson Gilford progeria syndrome

nuclear pore complexes

the major pathway for transport inside/outside of nucleus

what diffuses freely across nuclear envelope?

small stuff, such as nucleotides

no input of energy needed

what must be actively transported across nuclear envelope?

larger stuff, such as proteins and RNAs

input of energy required

nuclear localization signals (NLS)

peptide sequences that function as a “zip code” for proteins, they are recognizes by cytosolic nuclear transport receptors

what happens when a prospective nuclear protein dissociates from the NLS when inside the nucleus?

the receptor is transported back to the cytosol

what happens to proteins without NLS?

the protein remains in cytosol

NLS structure

short sequence rich in basic amino acids (Lys and Arg)

small G protein RAN

regulates nuclear transport

RAN in cytosol

RAN-GDP

RAN in nucleus

RAN-GTP

importin

binds NLS on cargo protein

process of nuclear import

1. importin binds NLS on cargo protein, the complex enters the nucleus

2. RAN-GTP binds to complex causing a conformational change releasing the protein

3. RAN-GTP-importin complex exits nuclear pore, where when in the cytosol it interacts with RAN GAP

4. RAN GTP → RAN GDP

5. importin dissociates from RAN GDP

recycling of RAN-GDP to get RAN-GTP inside nucleus

1. RAN-GDP binds RANs import receptor

2. once inside nucleus, RAN exchanges GDP → GTP by RAN GEF

RAN GAP

when inside cytosol, RAN GTP → RAN GDP

RAN GEF

when inside nucleus, RAN GDP → RAN GTP

molecules involved with nuclear export of a protein

• RAN GTP

• exportin

• nuclear export signals (NES)

regulation of nuclear transport example

NFkB phosphorylation/dephosphorylation

NFkB

transcription factor regulating the immune response

NFkB when there is no infection

NFkB binds to IkB (an inhibitor), which blocks NLS

NFkB when there is an infection

• IkB is phosphorylated and degraded

• NLS on NFkB is recognized by importin

• NFkB-importin complex enters nucleus, which activates the genes to fight infection

heterochromatin

condensed DNA that is not actively transcribed

heterochromatin location

inner nuclear envelope and nucleolus

euchromatin

actively transcribed DNA

euchromatin location

distributed throughout nucleus

ribonucleoprotein complex (RNP)

• how RNA gets out of the nucleus

• mRNA’s + escorter complex export out of the nucleus

• helicase prevents re-import of mRNA

snRNA synthesis

• made in nucleus

• exported to cytosol by exportin Crm1

• picks up proteins and returns to nucleus functional

snRNA function

mRNA splicing

chromosome organization

chromosomes have their own location in the nucleus, not random

replication factories

large complexes where DNA replication takes place

nuclear bodies

non-membrane bounds organelles within the nucleus that concentrate proteins and RNAs for specific processes

ex: nucleolus

three regions of the nucleolus

1. fibrillar center (FC)

2. dense fibrillar component (DFC)

3. granular component (G)

transcription of ribosomal RNA (5.8S, 18S and 28S)

transcribed as a single unit in a nucleolus by RNA pol I, yielding a 45S ribosomal precursor RNA

transcription of ribosomal RNA (5S)

occurs outside nucleolus and catalyzed by RNA pol III

how many copies of rRNA genes in cells?

multiple

processing of pre-RNA

processed by a series of cleavages

small nucleolar RNA (snoRNA)

modified the base of pre-rRNA

snoRNP

a complex of snoRNA with proteins, involved in directing base modification of pre-rRNA such as methylation by base pairing

assembly of ribosomes

• ribosomal proteins are imported from the cytoplasm to the nucleolus where they assemble with pre-rRNA prior to cleavage

• 5S rRNAs are assembled into pre-ribosomal particles elsewhere in the nucleolus

• additional ribosomal proteins and 5S rRNA are incorporated as cleavage and processing proceeds

• the two nascent ribosomal subunits are exported to cytoplasm

introns

regions of initial RNA transcript not expressed and removed by splicing

exons

joined together in mature mRNA

spliceosome

splices out pre-mRNA introns

cajal bodies

site of snRNP storage and maturation

proteins present in cajal bodies

coilin and fibrillarin

nuclear speckles

mRNA splicing occurs here, where the splicing machinery is transferred

where do mature snRNPs transfer from/to

from: cajal bodies

to: nuclear speckles

PML bodies

interact with chromatin, sites of accumulation of transcription factors, chromatin-modifying proteins and DNA repair enzymes

active origins of replication (ori)

a few thousand ori are active at any time, but they are located in a few hundred discrete clusters