Lec 12 - nucleo cytoplasmic transport

What separates transcription from translation in eukaryotic cells

The nuclear envelope separates nuclear genome/transcription machinery from cytoplasmic translation machinery.

What structure mediates traffic between nucleus and cytoplasm

Nuclear pore complexes (NPCs) spanning the nuclear envelope mediate bidirectional traffic.

Which NPC elements face the cytoplasm and nucleus respectively

Cytoplasmic filaments face the cytoplasm and the nuclear basket faces the nucleoplasm; NPCs also include cytoplasmic and nuclear rings and a central channel.

Which molecules passively diffuse through NPCs

Small molecules up to ~60 kDa (~10 nm) can diffuse; larger cargos need selective, active transport.

What encodes subcellular targeting information on proteins

Short amino acid “targeting sequences” (molecular zip codes) embedded in the protein sequence.

Name major post-translational vs co-translational routing hubs.

Post-translational targets include nucleus, mitochondria, and peroxisome; co-translational routing sends proteins into ER for the secretory/endolysosomal system.

Which transport receptor families move proteins through NPCs

Importins and exportins (collectively karyopherins) recognize NLSs and NESs on cargo.

What energy source powers directional nuclear transport

The Ran GTPase cycle and its nucleus–cytoplasm gradient provide directionality and energy coupling.

What ensures regulated access to DNA for transcription factors

Control of nuclear import/export regulates when transcription factors reach nuclear targets.

Which cargos typically require active transport

Folded proteins, RNPs (pre-ribosomal subunits, snRNPs), tRNA, pre-miRNA/miRNA, and mRNA complexes require transport receptors.

What is the key feature of a classical NLS

Enrichment in basic residues (lysine/arginine-rich short sequences).

What is a monopartite NLS

A single short basic stretch, e.g., SV40 large T antigen “PKKKRKV”.

What is a bipartite NLS

Two basic clusters separated by a spacer that come together upon protein folding (e.g., nucleoplasmin NLS).

Can an NLS function anywhere in the protein

Yes, NLS function is location-independent if it is accessible to importins.

Is the NLS removed after import

No, NLSs are not cleaved after import into the nucleus.

Do proteins need to unfold to pass through NPCs during import

No, nuclear import does not require unfolding; cargos are typically imported in folded states.

What kinds of NLS families exist beyond classical ones

PY-NLS, RS-NLS, arginine-rich NLSs, and atypical NLSs are recognized by specific importins/transportins.

Example of an arginine-rich NLS

HIV-1 Rev contains an arginine-rich NLS (e.g., “RQARRNRRRRWR”).

Example of an atypical NLS

Human rpL23a contains a complex NLS with multiple basic clusters within a longer sequence.

What experimental evidence showed an NLS is sufficient

Gold particles coated with NLS peptides were microinjected; particles localized through NPCs into nuclei over ~10–50 minutes.

What classic mutation demonstrated NLS necessity

A Lys→Thr mutation in SV40 T antigen NLS prevented nuclear accumulation, trapping it in cytoplasm.

What sequence hallmark helps distinguish NLS vs NES

NLSs are basic (Lys/Arg-rich) whereas NESs are typically leucine-rich hydrophobic clusters.

What is the hallmark of an NES

Short stretches of hydrophobic residues (often leucines) in specific patterns.

Provide a representative NES example.

“L-x(2,3)-L-x(2,3)-L-x-L” illustrates a leucine-rich export signal pattern.

Which exportin most commonly recognizes NES-bearing cargos

Crm1 (Exportin 1/XPO1) is the prototypical export receptor for proteins and some RNAs via adaptors.

Do all NESs look the same

No, multiple motif classes exist (e.g., class 1a–1d, 2–4) with hydrophobic spacing variations.

What are karyopherins

Importins and exportins that ferry cargos across NPCs by recognizing NLS or NES motifs.

What is Transportin (Karyopherin-β2/TNPO1) known to bind

PY-NLS motifs found in many RNA-binding proteins and mRNA processing factors.

What does Exportin-t (XPO3) export

tRNA and other structured RNAs.

What does Exportin-5 (XPO5) export

Pre-miRNA and some other RNAs.

What does CAS/XPO2 export

Importin-α re-export from the nucleus (Importin-α recycling).

What general cargo spectrum does CRM1/XPO1 handle

Broad protein cargos with NESs and RNAs via adaptors.

Where is Ran-GTP high vs low

Ran-GTP is high in the nucleus and low in the cytoplasm; Ran-GDP is high in the cytoplasm and low in the nucleus.

What establishes nuclear Ran-GTP

RCC1 (Ran-GEF) on chromatin exchanges GDP for GTP on Ran in the nucleus.

What maintains cytoplasmic Ran-GDP

Ran-GAP on the cytoplasmic side of NPCs stimulates GTP hydrolysis on Ran.

How does Ran-GTP control import

In the nucleus, Ran-GTP binds importins to displace cargo, promoting cargo release into nucleoplasm.

How does Ran-GTP control export

In the nucleus, Ran-GTP stabilizes exportin–cargo complexes for translocation to the cytoplasm.

How is Ran-GDP returned to the nucleus

NTF2 (a dedicated import receptor) imports Ran-GDP back into the nucleus for recharging by RCC1.

What is the key to transport directionality

The Ran-GTP/Ran-GDP gradient across the nuclear envelope.

Step 1 of classical import

Cytoplasmic importin binds a cargo’s exposed NLS.

Step 2 of classical import

Importin–cargo docks to FG-repeat nucleoporins and translocates through the NPC.

Step 3 of classical import

In the nucleus, Ran-GTP binds importin, displacing and releasing the cargo.

Step 4 of classical import

Importin–Ran-GTP recycles to cytoplasm, where Ran-GAP hydrolyzes Ran-GTP to Ran-GDP, freeing importin.

Is import post-translational

Yes, nuclear import occurs after translation in the cytosol.

Can an engineered NLS redirect a protein to the nucleus

Yes, NLSs are sufficient to drive nuclear import of heterologous proteins.

Step 1 of classical export

Nuclear Ran-GTP binds exportin, which binds NES-bearing cargo to form an export complex.

Step 2 of classical export

The Ran-GTP–exportin–cargo complex translocates through NPC to the cytoplasm.

Step 3 of classical export

Cytoplasmic Ran-GAP hydrolyzes Ran-GTP to Ran-GDP, releasing exportin and cargo.

How do exportins return to the nucleus

Exportins re-enter and reuse nuclear Ran-GTP for another cycle.

Which RNAs rely on exportins bound to Ran-GTP

tRNA, pre-miRNA/miRNA, rRNA and certain RNPs via appropriate exportins/adaptors (e.g., Crm1).

Is bulk mRNA export Ran-dependent

No, mRNA export is Ran-independent and uses the TREX (Transcription–Export) complex.

What ensures unidirectional mRNA export

A cytoplasmic RNA helicase at the NPC cytoplasmic face strips mRNA from export factors, preventing re-import.

What does TREX stand for

Transcription–Export complex that couples mRNA processing with export.

Do mRNPs contain proteins during export

Yes, mRNA is exported as an RNP and remodels upon cytoplasmic helicase action.

Name four nuclear bodies to recognize.

Nucleolus, Cajal bodies, nuclear speckles, and polycomb bodies.

Main function of nucleolus

Ribosome biogenesis (rRNA transcription, processing, and subunit assembly).

What do Cajal bodies do

Sites for assembling/storing snRNPs and telomerase components implicated in RNA processing.

What are nuclear speckles

Storage/accumulation sites for splicing factors associated with actively transcribed genes.

What are polycomb bodies

Repressive hubs enriched for histone methylation machinery linked to heterochromatin.

Where is heterochromatin often located

At the nuclear periphery (lamina-associated domains) and near nucleoli (nucleolus-associated domains).

How are active vs inactive chromatin positioned

Actively transcribed regions tend to occupy internal nucleoplasm; repressed regions cluster at lamina or nucleolus.

How can nuclear bodies be distinguished experimentally

By immunostaining known marker proteins (e.g., coilin for Cajal, fibrillarin for nucleolar components).

What are the three nucleolar subdomains

Fibrillar center (rDNA), dense fibrillar component (rRNA transcription), and granular component (ribosomal subunit assembly).

Where are rRNA genes located within the nucleolus

In fibrillar centers containing tandemly repeated rDNA.

Where does rRNA transcription occur

In the dense fibrillar component surrounding fibrillar centers.

Where does pre-ribosome assembly occur

In the granular component.

What polymerase transcribes the 45S pre-rRNA

RNA Polymerase I (Pol I) in the nucleolus.

What is the initial rRNA transcript

45S pre-rRNA that is processed into 18S, 5.8S, and 28S rRNAs.

What polymerase transcribes 5S rRNA

RNA Polymerase III (Pol III) in the nucleus but outside the nucleolus.

How many rDNA copies typically exist

Approximately ~200 copies distributed across several chromosomes.

What structural appearance reflects simultaneous rRNA transcription

“Christmas tree” morphology of tandemly transcribed rDNA units loaded with multiple RNA polymerases.

What guides pre-rRNA cleavage and modification

snoRNAs and snRNPs guide site-specific cleavages and modifications (methylation and pseudouridylation).

What is the functional benefit of pseudouridylation in rRNA

It increases rotational flexibility important for ribosome function.

What composes the 40S subunit

18S rRNA plus ~33 ribosomal proteins.

What composes the 60S subunit

5.8S rRNA, 28S rRNA, 5S rRNA plus ~40 ribosomal proteins.

Where are ribosomal proteins made

In the cytoplasm from mRNAs transcribed by Pol II.

How do ribosomal proteins reach the nucleolus

They are imported through NPCs (NLS-dependent) to assemble with rRNAs.

How do ribosomal subunits exit the nucleus

Pre-40S and pre-60S subunits are exported separately, commonly via Crm1.

What unique exporter handles mRNA during this process

The TREX complex mediates mRNA export independently of Ran.

What is NLS masking

A cytoplasmic inhibitor protein covers the NLS of a transcription factor, preventing import until a signal removes the inhibitor.

Example of NLS masking

NF-κB bound by IκB; upon IκB phosphorylation, NF-κB’s NLS becomes accessible for import.

What is modification-dependent NLS control

Phosphorylation of the TF near its NLS can block or allow import (e.g., yeast Pho4).

What is ligand-dependent nuclear import

Steroid hormones bind cytosolic receptors (e.g., glucocorticoid receptor), triggering conformational change/dimerization and import.

Why is transport regulation important

It times transcriptional programs to environmental cues and cellular needs by gating nuclear access.

What did the SV40 T antigen mutation show

Altering the NLS (Lys→Thr) abolished nuclear localization, proving NLS necessity.

What did NLS-coated gold particle microinjections show

NLS is sufficient to drive nuclear import of inert cargos through NPCs.

What key mechanistic insight emerged from these studies

Short amino acid signals encode nuclear targeting and are recognized by importins.

What mutated protein underlies Hutchinson–Gilford progeria syndrome (HGPS)

Lamin A with a mutation that removes the cleavage site needed after farnesylation; the mutant protein is called “progerin”.

Why is progerin clinically important

Progerin reflects a lamin A processing defect that disrupts nuclear lamina function and is a named target in clinical contexts and exams.

What modification drives nuclear envelope breakdown in mitosis

Phosphorylation of lamins triggers lamina disassembly; later dephosphorylation supports nuclear reassembly.

What happens if lamina phosphorylation/dephosphorylation cycles fail

Mitosis fails due to impaired envelope breakdown/reassembly and improper genome partitioning.

Where does a poly-A binding protein localize

Cytoplasm, because poly-A indicates mature mRNAs bound for translation.

Where does a heteronuclear RNA (hnRNA) binding protein localize

Nucleus, because hnRNA precedes cytoplasmic processing/export.

What is the size cutoff for simple diffusion through NPCs

Roughly ≤60 kDa or ~10 nm in dimension.

What are FG repeats in NPCs

Phe-Gly repeats in nucleoporins that form a selective permeability barrier and docking path for karyopherins.

Which polymerase transcribes 45S pre-rRNA in the nucleolus

RNA Polymerase I (Pol I).

Which polymerase transcribes 5S rRNA outside the nucleolus

RNA Polymerase III (Pol III).

What key sequence pattern helps spot an NLS

Short Lys/Arg-rich basic clusters.

What key sequence pattern helps spot an NES

Leucine-rich hydrophobic clusters in defined spacing.

What ensures import direction

Nuclear Ran-GTP abundance causing cargo release from importins in the nucleus.