Nuclear Import & Export/Ribosome Biogenesis

David M. Bedwell, Ph.D. Phone: 934-6593

Office: BBRB432 E-mail: dbedwell@uab.edu

General Reading: Alberts et al., Molecular Biology of the Cell (5th Ed.) Chapter 12, pp. 695-712

(2008).

Other References (if your interested): Komeili and O’Shea. New Perspectives on Nuclear Transport. Ann. Rev.

Genet. 35: 341-364 (2002). Strambio-De-Castillia et al. The Nuclear Pore Complex: Bridging Nuclear

Transport and Gene Regulation. Nat. Rev. Mol. Cell Biol. 11: 490-501 (2010). Tschochner and Hurt. Pre-Ribosomes on the Road From the Nucleolus to the

Cytoplasm. Trends Cell Biol. 13: 255-363 (2003). Other reviews and papers indicated in the slides.

Lecture Overview

Overview of cellular compartmentalization.

Features of nuclear pores.

Mechanism of nuclear Import.

Mechanism of nuclear export.

Ribosome assembly and export.

Overview of Cellular Compartmentalization

The Cellular Compartmentalization Problem

Protein Trafficking Mechanisms

There are two basic pathways of biosynthetic protein traffic:

Default localization

Signal-mediated localization gated transport transmembrane transport vesicular transport

gatedtransmembranevesicular

Cytosol

Endoplasmic Reticulum

Golgi Apparatus

Cell Surface

SecretoryVesicles

Lysosome

Early Endosome

Nucleus Peroxisome

Mitochondria Plastids

Late Endosome

Intracellular Protein Transport Mechanisms

NuclearArchitecture

Three-Dimensional Model of the Nucleus

Blue pseudo-coloring highlights the nuclear pore complexes, while green pseudo-coloring highlights the nuclear envelope together with the attached ribosomes.

Kiseleva, Nature Cell Biol. 6: 497 (2004)

Scanning EM of the Nucleus

Types of Traffic That Pass Through Nuclear Pores

Imported

RNA Polymerases

snRNPs

DNA Polymerases

Ribosomal Proteins

Histones

Transcription factors

Exported

40S ribosomal subunits

60S ribosomal subunits

tRNAs

mRNAs

snRNAs

Volume of Traffic Through Nuclear Pores

A single HeLa cell contains 10 million ribosomes, ~4000 nuclear pores, and divides every 24 hrs. This means a total of:

400,000 ribosomal proteins must be imported each minute (~100 r-proteins/pore).

12,000 ribosomal subunits must be exported each minute (~3 ribosomal subunits/pore).

If synthesizing DNA, need ~1 million new histone molecules every 3 minutes, so need to transport 100 histones/pore each minute.

Several hundred other proteins, RNAs, and RNPs move in and out of a single nuclear pore each minute.

Features of Nuclear Pores

Nuclear Pore Complex 8 fold rotational symmetry.

Size exclusion ranges from 9 nm (“closed”) to 26 nm (“open”).

The nuclear pore contains spoke and ring assemblies that are integrated into the two membranes of the nuclear envelope.

Nuclear Pores Embedded in the Nuclear Membranes

Nuclear Pore Complex (NPC) Composition

A single nuclear pore contains ~ 1000 proteins (total) and 60-100 different proteins. These nuclear pore complex (NPC) proteins are called nucleoporins.

Many nucleoporins are glycoproteins that carry O-linked N-Acetylglucosamine (Glc-NAc) residues.

Nuclear pore fibrils and other nucleoporins within the NPC channel contain phenylalanine and glycine (FG) repeats that facilitate binding of the nuclear import receptors to the nuclear pore complex during its translocation through the nuclear pore. These interactions allow transported molecules to pass bi-directionally through the 15nm long pore.

A relative size perspective: The NPC has a MW of 125 million Daltons. By comparison, a mammalian ribosome has a molecular weight of ~ 4 million Daltons.

Each nuclear pore complex (NPC) is a cylindrical structure comprised of eight spokes surrounding a central tube that connects the nucleoplasm and cytoplasm.

The outer and inner nuclear membranes (ONM and INM, respectively) of the nuclear envelope join to form grommets in which the NPC sits.

The NPC is anchored to the nuclear envelope by a transmembrane ring structure that connects to the core scaffold and comprises inner ring and outer ring elements.

Linker nucleoporins (Nups) help anchor the Phe-Gly (FG) Nups such that they line and fill the central tube.

Nuclear Pore Complex Structure

Strambio-De-Castillia et al., Nat. Rev. Mol. Cell Biol. 11: 490-501 (2010)

The Nuclear Pore Complex Functions as a “Virtual Gate”

Strambio-De-Castillia et al., Nat. Rev. Mol. Cell Biol. 11: 490-501 (2010)

The outer and inner nuclear membranes (ONM and INM, respectively) of the nuclear envelope join to form a ring-shaped pore where the nuclear pore complex (NPC) resides.

At the NPC, the nucleus and cytoplasm are connected by a channel, which is filled with flexible, filamentous Phe-Gly nucleoporins (FG Nups).

Spurious macromolecules are physically excluded from entering the densely packed FG Nup meshwork.

Nuclear transport factor (NTF)-bound cargo can enter the channel from either its cytoplasmic or nucleoplasmic side and hop between binding sites on the FG Nups until they return to the original compartment or reach the opposite side of the NPC.

Elbaum, Science 314: 766-767 (2006)

Two Models For Natively Disordered FG-Repeat Domains in the Transport Channel of the Nuclear Pore

Left: FG-repeat network may form a hydrogel, crosslinked by hydrophobic interactions between the phenylalanines.

Right: FG repeats could form a network of unlinked polymers whose thermally activated undulations create a zone of "entropic exclusion”.

Left: An aqueous solution with 26 mg/ml wild-type FG-repeat domain from Nsp1p (400 µM) was filled into a silicon tubing, where it completed gelling. The formed gel was pushed out of the tubing by gentle pressure, placed onto a patterned support (1 square = 1.4 mm2), and photographed. Note that the pattern shows clearly through this transparent gel. Inset illustrates how interactions between the hydrophobic clusters (shown in red) cross-link the repeat domains into a hydrogel.

The FG repeats can form a free-standing gel, and they measure elasticity comparable to 0.4% agarose.

Right: The FS mutated repeat domain remained liquid after identical treatment.

FG Repeats Can Form an Elastic Hydrogel in Aqueous Solution

Frey et al., Science 314: 815-817 (2006)

Frey et al., Science 314: 815-817 (2006); Burke, Science 314: 766-767 (2006)

Selective phase model for the passage of a nuclear transport receptor (NTR) through the permeability barrier of nuclear pore complexes.

Inter-repeat contacts between the hydrophobic clusters ( ) of FG-repeat-domains create a sieve-like barrier which restricts the passage of inert objects larger than the mesh-size.

NTRs can overcome this size-limit, because they possess binding sites ( ) for the hydrophobic clusters. They compete with inter-repeat contacts, thereby open adjacent meshes and dissolve within the barrier. Since the involved interactions are of low affinity, the NTR can leave the barrier on the other side.

Hydrogel Model of Nuclear Pore Function

Mechanism of Nuclear Import

Selective (Signal-Mediated) Nuclear Entry

Nuclear pores don't close completely - time required for proteins that lack a nuclear targeting signal to diffuse through the nuclear pore in living cells has been measured:

<5kD -seconds17kd -2 minutes44kD -30 min

>60kD -does not enter nucleus

Remarkably, even 20 nm gold particles coated with molecules having nuclear import or export signals can pass readily through the nuclear pore.

Outcomes of Nuclear Pore Function

N N N

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Nuclear Exclusion Nuclear Localization Diffusion-LimitedEquilibration

Characteristics of Nuclear Transport

Active transport through the nuclear pore complex (NPC) has the following features:

Energy dependent

Temperature dependent

Signal dependent

Saturable

These are features of a carrier-mediated process.

Nuclear Localization Signals

Two types of Nuclear Localization Signal (NLS):

Short basic sequences of 4-8 residues

[PPKKKRKV is the NLS of SV40 large T antigen]

Bipartite signals with two stretches of basic amino acids separated by ten less-conserved amino acids.

[KRPAATKKAGQAKKKK is the NLS of nucleoplasmin]

Both types of NLS are rich in the basic amino acids arginine and lysine and usually contain proline.

Location of Nuclear Localization Signals

Proteins don’t unfold during nuclear import. An NLS can be located anywhere in a protein, as long as they lie on the surface of the folded protein molecule where they can be recognized by an NLS receptor.

NLS

Methods Used to Identify NLSs

Microinjection studies- can be used to study nuclear targeting signals either in their natural context, when fused to passenger proteins, or when stuck to gold particles.

Deletion and gene fusion studies- Deletions can be used to identify regions necessary for nuclear import, while the fusion of these sequences to a passenger protein tests whether these sequences are sufficient for nuclear import.

Mutational analysis- Determine specific amino acid sequence necessary for nuclear localization.

Micro-injection Studies to Identify the Location

of an NLS

Electron micrograph showing nuclear entry of colloidal gold particles coated with

nucleoplasmin following microinjection.

Use of Electron Microscopy to Identify NLSs

The 8 amino acid SV40 NLS can target a cytosolic protein to the nucleus when introduced either genetically or by crosslinking.

Pyruvate Kinase Pyruvate Kinase plus SV40 NLS

Use of Immunofluorescence to Identify NLSs

Mutational analysis of the SV40 Large T antigen(90 kDa protein required for viral DNA replication)

Cytosolic Receptors Mediate Nuclear Protein Import

Mechanism of Nuclear Import

Importin-, a component of the nuclear localization signal (NLS) receptor complex binds to the NLS of a protein to be imported.

Importin-, the other subunit of the NLS receptor complex, mediates docking with the outer surface of the nuclear pore in a rapid, energy-independent fashion.

Translocation of the trimeric complex occurs along FG-repeat proteins within the nuclear pore in an energy-dependent manner. Importin- interacts with the FG-containing components of the pore complex.

Once the complex enters the nucleoplasm, Ran-GTP binds, releasing the cargo molecule from the complex.

Following the dissociation of the imported protein from the complex, the receptor components (with bound Ran-GTP) are then re-exported to the cytoplasm for another cycle.

Mechanism of Nuclear Import (cont)

Three important accessory proteins assist Ran function:

A cytosolic Ran Binding Protein (BP) dissociates Ran-GTP from the receptor.

The cytosolic Ran GTPase-Activating Protein (Ran-GAP) triggers GTP hydrolysis, converting Ran-GTP to Ran-GDP.

The Ran Guanine nucleotide Exchange Factor (Ran-GEF), which promotes exchange of GDP to GTP, is nuclear.

The nuclear location of the Ran-GEF maintains nuclear Ran in the GTP-bound form, providing directionality to nuclear transport.

Once cytosolic Ran-GTP is hydrolyzed to Ran-GDP by Ran-GAP, the Ran-GDP is then re-imported into the nucleus for another cycle.

Role of Ran in Nuclear Protein Import

Regulated Nuclear Import

In some cases, pre-synthesized transcription factors and cell cycle regulators are maintained in the cytoplasm and only translocate into the nucleus at specific times or in response to specific signals.

Mechanisms used to achieve regulated entry include:

A conformational change upon ligand binding.

Covalent modification (e.g., phosphorylation of NLS).

Attachment to a cytoplasmic structure to block import.

Binding of regulatory subunits that mask the NLS.

Ligand-Induced Activation of an NLS

Ligand-Induced Conformational

Change

NLSNLS

NucleusCytosol

Nuclear Transport

Regulation by Covalent Modification

Nuclear Transport

Nucleus

NLS

Dephos-phorylation

PO4

NLS NLS

Cytosol

NFAT (Nuclear Factor of Activated T cells) is a transcription factor that contains a nuclear localization sequence, but it is buried in the protein interior. 

Whether NLS or NES is masked depends on the phosphorylation state of specific serine residues in the regulatory domain.   Phosphorylation of these serine residues exposes an NES, whereas dephosphorylation exposes an NLS.

In resting cells, the NLS of the cytoplasmic NFAT is masked due to phosphorylation on these serine residues. 

In stimulated cells, an increase of intracellular calcium ions activates calcineurin, which then dephosphorylates the masking residues.  Consequently, the NLS is exposed and NFAT can be carried into the nucleus by the importin / complex. 

Inside the nucleus, NFAT may be re-phosphorylated by a protein kinase, exposing its NES so it can be exported by the exportin Crm1.

Regulation by Covalent Modification (cont)

Induced Activation of Nuclear Entry by the Level of Cytosolic Calcium

Nuclear entry of the transcription factor NFAT is induced when the level of cytosolic calcium increases.

Regulation by Cytosolic Retention

Release

NLS NLSNLS

NucleusCytosol

Nuclear Transport

CytoskeletalElements

NLS Masking by a Regulatory Subunit

Glucocorticoid Receptor

Why aren’t nuclear localization signals removed following import?

The Lamina Controls Nuclear Integrity

At the onset of mitosis, phosphorylation of nuclear lamins leads to the dissassembly of the lamina and the subsequent breakdown of the nuclear membrane.

Prior to nuclear re-assembly, dephophorylation of the lamins occurs. Lamin B, which remains associated with a specific receptor on nuclear membrane vesicles, is then rejoined by lamins A and C. This is followed by the reassembly of the lamina and the membrane in a GTP-dependent process.

Mutations in the gene encoding lamin A have been shown to be associated with at least six different diseases that are collectively called the laminopathies.

Repeated Nuclear Entry

Nuclear proteins are capable of repeated entry into the nucleus because nuclear localization signals are not removed when the protein enters the nucleus.

This is important, because when the cell undergoes mitosis, the nuclear membranes break down and nuclear proteins freely mix with cytosolic proteins.

Once mitosis is completed and the nuclear membranes re-form the nuclear proteins are imported again. This process can occur repeatedly.

Nuclear Membranes Break Down During Mitosis

Mechanism of Nuclear Export

Features of Nuclear Export

Nuclear export occurs by a mechanism analogous to nuclear import:

Protein to be exported contains a leucine-rich Nuclear Export Signal (NES).

A substrate to be exported is bound by an export receptor (such as Crm1) and Ran-GTP mediates its export from the nucleus.

Once in the cytosol, Ran-BP dissociates the exported substrate and its receptor.

Ran-GAP converts Ran-GTP to Ran-GDP.

Ran-GDP and the export receptor are then re-imported into the nucleus for another cycle of export.

Kutay and Güttinger, Trends Cell Biol. 15: 121-124 (2005)

CRM1-Mediated Nuclear Protein Export

(a) The CRM1 transport cycle.

In the nucleus, Ran-GTP stimulates binding of CRM1 to NES substrates.

After passage through the NPC, the CRM1/Ran-GTP/NES substrate complex is disassembled at the cytoplasmic filaments by the concerted action of Ran-BP1 and Ran-GAP.

The NES substrate is released to the cytoplasm and empty CRM1 is recycled back to the nucleus.

(b) Model of CRM1 export complex disassembly.

CRM1 is released into the cytoplasm and, for recycling into the nucleus, binds to a series of different, cargo-independent CRM1-binding sites.

Example of Nuclear Protein Export

PKI contains a Nuclear Export Signal (NES) [LALKLAGLDI]

PKI transport of PKA out of the nucleus is both temperature and energy dependent, indicating an active process.

Nucleus

PKA

PKAPKA

PKA

PKA

PKA

PKA

PKAPKA

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If you inject Protein Kinase A (PKA) and PKA Inhibitor (PKI)(†) into a cell nucleus, the PKI binds to PKA and transports PKA out of the nucleus by an active mechanism.

Many proteins and RNAs undergo export from the nucleus. Nuclear Export Signals (NES) mediate the export of protein and RNA species.

Nuclear Export of Various RNA Species

mRNAs, snRNAs and ribosomes are transported in or out of the nucleus as ribonucleoprotein complexes (RNPs).

Like protein transport, RNA transport is signal-dependent, carrier mediated, and occurs through the nuclear pore complex.

In general, nuclear export is mediated by adaptor proteins and export receptors (exportins).

Adaptor proteins bind the export signal and present it to the exportin, which facilitates transport of the complex through the nuclear pore complex.

However, different classes of RNAs utilize different adaptors and receptors. Not all require Ran-GTP.

Nuclear Export of mRNA

mRNA export occurs only following the attachment of the m7G cap at the 5´ end, splicing, poly(A) addition, and the attachment of various proteins during these steps.

Export requires the function of adaptor proteins that couple the mRNA to the exportin complex.

Nuclear mRNA export is mediated by the mammalian Tap-Nxt exportin complex (corresponds to Mex67-Mtr1 in yeast).

Ran-GTP is not involved in mRNA export (unlike the export of most other RNAs).

Certain viral RNAs contain a constitutive transport element (CTE) that eliminates the need for an adaptor protein.

Factors involved in Nuclear Export of mRNA

Cullen, J. Cell Sci 116: 587 (2003)

Recruitment of UAP56 to mRNA molecules, either by splicing or possibly via one of the other indicated mechanisms, likely represents the key initial step in inducing nuclear mRNA export.

UAP56 then recruits Aly, which in turn binds the Tap-Nxt nuclear RNA export factor.

In contrast, the Mason-Pfizer Monkey Virus (MPMV) CTE RNA can bind the Tap-Nxt heterodimer directly, thus obviating the need for upstream factors.

UAP56, Tap and Nxt are all essential for bulk mRNA nuclear export, but Aly is not, thus implying that a second, unknown factor may also mediate recruitment of the Tap-Nxt heterodimer by mRNA-bound UAP56 molecules.

Nucleocytoplasmic Trafficking of snRNAs

Like mRNAs, the m7G cap at the 5´ end of snRNAs is bound by a monomethyl cap-binding complex (CBC), which is important for its nuclear export.

Transport of the assembled snRNP particle back into the nucleus requires a two-component signal composed of the Sm proteins and a trimethyl G cap on the RNA (a cytosolic methylase hypermethylates the m7G cap to m3G).

Export is mediated by the Crm1 export receptor and Ran-GTP.

Nuclear Export of tRNAs

tRNA export mediated by exportin-t (and Ran-GTP).

All processing and modification must occur before the tRNA molecule can be transported from the nucleus.

Mutations that alter tertiary base pairs often effect both processing and transport, indicating that the mature conformation of the molecule is critical for each of these processes.

Rev-Mediated HIV-1 Genomic RNA Export from the Nucleus

For cellular RNAs, the presence of introns prohibits nuclear export. Similarly, unspliced HIV-1 RNA is not

exported in the absence of the Rev protein.

Nucleus

Rev-Response Element (signal in the HIV-1 RNA)

Rev-Mediated HIV-1 Genomic RNA Export from the Nucleus

The HIV-1 Rev protein functions as an export adaptor that mediates the Crm1-dependent export of unspliced HIV RNA through its leucine-rich NES [LPPLERLTL].

Rev (adaptor protein )

NucleusNuclear export

mediated by the Crm1 export receptor and

Ran-GTP.

Rev-Response Element (signal in the HIV-1 RNA)

Summary of Non-mRNA Export from the Nucleus

Cullen, J. Cell Sci 116: 587 (2003)

VA RNA represents

MicroRNAs

PHAX = Phosphorylated

Adaptor for RNA Export

Ribosome Assembly and Export

Fromont-Racine et al., Gene 313: 17-42 (2003)

Structure of the pre-rRNA 35S containing the mature rRNA, 18S, 5.8S and 25S.

Pre-RNA processing in Yeast

Schematic representation of the rRNA processing

pathway.

35S pre-rRNA synthesized by RNA polymerase 1, 5S rRNA made by RNA polymerase 3.

snoRNAs, ribosomal proteins and non-ribosomal factors form the 90S pre-ribosomal particle.

Cleavage of the 35S pre-rRNA splits the 90S precursor into 40S and 60S pre-ribosomes.

After export into the cytoplasm via the nuclear pores, additional maturation steps occur and the final non-ribosomal factors dissociate from mature 60S and 40S ribosomal subunits.

Tschochner and Hurt, Trends Biochem. Sci. 13: 255-263 (2003)

Simplified Scheme For Assembly, Maturation and Export of Pre-40S and Pre-60S Ribosomal Subunits

Fromont-Racine et al., Gene 313: 17-42 (2003)

Nucleolar processing/assembly events are highlighted in yellow.

The major role of U3 in processing 90S pre-ribosomes (part of a larger processome) is indicated.

Note that assembly of the small ribosomal subunit probably starts while the pre-rRNA 35S is still being transcribed. The early dichotomy of the 40S and 60S processing machinery is symbolized by the relative higher content of mature 40S ribosomal proteins in the 90S pre-ribosomes.

Synthesis of 60S ribosomes comprise early, middle and late steps based on protein and RNA composition.

Late steps include maturation of 40S and 60S ribosomes within the cytoplasm.

A number of factors know to control the synthesis of 40S and 60S ribosomes are depicted respectively in the left and right margins. The Rpl3-Rrb1 association is an example of regulatory protein-protein interactions.

The Ribosome Synthesis Pathway

Two Major Types of rRNA Modifications:

2’O methylation modifications are carried out by box C/D snoRNPs.

Pseudouridylation modifications of pre-rRNAs are guided by box H/ACA snoRNPs (basically, this is an isomerization of uridine).

These modifications primarily occur early in the biogenesis process in the 90S pre-ribosomal particle.

Schemes of the interactions established between the pre-rRNA and a box H/ACA snoRNP (left) or a box C/D snoRNP (right).

The modifications are carried out by snoRNPs, which contain proteins and “guide RNAs” that target the site of modification via base-pairing.

Dissociation of guide snoRNAs probably require helicase activities.

Henras et al., Cell. Mol. Life Sci. 65 2334 – 2359 (2008)

Post-Transcriptional Modifications during Ribosome Biogenesis

Schemes of export-competent pre-60S (A) and pre- 40S (B) particles with associated factors relevant to nuclear export.

The red protuberances on the adaptors Ltv1p and Nmd3p represent the nuclear export signals mediating interaction with Crm1p/Xpo1p.

Arrows between export receptors and the hydrophobic mesh of the NPCs refer to the reported ability of these factors to interact directly with the FG repeats of some nucleoporins.

Henras et al., Cell. Mol. Life Sci. 65 2334 – 2359 (2008)

Export of Pre-60S and Pre-40S Ribosomal Subunits Prior to Final Maturation

Fromont-Racine et al., Gene 313: 17-42 (2003)

For the large ribosomal subunit, two GTPases seem to play an important role. GTP hydrolysis of Efl1 accelerates nucleolar recycling of the anti-associating factor Tif6 whereas that of the putative Kre35/Lsg1 helps to recycle another exported nucle(ol)ar factor.

Tif6 release is thought to be mediated by Kre35/Lsg1.

Besides its role in pre-60S export, Nmd3 seems to play a role in recycling of mature free 60S subunits together with Lsg1.

Cleavage of the 20S pre-rRNA into mature 18S occurs in the cytoplasm. While the endonuclease in charge of this reaction is still unknown, Rio1 and Rio2 are thought to play a role in this process. Hcr1 is believed to influence the cleavage reaction and to play a role in translation initiation.

Late Cytoplasmic Steps of Ribosome Assembly

Proposed Pathway of 60S Maturation in the Cytoplasm

Maturation is initiated by the ATPase Drg1. Drg1 facilitates the replacement of Rlp24 by Rpl24, which then recruits Rei1. Rei1 enables the release of the export receptor Arx1, located near the polypeptide exit tunnel.

In parallel, Yvh1 enables replacement of Mrt4 with P0 to construct the ribosome stalk. Prior assembly of the ribosomal stalk is required for the release of Tif6.

Note that the stalk contains the GTPase Activating Center, or GAC. It normally recruits GTPases during translation. Interestingly, the GTPase Efl1 is required for the release of Tif6. Because Efl1 resembles the translation elongation factor eEF2 (EF-G in bacteria), assembly of the stalk may be required to recruit Efl1. Thus, this step in 60S biogenesis appears to mimic translocation, with Efl1 providing a mechanism to functionally check the nascent subunit.

Finally, The release of Tif6 activates Lsg1 to release the export adaptor Nmd3.

Lo et al., Mol. Cell 39, 196–208 (2010)

The stalk is made up of P0, P1 and P2 (corresponds to L10, L7/12 in bacteria).