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Nuclear pore complexes. Design, or evolution ?

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Nuclear pore complexes. Design, or evolution ?

http://reasonandscience.heavenforum.org/t2117-nuclear-pore-complexes-design-or-evolution

Nuclear pores are large protein complexes that cross the nuclear envelope, which is the double membrane surrounding the eukaryotic cell nucleus. There are about an average of 2000 nuclear pore complexes (NPCs), in the nuclear envelope of a vertebrate cell, but it varies depending on cell type and the stage in the life cycle. The proteins that make up the nuclear pore complex are known as nucleoporins.  Each NPC contains at least 456 individual protein molecules and is composed of 30 distinct proteins (nucleoporins). The other half show structural characteristics typical of "natively unfolded" or intrinsically disordered proteins, i.e. they are highly flexible proteins that lack ordered secondary structure.These disordered proteins are the FG nucleoporins, so called because their amino-acid sequence contains many phenylalanine—glycine repeats. 8



Nuclear Pore Complexes Perforate the Nuclear Envelope

Large and elaborate nuclear pore complexes (NPCs) perforate the nuclear envelope in all eukaryotes. Each NPC is composed of a set of approximately 30 different
proteins, or nucleoporins. Reflecting the high degree of internal symmetry, each nucleoporin is present in multiple copies, resulting in 500–1000 protein molecules
in the fully assembled NPC, with an estimated mass of 66 million daltons in yeast and 125 million daltons in vertebrates



Most nucleoporins are composed of repetitive protein domains of only a few different types. Some of the scaffold nucleoporins. The nuclear envelope of a typical mammalian cell contains 3000–4000 NPCs, although that number varies widely, from a few hundred in glial cells to almost 20,000 in Purkinje neurons. The total traffic that passes through each NPC is enormous: each NPC can transport up to 1000 macromolecules per second and can transport in both directions at the same time. How it coordinates the bidirectional flow of macromolecules to avoid congestion and head-on collisions is not known. Each NPC contains aqueous passages, through which small water-soluble molecules can diffuse passively. Researchers have determined the effective size of these passages by injecting labeled water-soluble molecules of different sizes into the cytosol and then measuring their rate of diffusion into the nucleus. Small molecules (5000 daltons or less) diffuse in so fast that we can consider the nuclear envelope freely permeable to them. Large proteins, however, diffuse in much more slowly, and the larger a protein, the more slowly it passes through the NPC. Proteins larger than 60,000 daltons cannot enter by passive diffusion. This size
cut-off to free diffusion is thought to result from the NPC structure. The channel nucleoporins with extensive unstructured regions form a disordered tangle (much like a kelp bed in the ocean) that restricts the diffusion of large macromolecules while allowing smaller molecules to pass. Because many cell proteins are too large to diffuse passively through the NPCs, the nuclear compartment and the cytosol can maintain different protein compositions. Mature cytosolic ribosomes, for example, are about 30 nm in diameter and thus cannot diffuse through the NPC, confining protein synthesis to the cytosol. But how does the nucleus export newly made ribosomal subunits or import large molecules, such as DNA polymerases and RNA polymerases, which have subunit molecular masses of 100,000–200,000 daltons? As we discuss next, these and most other transported protein and RNA molecules bind to specific receptor proteins that actively ferry large molecules through NPCs. Even small proteins like histones frequently use receptor-mediated mechanisms to cross the NPC, thereby increasing transport efficiency.

Nuclear Pore Complexes  are elaborate, specialized pores in the nuclear membranes that surround the nucleus of each cell in your body like a skin.  The pores look something like complex basketball hoops with rings and studs that act like electronic gates.  Their job is to control traffic in and out of the nucleus. Each nuclear pore complex works so fast, it can authenticate somewhere between 520 and 1000 pieces of cargo per second.  A typical nucleus has about 2000 to 4000 or more of these gates, which are made up of 30 or more very complex proteins.  They all have to be disassembled and reassembled every time a cell divides.  6

Where would you start in trying to work out the structure of a macromolecular machine consisting of 456 proteins? Taking a combined experimental and computational approach is one answer.

Consider a 1,000-piece jigsaw puzzle. There are millions of ways in which the pieces might fit together, yet there is only one solution.

This is just amazing  and it exemplifies  design in the cell very clearly . Watch the interview of the researchers which figured out the structure of the nuclear pore complex below, and how they describe it as extremely complex, but simple at the same time. Remarkable.

http://www.nature.com/nature/videoarchive/cellarchitecture/

https://www.youtube.com/watch?v=dJLeLRXIOj0







The Structure of the Cell Nucleus “Gatekeeper”

The massive NPC, which allows molecules such as RNAs and proteins to move in and out of the nucleus, is one of the largest macromolecular structures in the cell (containing about 5 million atoms). Its structure has been considered a "holy grail" in structural biology for many decades. The researchers from the Massachusetts Institute of Technology (MIT) found that the NPC scaffold forms an open lattice structure, similar to another membrane coating complex, called COPII. That complex has far fewer components and is involved in a completely different cellular process, called vesicular transport, by which materials move in, out and around the cell enclosed in vesicles.

The finding reveals a remarkable evolutionary story: The proteins that form the building blocks in the NPC and the COPII vesicle coats are so unique that they still have not been identified anywhere else in the cell. Once the models of the proteins were complete, it became clear that these two cellular structures share a common ancestor, dating back over one billion years, which already contained the same building blocks connected in very similar fashion. Second, discovering the specific relationship between the NPC and the much simpler, better understood COPII vesicle coat constitutes a significant step toward understanding the entire NPC assembly, which appears to be remarkably modular and most likely self-assembling.

Details Of Nuclear Pore Complex With Spin 1

A cell’s membrane-bound nucleus uses hundreds to thousands of nuclear pores as its gatekeepers, selective membrane channels that are responsible for regulating the material that goes to and from a cell’s DNA. Rockefeller scientists have nailed down the first complete molecular picture of this huge, 450-protein pore and their findings provide a glimpse into how the nucleus itself first evolved. Evolved ?!!

The group gathered and analyzed massive amounts of data to come up with a rough draft of the structure of the nuclear pore.

The scientists’ results have given them a peek into the early evolution creation of eukaryotic cells. Compartmentalization was made possible by membranes and coating complexes, which act “like little hands” to grab, shape and stabilize membranes.

“We think that once the cells gained this coating complex, they ran with it and started to duplicate it and specialize it,”

“Evolution is a process of duplication and divergence,” Rockefeller professor Michael Rout says. He and his colleagues saw clear evidence of this. For every protein, there was another one that looked quite similar. “These are evidence of duplication events, showing that the evolution of the complicated nuclear pore was a more straightforward affair than previously thought. It’s made of many different variations of a theme of just one unit.”

“The nuclear pore is the communication device that the nucleus uses to communicate with the rest of the cell. And if you don’t understand how that works, you don’t understand a key part of how the cell works. You have to see the cell as a machine and understand all of its parts.”

The authors state:

A cell’s membrane-bound nucleus uses hundreds to thousands of nuclear pores as its gatekeepers, selective membrane channels that are responsible for regulating the material that goes to and from a cell’s DNA.

Molecular Machine Turns Packaged Messenger RNA Into A Linear Transcript 23

For RNA, the gateway to a productive life outside the nucleus is the nuclear pore complex, an amalgamation of 30 kinds of proteins that regulates all traffic passing through the nuclear membrane. New research shows that one of these proteins magnetically couples with a special molecule -- a helicase -- to form a machine that unpacks balled-up messenger RNA particles so that they can be translated.

The work illuminates a previously unknown stage in the process by which genetic information is read and converted to proteins. In humans and other higher organisms, the genetic information that is encoded in the DNA is stored inside the nucleus, while the factories that convert DNA instructions into proteins are located in the surrounding cytoplasm. As those instructions — messenger RNA particles — pass through the nuclear membrane, numerous proteins that cover and protect the delicate messenger RNA molecules must be stripped off.

André Hoelz, a research associate in John D. Rockefeller Jr. Professor Günter Blobel’s Laboratory of Cell Biology, and his colleagues solved the crystal structure of a complex located on the cytoplasmic side of the nuclear pore — nucleoportin Nup214 coupled with helicase Ddx19. They then performed a series of biochemical experiments to further parse the interactions between these two molecules and to elucidate their mechanism of action. “We found that the messenger RNA protein package and Nup214 competitively bind to the helicase, one after the other,” Hoelz notes. Each time the helicase binds the ball of messenger RNA and protein, it strips one protein molecule off. “The process is akin to a ratchet mechanism for messenger RNA export.” The result, Hoelz speculates, is a linear messenger RNA transcript that travels on to the ribosome, where it delivers instructions for building proteins.

Science daily refers to coordination of independent parts.  DNA transcripts made of messenger RNA emerge from the nucleus in 3-D clumps.  These need to be “straightened out” into a linear code that can be read by the ribosome.  Research at Rockefeller University shows that one of the 30 kinds of proteins in the nuclear pore complex “magnetically” attaches to the transcript when it passes through the gate, joining an unwrapping machine called a helicase “to form a machine that unpacks balled-up messenger RNA particles so that they can be translated.”  Here’s how Andre Hoelz described the action: “We found that the messenger RNA protein package and Nup214 competitively bind to the helicase, one after the other.” Each binding strips one protein off as it passes through.  “The process is akin to a ratchet mechanism for messenger RNA export,” Hoelz said.  Failures in the mechanism, again, were said to be implicated in disease.  Once again, also, the article said nothing about evolution. 5

Structural and functional analysis of the interaction between the nucleoporin Nup214 and the DEAD-box helicase Ddx19

Key steps in the export of mRNA from the nucleus to the cytoplasm are the transport through the nuclear pore complex (NPC) and the subsequent remodeling of messenger RNA-protein (mRNP) complexes that occurs at the cytoplasmic side of the NPC. Crucial for these events is the recruitment of the DEAD-box helicase Ddx19 to the cytoplasmic filaments of the NPC that is mediated by the nucleoporin Nup214. Here, we present the crystal structure of the Nup214 N-terminal domain in complex with Ddx19 in its ADP-bound state at 2.5 Å resolution. Strikingly, the interaction surfaces are not only  conserved but also exhibit strongly opposing surface potentials, with the helicase surface being positively and the Nup214 surface being negatively charged. We speculate that the positively charged surface of the interacting ADP-helicase binds competitively to a segment of mRNA of a linearized mRNP, passing through the NPC on its way to the cytoplasm. As a result, the ADP-helicase would dissociate from Nup214 and replace a single bound protein from the mRNA. One cycle of protein replacement would be accompanied, cooperatively, by nucleotide exchange, ATP hydrolysis, release of the ADP-helicase from mRNA and its rebinding to Nup214. Repeat of these cycles would remove proteins from a mRNP, one at a time, akin to a ratchet mechanism for mRNA export.

Can evolution explain the origin of the Nuclear Pore Complex? 7

After nine years of work, three universities including a team at Rockefeller University completed a beautiful new model of the nuclear pore complex.  The story is told by Science Daily. The article attributed the origin of this exquisite gatekeeper of the nucleus to evolution: “their findings provide a glimpse into how the nucleus itself first evolved,” the article says.  How can this be?  The article claims that eukaryotic cells split off from the prokaryotes “when they developed a nucleus and other specialized organelles that allowed them to compartmentalize different aspects of cellular metabolism.
   This language suggests some kind of plan or intention – certainly not what neo-Darwinian theory allows.  Nevertheless, Michael Rout of Rockefeller continued the language of planning and purpose as he described himself visualizing an evolutionary progress from earlier structures: “We think that once the cells gained this coating complex, they ran with it and started to duplicate it and specialize it.”
   As for a mechanism that could generate complexity from simplicity, Rout invoked a kind of copying and tinkering algorithm: “Evolution is a process of duplication and divergence,” he said.  Because the nuclear pore complex contains multiple copies of similar structures arranged like a wheel, he thinks this is evidence they must have formed by gene duplication, even though wheels are usually built by intelligent agents:

He and his colleagues saw clear evidence of this when they color-coded the proteins in the pore.  One method of color coding revealed alternating stripes, like spokes on a wheel: For every protein, there was another one that looked quite similar.  Color coding a different way showed the same pattern in the pore’s outer and inner rings, one of which appears to be a slightly modified duplication of the other.  These are evidence of duplication events, Rout says, showing that the evolution of the complicated nuclear pore was a more straightforward affair than previously thought.  “It’s made of many different variations of a theme of just one unit.”

mRNA is needed to make the nuclear pore complex. But without the nuclear pore complex, mRNA cannot be prepared for translation in the Ribosome. Thats a catch22 situation....

If this observation is correct, to regulate, there must be:

a sensing mechanism
a feedback mechanism
a control mechanism.
a value against which the parameter is controlled




1) http://www.uncommondescent.com/intelligent-design/details-of-nuclear-pore-complex-with-spin/
2) http://www.sciencedaily.com/releases/2009/02/090213113340.htm
3) http://newswire.rockefeller.edu/2009/02/10/molecular-machine-turns-packaged-messenger-rna-into-a-linear-transcript/
4) http://www.pnas.org/content/106/9/3089.full
5) http://creationsafaris.com/crev200902.htm
6) http://creationsafaris.com/crev0603.htm
7) http://creationsafaris.com/crev200802.htm#20080202a
8 )https://en.wikipedia.org/wiki/Nuclear_pore

further readings :

http://www.nature.com/ncomms/2015/150114/ncomms6978/full/ncomms6978.html
http://www.ks.uiuc.edu/Research/npc/



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Nature Study Shows How Molecules Escape From the Nucleus 1

Protein synthesis is arguably the most important of all cellular processes. The instructions for making proteins are encoded in the Deoxyribonucleic acid (DNA) of genes, which reside on chromosomes in the nucleus of a cell. In protein synthesis, DNA instructions of a gene are transcribed, or copied, onto messenger RNA; these molecules of messenger RNA must then travel out of the nucleus and into the cytoplasm, where amino acids are linked together to form the specified proteins.

 But to our surprise, we observed that messenger RNA molecules pass rapidly through the nuclear pores, and that the slow events were docking on the nuclear side and then waiting for release into the cytoplasm.”
single messenger RNA molecules arrive at the nuclear pore and wait for 80 milliseconds (80 thousandths of a second) to enter; they then pass through the pore breathtakingly fast—in just 5 milliseconds; finally, the molecules wait on the other side of the pore for another 80 milliseconds before being released into the cytoplasm.

The waiting periods observed in this study, and the observation that 10 percent of messenger RNA molecules sit for seconds at nuclear pores without gaining entry, suggest that messenger RNA could be screened for quality at this point.

“Researchers have speculated that messenger RNA molecules that are defective in some way, perhaps because the genes they’re derived from are mutated, may be inspected and destroyed before getting into the cytoplasm or a short time later, and the question has been, ‘Where might that surveillance be happening?’,” said Dr. Singer. “So we’re wondering if those messenger RNA molecules that couldn’t get through the nuclear pores were subjected to a quality control mechanism that didn’t give them a clean bill of health for entry.”
In previous research, Dr. Singer studied myotonic dystrophy, a severe inherited disorder marked by wasting of the muscles and caused by a mutation involving repeated DNA sequences of three nucleotides. Dr. Singer found that in the cells of people with myotonic dystrophy, messenger RNA gets stuck in the nucleus and can’t enter the cytoplasm. “By understanding how messenger RNA exits the nucleus, we may be able to develop treatments for myotonic dystrophy and other disorders in which messenger RNA transport is blocked,” he said.




The Structure and Physiology of the Nuclear Pore Complex and its Role in Gene Expression and Human Disease 1

The Nuclear Pore Complex (NPC) is a protein channel that communicates and transports molecules between the cytoplasm and the nucleoplasm. It has a complex structure composed of many structural proteins, mainly nucleoporins and transporter proteins, and its biosynthesis is an extremely regulated and cell cycle phase dependent process. this structure could play a decisive role in gene expression and in the pathogenesis of several diseases. On the one hand, NPC affects gene expression by regulating epigenetic enzymes and affecting the nucleocytoplasmic balance of transcription factors and small nuclear RNAs. On the other hand, diverse studies show that NPC is involved in some diseases and pathological processes like aging, neurodegenerative diseases and extrapyramidal syndromes, cancer, cardiovascular diseases, infectious diseases and genetic syndromes. In conclusion, NPC might be an important element in the control of gene transcription and cell phenotype, and perhaps, it will become a future pharmacologic target for several diseases in which it is involved in.

INTRODUCTION

The Nuclear Pore Complex (NPC) is a cellular component that is located within the nuclear envelope. It is a protein channel that allows macromolecules to cross from the cytoplasm to the nucleoplasm and vice versa. This establishes an important physiological process known as nucleocytoplasmic transport (Jamali et al. 2011). NPCs are not only involved in transport but also have an important relationship with epigenetic enzymes and transcription factors, which are both proteins that participate in gene expression (Van de Vosse et al. 2011). Moreover, it has been demonstrated that the NPC is linked to many diseases such as aging, neurodegenerative diseases and extrapyramidal syndromes, cancer, cardiovascular diseases (arrhythmias and sudden death), infectious diseases and genetic syndromes. For example, in cancer or genetic syndromes, mutations exist in the proteins that form the NPC (Capelson and Hetzer 2009).

There are three structures within a NPC: nuclear basket, cytoplasmic filaments, and nuclear ring. They are all made by approximately 30 different proteins called nucleoporins.

The nucleoporins that form the NPC can be classified into three groups:

transmembrane nucleoporins (approximately 10%)
structural nucleoporins (50%)
and FG-nucleoporins (40% of the total population)


The deepest nucleoporins in the NPC project some of their peptide fragments into the pore hole. These peptide sequences are natively unfolded and hence are polypeptides without a secondary structure folding. They make up a peptide web within the pore complex hole. In this web there are nucleoporins containing

GLFG (glycine-leucine-phenylalanineglycine repeats)
FXFG (phenylalanine-x-phenylalanine-glycine repeats)
FG (the most common type) (phenylalanine-glycine repeats).


These FGs are extremely important for the NPC’s involvement in the nucleocytoplasmic transport of macromolecules, as FGs function as the anchoring points for the cargo, indicate the direction of transport and adapt to appropriate pore size (Denning et al. 2003; Terry and Wente 2009; Yang 2011)




The image below shows the main parts in which the NPC is divided and the specific proteins that compound them.

Nuclear basket (in orange) is composed of Nup153 and Nup50 which bind filaments of Tpr protein.
Cytoplasmic filaments (in purple) are made up by the nucleoporins Nup88, Nup214 and Nup358. The nuclear ring has four parts, namely,
the membrane ring (in brown)
the spoke ring (in red and grey)
the inner ring or nuclear ring
outer ring or cytoplasmic ring (both in green)

The membrane ring anchors the ring complex to the nuclear envelope and comprises nucleoporins gp210, Ndc1 and Pom121.
The spoke ring comprises the nucleoporins Nup53, Nup54, Nup58, Nup62 Nup93, Nup155, Nup188 and Nup205.
The outer and the inner rings are composed of nucleoporins Nup37, Nup43, Nup85, Nup96, Nup107, Nup133, Nup160, Sec13 and Seh1

Other important proteins are also show in the picture.









1) http://www.jyi.org/wp-content/uploads//June-The-structure-and-physiology-of-the-nuclear-pore-complex-and-its-role-in-gene-expression-and-human.pdf



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The Yeast Nuclear Pore Complex and Transport Through It 1

Exchange of macromolecules between the nucleus and cytoplasm is a key regulatory event in the expression of a cell’s genome. This exchange requires a dedicated transport system:

(1) nuclear pore complexes (NPCs), embedded in the nuclear envelope and composed of proteins termed nucleoporins (or “Nups”), and
(2) nuclear transport factors that recognize the cargoes to be transported and ferry them across the NPCs. This transport is regulated at multiple levels, and the NPC itself also plays a key regulatory role in gene expression by influencing nuclear architecture and acting as a point of control for various nuclear processes.

Here we summarize how the yeast Saccharomyces has been used extensively as a model system to understand the fundamental and highly conserved features of this transport system, revealing the structure and function of the NPC; the NPC’s role in the regulation of gene expression; and the interactions of transport factors with their cargoes, regulatory factors, and specific nucleoporins. Eukaryotic chromosomes are housed within the nucleus, which is delimited by the two parallel membranes of the nuclear envelope (NE). The evolution emergence of this physical barrier endowed eukaryotes with a critical control mechanism segregating the sites of gene transcription and ribosome biogenesis from the site of protein synthesis. This compartmentalization allows cells to strictly coordinate numerous key cellular processes, but it also presents cells with the challenge of selectively managing the transport of a bewildering number of proteins and RNAs between the nucleus and cytoplasm. This is accomplished by the presence of “nuclear pores,” which arise at points where the inner and outer NE membranes conjoin to form circular channels across the nuclear envelope. Within these pores sit large proteinaceous complexes, appropriately named nuclear pore complexes (NPCs), which, in conjunction with soluble transport factors, govern all biomolecular transport into and out of the nucleus. Beyond this fundamental control of transport, the NPC has adopted a host of other activities by acting as a spatial landmark or anchor site for many of the machineries that directly control gene activity and transcriptional processing. As a transporter, it must allow small molecules to pass as freely, prevent most macromolecules from crossing, and permit the quickest possible passage of selected macromolecules bidirectionally across the NE. As an anchor, it must allow free communication between the attached control machineries and the chromatin or transcripts that they regulate without hindering nuclear transport. One can thus also consider the NPC as a major way station in eukaryotes, interacting with and regulating DNA, RNA, and membranes and communicating between the cytoplasm, nucleoplasm, and ER lumen. Because of this, the subject of the nuclear pore complex and nuclear transport is a huge one, far beyond the scope of any single review. Our aim here is therefore to give an overview, including references to many excellent reviews that detail particular areas of study.



Diagrammatic representation of mRNA export. The SAGA complex is recruited to the promoter of a subset of inducible genes and promotes their transcription. SAGA and the NPC-associated TREX-2 complex may help the genes move to the vicinity of the NPC. The nascent transcripts recruit shuttling mRNA coating factors, THO, TREX, and, subsequently, the mRNA export factors Mex67p and Mtr2p, resulting in the formation of an export-competent mRNP; the association of the maturing mRNPs with components of the nuclear basket is strengthened in preparation for nuclear translocation, while nuclear basket-associated TRAMP and exosome complex-associated mRNP surveillance mechanisms ensure that the mRNP is correctly assembled for export. After translocation through the NPC, the release of mRNA export factors from mRNPs is induced by the combined action of Dbp5p and Gle1p, which are docked to NPC cytoplasmic filaments via interaction with Nup42p and Nup159p, respectively, and are thought to act as mRNP-remodelling factors. It is presumed that this process drives the directionality of mRNP export while at the same time priming mRNAs for translation initiation.


1) http://www.genetics.org/content/190/3/855.full.pdf

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Steps of mRNA export from nuclear transcription to the cytoplasm. 1



Components of transcription-export complex TREX, such as ALY, THO complex subunit 2 (THOC2) and THOC5, contribute to the selective export of a subset of mRNAs and thus to regulation of specific biological processes including maintenance of pluripotency, haematopoiesis, heat shock and safeguarding of genome integrity. TREX-2, through germinal centre-associated nuclear protein (GANP), also functions in the export of transcripts that are required for gene expression. Only the components of TREX and TREX-2 complexes that have been shown to contribute to selectivity are indicated. It is unknown whether the complete TREX and TREX-2 complexes contribute to selectivity, and thus they are represented as transparent in the figure. Although most mRNAs use TREX, TREX-2 and nuclear RNA export factor 1 (NXF1) receptors to transit through nuclear pore complexes (NPCs), a subset of mRNAs use chromosome region maintenance 1 protein homologue (CRM1), which is the main protein export receptor. Eukaryotic translation initiation factor 4E (eIF4E) and CRM1 preferentially export a subset of mRNAs that encode proteins involved in proliferation, survival, metastasis and invasion. Nucleoporins such as nuclear pore complex protein NUP96, which is a constituent of the NUP107–NUP160 complex, may contribute to the export of specific subsets of transcripts, such as those encoding cell cycle regulators and immune response factors. Whether NUP96 achieves this by modulating interactions of mRNA export factors at the NPC or in the nuclear interior, where a proportion of NUP96 is thought to localize, is unknown. Each of these potential pathways, together with the functional subset of transcripts that they export, is shown schematically. One important unresolved question is whether TREX and TREX-2 mediate alternative export routes, or whether they cooperate to export the same transcripts. IPMK, inositol polyphosphate multikinase; LRPPRC, leucine-rich PPR motif-containing protein; PtdIns(3,4,5)P3, phosphatidylinositol-3,4,5-trisphosphate.



The life cycle of a messenger ribonucleoprotein particle (mRNP) is shown, from its biogenesis and maturation into an export- competent form to its subsequent transport from sites of transcription and processing in the nucleus to the cytoplasm, where it is extensively remodelled. These steps are all coupled; for example, transcription-export complex TREX is recruited to the nascent mRNP by the splicing machinery. After a mature mRNP has been generated, the conserved nuclear RNA export factor 1 (NXF1) is recruited to the mRNP through direct interactions with several TREX components and SR splicing factors. Cargo mRNAs from both TREX and TREX-2 are transferred to NXF1 and its cofactor p15 for transit through the nuclear pore by interacting directly with the nucleoporins that line the pore. The kinetics of translocation of mRNAs from sites of transcription through nuclear pore complexes (NPCs) in mammalian cells suggest that transport to NPCs can take several minutes, whereas translocation through NPCs takes milliseconds. It is unknown whether TREX and TREX-2 cooperate to export the same transcripts or mediate alternative export routes and whether TREX-2 is recruited to transcripts in the nuclear interior as in the case of TREX. CBC, cap-binding complex; eIF4E, eukaryotic translation initiation factor 4E; EJC, exon–junction complex; InsP6, inositol hexakisphosphate; PABP, poly(A)-binding protein; Pol II, RNA polymerase II.



One of the final steps in the mRNA export pathway involves docking and translocation of messenger ribonucleoprotein particles through nuclear pore complexes (NPCs). A schematic of an NPC is shown, with its central structure, cytoplasmic pore filaments and nuclear basket. Interactions between mRNA export factors and specific NPC components during the export process are indicated. For example, nuclear RNA export factor 1 (NXF1) interacts directly with repeats of phenylalanine–glycine (FG) residues, which are present in a subset of the nucleoporins that line the central NPC transport channel. These include nuclear pore complex protein NUP153, which is associated with the nuclear basket, and cytoplasmic filament-associated NUP214 and NUP358. Simplified cartoon representations of transcription-export complexes TREX and TREX-2 are also shown, with their molecular composition based on available structural data. TREX is composed of the THO subcomplex (darker grey area), together with RNA-binding component ALY, DEAD-box type RNA helicase UAP56 and additional factors that have ATP-dependent interactions with UAP56. Several components of TREX interact directly with NXF1 and can shuttle between the nucleus and the cytoplasm. Mammalian TREX-2 interacts with the nuclear basket through NUP153 and nucleoprotein TPR, with all TREX-2 subunits binding to the scaffold of germinal centre-associated nuclear protein (GANP). In yeast Sac3 and in human GANP, α-helical and winged-helix domains bind to other components of TREX-2. TREX-2 also interacts directly with NXF1 through the amino-terminal domain of GANP, which contains regions of homology to nucleoporins, including a cluster of six FG repeats (indicated by dark grey lines). CETN, centrin; CHTOP, chromatin target of PRMT1 protein; ENY2, enhancer of yellow 2 transcription factor homologue; InsP6, inositol hexakisphosphate; PCID2, PCI domain-containing protein 2; PDIP3, polymerase δ- interacting protein 3; THOC, THO complex subunit; ZC11A, zinc finger CCCH domain-containing protein 11A.

NPC organization and nucleoporins with a role in RNA export 2





Nuclear pore complexes (NPCs) are large assemblies (approx125 nm in diameter, with a mass of 125 MDa in metazoa and 60 MDa in yeast) that are embedded in the nuclear envelope. NPCs have an eightfold symmetrical core structure (called the spoke complex) that is sandwiched between a cytoplasmic and a nuclear ring158. The 8 spoke units surround the centre of the NPC through which active transport takes place. A structure called the nuclear basket and 8 short cytoplasmic fibrils (only 4 are shown) are attached, respectively, to the nuclear and cytoplasmic ring of the NPC (see figure).

The NPC is formed by approx30 different nuclear pore proteins (nucleoporins) that exist in 8 or 16 (or sometimes 32) copies per NPC159, 160. Nucleoporins are grouped into three major classes.

The first class are the FG nucleoporins, which contain Phe-Gly-rich repeat domains that fill up the active transport channel and function directly in nucleocytoplasmic transport by mediating the passage of the soluble transport receptors161, 162.

The second class are nucleoporins that are devoid of FG-repeat sequences; these are the predominant structural constituents of the NPC.

The third class are Nups, which are integral membrane proteins and are thought to anchor the NPC in the nuclear membrane. FG nucleoporins can interact directly with the shuttling transport receptors161. Hydrophobic patches on the surface of these transporters bind transiently to the Phe residues that are part of the FG nucleoporin network in the active transport channel.

Most of the nucleoporins are located symmetrically on both sides of the NPC, but a few nucleoporins are found asymmetrically either on the cytoplasmic or nuclear side of the NPC. The asymmetrically located nucleoporins are thought to be involved in directional transport processes (initial receptor targeting or termination of transport) or to fulfil compartment-specific functions at the NPC (for example, interacting with chromatin or the transcription machinery, or as checkpoint proteins for quality-control steps during cargo export). Nucleoporins, which have been implicated in RNA export, are indicated (see main text for details). snRNA, small nuclear RNA; rRNA, ribosomal RNA. Orthologous proteins or complexes between yeast and metazoa are shown in the same colour.



1) http://www.nature.com/nrm/journal/v16/n7/full/nrm4010.html
2) http://www.nature.com/nrm/journal/v8/n10/box/nrm2255_BX1.html



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Gate-Crashing the Nuclear Pore Complex







Who would have thought a hundred years ago that a simple fungus contains high-tech security gates guarded by agents that authenticate cargo before letting it through? That's what advances in imaging are allowing scientists to observe. We must be among the most privileged in history to witness the foundations of biological life coming into focus in all their glory!
You got a simplified glimpse of this gate 13 years ago in the film Unlocking the Mystery of Life. It's that circular portal that allows the messenger RNA out into the cytoplasm. We've learned a lot since then. We know now that the nuclear pore complex (NPC) is one of the largest and most complex molecular machines in the cell. Each eukaryotic nucleus is studded with NPCs. They resemble portholes with nets, like basketball hoops, but much more sophisticated. They are the gateways for traffic in and out of the nucleus, but not just any molecule can pass through. Each macromolecule needs a ticket and an escort, represented by tags and accessory proteins that first authenticate the cargo then accompany it in or out.
New details of the NPC architecture came to light in a couple of papers inScience this month. Regarding these papers, Katharine S. Ullman and Maureen A. Powers state:


Nuclear pore complexes (NPCs), first observed by electron microscopy 65 years ago, mediate selective transport of macromolecules between the nucleus and cytoplasm of eukaryotic cells. Although the exact size and protein composition of NPCs can vary between species, these massive and complex machines are highly conserved in their overall organization, which consists ofmultiple copies of ∼30 nuclear pore proteins, or nucleoporins (Nups), in a symmetrical eightfold radial arrangement. Deciphering the structure of this immense complex has required ongoing multifaceted approaches. On page 106 and 56 in this issue, Chug et al. and Stuwe et al., respectively, have employed parallel approaches in very distant species and arrived at remarkably similar and informative structures of an essential subcomplex of the NPC.


What were the "very distant species" with conserved structures? The Chug team used a frog; the Stuwe team studied a thermophilic (heat-loving) fungus. Both teams looked at one element of the eight-fold radial structure at the central inner ring of the pore. They found that this domain has coils of protein that extend out into the pore and make contact with coils from the opposite side. These coils form a net that blocks entry unless the cargo has the right password, consisting of an "usher" of sorts called a nuclear transport receptor (NTR).
What's emerging is a different model from the previously-held vision of pores that dilate and constrict to allow passage. "Instead they suggest a rigid pore in which flexible domains called FG repeats fill the channel and form a barrier that can be traversed by receptors that carry cargos across," Valda Vinson explains in a brief summary of the papers in Science. FG repeats are so named for the phenylalanine-glycine amino acid patterns. "Interactions between NTRs and FG repeats are essential for facilitated translocation," Chug's team says.


NPCs grant free passage to small molecules but becomeincreasingly restrictive as the size of the diffusing species approaches or exceeds a limit of ≈30 kD in mass or 5 nm in diameter. This property is critical for keeping nuclear and cytoplasmic contents separateNTRs, however, are not bound by this size limit. They mediate facilitated NPC passage and ferry cargoes up to the size of newly assembled ribosomal subunits (≈25 nm in diameter) between the two compartments.


For size comparison, consider that the inner ring complex is 425 kilodaltons (kD) compared to the 30kD mass limit of unescorted cargo (one Dalton = one atomic mass unit). The specific order of amino acids in each Nup protein is critical to its structure and function. Nup62, for instance, requires "kinks" that fold it into the pore-traversing domain.
"The kink is stabilized by a network of highly conserved hydrophobic and hydrophilic contacts," the researchers say. Indeed, many of the residues show "extreme evolutionary conservation" in animals as diverse as amoebas, fungi, and higher animals. That's the only context in which Chug et al. mention evolution -- the lack of it.
Stuwe's team also mentioned evolution only in the context of conservation, except for a nonspecific comment in the opening paragraph:


One of the hallmarks of eukaryotic evolution is the enclosure of genetic information in the nucleus. The spatial segregation of replication and transcription in the nucleus from translation in the cytoplasm imposes the requirement of transporting thousands of macromolecules between these two compartments. Nuclear pore complexes (NPCs) are massive transport channels that allowbidirectional macromolecular exchange across the nuclear envelope (NE) and thus function as key regulators of the flow of genetic information from DNA to RNA to protein.


You can find the word conserved or conservation 16 times in the paper. In one case, structural conservation was found despite low sequence conservation. Mutation experiments, however, usually broke the function or reduced it dramatically; some cases were lethal.
The nuclear pore complex, therefore, is a highly selective filter for macromolecules. Why does that matter? Chug et al. explain:


For transport selectivity, preventing the passage of unwanted material is as important as allowing NTR•cargo species to pass. This is straightforward to explain by an adaptive barrier that seals around a translocating species, particularly because cohesive FG domains readily assemble such self-sealing barriers in the form of FG hydrogels.


But if anything and everything can attach NTRs to let it pass through, selectivity is also lost. "A 'general gate' would therefore stand open at all timesand consequently fail as a barrier," they realize. How do the NTRs know which cargo is legitimate? This point is not clear from the papers and probably will require more analysis. It appears that multiple interactions with other nucleoporins (Nups) take part in discrimination against unwanted molecules. But the mechanism is general enough to allow the FG domains to rapidly facilitate passage of a wide range of cargo sizes.
In a news item from Rockefeller University, Professor Michael P. Rout sheds some light on the tradeoff between selectivity and speed:

"Usually, binding between traditionally folded proteins is a time consuming, cumbersome process, but because the FG Nups areunfolded, they are moving very quickly, very much like small molecules. This means their interaction is very quick," explains Rout.

The disordered structure of the FG regions is critical to the speed of transport, allowing for quick loading and unloading of cargo-carrying transport factors. At the same time, because transport factors have multiple binding sites for FG Nups, they are the only proteins that can specifically interact with them -- making transport both fast and specific.

So in this case, "fuzzy" interactions are an asset. Rout thinks that this is "the first case where the 'fuzzy' property of an interaction is a key part of its actual biological function."

How fast are the NPCs? Chug et al. provide some details about the diffusion rate of large molecules through the pore:


Facilitated translocation is usually completed within 10ms[milliseconds].. NPCs can conduct ≈1000 facilitated translocation events or a mass flow of ≈100 MD [megadaltons] per pore per second, which further implies that NPCs can translocate numerous species in parallel.


With some 2,000 NPCs in a typical vertebrate cell (Wikipedia), that provides a huge capacity for facilitated, authenticated transport.
Not sufficiently impressed yet? Consider that the entire nuclear envelope breaks down at cell division, all the parts are duplicated (including all the genetic information, which is also error-checked), and the entire nucleus, NPCs and all, is reassembled in both daughter cells, ready to go sometimes in just minutes. That's intelligent design!


http://www.evolutionnews.org/2015/10/gate-crashing_t099941.html

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Biochemists solve the structure of cell's DNA gatekeeper







Composite structure of the symmetric core of the nuclear pore complex, shown in a cartoon representation. The composite structure was generated by sequential unbiased searches for each protein subunit. Distinct subunits are distinguished by …more
Caltech scientists have produced the most detailed map yet of the massive protein machine that controls access to the DNA-containing heart of the cell.

In a new study, a team led by André Hoelz, an assistant professor of biochemistry, reports the successful mapping of the structure of the symmetric core of the nuclear pore complex (NPC), a cellular gatekeeper that determines what molecules can enter and exit the nucleus, where a cell's genetic information is stored.
The study appears in the April 15, 2016 issue of the journal Science, featured on the cover.
The findings are the culmination of more than a decade of work by Hoelz's research group and could lead to new classes of medicine against viruses that subvert the NPC in order to hijack infected cells and that could treat various diseases associated with NPC dysfunction.
"The methods that we have been developing for the last 12 years open the door for tackling other large and flexible structures like this," says Hoelz. "The cell is full of such machineries but they have resisted structural characterization at the atomic level."
The NPC is one of the largest and most complex structures inside the cells of eukaryotes, the group of organisms that includes humans and other mammals, and it is vital for the survival of cells. It is composed of approximately 10 million atoms that together form the symmetric core as well as surrounding asymmetric structures that attach to other cellular machineries. The NPC has about 50 times the number of atoms as the ribosome—a large cellular component whose structure was not solved until the year 2000. Because the NPC is so big, it jiggles like a large block of gelatin, and this constant motion makes it difficult to get a clear snapshot of its structure.







Credit: Lance Hayashida/Caltech and the Hoelz Laboratory/Caltech
In 2004, Hoelz laid out an ambitious plan for mapping the structure of the NPC: Rather than trying to image the entire assembly at once, he and his group would determine the crystal structures of each of its 34 protein subunits and then piece them together like a three-dimensional jigsaw puzzle. "A lot of people told us we were really crazy, that it would never work, and that it could not be done," Hoelz says.
Last year, the team published two papers in Science that detailed the structures of key pieces of the NPC's inner and outer rings, which are the two primary components of the NPC's symmetric core. The donut-shaped core is embedded in the nuclear envelope, a double membrane that surrounds the nucleus, creating a selective barrier for molecules entering and leaving the nucleus.
By being able to piece these crystal structures into a reconstruction of the intact human NPC obtained through a technique called electron cryotomography—in which entire isolated nuclei are instantaneously frozen, with all of their structures and molecules locked into place, and then probed with a transmission electron microscope to produce 2-D images that can be reassembled into a 3-D structure—"we bridged for the first time the resolution gap between low-resolution electron microscopy reconstructions that provide overall shape and high-resolution crystal structures that provide the precise positioning of all atoms," Hoelz says.
With these structures known, the mapping of the rest of the NPC's symmetric core came quickly. "It is just like when solving a puzzle," he says. "By placing the first piece confidently, we knew that we would eventually be able to place all of them."
As described in the new paper, Hoelz's research group now has solved the crystal structures of the last remaining components of the symmetric core's inner ring and determined where all of the rings' pieces fit in the NPC's overall structure.
To do this, the team had to first generate a complete "biochemical interaction map" of the entire symmetric core. Akin to a blueprint, this map describes the interconnections and interactions of all of the proteins, as part of a larger cellular machine. The process involved genetically modifying bacteria to produce purified samples of each of the 19 different protein subunits of the NPC's symmetric core and then combining the fragments two at a time inside a test tube to see which adhered to each other.
The team then used the completed interaction map as a guide for identifying the inner ring's key proteins and employed X-ray crystallography to determine the size, shape, and position of all of their atoms. X-ray crystallography involves shining X-rays on a crystallized sample and analyzing the pattern of rays reflected off the atoms in the crystal. The team analyzed thousands of samples at Caltech's Molecular Observatory—a facility developed with support from the Gordon and Betty Moore Foundation that includes an automated X-ray beam line at the Stanford Synchrotron Radiation Laboratory that can be controlled remotely from Caltech—and the GM/CA beam line at the Advanced Photon Source at the Argonne National Laboratory.
"We now had a clear picture of what the key jigsaw pieces of the NPC looked like and how they fit together," says Daniel Lin, a graduate student in Hoelz's lab and one of two first authors on the study.
The next step was to determine how the individual pieces fit into the larger puzzle of the NPC's overall structure. To do this, the team took advantage of an electron microscopy reconstruction of the entire human NPC published in 2015 by Martin Beck's group at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany. The images from Beck's group were relatively low resolution and revealed only a rough approximation of the NPC's shape, but they still provided a critical framework onto which Hoelz's team could overlay their atomic high-resolution images, captured using X-ray crystallography. The NPC is the largest cellular structure ever pieced together using such an approach.
"We were able to use the biochemical interaction map we created to solve the puzzle in an unbiased way," Hoelz says. "This not only ensured that our pieces fit in the electron microscopy reconstruction, but that they also fit together in a way that made sense in the context of the interaction map."
Hoelz said his team is committed to solving the remaining asymmetric parts of the NPC, which include filamentous structures that serve as docking sites for so-called transport factors that ferry molecules safely through the pore and other cellular machineries that are critical for the flow of genetic information from DNA to RNA to protein.
"I suspect that things are going to move very quickly now," Hoelz says. "We know exactly what we need to do. It's like we're climbing Mount Everest for the first time, and we've made it to Camp 4. All that's left is the sprint to the summit."


http://phys.org/news/2016-04-biochemists-cell-dna-gatekeeper.html

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