New & Noteworthy

Acetylation Regulates the Nuclear Pore Complex

July 15, 2022

Several recent studies have done an excellent job characterizing the architecture of the yeast nuclear pore complex (NPC). With so much new information, researchers are now able to ask probing questions about how NPCs mediate communication between the nucleus and the rest of the cell. Considering that signals perceived from the environment need to reach the transcriptional machinery in the nucleus, and that mRNA transcripts made in response to these signals need to get back out to get translated, the NPC has a lot of communicating to do. A study in a recent issue of the EMBO Journal by Gomar-Alba et al. makes strong strides toward understanding how this communication is accomplished.

On the nuclear side of the NPC resides a substructure called the nuclear basket that has previously been shown to play roles in regulating gene expression and mRNA export. The nuclear basket also interacts with lysine acetyltransferases (KATs) and deacetylases (KDACs) that are best known for modulating transcription via reversible acetylation of histones in chromatin. These enzymes, however, can also act on non-histone proteins and have been linked to numerous cell processes, including DNA damage repair, cell division, and signal transduction.

From Gomar-Alba et al, 2022

Promotion of mRNA export is another function linked to acetylation, specifically by the NuA4 histone acetyltransferase complex, for which the catalytic subunit is Esa1p. Gomar-Alba et al. show in this recent study that Esa1p is the primary lysine acetyltransferase that promotes cell cycle entry—and also that it acetylates the nuclear pore protein Nup60p.

Acetylation of Nup60p promotes mRNA export, which in turn triggers fast entry into the Start phase of the cell cycle, thereby promoting cell division. Nup60p accomplishes this increased export by recruiting the TREX-2 transcription-export complex to the nuclear basket once Nup60p becomes acetylated. The deacetylated form of Nup60p has lower affinity for TREX-2 and thus mRNA export decreases. Deacetylation of Nup60p is performed by Hos3p, which acts in opposition to Esa1p in removing Esa1p-transferred acetyl residues.

Perhaps the most intriguing finding in this study is that Hos3p localizes primarily to daughter cells after cell division, causing displacement of the mRNA export complex and thus slowing G1/S phase transition. This action prevents premature division in the smaller daughter cells, as they require additional growth to meet the size control threshold for entry into a new cell cycle. Accomplishing this level of control with a single enzyme acting on a single nuclear pore protein is a simple, elegant solution.

As usual, studies in yeast make enormous impact on understanding cell division in other organisms.

Categories: Research Spotlight

Tags: acetylation, cell cycle control, mRNA export, nuclear export, nuclear pore, Saccharomyces cerevisiae

The Remarkable Nuclear Pore Complex

June 24, 2022

The nuclear pore complex (NPC) is a complicated assembly embedded in the nuclear envelope that has the ability not only to assemble and disassemble quickly, but to adapt to changing needs for transport of macromolecules. The critical function of this elaborate complex has led researchers to invest intensive study, which has recently yielded remarkable new understanding.

In a January study in Cell, Akey et al. describe resolving the yeast nuclear pore complex to astounding detail. Using both in situ and isolated complexes, they dissect the layered organization of the pore to characterize the flexible inner ring, the adaptin-like central layer, and then the membrane-interacting layer that anchors the complex.

from Akey et al., 2022

Each of these layers employs complex protein connections that together form “spokes” in the pore. The authors speculate that the multiple layers and flexible connectors provide the means for NPCs to assemble and disassemble as quickly as they do, giving the ability to react to cell cycle stages and environmental conditions.

from Akey et al., 2022

Interestingly, upon close examination of crystalized structures, the authors observed that yeast has both single and double outer rings. The double outer ring has been observed in two other fungi to date and appears to represent a functional variant.

The double outer ring in a subset of yeast NPCs, from Akey et al., 2022

Upon further examination, the authors identified a third variant in yeast and were able to show that the variants co-exist in cells. One variant has two single outer rings that frame the inner ring, a second form has a single ring on the cytoplasmic surface and a double ring on the nuclear surface (both with nuclear baskets); while a third variant has two single rings, no baskets, and is specifically enriched over the nucleolus.

NPC variants in the same cell, from Akey et al., 2022

These variants provide further clues as to how the NPC might assemble as modular structures with multiple forms that adapt to different conditions. Further, the inner ring appears to have the ability to dilate and contract to allow smaller versus larger macromolecules to pass through, thereby adding another means of adaptability.

Understanding the yeast nuclear pore complex provides a foundation for understanding the eukaryotic NPC in general. A paper released this past week by Petrovic et al. in Science looked closely at just this set of relationships, comparing the human NPC to both the S. cerevisiae and Chaetomium thermophilum fungal NPCs. They show how, despite low conservation of sequence among nucleoporins and the other components of the pore complex, there is strong evolutionary conservation of the linker-scaffold architecture between humans and fungi. Once more, studies on model organisms throw bright light on the inner workings of our own cells.

Categories: Research Spotlight

Tags: nuclear pore, nucleocytoplasmic transport, Saccharomyces cerevisiae, yeast model for human cells

Clearing Customs in the Nucleus

January 06, 2016

Getting through the nuclear pore is like going through customs at the airport. And now we can see the mRNA make this journey in real time. Image from Wikimedia Commons.

Going through customs at the airport is a necessary evil. Once off the plane, you need to stand in line, scan for an open station, have various forms looked over and possibly stamped before you can pass through the airport doors and get into a new country.

And of course if there is anything wrong, you can be sent back to get your papers in order. A pain but it does help protect people.

Things work pretty similarly in the nucleus. The mRNA disembarks off the DNA, gathers up a set of proteins, and heads for the nuclear pore. There its proteins are checked and if everything is in order, it is allowed to proceed to the cytoplasm. And if there are problems, it is denied entry.

A couple of new studies out in the Journal of Cell Biology use imaging microscopy to give us a close up view of the bustling airport that is the nucleus of a yeast cell. It is utterly fascinating.

Both studies showed that mRNAs often hang out at the nuclear envelope, pausing at a nuclear pore and then sometimes moving to a new one. And that factors both in the nuclear pore and bound to the mRNA affect this scanning of the nuclear envelope.

The basic strategy with both studies is to fluorescently label specific mRNAs in a live yeast cell and follow its journey from the nucleus to the cytoplasm. To do this, they also needed to fluorescently label the nuclear pores, the custom stations in the nuclear envelope.

They labeled the mRNA using the bacteriophage PP7 RNA-labeling system. Basically, they load up the untranslated region (UTR) of a specific gene with sequences that form specific loops. Once transcribed, these loops are then bound by fluorescently labeled PP7 coat protein. Now they can track this labeled mRNA.

To more easily track mRNAs, they chose low expressing genes. That way they could follow a single mRNA more easily. They also needed to get rid of the yeast cell wall so they could see inside the cell better.

Overall they found that at least in yeast, the mRNA takes around 200 milliseconds to get exported to the cytoplasm. Very little of this time is spent in the nucleoplasm; the mRNA very quickly makes its way to a nuclear pore.

Once there things slow down. The mRNA stays at a nuclear pore or slides along the nuclear envelope to a different pore in a process the authors call scanning. Eventually the lucky successfully make it through the pore to the cytoplasm where they can seek out a ribosome for translation. Around 90% of the mRNAs they studied made it through.

They had a couple of different ideas about why the mRNA hangs around the nuclear envelope for so long. One is that the extended stay at the pore is to make sure everything is in order with the mRNA. It can’t pass through customs unless all of the right forms have been filled out properly.

Another possibility is that by scanning it is looking for a nuclear pore that is competent for exporting. It has to search for an available customs agent.

Now that the authors had established a system to look at mRNA export, they next set out to see which factors play important roles. As you might guess, mucking with parts of the nuclear pores or the proteins that bind the mRNA can throw a monkey wrench into the process.

In the first study, Smith and coworkers looked at what happens to the process when one of the key mRNA binding proteins, Mex67p is mutated. This protein is known to interact with the nuclear pore.

Losing the nuclear basket means mRNAs fall away from nuclear pores more easily. It is like getting to the head of the line and then having it close for lunch. Image from thornet on flickr.

It has also been proposed that Mex67p is important in making sure the trip through the pore is one way. Once the mRNA goes through, it releases Mex67p which makes the mRNA let go of the cytoplasmic side of the nuclear pore. The imaging studies here confirmed that Mex67p is indeed important for mRNA directionality.

Using a temperature sensitive mutant of Mex67p the researchers found that the mRNA they tracked stayed at the nuclear envelope about three times longer than in a wild type strain. The process was also much less efficient with only 32% making it to the cytoplasm instead of the 90% seen in the wild type strain. And of the 14 mRNAs which failed to make it through the pore, 7 headed back through the pore to the nucleus.

In the second study, Saroufim and coworkers concentrated on a part of the nuclear pore called the nuclear basket. This is the first part of the nuclear pore that the mRNP, the mRNA plus its proteins, encounters.

They found that deleting or mutating two key parts of the nuclear basket, MLP1 and MLP2, made the mRNA linger for a shorter time at the pore. The mRNA no longer scans the nuclear envelope.

But that didn’t mean the mRNA passed through to the cytoplasm more quickly. No, it just tended to fall back into the nucleoplasm and then have to reattach more often.

It is as if you had to deal with a customs agent who keeps sending you back into the airport. Or agents who keep putting up the “Out to Lunch” sign as soon as you get to the head of the line.

These two studies give researchers a way to study mRNA export in live cells in real time. As we piece together which proteins play what role, we will get a better handle on this important part of gene expression.

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: mRNA export, nuclear pore, Saccharomyces cerevisiae