January 06, 2016
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.
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.
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