CELL BIOLOGY

Study Adds Feather to mRNA Cap

Understanding Cap’s Role in Export Could Facilitate Protein Production in Biotech Industry

Almost every aspect of the cell can trace its beginning to that moment when a lone strand of mRNA slips through the nuclear membrane into the arms of a ribosome, where it is translated into a protein. Over the past few years, researchers have been piecing together a story of how mRNAs make their exodus from the nucleus into the cytoplasm. A team of HMS researchers has recently added a surprising twist to the tale.

Photo by Graham Ramsay

“It costs a lot of money to make proteins. If we could enhance the export efficiency, and that turns out to enhance overall production of the protein, that could have a huge benefit for everybody’s research—not just for biotech companies but for us,” said Robin Reed (center), shown with Hong Cheng (right) and Kobina Dufu.

Single-stranded though they may be, mRNAs are hardly lonely molecules. From the moment they are copied from the DNA, they are surrounded by retinues of proteins that successively groom, inspect, and shepherd them out of the nucleus. These helper proteins turn out to be highly versatile. Nascent mRNAs are unwieldy and vulnerable molecules that must be equipped with a protective cap and tail. Large stretches of noncoding sequences, or introns, must be cut from their middle and the ends rejoined. Finally, the messengers have to be checked to be sure they carry the correct genetic instructions. Some of the proteins surrounding the cap have been shown to perform extra duties, helping to splice and even inspect the mature mRNAs. But their multitasking was thought to stop there.

It now appears that the cap and its associated proteins also play a role in the export of mRNA, and a fairly direct one at that. Hong Cheng, Robin Reed, and colleagues, working in human cells, found that the cap not only recruits but is physically linked to a critical set of export proteins, known collectively as the transcription export (TREX) complex. The findings by Cheng, HMS research fellow in cell biology, Reed, HMS professor of cell biology, and colleagues appear in the Dec. 29 Cell.

“There were some hints that the cap might contribute to export, but it had not been tied to this TREX complex,” said Stephen Buratowski, HMS professor of biological chemistry and molecular pharmacology, who was not involved with the research.

Though unexpected, the findings exemplify a growing consensus in the field of mRNA biology. Over the past decade, researchers have discovered that the various phases of mRNA processing, such as capping, splicing, and inspection, are functionally linked. “The messenger RNA does not just float around from place to place to pick up modifications and the proteins that it needs to be exported,” said Buratowski. “There is a very direct series of steps, and one step in a very physical way leads to the next one.”

Increasing Export
Tracing TREX’s role in this sequence of events could have practical ramifications. “For biotech companies or anyone who wants to express large amounts of protein the question is, could better recruitment of the TREX allow you to export more RNA and therefore make more protein,” said Reed.

In addition, defects in one of the TREX proteins have been associated with disease, specifically, some breast cancers. A better understanding of how TREX functions could lead to new approaches to understanding and possibly treating such cases.

Reed has been following the comings and goings of mRNA molecules and their associated helper proteins for years. In the late 1990s, she and colleagues, following up on work in yeast, identified the human TREX complex and set out to see how exactly it worked. Reed suspected that TREX—which consists of two proteins, Aly and UAP56, and a set of five proteins known collectively as THO—might be linked in some way to the mRNA splicing machinery. Soon after, she and colleagues found Aly in the spliceosome, the complex of proteins that snips out introns (see Focus, Sept. 29, 2000).

Joint Interest
Meanwhile, researchers at Brandeis University had discovered another intriguing set of proteins, this one located close to where the newly spliced mRNA exons are joined. Some of the proteins in this group, the exon junction complex (EJC), were shown to play a role in inspecting the newly spliced mRNA, a process called nonsense-mediated decay (NMD). “There was so much excitement about the EJC,” said Reed. “Everybody wanted to know what else was in it.” Thinking that mRNA inspection and export were linked, many began looking for TREX proteins in the EJC. It was not long before researchers in other labs reported that Aly and UAP56 were present.

No longer under the hat. Proteins surrounding the mRNA cap play an unexpected role in exporting the mRNA. After the pre-mRNA is spliced, the cap recruits the export complex TREX to the 5' end of the mRNA molecule. One of the TREX proteins, Aly, is bound by a cap-binding protein (CBP), which may play a role in ushering the mRNA through the nuclear pore and into the arms of the ribosome.

“We wanted to add to that and say THO is there too,” said Reed. Following previous researchers, Cheng decided to cut human mRNA into bits, including an EJC-containing fragment, mix them with antibodies for THO, and see which ones coprecipitated. For controls, she used antibodies to Aly and to a protein found in the NMD complex. The NMD antibody coprecipitated with the EJC all right, but to her surprise, the Aly control did not. Instead, it appeared with a cap-associated protein (CAP) fragment. “We thought that was really strange,” Cheng said.

It turned out, UAP56 and THO also associated only with the CAP fragment. “We were really puzzled,” said Reed. Confusing matters even more, previous work had suggested that the cap was not required for the export of mRNA. These studies, it turns out, had a checkered history. Initial experiments, carried out on spliced mRNAs, showed that the cap was required for export. It was only in subsequent studies—performed, as had become the fashion, on cDNAs—that the cap appeared not necessary.

Reed and Cheng, working with Kobina Dufu, decided to repeat the studies. Sure enough, when the cap was removed, TREX did not bind to the spliced mRNA. In the case of cDNAs, TREX bound whether or not the cap was present. “We were stunned,” Reed said. “They really had the story right in the very beginning.” Dufu, an HMS graduate student, then showed that there is a direct physical interaction between one of the TREX proteins, Aly, and one of the cap-associated proteins, cap binding protein 80 (CBP80). “Again we were stunned because it was just what we would’ve expected,” she said.

It is an elegant arrangement, said Buratowski. “The cap is the first really distinctive mark of a messenger RNA. It is a very nice handle for all these subsequent steps to latch on to,” he said.

For Reed and colleagues, the lesson of their work—which looked for one thing and found something else entirely—is also quite simple. “The most important thing is to not make assumptions,” Reed said. “That gets you into a lot of trouble. If you discover something and really want it to be that way, you can find it to be true just by the experiments you do. We are very cautious not to do that. We look from as many directions as we can.”

—Misia Landau

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