Scientists Probe Role of Nucleus in Protein Synthesis

EXPORT MECHANISM: The tRNA must mature inside the nucleus before export. tRNAs are aminoacylated, and only tRNAs charged with an amino acid are exported efficiently. Export occurs when the tRNA is carried through the nuclear pore by a complex of exportin-t and Ran-GTP. As the project to sequence the human genome moves relentlessly ahead, molecular biologists are hard at work posing the next important questions about protein synthesis. Soon scientists will know every single gene and every RNA, notes Anita Hopper, professor of biochemistry and molecular biology at the Pennsylvania State University College of Medicine in Hershey, Pa. "But then [learning] how this is used within the architecture of the cell--the cytosol and the nucleus--is much more difficult," she says. Questions being explored include how the RNAs get out of the nucleus and how they get to the ribosomes, organelles in the cytoplasm where the proteins are actually assembled. Many scientists are particularly interested in transfer RNA, or tRNA--RNA molecules that assemble the proteins specified by DNA in the ribosomes. A major focus of such work is understanding the role of the nucleus. "These steps of gene expression have historically been the most poorly understood," observes Christine Guthrie, professor of biochemistry at the University of California, San Francisco. It's been only in the past three to five years, she points out, that researchers have begun to focus on such issues as transport out of the nucleus into the cytosol. Green fluorescent proteins and confocal microscopy are technological advances that allow observation of protein movement in real time. "The part of transport that was historically most lagging is RNA transport. I think that had been lagging because people have not had the wherewithal to look at the RNA directly," she says. Research on HIV led to the exploration of the role of the nucleus in exporting RNA to the cytosol, notes Guthrie. "One of the key viral strategies was to produce a viral protein called rev, whose specific task seems to be to transport a class of RNA that is normally never transported: unspliced viral RNA. That really set up ... [what] is still kind of the paradigm in the RNA export field. Once you have a viral paradigm for something, that suggests the cell has machinery to do a similar thing that the virus has exploited," Guthrie comments. Christine Guthrie Understanding the process of molecular transport through the nucleus means understanding a basic cellular process, says Hans Ris, professor emeritus of zoology at the University of Wisconsin, Madison. Not only do molecules necessary for cellular differentiation pass through the membrane, but infection-causing viruses also use the portals in the membrane. James Dahlberg Moreover, notes Ris, the transport process is highly conserved. Consequently studies of it in the yeast Sacchromyces cerevisiae, such as the one being done by Guthrie, and in Xenopus by James Dahlberg, professor of biomolecular chemistry at the University of Wisconsin, Madison, have implications for mammalian cells. Using oocytes from the frog (Xenopus laevis), Dahlberg and postdoctoral fellow et Lund,1 found that aminoacylation of tRNA occurred in the nucleus prior to its moving to the cytoplasm, where proteins are assembled. Aminoacylation is the adding of an amino acid to the tRNA molecule. This is part of the process of making a mature tRNA. et Lund "They [Lund and Dahlberg] show that amino acids [go on] tRNA in the nucleus, and that was always thought to be restricted to the cytosol," says Hopper.2 Guthrie called the finding "a stunning surprise." "If Lund and Dahlberg's report is right, clearly we have to rethink what a nucleus does," adds Hopper. One key aspect of the research, notes Dahlberg, is the finding that the nucleus acts as a proofreader to make sure that aminoacylated tRNAs get to the cytoplasm. "If [tRNA] goes out in its damaged form it can do more harm than good because it might actually block the cell's ability to carry out the normal cytoplasm functions," says Dahlberg. If mutant tRNAs got out and into the ribosome and blocked good tRNAs from binding, the system would gradually deteriorate. This scenario hasn't been studied yet. "That obviously is an implication," affirms Paul Schimmel, professor and member of the Skaggs Institute for Chemical Biology at the Scripps Research Institute. "Certainly in mitochondria there are many pathologies associated with mitochondrial tRNA mutations. So we imagine by extension there certainly may be such uncharacterized pathologies resulting from this sort of thing. Or they may be lethal, so you never see them," he adds. But other recent research by Gert-Jan Arts, et al.3 suggests that aminoacylation in Xenopus is not essential for tRNA to leave the nucleus. And Lund and Dahlberg also claim that aminoacylation is "not essential" and argue that unaminoacylated tRNA can be exported from the nucleus, "albeit more slowly." Paul Schimmel "It's a luxury to be able to proofread. Maybe you can live without that luxury," says Hopper. Schimmel notes this research is all in vitro. The question of whether aminoacylation in the nucleus is essential in a whole organism has not yet been asked. Aminoacylation involves 20 amino acids and an enzyme for each to put it on the appropriate tRNA, explains Hopper. The enzymes are known as aminoacylation tRNA synthetases. But there may be 40 tRNAs, with several requiring leucine, for example. "One enzyme will recognize all the right tRNAs and put the right amino acid on the right tRNA. They have to be very smart enzymes. They have to select the right tRNA and the right amino acid," she says. Aminoacylation establishes the genetic code, notes Schimmel. Beyond establishing a proofreading function in the nucleus, Lund and Dahlberg's findings suggest something quite significant, he says. "It implies that the enzymes, the aminoacylation tRNA synthetases, must somehow get into the nucleus [from the cytoplasm]. It's shocking to think these proteins, which have always been thought of as restricted to the cytoplasm, must be getting into the nucleus." Anita Hopper While Lund and Dahlberg haven't demonstrated that these enzymes do in fact get into the nucleus, it's clear that must happen for the aminoacylation to occur, says Schimmel. "It suggests there's a huge amount of traffic going in and out of the nucleus. It's like some kind of freeway," he remarks. And that of course raises more questions. Those enzymes are made in the cytoplasm, Hopper comments. And they get into the nucleus. "Nobody has a clue how that happens. Nobody has a clue if that's regulated in any way," she observes. "I'm sure that will be a very active area of research coming up." "That whole business is one area of exciting research right now," says Schimmel. Already at least one pathway has been found. "One of these pathways is thought to possibly involve a protein called Arc1p, which appears to stick to one of the RNA synthetases," he reports. The protein was first reported to function this way by Simos et al.4 Another molecule in the nucleus, Ef1 * , is also of interest, notes Schimmel. It may play a role in exporting tRNA. The explosion of information from the Human Genome Project will also provide information for researchers exploring nuclear transport, remarks Schimmel. Part of that information deluge will include protein sequences. But the question that needs to be answered is, "What do these sequences do?" "One of the first things you do is ask if these sequences are related to sequences of other proteins that we do know something about. As you see these things, you'll ask if they are involved in trafficking the synthetases. That way you build a broader and broader platform to work your way through the system," he explains. Another genome-related approach leads to using protein structure to explore the nuclear transport proteins. Instead of working directly on protein sequences, says Schimmel, scientists can use protein structure. "Why not get a three-dimensional structure of those proteins? Maybe you can make structural relationships. Maybe you start to guess the function if you knew the structure. I think there's going to be a burgeoning role for X-ray crystallography to determine a lot of these structures. Some of these will be proteins involved in this nuclear trafficking," he comments. Another area of research concerns understanding the proofreading mechanism itself. "What is it about aminoacylation that is important for the proofreading?" asks Dahlberg who says that research is now under way in his lab. It seems likely as scientists continue to probe the role of the nucleus and as information from the Human Genome Project comes in that more surprises such as those uncovered by Dahlberg will result. Harvey Black (73773.2227@compuserve.com) is a freelance science writer in Madison, Wis. E. Lund, J.E. Dahlberg, "Proofreading and aminoacylation of tRNAS prior to export from the nucleus," Science, 282:2082-5, Dec. 11, 1998. A. Hopper, "Nuclear functions charge ahead," Science, 282:2003-4, Dec. 11, 1998. G-J. Arts et al., "The role of exportin -t in selective export of mature tRNAs," The EMBO Journal, 17:7430-41, Dec. 1998. G. Samos et al., "The yeast protein Arc1p binds to tRNA and functions as a cofactor for the methionyl-and glutamyl-tRNA synthetases," The EMBO Journal, 15:5437-48, 1996.

Scientists Probe Role of Nucleus in Protein Synthesis

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